SIGNIFICANCE AND RISKS OF CELL OXIDANTS [REVIEW WORKS] Ph ton eBooks

SIGNIFICANCE AND RISKS

OF CELL OXIDANTS

[REVIEW WORKS]

Author: Dr. Caser Ghaafar Abdel, PhD

Iraq

Ph ton eBooks

All Rights Reserved with Photon.

UBN: 015-A94510112002 Edition: I Impact Index: 3.47

Preface

Recently, it is very difficult for researcher, student, and even instructors to cope with numerous numbers of published manuscripts, owing to the sites where these manuscripts are produced, besides their languages have been published by. This book aimed to introduce manuscripts related to the significance and risk of cell oxidants to aid workers in this field of science. Therefore, this book included the following subjects: 1. Homeostasis mechanisms of cell oxidants at normal plant growth to achieve the optimal development, the balance between oxidants and antioxidants. 2. Oxidant roles in organs developments. 3. Oxidant roles on cells and organ functions. 4. Oxidant roles on genome expressions to match with the overwhelming environments. 5. Oxidant roles on plants hormones homeostasis under fluctuated environments. 6. Oxidants roles on plant defense systems to adapt and tolerate stresses. 7. Oxidant roles on programmed cell death and apoptosis. 8. Mechanisms of DNA damage by oxidants and their consequences. 9. Mechanisms of RNA damage by oxidants and their consequences. 10. Mechanisms of cellular membrane damage by oxidants and their consequences. I hope that readers will appreciate these review collections, and I really do apologize to authors that I failed to get their permissions for including their finding, ideas and conclusions.

Dr. Caser G. Abdel

Ph ton eBooks

Author’s Affiliations

Book Title: Significance and Risks of Cell Oxidants [Review Works]

Dr. Caser Ghaafar Abdel, PhD was borne in Jadedat Alshat 25Km north the Iraqi Capital Baghdad. BSc, Baghdad University, Agriculture College 1977, MSc, Bath University, England, UK, School of Biology and Biochemistry, 1983, and PhD. From Mosul University, Agriculture College 1997. Thirty three years’ experience in teaching and research doing at several Iraqi Universities. 70 published manuscripts, supervising 6 graduated MSc and four graduating PhD and two published books in Abiotic Stresses.

Contents

CHAPTER 1 Page No.

Stresses Causes Programmed Cell Death (PCD)

1. STRESSES CAUSES PROGRAMMED CELL DEATH (PCD) 1

1. A. WALL DEGRADATIONS 1

1. A. I. POLYSACCHARIDES 2

1. A. II. POLYPHENOLIC 4

1. B. PROTOPLAST DEGRADATIONS 4

1. B. I. CHLOROPLAST DAMAGE BY OXIDANTS 4

1. B. I. 1. PHOTOSYNTHETIC (psbA) AND NONPHOTOSYNTHETIC (trnQ)

GENES

1. B. I. 2. PLASTID TRANSCRIPTIONAL KINASE (PTK) 5

1. B. I. 3. PLASTID ENCODED POLYMERASE (PEP) 6

1. B. I. 4. SPECIFIC SENESCENCE ASSOCIATED GENE (SAGS) 6

1. B. II. REPAIR AND DAMAGE IN CHLOROPLAST DNA 7

1. B. III. CHLOROPLAST ROLE IN PROGRAMMED CELL DEATH 8

1. B. IV. CHLOROPLAST APOPTOTIC-LIKE PCD 9

1. B. V. PHYTOCHROMES INITIATE PCD 10

1. C. MITOCHONDRIA, 10

1. C. I. TRICARBOXYLIC ACID CYCLE (TCA) 11

1. C. II. SENESCENCE OF MITOCHONDRIA INDUCES PCD 11

1. C. III. MITOCHONDRIAL ENZYMES LEAD TO PCD 12

1. C. III. 1. PROTEIN KINASES 12

1. C. III. 2. CYTOCHROME C RELEASE 12`

REFERENCES 13

CHAPTER 2

Stresses Damaged DNA

2. STRESSES DAMAGED DNA 21

2. A. OXIDATIVELY DAMAGED DNA 21

2. A. I. PRODUCT OF PHOTO DAMAGED DNA 21

2. A. II. PRODUCT OF OXIDATIVELY DAMAGED DNA 22

2. A. III. FRAGMENTATION OF DNA BASES 23

2. A. III. 1. DNA FRAGMENTATION BY OXIDANT 24

2. A. III. 2. DNA FRAGMENTATION MECHANISMS 24

2. A. III. 3. DNA FRAGMENTATIONS ROLE IN PCD 25

2. A, III. 4. ATP ROLE ON DNAs FRAGMENTATIONS 26

2. A. IV. CROSSLINKS DAMAGE 26

2. A. IV. 1. INTERSTRAND AND INTRASTRAND CROSS LINKS 27

16. A. V. POLYPLOIDITY DAMAGE 29

2. A. VI. DEMONSTRATED RISK OF DAMAGED NUCLEIC ACIDS 30

2. A. VII. AGENT CAPABLE TO INDUCE DNAs DAMAGE 31

2. A. VIII. DNAs DAMAGE BY CADMIUM METAL 32

2. A. IX. DNA DAMAGED BY REACTIVE OXYGEN SPECIES 33

2. A. X. OXIDATIVELY DAMAGED MITOCHONDRIAL DNAs 34

2. B. OXIDATIVELY DAMAGED RNA 34

2. B. I. RIBNUCLEOTIDE MODIFICATIONS 36

2. B. II. NITRIC OXIDE ROLE IN RNA DAMAGE 37

2. B. III. ALKYLATION DAMAGE OF RNA 37

2. B. IV. DEGRADATION MECHANISM FOR DAMAGED RNAs 38

2. B. IV. 1. EXONUCLEASES 38

2. B. IV. 2. POLY (A) POLYMERASES AND HELICASES 38

2. C. DNA DAMAGE PROTECTION AND REPAIR RESPONSES 40

2. C. I. REPAIR OF OXIDATIVELY DAMAGED DNAs 41

2. C. II. LIGASES ENZYMES 41

2. D. ALKYLATION REPAIR MECHANISM OF DAMAGED RNA 46

2. D. I. PHOTOLYASES REPAIR OF CYCLOBUTANE AND PYRIMIDINE 46

2. D. II. REPAIR MECHANISM FOR OXIDIZED NUCLEOTIDES 46

2. D. III. DEGRADATION OF DAMAGED RNAs 47

2. D. IV. CYTOPLASMIC REPAIR OF ULTRA-VIOLET OXIDATIVELY DAMAGED

RNAs 47

2. D. V. PROTEIN ROLE IN REPAIRING OXIDATIVELY DAMAGED RNAs 48

2. E. PROTECTION OF DNAs DAMAGE 49

2. E. I. DNA PROTECTIONS FROM ROS 50

REFERENCES 51

CHAPTER 3

Altrastructural Alteration of Bichemical Compounds Caused by Abiotic

Stresses

3. ALTRASTRUCTURAL ALTERATION OF BICHEMICAL COMPOUNDS CAUSED

BY ABIOTIC STRESSES 66

3. A. LIPID PEROXIDATIONS 66

3. A. I. INTRODUCTION

3. A. II. LIPID PEROXIDATION IN CELL ORGANELLES 67

3. A. III. 4-HYDROXY-2-NONENAL THE PROTEIN INHIBITOR 68

3. A. IV. MINERAL ROLES IN LIPID OXIDATIONS 71

3. A. V. MEMBRANE LIPID OXIDATION 72

3. A. V. 1. MEMBRANE COMPOSITIONS 72

3. A. V. 2. MEMBRANE OXIDANTS 72

3. A. V. 3. LIPID OXIDATION DURING GERMINATIONS 74

3. A. V. 4. LIPID PEROXIDATIONS 74

3. A. V. 5. LIPID PEROXIDATION CAUSES PCD 76

3. B. PROTEIN DETERIORATIONS 76

3. B. I. PROTEIN BONDING DAMAGE 76

3. B. II. PROTEIN DETERIORATIONS BY OXIDANTS 77

3. B. III. CRYPTOGEIN 78

3. B. IV. PROHIBITINS 78

3. C. ENZYME ROLES 79

3. C. I. PROGRAMMED CELL DEATH INHIBITORS 79

3. C. II. ROS INDUCED AMELIORATING GENES 79

3. C. III. GRIM REAPER (GRI) 80

3. C. IV. MITOGEN ACTIVATED PROTEIN KINASE (MAPK) 81

3. C. V. KINASE GENES 81

3. C. VI. CASPASES 82

3. D. ANTIBIOTICS 83

3. E. APOPTOTIC-LIKE PCD (AL-PCD) 83

3. F. HORMONAL AND OXIDANT ROLES IN DEVELOPMENTAL CELL

APOPTOSIS 85

3. F. I. GENERAL CONCEPTS 85

3. F. II. HORMONAL REGULATIONS 85

3. F. II. 1. SALICYLIC ACID (SA) 85

3. F. II. 2. ETHYLENE ROLE 86

3. F. II. 3. JASMONIC ACID AND METHYL JASMONATE 87

3. F. III. HORMONAL COALITIONS 90

REFERENCES 91

CHAPTER 4

Stresses Implicated In Growth Limiting Factors 109

4. STRESSES IMPLICATED IN GROWTH LIMITING FACTORS 109

4. A. OXIDANT ROLES IN PROGRAMMED CELL DEATH 109

4. A. I. REACTIVE OXYGEN SPECIES (ROS) 109

4. A. I. 1. REACTIVE OXYGEN SPECIES IN TRIGGERING PCD 109

4. A. I. 2. ROS ROLE IN INITIATION AND SPREAD OF PCD 110

4. A. II. REACTIVE NITROGEN SPECIES (RNS) TRIGGERING PCD 111

4. A. III. COALITION OF ROS AND RNS IN TRIGGERING PCD 113

4. B. TEMPERATURE STRESSES 113

4. B. I. REACTIVE OXYGEN SPECIES ROLE IN TEMPERATURE STRESSES 113

4. B. II. REACTIVE NITROGEN SPECIES ROLE IN TEMPERATURE

STRESSES 113

4. C. WATER RELATION AND DROUGHT STRESS 115

4. C. I. OXIDANT ROLES IN DROUGHT STRESS 116

4. C. II. PLASMA MEMBRANE HYDRULIC CONDUCTIVITY UNDER LOW

TEMPERATURE 117

4. C. II. 1. H+ ATPase Activity 117

4. C. II. 2. AQUAPORIN 119

4. D. SALT STRESS 120

4. D. I. OXIDANT ROLES IN SALT STRESSES 120

4. E. LIGHT STRESS 121

4. E. I. DARKNESS ROLES ON TISSUE GENERATED ROS 122

4. E. II. HIGH LIGHT INTENSITY 123

4. E. III. PHOTORESPIRATION 124

4. E. IV. CHLOROPHYLL DESTRUCTIONS 125

4. E. V. UV CELL DIVISION 126

4. E. VI. CADMIUM STRESS 126

4. E. VI. 1. ROS PRODUCTION BY CADMIUM 126

4. E. VI. 2. CADMIUM INDUCE HORMONE BIOSYNTHESIS 128

4. E. VI. 3. MINERALS COMPETING CADMIUM ABSORPTION 129

4. E. VI. 4. GENE REGULATIONS 129

4. F. HERBICIDES IMPACTS 130

REFERENCES 130

CHAPTER 5

Oxidant Roles in Signaling 144

5. OXIDANT ROLES IN SIGNALING 144

5. A. OXIDANT SIGNALING ADVANTAGES 144

5. B. SIGNALING MECHANISMS 145

5. C. PROTEIN NITROSYLATIONS 147

5. D. ANNEXINS 149

5. D. I. ANNEXIN FUNCTIONS 149

5. D. II. ANNEXIN LOCALIZATIONS 149

5. E. OXIDANT SIGNALING IN CELL ORGANELLES 150

5. E. I. OXIDANT SIGNALLING IN PEROXISOMES 150

5. E. II. OXIDANT SIGNALLING IN CHLOROPLASTS 151

5. F. OXIDANT SIGNALING FOR GROWTH AND DEVELOPMENTS 151

5. F. I. NITROGEN FIXATION 151

5. F. II. ORGAN DEVELOPMENT 152

5. F. III. OXIDANT MEDIATING DEVELOPMENTAL SIGNALING 152

5. G. OXIDANT SIGNALING FOR BIOTIC STRESS RESISTANCE 153

5. H. OXIDANT SIGNALING FOR ABIOTIC STRESS RESISTANCE 154

5. I. OXIDANT SIGNALLING FOR STOMATA BEHAVIOUR 156

5. I. I. REACTIVE OXYGEN SPECIE SIGNALLING FOR STOMATA

BEHAVIOUR 156

5. I. II. REACTIVE NITROGEN SPECIE SIGNALLING FOR STOMATA

BEHAVIOUR 156

5. J. OXIDANT SIGNALING FOR HORMONAL METABOLISMS AND

FUNCTIONS 157

5. J. I. OXIDANT SIGNALING FOR AUXIN METABOLISMS AND

FUNCTIONS 157

5. J. II. OXIDANT SIGNALING FOR ABSCSIC ACID METABOLISMS AND

FUNCTIONS 161

5. J. III. OXIDANT SIGNALING FOR ETHYLENE METABOLISMS AND

FUNCTIONS 164

5. J. IV. OXIDANT SIGNALING FOR JASMONIC ACID METABOLISMS AND

FUNCTIONS 165

REFERENCES 165

CHAPTER 6

Oxidants Role in Developmental Processes 183

6. OXIDANTS ROLE IN DEVELOPMENTAL PROCESSES 183

6. A. OXIDANT INITIATE GENE TRANSCRIPTION AND

RELATIVE PROTEIN 183

6. A. I. ANNEXINS 183

6. A. I. 1. ANNEXINS IN CELL MEMBRANES 184

6. A. I. 2. ENVIRONMENT STIMULATE ANNEXINS 185

6. A. II. OXIDANT INDUCTIONS OF CALMODULIN GENES 185

6. A. III. OXIDANT INDUCTIONS OF DIAMINE OXIDASE 186

6. A. IV. NITRIC ACID AND NITRATE REDUCTASE 187

6. A. V. PROHIBITINS 188

6. A. VI. ASCORBATE PEROXIDASE 189

6. A. VII. METALLOTHIONEINS 190

6. A. VIII. CATALASES 192

6. B. OXIDANT MEDIATE DIFFERENCIATION OF REPRODUCTIVE

ORGANS 192

6. C. ROLE OF OXIDANTS IN SYMBIOSIS 195

6. C. I. NODULATION FACTOR 195

6. C. II. ROLE OF NITROGEN REACTIVE SPECIES (RNS) IN NITROGEN

FIXATION 196

6. C. III. ROLE OF REACTIVE OXYGEN SPECIES IN NITROGEN

FIXATION 197

6. C. IV. OXIDANT ROLE ON MYCORRHIZAL 198

6. C. V. ROS HOMESTASIS FOR OPTIMAL SYMBIOTIC 199

6. D. MESCELLENUOUS ROLES OFH2O2 201

REFERENCES 202

CHAPTER 7

Oxidant Homeostasis Inititates the Defense System 217

7. OXIDANT HOMEOSTASIS INITITATES THE DEFENSE SYSTEM 217

7. A. OXIDANT HOMEOSTASIS 217

7. A. I. ESCAVENGING ENZYMES 217

7. A. II. ESCAVENGING SOLUBLE PHENOLIC COMPOUND 218

7. A. III. THIOL ESCAVNGING OF SUGAR PRODUCEDROS 219

7. B. OXIDANT ROLE IN GENE EXPRESSIONS AND ENZYMES 220

7. B. I. ROLE OF REACTIVE OXYGEN SPECIES (ROS) 219 7. B. II. NITRIC OXIDE (●NO) 221

7. C. HYDROXYPROLINE CONTAINING PROTEINS 222

6. D. OXIDANT REGULATES DEFENSE GENES 222

7. E. OXIDATIVE BRUST 223

7. E. I. REGULATION OF OXIDATIVE BRUST 223

7. E. II. OXIDATIVE BRUST SIGNIFICANCE 223

7. E. III. PATHOGEN INFECTIONS INITIATE OXIDATIVE BRUST 224

7. E. IV. DEVELOPMENTAL OXIDATIVE BRUST 225

7. E. IV. I. REACTIVE OXYGEN SPECIES 225

7. E. IV. II. REACTIVE NITROGEN SPECIES 226

7. E. V. HYPERSENSITIVE RESPONSE 226

7. F. 8. F. OXIDANTS INDUCTION OF TOLERANCE 227

REFERENCES 228

CHAPTER 8

Oxidants Ameliorate the Adversity of Stresses 242

8. OXIDANTS AMELIORATE THE ADVERSITY OF STRESSES 242

8. A. CADMIUM DETOXIFICATIONS 242

8. B. CALCIUM MITIGATES STRESSES RISK 243

8. B. I. CALCIUM MITIGATES CADMIUM RISK 243

8. B. II. CALCIUM AND NITRIC ACID MITIGATE CADMIUM RISK 244

8. B. III. ROS, NO and Ca MITIGATE Cd ADVERSITY 245

8. B. IV. CALCIUM BINDING ANNEXINS 246

8. C. MECHANISM OF STRESS AMELIORATIONS 247

8. C. I. CYTOSOLIC CALCIUM 247

8. C. II. CALCIUM AND NITRIC ACID 248

8. C. III. CELLULAR MEMBRANES MODULATIONS BY OXIDANTS AND

CALCIUM 248

8. C. IV. PHOSPHORYLATIONS 249

8. C. V. INOSITOL TRIPHOSPHATE (IP3) 250

8. C. VI. CALCIUM CHENNEL 250

8. D. INDUCTION OF SALT TOLERANCE 250

8. E. INDUCTION OF COLD ACCLIMATION 251

8. F. INDUCTION OF DROUGHT TOLERANCE 252

8. G. INDUCTION OF OZONE TOLERANT 253

8. H. LIGHT ACCLIMATION 253

REFERENCES 253

GLOSSARY AND IDOMS 261

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1. Stresses Causes Programmed Cell Death

(PCD)

1. A. Wall Degradations

In lace plant, thinning of the cell wall is one of the first indicators of wall alteration during early stage 2 of PCD. Simply it might reflect an absence of the normal processes of wall loosening and deposition of wall components in living adjacent cells. Late in stage 2, however, scanning electron micrographs of perforation zones indicate that matrix wall components have been degraded, exposing a fibrillar material, and transmission electron micrographs give the appearance of ‘‘beads on a string’’ that correspond to the dimensions of the larger fibrillar material seen in the SEM. These images are similar to those for differentiating tracheary elements, where the early stages of perforation formation (degradation of the end wall of a single cell in this case) give first a pitted and then a fibrillar appearance before disappearing completely (Nakashima et al., 2000; Ohdaira et al., 2002). Electron micrographs of differentiating vessel elements indicate that the remaining primary cell wall of a differentiating tracheary element is also highly modified, becoming more fibrillar and less electron dense between the secondary wall thickenings (O’Brien, 1970). Similarly, during the formation of cortical aerenchyma in maize roots, degrading walls become thinner, fewer electrons dense, and more fibrillar (Campbell and Drew, 1979; Webb and Jackson, 1986), as do cell walls in the abscission layer of Phaseolus. This suggests that cell wall matrix components such as pectins and hemicelluloses are degraded early, initially exposing the fibrillar cellulose component (Sexton and Hall, 1974). The electron micrographs illustrating a progression of cell wall breakdown suggest that enzymatic degradation of pectin would be an early event during perforation formation in lace plant leaves. Gel diffusion assays showed that leaves at all stages of development had

pectinase activity comparable to that of the exogenous pectinase positive control, in contrast to the distilled water, boiled enzyme extracts, and non-senescent Arabidopsis leaf negative controls. Therefore, some pectinase activity is present in the leaf tissues of lace plant even at stage 1, before other physical evidence of PCD was detected. Pectinase activity during stages 2 and 3 corresponds well with the hypothesized removal of wall matrix components. Continued endogenous pectinase activity during stages 4 and 5 may reflect the degradation of cell walls of late-dying cells at the periphery of the perforation and continued activity, perhaps turnover, even in leaves judged to have completed expansion (Arunika et al., 2007). Alcian blue staining of whole mounts of living leaves also indicates the presence of pectins throughout the perforation formation process, as is seen for differentiating tracheary elements (Ohdaira et al., 2002). Similarly, immuno staining indicates the presence of both esterified and de-esterified pectins within the perforation zone (stages 2 and 3) and at the periphery of the zone (stages 4 and 5) (Willats et al., 2000; Clausen et al., 2003). No shift in the relative proportions of esterified and de-esterified pectins or differences between dying and healthy adjacent tissues was detected, indicating that de-esterification may not be a predominant pathway for pectin removal. Rather, other enzymes with pectinase activity, such as polygalacturonase and pectic lyase, may be operating, as often observed for the removal of pectins during fruit ripening (Stolle-Smits et al., 1999; Karakurt and Huber, 2002; Rose et al., 2003). In combination with the electron micrographs, these results indicate that pectinase activity could contribute to the disassembly of lace plant cell walls during stage 2 of perforation formation and could underlie the mechanical weakening of the cell walls that

Ph ton 2

allows rupture during stage 3 (Arunika et al., 2007). In addition, pectinase activity could contribute to cell wall removal during the late stages of perforation expansion. However, the lack of specificity of pectinase activity to stages 2 and 3, when wall degradation is most conspicuous, indicates that it is not the primary pathway for wall removal. Under natural growth conditions, numerous microorganisms are associated with lace plant leaves undergoing PCD during perforation formation, and these may contribute to wall removal (Gunawardena et al., 2004). Plants grown in sterile culture their cell wall degradation reflected endogenous cellular processes (Arunika et al., 2007). Using gel diffusion assays indicated that cellulase activity is present in stages 2 and 3 lace plant leaves developments, at a level similar to the cellulase positive control, but is absent earlier (stage 1) and later (stages 4 and 5). While it is perhaps surprising that detectable cellulase activity does not follow the same time course as pectinase activity, cellulose must be disassembled for mechanical disruption of the perforation site at stage 3. Once the cell walls in the perforation site are weak enough for rupture, cellulase activity may no longer be required, and the cell walls may disassemble more gradually over later stages through the activity of an unidentified pectinase or other enzymes. If cellulose microfibrils short and/or have weakened hemicelluloses cross-linking, they might be released into the aquatic medium in nature or into the culture medium under experimental conditions. If so, analysis of the carbohydrate content of the liquid medium might be useful for determine the products of cell wall breakdown, as was done for differentiating tracheary elements in the Zinnia mesophyll system (Nakashima et al., 2000). Additionally, cellulose activity might be detected with the viscometric method used to detect cellulase activity during formation of aerenchyma in maize root (He et al., 1996) and the abscission layer in bean (Del Campillo et al., 1990). Cellulase activity could be detected by immunolocalization, which has been used to detect cellulase activity during abscission layer formation in Phaseolus vulgaris (Gonzalez-Carranza et al., 1998). Deposition of suberin in cell walls at the perforation border perhaps the most intriguing feature of developmentally regulated PCD is that specific cells and tissues

seem to be fated to die, while adjacent cells do not perceive or respond to the cell death signals. This is most dramatic in instances where, prior to PCD, the tissue appears homogeneous, as in gynoecium primordial in male flowers of maize (Cheng et al., 1983; Calderon-Urrea and Dellaporta, 1999) or in the unfurling leaves of lace plant (Gunawardena et al., 2004). Sergueeff (1907) noticed that perforation zones are ringed with cells with brown-appearing cell walls. Because these brown cell walls resisted sulfuric acid treatment, Sergueeff hypothesized that the walls were modified by the deposition of suberin and that isolation of the perforation zone cells from surrounding living cells by the suberin barrier might cause the cell death within the perforation zone. 1. A. I. Polysaccharides

Sclerenchyma cells are dead because thick cell walls perform the mechanical function. Cork is constituted of characteristic cells with thick suberized layer of the cell wall. Suberin combined with lack of intercellular spaces, protects internal tissues against desiccation. The protoplast is no longer needed, and therefore, it is eliminated. The continuous growth of the stem is also result with the cell death. Cell division in the cambium layer causes cell death in the cork layer that is replaced with the ruptured epidermis and in parenchyma cells at the stem pith (PalaVan-Unsal et al., 2005). The enzyme polygalacturonase hydrolyzes the α-1,4-D-galacturonan backbone of pectic polysaccharides, and this increased activity coincides with pectin depolymerization (Hadfield and Bennett, 1998). During growth, pectin is secreted from the Golgi apparatus in an esterified form. Then,it undergoes deesterification as required at their destination in the wall, catalyzed by the activity of pectin methyl esterase. Deesterification significantly affects the physical properties of pectin, and the creation of free carboxyl groups during deesterification increases the charge density in the wall, influencing the activities of wall enzymes such as polygalacturonase and pectic lyase (Wakabayashi et al., 2000). Endo-β-1,4-glucanase can hydrolyze the 1, 4-β-D linkages between un-substituted linear glucans, and thus cellulose and xyloglucan within plant cell walls are potential substrates (Rose et al., 2003).

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Expansion enzymes weaken glucan–glucan interactions by disrupting hydrogen bonds at the cellulose–cross-linking glucan interface and appear to act in wall disassembly as well as in cell growth (Cosgrove, 2000). Similarly, cleavage and re-ligation of xyloglucans through the activity of xyloglucan endotransglycosylase may also function during cell wall degradation (Rose et al., 2003). In addition to these and other enzymes, non-enzymatic factors have been identified that may play a role in cell wall degradation. Two groups of reactive oxygen species (ROS), hydroxyl radicals (Fry, 1998; Fry et al., 2001) and hydrogen peroxide (H2O2) (Miller, 1986), may disrupt pectin and hemicellulose backbones. It was found that ROS degrade cell wall polysaccharides in vitro (Brennan and Frenkel, 1977; Fry, 1998; Fry et al., 2001), and evidence of ROS-mediated breakage of chemical bonds has been observed during fruit ripening (Fry et al., 2001) and during wall expansion in growing leaves (Rodriquez et al., 2002) and roots of maize (Liszkay et al., 2004). 1. A. II. Polyphenolic

PCD is typically localized in space and restricted in time, and tissues undergoing PCD can be directly adjacent to tissues that do not respond to the cell death signals. This is particularly striking when specific cell walls are degraded, yet neighboring walls retain structural integrity, such as seen for xylem differentiation in vivo (O’Brien, 1970), floral organ abortion (Cheng et al., 1983) and for perforation formation in lace plant. In lace plant, a cell wall modification was described that might play a role in resistance to wall-degrading enzymes (Gunawardena et al., 2004). Sergueeff (1907) reported that, before perforations form, a brown pigment is deposited that demarcates the future perforation site. The substance was identified as suberin because the brown regions were the only areas that resisted sulfuric acid digestion. Therefore, Sergueeff (1907) hypothesized that the suberized zone actually induces perforation formation because the suberin blocks the cells to the interior from receiving adequate nutrients, thus leading to their death. Suberin is a hydrophobic material impregnates the walls of specialized cells (Kolattukudy, 1984; Bernards, 2002). It is thought to prevent the passage of water and other materials through the apoplast. It consists of both a suberin polyphenolic domain and a

suberin polyaliphatic domain (Bernards et al., 2004).

Specific isoforms of peroxidase have been associated with wound-induced suberization (Bernards et al., 1999; Quiroga et al., 2000; Lucena et al., 2003). Bernards et al. (2004) suggested that the assembly of suberin polyphenolic domain involves cell wall-associated peroxidase enzymes that use hydrogen peroxide (H2O2) to oxidize phenolic suberin monomers, resulting in their polymerization. In lace plant, one of the programmed cell death processes has a highly unusual developmental function. It results in the formation of tiny openings in young, expanding leaves that later enlarge 10–20-fold to form conspicuous holes or perforations in mature leaves. These perforations are formed in highly predictable positions with respect to the lattice-like vein pattern and at a predictable stage of leaf expansion, making them suitable material for studying developmentally regulated PCD in plants (Gunawardena et al., 2006). PCD is initiated in a localized population of about 100 epidermal and mesophyll cells. It propagates to neighboring cells at the periphery of the perforation site. Finally is arrested about five cells from the vascular tissue. The developmental environment for perforation formation by PCD in lace plant is striking. Since, PCD is precisely localized adjacent to and concomitant with cells that are proliferating and growing (Gunawardena et al., 2004). Thus, cells outside the perforation site are undergoing wall expansion and remodeling, presumably through the activity of wall-loosening enzymes (Cosgrove, 2000), but they appear not to be affected by the processes that result in the complete wall degradation accompanying PCD within the perforation zone. Protoplast alteration consistent with the initiation of PCD and features consistent with the initiation of wall degradation occur coincidentally. Perforation zones are first evident microscopically as the loss of vacuolar anthocyanin coloration and an erratic pattern of cytoplasmic streaming, both thought to reflect changes in tonoplast permeability and a resultant lowering of cytoplasmic pH (Gunawardena et al., 2004). Arunika et al. (2007) found that the walls of cells forming the margin of the perforation were brown, unlike the colorless walls of living cells to the exterior. The same walls were resistant to acid digestion and positive for Sudan 7B and

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fluorol yellow 088, and they had weak auto fluorescence, indicating that both the aliphatic and phenolic components of suberin, respectively, were present. Staining for flavonoids with Naturstoffreagenz A, for lignin with phloroglucinol, and for tannins with DMB were all-negative, indicating that the primary wall modification is the deposition of suberin. In addition, suberized walls appeared more electron dense than no suberized walls in adjacent tissue, but there was no indication of a suberin lamella as in suberized endodermal, bundle sheath, or cork tissues (Kolattukudy, 1984; Schreiber et al., 2005). Although Arunika et al. (2007) found additional evidence for the presence of suberin in the walls of cells at the perforation periphery, the timing of suberin deposition does not support the hypothesis that suberization plays a causal role in the death of cells within the perforation zone (Sergueeff, 1907). Thus, it is more likely that suberin deposition plays other roles, such as forming an apoplastic barrier against the loss of organic solutes from the leaf apoplast or an antimicrobial barrier against microorganisms attracted to the nutrients that escape into the surrounding medium from cells undergoing PCD. Wall suberization may protect against endogenous cell wall disassembly enzymes, such as pectinase, that appear to act throughout perforation formation and leaf growth (Bernards, 2002; Razem and Bernards, 2003).

ROS are closely associated with PCD and may both provide an inductive signal and be one of the first manifestations of PCD (Orozco-Cardenas and Ryan, 1999; Houot et al., 2001; Dat et al., 2003; Gechev and Hille, 2005). In lace plant, histochemical evidence suggests that ROS are present within the perforation site at early stage 2 and throughout the perforation zone by late stage 2. Staining is especially strong in cells at the periphery of the perforation during stage 4, the stage at which suberin deposition appears to be initiated, and is still present in stage 5 leaves, indicating that H2O2 may be involved in the oxidation of phenolic suberin monomers, resulting in their polymerization. Thus, ROS could have multiple roles, including PCD initiation, wall degradation, and wall suberization in perforating lace plant leaves (Razem and Bernards, 2003; Bernards et al, 2004).

1. B. Protoplast Degradations

Ultra structurally, walls of cells within the perforation zone have thinned. During late stage 2, cytoplasmic streaming ceases in cells at the center of the perforation zone, and the protoplast collapses, although the nucleus and organelles initially remain intact. Nuclei in cells in the perforation zone become positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), an indicator of DNA fragmentation, a characteristic feature of PCD (Gunawardena et al., 2004). At this stage of development (early stage 2), cell walls in the perforation zone have signs of surface erosion and loss of Sudan 7B staining, indicating that cuticular material has degraded (Arunika et al., 2007). Cell walls show striking signs of degradation at the ultrastructural level and presumably represent zones of mechanical weakness, because the perforation sites rupture during stage 3, forming regular breaks in the previously continuous leaf blade. Wall remnants of late dying cells at the periphery of the perforation may be retained in mature leaves, but cell walls of the initial population of cells undergoing PCD in each perforation zone ultimately are entirely degraded. The complete wall degradation is initiated at a comparatively early stage and is an integral part of lace plant PCD, as shown for the formation of cortical aerenchyma (Campbell and Drew, 1979; He et al., 1994; Gunawardena et al., 2001), abscission and differentiation of xylem vessel elements (O’Brien, 1970; Fukuda, 2000; Ohdaira et al., 2002). This is not surprising because the process of wall degradation depends entirely on the production and secretion of wall-mobilizing enzymes by the protoplast. This synthesis and Delivery must occur before the death of the cell. 1. B. I. Chloroplast Damage by Oxidants

1. B. I. 1. Photosynthetic (psbA) and

nonphotosynthetic (trnQ) Genes

Under low light conditions environmental stresses result in excess excitation energy within the chloroplast, making the chloroplast a likely candidate for a sensor of stress and an initiator of cellular signaling that leads to stress responses (Mullineaux and Karpinski, 2002). Studies that test chloroplast involvement in plant PCD vary widely according to the involvement

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nature. Mittler et al. (1997) revealed that changes in chloroplast ultrastructure occurred after hypersensitive response progression. It was emphasized that redox regulation of plastid gene expression was first demonstrated to play a role in the case of translation initiation in Chlamydomonas reinhardtii. This process was shown to depend on the activity state of a redox-responsive oligomeric protein complex which is capable of binding to the 5’-untranslated region of photosynthetic gene (psbA) mRNA (Danon and Mayfield, 1994). Identification and function of photosynthetic gene (psbA) components now becoming fully resolved, the mRNA binding complex can serve as a paradigm for redox-regulatory mechanisms in eukaryotic photosynthetic organisms (Bruick and Mayfield, 1999; Trebitsh et al., 2000). Steps of chloroplast gene expression are subjected to photosynthetic redox control, where these steps include translation elongation (Kuroda et al., 1996; Kettunen et al., 1997; Zhang et al., 2000), RNA degradation (Liere and Link, 1997; Salvador and Klein, 1999), and RNA splicing (Deshpande et al., 1997). Available evidence suggests that transcription, the very first step in chloroplast gene expression, is also under redox control. Pool sizes of specific plastid RNAs respond to light intensity in vivo (Kettunen et al., 1997). General transcriptional switch in vitro experiments showed that the kinase not only affects the transcription of a photosynthetic gene (psbA); but also that of a non-photosynthetic gene (trnQ); the polymerase-kinase complex revealed specific differences in the phosphorylation state of polypeptides depending on the light intensity to which the seedlings had been exposed prior to chloroplast isolation (Baena-Gonzalez et al., 2001). Taken together, these data are consistent with GSH and phosphorylation-dependent regulation of chloroplast transcription in vivo. Phosphorylation of the RNA polymerase leads to decreased transcriptional activity from the psbA promoter in vitro (Baginsky et al., 1999). To decide whether this inhibitory effect was restricted to this particular promoter, the role of plastid transcription kinase (PTK) in the same in vitro system was tested, yet with the no photosynthetic gene (trnQ) promoter, i.e. a chloroplast promoter for a non-photosynthetic gene encoding tRNAGln (Sugita and Sugiura, 1996). Phosphorylation of the polymerase prior to RNA synthesis resulted in a decrease of specific transcript formation as indicated by the

loss of correctly sized S1-resistant material. Although this effect was most pronounced for the trnQ transcripts, the amount of specific psbA transcripts also was reduced when the polymerase was phosphorylated (Baena-Gonzalez et al., 2001). Suggested that PTK mediated control of plastid transcription is not restricted to a single class of chloroplast genes, but applies to both photosynthetic (psbA) and no photosynthetic (trnQ) genes. 1. B. I. 2. Plastid Transcriptional Kinase (PTK)

Polymerase associated enzyme, named plastid transcription kinase (PTK), was found to respond to changes in thiol/disulfide redox state mediated by glutathione (GSH), indicating that it may serve as a component of a signal transduction pathway that connects photosynthetic electron transport (via the production of reducing equivalents) with chloroplast transcription (Link et al., 1997). The psbA promoter was specifically upregulated both by Ser/Thr kinase inhibitors and reduced GSH (Baginsky et al., 1999). Although these in vitro data seemed to imply that PTK might be a redox-regulatory component of chloroplast transcription also in vivo (Baena-Gonzalez et al., 2001). It was found that GSH to be an effective inhibitor of the plastid transcription kinase (PTK) at a concentration of 20 to 30 mM (Baginsky et al., 1999). On the other hand, the organellar GSH concentration was estimated to be within a range of only 1 to 10 mM, i.e. a concentration too low to be significantly inhibitory in vitro under the previously used assay conditions. The GSH concentration of 5 mM that consistently gave strong inhibition of kinase activity in vitro is in good correlation with the expected in vivo concentration 1–10 mM. To help resolve this apparent discrepancy, we investigated conditions under which the in vitro assay would more closely resemble the in vivo situation (Noctor and Foyer, 1998). Alterations in the concentration of the ATP phosphor donor gave the most significant effect where decreasing ATP levels at a constant ratio of labeled to unlabeled ATP, it was found that an increasing inhibitory effect of GSH on the kinase activity. Approximately 90% inhibition was noticeable with 10 µM ATP in the presence of 5 mM GSH in this experiment, and at least 80% to 90% inhibition was found in two other independent experiments that were carried out with different

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PTK preparations (Baena-Gonzalez et al., 2001). There is accumulating evidence supporting a scenario in which the major chloroplast RNA polymerase PEP-A is controlled by an associated Ser/Thr kinase of the CK2 type (Baginsky et al., 1997). The identified enzyme, named PTK, controls chloroplast transcription via phosphorylation of sigma-like transcription factors and several other polypeptides that are associated with the plastid transcription apparatus (Baginsky et al., 1999; Pfannschmidt et al., 2000). PTK has been shown previously to be subject to thiol (SH) group regulation by GSH, i.e. a major redox mediator in chloroplasts (Karpinski et al., 1997; Noctor and Foyer, 1998). Transgenic plants with a modified GSH pool is used (Creissen et al., 1999). Baena-Gonzalez et al. (2001) suggested a mechanism by which changes in the SH group redox state of the organelle could be transducer into transcriptional responses. Chloroplasts are the sites of photosynthesis in plants, and they contain their own multicopy, requisite genome. Chloroplasts are also major sites for production of reactive oxygen species, which can damage essential components of the chloroplast, including the chloroplast genome (Gutman and Niyogi, 2009). 1. B. I. 3. Plastid Encoded Polymerase (PEP)

It was found that plastids contain two different types of RNA polymerase, a single-subunit (phage-type) enzyme of nuclear origin (NEP) and a multisubunit bacterial type enzyme with chloroplast-encoded catalytic subunits plastid encoded polymerase [PEP] (Maliga, 1998). As shown for mustard (Sinapis alba), the plastid-encoded polymerase (PEP) exists in two sub forms, PEP-A and PEP-B, which can be distinguished on the basis of their size and complexity, their biochemical properties and sensitivity to transcription inhibitors, and their relative abundance in different plastid types (Pfannschmidt and Link, 1994). The rifampicin-sensitive plastid encoded polymerase (PEP-B) form predominates in etioplasts and perhaps other non-green plastid types, the rifampicin-resistant PEP-A form is the major chloroplast RNA polymerase in functional chloroplasts (Pfannschmidt and Link, 1997). The rifampicin-resistant PEP-A enzyme seems to be responsible for the transcription of most plastid genes, including those for proteins involved in

photosynthesis (Hajdukiewicz et al., 1997; Maliga, 1998). Rifampicin-resistant PEP-A shares a common catalytic core with PEP-B, but contains a number of accessory proteins, the identification of several of which has recently been achieved by using protein-sequencing techniques (Pfannschmidt et al., 2000). 1. B. I. 4. Specific Senescence Associated Gene

(SAGS)

Rosenwasser et al. (2011) employed different bio informatics techniques combined with cellular redox-sensitive probes to establish the occurrence of ROS-related events at the early pre-symptomatic stages of extended dark stress. Treatment of detached rosettes of Arabidopsis by extended darkness leads to gradual loss of chlorophyll, massive changes in gene expression, and ultimately cell death. ROS stress has been described during the later stages of senescence-induced cellular disintegration processes, and its significance at early stages of dark treatment is shown here (Del Rio et al., 1998; Jimenez et al., 1998; Guo and Crawford, 2005; Zentgraf and Hemleben, 2008; Rosenwasser et al., 2010). Prior to tissue degeneration there are changes in peroxisomal and chloroplast metabolism and in chloroplast structure (Thomas and Stoddart, 1980; Zimmermann and Zentgraf, 2005). Such regulated process is characterized by a decrease in expression of photosynthesis-associated genes (PAGs) and induction of specific senescence-activated genes (SAGs) generally involved in macromolecule degradation (Buchanan-Wollaston et al., 2003; Gepstein et al., 2003; Lin and Wu, 2004; Buchanan-Wollaston et al., 2005). In Pelargonium cuttings, ROS levels increased in epidermal cytoplasm and chloroplasts concomitantly with a decrease in chlorophyll levels and increase in SAGs (Rosenwasser et al., 2006; Rosenwasser et al., 2010). Recently, some specific senescence activated genes (SAGs) were expressed; however, others were induced later. The transcript levels of the 32 general oxidative markers were next examined in the extended night data of experiments generated in non-detached rosettes. The analysis showed that 48 h after the ‘end of the night’ 24/32 transcripts were upregulated, while shorter dark periods induced the expression of only a few of the genes. Thus,

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the use of different experimental platforms such as non-detached darkened rosette or detached rosettes (Usadel et al., 2008). These results support the conclusion of the occurrence of oxidative stress due to the dark treatment.

1. B. II. Repair and Damage in Chloroplast DNA

Relatively little is known about the potential to repair oxidative DNA damage in chloroplasts. This provides evidence of DNA glycosylase-lyase/endonuclease activity involved in base excision repair of oxidized pyrimidines in chloroplast protein extracts of Arabidopsis thaliana. Three base excision repair components two endonuclease III homologs and an apurinic/apyrimidinic endonuclease that might account for this activity were identified by bioinformatics. Transient expression of protein-green fluorescent protein fusions showed that all three are targeted to the chloroplast and co-localized with chloroplast DNA in nucleoids. The glycosylase-lyase/endonuclease activity of one of the endonuclease III homologs, AtNTH2, which had not previously been characterized, was confirmed in vitro. T-DNA insertions in each of these genes were identified, and the physiological and biochemical phenotypes of the single, double, and triple mutants were analyzed. This mutant analysis revealed the presence of a third glycosylase activity and potentially another pathway for repair of oxidative DNA damage in chloroplasts (Gutman and Niyogi, 2009). The TG glycosylase-lyase/ endonuclease activity observed in chloroplast extracts of wild type Arabidopsis represents the first time that Base excision repair (BER) activity has been reported targeting oxidative damage in the chloroplast. It is only the second time any glycosylase activity has been observed in chloroplasts, after the finding of uracil-DNA glycosylase in maize (Bensen and Warner, 1987). AtNTH1, AtNTH2 and ARP have enzymatic activities in vitro that might be involved in TG glycosylase-lyase activity in vivo, and a T-DNA insertion in the ARP gene eliminated the activity in chloroplast extracts. All three enzymes were co-localized to nucleoids within Arabidopsis chloroplasts. Taken together, these results provide strong evidence for the occurrence of BER in chloroplasts (Gutman and Niyogi, 2009).

AtNTH1, AtNTH2, and ARP fusion proteins to green fluorescent protein (GFP) are not only

targeted to chloroplasts, they are localized specifically in chloroplast nucleoids that stain with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). The observation of nucleoid colocalization supports the in vitro activity assays indicating that these proteins do in fact interact with and repair DNA. The punctate appearance is very similar to that seen in other studies of nucleoid proteins (Sato et al., 2001). It is notable that there are two Nth homologs in the chloroplast, perhaps indicating an especially robust repair framework to cope with oxidative stress. However, the finding that both proteins are chloroplast targeted also raises the question of what is catalyzing this type of repair in the nucleus. It is possible that there are splices or other variants of these genes that are also targeted to the nucleus (Gutman and Niyogi, 2009). There is expressed sequence tag support for transcription initiation variation in the AtNTH1 gene, which would allow for translation from a downstream ATG to generate a predicted protein lacking a chloroplast transit peptide. Splice-dependent localization of plant DNA repair enzymes has been postulated for one of the purine-specific glycosylases (Sunderland et al., 2006). Although both AtNTH1 and AtNTH2 have Nth-type glycosylase-lyase activity in vitro, the T-DNA insertional mutants of each of these genes do not affect the activity detected in this qualitative assay of chloroplast extracts. Because atnth1 does not appear to be a null mutant, there might be residual AtNTH1 activity appearing in these lines. However, a T-DNA disruption of the ARP gene eliminated the TG glycosylase-lyase/endonuclease activity detected in wild-type Arabidopsis chloroplasts, suggesting that AtNTH1 and AtNTH2 are not responsible for the major chloroplast glycosylase activity against TG and that there must be an additional TG glycosylase activity in Arabidopsis chloroplasts (Gutman and Niyogi, 2009). Alternatively, this result could be explained by the presence of AP sites in the OsO4-treated substrate DNA; however, this possibility was eliminated by control experiments using the AP lyase activity of human Ape1 or T4 endonuclease V (pyrimidine dimer glycosylase). The additional TG glycosylase activity in chloroplasts might be a mono functional glycosylase enzyme that requires the AP endonuclease activity of ARP. The low apparent activity of AtNTH1 and AtNTH2 in chloroplast extracts might be explained in a number of

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ways. They might be present at much lower protein levels or have lower stability in our chloroplast protein extracts. The lack of a growth or developmental phenotype in the T-DNA mutants was something of a surprise, given that the arp mutation blocked the major TG glycosylase pathway detected in chloroplasts. DNA repair mutants often exhibit a detectable phenotype only when more than one gene is disrupted (Jiang et al., 1997; Saito et al., 1997), so the lack of a visible phenotype in the Arabidopsis single mutants were expected. However, even the atnth1 atnth2 arp triple mutant did not exhibit a clear whole-plant phenotype, implying that chloroplast repair of TG lesions is unnecessary, that it is necessary on a time scale of generations, and/or that there is a redundant non-BER pathway masking the phenotype. A similar lack of phenotype was found for an ogg fpg double mutant in Arabidopsis apparently blocked in nuclear purine BER (Murphy, 2005). The observed multiplicity of chloroplast enzymes for BER of TG lesions (two AtNTH proteins and a third activity) would seem to argue for the importance of this repair pathway. However, it is likely that there are other DNA repair pathways, such as nucleotide excision repair (Hays, 2002; Netrawali and Nair, 1984), recombinational repair (Cerutti et al., 1992), or gene conversion (Khakhlova and Bock, 2006) that are protecting chloroplast DNA in the absence of BER, at least in the short term. Over multiple generations, it is possible that phenotypes might emerge as mutations and/or damage accumulates in the plastome, similar to what has been observed for the nuclear genome in mismatch repair-deficient mutants (Hoffman et al., 2004).

1. B. III. Chloroplast Role in Programmed Cell

Death

Protecting mitochondria against ROS might improve plant performance under non-stress and water stress conditions. While, most of the attention has so far focused on chloroplasts, damage to mitochondria may be equally important. Since they play many important functions in plant metabolism, for example, supply of ATP and carbon skeletons for the biosynthesis of several compounds, participation in photorespiration and programmed cell death (McCabe et al., 2000), optimization of photosynthesis (Padmasree et al., 2002), and

synthesis of Ascorbic Acid (AA) (Bartoli et al., 2000; Millar et al., 2003). Seo et al. (2000) found that the hypersensitive response was accelerated by the loss of chloroplast function and although Dzyubinskaya et al. (2006) found that light accelerated cyanide-induced PCD in chloroplast-containing guard cells, PCD was highly inducible by cyanide in epidermal cells, despite their lack of chloroplasts. Subsequently, it has been suggested that chloroplasts might play significant roles in plant PCD. For example, on epidermal peels of pea leaves, cyanide induces guard cell death (containing chloroplasts and mitochondria) but not epidermal cell death (containing mitochondria only) in the presence of light (Samuilov et al., 2002; Samuilov et al., 2003a; Samuilov et al., 2003b). Typical leaf senescence symptoms are loss of chlorophyll, the decline in net photosynthesis rate (Pn), the repression of genes related to photosynthesis at the transcriptional and translational levels and the degradation of the subunit of ribulose bisphosphate carboxylase/ oxygenase (Rubisco). These symptoms are promoted by MeJa treatment in mature leaves (Muller-Uri et al., 1988; Reinbothe et al., 1994; Rakwal and Komatsu, 2000; Wierstra and Kloppstech, 2000). These data suggested that an active involvement of chloroplast-derived signals is also essential during plant PCD induced by numerous stimuli. Developmental senescence might be triggered by ROS (Miao et al., 2004), which has been suggested as the cause of early death symptoms in the mutant cpr5/old1, in which ROS-related stress was observed in pre symptomatic leaves (Jing et al., 2008). However, similar data for dark-induced senescence in normal leaves is lacking (Rosenwasser et al., 2011). Bioinformatics or biochemical evidence for the involvement of ROS or modulation of redox potential in the induction of senescence due to dark treatment is not available, and the temporal analysis of events upon transfer to darkness can reveal early events, which may act as primary triggers for senescence Rosenwasser et al. (2011). They found that a decrease in chlorophyll content was apparent in cut leaf after 2 days under darkness, and cell death, as determined by staining with Evans Blue, was apparent after 5 days. For transcript profiling, samples were harvested following 2, 3 and 5 days under darkness and at the same hour, to minimize circadian effects. They

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revealed that the expression of genes was determined using the dark induced transcriptome, 82/152 of the genes showed 2-fold increase or decrease compared to the wild type level at any one of the days during darkness. Of those transcripts, 47 of the gene products have been assigned to a specific subcellular localization and the rest 35 transcripts, which were not clearly assigned to a specific subcellular localization, are presented in transcript levels on days 2, 3, and 5 in relation to day 0. Due to the photosynthetic reduction of oxygen and the existence of chlorophyll and its derivatives, producing large amounts of ROS is an inevitable consequence in chloroplasts. ROS, in turn, promote growth retardation or cell death (Tanaka and Tanaka, 2006). Analysis of the Arabidopsis conditional fluorescence mutant has clearly demonstrated that singlet oxygen (1O2) generated from excess protochlorophyllide can function as a signal leading to cell death. Seedling lethality and growth inhibition by protochlorophyllide-generated 1O2 resulted from a genetic program (Wagner et al., 2004). Similar cell death phenotypes have also been observed in various other species in which chlorophyll intermediates are in excess, and the same genetic program might operate in these plants (Tanaka and Tanaka, 2006). Moreover, precise analysis of the subcellular ultrastructure using transmission electron microscopy has demonstrated that strong H2O2 production was found along not only the mitochondrial outer membranes but also the chloroplast outer membranes in the Arabidopsis Accelerated Cell Death2 mutant leaf tissue undergoing spontaneous cell death (Yao and Greenberg, 2006). These findings suggested that chloroplast-derived signals could trigger the cell death program, and the process involved the generation of ROS. In this case, ROS accumulation was found in the Arabidopsis protoplasts under MeJa treatment. Upon exposure to adverse stimuli, such as temperature extremes, drought or salt stress, the delicate redox balance is easily disturbed, causing ROS accumulation and further oxidative damage, as well as a reduction in photochemical efficiency. As mentioned earlier, MeJa-promoted senescence-associated cell death is accompanied by a decline in photosynthetic activity, which is closely related to the decrease in the levels of Rubisco and chlorophyll (Rakwal and Komatsu 2001). The decline in

photochemical efficiency was ROS dependent, and occurred upstream of the alterations in chloroplasts structure and evident damage to whole seedlings. These results allow us to be confident that the dysfunction of the photosynthetic apparatus is an early and plant-specific indicator of subsequent MeJa-induced cell death. The picture emerging from this contribution is, then, as follows. After MeJa treatment, the production of ROS first occurred in mitochondria and subsequently in chloroplasts. As result of ROS production, alterations in mitochondrial dynamics took place including the abnormality and change in morphology, the irregular distribution, the cessation of mitochondrial movement and the loss in MPT. Thereafter, the photochemical efficiency dramatically declined before obvious distortion of chloroplast morphology, which occurs prior to MeJa-induced cell death in protoplasts or intact seedlings. Protoplasts treated with MeJa for 5 h started to show a decline in photochemical efficiency, which took place before the alterations in the structure of chloroplasts and cell morphology (Zhang and Xing, 2008). Moreover, the decline in photochemical efficiency could be inhibited by pre-incubating the protoplasts with antioxidants. In addition, using a range of concentrations of MeJa to treat the intact seedlings of V. faba and rice plants, an obvious decline in photochemical efficiency could be found in the absence of visible effects on seedling morphology. 1. B. IV. Chloroplast Apoptotic-Like PCD

Transmission electron microscopy, culture pigment analysis, and PAM fluorometry all provided evidence that functional chloroplasts were present in light-grown cultures but absent from dark-grown cultures. PAM fluorometry is used to measure the photosynthetic potential of photosystem II and the fluorescence yield of healthy leaves should measure around 0.8 (Maxwell and Johnson, 2000). The fluorescence yield of light-grown culture cells measured around 0.4, which, although much lower than that of healthy leaves, was significantly higher than that of dark-grown culture cells, which measured just 0.1 (Doyle et al., 2010). Black and Osborne (2004) used PAM fluorometry to measure the fluorescence yield of photosynthetic cyanobacterial (Nostoc sp.) cells from suspension cultures. The fluorescence yield of free living cyanobacteria measured

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around 0.4, which was reduced to around 0.1 when the cyanobacteria were in symbiosis with their plant partner (Gunnera tinctoria) and no longer needed to photosynthesize (Doyle et al., 2010). Although the fluorescence yield measurements were found much lower than those measured in healthy leaves, the free-living cyanobacteria were grown in the absence of a carbon source and therefore must have been photosynthetically active, despite a fluorescence yield of just 0.4. In the present study, the fluorescence yield results suggested that although photosynthesis is not necessary for their survival, A. thaliana, culture cells produce functional chloroplasts when grown in the light. Chloroplasts are important sources of ROS in plant cells (Foyer and Noctor, 2003), chloroplasts may also regulate PCD in plants. This would be difficult to test in planta due to the complexity of plant tissues; however, A. thaliana, suspension cultures provide an ideal system, due to their easily accessible, undifferentiated cells. A number of studies have shown that plant Apoptotic-like PCD (AL-PCD), including hypersensitive response progression, is affected by light (Genoud et al., 2002; Danon et al., 2004; Zeier et al., 2004; Dzyubinskaya et al., 2006). It has been found that the hypersensitive response was accelerated by the loss of chloroplast function (Seo et al., 2000) and several recent studies have linked chloroplast-produced ROS with the hypersensitive response (Mur et al., 2008). Apoptotic-like PCD (AL-PCD) can be initiated in undifferentiated Arabidopsis thaliana cells in suspension culture and may easily be observed and quantified using the hallmark features of condensed cell morphology and fragmented DNA (McCabe and Leaver, 2000). Cell suspension cultures are often used in plant PCD studies, but because the cells are grown in sucrose medium, they may be grown in the dark and usually do not contain chloroplasts even when grown in the light. Therefore, many of these cell culture-based studies cannot test any possible effects of chloroplasts on the regulation of plant PCD. However, A. thaliana cultures are green in colour if grown in the light and it is shown here that growth in the light stimulates the biosynthesis of functional chloroplasts (Doyle et al., 2010).

1. B. V. Phytochromes Initiate PCD

phytochrome signaling during the establishment

of the Hypersensitive Response (HR) has implicated the need for a chloroplast factor in the pathway leading to the HR-associated cell death (Karpinski et al., 2003). Another line of proof for the role of chloroplasts in cell death comes from the ectopic expression in the chloroplasts of mammalian anti-apoptotic Bcl-2 family members, which protect transgenic tobacco plants from PCD induced by chloroplast-targeted herbicides (Chen and Dickman, 2004). To recognized participation of mitochondria in cell death, an active involvement of chloroplast-derived signals is also important during plant cell death (Zuppini et al., 2007; Gao et al., 2008). Like mitochondria under normal conditions, a number of complex and refined antioxidant regulatory mechanisms, thus establishing redox homeostasis (Apel and Hirt, 2004), minimizes the formation of ROS in chloroplasts. 1. C. Mitochondria Chloroplasts, mitochondria and peroxisomes are important sources of reactive oxygen species (ROS) the relative extent of oxidative damage to these organelles has not been studied. These organelles are most vulnerable to oxidative damage under stress conditions might help to design stress tolerant crops through targeted over expression of antioxidant enzymes (Bartoli et al. 2004). Many plant cell compartments are capable of producing ROS for instance thylakoid membranes constitute an important site of ROS production in leaves exposed to light, partly because the triplet excited state of chlorophyll interacts with ground state O2 forming the dangerous, ●O2 (Foyer and Fletcher, 2001). Then, ●O2 rapidly reacts with nearby molecules causing oxidative damage to proteins, lipids, and DNA. Peroxisomes are another important source of ROS, especially in C3 plants where photorespiration contributes large amounts of H2O2 (Corpas et al., 2001; Noctor et al., 2002). Photosynthesis, respiration, and other processes produce reactive oxygen species (ROS) that could cause oxidative modifications to proteins, lipids, and DNA. The production of ROS increases under stress conditions, causing oxidative damage and impairment of normal metabolism. An imbalance between the ROS production and antioxidant defenses can lead to an oxidative stress condition. Increased levels of ROS may be a consequence of the action of plant hormones, environmental stress,

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pathogens, or high levels of sugars and fatty acids (Bolwell et al., 2002; Couee et al., 2006; Gechev et al., 2006; Liu et al., 2007; Rhoads and Subbaiah, 2007). These conditions may lead to storage deterioration or impairment of seedling growth decreasing on crop yield. To avoid the harmful accumulation of ROS or to fine-tune the steady-state levels of ROS, various enzymatic systems control the rate of ROS production in mitochondria (Schreck and Baeuerle, 1991; Moller, 2001).

In apoptosis, release of Cytochrome c (Cyt c) from the mitochondria into the cytosol precedes any morphological changes (Yang et al., 1997). It was found that the translocation of Cyt c into the cytosol started at day 0d of petal opening following the disappearance of chlorophyll. The translocation of Cyt c into the cytosol was detected during stress-induced PCD of other plants (Balk et al., 1999; Stein and Hansen, 1999; Tiwari et al., 2002). In PET1 cytoplasmic male sterility of sunflower, there is Cyt c release into the cytosol followed by changes in cell morphology, loss of outer mitochondrial membrane integrity, and fall in the respiratory control ratio (Balk and Leaver, 2001). Many studies have shown that mitochondria are important regulators of animal apoptosis (Desagher and Martinou, 2000) and plant PCD (Diamond and McCabe, 2007). Mitochondria are important sources of ROS, which are thought to act as signals during plant PCD regulation (Diamond and McCabe, 2007). 1. C. I. Tricarboxylic Acid Cycle (TCA)

Direct inhibition of mitochondrial enzymes a number of mitochondrial proteins have been inhibited or degraded by AOS exposure in mammals (Taylor et al., 2004). These include the mitochondrial NADH dehydrogenase complex I, succinate dehydrogenase complex II, and ATP synthase complex V (Zhang et al., 1990). In plant, however, direct inhibition of mitochondrial protein by AOS was through a known mechanism; mitochondrial aconitase (EC 4.2.1.3) (Verniquet et al., 1991). Aconitase catalyzes the reversible hydration of cis-aconitate to either citrate or isocitrate and is a component of the TCA cycle. Mitochondria contain biochemical pathways and components, which link the cellular processes of carbon and nitrogen metabolism in plants. The tricarboxylic acid cycle (TCA cycle) links both carbon and

nitrogen metabolism by the oxidation of organic acids from glycolysis and the export of either α-ketoglutarate directly or citrate, which can be converted to α-ketoglutarate in the cytosol via cytosolic isoforms of aconitase and isocitrate dehydrogenase, as carbon skeletons for amino acid synthesis (Hodges, 2002). Verniquet et al. (1991) showed that H2O2 was able to inhibit citrate-stimulated O2 consumption in potato mitochondria, but that O2 consumption could be recovered following the addition of isocitrate. They also demonstrated similar inhibition following exposure of the isolated enzyme, and changes in Electro Paramagnetic Resonance (EPR) spectra of aconitase indicated modification of the 4Fe−4S cluster. They also noted no significant effects on the rates of succinate, NADH and 2-oxoglutarate-dependent respiration in the presence of H2O2 and concluded that aconitase is the major intra mitochondrial target for inactivation by H2O2. Their data further suggest that other likely targets of direct inhibition by AOS are proteins containing Fe−S clusters, due to the high reactivity of AOS with Fe2+ found in these proteins. In a proteomic study of the impact of oxidative stress on Arabidopsis mitochondria (Sweetlove et al., 2002), decreased abundances of a series of specific proteins were found. These included aconitase, Fe−S centres of the NADH dehydrogenase (complex I) and core subunits of ATP synthase. These data are clearly in line with the inhibitions reported in mammalian mitochondria in response to oxidative stress (Zhang et al., 1990). 1. C. II. Senescence of Mitochondria Induces

PCD

Plant cell death has been linked with the enhanced production of reactive oxygen species (ROS), especially hydrogen peroxide (H2O2) (Levine et al., 1994). Numerous stresses such as high temperature and ultraviolet-C exposure can raise ROS levels due to perturbations of chloroplast and mitochondrial metabolism as well as cellular redox equilibrium, leading to oxidative damage and subsequent cell death (Apel and Hirt 2004; Vacca et al. 2004; Vacca et al. 2006; Gao et al. 2008). It was shown that mitochondrion-derived H2O2 and not chloroplast H2O2 plays an important role in acd2-induced cell death (Yao and Greenberg, 2006). Further support for the involvement of mitochondria in

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dark-induced senescence comes from the work of disrupting the electron transport chain by damaging the electron transfer flavoproteins (ETF). Dark-induced senescence was enhanced in these mutant plants (Ishizaki et al., 2006). The finding that early responses to extended dark are centered in the mitochondria and peroxisome, lead to the notion that these organelles are the source of senescence signaling (Rosenwasser et al., 2011). Numerous reports have shown that senescence and cell death involve ROS and oxidative damage (Jing et al., 2003; Overmyer et al., 2003; Wendehenne et al., 2004). They also involve mitochondria, because mitochondrial metabolism is altered during senescence and mitochondria can contribute to cell death (Robson and Vanlerberghe, 2002; Saviani et al., 2002; Fridovich, 2004; Laloi et al., 2004; Vacca et al., 2004; Yao et al., 2004). Mitochondria also contribute to ROS production (Moller, 2001; Tiwari et al., 2002; Overmyer et al., 2003; Mittler et al., 2004). NO can affect mitochondrial metabolism and modulate ROS accumulation, senescence, and cell death (Tiwari et al., 2002; Jing et al., 2003; Overmyer et al., 2003; Vacca et al., 2004). ROS production, oxidative damage, and altered mitochondrial metabolism (Sweetlove et al., 2002; Tiwari et al., 2002; Jing et al., 2003; Overmyer et al., 2003; Vacca et al., 2004). For example, ROS production in mitochondria increases significantly in dark-induced senescent pea leaves (Jimenez et al., 1998) and in aged potato (Solanum tuberosum) tubers (Boveris et al., 1978). 1. C. III. Mitochondrial Enzymes Lead to PCD

1. C. III. 1. Protein Kinases It has been proved that the alterations of mitochondrial morphology and motility are the early indicators of cell death and are the necessary components of the progress of cell death (Logan, 2003; Yao et al., 2004; Yao and Greenberg, 2006; Gao et al., 2008; Scott and Logan, 2008). Unlike necrotic cell death caused by excessive phytotoxics, PCD is a cell death that involves many reversible molecular processes and cellular machineries. The cellular components for both ROS and ●NO signaling pathways leading to cell death include Ca2+

spiking, Ca2+‑binding proteins, protein kinases

such as MAPKs, caspase or caspase‑like

proteases, lipid messengers such as phosphatidic acid and fatty acid hydroperoxides (Montillet et al., 2005; Laxalt et al., 2007). The apoptosis and necrosis in animals all are originated from the mitochondria, where ROS and ●NO can be excessively generated during pathogenesis (Borutaite et al., 2003). The mitochondrial dysfunction such as permeability transition, cytochrome c release and respiration inhibition caused by ROS and RNS stresses, as well as their interaction results (Vacca et al., 2006; Morimoto et al., 2007). Cytochrome c release is necessary for caspase activation that precedes mitochondrial permeability transition, nuclear condensation, and other hallmarks of apoptosis in animals (Borutaite et al., 2003). Mitochondria also serve as an essential place for launching plant cell death because ROS and NO● stresses are amplified in mitochondria to trigger cytochrome c release through mitochondrial transition pore opening and morphological changes (Vacca et al., 2006; Morimoto et al., 2007). One of the interesting observations for elicitor induced PCD in C. lusitanica, cell culture is treachery element differentiation. This xylogenesis PCD may involve ●NO and ROS signaling as reported by others (Gabaldon et al., 2005). ●NO and ROS often show some overlapping and synergistic functions, particularly in cell death induction through interactions, which are determined by their reactive natures. The production balances of and diverse interactions between ROS and ●NO under different physiological environments form a complex signaling cellular network to determine if plant cells continue to survive or are directed to death. These redox signaling and complex cellular processes play essential roles in innate immune response and other defense systems of plants (Zhao, 2007). 1. C. III. 2. Cytochrome C Release

The certain aspects of cellular energy metabolism that might be assumed to play a key role in PCD is given that the involvement of mitochondria cytochrome c releases (Balk et al., 1999), and PCD in the hypersensitive response (Lam et al., 2001). Glucose oxidation is impaired during PCD in a manner completely prevented by ROS scavengers. It is important to note that oxidation of Glc was already impaired under conditions where cell viability was unaffected; that is, the impairment of energy metabolism is a

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process occurring in the early phase of PCD, when the cells are still viable. In this regard, plants resemble mammalian cells (Atlante et al., 1998). Cryptogein has been shown to inhibit Glc transport in tobacco cells, whether this occurs in cells remain to be established (Bourque et al., 2002). The increase in concentration of cellular NAD(P)H in PCD cells showed that the cells can take up Glc and that mitochondrial NAD(P)H oxidation is impaired in PCD. Consistently, it was found that mitochondria isolated from 4-h PCD cells were unable to generate a membrane potential or to increase oxygen consumption in third state of respiration. Since mitochondrial coupling appeared to be completely impaired and mitochondrial, damage occurs in the early phase of PCD, while cell viability was almost unaffected. After heat shock, as a result of the immediate production of ROS, the death program starts and evokes biosynthetic processes, imbalance in ASC dependent H2O2 scavenging, and impairment of oxidative mitochondria metabolism as early events. In addition to genetic control, changes in enzyme kinetics are suggested to contribute to regulation of PCD, thus integrating the regulatory mechanisms acting at transcriptional and post transcriptional levels (Vacca et al., 2004). Mitochondria were isolated by protoplast fractionation and lysis, followed by differential centrifugation essentially as described by de Pinto et al. (2000). The mitochondrion has been shown to regulate PCD in a number of ways; for example, cytochrome c is released early in the PCD process (Balk et al., 1999). Cytochrome c is a component of the electron transport chain and one effect of its release is the production of reactive oxygen species (ROS), which have been implicated as regulators of PCD in both animal and plant cells (Jabs, 1999; Jones, 2001). Many studies have shown that mitochondria are important regulators of animal apoptosis (Desagher and Martinou, 2000) and plant PCD (Diamond and McCabe, 2007). Mitochondria are important sources of ROS, which are thought to act as signals during plant PCD regulation (Diamond and McCabe, 2007). As chloroplasts are also important sources of ROS in plant cells (Foyer and Noctor, 2003), chloroplasts may also regulate PCD in plants. This would be difficult to test in planta due to the complexity of plant tissues; however, A. thaliana, suspension cultures provide an ideal system, due to their

easily accessible, undifferentiated cells. Antibiotic treatment did not affect culturegrow References

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Webb J. and M. B. Jackson (1986). A transmission and cryo-scanning microscopy study of the formation of aerenchyma in adventitious roots of rice (Oryza sativa). Journal of Experimental Botany 37: 832-841. Wendehenne D., J. Durner and D. F. Klessig (2004). Nitric oxide: A new player in plant signaling and defense responses. Curr. Opin. Plant Biol. 7, 449-455. Wierstra I. and K. Kloppstech (2000). Differential effects of methyl jasmonate on the expression of the early light-inducible proteins and other light-regulated genes in barley. Plant Physiol. 124: 833-844. Willats W. G. T., G. Limberg, H. C. Buchholt, G. J. Van Alebeek, J. Benen, T. M. I. E. Christensen, J. Visser, A. Voragen, J. D. Mikkelsen and J. P. Knox (2000). Analysis of pectic epitopes recognized by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation. Carbohydrate Research 327: 309-320. Yang W. H. and D. B. Bloch (2007). Probing the mRNA processing body using protein macroarrays and “autoantigenomics”. RNA, 13:704-12. Yao N. and J. T. Greenberg (2006). Arabidopsis Accelerated Cell Death2 modulates programmed cell death. Plant Cell 18: 397-411. Yao N., B. J. Eisfelder, J. Marvin and J. T. Greenberg (2004). The mitochondrion—an organelle commonly involved in programmed cell death in Arabidopsis thaliana. Plant J. 40, 596-610. Zeier J., M. Delledonne, T. Mishina, E. Severi, M. Sonoda and C. Lamb (2004). Genetic elucidation of nitric oxide signaling in incompatible plant-pathogen interactions. Plant Physiol. 136, 2875-2886. Zentgraf U. and V. Hemleben (2008). Are reactive oxygen species regulators of leaf senescence? In Progress in Botany, Vol 69. Springer-Verlag, Berlin, pp 117-138. Zhang L. R. and D. Xing (2008). Rapid determination of the damage to photosynthesis caused by salt and osmotic stresses using Delayed fluorescence of chloroplast. Photochem. Photobiol. Sci. 7: 352-360. Zhang L., A. Ohta, M. Takagi and R. Imai (2000). Expression of plant group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed functional divergence among LEA proteins. J Biochem (Tokyo). 127: 611–616 Zhang Y., O. Marcillat, C. Giulivi, L. Ernster and K. J. Davies (1990). The oxidative inactivation of mitochondrial electron transport chain components

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2. Stresses Damaged Nucleic Acids

2. A. Oxidatively Damaged DNA

DNA is a biological macromolecule bearing the genetic information in all living organisms. In eukaryotic cells, DNA is located inside cell nuclei. It codes all information concerning the function and structure of the organism. The DNA structure is composed by nucleotide units (base + sugar + phosphate) arranged as a double helix. The double helix arrangement is formed by the characteristic pairs of bases which are connected either by two (adenine-thymine) or three (guanine-cytosine) H-bonds (Murray et al., 2003). It was found that ROS-mediated lipid peroxidation, oxidation of proteins, and DNA damage are well-known outcomes of oxygen-derived free radicals, leading to cellular pathology and ultimately to cell death. The mechanism of ROS-mediated oxidative damage of lipids, proteins, and DNA has been extensively studied. The site-specific oxidative damage of some of the susceptible amino acids of proteins is now regarded as the major cause of metabolic dysfunction during pathogenesis (Bandyopadhyay et al., 1999). ROS have also been implicated in the regulation of at least two well-defined transcription factors, which play an important role in the expression of various genes encoding proteins that are responsible for tissue injury. One of the significant benefits of the studies on ROS will perhaps be in designing of a suitable antioxidant therapy to control the ROS mediated oxidative damage, and the disease processes (Bandyopadhyay et al., 1999). Perception of ROS triggers several signal transduction pathways involved in responses to oxidative stress. Plant hormones are involved in controlling programmed cell death (PCD), ROS formation, and the extent of lesion propagation in response to O3 exposure. The ROS serve as signaling molecules, and their levels are tightly controlled in plants (Laloi et al., 2004; Mittler et al., 2004; Foyer and Noctor, 2005; Pitzschke et al., 2006). However, ROS are highly reactive molecules, and uncontrolled production of ROS can damage cellular structures and molecules. Of particular importance are oxidative DNA

lesions caused by ROS, as accumulation of high levels of DNA damage can lead to cell death due to blockage of key nuclear functions (e.g., transcription and replication) and to accumulation of Deleterious mutations (Britt, 1996; Bray and West, 2005).

2. A. I. Product of Photo Damaged DNA

It is not clear whether these RNA photoproducts observed in vitro are formed in vivo under physiologic conditions. One important consideration is the wavelength of UV. The early in vitro studies of RNA photo damage primarily used UVC (200–290 nm), which is the highest energy form of UV irradiation but is not environmentally relevant as it is absorbed by the ozone layer in Earth’s atmosphere. However, the lower energy UVB (290–320 nm) wavelengths do reach the Earth’s surface. Additionally, UVA (320–400 nm) wavelengths are of even lower energy, but reach the Earth’s surface at levels ~10–100 times that of UVB and can travel farther into tissue (Kaminer, 1995). Direct absorption of UVB by nucleic acids only very inefficiently creates photoproducts, and UVA absorption by nucleic acids is even weaker. Thus, the formation of photoproducts at these wavelengths, particularly for UVA, may also involve the action of photosensitizer molecules within the cell. Although this is an area of ongoing investigation, it is speculated that molecules such as NADPH, flavins, or porphyrins could absorb UV and then in turn react with nucleic acids (Cadet et al., 1992; Cadet et al., 2005; Mitchell, 2006; Mouret et al., 2006). In early investigations to detect RNA photoproducts, isolated RNA was irradiated and hydrolyzed enzymatically and then, separated by chromatography to resolve photoproducts such as modified nucleotides and dimers (Small et al., 1968). With this technique, in vitro

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irradiation of synthetic poly (U) and poly(C) or isolated tobacco mosaic virus (TMV) RNA was observed to induce cyclobutane pyrimidine dimers, uridine hydrate, and cytidine hydrate (Singer, 1971; Miller and Cerutti, 1968). These studies identified commonly formed photoproducts as well as general principles about the photo reactivity of RNA (Gordon et al., 1976). In particular, single-stranded RNA is more likely to form photoproducts than double stranded RNA, as seen in the comparisons of poly (U) to a complex of poly (U) and poly (A) (Pearson and Johns, 1966). The presence of Mg2+, which can promote the folding of RNAs, also leads to decreased susceptibility of RNA to photo damage (Singer, 1971). Additionally, studies on purified RNAs and viral particles have shown that protein binding can alter photo reactivity (Remsen et al., 1970; Gordon et al., 1976). DNA is one of the key targets for ultra-violet (UV) induced damage in a variety of organisms ranging from bacteria to human. Among ultraviolet radiation, UV-B (280–315 nm) is the most Deleterious that induces two of the most abundant mutagenic and cytotoxic DNA lesions, such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs) and their Dewar valence isomers, that ultimately block the movement of DNA polymerases on DNA template (Sinha and Hader, 2002; Hader and Sinha 2005). Progression of mammalian RNA polymerase II is also interrupted by cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) (Protic-Sabljic and Kraemer, 1986). In contrast, UV-A (315–400 nm) radiation is less efficient in inducing DNA damage because it is not absorbed by native DNA. However, it can still produce secondary photoreactions of existing DNA photoproducts or damage DNA via indirect photosensitizing reactions (Hargreaves et al., 2007). Nature of bases and the flexibility of DNA play a major role to the extent of DNA damage that can occur. Sequences that facilitate bending and unwinding are favorable sites for damage formation e.g. cyclobutane-pyrimidine dimers (CPDs) are produced at higher rates in single stranded DNA and at flexible ends of poly d(A)-d(T) tracts, but not in their rigid center (Becker and Wang, 1989; Lyamichev, 1991). Other than UV radiation, there are number of factors such as ionizing radiations (X-rays, γ-rays, alpha particles), acridine dye, mustard gas

and bleomycin that are known to cause DNA damage. Acridine dye and acriflavin are mutagenic for bacteria and higher plants (Sugino, 1966) whereas proflavin is mutagenic for phages (Crick et al., 1961). They induce Deletion and insertion of single base pair in DNA helix which result damage in the native structure of DNA. γ-Rays induce DNA damage directly (as a result of deposition of energy in cells) or indirectly (as a result of free radical formations and oxidative damage). The main lesions produced by the physico-chemical interaction between ionizing radiation and DNA are single and double strand breaks, DNA–DNA and DNA–protein cross-links, alkali labile sites and damage to purine and pyrimidine bases (Cadet et al., 1997). All these types of DNA damage can be detected by the alkaline comet assay (Collins, 2004; Tice et al., 2000).

2. A. II. Product of oxidatively Damaged DNA

In recent years, many articles have been published on oxidative stress. Perhaps the knowledge about free radicals likes reactive oxygen and nitrogen species metabolism; definitions of markers for oxidative damage provide evidences linking chronic diseases and oxidative stress. Identification of flavonoids and other dietary polyphenol antioxidants present in plant foods and data supporting the idea that health benefits are associated with fruits, vegetables and red wines in the diet are probably linked to the polyphenol antioxidants they contain. Excessive reactive oxygen species may cause irreparable DNA damage, leading to mutagenesis and perhaps cancer (Nisha and Deshwal, 2011).Therefore, DNA damage caused by exposure to reactive oxygen species is one of the primary causes of DNA decay in most organisms. In plants, endogenous reactive oxygen species (ROS) are generated not only by respiration and photosynthesis, but also by active responses to certain environmental challenges, such as pathogen attack. Significant extracellular sources of activated oxygen include air pollutants such as ozone and oxidative effects of UV light and low-level ionizing radiation. Plants are well equipped to cope with oxidative damage to cellular macromolecules, including DNA. Oxidative attack on DNA generates bases alteration and sugar residues damage that underwent fragmentation and lead to strand breaks. Recent advances in the study of DNA repair in higher plants show that they use mechanisms similar to those present in

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other eukaryotes to remove and/or tolerate oxidized bases and other oxidative DNA lesions. Therefore, plants represent a valuable model system for the study of DNA oxidative repair processes in eukaryotic cells (Roldan-Arjona and Ariza, 2009). The Fenton reaction is a major physiological source of ●OH, which is produced near DNA molecules in the presence of transition metal ions such as iron and copper (Wiseman and Halliwell, 1996). UV-C did not influence Arabidopsis sensitive to nitrogen mustard protein (AtSNM1) expression in the first 24 h following the treatment, whereas its expression was found to be induced 24 h after bleomycin (BLM) treatment and 2 h after methyl methane sulphonate (MMS) and H2O2 treatments. AtSNM1 expression was found to induce on exposure to the elicitor xylanase (6 h) and flagellin, which provoke an oxidative burst and defense responses (Enkerli et al, 1999; Felix et al, 1999). Reactive oxygen species (ROS), such as H2O2, mediate the plant response to pathogen attack (Apel and Hirt, 2004) and cause oxidative DNA damage (Sekiguchi and Hayakawa, 1998). These expression data confirm microarray analyses of WT Arabidopsis plants exposed to several different genotoxic and environmental stresses. Therefore, suggest a role for Arabidopsis sensitive to nitrogen mustard protein (AtSNM1) in the response to genotoxic stress, including that arising from pathogen attack (Molinier et al, 2004).

2. A. III. Fragmentation of DNA Bases

Several types of DNA damage such as single strand break (SSB), double strand break (DBS), cyclobutane pyrimidine dimmers (CPDs), 6-4 photoproducts (6-4PPs) and their Dewar valence isomers have been identified that result from alkylating agents, hydrolytic deamination, free radicals and reactive oxygen species formed by various photochemical processes including UV radiation (Kumari et al., 2008). Polymerase chain reaction (PCR), comet, halo, terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, HPLC-Electrospray tandem mass spectrometry, Fluorescence in situ hybridization (FISH); Flow cytometry (FCM), annexin V labeling, immunological assays including immunofluorescent and chemiluminescence thymine dimer detection, immuno histochemical assay, Enzyme-linked

immunosorbent assay (ELISA), Radio immunoassay (RIA), Gas chromatography-mass spectrometry and electrochemical methods are main stratiges adopted to detect DNA damage in various organisms. The main aim of this review is to present a brief account of the above-mentioned DNA damage detection strategies for the convenience of interested readers (Kumari et. al., 2008). Notorieties of free radicals such as ROS and reactive nitrogen species (RNS) are well identified (Gutteridge, 1994).Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane (Gilbert, 1981).Cells may function poorly or die if this occurs. They are highly reactive that have short half-life and strong damaging activity towards macromolecules like DNA, Proteins lipids etc due to their oxidation properties. Free radicals have been implicated in the etiology of several human diseases as well as ageing (Halliwell and Dizdaroglu, 1992; Youshikawa et al., 2000; Duarte and Lunec, 2005). Free radicals resulted by exogenous factors like exposure to ionizing radiation or heat or by desiccation or mechanical shearing or smoking as well as endogenous processes during normal cell metabolism. Excess oxidative stress can lead to oxidative damage of DNA causing significant base damage, strand breaks, altered gene expression, and ultimately mutagenesis. Continuous oxidative damage to DNA was believed to be a significant contributor to the age-related development of many cancers, such as those of the breast, colon/rectum, and prostate (Block et al., 1992; Byers, 1993). Programmed cell death (PCD) is a cellular process in plants that is an essential component of several developmental programs. It can occur in response to environmental stresses and is often a part of the defense mechanism against pathogen attack via the hypersensitive response (Jones, 2001; Williams and Dickman, 2008). Morphologically, PCD, known as apoptosis, is generally characterized by a subset of changes such as chromatin and cytoplasm condensation (Vaux, 1993). Little is known about apoptosis in plants including the morphological changes (Danon et al., 2000). Although some accumulating evidence suggests that some features of plant apoptosis such as nuclear disintegration and chromatin condensation triggered endogenously or environmentally are

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similar to those in animals, other features such as cytoplasm shrinkage, nuclear periphery and the formation of apoptotic bodies have not been universally identified (Danon et al., 2000; Vaux and Korsmeyer, 1999). It seems that chromatin cleavage is the most characteristic feature of PCD. The morphological hallmarks of apoptosis include cytoplasmic shrinkage, nuclear condensation, and membrane blabbing the biochemical events involve calcium influx, exposure of phosphatidylserine, and activation of specific proteases and DNA fragmentation, first to large 50-kb fragments and then to nucleosomal ladders (McConkey and Orrenius, 1994; Wang et al., 1996). The stimuli that activate apoptosis are similar in plant and animal cells (O'Brien et al., 1998). Although, it should be noted that not all of the events were demonstrated in the same plant system, taken together these results infer a common basic cell death process in plants and animals.

2. A. III. 1. DNA Fragmentation by Oxidants Breaks in DNA may also result from damaged DNA replication forks or from oxidative destruction of deoxyribose residues. Double strand breaks are lethal as they affect both strands of DNA and lead to the loss of genetic information (Altaf et al. 2007). Low pH causes depurination and backbone breakage of DNA (Shchepinov et al., 2001). Oxidative cytosine derivatives are the most abundant and mutagenic DNA damage induced by oxidative stresses (Daviet et al. 2007). Recent advances in the study of DNA repair in higher plants show that they use mechanisms similar to those present in other eukaryotes to remove and/or tolerate oxidized bases and other oxidative DNA lesions. Therefore, plants represent a valuable model system for the study of DNA oxidative repair processes in eukaryotic cells (Teresa and Rafael, 2008). Due to DNA fragmentation and strand breakage, the 3'-OH termini becomes free which are enzymatically labeled with a modified nucleotide dUTP and digoxigenin, and then anti-digoxigenin antibody is used for signal detection. However, it has limitation in sensitivity and specificity (Pulkkanen et al., 2000). The monofunctional alkylating agent ethylmethanesulphonate (EMS) generates alkylated DNA bases that may be sites of DNA excision repair. Incomplete excision repair sites are a source of DNA strand breaks that are detected in the comet assay. Alkylated bases are also substrates for DNA glycosylases, which

lead to apurinic or apyrimidinic sites (AP sites). Under the conditions of the alkaline comet assay, these AP sites are converted into single-strand DNA breaks via β-elimination (Friedberg et al., 1995). Ethylmethanesulphonate (EMS) is known to induce DNA damage in plants measurable by the comet assay (Gichner and Plewa, 1998). 2. A. III. 2. DNA Fragmentation Mechanisms

A number of endonucleases catalyzes DNA fragmentation in both animals and plants,, and even a single DNAse is sufficient to degrade single- or double-stranded DNA (Krishnamurthy et al., 2000; Xu and Hanson, 2000). The bivalent cation dependent DNAse activity is a feature in the floral senescence of many plants (Rubinstein, 2000; Xu and Hanson, 2000). However, the timing of DNAse expression in natural petal senescence may be species specific (Langston et al., 2005). The DNAse activity observed from day 3, may be related to the onset of DNA degradation that was detected at day 6 (Azad et al., 2008). The terminal phase of leaf senescence is characterized by DNA fragmentation, membrane deterioration, disintegration of the nuclei and mitochondria; and massive release of free radicals (Yoshida, 2003; Zimmermann and Zentgraf, 2005). The most animal paradigms of apoptotic cell death is the loss of mitochondrial transmembrane potential (MPT), which occurs before cells exhibit nuclear DNA fragmentation, chromatin condensation or other biochemical changes leading to cellular demise (Kroemer et al., 1997). DNA is under constant attack from many sources such as radiation, ultraviolet light, and contaminants in our food and in our environment can all wreak havoc on our genetic material, potentially leading to cancer and other diseases (Kang, 1998).A convenient biomarker of oxidative stress is the extent of oxidation of bases in DNA (8-Oxo-7,8-dihydroguanine). This oxidation was confirmed by chromatographically by gas chromatography-mass spectrometry, HPLC with electrochemical detection, or HPLC-tandem mass spectrometry (Loft and Poulsen, 1996), or enzymically, with the use of the enzyme formamidopyrimidine DNA glycosylase to convert 8-oxo-7,8-dihydroguanine to DNA breaks, which are detected with alkaline elution, alkaline unwinding, or the comet assay (Keli et al., 1996). DNA damage induction is a fundamental unavoidable process and plays a

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key role in cancer development and the induction of heritable genetic defects. DNA damaging agents (genotoxin) are nearly always identified as carcinogens. Humans are constantly exposed to natural DNA damaging agents such as sunlight, dietary agents, such as cooked meat, acrylamide and endogenously formed oxygen free radicals (Kalim et al., 2010). Both exogenous and endogenous agents can damage cellular DNA, RNA, proteins and lipids. Therefore, cellular components are exposed to ultraviolet light, reactive oxygen species and other oxidants, hypohalous acids, nitric oxide, and alkylating agents, all of which are known from in vitro experiments to cause chemical modification or crosslinking of nucleic acids. For RNA, modification can in theory impact the function of both messenger and noncoding RNAs in numerous ways, such as interfering with base-pairing interactions in the transcription and translation of mRNAs or by altering the chemical properties of ribosomal RNA nucleotides necessary for RNA-driven catalysis in the ribosome. Many types of RNA damage that were first described in vitro also occur in vivo under physiologic conditions, so too have observations begun to indicate that the damage interferes with RNA function. It is also becoming clear that the cell may respond to RNA damage with a number of different defense mechanisms, including repair mechanisms specific forms of damage as well as normal turnover pathways (Wurtmann and Wolin, 2009). DNA damage is caused due to interaction of DNA with ROS or RNS where, free radicals such as ●OH and H● react with DNA by addition to bases or abstractions of hydrogen atoms from the sugar moiety. The C4-C5 double bond of pyrimidine being sensitive to attack by ●OH, generates a spectrum of oxidative pyrimidine damage products, including thymine glycol, uracil glycol, urea residue, 5-hydroxydeoxyuridine, 5-hydroxydeoxycytidine etc. Similarly, the interaction of ●OH with purines generates 8-hydroxydeoxyguanosine (8-OHdG), 8-hydroxydeoxyadenosine, formamidopyrimidine and other purine oxidative products (Nisha and Deshwall, 2011). Moreover, ultraviolet (UV) irradiation can cause several types of damage to RNA for instance photochemical modification, crosslinking, and oxidative damage. Although, much of the work describing UV damage to RNA has been carried out in vitro, there are few studies suggesting damage may also occur in

vivo under physiologic conditions. Therefore, the extensive study of UV damage to DNA is useful in suggesting the effects that UV damage could have on RNA, as the photochemistry of nucleic acids occur primarily DNA photo damage at the nucleobase (Cadet et al., 2005; Ravanat et al., 2001). 2. A. III. 3. DNA Fragmentations Role in Pcd

PCD can be subdivided into three stages: Signaling phase, execution phase and dismantling phase (Depreatere and Golstein, 1998). 30 new molecules have been found that initiate and regulate apoptosis. 20 other molecules associated with signaling or DNA replication, transcription or repair have also been discovered as apoptosis regulators (Willie, 1998), DNA fragmentation is a characteristic marker of PCD in many plants (Green and Kroemer, 2004; Rogers, 2005) and has been observed in the flowers of Actinidia Deliciosa (Coimbra et al., 2004), the petal and ovary of Pisum sativum (Orzaez and Granell, 1997a), and petals of Alstroemeria peruviensis (Wagstaff et al., 2003) and Petunia inflata (Xu and Hanson, 2000). Although DNA fragmentation is observed during PCD in many plants, some plant cells show DNA degradation rather than classical DNA laddering (Yamada et al., 2006). The haploid mega gametophyte of white spruce (Picea glauca) seeds is a living nutritive tissue that encloses the embryo at seed maturity. With the exception of the micropylar mega gametophyte cells, most cells of this tissue undergo programmed cell death (PCD) after seed germination. Distinct morphological and biochemical features, such as DNA fragmentation, and induction of proteases including caspase-like protease (CLP) activities (He and Kermode, 2003a), characterize this process. Inhibition of caspase-like protease (CLP) activity Delays the onset of DNA fragmentation (He and Kermode 2003b). DNA fragmentation is a hallmark trait for PCD in many plant cells, including pollination-induced petal senescence in Petunia inflata (Xu and Hanson, 2000), natural petal senescence in Pisum sativum (Orzaez and Granell, 1997b) and Sandersonia aurantiaca (Eason and Bucknell, 2001), and harvest-induced senescence in Brassica oleracea (Coupe et al., 2004) and Asparagus officinalis (Eason et al., 2002). However, the time at which DNA fragmentation begins may vary depending on the type of plant

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and plant organ, and becomes apparent earlier in the induced senescence than in natural senescence (Coupe et al., 2004). In natural petal senescence of P. inflata (Xu and Hansan, 2000), DNA fragmentation was evident at day 6 after flower opening, and, prior to visible wilting, DNA degradation was reported in petals of Antirrhinum majus, Argyranthemum frutescens, and Petunia hybrida (Yamada et al., 2006). It was possible to detect the onset of DNA degradation at day 6 when petal movement capability in the intact plant was very low. These results indicate that DNA fragmentation or degradation may be a common feature associated with petal senescence (Azad et al., 2008).

2. A. III. 4. ATP Role on DNAs Fragmentations

The signals for apoptosis are a decrease in mitochondrial transmembrane potential, irrespective of any apoptosis-inducing stimulus. The early marker of apoptosis is aberrant exposure of phosphatidylserine in the plasma membrane (Kroemer et al., 1998). These events are followed by the activation of proteases, phospholipases and phosphatases. The role of calcium was well-documented (Schwartzman and Cidlowski, 1993). The activation of nucleases leads to cleavage of nuclear DNA (Bayly et al., 1997). Internucleosomal DNA cleavage results in the formation of small fragments (Oberhammer et al., 1993). During apoptosis in animal cells, glucose deprivation, leading to ATP depletion and a fall in energy charge potentials, has been reported as a signal to trigger DNA fragmentation and mitochondrial dysfunction (Comelli et al., 2003; Liu et al., 2003). These results suggest that intracellular energy depletion, rather than oxidative stress or ethylene production, may be the very early signal to trigger PCD in tulip petals (Azad et al., 2008). Therefore, petal senescence, an active process, is a useful model system for studying the molecular mechanisms of organ senescence and requires de novo gene expression at both the transcriptional and translational levels (Lawton et al., 1990; Nooden et al., 1997). The release of Cyt c from mitochondria to the cytosol is an indicator of mitochondrial disintegration. The decrease in activity of Cyt c oxidase, an indicator for the activity of respiration, was observed in petals on day 3, which suggests the inactivation of photosynthetic as well as respiration activities

after flower opening. ATP is necessary to prevent DNA fragmentation (Nakamura and Wada, 2000), and to maintain mitochondrial transmembrane potential (Liu et al., 2003) and cell volume regulation by an ATP-regulated ion channel. However, all these events are evident only in animal cells (Okada et al., 2001). DNA degradation and cytochrome c (Cyt c) release were clearly observed in 6-d-old flowers. Oxidative stress or ethylene production can be excluded as the early signal for petal PCD. In contrast, ATP was dramatically depleted after the first day of flower opening. The onset of DNA degradation, Cyt c release, and petal senescence was also delayed by sucrose supplementation to cut flowers. Once the cascade is activated by the cell death stimuli (e.g. TNF-α), caspases-8 and -10 are cleaved to their active forms and mitochondria release cytochrome c and Apoptosis Inducing Factor into the cytoplasm (Reed and Green, 2002). Released cytochrome c then activates the initiator caspase-9, which consequently activates the effector caspases-3 and -7. The initiator caspases-8 and 10 also directly activate these effector caspases. Prolonged oxidative stresses as well as DNA damage are some of the triggers of apoptosis that have been identified (Hruda et al., 2010).

2. A. IV. Crosslinks Damage

High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification of pyrimidine dimer formation in human skin after low doses of UVB demonstrated a dimer formation rate of ~0.0520 lesions per 106 normal bases per J/m2. Furthermore, the exposure to low doses of UVA results in a rate of pyrimidine dimer formation of ~0.000008 lesions per 106 normal bases per J/m2 (Mouret et al., 2006). Minimal erythema dose (MED) values were used to evaluate these rates of lesion formation in DNA; it appears that relevant levels of UVA and UVB exposure could cause damage approximately 105 lesions per cell. It is thus possible that significant RNA damage also occurs, as average prokaryotic and eukaryotic cells contain ~4-6 times more RNA than DNA, most of which is noncoding RNA with a half-life. In addition to modification of nucleobases, UV can induce long-range covalent crosslinks involving RNA. This was first suggested from the experimental use of UV irradiation to probe ribosome structure, where both RNA-RNA and RNA-protein crosslinks

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were observed. RNA-RNA crosslinks can be induced both by UVC and by UVB irradiation (Zwieb et al., 1978; Wilms et al., 1997). The frequency of crosslinking between nucleotides is correlated with the distance and angle between nucleotides as well as the ability of the nucleotides to undergo transient conformational change to a conformation that would allow crosslinking within the timescale of photoexcitation (Huggins et al., 2005). For RNA-protein crosslinks, the likelihood of crosslinking is also determined by the geometry and photoreactivity of the nucleotide and amino acid (Smith, 1976). As with RNA-RNA crosslinks, there is a long history of using RNA-protein crosslinking experimentally to study ribonucleoprotein (RNP) complexes including the ribosome (Moller et al., 1978). These studies provide insight into the types of crosslinks that are possible. For example, it has been noted that crosslinking does not normally occur in double-stranded regions (Noah et al., 2000). Thus, certain cellular RNPs may be more or less predisposed by RNA structure to UV crosslinking damage. Above all, the experimental use of UV to generate RNA-protein crosslinks reflects the inherent photoreactivity of RNPs (Wurtmann and Wolin, 2009). RNA-RNA crosslinking occurs within tRNAs in Escherichia coli treated with relevant doses of broad spectrum UVA light (320–405 nm). The observed crosslinks occur between cytidine and the naturally modified pseudouridine nucleotide (Ramabhadran et al., 1976). In vitro experiments showed that tRNAs containing this crosslink had lower rates of aminoacylation and caused inefficient amino acid incorporation in translation assays (Ramabhadran et al., 1976; Chaffin et al., 1971). RNA-protein crosslinks have been described in UVB-irradiated insect eggs, although the RNAs involved were not identified (Jackle and Kalthoff, 1979), and in maize (Casati and Walbot, 2004). In the maize study, environmentally relevant doses of UVB caused the formation of covalent crosslinks between protein and RNA within the ribosome. The observed crosslinks occurred at locations where there is close proximity between RNA and protein in the 3D structure of the ribosome. Furthermore, this crosslinking damage may have functional consequences for the cell. As the duration of UVB exposure increased, the accumulation of crosslinked products increased, and the total cellular level of protein synthesis decreased. This correlation suggests that the

protein-RNA crosslinks may have contributed to the loss of ribosome function, although other UV-induced mechanisms for inhibition of translation are also possible (Wurtmann and Wolin, 2009).

2. A. IV. 1. Interstrand and Intrastrand Cross

Links

Interstrand and intrastrand crosslinks (ICLs) are a highly toxic form of DNA lesion. ICLs can be induced by UV radiation, furocoumarins and chemotherapeutics (Dronkert and Kanaar, 2001). ICLs prevent DNA strand separation, in turn blocking transcription, replication and chromosomal segregation, and thus triggering cell apoptosis (Li and Moses, 2003). Many processes are involved in ICL repair, suggesting interactions between different repair pathways. The initial step of ICL repair involves the removal of the lesion by the nucleotide excision repair (NER) machinery (McHugh et al, 2000). In yeast and mammals, homologous recombination (HRc) also has an important role in the repair of ICLs (Jachymczyk et al, 1981; de Silva et al, 2000). Therefore, the balance between the non-homologous end joining (NHEJ) and homologous recombination (HRc) pathways is not only critically important in the repair of double strand breaks (DSBs) but also has a strong influence on the outcome of ICL repair. The sensitive to nitrogen mustard (SNM1 or ARTEMIS) protein is involved in ICL repair in yeast (Wolter et al, 1996). The mammalian and yeast SNM-related proteins are nuclear proteins containing a conserved metallo-β-lactamase domain (Callebaut et al, 2002; Ma et al, 2002; Li and Moses, 2003). This domain is essential for the nuclease activity of SNM1 and is required for repair of DNA double-strand breaks (DSBs) resulting from ICL removal (Li and Moses, 2003). Mouse and human sensitive to nitrogen mustard protein (SNM1) homologues are also involved in the repair of ICL-mediated damage (Dronkert et al, 2000). One of the human SNM-related proteins is also known as ARTEMIS. Mutations in ARTEMIS are associated with severe immunodeficiency syndrome due to a defect in V (D) J recombination. ARTEMIS interacts with the DNA-dependent protein kinase and is responsible for opening the hairpin and for overhang processing during the V (D) J recombination process (Ma et al, 2002).

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ARTEMIS mutants are also hypersensitive to DNA damaging agents such as bleomycin (BLM) and g-rays (Moshous et al, 2001). ARTEMIS therefore seems to have a general role in NHEJ, in addition to a specific role in V (D) J joining. Taken together, these studies suggest that SNM-related proteins have a variety of roles in DSB repair in different organisms. The study of the closest Arabidopsis homologue of the yeast and mammalian sensitive to nitrogen mustard protein (SNM1) also suggests a broad role of SNM1in DSB repair in plants. However, contrary to expectation, the AtSNM1-deficient plants were not hypersensitive to the two ICL forming agents cisplatin and mitomycin C (MMC), but showed a moderate sensitivity to the chemotherapeutic agent bleomycin (BLM) and to hydrogen peroxide (H2O2). In AtSNM1 mutant plants exposed to H2O2 and to the bacterial elicitor flagellin, both inducing oxidative stress, the frequency of somatic homologous recombination (HRF) was not enhanced as compared with the control plants. Therefore, Molinier et al. (2004), suggest the existence in plants of an SNM1-dependent recombination repair process of oxidatively induced DNA damage. The Arabidopsis sensitive to nitrogen mustard (SNM), Arabidopsis sensitive to nitrogen mustard protein (AtSNM1) studied is predicted to be 484 amino acids long. SNM homologues range from 484 amino acids long in Arabidopsis to 1,040 amino acids long in mammals. SNM proteins contain a conserved metallo-β-lactamase domain. This domain, although variable in size, always retains a conserved motif of 10 amino acids, which has been shown to be essential for SNM1 function in Saccharomyces cerevisiae. In the species mentioned, the region surrounding the metallo-β-lactamase domain shares 20–90% identity at the protein level (Li and Moses, 2003). The Arabidopsis genome encodes three SNM-like proteins sharing 36 and 32% identity among them. Arabidopsis sensitive to nitrogen mustard protein (AtSNM1) shares around 48% identity with the mammalian and Drosophila SNM1 proteins are involved in inter strand and intra strand crosslink (ICL) repair. Therefore, expression of the AtSNM1 gene expected to be induced on exposure to the two ICL-inducing agent cisplatin and MMC, and SNM1-defective plants were expected to be sensitive to the two ICL-inducing agents as in yeast and mammals (Wolter et al, 1996; Dronkert et al, 2000).

No significant differences in development were noticed between the Atsnm1 plants and the control plants exposed to methyl methane sulphonate (MMS) or the two Inter and/or intra cross-link (ICL)-inducing agent mitomycin C (MMC) and cisplatin. Although sensitive to nitrogen mustard protein (SNM1) functions in ICL repair are partially conserved between yeast and mammals, the relationship between SNM1 structure and function seems to be different in plants, suggesting that this particular Arabidopsis sensitive to nitrogen mustard protein (AtSNM1) protein is not essential for the general Inter and/or intra cross link (ICL) repair process in Arabidopsis. AtSNM proteins may have redundant functions in the cellular response to DNA damaging agents, may differ in tissue specificity of DNA repair, or may be involved in other cellular processes (Molinier et al., 2004). The involvement of the two other Arabidopsis sensitive to nitrogen mustard protein (SNM)-like proteins in ICL repair remains to be investigated. AtSNM1 transcripts remain undetectable in snm1-1 and snm1-2 mutant plants following exposure to bleomycin (BLM) (24 h) or H2O2 (2 h). These results confirm that snm1-1 and snm1-2 lines are defective in AtSNM1 expression. Plants in which RNA-mediated interference (RNAi) was used to down regulate the SNM1 steady-state level also did not produce detectable levels of AtSNM1 under any condition tested. SNM1-defective plants were retarded in growth at 10-7 and 10-6M BLM compared with the control. However, in yeast and mice, hypersensitivity to bleomycin (BLM) was observed in ARTEMIS-defective cells but not in SNM1-defective cells (Wolter et al, 1996; Moshous et al, 2001; Rooney et al, 2003). A moderate sensitivity of the mutant plants compared with the control plants was also noticed in the presence of 0.6 and 1.2 mM H2O2. Hypersensitivity of SNM1-deficient cells to agents inducing oxidative damage has not been reported in other organisms. These results suggest a nonessential role for AtSNM1 in the repair process of bleomycin (BLM)- and H2O2-induced DNA damage. Therefore, to determine whether AtSNM1 has a direct role in the repair of DNA damaged by ROS, the repair efficiency of Atsnm1 plants was evaluated using cell extracts and an in vitro DNA repair assay. A linearized plasmid was treated with methylene blue plus visible light, which causes oxidative damage (mainly 8-oxo-G), that serves as a substrate for the DNA synthesis-dependent

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repair such as base excision repair (BER) of oxidative DNA damage (Li et al, 2002). A significant Delay in the incorporation of digoxigenin (DIG)-labelled dUTP in the mutant plants compared with the control was observed. These data suggested that Atsnm1 plants are delayed, but not impaired in DNA-synthesis-dependent repair of oxidative DNA damage. Therefore, AtSNM1 seems to encode a protein involved in the processing of DNA lesions formed after exposure to oxidative stress. Recombination repair of ROS-induced DNA damage in yeast and mammals, SNM-related proteins were shown to be involved in the processing of ICL-induced DNA DSBs and in the V(D)J recombination process, respectively (Wolter et al, 1996; Dronkert et al, 2000; Ma et al, 2002). To determine whether the studied AtSNM1 protein could have a role in DSB repair, AtSNM1-defective plants were monitored for recombination DNA repair using an in planta recombination assay (Swoboda et al, 1994; Molinier et al, 2004a). Somatic homologous recombination (HRF) was monitored in RNAi plants and in snm1-1 plants crossed with Arabidopsis lines containing recombination substrates, after exposure to the DNA-damaging agents UVC, MMS, cisplatin, MMC, BLM and H2O2. Somatic homologous recombination (HRF) was increased to the same extent in WT and AtSNM1-defective plants treated with UVC, BLM, MMS, cisplatin and MMC when compared with untreated control plants (Molinier et al., 2004). To test the biological relevance of AtSNM1-mediated repair of oxidatively induced DNA damage, H2O2 and a bacterial elicitor mimicking natural stress responses were used. FLG22, a 22 amino acids long synthetic peptide derived from flagellin, the most abundant protein of bacterial flagella, is known to elicit plant defense responses and to induce a rapid oxidative burst. As a control, a 22 amino acids-long peptide derived from Agrobacterium tumefaciens was used. It differs from the active FLG22 by 1 amino acid and does not induce oxidative burst and defense responses (Felix et al, 1999). Somatic HRF was enhanced by five- to tenfold after treatment of control plants with H2O2 and active FLG22. In contrast, in both RNAi and insertional mutant plants, somatic HRF remained almost unchanged after treatments. These results suggest that HRc-mediated repair of these two ROS inducing treatments specifically requires the activity of SNM1,

whereas the repair of UVC, cisplatin or MMC DNA damage is independent of this activity. One scenario that may explain the specific role of AtSNM1 in recombination DNA repair borrows ideas from the nucleolytic activity of SNM proteins (Jeggo and O’Neill, 2002; Li and Moses, 2003). If AtSNM1 can process DSB intermediates as suggested for yeast and mammalian homologues, it might be predicted to be of special importance in reacting to oxidative lesions within single-stranded DNA overhangs. The role of SNM1 in somatic HRc may be of special importance for the evolution of disease resistance genes, as they can be arranged as clustered loci (Hulbert et al, 2001; Leister, 2004). Oxidative damage, occurring during pathogen infection, may speed up the generation of new resistance gene assortments (Lucht et al, 2002; Kovalchuk et al, 2003). Therefore, it was clearly documented the existence of an SNM1-dependent repair process of ROS-induced DNA damage in Arabidopsis (Molinier et al., 2004). 2. A. V. Polyploidity Damage Distribution profiles of DNA ploidy levels assessed in 9-d-old plants revealed a small but significant increase in 4C/2C cell ratios in apx1/cat2 plants, indicating an impairment of cell-cycle progression at the G2-to-M transition. In accordance, apx1/cat2 plants displayed a decreased average cell number and area per leaf. At the G2 checkpoint, WEE1 kinase is a critical downstream target of the ATM/ATR signaling cascade (Harper and Elledge, 2007). In cells suffering DNA damage, WEE1 halts cell-cycle progression upon cessation of DNA replication, thereby coupling mitosis to DNA repair (De Schutter et al., 2007). To evaluate the role of WEE1 in apx1/cat2 double mutants, apx1/cat2/wee1 plants were generated. During the production of these triple mutants, we observed that the RNAi effect that down regulated the CAT2 transcript and subsequent activity levels was lost recurrently in individual progeny plants. These findings indicate that, in individual apx1/cat2/wee1 mutants, unknown mechanisms are activated that interfere with RNAi-dependent gene silencing. Nevertheless, several individual catalase-deficient triple mutants could be identified through PCR-based genotyping and CAT activity assays (Vanderauwera et al., 2011). Some individual triple mutants all showed restored growth capacity compared with apx1/cat2 double mutants and abolished transcriptional induction

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of the DNA damage response (DDR). Together with the DNA ploidy assays, these results revealed that WEE1-dependent checkpoints are activated as part of the H2O2-dependent DNA damage response (DDR) of apx1/cat2 plants. 2. A. VI. Demonstrated Risk of Damaged

Nucleic Acids

Germination is defined as the events that occur in the embryo upon imbibition’s (water uptake) by the quiescent seed, and which are completed upon emergence of the radical from the seed coat (Bewley, 1997). Dehydration and rehydration during seed development and germination is associated with high levels of oxidative stress, resulting in DNA damage, including base modification and DNA strand breaks (Dandoy et al., 1987). Genome damage also occurs during seed storage, and is exacerbated by adverse environmental conditions (typically high temperature and relative humidity) that accelerate seed ageing, leading initially to a loss of seed vigour and then to a loss of viability (Cheah and Osborne, 1978). Dehydration and rehydration during seed development and imbibition is associated with high levels of DNA damage (Dandoy et al., 1987). Genome damage also occurs during seed storage, and loss of seed viability correlates with the accumulation of DNA breaks and chromosome rearrangements, clearly establishing a link between DNA damage incurred during seed ageing and reduced germination potential (Roberts, 1972; Cheah and Osborne, 1978; Osborne, 1982). Seeds of Arabidopsis wild type and mutant lines were aged at 45° C over KCl to a relative seed moisture content of 10.8% for 8 days. Seeds were either plated directly on MS plates or stored at 2° C prior to plating. This ageing regime resulted in Delayed radical emergence in the wild-type seeds without affecting viability, but greatly reduced seed viability and vigour in the atlig4-5, atlig6-1 and atlig4-5 atlig6-1 double mutants (Hay et al., 2003; Waterworth et al., 2010). At 8 days, almost 100% of the wild-type seeds had germinated, as opposed to around 50% of atlig6-1 and 75% of atlig4-5. Strikingly, almost no atlig4-5 atlig6-1 seeds germinated at 8 days imbibition. Germination scores were continued up to day 26, after which no further seeds germinated, with final germination percentages of 93% for wild type, 80% for atlig4-5, 70% for atlig6-1 and 40% for atlig4-5 atlig6-1

(Waterworth et al., 2010). This indicates that deficiencies in AtLIG4 and AtLIG6 result in both decreased seed viability and seed germination vigour with seed ageing. That the most detrimental effects on seed viability were in atlig4-5 atlig6-1 double mutants suggests that either AtLIG4 and AtLIG6 function in distinct pathways with different specificities for DNA lesions, or that both enzymes are needed in pathways to remove the levels of DNA damage encountered in aged seeds. In Arabidopsis, a resumption of cell cycle activity in the root is required for germination, with important roles for the cyclins CYCD4; 1 and CYCD1; 1. In particular, Arabidopsis cycd1; 1 mutants display greatly Delayed radical emergence, with an approximate 80–90% reduction in germination at the 24-h time point, which correlates with Delayed cell division in the embryo root apex (Masubelele et al., 2005). Thus, the detection and signaling of DNA damage in aged or mutant seeds may limit mitotic activation, possibly through the WEE1-mediated G2/M checkpoint (De Schutter et al., 2007), or may activate a G1/S checkpoint as observed in irradiated wild-type and atlig4 seedlings (Hefner et al., 2006). A useful reference point for UV doses is the minimal erythema dose (MED) or the dose where sunburn is first apparent. The UVB MED has a mean of 704 ± 188 J/m2, and the MED for UVA has a median of 207,000 J/m2 as measured on Caucasian subjects (Gambichler et al., 2006); these doses can be reached within an hour during the summer in Europe (Ambach and Blumthaler, 1993). Considering these criteria, some observations have been made that are consistent with RNA photoproduct formation as result of physiologically relevant irradiation. Early studies in insect eggs used chromatography to detect pyrimidine dimers in RNA following irradiation at a UVB wavelength of 295 nm (Jackle and Kalthoff, 1978). Conversion of ~0.15% of pyrimidines to dimers occurred at a dose of 110 J/m2, which although strong enough to cause inactivation of the eggs, is within a relevant exposure range for humans. Furthermore, rRNA damage caused by UV in cultured mammalian cells is consistent with formation of RNA photoproducts (Iordanov et al., 1998). At either a UVB dose of 600 J/m2 or high doses of UVC, lesions in the 28S rRNA were detected by a primer extensions assay. The nature of the lesions was not determined, but the lesions occur primarily at adjacent pyrimidine nucleotides, which is consistent with the

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formation of cyclobutane pyrimidine dimer. Interestingly, the UV-induced lesions occurred in a site-specific, biased pattern within the 28S rRNA, particularly affecting the active site of the ribosome, and a decrease in translation activity was observed (Iordanov et al., 1998). Further, this damage to actively translating ribosomes is associated with the activation of a kinase-mediated stress response. Although the relative susceptibility of RNA and DNA to UV-induced lesion formation in vivo is unknown, there is evidence that environmentally relevant doses of UVB and UVA lead to photoproduct formation in DNA at levels that could cause cellular consequences.

2. A. VII. Agent Capable to Induce DNAs

Damage

Numerous chemicals (organics and metals) were tested: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), mitomycin C, cycloheximide, cadmium chloride, potassium dichromate, and chromium trichloride. With the exception of cycloheximide, all agents induced a significant increase in DNA migration. With cycloheximide, there was a suppression of DNA migration, which could reflect crosslinking of the DNA. It was found that the exposure of cells to tert-butylhydroperoxide (tBH) resulted in increased number of apoptotic cells, and this effect was prevented by administration of an antioxidant N-Acetyl cysteine. Rising concentrations of glucose added to the toxic effect of tert-butylhydroperoxide (tBH); some toxic effect of fructose and no effect of glutamine was observed. They found higher susceptibility to hydrogen peroxide induced DNA damage with 30 mM glucose concentration. Hyperglycemia increases the cell’s susceptibility to oxidative stress and it amplifies oxidative DNA damage. They confirmed that Glutamine – when used as a sole energetic substrate – showed no protective effect against oxidative stress (Hruda et al., 2010). Gaseous was also categorized with agents that capable to inducing DNAS damage. Tai et al. (2010) abstracted that exposure to O3 and CO2 in combination with O3 increased DNA damage levels above background as measured by the comet assay. Ozone-tolerant clones 271 and 8L showed the highest levels of DNA damage under elevated O3 compared with ambient air; whereas less tolerant clone 216 and sensitive clones 42E and 259 had comparably lower levels of DNA damage with no significant

differences between elevated O3 and ambient air. Clone 8L was demonstrated to have the highest level of excision DNA repair. In addition, clone 271 had the highest level of oxidative damage as measured by lipid peroxidation. These results suggest that variation in cellular responses to DNA damage between aspen clones may contribute to O3 tolerance or sensitivity. The expression profile of the Arabidopsis sensitive to nitrogen mustard protein (AtSNM1) gene was first analyzed in wild type (WT) plants exposed to MMC and cisplatin. Surprisingly, neither mitomycin C (MMC) nor cisplatin influenced AtSNM1 expression in the first 24 h following the treatment. Plants were exposed to bleomycin (BLM), which is known to induce DNA strand break (DSBs) by means of an oxidation-mediated reaction and other DNA oxidative lesions (Povirk and Steighner, 1989), to UVC, methyl methane sulphonate (MMS) or H2O2, which causes oxidative damage. Radical chlorine and nitrogen species arise from multiple intracellular sources and can cause damage to RNA. Phagocytic cells produce a range of radical species including hypochlorous acid (HOCl), nitric oxide (NO●), and peroxynitrate (ONOO−) to fight infections, and many cell types use nitric oxide as a signaling molecule. However, these radical species can lead to the chlorination, nitration, and oxidation of biological molecules including RNA (Hawkins et al., 2007). Reaction of HOCl with DNA nucleotides in stop-flow experiments shows that guanine and thymine are especially reactive, most likely due to chlorination at the heterocyclic nitrogen (Prutz, 1996). In cellular RNA from a human monocytic cell line treated with HOCl, HPLCMS/MS has been used to detect 5-chlorocytidine, 8-choloroguanosine and to a lesser extent, 8-chloroadenosine and to show that chlorination of RNA occurs at higher levels than for DNA (Badouard et al., 2005). Interestingly, denaturation of double-stranded DNA is also seen in vitro upon HOCl treatment, suggesting that RNA base pairing and folding could also be disrupted by chlorination (Prutz, 1996). To determine whether well-known reactive oxygen species (ROS)-generating agents can induce DNA damage in a simple chemical system with or without Fenton reaction components (iron and reducing agents), and to explore whether antioxidants which normally

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exist in the cellular environment can modify such damage, i.e. to determine chemical reactions of relevance to biological systems. A neutral electrophoresis technique was used to investigate DNA double stranded breaks (DSBs) caused by chemical treatments of λ-DNA in Eppendorf tubes by various ROS-generating compounds and the degree of DNA damage was categorized by analysis of enhanced digital images. Double strand breaks were induced by hydroquinone (HQ), benzoquinone (BQ), benzenetriol (BT), hydrogen peroxide (H2O2), bleomycin (BLM) and sodium ascorbate (Vit C). Various agents including catalase (CAT), superoxide dismutase (SOD), desferoxamine mesylate (DFO), ferrous chloride (FeCl2), reduced glutathione (GSH), trolox, silymarin and myricetin modulated DNA damage. Individual chemicals (except BLM) at the concentration of 1 mM did not induce large numbers of DSBs without iron [Fe (II) or Fe (III) at 25 µM]. GSH enhanced the damaging effect of HQ, BT and Vit C, did not alter the non-damaging effect of H2O2, but had a small protective effect on BLM. When compared with the non-enzyme protein, bovine serum albumin (BSA), SOD had a protective effect against BT, H2O2 and BLM; in the presence of GSH, SOD diminished the effect of HQ, BQ and Vit C but enhanced the effect of BT, H2O2 and BLM. With both GSH and Fe and compared with BSA, SOD enhanced the effect of HQ, BQ and BLM, ameliorated the effect of H2O2, and did not affect the others. CAT showed a protective effect for almost all examined compounds, but had little effect on BLM. With GSH alone, DFO enhanced the effect of HQ, BQ, H2O2and ameliorated the effect of BT, BLM and Vit C and trolox was largely protective. With GSH and Fe, DFO was protective for all compounds except doxorubicin (Dox), trolox was protective for all compounds except Dox and BLM, silymarin was protective except that it had little effect on BLM and Dox, but myricetin did not show any protective effect. In conclusion, the results from the present study have further highlighted the adverse potential of reducing agents and redox cycling agents, and the need for a cautious view of antioxidants (Yu and Anderson, 1997). 2. A. VIII. DNAs Damage by Cadmium Metal

Cadmium is a widespread heavy metal, released into the environment from power stations, heating systems, waste incinerators, and metal working industries and from many other sources.

Accumulation of cadmium in soil can become dangerous to all kinds of organisms, including plants. Even though the toxic effects of cadmium compounds in plants have been studied over many years, inconsistent results have been obtained about their genotoxic properties (Koppen and Verschaeve, 1996; Steinkellner et al., 1998). The possible pathway(s) of cadmium-induced genotoxicity are still unknown, but may involve the interaction of this metal with DNA, either directly or indirectly via the induction of oxidative stress. Cadmium chloride induced DNA damage as evaluated by the comet assay in broad bean (Vicia faba L.) and tobacco roots, but not in tobacco leaves after 2-72 h treatments (Koppen and Verschaeve, 1996; Gichner et al., 2004). After 2 and 24 h treatments of the rooted cuttings with the heavy metal cadmium (Cd2+), a dose response increase in DNA damage was noted versus controls in root nuclei. With a 24 h recovery period, the Cd2+-induced DNA damage in Solanum tuberosum L. cultivar Korela roots increased significantly. No significant increase in DNA damage was demonstrated in leaf nuclei after 24 h Cd2+ treatments, but continuous Cd2+ treatments for 2 weeks resulted in an increase in leaf DNA damage. This increase may be however associated with necrotic, apoptotic DNA fragmentation, as the affected plants had inhibited growth, and distorted yellowish leaves. For comparison, the monofunctional alkylating agent ethyl methanesulphonate, and γ-rays were assessed for induced DNA damage. Analysis of the accumulation of cadmium by inductively coupled plasma optical emission spectrometry demonstrates that roots accumulate almost 9-fold more cadmium than aboveground parts of the rooted potato cuttings. This may explain the absence of Cd2+ genotoxicity in leaves after short-term treatments (Gichner et al., 2008). Performing the comet assay in tobacco (Nicotiana tabacum L.) plants, a 15 min unwinding and a 25 min electrophoresis provides the best results (Gichner et al., 2006). The heavy metal cadmium (Cd2+) applied on tobacco roots in the form of cadmium chloride, induced significantly higher levels of DNA damage as measured by the cellular Comet assay than did treatment of isolated root nuclei, analyzed by use of the a cellular Comet assay. DNA damage induced by Cd2+ in roots of a transgenic catalase-deficient tobacco line

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(CAT1AS) was higher than in wild-type tobacco (SR1) roots. In contrast to treatment with the positive control ethyl methanesulphonate, Cd2+ induced no significant DNA damage in leaf nuclei, and neither somatic mutations, nor homologous recombination as measured by the GUS gene reactivation assay, were observed in leaves. Analysis of the accumulation of cadmium by inductively coupled plasma optical emission spectrometry demonstrates that roots accumulate almost 50-fold more cadmium than above ground parts of the tobacco seedlings. This may explain the absence of Cd2+ genotoxicity in leaves (Gichner et al., 2004). Compared to roots, leaf cells are better equipped with antioxidant defense system that might protect the nuclear DNA in leaf cells from Cd-induced oxidative stress. It was demonstrated that the activity of catalases, the principal H2O2 scavenging enzymes, is about 30 times higher in tobacco leaves than in roots (Gichner et al., 2004). Consequently, the high content of catalases, and other enzymes inactivating ROS in leaves, prevents the reaction of ROS with leaf nuclear DNA. By contrast, the direct acting monofunctional alkylating agent EMS induced a dose–response increase in DNA damage in both leaves and roots of potato (Gichner et al., 2008). Cd2+- and EMS-induced DNA damage in roots increases after a 24 h recovery period in water. By contrast, most of the γ-ray-induced DNA damage is repaired within 24 h. The data indicate that the standard alkaline protocol of the comet assay may not be suitable for bio monitoring of increased levels of acute radiations, as the induced DNA damage is rapidly repaired. By contrast, the comet assay may be more suitable for monitoring genotoxic effects of environmental chemical pollutants, where the induced DNA damage may persists for a longer period (Gichner et al., 2008).

2. A. IX. DNA Damaged by Reactive Oxygen

Species

DNA lesions induced by ROS have been documented to include formation of oxidative adducts, cleavage of bases, and strand breakage (Aust and Eveleigh, 1999; Tuteja et al., 2001; Bjelland and Seeberg, 2003). The ROS also attack lipids, initiating a process of lipid peroxidation that degrades cell membranes and other lipid structures and forms byproducts that damage DNA (Tuteja et al., 2001; Oksanen

et al., 2003). After an appropriate glycosylase cleaves the N-glycosyl bond attaching a damaged base to deoxyribose, leaving an abasic site, the sugar-phosphate backbone is nicked. Bifunctional glycosylases also have an apyrimidinic (AP) lyase activity that cleaves on the 3’side of the AP site. However, the site still requires the function of a separate AP endonuclease that cuts on the 5’side of the AP site to remove the 3’-deoxyribose residue at the nick site (Tell, 2005) before repair can continue. In the case of a monofunctional glycosylase, an AP endonuclease nicks the strand on the 5’side of the AP site. Escherichia coli has two unrelated AP endonucleases, exonucleases III (Xth) and endonuclease IV (Nfo). In humans, Ape1/Ref-1 is an Xth homolog, and in yeast Apn1p is an Nfo homolog (Demple and Harrison, 1994; Sung and Demple, 2006). Following generation of the adenosine phosphate (AP) site and nicking of the backbone, the gap is filled by a polymerase in either a short or a long patch then it is sealed by a ligase. Base excision repair (BER) of oxidative DNA lesions such Thymine glycol (TG) has been studied intensively in E. coli, yeast, and mammals, whereas comparatively little is known about BER in plants. For example, only two genes involved in BER of oxidized pyrimidines have been characterized previously in the model plant Arabidopsis thaliana (Babiychuk et al., 1994; Roldan-Arjona et al., 2000), and their localization within the plant cell is unknown. An Nth homolog in Arabidopsis, AtNTH1 (At2g31450), has the expected bifunctional glycosylase-lyase activity in vitro (Roldan-Arjona et al., 2000). The ARPgene (At2g41460) in Arabidopsis encodes an enzyme with AP endonuclease activity (Roldan-Arjona et al., 2000). The chloroplast localization of ARP, AtNTH1, and AtNTH2, a second Arabidopsis homolog of Nth, was tested experimentally, and the predicted activity of AtNTH2 was confirmed in vitro. In addition, an analysis of T-DNA insertion mutants affecting each of these three BER genes was performed (Gutman and Niyogi, 2009). Wild-type Arabidopsis Chloroplasts Contain DNA Glycosylase-Lyase/Endonuclease Activity of chloroplasts were isolated from wild-type Arabidopsis leaves, and a soluble chloroplast protein extract was prepared by a method used previously to determine nucleotide excision repair and possible Base excision repair (BER) activity in Arabidopsis whole-cell extracts (Li et al., 2002).

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2. A. X. Oxidatively Damaged Mitochondrial

DNAs

Plant cells produce harmful reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), singlet oxygen (●O2), the superoxide anion (●O−

2), and the hydroxyl radical (HO●), as by-products of their normal metabolism (Mittler, 2002). ROS may react with proteins, lipids, and DNA causing oxidative damage and, thereby, impairing the normal functions of cells. The Deleterious effects of many environmental stresses are due to an increase in ROS production and therefore, to oxidative damage to macromolecules (Foyer and Fletcher, 2001).The impact of ROS on mtDNA is well documented in animals and humans, where it leads to a number of different diseases and may contribute to ageing. Such damage is also likely to occur in plant mitochondria, but this area has received little attention to date (Wallace, 1999). The targets of active oxygen species (ROS) or damage by lipid peroxidation product, it is a challenge to distinguish between targets whose damage causes a large decrease in biological efficiency, and therefore has a high cost to the cell, and those that may be acting as sacrificial components or even scavengers of these toxic compounds. It has generally been assumed that small molecular mass targets for instance ascorbate, glutathione and tocopherol are the sacrificial components, and large molecular mass molecules such as proteins and DNA are targets for damage. However, this clear-cut distinction may not always be the case, and it would be well to keep an open mind on this issue (Elstner, 1982; Noctor and Foyer, 1998).

2. B. Oxidatively Damaged RNA

Oxidative damage to RNA has been reported in animal models of aging, human neurodegeneration and UV-irradiated cells. There is also growing evidence that oxidative RNA damage can lead to defects in protein synthesis, including decreased rates of protein synthesis and the production of aggregated and truncated peptides. Under most conditions, the bulk of cellular oxidative damage is a result of reactive oxygen species (ROS) formation due to metabolic reactions (Wurtmann and Wolin, 2009). Multiple steps in the electron transport chain involve a free radical semiquinone anion (●Q−) that can react with oxygen to form the superoxide radical (●O2

−). The cell eliminates

this superoxide radical through the action of superoxide dismutase to form hydrogen peroxide (H2O2), followed by enzymatically catalyzed reduction of H2O2 to H2O and O2 (Finkel and Holbrook, 2000). However, the reaction of H2O2 with intracellular iron in the form of Fe2+ and Fe3+ can lead to a cascade of radical chemistry through the Fenton and Haber-Weiss reactions and the production of ROS, including the highly reactive hydroxyl radical (●OH) (Halliwell and Gutteridge, 1984). The reaction of ROS with free nucleobases, nucleosides, nucleotides or oligonucleotides can create numerous distinct modifications in RNA (Barciszewski et al., 1999). In studies of DNA, guanine bases have been shown to be particularly reactive, and a major product observed in vitro and in vivo is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) (Cadet et al., 2007; Ravanat et al., 2001). In vivo studies of RNA oxidation have also detected 8-oxo-7,8-dihydroguanosine (8-oxoG) in RNA through the use of HPLC-MS/MS and immunocytochemistry, indicating that this common oxidative modification to DNA occurs in RNA as well. The formation of 8-oxoG occurs by reaction of guanine with the ●OH radical followed by oxidation, or by reaction of guanine with singlet oxygen ●O2 followed by reduction. Oxidation of other nucleobases is also possible (Cadet et al., 2007), and at least two other modifications have been identified in yeast RNA (Yanagawa et al., 1992). Radical anion species can be formed from nucleobases due to the direct absorption of UVB light by a nucleobase; the radical anion species can then undergo further reaction to generate oxidized nucleotides. Oxidative damage can also occur from UVA, mediated either by photosensitizer molecules or by (ROS) formation (Cadet et al., 2007). UVB- and UVA-induced DNA oxidation, most commonly result in the formation of (8-oxodG), although photo oxidation of adenine, cytosine, and thymine bases is also detected in DNA from irradiated cells (Cadet et al., 2005). Importantly, much of the chemistry described for photo oxidation of DNA is also applicable for RNA, as is borne out by measurement of UV-induced oxidative damage to RNA in cells. UV-induced oxidative damage to RNA has been demonstrated in experiments with human skin fibroblasts. Exposure of fibroblasts to UVA at sub lethal doses led to significantly increased levels of (8-oxoG) in RNA as measured by (HPLC-EC) analysis of total

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RNA (Wamer and Wei, 1997). Increasing intensity of UVA irradiation caused a dose-dependent increase in 8-oxoG. Interestingly, as seen in other examples of oxidative stress, the RNA damage was greater than the DNA damage observed. Importantly, UVA is used at doses comparable to the MED, supporting the physiologic relevance of UVA-induced oxidative damage to RNA (Wurtmann and Wolin, 2009). Studies of oxidative damage, a common finding is that damage levels are higher in total RNA than in total DNA. This observation has been made by comparing the level of 8-oxoG immune staining after RNase or DNAse treatment, such as in carcinogen treatment of rats (Fiala et al., 1989) and studies of neurodegeneration (Nunomura et al., 1999). Livers of rats treated with doxorubicin to induce oxidative stress, RNA and DNA was isolated untreated animals, the ratio of 8-oxoG: guanosine in RNA was 1.4-fold higher than the ratio of 8-oxodG: 2′-deoxyguanosine in DNA. Further, RNA damage increased significantly upon doxorubicin treatment while DNA damage did not (Wurtmann and Wolin, 2009). The level of RNA damage per nucleoside was measured to be 14–25 times greater than that in DNA. One explanation for the higher levels of oxidized nucleosides in RNA compared to DNA under normal cellular conditions is that there is a higher incidence of damage to molecules in closer proximity to mitochondria. This is supported by observations that levels of oxidized nucleosides are higher in mitochondrial DNA than in nuclear DNA (Shen et al., 2000). Differential susceptibility of RNA and DNA to oxidation could result from differences in protein association and in the degree of single strandedness. The higher levels of oxidization observed for RNA could be a consequence of different rates of removal of RNA and DNA damage. RNA susceptibility to oxidative damage may also vary between noncoding and coding RNAs, among different classes of noncoding RNAs, and among different mRNA species. The suggested factor is the degree of protein association, which may serve to protect RNAs from damage. Indeed, different RNAs have varying degrees and temporal patterns of association with proteins, from the stable coating of rRNAs with ribosomal proteins to the progressive association of mRNAs with transcriptional machinery, splicing and export factors, and eventually polyribosomes. The

basis behind the differences in damage is not yet fully understood. It has been reported that different mRNA transcripts show differing levels of oxidative damage (Wurtmann and Wolin, 2009). The consequences of oxidative RNA damage include impairment of translation due to damage to mRNAs and due to decreased ribosome function. The effect of oxidatively damaged mRNA on translation has been tested by introducing in vitro oxidized luciferase mRNA into cultured human HEK293 cells (Shan et al., 2003). The oxidized transfected mRNA was not degraded over the time course of the experiment, but compared to undamaged controls, luciferase activity and protein level from the damaged mRNA were strongly reduced. Additionally, proteins translated from the oxidized mRNA were shown to form aggregates suggestive of misfolding. In a similar study, transfected oxidized luciferase mRNA was shown to have normal polysome association, yet the expression of full-length protein decreased with a dose-dependent relationship to level of mRNA oxidation. The use of protease inhibitors revealed that short polypeptides result from the translation of the oxidized mRNAs, perhaps due to both premature termination and protease degradation of aberrant full-length protein. Similar effects were seen when HEK293 cells were treated with paraquat to induce oxidative damage, demonstrating that following cellular oxidative stress, mRNAs are translated yet often generate truncated proteins that must be degraded by the proteasome (Tanaka et al., 2007). Another consequence of mRNA oxidation may be ribosome stalling, as the size of polyribosome complexes formed on in vitro oxidized mRNAs increases in rabbit translation systems of reticulocyte, consistent with a slower rate of elongation (Shan et al., 2007). The types of damage described for DNA from nitration include base deamination leading to depurination and strand breakage, DNA-DNA and DNA-protein crosslinking, and numerous base modifications including 8-oxodG and 8-nitro-2′-deoxyguanosine; these lesions and modifications are chemically possible for RNA as well (Reiter et al., 2009; Ohshima et al., 2006). Indeed, in vitro deamination of purified adenine, guanine, yeast tRNA and bovine liver tRNA by nitric oxide has been measured (Nguyen et al., 1992). Moreover, in vitro exposure of purified total RNA to nitrogen radical species including ONOO− leads to stable 8-

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nitroguanosine and 8-oxoG formation. Treatment of cultured human lung cancer cells with ONOO− also is associated with the formation of 8-nitroguanosine (Masuda et al., 2002). 2. B. I. Ribnucleotide Modifications

Oxidized RNA has been measured in vivo in a growing number of contexts, both as result of experimental treatment of cells with oxidative agents and in diseased tissue. Treatment of E. coli cultures with H2O2 has been shown to cause 16S rRNA damage that can be detected by a reverse transcription polymerase chain reaction (RT-PCR) assay and is correlated with an increase in the 8-oxoG content in the cell (Gong et al., 2006). Experimental treatment has also been shown to result in RNA damage in animals. Exposure of rats to ethanol is associated with increased 8-oxoG RNA immuno-staining in the pituitary gland (Ren et al., 2005), while ammonia exposure triggers RNA oxidation in the rat brain (Gorg et al., 2008). Additionally, in a study of the carcinogen 2-nitropropane (2-NP), intraperitoneal injection of rats with 2-NP led to a significant increase in the levels of 8-oxoG in liver RNA. In addition to increased 8-oxoG levels, HPLC-electrochemical detection (HPLC-EC) also detected other forms of unidentified modified ribonucleotides, indicating that oxidative damage to RNA in vivo may lead to the formation of numerous chemical modifications (Fiala et al., 1989). Moreover, Oxidative RNA damage to cells under normal physiologic conditions has been suggested by detection of the oxidized ribonucleoside 8-oxoG in the urine of rats and humans as well as in human blood plasma (Park et al., 1992; Weimann et al., 2002). Metabolic reactions are major sources of ROS, metabolic rate as well as mitochondrial dysfunction can be significant factors in oxidative damage. One model system is aging rats, which display mitochondrial dysfunction. In the brains of such rats, levels of 8-oxoG in RNA are significantly higher than levels in young rats (Liu et al., 2002). Indeed, treatment of old rats with metabolites that boost mitochondrial function resulted in oxidized RNA levels comparable to those of younger rats, demonstrating a link between mitochondrial function and intracellular RNA damage. Additionally, studies of aging muscle displaying deregulation of iron homeostasis are consistent

with the role of intracellular metals in generating ROS through Fenton chemistry, and in turn oxidative damage (Hofer et al., 2008). Muscle from 32-month old rats was shown to have higher levels of 8-oxoG levels in total cellular RNA as well as higher free, non-heme iron levels compared to 6-month old rats. Additionally, imposing disuse atrophy on the older rats further elevated both the levels of free, non-heme iron and 8-oxoG. Thus, both the mis-regulation of metabolism and the loss of metal homeostasis that occur with age are correlated with increased oxidative damage to RNA. Other biological processes may also generate reactive oxygen species that can contribute to RNA damage. For example, as a part of the immune response to pathogens, neutrophils and eosinophils use NADH oxidase to produce ●O2

− and heme proteins to generate hypohalous acids, both of which can then lead to ●OH formation (Wurtmann and Wolin, 2009). In a study designed to approximate conditions generated at sites of inflammation by the activation of neutrophils and eosinophils, fibroblasts were treated with the hypohalous acid HOCl in combination with hyperoxia. Such treatment resulted in increased 8-oxoG to levels of 7–8 per 105 G in poly (A) RNA and in the free nucleotide pool, as compared to 1 per 105 G in control cells. Yet, most treated cells remained viable, indicating that cells are able to survive this level of damage (Shen et al., 2000). Notably, other tissues also express NADH oxidases. The catalytic subunit of the phagocytic NADH oxidase has shown to be expressed in a variety of tissues, including colon, reproductive tissues, and muscle (Suh et al., 1999), while the kidney expresses an NADH oxidase called Renox that is proposed to be an oxygen-sensing protein. Thus, many tissues may have a heightened requirement for cellular systems that respond to oxidative RNA damage (Geiszt et al., 2000). UV is a source of oxidative damage to RNA where, numerous pathways of excitation and oxidation are possible for nucleobases, as has been studied extensively in research on DNA damage (Ravanatet al., 2001). Purifed poly (A) RNA and oxidized mRNAs were isolated by immune precipitation with an antibody against 8-oxoG. RT-PCR for mRNAs of interest found that not all mRNAs were present in the pool of oxidized RNA, independent of transcript abundance. Further, even for mRNAs identified

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as oxidized, the prevalence of damage differed for particular mRNAs, in the range of ~54–75% damage-containing transcripts (Shan and Lin, 2006). Therefore, different cellular RNAs may be unequally susceptible to oxidative damage or certain damaged transcripts may be preferentially degraded or repaired. Factor that may render some RNAs particularly susceptible to oxidative damage is association with iron, as iron catalyzes Haber-Weiss and Fenton reactions that produce ROS (Halliwell and Gutteridge, 1984). In vitro experiments correlated binding of redox-active iron with oxidative damage to rRNAs: rRNA was found to have higher in vitro iron-binding than tRNA or mRNA, and this iron-rich rRNA had a 13-fold greater formation of 8-oxoG in Fenton reaction oxidation experiments as compared to the iron-poor tRNA. Thus, iron-binding properties of certain classes of RNAs may correlate with vulnerability to oxidative damage under basal conditions and especially in disease states involving perturbation of intracellular metal homeostasis (Wurtmann and Wolin, 2009). Translation defects after oxidative stress may also result from damage to the ribosome. In vitro, oxidative damage to purified ribosomes has been shown to result in decreased translation of an undamaged poly (U) mRNA in a translation assay (Honda et al., 2005). Similarly, in vivo defects in ribosome function are suggested by the finding that for AD patients, polyribosomes isolated from regions of the brain known to incur oxidative damage show a reduced rate of protein synthesis in an in vitro assay compared to polyribosomes from non-diseased regions within AD patient brains or from control patients (Ding et al., 2005). At least two types of damage have been observed in ribosomes under oxidizing conditions. First, studies of 8-oxoG levels in rRNAs from affected regions of AD brains have revealed that rRNA is oxidized under conditions of oxidative stress (Ding et al., 2006). A second form of oxidative damage to ribosomes is the crosslinking of rRNA to ribosomal proteins. This has been observed in H2O2-treated yeast cells, where 85% of ribosomal proteins were seen to be oxidized and HPLC-MS/MS identified numerous sites of ribosomal protein nucleotide crosslinking (Mirzaei and Regnier, 2006).

2. B. II. Nitric Oxide Role in RNA Damage

In vivo, nitric oxide is generated as a part of the inflammatory response, and this has been observed to cause a concomitant increase in 8-nitroguanosine levels in the RNA from lungs of mice infected with virus. Cytoplasmic immuno-staining for 8-nitroguanosine detection were found in booth the infected of hamsters with parasites (Pinlaor et al., 2003), and in human patients with Helicobacter pylori infection (Ma et al., 2004). Although controls were not performed in these experiments to conclusively identify RNA rather than DNA as the source of the cytoplasmic staining. These results suggest a pattern of damage to RNA from nitration in cases of infection, and further studies may extend the current understanding of the scope of chlorination and nitration under physiologic conditions. 2. B. III. Alkylation Damage of RNA

RNA, like DNA, can undergo alkylation by SN1 or SN2 nucleophilic reactions with methylation agents (Sedgwick, 2004). Endogenous sources of methylation are still under investigation but include methyl halides, compounds arising from the nitrosation of amines, and S-adenosylmethionine. Methylation can occur at nucleophilic N and O atoms on all of the ribonucleobases as well as at the O atoms of the phosphate backbone. As single-stranded nucleic acids are more vulnerable to methylation, many RNAs may be more susceptible than DNA to this damage. While methylation is a naturally occurring rRNA modification, only specific, largely conserved positions are methylated in a process requiring a large class of small nucleolar ribonucleoproteins (snoRNPs) to guide methylation. Indeed, experimental re-engineering of snoRNPs to direct methylation of normally unmethylated sites in yeast rRNA can cause growth defects and inhibition of protein synthesis (Liu and Fournier, 2004).When alkylation occurs at random, one source of deleterious effects is the inhibition of base-pairing interactions. For example, methylation can interfere with tRNA-rRNA interactions in translation (Yoshizawa et al., 1999) and mRNA-tRNA interactions in translation (Ougland et al., 2004) and can theoretically interfere with any other RNA function that relies on base pairing, such as siRNA and miRNA function. Additionally, loss of base pairing interactions can cause structured RNAs to misfold. Protein

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recognition of RNA bases, such as in the amino acylation of tRNAs, can also be inhibited by methylation (Ougland et al., 2004). 2. B. IV. Degradation Mechanism for Damaged

RNAs

2. B. IV. 1. Exonucleases

In eukaryotes, an important candidate for degradation of damaged RNA is the exosome complex of 3′-5′ exonucleases. As this complex acts in the nuclear degradation of aberrant precursor mRNAs, tRNAs, and rRNAs as well as in the cytoplasmic degradation of mRNAs that contain premature stop codons (Houseley et al., 2006). The exosome may be recruited to damage RNAs. Interestingly, yeast strains mutant for exosomal proteins show increased sensitivity to 5-fluorouracil (5FU) treatment, which may be consistent with a role for the exosome in turning over RNAs containing that unnatural base (Fang et al., 2004). In bacteria, several exoribonucleases have been implicated in the decay of damaged RNAs. In Deinococcus radiodurans, polynucleotide phosphorylase protein (PNPase) is important for viability following UV irradiation and for growth on H2O2-containing media, indicating a potential role for that nuclease in degradation of damaged RNAs (Chen et al., 2007). 3′-5′ exoribonuclease, RNase R, which is remarkable for its ability to process degrade through regions of significant secondary structure, shows increased activity in E. coli in a number of stresses such as growth in minimal media, starvation, and cold shock (Chen and Deutscher, 2005). There is evidence that PNPase and RNase R function in the turnover of aberrant RNAs has been provided by experiments in E. coli. At 42° C, the PNPtsR− strain accumulates fragments of the 16S and 23S rRNAs and is deficient in 70S ribosome assembly (Cheng and Deutscher, 2003). This suggests that these two nucleases function to degrade defective rRNAs that cannot be incorporated into ribosomes. Furthermore, PNPase has been shown to degrade mutant precursor tRNAs (Li et al., 2002). These findings suggest that these nucleases may also act to clear the cell of defective noncoding RNAs after RNA damage. While, different nucleases are known for having varying tolerance for RNA secondary structure (Deutscher, 2006).

It is unclear whether different nucleases may show particular tolerance or sensitivity toward unusual structures formed by RNA damage such as RNA-RNA or RNA protein crosslinks or modified nucleotides. This point has not been addressed directly, but interestingly, the RNase R homolog from Mycoplasma genitalium stalls at sites of 2′-Omethylation, while the E. coli RNase R does not show this sensitivity (Lalonde et al., 2007). The activity of different nucleases in degrading through crosslinks or other nucleotide modifications resulting from RNA damage may determine what nucleases are most active in the turnover of damaged RNAs. It is notable that both prokaryotes and eukaryotes have mechanisms for large-scale turnover of rRNA. In E. coli, starvation induces degradation of most of the rRNA in the cell, presumably in order to scavenge nucleotides, although the mechanism is not well-understood (Jacobson and Gillespie, 1968). In S. cerevisiae, starvation causes ribosomes to be targeted to the lysosome for degradation in a process termed ‘ribophagy’. While the extensive rRNA degradation seen during starvation has not been reported for conditions known to damage RNA, it is possible that cells use the same underlying mechanisms to target damaged RNAs for degradation (Kraft et al., 2008). 2. B. IV. 2. Poly (A) Polymerases and Helicases The activities of some prokaryotic nucleases and the eukaryotic exosome are enhanced by poly (A) polymerases and helicases. In prokaryotes, the degradation of mRNAs and noncoding RNAs is stimulated by the addition of poly (A) tails by poly (A) polymerase (Deutscher, 2006). However, in eukaryotes, polyadenylation of RNAs by the TRAMP complex promotes degradation by the nuclear exosome (LaCava et al., 2005; Vanacova et al., 2005). The TRAMP complex consists of the poly (A) polymerase Trf4 as well as an RNA helicase Mtr4 and one of two closely related RNA-binding proteins, Air1 and Air2. Polyadenylation may play a role in targeting certain RNAs for degradation, as the TRAMP proteins have been reported to preferentially polyadenylate incorrectly folded tRNAs in vitro (Vanacova et al., 2005). It is speculated that this specificity arises from greater accessibility of the 3′ end (Reinisch and Wolin, 2007). However, it is unclear how this preference may extend too many types of damaged RNAs. Interestingly, polyadenylated rRNA precursors accumulate in yeast treated

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with 5-fluorouracil (5FU), especially in strains mutant for the exosomal subunit Rrp6 (Fang et al., 2004). As 5FU-containing RNAs can form stable RNA-protein adducts with the modifying enzyme pseudouridylase, the accumulating defective rRNA precursors are hypothesized to require TRAMP/exosome-mediated degradation (Hoskins and Butler, 2008). Helicases are thought to aid degradation by facilitating translocation of the nucleases over the RNA, removal of RNA secondary structure, and removal of proteins from the RNA. The helicase activity of the TRAMP complex is required for the degradation of substrates including aberrant tRNAs (Wang et al., 2008), while the cytoplasmic exosome interfaces with the Ski complex, of which Ski2 has helicase activity (Houseley et al., 2006). In bacteria, polynucleotide phosphorylase protein (PNPase) can function with at least two different helicases, one of which may be a cold adaptation, suggesting that different accessory proteins may function to adapt nuclease activity to different environmental stresses (Prud’homme-Genereux et al., 2004). Cells have multiple mRNA quality control pathways that eliminate faulty transcripts, including nonsense-mediated decay of mRNAs containing premature stop codons, non-stop decay of mRNAs lacking stop codons, and no-go decay of mRNAs stalled in translation (Doma and Parker, 2007; Isken and Maquat, 2007). These mechanisms may also participate in the handling of some forms of RNA damage. For example, the production of truncated protein products by the translation of oxidized mRNAs (Tanaka et al., 2007) could potentially trigger nonsense-mediated decay (NMD), and likewise, stalling of elongation in the translation of oxidatively damaged mRNAs could lead to mRNA degradation by no-go decay (Shan et al., 2007). There is evidence that certain types of stress may activate nonsense-mediated decay (NMD) activity. One regulator of NMD activity is hSMG-1 kinase, which phosphorylates hUpf1 to promote nonsense-mediated decay (NMD) activity. Interestingly, activity of the hSMG-1 kinase increases with UV or ionizing radiation (IR), and higher NMD activity was observed after IR. However, it has not yet been demonstrated that the increased NMD activity is required for the degradation of damaged RNA, rather than another function (Brumbaugh et al., 2004).

The Ro auto antigen, a ring-shaped RNA binding protein, it has roles in both RNA quality control and cell stress response. In vertebrate cells, the Ro protein associates with misfolded pre-5S rRNAs (O’Brien and Wolin, 1994), and aberrant U2 snRNAs (Chen et al., 2003). It is thought to act in quality control for these RNAs, most likely by a scavenger mechanism of binding to RNAs that fail to properly associate with processing factors or incorporate into mature RNPs (Fuchs et al., 2006). Further, in the bacterial species D. radiodurans, the Ro homolog Rsr immune precipitates with PNPase and acts with the exoribonucleases RNase PH and RNase II in 23S rRNA maturation (Chen et al., 2007). This suggests that Ro proteins could act with nucleases in other processes as well, such as the turnover of damaged RNAs. Consistent with this hypothesis, Ro contributes to survival after UV in D. radiodurans (Chen et al., 2000) and in mammalian cells, where an accumulation of Ro is seen in the nucleus during recovery from UVC irradiation (Chen et al., 2003). Ro also exhibits genetic interactions with PNPase in both UV and oxidative stress in D. radiodurans (Chen et al., 2007). It will be of interest to determine what RNAs are bound by Ro during recovery from UV and other stresses. How Ro binding may lead to RNA degradation; and how Ro activity and subcellular localization is regulated during stress conditions. The host factor I (Hfq) protein is another bacterial ring-shaped RNA binding protein and, like components of the Lsm1–7 ring, is a member of the Sm-like family of proteins. Hfq has a variety of functions, including modulation of several steps in RNA decay. Hfq protects some RNAs from RNase E cleavage, which often functions as an initiating event in RNA degradation (Moller et al., 2003). Hfq also promotes polyadenylation by poly(A) polymerase I (PAPI), which in turn increases mRNA turnover by making mRNAs better substrates for PNPase-mediated degradation (Mohanty et al., 2004). Such activities could be critical to promoting or regulating the turnover of damaged RNAs. The hfq insertion mutants show sensitivity to UV (Tsui et al., 1994) and H2O2 (Muffler et al., 1997), among other growth phenotypes. However, H2O2 sensitivity was correlated with another Hfq function, the translation of the RNA polymerase σs subunit responsible for transcription of stress-response genes (Muffler

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et al., 1997), and could also result from the action of Hfq in the post-transcriptional regulation of some mRNAs (Zhang et al., 2002). Therefore, it is not currently possible to conclude whether Hfq modulation of RNA decay is important for survival following stress. Additionally, multiple ribosomal proteins have secondary activities that suggest roles in response to RNA damage. In Arabidopsis, one part of the UV-induced stress response is the rapid degradation of bulk mRNA, but not noncoding RNA (Revenkova et al., 1999). This extreme turnover of mRNA may allow more responsive upregulation of stress response genes as well as function to degrade damaged mRNAs. Disruption of the promoter of one isoform of ribosomal protein S27 inhibits this mRNA degradation and causes UVC sensitivity. Additionally, in Drosophila melanogaster, the small ribosomal subunit protein S3 and the ribosomal stalk protein P0 have been reported to have activities that are a part of DNA base excision repair (Yacoub et al., 1996; Yacoub et al., 1996). S3 was shown to have endonucleolytic activity on 8-oxodG-containing DNA, and both S3 and P0 were reported to have cleavage activity on basic sites. The activity of P0 on single-stranded as well as double stranded DNA raises the possibility that these proteins might act on RNA lesions as well. 2. C. DNA Damage Protection and Repair

Responses The DNA damage response (DDR) induced in apx1/cat2 plants correlated with increased amounts of DNA damage were determined to examine both the levels of phosphorylated histone H2AX (γ-H2AX), a reliable indicator of DNA double-strand breaks (Rogakou et al., 1998; Friesner et al., 2005), and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo), hallmark of oxidative DNA base damage (David et al., 2007). No increase in γ-H2AX was observed in any of the lines. However, cat2 plants grown in ambient air, apx1/cat2 plants did not accumulate 8-oxo-dGuo, suggesting that activation of the DNA damage response (DDR) pathway might not require DNA damage or, alternatively, that DNA lesions are repaired efficiently, leading to levels of basal DNA damage similar to those in Wild Type (WT) and apx1 plants. The lack of DDR activation in cat2 grown under ambient conditions suggests that DNA damage response (DDR) activation is

highly regulated and requires more than one type or source of ROS signal(s) (Hammond et al., 2007; Soutoglou and Misteli, 2008). Treatment with the DNA stress-causing agent aphidicolin revealed that apx1/cat2 plants were more tolerant than WT, apx1, or cat2 plants, for tolerance to mitomycin C (MMC), indicating that the constitutively activated DNA damage response (DDR) in apx1/cat2 plants is functional (Vanderauwera et al., 2011). An inherent part of the DNA damage response (DDR) is the activation of checkpoints to arrest cell-cycle progression and to allow time for repair, thus preserving genome integrity. Conserved key regulators for these checkpoint pathways are the ataxia telangiectasia-mutated (ATM), ATR, and WEE1 kinases (Harper and Elledge, 2007; Coolsand De Veylder, 2009). DNA is strongly protected against changes, which can cause serious change in the function of cells. Damages in structure may affect the right transcription of DNA and thus cause mutagenic changes. The reactions, which contribute to DNA damage, are oxidation, methylation, depurination or deamination (Marnett, 2002; Cooke et al., 2003; Wiseman and Halliwell, 1996). Generally, the reactive species are the most possible initiators for DNA changes. They cause mutation in base structure, Deletion or insertion of chemical groups. The different reactive species have different effects on DNA structure. The most serious damage is caused by hydroxyl radical, which can change all bases in DNA structure. Singlet oxygen reacts only with guanine and superoxide radical or hydrogen peroxide do not react (Cooke et al., 2003; Wiseman and Halliwell, 1996). Other serious damages can be caused by condensation of aldehydes with adenine, guanine or cytosine. The most common is Malondialdehyde, which is one of the major products of lipid peroxidation (Marnett, 2002). Most of changes can have serious consequence. Therefore, the cells have sophisticated mechanism how repair such changes (Cooke et al., 2003; Laval, 1996). The involvement of the DNA damage response (DDR) in cellular protection from oxidative stress was evident from the enhanced sensitivity to high light (HL). Oxidative stress of the DNA stress checkpoint mutants atr-2, E2F target gene 1 (etg1-1), and wee1-1and from the identification of these DNA damage response (DDR) transcripts in plants treated with the herbicide norflurazon or infected with different

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pathogens (Zimmermann et al., 2004). These results demonstrate the broad biological role of the DNA damage response (DDR) in protecting plants from different environmental stresses (Vanderauwera et al., 2011). The activation of the DNA damage response (DDR) in apx1/cat2 plants also was coordinated with the accumulation of transcripts encoding Bcl2-associated X protein (Bax) inhibitor 1 (BI-1), a plant anti-programmed cell death (PCD) protein that suppresses the endoplasmic reticulum (ER)–PCD pathway (Watanabe and Lam, 2009). Application of agents that block the ER–PCD pathway rescued cat2 under high light (HL) conditions, and mutants deficient in BI-1 were highly sensitive to HL, indicating that BI-1 is an important component of the pathways activated in apx1/cat2 double mutants (Vanderauwera et al., 2011). DNA repair of oxidized bases, such as thymine glycol (TG) or 8-oxoguanine, can be hypothesized as an important element of chloroplast photo protection. Although there is considerable overlap in both the types of DNA lesions caused by different insults and the targeting of different DNA repair mechanisms, baseexcision repair (BER) is considered to be the main repair pathway for oxidative DNA damage, at least in the nucleus and mitochondrion (Cadet et al., 2000). Base excision repair (BER) repairs single damaged bases (because of oxidation, deamination, alkylation, etc.) in DNA by removing them, breaking the phosphor diester backbone, excising the sugar residue at the a basic site, and filling the gap (Demple and Harrison, 1994; Huffman et al., 2005). Base excision repair (BER) begins with a DNA glycosylase or glycosylase-lyase. However, there are many types of glycosylases in any given organism and across taxa, and they are distinguishable by their substrate specificity, whether they are monofunctional (glycosylase activity only) or bifunctional (glycosylase plus apurinic/apyrimidinic (AP) lyase activities), by the phylogenetic family in which they reside, and/or by conserved structural characteristics (Huffman et al., 2005; Krokan et al., 1997). The glycosylases involved in Base excision repair (BER) of oxidative DNA damage can be roughly divided into those that target either oxidized purines or oxidized pyrimidines (Dizdaroglu, 2005; Wallace, 2002). Thymine glycol (TG) is a common type of oxidized pyrimidine, which is removed primarily by endonuclease III (Nth), endonuclease VIII (Nei), or their homologs

(Bandaru et al., 2002). TGis only poorly mutagenic, but it strongly blocks polymerases, inducing cell cycle arrest and potentially cell death if it is not removed (Gutman and Niyogi, 2009). 2. C. I. Repair of Oxidatively Damaged DNAs

All living organisms are constantly exposed to environmental stresses that cause damage. Plants, because of their sedimentary lifestyle, are especially susceptible to damage caused by environmental factors. To preserve genome integrity, plants use different DNA repair pathways, such as direct repair, base excision repair (BER), nucleotide excision repair (NER) or mismatch repair (MMR). Homologous recombination (HRc) and non-homologous end joining (NHEJ) are the two pathways used for repairing DNA double-strand breaks (DSBs). HR contributes to the preservation of genome integrity, whereas NHEJ seals DNA ends directly and can lead to sequence alterations (Vonarx et al, 1998; Britt, 1999). Therefore, there are a number of repair mechanisms such as photoreactivation (Pre), nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), recombinational repair (Rre) and apoptosis which are operative in organisms to enable them to withstand the damage. In humans, failure of these mechanisms leads to serious hereditary diseases such as xeroderma pigmentosum and non-polyposis colon cancer as well as non-hereditary disease such as breast cancer (Waterworth et al., 2010) . DNA Damages are physical abnormalities in the DNA, such as single and double strand breaks, 8-hydroxydeoxyguanosine residues and polycyclic aromatic hydrocarbon adducts. Enzymes can recognize DNA damages, and thus they can be correctly repaired if information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying (Nisha and Deshwall, 2011). Several repair pathways repair DNA damage. 8-OHdG has been implicated in carcinogenesis and is considered a reliable marker for oxidative DNA damage. DNA damage accumulates in brain, muscle, liver, kidney, and in long-lived stem cell. These accumulated DNA damages are the likely cause of the decline in gene expression and loss of functional capacity

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observed with increasing age (Nisha and Deshwall, 2011). Methods like HPLC-ECD, LC-MS/MS, total antioxidant capacity (FRAP, ORAC) are used to determine antioxidant defenses and biomarkers19 of oxidative stress in vitro and in vivo including antioxidant nutrients (ascorbate and tocopherol) and phytochemicals carotenoids and flavonoids (Nisha and Deshwall, 2011). It was demonstrated that Xeroderma pigmentosum is a rare sun-sensitive hereditary disease (Friedberg, 2003; Masutani et al., 2000) in which organism has 10,000 fold-increased risk of skin cancer on sunlight exposure. Numerous studies have demonstrated a requirement in plants for repair of DNA damage arising from either intrinsic or extrinsic sources. Investigations also have revealed a capacity for repair types of DNA damage, and conversely, identified mutants apparently defective in such repair. This article provides a concise overview of nuclear DNA repair mechanisms in higher plants, particularly those processes concerned with the repair of UV-induced lesions, and includes surveys of UV-sensitive mutants and genes implicated in DNA repair (Edward et al., 1998).

2. C. II. DNA Repair Ligases Enzymes

DNA repair is important for maintaining genome integrity. Therefore, in plants, DNA damage accumulated in the embryo of seeds is repaired early in imbibition, and is important for germination performance and seed longevity. An essential step in most repair pathways is the DNA ligase-mediated rejoining of single- and double-strand breaks. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. The characterization of DNA LIGASE VI is only found in plant species. The primary structure of this ligase shows a unique N-terminal region that contains a b-CASP motif, which is found in a number of repair proteins, including the DNA double-strand break (DSB) repair factor Artemis. Phenotypic analysis revealed a Delay in the germination of atlig6 mutants compared with wild-type lines, and this Delay becomes markedly exacerbated in the presence of the genotoxin menadione. Arabidopsis atlig6 and atlig6 atlig4 mutants display significant hypersensitivity to controlled seed ageing, resulting in Delayed germination and reduced seed viability relative to wild-type lines. In

addition, atlig6 and atlig6 atlig4 mutants display increased sensitivity to low-temperature stress, resulting in Delayed germination and reduced seedling vigour upon transfer to standard growth conditions. Seeds display a rapid transcriptional DNA DSB response, which is activated in the earliest stages of water imbibition, providing evidence for the accumulation of cytotoxic DSBs in the quiescent seed. These results implicate AtLIG6 and AtLIG4 as major determinants of Arabidopsis (Waterworth et al., 2010). DNA damage must be repaired during imbibition and prior to the initiation of cell division in order to minimize growth inhibition and mutagenesis in subsequent seedling development (Waterworth et al., 2010). DNA damage usually results in the production of single and double-strand breaks, either directly through the disruption of the sugar-phosphate backbone of DNA or indirectly through the process of excision repair. These breaks are re-joined by the action of DNA ligases (Wood, 1996). Eukaryotes possess several ATP-dependent DNA ligase activities, each of which, have specific roles in cellular DNA metabolism: DNA ligases are essential for the replication and repair of the nuclear and organellar genomes (Bray et al., 2005). Three DNA ligase genes have been identified in mammals, termed LIG1, LIG3 and LIG4. DNA ligase I joins Okazaki fragments as part of the DNA replication complex, and LIG1 genes are essential in humans (Homo sapiens), Saccharomyces cerevisiae and Arabidopsis thaliana (Barnes et al., 1992; Tomkinson et al., 1992; Babiychuk et al., 1998; Taylor et al., 1998a). DNA ligase I has roles in single-strand break (SSB) repair in all eukaryotes, and additional roles in DNA double-strand break (DSB) repair in plants and animals (Liang et al., 2008; Waterworth et al., 2009). LIG1 also encodes mitochondrial DNA ligase activity in S. cerevisiae and Arabidopsis, with the nuclear and mitochondrial DNA ligase I isoforms arising from alternative translation start sites in a single mRNA transcript, producing DNA ligase protein isoforms either with or without a mitochondrial targeting pre-sequence (Willer et al., 1999; Sunderland et al., 2006). LIG4 is also present in yeast, mammals and plants (West et al., 2000; Ellenberger and Tomkinson, 2008). DNA ligase IV has specific roles in the non-homologous end joining (NHEJ) pathway of DSB repair (Critchlow et al., 1997) and is important in the survival of Arabidopsis

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under conditions of genotoxic stress (Van Attikum et al., 2003; Friesner and Britt, 2003). Protein complexes of KU70-KU80, LIG4-XRCC4 and the MRE11-RAD50-NBS1 (MRN) complex (Bray and West, 2005), mediate NHEJ in plants. In this pathway DNA, rejoining is independent of the DNA sequence, and is the predominant DSB repair mechanism in most plant tissues. Homologous recombination (HRc) requires a homologous template for repair, which is catalysed by the RAD52 epistasis group, including the recombinases protein RAD51. DSB detection is linked to intracellular signaling, orchestrated by the protein kinases ATM and ATR, which phosphorylate a number of cellular targets, including the histone variant H2AX (Falck et al., 2005; Friesner et al., 2005). Mammals possess a third DNA ligase gene, termed LIG3, which is expressed as two splice variants (Ellenberger and Tomkinson, 2008). DNA ligase IIIα joins SSBs in DNA, is associated with base excision repair and binds to XRCC1, a protein required for the repair of SSBs induced by c-irradiation and alkylating reagents (Taylor et al., 1998b). LIG3β is expressed only in the testis and may have a role in meiotic recombination (Chen et al., 1995). Human LIG3 also encodes a mitochondrial form, produced by translation initiation at an alternative upstream translation start site in the LIG3a transcript, resulting in a protein with a mitochondrial targeting sequence (Lakshmipathy and Campbell, 2000). Plants, as sessile photosynthetic organisms, are necessarily exposed to high levels of environmental stresses, including UV-B, gamma irradiation and heavy metals, which have necessitated the evolution of a highly effective DNA damage response to counteract continuous genome damage. Subsequently, plants require multiple DNA ligase activities to support replicative, repair and recombination activities in the three genomes localized to the nucleus, mitochondrion and chloroplast, leading to the likelihood that multiple DNA ligase genes are present in plants. Analysis of Arabidopsis genome sequence confirmed the ligase genes. Three DNA ligase sequences were identified, two sequences homologous with animal/yeast DNA ligases I and IV, AtLIG1 and AtLIG4. The other is an uncharacterized DNA ligase termed AtLIG6, which encodes a protein with a domain structure unique to plant species and distinct from those of DNA ligases I, III and IV (West et

al., 2000; Molinier et al., 2004; Bonatto et al., 2005; Sunderland et al., 2006). The functional characterization of DNA LIGASE VI from A. thaliana (AtLIG6) revealed that AtLIG6 is not only required for rapid seed germination under optimal conditions but also becomes an important determinant of germination performance and seed quality in terms of seed viability, vigour and longevity under adverse germination conditions, including accelerated seed ageing, genotoxic stress and low temperature. It was demonstrated that rapid and strong DNA-DSB response is activated at the earliest stages of seed imbibition even, in high-quality seeds implicating repair of cytotoxic DSBs accumulated in the quiescent seed as an important integral component of the germination process (Waterworth et al., 2010). Repair of accumulated DNA damage is initiated in the earliest stages of imbibition, observed as high levels of unscheduled de novo DNA synthesis in seeds several hours before activation of the cell cycle and entry into the S phase (Osborne et al., 1984). Higher levels of repair synthesis occur in aged seeds, often accompanied by a Delay in the onset of replicative DNA synthesis and Delayed germination or loss of viability (Osborne et al., 1984). The expression of AtLIG6 observed in Cvi and Col-0 seeds led to the hypothesis that DNA repair activities also occur in the dormant, imbibed seeds, as previously observed in Lactuca sativa (lettuce) seeds (Villiers, 1974). Seed priming improve seed germination performance and have been shown to be advantageous in producing early and uniform seed germination and seedling emergence, with benefit to seedling establishment in the field. One such priming treatment termed osmopriming (Heydecker et al., 1975), involves controlled hydration of seeds to bring them to the brink of germination, whilst preventing their entry into the final phase of germination (Karssen et al., 1990). Significant levels of nuclear DNA repair synthesis in the absence of any detectable nuclear DNA replication or cell division has been demonstrated in the embryos of both high- and low-vigour Allium porrum L. (leek) seeds during osmopriming, consistent with the importance of DNA repair processes to germination performance (Ashraf and Bray, 1993). However, prolonged imbibition of seeds in water under conditions unfavourable for germination may result in the extended exposure of the seed embryo genome to attack

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by reactive oxygen species (ROS) generated upon water uptake, leading to a requirement for DNA ligase mediated DNA repair. Germination analysis of high quality (un-aged) wild-type seeds and atlig6 mutant seeds revealed Delayed germination in the AtLIG6-deficient lines. Radicle emergence was delayed by 6 h in seeds of atlig6-1 mutants compared with wild-type Col-0 seeds; atlig6-1 seeds reached 50% germination after 35 h of imbibition, compared with wild-type Col-0 lines, which attained 50% germination by 29 h of imbibition (Waterworth et al., 2010). These results indicate that even in the absence of environmental conditions that adversely affect seed germination performance, AtLIG6-mediated DNA repair pathways are still necessary to facilitate rapid germination. However, significant differences were not detected in final seed viability scores between mutant and wild type lines, with all lines studied displaying near 100% germination after 48 h. The results obtained with both atlig6 alleles (atlig6-1 and atlig6-2) germination performances, suggesting that the AtLIG6 has roles in repairing DNA damage accumulated during seed development, storage and/or imbibition (Waterworth et al., 2010). In contrast with previously published data (Friesner and Britt, 2003), atlig4 mutants also displayed a Delay in germination relative to wild-type lines, with reproducible Delays observed in two independent alleles atlig4-5 and atlig4-2, which may reflect differences in germination or growth and seed harvest/storage conditions between the two studies. The atlig4-5 atlig6-1 double mutant displayed a similar germination rate to the atlig6-1 line. DNA ligases are likely to be required for the repair of DNA damage that accumulates in seeds, which correlates with a loss of seed viability (Cheah and Osborne, 1978), and is present in high-quality seeds resulting in DNA repair synthesis early in imbibition (Ashraf and Bray, 1993). DNA ligases are important for seed longevity where, the Delayed germination exhibited by atlig6 mutants suggests that DNA damage and repair pathways are important to seed germination and seed quality. Seed longevity or storability is an important agronomic aspect of seed quality, and is of importance for the conservation of plant genetic resources. Increases in seed moisture content and temperature during storage resulted in quantifiable and predictable decreases in seed viability (Roberts, 1973). Cytological studies,

quantifying aberrant products of Double Strand Break (DSB) repair in seeds, concluded that even small losses of seed viability through ageing are inevitably associated with an increase of chromosome damage (Dourado and Roberts, 1984). The Delayed germination of aged seeds may result from the operation of checkpoints prior to the commencement of DNA replication that control progression through germination in response to the accumulation of damage to the genome. This is analogous with the effects of genotoxic agents or deficiencies in repair capacity that slow germination. Therefore, the response of atlig6-1, atlig4-5 and atlig4-5 atlig6-1 mutants to accelerated ageing was investigated (Waterworth et al., 2010). Measurements of root length in germinated seedlings from each seed lot at 8 and 10 days show substantially reduced seedling vigour in the mutant lines. These results indicate that both NHEJ (involving AtLIG4) and an uncharacterized repair pathway (involving AtLIG6) are important contributors to seed longevity. These results implicate the capacity of the embryo to repair DNA damage that accumulates during storage as being a determinant of seed resilience to deterioration during storage, and the subsequent germination vigour of stored seed lots (Waterworth et al., 2010). Thus, although AtLIG6 facilitates germination in unstressed seeds, it becomes an increasingly important determinant of the quality of aged seeds. DNA ligase 6 is important for germination vigour under cold temperature stress. The germination performance of wild type and DNA ligase deficient lines was evaluated under suboptimal conditions using low temperature stress (germination at 2° C). Germination at low temperatures substantially slows radical emergence, and is a stress that can be encountered by field-grown crops sown in early spring and late autumn. We analyzed seed quality by measuring germination rates, whereas seedling vigour was assessed by the measurement of root elongation rates. DNA ligase 6 is important for germination under oxidative stress previous analysis of atlig4-1 mutants had identified roles for AtLIG4 in the germination of seeds subjected to irradiation by X-rays (Friesner and Britt, 2003). Oxidative damage generated by ROS is believed to be the major source of DNA damage in germination under normal growth conditions (Bray and West, 2005). Germination of seeds

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imbibed in the presence of the genotoxin menadione, which induces oxidative damage to DNA, was used to examine the roles of AtLIG6 in germination under conditions of genotoxic stress. Seeds were imbibed at 4° C for 24 h in the presence of menadione in order to ensure the uptake of the toxin prior to the onset of germination. Wild-type Arabidopsis seeds displayed a progressively Delayed time to radical emergence (51% germination at 4 days in 50 µM menadione) and decline in seed viability to 52%. The results indicate that the Delayed germination of the atlig6-1 and atlig6-2 lines is exacerbated in the presence of menadione, with the mutant lines taking 3.5–5.5 days longer to reach 25% germination. This reduced vigour in the presence of menadione indicates that the AtLIG6-dependent DNA repair pathway is important for germination in the presence of genotoxic stress, and emphasizes the relationship between DNA damage, repair capacity and timing of germination (Waterworth et al., 2010). The genetic interaction between AtLIG4 and AtLIG6 is unclear and the additive nature of the mutations is consistent with partial redundancy between the ligases, whereas the major role of AtLIG4 in the repair of X-ray-induced DNA damage, with only a minor role for AtLIG6. This may reflect difference in substrate specificity between the ligases, or differences in activity between tissue types. Evidence of DNA damage in germinating seeds demonstrates roles for both AtLIG6 and AtLIG4 in germination. This requirement for DNA repair pathways for rapid germination suggests that even in high-quality seeds that have been harvested, stored and germinated under optimal conditions, DNA damage still accumulates and needs to be repaired during imbibition (Waterworth et al., 2010). To investigate the appearance of DSBs in non-aged seeds (Waterworth et al., 2010), examined the DNA damage response in imbibing seeds using an analysis of publically available microarray data (Nakabayashi et al., 2005;Winter et al., 2007). When exposed to clastogens, plants display a very well characterized, ATM dependent transcriptional induction, specifically in response to the presence of DSBs (Culligan et al., 2006; Ricaud et al., 2007). Microarray analysis of temporal gene expression during Arabidopsis seed germination identified coordinated up regulation of a number of DSB-responsive genes in the earliest stages of imbibition, coincident with the

reported incidence of DNA repair synthesis (Nakabayashi et al., 2005). This DSB-induced DNA damage response resulted in the significant up regulation of transcripts within 3 h of imbibition, including RAD51 and PARP2, a protein that plays a number of roles in the DNA damage response. RAD51 expression patterns were verified by semi-quantitative RT-PCR and real-time PCR, confirming the transcriptomic data. In contrast, AtLIG4 and AtLIG6 transcripts showed no significant transcriptional upregulation. Nuclear DNA replication occurs later than DNA repair, around the time of radical emergence (Barroco et al., 2005). In Arabidopsis, it is likely that the S phase and cell division precede radical emergence, as cell cycle activity is required for rapid germination (Masubelele et al., 2005). The repair of these lesions is an important aspect of germination, and provides an explanation for the accumulation of chromosomal aberrations associated with a loss of viability during seed ageing (Dourado and Roberts, 1984). The DNA damage response, indicated by induction of the AtRAD51 transcript, was significantly higher in both the atlig4 and atlig6 mutant lines, compared with Col-0. This suggests that deficiency in either DNA ligase leads to an accumulation of DNA damage in seeds, and is consistent with the observed Delay in germination in both mutant lines. Previous studies of atku80 mutants revealed a constitutive up regulation of DNA damage in seedlings in the absence of external stresses (West et al., 2004), and hypersensitivity to methyl methanesulfonate-induced DNA damage specifically during germination (Riha et al., 2002). In plants, the accumulation of DNA strand breaks during storage of orthodox seeds (i.e. seeds that survive drying to low moisture content) reduces viability and vigour, although the specific DNA lesions responsible for impaired germination and their repair pathways remain poorly defined. Recent studies point to a role for the DNA damage response protein poly (ADP-ribose) polymerase (PARP) in germination (Hunt et al., 2007), although the loss of base excision repair factors did not affect germination (Murphy and George, 2005), suggestive that in plants, as in other organisms, high levels of base damage can be tolerated. Following irradiation of seeds, the Arabidopsis mutant’s atku70 and atlig4 displayed Delayed germination relative to irradiated wild-type controls, supportive of roles for NHEJ in the repair of DNA

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double-strand breaks (DSBs) as a necessary prerequisite for the germination of irradiated seeds (Friesner and Britt, 2003). Phenotypic analysis of irradiated kernels of maize rad51 (an HR pathway component) mutant lines also revealed a severe Delay in seed germination relative to wild-type lines (Li et al., 2008). Waterworth et al. (2010) demonstrated that the accumulation of DNA double-strand breaks (DSBs) and the capacity to repair these DSBs influences the germination rate of seeds. This indicates that genome integrity is sensed during imbibition and is part of the control mechanism that determines the timing of germination, as measured by radical emergence. 2. D. Alkylation Repair Mechanism of

Damaged RNA The current understanding of turnover or repair mechanisms for damaged RNA varies for different forms of damage. For some, such as alkylation, a clear mechanism has been identified. For most other types of damage, the roles of normal RNA turnover mechanisms and alternate pathways are still under investigation (Wurtmann and Wolin, 2009). Alkylated nucleic acids can be repaired by enzyme-catalyzed oxidative demethylation. The E. coli AlkB enzyme and one of the human oxidative demethylases, hABH3, demethylate 1-methyladenine and 3-methylcytosine in RNA in addition to having activity on DNA (Aas et al., 2003). In vivo, the action of AlkB and hABH3 can lead to the restoration of RNA function, as exemplified by reactivation of alkylated RNA bacteriophages (Aas et al., 2003), and tRNA (Ougland et al., 2004). Interestingly, because these oxidative demethylases only function to remove methyl groups from 1-methyladenine and 3-methylcytosine, certain classes of RNAs may be more likely to accumulate alkylation damage that cannot be repaired. Alternatively, demethylases with other specificities may remain to be identified (Wurtmann and Wolin, 2009). The efficacy of AlkB in restoring tRNA function as measured by in vitro assays is less than that for mRNA, consistent with the more double stranded nature of tRNA, which would lead to alkylation damage predominately at positions not repaired by AlkB (Ougland et al., 2004). This suggests that in vivo certain classes of RNAs may show higher capacity for repair, while others may be targeted by other mechanisms for

decay. Finally, it has been noted that AlkB has only weak affinity for DNA and may interact with other DNA-binding proteins in order to increase its affinity in vivo. This may be true for RNA as well, and it will be of interest to identify proteins that function with these demethylases (Yang et al., 2008). 2. D. I. Photolyases Repair of Cyclobutane and

Pyrimidine

Cyclobutane pyrimidine dimers are actively repaired in some organisms by photolyase enyzmes that catalyze light-dependent dimer splitting. Whereas, many prokaryotes and eukaryotes have DNA photolyases, the physiological relevance of DNA photolyases in RNA repair is most likely minimal, as exemplified by the poor RNA binding of the E. coli photolyase (Kim and Sancar, 1991). RNA photolyase activity has only been observed in plants (Gordon et al., 1976) and insects (Jackle and Kalthoff, 1980). Therefore, it is unclear to what extent most organisms actively repair cyclobutane pyrimidine dimers in RNA. The rRNA-protein crosslinking observed in maize subjected to UVB irradiation (Casati and Walbot, 2004), a cellular handling mechanism is implied by the disappearance of the crosslinked products in a recovery period following UVB exposure. However, the mechanism that recognizes and degrades the crosslinked RNAs and proteins is not yet known (Wurtmann and Wolin, 2009). 2. D. II. Repair Mechanism for Oxidized

Nucleotides

A mechanism has been described for the turnover of oxidized free nucleotides. Clearing oxidatively damaged free nucleotides can prevent incorporation of these nucleotides into newly synthesized RNAs. Besides triggering problems caused by the presence of oxidized nucleotides in a particular RNA, 8-oxoG can be incorporated opposite adenosine by RNA polymerase and thus cause mutations at the level of transcription. Such mutations can cause errors in protein synthesis if they occur in mRNAs, affect the folding, or function of noncoding RNAs. In E. coli, the MutT protein hydrolyzes 8-oxoguanosine triphosphate (8-oxoGTP) to 8-oxoguanosine monophosphate (8-oxoGMP) in addition to catalyzing the same

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hydrolysis reaction on 8-oxo-2′-deoxyguanosine triphosphate (Taddei et al., 1997). 2. D. III. Degradation of Damaged RNAs

There is also evidence for the turnover of damaged mRNAs and noncoding RNAs where the quantification of 8-oxoG in RNA after oxidative treatment of cultured human lung epithelial cells shows that approximately a third of the 8-oxoG in RNA is cleared by 3 hours after removal of oxidative stress (Hofer et al., 2005). As 95% of cellular RNA is noncoding RNA with a normal half-life much greater than 3 hours, mechanisms must exist to rapid degrade some damaged RNAs. Interestingly, after the 3 hour time point, the turnover of 8-oxoG slows, leading to an overall half-life of ~12.5h for 8-oxoG in RNA. Similar observations have been made for Hela cells treated with sub-lethal doses of H2O2 (Wu and Li, 2008). 8-oxoG levels in RNA is rapidly peaked following a pulse of oxidative stress, were reduced by half within 30 minutes of recovery, and then slowly reached basal levels by 24h. Additionally, turnover or degradation has been reported in total RNA isolated from rat astrocytes subjected to ammonia stress (Gorg et al., 2008). However, elevated levels of oxidized RNA measured in studies of aging and neurodegeneration imply that at least some fraction of damaged RNA accumulates in those contexts (Liu et al., 2002; Nunomura et al., 1999). The rRNA in particular, there are reports of degradation following oxidative damage. In human atherosclerotic plaques, reduction of 18S and 28S rRNA levels coincides with high 8-oxoG RNA levels (Martinet et al., 2004). Isolation of total RNA and ribosomes from affected regions of AD patient brains has shown decreased levels of all four individual rRNAs (5S, 5.8S, 18S, and 28S) and in turn decreased levels of the 40S and 60S ribosomal subunits and mature 80S ribosomes (Ding et al., 2005; Ding et al., 2006). Degradation of the 25S and 5.8S rRNAs is also seen in yeast under oxidative stress (Mroczek and Kufel, 2008; Thompson et al., 2008). However, for these observations, it is unclear to what extent the rRNA degradation was an effect of ongoing apoptosis, rather than a specific mechanism to degrade damaged RNAs. Oxidative stress has also been noted to induce the accumulation of cleaved tRNA halves in yeast, Arabidopsis thaliana, and human cells (Thompson et al., 2008).

2. D. IV. Cytoplasmic Repair of Ultra-Violet

Oxidatively Damaged RNAs

Conditions that cause RNA damage can also lead to translational arrest and induction of two sites of mRNA accumulation within the cytoplasm, stress granules and processing bodies [P bodies] (Anderson and Kedersha, 2008). The role of these foci in the turnover of damaged RNA is just beginning to be understood. The first step in the formation of stress granules occurs through pathways that converge on eukaryotic initiation factor 2α (eIF2α) to stall translation. For example, arsenite-induced oxidative stress leads to heme-regulated initiation factor 2α kinase (HRI) inactivation of eIF2α, the inhibition of protein synthesis, and the formation of stress granules (McEwen et al., 2005). UV irradiation also leads to stress granule formation, possibly because UV irradiation activates protein kinase R, which is a regulator of eukaryotic initiation factor 2α (eIF2α) (Kedersha et al., 1999; Anderson and Kedersha, 2008). Ribosomes blocked at initiation accumulate on mRNA as 48S structures, which then begin to nucleate stress granules through the binding of a variety of aggregate-forming RNA-binding proteins (Kedersha et al., 1999). Translational arrest of mRNAs that had been undergoing translation during normal growth conditions may allow cells to degrade damaged RNAs and alter the profile of mRNA translation in response to stress. mRNAs are thought to exit stress granules by re-initiation of translation or transfer to processing bodies (P-bodies) (Kedersha et al., 2005). P-bodies are sites of mRNA decapping and decay and are found in cells under normal growth conditions but are further induced by stress (Eulalio et al., 2007). Decay of a de-adenylated mRNA via the 5′-3′ pathway is initiated by removal of the 5′ cap structure by the de-capping complex Dcp1-Dcp2. Numerous activators of de-capping also act at this step, including the Sm-like (Lsm) 1–7 complex, Pat1, and the helicase Dhh1. After decapping, the mRNA is degraded by the 5′-3′ exo-ribonuclease Xrn1. The proteins involved in the 5′-3′ decay pathway are all localized to P-bodies (Sheth and Parker, 2003). The decay of mRNA has been demonstrated to occur within these foci, as yeast strains that lack 5′-3′ exo-ribonuclease (Xrn1) have enlarged P-bodies that

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contain accumulating mRNA. Interestingly, at least one type of mRNA quality control, nonsense-mediated decay (NMD), is thought to use P-bodies as a site of degradation of defective mRNAs. Several factors essential for NMD are localized to P-bodies (Fukuhara et al., 2005; Unterholzner and Izaurralde, 2004) and mRNAs containing premature stop codons are recruited to P-bodies by the nonsense-mediated decay (NMD) protein Upf1 (Sheth and Parker, 2006). The nonsense-mediated decay (NMD) may occur in P-bodies raises the possibility that P-bodies may be sites of turnover for other types of aberrant mRNAs. The function of P-bodies in the handling of damaged RNA is supported by the increased sensitivity of cells lacking certain P-body proteins to conditions that cause RNA damage. Notably, a mutant xrn1 strain displays UV sensitivity (Tishkoff et al., 1991). This is consistent with the UV-sensitive phenotypes for strains lacking Pat1 and strains lacking Lsm1 (Birrell et al., 2001; Wang et al., 1999). Interestingly, in addition to the UVC sensitivity of the lsm1∆ Deletion strain, both the lsm1∆ and lsm6∆ Deletion strains are sensitive to the alkylating agent methyl methanesulfonate (MMS) (Chang et al., 2002). These phenotypes suggest that P-bodies may be important for the decay of several different types of damaged mRNAs and that failure to degrade damaged RNAs can impact cell growth. Moreover, in addition to these stress sensitive phenotypes of P-body proteins, the role of P-bodies in conditions that cause RNA damage is indicated by findings that while P-bodies exist under normal cellular conditions, they enlarge or change subcellular localization in response to certain stress conditions. Yeast cells exposed to UV light show intensified localization of the RNA helicase Dhh1 and the decapping protein Dcp2 to P-bodies during recovery following treatment (Teixeira et al., 2005). Following arsenite-induced oxidative stress of mammalian cells, stress granules form and P-bodies localize adjacent to stress granules, possibly to expedite the degradation of damaged mRNAs accumulating in stress granules (Yu et al., 2005; Kedersha et al., 2005). Distinct cytoplasmic bodies named UV-induced mRNA granules (UVG) have also been proposed (Gaillard and Aguilera, 2008). Following UV treatment of yeast cells, over expressed reporter mRNAs were observed to be

stabilized and to accumulate in UVGs, which do not colocalize with P-body or stress granule components. It is possible that such localization serves to sequester mRNAs from translation, but it is not yet known how sorting to UVGs occurs or whether these RNAs contain UV damage. 2. D. V. Protein Role in Repairing Oxidatively

Damaged RNAs

Another candidate for involvement in turnover of oxidatively damaged RNA that is also localized to P-bodies and stress granules (Yang and Bloch, 2007) is the Y-box-binding protein (YB-1). A member of the cold-shock domain super family, YB-1 participates in a wide range of processes, including transcription regulation, translation regulation, and DNA repair (Kohno et al., 2003). In vitro experiments suggest that YB-1 may have specific binding for 8-oxoG-containing oligonucleotides. A role for YB-1 in removal of oxidatively damaged RNAs is also consistent with the observation that expression of human YB-1 in E. coli increases resistance to paraquat-induced oxidative stress (Hayakawa et al., 2002) while YB-1 knockdown induces UV sensitivity in human epidermoid cells (Ohga et al., 1996). The numerous roles of YB-1, as stress resistance could also be a result of YB-1 regulation of transcription. One additional indication of a role for YB-1 in handling damaged nucleic acids is that YB-1 has the capacity to stimulate the activity of base excision repair of the NEIL2 protein, which removes oxidized bases from DNA. While, oxidatively damaged RNAs are not thought to repair. This finding suggests a model wherein YB-1 binding to 8-oxoG-containing RNAs may assist turnover of these RNAs by other proteins (Das et al., 2007). Much research has focused on the human polynucleotide phosphorylase protein (hPNPase), a homolog of the well-characterized bacterial 3′-5′ exoribonuclease. In bacterial cells, PNPase participates in mRNA turnover as well as the degradation of some noncoding RNAs, including aberrant precursor tRNAs and rRNAs (Deutscher, 2006). Human polynucleotide phosphorylase protein (hPNPase) emerged as a candidate for turnover of oxidatively damaged RNAs when in vitro binding experiments suggested hPNPase has some specificity for oxidatively damaged RNA (Hayakawa et al., 2001). Additionally, overexpression of hPNPase lowers levels of 8-oxoG RNA while siRNA

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knockdown of hPNPase raises 8-oxoG levels (Wu and Li, 2008). The possible roles for hPNPase in degrading oxidized RNAs must be reconciled with the localization of hPNPase to the mitochondrial inter membrane space, which is not known to contain RNA. While hPNPase mobilization into the cytosol is seen in conditions that induce apoptosis, it is not clear whether hPNPase is also released into the cytosol under other stress conditions (Chen et al., 2006). 2. E. Protection of DNAs Damage

It was demonstrated that polyphenol rich diets might decrease the risk of chronic diseases by reducing oxidative stress. The Fenton reaction is prevented by hydroxyl radical-scavenging flavonoids (Husain et al., 1987). Here, the capacities of all detected ten plant extracts to protect against oxidative DNA damage were checked against DNA strand scission by ●OH generated in Fenton reactions on pBluescript II SK (–) DNA. Polyphenol rich diets may decrease the risk of chronic diseases by reducing oxidative stress (Kim et al., 2003). The Fenton reaction is prevented by hydroxyl radical-scavenging flavonoids (Husain et al., 1987). Here, the capacities of all ten plant extracts to protect against oxidative DNA damage were checked against DNA strand scission by ●OH generated in Fenton reactions on pBluescript II SK (–) DNA. Kalim (2010) conclude that a significant contributor to DNA damage prevention is the scavenging of ●OH by the extracts of Celome scariosus, Celome icosandra, Rosa damascena and Holarrhena antidysenterica at 0.13µg/ml, 0.16 µg/ml, 0.2 µg/ml and 0.28 µg/ml, respectively; this was corroborated by densitometric analysis. The effective extracts, viz. Celome icosandra, Rosa damascena and Cyperus scariosus, were not cytotoxic in comparison to doxorubicin, and this appears consistent with their long history of use in the Unani system of medicine. Unani plants like Celome icosandra, Rosa. damascena and Cyperus scariosus showed significant oxidative DNA damage preventive activity and antioxidant activity.70% methanol extract of the fruits of Terminalia chebula, Terminalia belerica and Emblica officinalis, imposes the fact that they might be useful as potent sources of natural antioxidant. Studies indicated that Gingko biloba, Centella asiatica, Hippophae rhamnoides, Ocimum sanctum, Panax ginseng, Podophyllum hexandrum, Amaranthus

paniculatus, Emblica officinalis, Phyllanthus amarus, Piper longum, Tinospora cordifoila, Mentha arvensis, Mentha piperita, Syzygium cumini, Zingiber officinale, Ageratum conyzoides, Aegle marmelos and Aphanamixis polystachya, and Paeonia mffiticosa were protected against radiation-induced lethality, lipid peroxidation and DNA damage (Akhilesh, 2007; Jagetia, 2007). Investigation into the nature of DNA damage and repair have provided valuable insight into aging, human genetics and cancer. Now, there is deep interest in identifying free radical scavengers or antioxidants that inhibit oxidative DNA damage. DNA damage due to oxidative stresse, besides the role of potent antioxidants in its protection was investigated by Nisha and Deshwal (2011). Initially a Fe3+ dependent system is designed to test the scavenging activity of plant extracts on radicals generated by iron, because hydroxyl radicals are known to be the most reactive of all the reduced forms of dioxygen and are thought to initiate cell damage (Mello-Filho et al., 1991; Duarte and Lunec, 2005). The scavenging effect of plant extract on Fe3+ dependent hydroxyl radicals was investigated, whether the extract reduced Fe3+ dependent DNA nicking. When pBR322 plasmid DNA was dissolved in the reaction mixture, a time-dependent increase in the formation of single-stranded nicked DNA (Form II) and of double-stranded nicked and linear DNA (Form III) was observed. However, the addition of 20 íg of Opuntia ficus-indica (OFS) extract to the nicking reaction mixture increased Form I DNA formation. Consequently, the treatment caused Fe3+-mediated Form III DNA formation to disappear and reduced Form II DNA formation. This OFS ethanol extract-mediated antioxidant activity was similar to that of 2 U of superoxide dismutase (SOD) and 5 U of catalase. These results indicate that the Opuntia ficus-indica (OFS) ethanol extract effectively mitigates the oxidative stresses on susceptible biomolecules, such as DNA (Chevion, 1988). Similarly, extracts of Curcumin is a non-toxic, highly promising natural antioxidant compound having a wide spectrum of biological functions. In addition, sunflower, Trapa, Onion, lotus extracts…etc showed very good protection from DNA damage (Hofer et al., 2006). Lycopene probably acted by suppressing the oxidative stress caused due to high levels of iron that may be carcinogenic. Sies (1996) demonstrated that rats treated with a high level of iron (ferric nitrilotriacetate) significant increased DNA damage and Malondialdehyde

Ph ton 50

(indicator of lipid oxidation) level in the prostate. The pre-treatment of the rats with lycopene and beta-carotene reversed these effects and DNA damage was almost completely prevented. Jeong et al. (2002) reported that n-Butanol soluble fraction (PE) derived from methanol extract of Mentha spicata Linn., exhibited significant protecting activity against DNA strand scission by ●OH on pBluescript II SK(–) DNA. 2. E. I. DNA Protection from ROS

Eukaryotic organisms evolved under aerobic conditions subjecting nuclear DNA to damage provoked by reactive oxygen species (ROS). Although ROS are thought to be a major cause of DNA damage, little is known about the molecular mechanisms protecting nuclear DNA from oxidative stress (Vanderauwera et al., 2011). Reactive oxygen species (ROS) are toxic molecules continuously produced in cells during aerobic metabolism. In plants ROS are produced mainly in peroxisomes during photorespiration, in chloroplasts during photosynthesis, and in mitochondria during respiration (Apel and Hert, 2004; Mittler et al., 2004). During aerobic metabolism, the reactive oxygen species (ROS) are formed. Because of possible negative impact of ROS on organisms, the cells have set up strategies for their elimination. An imbalance between the production of ROS and their elimination may cause tissue damage. This imbalance is called “oxidative stress” (Sies, 1997). Damage could affect all types of biological molecules, i.e., DNA, (Cooke et al., 2003), lipids (Halliwell and Chirico, 1993; Gutteridge, 1995), proteins (Berlett and Stadtman, 1997) and carbohydrates. Protection of nuclear DNA in plants requires a coordinated function of ROS-scavenging pathways residing in the cytosol and peroxisomes, demonstrating that nuclear ROS scavengers such as peroxiredoxins and glutathione are insufficient to safeguard DNA integrity. Therefore, both catalase (CAT2) and cytosolic ascorbate peroxidase (APX1) play a key role in protecting the plant genome against photo respiratory-dependent H2O2-induced DNA damage. In apx1/cat2 double-mutant plants, a DNA damage response is activated, suppressing growth via a WEE1 kinase-dependent cell-cycle checkpoint. This response is correlated with enhanced tolerance to oxidative stress, DNA stress-causing agents, and inhibited programmed cell death (Vanderauwera et al.,

2011). A genome-wide transcriptome analysis of WT, apx1, cat2, and apx1/cat2 plants grown under ambient air and exposed to high light (HL) for 0 or 1 h identified 381 transcripts that specifically and constitutively accumulated in apx1/cat2 plants. In contrast, no apx1/cat2-specific HL-induced transcripts were identified. Interestingly, none of the 381 constitutively accumulating transcripts found in apx1/cat2 plants corresponded to known enzymes with superoxide- or H2O2-scavenging activities (Mittler et al., 2004). However, a significant enrichment for genes that previously had been reported to be induced by different genotoxic stresses, such as γ irradiation and hydroxyurea, including BRCA1, PARP2, B-type cyclin (CYCB1;1), and RAD51, which are typical hallmarks of the DNA damage response (DDR) (Cullings et al., 2006). When plants were grown under high CO2 levels, which suppressed ROS accumulation because of CAT2 deficiency, the accumulation of DNA damage response (DDR) transcripts in apx1/cat2 plants was suppressed. In time-course experiments in which apx1/cat2 plants were released from a high CO2 environment to ambient air, DNA stress-responsive transcripts accumulated within 1 d, corresponding with the induction of the high light (HL) resistance phenotype in apx1/cat2 plants (Vanderauwera et al., 2011). DNA damage and DNA damage response (DDR) in plants were studied mainly in response to exogenously applied DNA damaging agents such as bleomycin and hydroxyurea or ionizing irradiation (Culligan et al., 2006). Vanderauwera et al., (2011) demonstrate that a DNA damage response (DDR) is induced in Arabidopsis thaliana double mutants lacking APX1 and CAT2 and that this response is correlated with an increased tolerance for agents causing oxidative stress and DNA stress. These results indicate that a coordinated function of ROS-scavenging pathways in the cytosol and other cellular compartments is required for the protection of nuclear DNA, demonstrating that alternative nuclear ROS scavengers such as 1-cysteine peroxiredoxins, glutathione (GSH), and flavonoids are insufficient to safeguard DNA integrity. Unless detoxified by specialized enzymes and low molecular antioxidants, ROS can lead to protein, lipid, and DNA oxidation and to cell death (Apel and Hert, 2004; Mittler et al., 2004). Reactive oxygen species (ROS) such as ●O2

–, H2O2 and ●OH are highly toxic to cells. Cellular antioxidant

Ph ton 51

enzymes and the free-radical scavengers normally protect a cell from toxic effects of the ROS. However, when generation of the ROS overtakes the antioxidant defense of the cells, oxidative damage of the cellular macromolecules (lipids, proteins, and nucleic acids) occurs, leading finally to various pathological conditions (Bandyopadhyay et al., 1999). Plants contain a large network of genes encoding different pathways involved in ROS scavenging and production, with a key role in managing the overall steady-state level of ROS in cells (Mittler et al., 2004). Similar to genotoxic agents or ionizing radiation, ROS-derived DNA oxidation leads to altered bases and damaged sugar residues, resulting in DNA single- and double-strand breaks (Amor et al., 1998; Roldan-Arjona and Ariza, 2009). Strand breaks trigger a DNA damage response (DDR) by inducing the expression of molecular markers associated with DNA damage repair, such as poly(ADP ribose) polymerase (PARP), RAD51, and BREAST CANCER (BRCA) family members (Doutriux et al., 1998; Siaud et al., 2004). Upon DNA stress, the ataxia telangiectasia-mutated (ATM) and the ataxia telangiectasia and Rad3-related (ATR) signaling kinases are activated and lead, via the WEE1 serine/threonine kinase, to a transient cell-cycle arrest that allows cells to repair DNA before proceeding into mitosis (De Schutter et al., 2007). Although, oxidative DNA base damage has shown to initiate a DNA damage response (DDR) in mammalian and yeast cells (Hammond et al., 2007; Liu et al., 2009), reports in plants on either the sources of oxidative stress that cause DNA damage or the subsequent induction of a DNA damage response (DDR) directly through ROS remain scarce (Stavera and Gichner, 2002). ROS toxicity and its involvement in different human pathologies underline the need to identify new pathways and proteins that can mitigate the adverse effects of ROS accumulation (Graves et al., 2009). Despite the widely assumed involvement of ROS in DNA damage in plants, no transcriptional DNA damage response (DDR) has been observed in various genome-wide transcript profiling studies that directly monitored ROS- or abiotic stress-mediated responses in Arabidopsis (Gadjev et al., 2006). Oxidative lipid and protein modifications that occur upon ROS accumulation in cells, the lack of a detectable DNA damage response (DDR) might reflect the existence of a highly efficient mechanism that

preserves the nuclear plant genome against oxidative stress (Hardin et al., 2009). Vanderauwera et al. (2011) demonstrated that ROS produced in specific cellular compartments might reach the nuclei and trigger a DNA damage response (DDR) that is accompanied by activation of a cell-cycle checkpoint and that is essential to safeguard cells from oxidative stress. This protective response requires a coordinated balance between different ROS-removal mechanisms residing outside the nuclei, i.e., in the cytosol and peroxisomes. Extra nuclear protection of chromosomal DNA, BI-1, and the DNA damage response (DDR) therefore are important for the survival and growth of eukaryotic organisms under aerobic conditions. At 0.1 mg/ml, the ethyl acetate extract (EAE) of the crude 85% methanolic extract (CAE) of Stevia rebaudiana leaves exhibited preventive activity against DNA strand scission by ●OH generated in Fenton's reaction on pBluescript II SK (-) DNA. Its efficacy is better than that of quercetin. The radical scavenging capacity of CAE was evaluated by the DPPH test (IC50=47.66+/-1.04 µg/ml). EAE was derived from CAE scavenged DPPH (IC50=9.26+/-0.04 µ/ml), ABTS+ (IC50=3.04+/-0.22 µ/ml) and ●OH (IC50=3.08+/-0.19 µ/ml). Additionally, inhibition of lipid peroxidation induced with 25 mM FeSO4 on rat liver homogenate as a lipid source was noted with CAE (IC50=2.1+/-1.07 mg/ml). The total polyphenols and total flavonoids of EAE were 0.86mg gallic acid equivalents/mg and 0.83 mg of quercetin equivalents/mg, respectively. Flavonoids, isolated from EAE, were characterized as quercetin-3-O-arabinoside, quercitrin, apigenin, apigenin-4-O-glucoside, luteolin, and kaempferol-3-O-rhamnoside by LC-MS and NMR analysis. These results indicate that Stevia rebaudiana may be useful as a potential source of natural antioxidants. References

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Zwieb C., A. Ross, J. Rinke, M. Meinke and R. Brimacombe (1978). Evidence for RNA-RNA cross-link formation in Escherichia coli ribosomes. Nucleic Acids Res, 5: 2705-20.

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3. Altrastructural Alteration of Bichemical

Compounds Caused by Abiotic Stresses

3. A. Lipid Peroxidations

3. A. I. General Concepts

Three different mechanisms are able to induce lipid peroxidation: autoxidation (Halliwell and Gutteridge, 1989), photooxidation (Aro et al., 1993), and enzyme catalysis via lipo- or cyclo-oxygenases (Feussner and Wasternack, 2002; Montillet et al., 2002). While lipoxygenase-mediated peroxidation and photo-oxidation are considered very significant pathways in chloroplasts, due to the absence of photosensitizers or lipoxygenase activity in mitochondria, free radical autoxidation is the primary route of lipid peroxidation (Siedow and Girvin, 1980). Glyceraldehyde 3-phosphate dehydrogenase, a well-defined redox-sensitive glycolytic enzyme, was found to be a surface-associated fibronectin and laminin-binding protein in Streptococcus pyrogenes, Schistosoma mansoni and Candida albicans (Gozalbo et al., 1998). In plants, the activation of lipoxygenases, which produce fatty acid hydroperoxides from polyunsaturated fatty acids, leads to the formation of the bioactive compounds called oxylipins, which have diverse roles in signaling in biotic and abiotic stresses (Porta and Rocha-Sosa, 2002). Hydroxyl radical can cause the peroxidation of membrane lipids. To ameliorate the damage caused by hydroxyl radical formed superoxide radical and hydrogen peroxide, organisms have evolved mechanisms to control the concentration of the two reactants (Campana et al., 2004). The aldehydes produced during the lipid peroxidation can react with proteins residues and thus change their structure. This is not a direct reaction of ROS, but it can also cause serious damage of proteins (Refsgaard et al., 2000). The interaction of AOS, specifically ●OH, which is sufficiently reactive to facilitate autoxidation

with polyunsaturated fatty acids initiates lipid peroxidation. First-chain initiation involves the attack of ●OH on the methylene (-CH2-) of a polyunsaturated fatty acid, to abstract a hydrogen atom. This abstraction of an H from -CH2- group leaves behind an unpaired electron on the carbon (-CH-). The presence of a double bond in the fatty acid weakens the C-H bonds on the carbon atom adjacent to the double bond and so makes removal of the H easier. The carbon radical tends to be stabilized by a molecular rearrangement to form a conjugated diene. These can undergo various reactions, the most common being, in aerobic conditions, to combine with O2, giving rise to a (1st) peroxyl radical (CHOO●). Propagation of the chain reaction continues by the (1st) peroxy radical abstracting an H from another adjacent polyunsaturated fatty acid. The carbon radical (CH●) formed can react with O2 to form another (2nd) peroxyl radical and so the chain reaction of lipid peroxidation continues. The initial (1st) peroxyl radical combines with the hydrogen it had removed to yield a lipid hydroperoxides. The hydroperoxides are then degraded non-enzymatically to yield carbonyl compounds, many of which are aldehydes (Noordermeer et al., 2000; Schneider et al., 2001). This decomposition is enhanced by the presence of metal ions, such as Fe2+ (Poli and Schaur, 2000). Fats and oils are energy sources that are composed mostly of triacylglycerols. Lipid profiles are risk indicators of coronary heart disease. Various types of lipoproteins exist, but the two most abundant are Low-density Lipoprotein (LDL) and High-density Lipoprotein (HDL). Lipid peroxidation is the introduction of a

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functional group containing two catenated oxygen atoms into unsaturated fatty acids in a free radical reaction. Life in oxygen has led to the evolution of biochemical adaptations that exploit the reactivity of Active Oxygen Species (AOS). Antioxidant enzymes are an important protective mechanism ROS (Dauqan et al., 2011). Inhibition of lipid peroxidation in liver and kidney were observed as 15.92 ± 3.01% and 17.10 ± 3.48% in ethanolic extracts of Anacardium occidentale bark and leaves respectively while it was 30.67 ± 0.47% for Carica papaya Linn. The water extract of Azachiractha indica bark inhibited liver lipid peroxidation by 8.70 ± 0.32% while that of Anacardium occidentale bark inhibited kidney lipid peroxidation by 11.78 ± 1.08%. These results suggested a need for further examination of the water extract of Anacardium occidentale bark as this part of the plant appears to be critical in the phytotherapy of malaria infection (Iyawe and Azih, 2011). The aqueous extract of Anacardium occidentale and Azachiractha indica barks had the highest degrees of lipid peroxidation in kidney and liver tissues. In most of malaria endemic areas, the aqueous extracts of these plants and not the ethanolic extracts are most often used to treat malaria infection (Iyawe and Azih, 2011). Most biologically relevant free radicals are derived from oxygen and nitrogen and the so-celled ROS and Reactive Nitrogen Species (RNS). Both these elements are essential but in certain circumstances are converted into free radicals which are highly unstable and their reactive capacity makes them capable of damaging biologically relevant molecules such as proteins, lipid or carbohydrates (Peter, 2007). Hydroxyl radical can cause the peroxidation of membrane lipids. To ameliorate the damage caused by hydroxyl radical formed superoxide radical and hydrogen peroxide, organisms have evolved mechanisms to control the concentration of the two reactants (Campana et al., 2004). In nodules of several legumes exposed to drought (Gogorcena et al., 1995), nitrate (De Lorenzo et al., 1994; Escuredo et al., 1996), prolonged darkness (Gogorcena et al., 1997; Hernandez-Jimenez et al., 2002), or pro-oxidants such as Cd or H2O2 (Loscos et al., 2008), there was accumulation of lipid peroxides or oxidized proteins concomitant with a decline in antioxidant protection. Wei and Shibamoto (2007) studied the antioxidant activities of major essential oils from several plants and reported

that myristicin from parsley seeds, patchouli alcohol from patchouli, and citronellol from roses showed high antioxidant activities. An increase in hydroxyl radical production and catalytic Fe was detected (Becana and Klucas, 1992; Gogorcena et al., 1995). These observations were also interpreted in terms of oxidative damage in nodules as result of an increase in ROS production and ⁄ or a decrease in antioxidant defenses. Recent work indicated that the application to pea roots of paraquat, a compound that exacerbates formation of superoxide radicals, caused similar effects to those produced by drought (Marino et al., 2006). 3. A. II. Lipid Peroxidation in Cell Organelles

The sensitivity of key mitochondrial enzymes to damage by AOS and lipid peroxidation products has the potential to impair primary metabolism significantly. This is an aspect of environmental stress that has been highlighted by the work of some especially that with regard to photorespiration (Wingler et al., 2000; Bauwe and Kolukisaoglu, 2003). Plant mitochondria link the cellular processes of carbon and nitrogen metabolism through the tricarboxylic acid cycle and the photorespiratory cycle, where environmental stresses lead to damage of specific mitochondrial targets through the direct action of reactive oxygen species (ROS) and indirect action of lipid peroxidation products. Lipid peroxidation is broadly defined as the oxidative deterioration of polyunsaturated lipids, but in a mitochondrial context, it principally refers to the polyunsaturated fatty acids of membrane lipids such as linoleic acid, linolenic acid and arachidonic acid, which yield various cytotoxic aldehydes, alkenals and hydroxyalkenals. Damage to Fe-S centres and proteins containing lipoic acid moieties appear to predominate in current reports. Substantial evidence that both TCA cycle and photorespiratory capacity of mitochondria are sensitive sites for damage is highlighted and the implications for mitochondrial-dependent carbon and nitrogen metabolism are discussed (Taylor et al., 2004). The components of the photorespiratory cycle in mitochondria decarboxylate and deaminate glycine to produce serine, which feeds into the resynthesis of phosphoglycerate for the Calvin cycle. This process also releases ammonia that becomes a source for nitrogen assimilation, yielding amino acids in plants. Both the TCA and

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photorespiratory cycles produce the bulk of NADH in mitochondria required for fuelling respiratory electron transport and thus ATP production. A disruption in the normal function of the mitochondria will therefore, have serious consequences for plant carbon and nitrogen metabolism and cellular biosynthetic reactions (Taylor et al., 2004). Active oxygen species (AOS) are able to damage proteins, lipids and nucleic acids in plants and thus their production and removal must be strictly controlled (Mittler, 2002; Moller, 2001; Noctor and Foyer, 1998). In animal cells, mitochondria are major sites of AOS formation and major targets of AOS-induced damage (Skulachev, 1996; Kowaltowski and Vercesi, 1999). However, in plant cells, especially in photosynthetic cells, the plastids and peroxisomes are also likely to produce large quantities of AOS, but mitochondrial enzymes are nonetheless susceptible to this and their response may affect leaf metabolism significantly. Respiration of isolated plant mitochondria can be disrupted by the generation of AOS (Verniquet et al., 1991) or the production of toxic lipid peroxidation end-products following AOS accumulation (Millar and Leaver, 2000; Taylor et al., 2002). Peroxisomes are cell compartments involved in many physiological functions, such as lipid mobilization, photorespiration, and hormone biosynthesis under normal and stress conditions (Del Rio et al., 1992; Hayashi and Nishimura, 2003; Reumann, 2004; Pracharoenwattana and Smith, 2008; Corpas et al., 2009). Peroxisomal proteins are selectively targeted for import from the cytosol post translationally by either a peroxisomal targeting sequence or protein-protein associations. So-called PEX genes are also involved in regulating peroxisomal biogenesis. At least 22 PEX genes have been identified in Arabidopsis (Nito et al., 2007), and it has been demonstrated that APM2 and APM4 encode proteins homologous to peroxins Pex13 and Pex12, respectively (Mano et al., 2006). Pex12 is an integral membrane protein containing a RING-finger domain that functions as an ubiquitin ligase, an essential component of a multi protein complex for peroxisomal matrix protein import (Albertini et al., 2001; Mano et al., 2006). It has also been suggested that Pex13 as well as Pex14 and Pex17 are membrane bound peroxins that act as a docking complex to import proteins into the peroxisomal matrix (Mullen et al., 2001). Thus, Arabidopsis mutant defects in the PEX13 gene cause loss of peroxisomal

function due to misdistribution of peroxisomal matrix proteins in the cytosol (Mano et al., 2006). The Pex13 and Pex14 proteins appear to operate stoichiometrically in vivo, acting as the ideal docking proteins for the receptor-cargo complexes (Azevedo and Schliebs, 2006). Furthermore, it has been demonstrated that apm2/4 mutants have also disturbed the PTS2-dependent protein transport mechanism (Mano et al., 2006), indicating that the apm2/4 mutants are characterized by defective targeting of both PTS1- and PTS2-containing proteins. The nuclear restorer gene of maize T cytoplasm male sterility (CMS) as an aldehydes dehydrogenase (Cui et al., 1996) has excited researchers to consider that restoration may be due to the alleviation of lipid peroxidation stress induced by URF13 accumulation in the T cytoplasm background (Liu and Schnable, 2002; Moller, 2001). These aldehydes dehydrogenases might be part of the plant mitochondrial-lipid peroxidation product detoxifying system. Such dehydrogenases have been found in proteome studies of pea, rice and Arabidopsis mitochondria (BarDelet al., 2002; Heazlewood et al., 2003; Kruft et al., 2001; Millar et al., 2001). It was suggested that T cytoplasm pollen abortion in male sterility is due to elevated lipid peroxidation (Liu and Schnable, 2002). While the restoring aldehydes dehydrogenase (rf2a) does appear to use HNE as a substrate, this is a very slow reaction and the dehydrogenase is much more active against other classes of aldehydes (Liu and Schnable, 2002). 3. A. III. 4-Hydroxy-2-Nonenal the Protein

Inhibitor

In plants, the 3Z-alkenals are then oxygenated to form 4-hydroxy-2-alkenals by a non-enzymatic process (Noordermeer et al., 2000). These 4-hydroxy-2-alkenals such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxy-2-hexanal (HHE) are examples of lipid peroxidation products, which have toxic effects on both plant and animal cells. Probably the most cytotoxic and the most studied lipid peroxidation end-product is HNE (Taylor et al., 2004). HNE (4-hydroxy-2-nonenal) was first discovered in 1964 in natural fats and was thought to be a by-product of the autoxidation of unsaturated fatty acids (Schauenstein, 1967). It is potentially able to undergo a number of reactions with proteins, phospholipids and nucleic acids. Since its

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discovery it has been shown to accumulate in mammalian cells under both normal and stress conditions (Esterbauer et al., 1991) and in plants during the oxidative burst (Deighton et al., 1999). In mammals, it has been implicated as being causally involved in the pathogenesis of a number of inflammatory and degenerative diseases (Poli and Schaur, 2000). In plants, two other pathways of HNE metabolism have been identified, which may prevent HEN accumulation and subsequent damage to proteins, although to date neither has been localized to mitochondria. The first of these is a NADPH: quinone oxidoreductase called P1-ζ-crystallin (P1-ZCr), which is able to catalyze the reduction of 2-alkenals by α-β-hydrogenation (Mano et al., 2002). The highest specific activity of this enzyme is for HNE (kcat=88 s-1 and Km=13.4 mM), but it also reacts with HHE (kcat=42 s-1 and Km=145 mM) and acrolein (kcat=40 s-1 and Km=4.65 mM) (Mano et al., 2002). The second is an NADPH dependant aldo-keto reductase identified by Oberschall et al. (2000). They isolated a gene MsALR from alfalfa encoding an aldose/aldehydes reductase that reduces the aldehyde. This enzyme exhibited characteristics homologous to the human enzyme involved in HNE metabolism. However, it has a significantly lower kcat (8. 9 s-

1) and higher Km (740 mM) than P1-ZCr. This indicates P1-ZCr may have a greater role in HNE metabolism than the MsALR gene product. However, in mammals, two additional mechanisms have been identified for HNE detoxification, one an oxidation of the aldehydes by an alcohol dehydrogenase (Sellin et al., 1991) and the second, the conjugation of the aldehydes to GSH by the GST A4-4 (Hubatsch et al., 1998). Recent research has identified a wider range of proteins, or pathways that are damaged or inhibited by 4-hydroxy-2-nonenal (HNE). In some cases, lipoic acid moieties are not involved and 4-hydroxy-2-nonenal (HNE) acts directly by covalent modification of amino acid residues such as Cys, Lys, His, Ser, and Tyr or acts non-covalently as a substrate analogue of other aldehydes (Esterbauer et al., 1991). Cytochrome c oxidase is also inhibited by binding of 4-hydroxy-2-nonenal (HNE) to histidine residues near the functional core of this enzyme (Chen et al., 1998). In plants, however, NAD-malic enzyme is inhibited irreversibly by 4-hydroxy-2-nonenal (HNE), and it has been

proposed that modification of a critical cysteine residue near the active site might be responsible (Millar and Leaver, 2000). 4-hydroxy-2-nonenal (HNE) inhibition of mitochondrial aldehydes-dependent enzymes, the class 2 aldehydes dehydrogenase and the succinic semi-aldehydes dehydrogenase, have also been reported in mammals (Nguyen and Picklo, 2003). These are examples of 4-hydroxy-2-nonenal (HNE) acting as a competitive inhibitor, preventing breakdown of the other aldehydes. These suggested that all proteins with Cys, Lys, His, Ser, and Tyr in crucial places in their active sites are potential targets for 4-hydroxy-2-nonenal (HNE). New lipid peroxidation targets in plant mitochondria, as the list of protein targets for 4-hydroxy-2-nonenal (HNE) steadily grows in mammalian mitochondrial systems, it is imperative to consider that many more plant targets remain unidentified (Taylor et al., 2004). Glycine dehydrogenase (GDC) is a multienzyme complex composed of four component enzymes, the P-protein, H-protein, T-protein, and L-protein, and is responsible for the conversion of glycine produced in the peroxisome to serine in the mitochondria, during the operation of the photorespiratory cycle (Douce et al., 2001). The H-protein plays a pivotal role as a mobile substrate which commutes between the other subunits allowing its lipoic acid arm to visit the active sites of the other three components (Vauclare et al., 1996). It was shown that HNE inhibits glycine-dependent respiration of isolated pea leaf mitochondria and that the site of inhibition is the H-protein. The H-protein subunit of Glycine dehydrogenase (GDC) was substantially more sensitive to modification than the lipoic acid-containing subunits of related 2-oxo acid dehydrogenases (Taylor et al., 2002). A substantial decline in GDC activity was also observed following the induction of in vivo oxidative stress, using the herbicide paraquat. A similar result was also observed when plants were exposed to the environmental stresses of drought and chilling. Taken together, the results obtained suggest that environmental stress leads directly to lipid peroxidation, the products of which can then inhibit mitochondrial function and, in particular, GDC (Taylor et al., 2004). This photorespiratory enzyme is thus a major target for oxidative damage in mitochondria in leaves (Taylor et al., 2002). Increased steady-state levels of 4-hydroxy-2-alkenals such as 4-hydroxy-2-nonenal (HNE)

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have been detected in a wide range of human diseases including Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, deep venous thrombosis, diabetes mellitus, and mitochondrial complex 1 deficiency (Poli and Schaur, 2000). HNE modifies lipoic acid moieties HNE has been shown to inhibit the activities of potato mitochondrial pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) (Millar and Leaver, 2000) via the modification of the lipoic acid residues found on the E2 subunits of these enzymes. These modifications by HNE results in the formation of an HNE-Michael adduct, which no longer allows the normal function of the essential E2 catalytic subunit. In plant mitochondria, the effect of 4-hydroxy-2-alkenals such as 4-hydroxy-2-nonenal (HNE) on the most abundant lipoic acid-containing protein and the glycine decarboxylase complex (GDC) were examined. In photosynthetic tissue, this enzyme can account for up to 50% of matrix protein, and is responsible for the most prominent metabolic activity in the mitochondria of illuminated leaves, photorespiration (Douce et al., 2001). There is evidence for enzymes from both these general classes in plant mitochondrial preparations, however, there is no evidence yet that they are capable of detoxifying HNE. Once damage has occurred mechanisms may exist to repair damage, avoiding costly resynthesis of whole polypeptides in order to repair single residue defects. For example, there may be enzymes, which can remove HNE once it has attached to Cys, Lys, His, Ser or Tyr residues. In the case of lipoic acid moieties, there may be a lipoate protein lyase which is able to remove damaged moieties, regenerating the apoprotein and allowing reattachment of unmodified lipoate by the established lipoate transferase pathway (Gueguen et al., 2000). As far as is known, none of these proposed enzymes has been identified in plants or animals. Alternatively, damaged proteins could simply be targeted for degradation and new protein synthesized. Recently, it was demonstrated that the mammalian 26s proteasome responsible for the degradation of abnormal proteins, including proteins with carbonyl residues generated by oxidative stress, is itself readily inhibited by HNE (Taylor et al., 2004). Inhibition of the proteasome tends to increase the levels of ubiquinated and carbonylated proteins. It was implicated in rendering cells sensitive to oxidative stress and apoptotic death (Hyun et al., 2002). In plants,

inhibition of the 26s proteasome has very recently been shown to lead to an accumulation of ubiquinated proteins and induction of programmed cell death, however, a direct link to HNE inhibition still awaits to be elucidated. Clearly, a pathway for HNE damage repair is likely to exist in eukaryotes, but the mechanism is still unresolved (Kim et al., 2003). The components of the protein import machinery of plant mitochondria, yet to be identified, are inhibited during environmental stress. This may be another example of a target of 4-hydroxy-2-nonenal (HNE), and this possibility is currently being investigated (Taylor et al., 2003). Four plant mitochondrial enzymes were experimentally detected. These were inhibited by 4-hydroxy-2-nonenal (HNE). All of these are involved in the major metabolic pathways, one in photorespiration, and the others in the TCA cycle. Their inhibition will directly affect the activity of the mitochondrial electron transfer chain through depletion of matrix NADH pools (Taylor et al., 2004). Identifying more targets and understanding their significance, and how can it be known that 4-hydroxy-2-nonenal (HNE) is the only or indeed the major lipid peroxidation cytotoxin in plants. Sweetlove et al. (2002) used Arabidopsis cell cultures under chemically induced oxidative stress, they showed an increase in the presence of lipid peroxidation end-products occurred and a significant number of mitochondrial protein subunits were degraded by these stresses. These subunits were identified by IEF/SDSPAGE 2D gel separation and mass spectrometry. Those identified included expected components such as pyruvate and 2-oxoglutarate dehydrogenases, aconitase and complex I Fe−S centres, but also unexpected components such as succinyl CoA ligase, methylmalonate semialdehyde dehydrogenase, fumarase, and GABA aminotransferase (Taylor et al., 2004). A number of proteins increased during these stresses, including a thioredoxin-dependent peroxidase, a thioredoxin reductase-dependent protein disulphide isomerase and a glutathione-S-transferase. These proteins may represent targets of damage, responses to stress, or detoxification systems (Taylor et al., 2004). Recently, the use of anti-HNE antibodies immobilized on CNBr-activated sepharose allowed the selective enrichment of HNE adducted peptides in biological samples. These samples were then tryptic digested and analyzed by electro spray ionization mass

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spectrometry, allowing the identification of these modified peptides at a substantially lower detection limit than previously possible (Fenaille et al., 2002). HNE is probably the most studied lipid peroxidation end-product a number of other end products have also been shown to be cytotoxic through inhibitory effects on proteins and biochemical pathways. These include the 4-hydroxyalkenals of various chain lengths such as 4-hydroxy-pentanal, which inhibits mammalian glucose-6-phosphate, succinate dehydrogenase, RNA synthesis, and phosphate transport (Taylor et al., 2004). 4-hydroxyalkenals including 4-hydroxy-hexanal and 4-hydroxy-octanol have been shown to have genotoxic effects and to inhibit anaerobic glycolysis. Malondialdehyde (MDA), a commonly studied marker of oxidative stress, and acrolein, both produced from the peroxidation of polyunsaturated fatty acids, have been shown to have various DNA damaging effects (Esterbauer et al., 1991). Malondialdehyde (MDA) can modify proteins by Schiff base addition (Fenaille et al., 2002). There is also the possibility of other, yet undetermined, products of lipid peroxidation that accumulate during stress and may inhibit proteins or disrupt biochemical processes within plant mitochondria. The lipid peroxidation product, 4-oxo-2-nonenal was identified as a major product of lipid peroxidation in the presence of low Fe2+ concentrations, where the cytotoxic effects of this compound are yet to be examined (Lee and Blair, 2000). Hence, analyzing which products of lipid peroxidation predominate in plant mitochondria under in vivo conditions remains a challenge. The potential damage occurring to plant proteins seem only logical that there must be mechanisms present either to prevent this type of damage occurring or to repair damage once it has occurred. A series of different enzymes was proposed, as HNE-detoxifying systems. Therefore, ranges of repair options for HNE-induced damage have been highlighted (Taylor et al., 2004). 3. A. IV. Mineral Roles in Lipid Oxidations Several studies confirmed the relation among minerals and lipid peroxidation, particularly potassium (K+), which is the most abundant cation in plant cells and is responsible for numerous physiological functions. In saline environment, similarity of Na+ and K+ causes an unbalance in K+ uptake and disorder in its

functions. In terms of chemical properties, K+ and Na+ are similar. Therefore, in high concentration of Na+, this ion replaces K+ and consequently appears K+ deficiency syndrome. Homeostasis of Na+/K+ ratio is very important specially, for functional regulation of membrane carriers and channels that are associated in the plant cells for K+ influx and Na+ efflux (Amtmann et al., 1999). It was found that potassium deficiency reduced Malondialdehyde (MDA) and chlorophyll b content. Alikhani et al. (2011) measured the highest amount of glycinebetaine in the presence of 600 mM NaCl in the company of 100 mM K+. Subsequently it can be concluded that chlorophyll oxidation was occurred in K+ deficiency as consequence of lipid peroxidation increases and disruption of protein-pigment complexes. The Malondialdehyde (MDA) concentration was measured in leaves and roots as an indicator of oxidative stress in plants. Our results were showed that in natural condition, lipid peroxidation of the shoot was about 2.5 fold of the root in A. lagopoides but in media containing low concentration of K+ (K0 and K1.75 treatments), lipid peroxidation in the shoot were 22.4 and 8.1 times greater than the root, respectively (Alikhani et al., 2011). On the contrary, by increasing of K+ concentration in the media (100 mM K+), peroxide production decreased significantly in shoot compared with the root. Minor increasing of the Malondialdehyde (MDA) in both organ tissues of A. lagopoides take placed in the presence of 600 mM NaCl that exhibited resistance of the membrane of this halophytic plant to salinity and the more sensitivity to the K+ deficiency. Producing of the lipid peroxids in the seedling were markedly increased under salt stress, because of Na+ accumulation (15-16µg against to 0.07µg per mg FW in the control). In this regard, presence of the high concentrations of K+ was not significant effect on the root MDA content. The produced MDA in the roots by 1.75 and 100 mM K+ treatments under saline condition were 0.08 and 0.04 µg.mg-1 FW, about 6.5 and 3 fold of the control, respectively. But in the shoot tissues, low concentration of K+ was dramatically effected on degradation of the lipids (Alikhani et al., 2011). Jasmonic acid (JA) is obtained from linolenic acid, and its production is associated with lipid peroxidation and membrane damage (Rodriguez-Serrano et al., 2006). Sandalio et al. (2001) demonstrated that growth of pea plants with Cd induced lipid peroxidation in leaves

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which would explain the increase observed in this work in JA production. The activation of the pathogen-dependent JA receptor is linked to the ion channel stimulation and ROS production and these conditions come together under Cd stress (McDowell and Dangl, 2000; Garrido et al., 2003). The increase of JA could also contribute to metal toxicity through the activation of lipoxygenase activity, H2O2 production, and lipid peroxidation (Wang and Wu, 2005; Maksymiec et al., 2007).Aldehydes that obtained from the lipids peroxidation of the membrane fats to the plants, which were kept under salt stress. However, the treatment of barely seedlings with cadmium (Cd) through the activation of antioxidant system decreased the amount of Malondialdehyde MDA.

3. A. V. Membrane Lipid Oxidation

3. A. V. 1. Membrane Compositions

Membranes are highly viscous liquids, which play important role in cell life. They form the boundaries of cells. Many processes occur through membranes, e.g., signal transfers between cells and exchanges of ions and other molecules from (to) the extra- to (from) the intracellular domains. Mammalian membranes consist mainly of lipids (phospholipids, cholesterols, glycolipids), then proteins and other smaller hydrocarbon compounds (Bretscher, 1973; Mouritsen and Jorgensen, 1998; Spector and Yorek, 1985). Phospholipids constitute the major part of the mass of membrane (Figure, 17.1). In membranes, there are four major phospholipid types namely phosphatidylcholine, phosphotidyl ethanolamine, phosphatidylserine and sphingomyelin (Kosinova, 2011). Other types of phospholipids are also present in small quantities. Even if their concentration is not so high, they influence the membrane properties and they play an important role in its function (Alberts et al., 1994). A phospholipid unit consists of one polar head group and two hydrophobic hydrocarbon tails (fatty acids). This structure gives them amphipathic character. Due to amphipathic character, the individual phospholipids are organized into bilayers, the hydrophobic chains are oriented “head to head” to form the inside part of membrane. The hydrophilic moieties are located in outside part of the bilayers making

internal and external surfaces. Thus, lipid bilayers are thermodynamically stabilized by hydrophobic effect (Kosinova, 2011). Membranes may differ from each other by their composition (e.g., type and concentration of the individual proteins, which they contain, asymmetry in phospholipid composition, and presence of cholesterol). Composition, temperature, length and degree of unsaturation of fatty acids influence the fluidity of membranes. Unsaturated lipid chains disfavour membrane packing, which indirectly induces a higher fluidity (Murray et al., 2003). Changes in membranes can cause perturbations in their structure, which can affect intracellular processes. Some of such changes are attributed to reactions of reactive species with lipid chains. This reaction is known as lipid peroxidation (Kosinova, 2011).

Figure 17.1: Example of phospholipid (phosphatidylcholine) and the structure of membrane

N+

OPO

O

OOOO

Ac

3. A.V. 2. Membrane Oxidants

Reactive oxygen species (ROS) are produced in aerobic organisms within the cell and are normally in balance with antioxidant molecules. Oxidative stress arises from an imbalance between generation and elimination of ROS. These cytotoxic activated ROS can seriously disrupt normal metabolism through oxidative damage to lipids, protein and nucleic acids (Mittler et al., 2004). This can lead to changes in the selective permeability of bio-membranes, causing membrane leakage and changes in the activity of membrane-bound enzymes (Apel and Hirt, 2004). Lipid peroxidation stimulates degradation of membrane structures (Laster and Stein, 1993) and because of electrolyte leakage (Tiburcio et al., 1994) alters selective permeability of the membrane (Weckx et al., 1993). So, in different stress, stability of the cell

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membranes in sensitive and tolerate plants is widely depending on integrity of these structures (Aziz and Larher, 1998; Liang et al., 2003). The carotenoids contents as antioxidant compounds had increased considerably under saline conditions and in the absence of K+. Carotenoids are pigments that protect plant against light inhibitory induced by activated oxygen species (AOS) during oxidative stress, also they contribute to stability of lipid membranes (Kitahata et al, 2006; Lobato et al, 2009). These phenomena take placed via two ways: function of xanthophyll cycle which turn away absorbed light energy to heat and fluorescence quenching of triplet chlorophyll molecules that reduces peroxidation of the thylakoid lipids and chlorophyll oxidation; therefore reduce photosystem injury and photosynthetic devices will be protects against AOS (Pogson and Rissler, 2000; Tardy and Havaux, 1996). Malondialdehyde (MDA) is considered sensitive marker commonly used for assessing membrane lipid peroxidation (Bailly et al., 1996; Goel and Sheoran, 2003). It is well known that ROS induced lipid peroxidation of membranes is a reflection of stress induced damage at the cellular level. The change in Malondialdehyde (MDA) contents, especially in oil rich seeds, is often used as an indicator of oxidative damage (Sung, 1996). Significant increase in the electrolyte leakage and lipid peroxidation of rosemary leaves. Salinity can cause oxidative damage to cell membrane and lipids (lipid peroxidation), leading to an increase in electrolyte leakage (Amor et al., 2005; Kaya et al., 2007; Arvin and Donnelly, 2008). The electrolyte leakage was positively correlated with the MDA content, indicating that the membrane injury induced by salt stress is a result of oxidative damage. The increase in electrolyte leakage and lipid peroxidation by salinity became more severe at 0 µM Cu2+ than 0.5 and 1.0 µM Cu2+. Since copper is directly involved in both gene expression and protein synthesis, it seems that copper deficiency inhibits the activities of CAT, resulting in extensive oxidative damage to membrane lipids (Marschner, 1995). Enhanced production of oxygen free radicals is responsible for peroxidation of membrane lipids and the degree of peroxides damage of cell was controlled by the potency of peroxidase enzyme system (Sairam and Tyagi, 2004). Reactive

oxygen species (ROS) such as the superoxide radical, hydrogen peroxide and hydroxyl radical can cause lipid peroxidation and consequently membrane injury which leads to leakage of cellular content, protein degrading, enzyme inactivation, pigment bleaching and disruption of DNA strands and thus cell death (Scandalios, 1993). Allen (1995) also reported that much of injury to plants caused by various stresses is associated with oxidative damage at cellular level such as cell membrane damage. Salt stress imposed at various stages of crop growth resulted in an increase in lipid peroxidation and decrease in membrane stability, chlorophyll fluorescence ratio (fv/fm) and chlorophyll and carotenoid contents (Gomathi and Rakkiyapan, 2011). Stress conditions that cause ROS generation such as pathogen attack, drought, salinity, cold, hypoxia, and nutritional restriction prompt annexin accumulation or relocation to membranes. However, it is as yet unclear whether ROS or elevated [Ca2+]cyt trigger annexin responses. Membrane oxidation increases membrane binding of animal annexins (Balasubramanian et al., 2001). Peroxide can induce the channel-forming vertebrate AnxA5 to be inserted into membranes in vitro, and peroxide-induced Ca2+ influx in vivo in DT40 pre-B cells requires AnxA5 (Kubista et al., 1999). From this it follows that channel-forming plant annexins such as AnxAt1 are candidates for the ROS-activated channels identified in several plant cells (Foreman et al., 2003). Recently, annexins have been identified as protein components of an M. truncatula plasma membrane lipid raft alongside signaling and redox proteins (Lefebvre et al., 2007). In measuring the lipids peroxidation, it was observed that drought stress caused the augmentation of lipids per oxidation in Satureja hortensis. The cellular membrane and the other internal membranes of chloroplast membrane and mitochondrial were formed from two layers of phospholipids. The produced super oxide radicals from the drought stress caused the lipids' per oxidation (Dewlin and Withman, 2002). The production of membrane lipids peroxidation would be some compounds such as Malondialdehyde (MDA), propanol, botanal, hexanal, heptanal and propanal de methylastal and these compounds were used as an index for measuring the amount of lipids per oxidation. Increasing the lipids per oxidation was

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considered as an index for increasing the oxidative stress (Meirs et al., 1992). 3. A. V. 3. Lipid Oxidation during Germinations

In plants, there are several types of peroxisomes, which are specialized in certain metabolic functions. Glyoxysomes are specialized peroxisomes, occurring in the storage tissues of oil seeds that contain the fatty acid β-oxidation and glyoxylate cycle enzymes to convert the seed reserve lipids into sugars, which are used for germination and plant growth (Tolbert, 1981; Beevers, 1982). Leaf peroxisomes are specialized peroxisomes present in photosynthetic tissues that carry out the major reactions of photorespiration (Douce and Heldt, 2000). In plants, the cellular population of peroxisomes can proliferate during senescence and under different stress conditions produced by xenobiotics, ozone, cadmium, and H2O2. Peroxisome proliferators activated receptor, the transcription factor involved in peroxisomal proliferation and induction of peroxisomal fatty acid β-oxidation in animal tissues, recently was demonstrated to be functional in transgenic tobacco (Nicotiana tabacum) plants (Nila et al., 2006). Peroxisomal MDAR1 transcripts were induced in pea leaves sprayed with the herbicide 2, 4-dichlorophenoxyacetic acid (Leterrier et al., 2005). Malondialdehyde (MDA) content in endosperms was increased during 10 day of germination, while in cotyledons it was increased during six day of germination and then decreased (Cai et al., 2011). Malondialdehyde (MDA) content increased in parallel with the increase in activities of SOD, POD and CAT. The changes of Malondialdehyde (MDA) content and antioxidant enzymes activity in the degrading endosperms and developing cotyledons of J. curcas observed appear to be more closely related to germination process and plant tissues. Changes in antioxidant enzymes activity might be regulated by different responses of SOD, POD and CAT, not only by changing enzyme activities, but also by alteration in isoenzymes patterns (Cai et al., 2011). Elevated Malondialdehyde (MDA) contents mediated by free radicals and peroxides are considered to be one of the likely explanations for lipid peroxidation during germination (Schopfer et al., 2001). Oxidative damage to tissue lipid was estimated by Malondialdehyde (MDA) content.

Increased MDA contents in endosperms and cotyledons during seed germination suggest that lipid peroxidation increases during germination process. Cai et al. (2011) indicated that lipid peroxidation occurred during seed germination and early seedlings growth. The control of steady-state ROS levels by SOD is an important protective mechanism against cellular oxidative damage, since ●O2 acts as a precursor of more cytotoxic or highly relative ROS (Mittler et al., 2004). 3. A. V. 4. Lipid Peroxidations

Lipid peroxidation was measured as the amount of thiobarbituric acid reactive substance (TBARS) and it was determined by the method of Buege and Aust (1978). Lipid peroxidation was identified as an oxidative process, which has a chain character. As all radical chain reactions, the whole process is divided into three steps – initiation, propagation and termination (Halliwell and Chirisco, 1993; Cheeseman, 1993). Lipid peroxidation is initiated by the attack of the reactive species, which have the capacity to abstract H-atom from fatty acid chains. The ●OH radical is the most probable initiator in the living cells. The most sensitive positions of the polyunsaturated fatty acids for HAT are the methylene groups close to double bonds, which help the relative stabilization of the subsequent carbon-centred radical (R●) by π-electron conjugation, R-H + ●OH → R● + H2O, (Halliwell and Chirisco, 1993; Gutteridge, 1995). Because of the instability of carbon-centred radicals, the initiation step is followed by the fast reaction with dioxygen molecules. During this reaction, a peroxyl radical of fatty acid (ROO●) is formed. [R●+ O2 → ROO●], ROO● may react by HAT with adjacent fatty acids to produce lipid peroxides and new carbon-centred radicals, ROO● + RH → ROOH + R● (Kosinova, 2011). The cycle is repeated until termination. The number of cycles depends on many factors (e.g. concentration of oxygen, type of fatty acid).Termination occurs when two radicals react together. Lipid peroxidation can be terminated by several ways: two peroxyl radicals meet: 2 ROO●

→ ROOR + O2, two carbon-centred radicals meet: 2 R● → R-R and peroxyl radical and carbon-centred radical meet: ROO● + R● → ROOR. These three possibilities can occur if the concentration of radicals is high enough to meet. To protect against membrane damage, the membranes include different chain-breaking molecules (antioxidants (A)) which also

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interrupt the chain reaction by providing an H-atom (Kosinova, 2011). ROO● + AH → A● + ROOH, The radical derived from the antioxidant is not sufficiently reactive to attack new fatty acids and thus the chain reaction is terminated. Lipid peroxidation can be generated by ROS directly attacking membrane lipids, or by lipoxygenase activated after oxidative stress (Maccarrone et al., 2001; Gobel et al. 2003). Degradation of O3 in the apoplast leads to formation of various ROS. Among these, H2O2 is not very toxic to plants cells, which are capable of detoxifying substantial amounts of it. However, formation of hydroxyl radicals (●OH) in the apoplast from H2O2 and superoxide (●O2) can initiate lipid peroxidation that results in lipid hydroperoxides (LOOH) formation (Schraudner et al., 1997). Various lipid hydroperoxides and other lipid-based signaling molecules, such as jasmonate are biologically active substances that control a variety of downstream processes. Thus formation of these plasma membrane lipid-based molecules as a result of O3 action in the apoplast may also be regarded as O3-perception (Kangasjarvi et al., 2005). It has been shown that oxidative damage can cause non apoptotic cell death resembling necrosis. Where, death is due to oxidative membrane lipid damage and it is suppressed in most circumstances by the activity of caspases, the major enzymatic mediator of apoptosis (Yu et al., 2006). Sugar deprivation caused by stresses can cause mitochondrial oxidation, and reduce mitochondrial capacity, possibly leading to the increase in peroxisome-based ROS signals. β-oxidation of fatty acid and the glyoxylate cycle enzymes are localized in peroxisomes and may provide energy in the dark. Indeed, the expression of genes encoding fatty acid oxidation enzymes was increased on the second day under darkness. Thus, increase in probe oxidation in peroxisomes during darkness can results from H2O2 generation occurring due to enhanced fatty acid oxidation (Del Rio et al., 1998). Sarvajeet and Narendra, (2010) reported that the ROS affect many cellular functions by damaging nucleic acids, oxidizing proteins, and causing lipid peroxidation and it is important to note that whether ROS will act as damaging, protective or signaling factors depends on the Delicate equilibrium between ROS production and scavenging at the proper site and time. Oxidative stress occurs when this critical

balance is disrupted due to depletion of antioxidants or excess accumulation of ROS, or both (Scandalios, 2005). Once accumulation of AOS occurs, damage to cellular components begins. This includes direct inhibition of enzymes by AOS, protein oxidation reactions, membrane lipid peroxidation yielding toxic products, and oxidative DNA and RNA damage (Elstner, 1982). In addition to their effects as oxidants, increased production of H2O2 and other ROS may constitute an alarm signal that regulates gene expression for the development of a coordinated protection against harmful environments (Pastori and Foyer, 2002). Plant cells may use this increase in ROS production to monitor the extent of environmental stress, the higher steady state levels of ROS may lead to oxidative damage and trigger programmed cell death (Fath et al., 2001; Tiwari et al., 2002). Corn oil presents a relatively high concentration of polyunsaturated fatty acids (PUFA). Due to the high levels of unsaturation, these lipids are highly susceptible to free radical oxidative reactions, giving rise to the formation of lipid peroxides. Many investigations suggested that a large number of polyunsaturated fatty acids produces more lipid peroxides and may have mutagenic activity (Valls et al., 2003). In fact, corn oil is the most unsaturated oil among widely consumed oil. It is rich in oleic, linoleic and linolenic acids. Therefore, it is easily affected by free radical reactions, which results in the formation of oxidized LDL. This particle showed another significant risk factor for atherosclerosis (Recep et al., 2000). Lipid peroxidation is the most extensively studied manifestation of oxygen activation in biology. It is broadly defined as oxidative deterioration of polyunsaturated fatty acid (PUFA) which is fatty acids that contain more than two carbon double bonds (Maneesh and Jayalekshmi, 2006). Lipid peroxidation is the introduction of a functional group containing two catenated oxygen atoms, O-O, into unsaturated fatty acids in a free radical reaction. Polyunsaturated fatty acids susceptible to free radical attack are initiated by the formation of a carbon-centered radical by the abstraction of a hydrogen atom at one of the double bonds of the lipid. Lipid peroxidation is also one of major causes of quality deterioration during the storage of fats, oils or other lipid-rich foods (Wang, 2005). Lipids when reacted with free radicals can undergo the highly damaging chain reaction of lipid peroxidation leading to both direct and indirect effects (Devasagayam et

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al., 1991). Peroxidation of lipids is a binding process connected with the formation of aldehydes. Malondialdehyde (MDA) (Niedworok and Bielaszka, 2007). It is the product of lipid peroxidation, is a good marker of free radical-mediated damage and oxidative stress (Atip et al., 2010). It has been utilized as a suitable biomarker for lipid peroxidation (Adriano et al., 2004).

3. A. V. 5. Lipid Peroxidation Causes PCD

The role of lipid peroxidation in mega gametophyte cell death awaits further investigation. This process has been implicated in the PCD of animal as well as plant cells (Sandstrom et al., 1995; Aoshima et al., 1997; Maccarrone et al., 2001; Gechev et al., 2002). Cell death during the lesion spread creates an increasing concentration of products of membrane lipid peroxidation, which serve as a substrate for jasmonate biosynthesis. Accordingly, only in the sensitive accessions is high JA accumulation observed (Kangasjarvi et al., 2005). High Fe2+ in culture medium induced cell death and strong lipid peroxidation through Fenton reaction, and induced β‑thujaplicin biosynthesis. However, glutathione enhanced both H2O2 and Fe2+

‑induced biosynthesis although it reduced lipid peroxidation, which was proposed to lead to oxylipin signaling for β‑thujaplicin induction. This may suggested GSNO from NO●− glutathione played roles in β‑thujaplicin biosynthesis (Zhao et al., 2005). RNS, ROS and lipid free radicals often form self-propagated chains and generated a large number of toxic oxidants, which cause various cellular effects from modulations of cell signaling to overwhelming oxidative injury on lipids, DNA and proteins as well as integrity of both plasma or end membranes, and eventually commit cells to necrosis or PCD (Pacher et al., 2007). 3. B. Protein Deteriorations

3. B. I. Protein Bonding Damage

The induction of pathogen resistance (PR) genes under biotic and abiotic conditions suggests that different stresses induce the same set of genes by sharing effectors of gene regulation. ROS are involved in signalingin different processes such as growth, development, and responses to biotic and

abiotic stresses. The signaling process is controlled through the balance between ROS production and scavenging (Bailey-Serres and Mittler, 2006). Proteins are other basic structural component of all living organisms. They belong to biopolymers that are made of amino acids. Individual amino acids are connected by the peptide bond (-NH-CO-). Several hundreds of naturally occurring amino acids, however, only 20 L-α-amino acids are structural units for proteins (Kosinova, 2011). It has been proved that the reactive species may lead to serious changes that inhibit their function. ROS can directly attack the polypeptide backbone to induce oxidation processes. In the presence of molecular dioxygen, peroxides or hydroxyl derivatives are formed. If dioxygen is absent, two carbon centered radicals may react together to form protein–protein cross-linked derivatives (Berlett and Stadtman, 1997). Alkoxyl radicals may cleave peptide bonds by either the diamide or the α-amidation pathways. In the former mechanism, CO2, NH3 and carboxylic acid are formed. The latter pathway yields NH3 and free α-ketocarboxylic acid. Cleavage of peptide bond can also occur by ROS attack on glutamyl, aspartyl and prolyl side chains. All amino acids are sensitive to the direct oxidation by ROS (Figure, 17.2). Probably the most sensitive are residues containing sulphur as cysteine and methionine. Cysteine is converted to disulfides and methionines are converted to methionine sulfoxide. These changes (Table, 17.1) can be easily repaired because cyclic oxidation-reduction of sulphur residues is one of antioxidant mechanism. The other amino acids, e.g., with aromatic rings (Phe and Tyr) are also sensitive to ROS attacks (Kosinova, 2011).

Table 17.1: Possible oxidation changes in structure of amino acids. Amino acid

Products of oxidation

Trp Hydroxytryptophans, nitrotryptophan, kynurenins.

Phe Hydroxyphenylalanines. Try Dihydroxyphenylalanine, tyr-tyr

cross-linkage, tyr-O-tyr cross-linkage. His Oxohistidine, asparagine, aspartic

acid Arg Glutamic semialdehyde Lys Aminoadipic semialdehde Pro Pyrrolidone, hydroxyproline, glutamic

semialdehyde. Glu Oxalic acid, pyruvic acid.

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Figure 17.2: Protein peptide bond cleavage by reactive oxygen species (ROS)

NH

R

H O

OH H2O

NH

R

O

O2

NH

R

O

OO

HO2 + Fe+2

O2 + Fe+3

NH

R

O

OOHHO2

Fe+2O2+H2O Fe+3

NH

R

O

OHO2

Fe+2

O2

Fe+3

NH

R

O

OH

protein-cross linkage

peptide bond cleavage

3. B. II. Protein Deteriorations by Oxidants

Oxidative damage to various subcellular compartments (i.e. chloroplasts, mitochondria, and peroxisomes) was studied in two cultivars of wheat differing in ascorbic acid content, and growing under good irrigation or drought. In well-watered plants, mitochondria contained 9-28-fold higher concentrations of oxidatively modified proteins than chloroplasts or peroxisomes. The high level of carbonylated proteins in mitochondria is due to a high ROS production, or to comparatively lower levels of antioxidant defenses remains to be determined. In general, oxidative damage to proteins was more intense in the cultivar with the lower content of ascorbic acid, particularly in the chloroplast stroma. Water stress caused a marked increase in oxidative damage to proteins, particularly in mitochondria and peroxisomes (Bartoli et al. 2004). Tiwari et al. (2002) they revealed that mitochondria contain higher levels of oxidatively damaged proteins than chloroplasts or peroxisomes, and that protein carbonylation increases significantly in mitochondria of water-stressed plants also confirmed this notion. The increase in the production of ROS, for example, H2O2, by mitochondria might be an important alarm signal up-regulating antioxidant defense systems (Prassad et al., 1994) or triggering programmed cell death (PCD) if oxidative stress becomes rampant. These results indicate that mitochondria are the main target for oxidative damage to proteins under well irrigated and drought conditions. As a result, ROS-producing pathways may be either stimulated or repressed (Couee et al., 2006). Unlike PUMP activity,

which is activated by an excess of free fatty acids, a specific mechanism for mitochondrial ROS production caused by an excess of hexose remains elusive. In plants growing under normal, non-stressful conditions, mitochondria contained the highest concentrations of oxidatively modified proteins. The concentrations of protein carbonyl groups in mitochondria were at least 9 times higher than in the other compartments tested i.e. chloroplast stroma, thylakoids, and peroxisomes (Bartoli et al., 2004). ROS can react with proteins, lipids, and DNA causing decreased enzyme activities, increased membrane permeability and mutations, respectively. For example, as a result of ROS production, both the amount of the lipid cardiolipin and cytochrome c oxidase (CCO) activity in isolated bovine heart submitochondrial particles (SMP) decreased by 40% in 60 min., cytochrome c oxidase (CCO) activity was restored with cardiolipin but not with peroxidized cardiolipin (Paradies et al., 2000). Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of free cytochrome c in the inter membrane space, which is subsequently released into the cytosol upon permeabilization of the outer mitochondrial membrane (Orrenius et al., 2007; Ott et al., 2007). Thus, the production of ROS must be avoided or minimized and, if produced, ROS should be detoxified efficiently. In plants, oxidative damage to mitochondria may be crucial in determining the survival of cells exposed to different kinds of stress. For example, pre-acclimation treatments at moderately low temperatures improved survival after a chilling period in maize, probably by triggering the induction of mitochondrial targeted antioxidant enzymes. Survival after a chilling episode correlated with enhanced recovery of mitochondrial function, and particularly of the cytochrome c oxidase (CCO) pathway (Prasad et al., 1994). Likewise, exposure to ozone triggered oxidative stress in birch leaves, with H2O2 accumulation in mitochondria. Hydrogen peroxide accumulation in mitochondria coincided with disintegration of the mitochondrial matrix and the appearance of the first symptoms of leaf damage (Pellinen et al., 1999). However, plant mitochondria have mechanisms for minimizing ROS production, such as that involving the alternative oxidase (AOX), as well as several different systems for removing ROS once formed (Moller, 2001). The significance of the mitochondrial involvement in phenotypes and

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further uncovering plant pathways of repair and avoidance of protein damage will provide tools for enhancing plant performance through the manipulation of mitochondrial function in the future (Taylor et al., 2004). The induction of a mitochondrial protease specific for oxidatively modified proteins has been documented in mammals (Marcillat et al., 1988). Preliminary evidence for the presence of a similar protease has been presented for plant mitochondria (Sweetlove et al., 2002). Such a proteolytic mechanism might indicate that longer-term losses in activity are due to degradation of modified proteins rather than direct oxidative inactivation of enzymatic function. The direct effects of AOS on proteins observed to date, both in mammalian mitochondria and in the limited examples that there are in plants, have usually involved the direct application of vastly different concentrations of AOS. Some of these effects may have little physiological significance and it is important to distinguish these from the effects of physiological concentrations of AOS. Few reliable measurements of steady-state AOS concentrations in plant tissues have been reported and thus the likely range of physiologically significant concentrations is largely unknown (Dat et al., 2000; Moller, 2001). Protein carbonylation is widely used as a marker for oxidative stress and aging (Chevion et al., 2000; Stadtman and Levine 2000; Levine, 2002). Carbonylated proteins were detected before the stages associated with extensive mega gametophyte cell death. Protein carbonylation during the induction of apoptosis may result in the modification of a restricted number of proteins (England et al., 2004). A reduction in glycolysis due to the carbonylation of glycolytic enzymes occurs in human leukemia (HL) 60 cells, in which apoptosis is induced by treatment with etoposide [VP-16, a topoisomerase II inhibitor that acts as a DNA-damaging agent] (England et al., 2004). Exogenous salicylic acid treatment leads to a marked increase in the carbonylation of the α-subunits of 12S cruciferin storage proteins in Arabidopsis seeds, a process that may facilitate storage protein mobilization. It is possible that ROS-induced protein carbonylation in mega gametophyte cells has dual roles: facilitating storage protein mobilization and signaling cell death (Rajjou et al., 2006). The plant heterotrimeric G protein complex is involved in

cell death signaling during the unfolded protein response and in ozone-induced activation of NADPH oxidases. Because of its involvement in light transmission, hormone signaling, and regulation of ion channels, this complex is a potential interface between ROS and these processes (Wang et al., 2007). H2O2 has been found to activate G protein signaling through the promotion of dissociation of the Ga-subunit from the G protein macromolecular complex (Wang et al., 2008). 3. B. III. Cryptogein

The downstream of the interaction between cryptogein and plasma membrane binding sites (Bourque et al., 1999), the occurs protein phosphorylation followed by Ca2+ influx (Tavernier et al., 1995; Lecourieux-Ouaked et al. (2000). Procession of events, including activation of a mitogen-activated protein kinase (MAPK) identified as salicylic-induced protein kinase (SIPK), and of the wound-induced protein kinase (WIPK) (Lebrun-Garcia et al., 1998; Zhang et al., 2000). Cryptogein induced cell death through complex processes involving anion channel activity independent of ROS production (Binet et al., 2001; Lecourieux et al., 2002; Wendehenne et al., 2002) required several processes. These are mobilization of intracellular Ca2+ (Lecourieux et al., 2002), activation of anion channels upstream of a plasma membrane NADPH oxidase that are responsible for ROS production, cytosol acidification (Pugin et al., 1997; Simon-Plas et al., 2002; Wendehenne et al., 2002). Besides the alkalinization of extracellular mediumdisruption of the microtubular cytoskeleton (Binet et al., 2001), inhibition of Glc uptake (Bourque et al., 2002), defense related gene expression (Lecourieux et al., 2002), and phytoalexin synthesis (Tavernier et al., 1995). Cell death event involves early root to shoot signaling and shoot regulatory feedback on a root specific redox regulation, emphasizing the interplay between ROS and redox (Muhlenbock et al., 2007).

3. B. IV. Prohibitins

Prohibitins were first identified in a screen for regulators of human cell proliferation (McClung et al., 1989). These protein type hibitins are an extensively studied family of proteins that are highly conserved between animals and plants

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(Nadimpalli et al., 2000; Mishra et al., 2006). Subsequently, prohibitins were shown to serve many functions in such diverse processes as apoptosisand aging, cell cycle progression, tumorigenesis, transcriptional regulation, signaling, oxidative damage, respiration, and mitochondrial biogenesis (Mishra et al., 2006; Artal-Sanz and Tavernarakis, 2009). The biochemical function(s) of prohibitins is not clear, but they interact with receptors on the plasma membrane and with transcriptional regulatory components in the nucleus. In the mitochondria, PHB1 and PHB2 form a complex at the inner mitochondrial membrane and act as a chaperone that influences mitochondria ultrastructure, respiratory complexes, and cellular senescence (Artal-Sanz and Tavernarakis, 2009). In plants, the initial identification and characterization of prohibitins showed a high degree of conservation with animal genes with >70% similarity at the amino acid level between plants and mammals and plants and yeast (Snedden and Fromm, 1997).The best studied are the soya saponins both in terms of epidemiology and in vitro and in vivo systems. Additional mechanistic studies indicate that there is evidence for saponins regulation of the apoptosis pathways enzymes (AkT, Bcl and ERk1/2), leading to programmed cell death of cancer cells (Ellington et al., 2006; Xiao et al., 2007; Zhu et al., 2005). 3. C. Enzyme Roles

3. C. I. Programmed Cell Death Inhibitors

ROS-scavenging enzymes are key players in regulating levels of ROS in cells; these enzymes appear to be downregulated before or during cell death in plants (Mittler et al., 1999; Fath et al., 2001). It was demonstrated that one CAT in the mega gametophyte was ubiquitinated and degraded by the proteasome before extensive cell death occurred. The tyrosine-phosphorylated CAT appeared to be a major target of ubiquitination and the degree of ubiquitination was progressively increased at stages associated with high endogenous H2O2 levels. In addition, treatment of germinated white spruce seeds with the proteasome inhibitor MG132 was associated with increased CAT activity from d1 to d4 of the treatment. MG132 treatment also delayed cell death, suggesting that degradation of CAT by the ubiquitin proteasome pathway is an integral mechanism

facilitating H2O2 accumulation and thus promoting PCD (He and Kermode, 2010). Actinomycin D and cycloheximide are inhibitors of transcription and translation, respectively, also prevented cell death, but with a lower efficiency. Induction of PCD resulted in gradual oxidation of endogenous ascorbate (ASC); this was accompanied by a decrease in both the amount and the specific activity of the cytosolic ascorbate peroxidase (cAPX). A reduction in cAPX gene expression was also found in the late PCD phase. Moreover, changes of cAPX kinetic properties were found in PCD cells (Vacca et al., 2004). The important regulators of apoptosis are the inhibitor of nuclear factor- κ B kinase, which have been identified as substrates of the proteasome. The involvement of the ubiquitin–proteasome pathway in apoptosis of animal cells has been well characterized. The Bcl-2 family of proteins, the IAPs, p53 and the inhibitors of nuclear factor-B and of nuclear factor-B kinase are all ubiquitin–proteasome substrates (Jesenberger and Jentsch, 2002). The roles of the ubiquitin-proteasome pathway in plant PCD are only beginning to beelucidated; nevertheless, the limited data available suggest its involvement. In transgenic tobacco plants, virus-induced gene silencing of two different subunits of the 26S proteasome, the α6 subunit of the 20S proteasome and the RPN9 subunit of the 19S regulatory complex, leads to PCD (Kim et al., 2003).

3. C. II. Ros Induced Ameliorating Genes

In mammals, the mitochondria controlled program Cell Death (PCD) response involves the pro-apoptotic Bax family of proteins and anti-apoptotic Bcl-2 and Bcl-XL family. Although Bax, Bcl-2 and Bcl-XL homologues have not yet been found in plants, expression of mammalian Bax causes death while that of mammalian Bcl-XL or Bl-1 suppresses cell death in plant cells challenged with elicitors, suggesting that elements of mammalian PCD processes are also found in plants (Matsumura et al., 2003). Moreover, mammalian Bcl-2 family members localize to the mitochondrial, chloroplast and nuclear fractions when expressed in plants, where they prevent herbicide and ROS-induced apoptosis (Chen and Dickman, 2004). The most potentially Deleterious effect of ROS under most conditions is that at high concentrations they trigger genetically programmed cell suicide

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events. Moreover, because plants use ROS as second messengers in signal transduction cascades in processes as diverse as mitosis, tropisms and cell death, their accumulation is crucial to plant development as well as defense (Foyer and Noctor, 2005). Over the last 20 years, a large body of evidence has demonstrated unequivocally that H2O2 is a key signaling molecule in plants, as it is in other eukaryotes (Drogue 2002), and that dedicated ROS-producing NADPH oxidases and peroxidases are activated to control processes as diverse as gene expression, stomata closure, root growth and programmed cell death (PCD). The generation of hydrogen peroxide and other ROS has been shown to result in a type of cellular suicide known as the hypersensitive response (Mehdy et al., 1996). H2O2 also activates mitogen-activated protein kinases (MAPKs), conserved signaling kinases that modulate gene expression and transduction of cellular responses to extracellular stimuli (Samuel et al., 2000). Furthermore, several studies indicate that H2O2 is a key factor mediating programmed cell death (PCD) in response to pathogens, elicitors, and hormones (Solomon et al., 1999). Increasing evidence indicates that H2O2 functions as a stress signal in plants, mediating adaptive responses to various stresses. A regulatory role for ROS such as H2O2 has been implicated during senescence (Pastori and Del Rio, 1994), it was shown that H2O2 induces the expression of a senescence-related gene (Desikan et al., 2000). Thus, it is not surprising that there are genes that are induced by both oxidative stress and senescence. The expression of genes encoding a mitochondrial uncoupling protein, Pyruvate decarboxylase, and a myb-related transcription factor were induced by H2O2. Mitochondrial uncoupling proteins are key factors regulating ATP synthesis and generation of ROS in mitochondria, this redox balance affecting the longevity of organisms (Finkel and Holbrook, 2000). Moreover, a gene encoding such a protein was found to be highly upregulated in mammalian cells induced to undergo PCD (Voehringer et al., 2000). Transcription factors have been reported to induce rapidly during defense responses (Durrant et al., 2000). Among the transcription factors induced by H2O2, EREBP and DREB2A are important ones that regulate gene expression during various stresses (Liu et al.,

1998). Other transcription factors induced by H2O2 include a myb related TF, several zinc finger proteins, and a heat shock transcription factor. Zinc finger proteins have wide-ranging functions and several types exist in plants (Takatsuji, 1999). The involvements of stress response factors of zinc finger transcription were reported. For example, during barley powdery mildew interactions, a zinc finger protein was identified as a key mediator of R gene-induced resistance responses such as H2O2 generation (Shirasu et al., 1999). Furthermore, during Avr-9: Cf-9 interactions in tomato, a gene encoding a zinc finger protein was induced (Durrant et al., 2000), and other stresses such as UV, high salinity, ozone, and wounding induce this class of genes (Takatsuji, 1999). 3. C. III. Grim Reaper (GRI)

GRIM REAPER, a protein that is involved in the regulation of cell death, GRI display an ozone sensitive phenotype. It was considered as a protein, which is an Arabidopsis ortholog of the tobacco stigma-specific protein 1 (STIG1). The RT-PCR analysis of GRI transcript showed that a 5’ part of the GRI transcript was still present in gri and, thus a truncated GRI protein, similar in size to the fragment that is released from the N terminus of GRI, could be produced. Infiltration of a recombinant peptide corresponding to this truncated protein induced cell death in Arabidopsis leaves (Wrzaczek et al., 2009). This suggests that the GRI N-terminal part is involved in the regulation of cell death. This, however, poses the question of how gri plants survive under normal growth conditions in the presence of a potentially lethal truncated GRI protein. Hence, GRI-peptide needs an additional signal for the cell death induction. However, it is possible that gri plants facing the challenge of living in the presence of the peptide will survive through upregulation of negative regulators of cell death. ROS are involved in the regulation of various types of cell death. Removal of superoxide by co-infiltration of the GRI peptide with SOD reduced cell death to control levels. A weaker reduction was observed by H2O2 removal with catalase. Therefore, plant respiratory burst oxidases (Atrboh) are proposed to be a major source of extracellular superoxide (Overmyer et al., 2003). Torres et al. (2002) reported that AtrbohD and AtrbohF are the highest expressed members in leaves. Interestingly, cell death induced by GRI peptide was eliminated in atrbohD (and the double

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atrbohD/F) but not in atrbohF. This suggests that superoxide derived from AtrbohD is required for GRI peptide induced cell death. The differential response of atrbohD and atrbohF is consistent with earlier results showing different functions for these mutants; atrbohD lacked most ROS production in response to a fungal pathogen whereas atrbohF allowed for enhanced cell death and improved pathogen resistance (Torres et al., 2002). 3. C. IV. Mitogen Activated Protein Kinase

(MAPK)

ROS activation of Arabidopsis AtMPK6 and AtMPK3 by constitutively active forms of the upstream MAPKKs, AtMKK4 and AtMKK5 induced endogenous hydrogen peroxide production and cell death (Ren et al., 2002). Mitogen-activated protein kinase (MAPK) cascades are a conserved signal transduction system in all eukaryotes and their importance for plants is well known (Jonak et al., 2002; Baier et al., 2005). The universal structure of this signaling module consists of a MAP kinase kinase kinase (MAPKKK) that phosphorylates a MAP kinase kinase (MAPKK) that in turn phosphorylates a MAP kinase (MAPK) (Kangasjarvi et al., 2005). Of the 20 MAPKs in the Arabidopsis genome (MAPK Group 2002), the two primary oxidative stress-related are AtMPK6 and AtMPK3. They, and their orthologue in tobacco salicylic-induced protein kinase (SIPK) and wound induced protein kinase (WIPK), respectively, are induced, among other stresses, by ozone (Samuel et al., 2000; Samuel and Ellis, 2002; Ahlfors et al., 2004b; Joo et al., 2005). AtMPK6/SIPK and AtMPK3/WIPK are activated by hydrogen peroxide and superoxide (Kovtun et al., 2000; Samuel et al. 2000; Moon et al. 2003). In addition to being activated by ROS, the activation of Arabidopsis AtMPK6 and AtMPK3 by constitutively active forms of the upstream MAPKKs AtMKK4 and AtMKK5 induced endogenous hydrogen peroxide production and cell death (Ren et al., 2002). Mitogen-activated protein kinases (MAPKs) act both upstream and downstream of the oxidative burst, and the regulation of their activity is necessary to adequately trans-duce stress signals to downstream targets. The Arabidopsis Mitogen-activated protein kinases (MAPKs) kinase MEKK1 protein levels are both stabilized and activated by H2O2 in a proteasome dependent manner and thereby positively regulate ROS-induced activation of the

MAPKMPK4 (Nakagami et al., 2006). A Mitogen-activated protein kinases (MAPKs) phosphatase (AtMKP2) deactivates MAPK3 and MAPK6 and, hence, serves as a regulator of the plant response to oxidative stress (Lee and Ellis, 2007). Although both AtMPK6 and AtMPK3 are rapidly activated by O3, the regulation of their activity by O3 is different. AtMPK3 was up-regulated by ozone on transcriptional, translational as well as on post-translational levels, whereas only post-translational activation of the kinase activity of AtMPK6 was detected (Ahlfors et al., 2004b). The activation of AtMPK3 lasted longer than that of AtMPK6. Plant ozone sensitivity and the expression of antioxidant genes is affected by these kinase classes, since both suppression and overexpression of the tobacco SIPK (the AtMPK6 orthologue) led to increased ozone sensitivity and changes in the expression of APX and GST (Samuel and Ellis, 2002). Interestingly, at least in tobacco, SIPK also seems to regulate the activity of WIPK (the AtMPK3 orthologue), since in the SIPK over expression lines, WIPK activity induced by ozone was significantly reduced, whereas the opposite was true for the SIPK suppression line (Samuel and Ellis, 2002). Activation of MAP kinases by phosphorylation generally leads to nuclear localization and activation of transcription factors by the MAPK. Accordingly, the phosphorylated AtMPK3 and AtMPK6 were translocated rapidly to the nucleus within 30 min of initiating O3 exposure (Ahlfors et al. 2004b). The role and function of these MAP kinases in O3-exposed plants is still, however, unknown, although there is recent evidence that they might be connected to the hormonal responses. ABA, salicylic acid (SA), ethylene, and jasmonic acid (JA) are important in determining the degree of plant ozone sensitivity and O3-lesion initiation, propagation and containment (Overmyer et al., 2003).

3. C. V. Kinase Genes

Lesions simulating disease resistance 1 (LSD1), the first negative regulator of plant cell death identified, seems to act as a cellular hub that keeps a positive cell death regulator, the Arabidopsis basic Leu zipper (bZIP) transcription factor AtbZIP10, outside the nucleus under oxidative stress conditions. Hence, in lsd1 mutants, AtbZIP10 transits freely into the nucleus and triggers the uncontrolled cell death phenotype (Kaminaka et al., 2006). The

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oxidative signal-inducible 1 (OXI1) Ser/Thr kinase is required for oxidative burst-mediated signaling and first was characterized as a downstream signaling component of the phosphorinositide-dependent protein kinase 1 (PDK1). Another Ser/Thr kinase, PTI-2, was identified as an OXI1 substrate. Specific lipid signals have been shown to activate the OXI1-PTI-2 tandem through PDK1, whereas H2O2 signals to OXI1-PTI-2 via a PDK1-independent pathway (Anthony et al., 2006). ROS accumulation in response to elicitation was observed in nuclei (Ashtamker et al., 2007), chloroplasts (Liu et al., 2007), and mitochondria (Vidal et al., 2007). The distribution of iron within the different subcellular compartments was linked to ROS production and activation of plant defenses during the oxidative burst (Liu et al., 2007). These studies suggested that ROS production during pathogen responses occurs at multiple subcellular locations (and not exclusively at the apoplast) and that ROS production at these subcellular compartments has an important function in the activation of defense responses and programmed cell death (PCD).

3. C. VI. Caspases

The crucial biochemical effectors of apoptotic cell death are the caspases, a family of cysteine proteases. These enzymes are activated during apoptosis, are responsible for the observed morphological changes, and lead to cell death. The release of cytochrome c (Cyt c) from mitochondria to the cytosol is considered as prerequisite for apoptosis. Following its release, the cell will die due to the collapse of electron transport, the generation of reactive oxygen species (ROS), and a reduction in ATP generation (Yang et al., 1997; Green, 1998). While these events have been observed in plant PCD (Balk et al., 1999; Kim et al., 2003; Vacca et al., 2004), they are not associated with every type of PCD in plants (Pennell and Lamb, 1997). Apoptosis, the most thoroughly studied form of programmed cell death is defined by dependence on a family of proteases known as caspases. Activation of caspases leads to distinct morphological features in the cell, such as nuclear condensation, membrane blabbing, and cell shrinkage (Salvesen et al., 1999). Several studies have identified cell death programs that are clearly distinct from apoptosis (Fiers et al., 1999). These non-apoptotic cell

death programs are genetically regulated and often have morphological features resembling necrosis, yet their underlying molecular mechanisms are unclear. Molecular insights into alternative cell death programs could lead to a better understanding of their role in normal cellular homeostasis as well as disease processes (Yu et al., 2006). Inhibition of caspase 8 by treatment with either benzyloxycarbonyl-valyl-alanyl-aspartic-acid(O-methyl)-luoromethylketone (zVAD) or specific caspase 8 RNA interference (RNAi) triggers autophagy and cell death (Yu et al., 2004). Benzyloxycarbonyl-valyl-alanyl-aspartic-acid (O-methyl)-fluoromethylketone (zVAD)-induced death was arrested by either chemical autophagy inhibitors or by knocking down the protein products of key autophagy genes. This finding provided definitive evidence of a signaling pathway leading to autophagic cell death. However, the molecular mechanism by which autophagy causes cell death remains a key unanswered question (Yu et al., 2006). They showed that zVAD treatment causes selective autophagic degradation of one of the main cellular antioxidants, catalase. The depletion of catalase causes a severe imbalance of ROS metabolism, leading to dramatic ROS accumulation, lipid peroxidation, and ultimately non-apoptotic death. Yu et al. (2006) proposed a model for benzyloxycarbonyl-valyl-alanyl-aspartic-acid (O-methyl)-fluoromethylketone (zVAD)-induced autophagic cell death in which caspase 8 inhibition activates a pathway involving RIP and JNK that eventually leads to autophagy (Yu et al., 2004). However, the molecular mechanism behind autophagic cell death was not clear. Yu et al. (2006) showed that autophagy could lead to the degradation of catalase, a key enzymatic ROS scavenger, which disrupts the intracellular ROS balance. The resulting accumulation of ROS in the cell leads to membrane peroxidation, loss of membrane integrity, and eventually cell death. Directly varying the cellular level of catalase recapitulates the mechanism of autophagic cell death. Where, a reduction in cellular levels of catalase leads to cell death even if the cell has been treated with wortmannin, an autophagic inhibitor (Yu et al., 2006). By contrast, an increase in the cellular levels of catalase protects against autophagic cell death. Therefore, the regulatory effect of autophagy on catalase and thereby ROS and cell fate is direct and rate limiting.

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Apoptosis is energy-demanding cellular death, which is regulated in several ways (Steven, 1998). Mitochondria play a key role in the signaling pathway resulting in apoptosis. Mitochondria play a key role in the signaling pathway resulting in apoptosis (Cavalli and Liang, 1998). Once the cascade is activated by the cell death stimuli (e.g. TNF-α), caspases-8 and -10 are cleaved to their active forms and mitochondria release cytochrome C and Apoptosis Inducing Factor into the cytoplasm (Reed and Green, 2002). Released cytochrome c then activates the initiator caspase-9, which consequently activates the effectors caspases-3 and -7. These effectors caspases are also directly activated by the initiator caspases-8 and -10. Prolonged oxidative stress as well as DNA damage is some of the triggers of apoptosis that have been identified (Hruda et al., 2010). Diphenyliodonium (DPI) suppressed caspase-like protease (CLP) activity and Delayed DNA fragmentation. Therefore, the mechanism by which ROS mediate cell death is at least partially via the activation of CLPs. Interestingly, the onset and rate of cell death was not significantly changed in the presence of the hydroxyl radical scavenger, 1mM dimethyl thiourea (DMTU), suggesting that this specific ROS may not be involved in mega gametophyte cell death (He and Kermode, 2010). 3. D. Antibiotics

Spectinomycin and lincomycin are antibiotics that inhibit prokaryotic and organellar protein synthesis but does not affect cytosolic translation (Thomson and Ellis, 1972); however, they may indirectly result in a decrease in the expression of nuclear genes that regulate chloroplast biosynthesis and function (Gray et al., 2003; Mulo et al., 2003). Spectinomycin and lincomycin block plastid protein synthesis, which is necessary for correct thylakoid membrane structure (Thomson and Ellis, 1972) and transmission electron microscopy revealed that the cells of these cultures only contained very simple proplastids. These cultures responded to heat treatment with significant increases in levels of Apoptotic-like PCD (AL-PCD) compared with untreated light-grown cultures and an increase in the extent of cell condensation, again suggesting that in light-grown cultures, the presence of functional chloroplasts attenuates Apoptotic-like PCD (AL-PCD) progression (Doyle et al., 2010).

Cycloheximide blocks nuclear protein synthesis by inhibiting cytoplasmic translation only (Mulo et al., 2003). Cycloheximide treatment of light-grown cultures resulted in a temporal promotion of cell viability and repression of AL PCD compared to untreated controls. However, these effects were less pronounced in dark-grown cultures, besides they did not occur in antioxidant-treated light-grown cultures, as well. Although they did occur in norflurazon cultures (Doyle et al., 2010). Studies on the effects of cycloheximide on plant PCD have produced conflicting results, including PCD suppression (Desikan et al., 1998; Solomon et al., 1999; Vacca et al., 2004), no effect on PCD (Mazel and Levine, 2001; Elbaz et al., 2002) and PCD promotion (Dzyubinskaya et al., 2006). Doyle et al. (2010) reported that whether the cells were light grown or dark-grown, and whether the cells were chloroplast containing or not. It appears that plant ALPCD can precede in the absence of de novo nuclear protein synthesis, but that a complicated balance between protein synthesis, light, chloroplast activity, and ROS exists, which could somehow explain a cycloheximide-induced attenuation of Apoptotic-like PCD (AL-PCD), under certain conditions only. Light and the presence of functional chloroplasts have been shown to have significant effects on many forms of plant PCD, including UV-induced (Danon et al., 2004), mycotoxin-induced (Asai et al., 2000) and wounding-induced PCD (Gray et al., 2002), leaf senescence (Zapata et al., 2005), and the hypersensitive response (Genoud et al., 2002; Zeier et al., 2004; Chandra-Shekara et al., 2006). 3. E. Apoptotic-Like PCD (AL-PCD)

The morphological hallmarks of apoptosis include cytoplasmic shrinkage, nuclear condensation, and membrane blabbing (Earnshaw, 1995); the biochemical events involve calcium influx, exposure of phosphatidylserine, and activation of specific proteases and DNA fragmentation, first to large 50-kb fragments and then to nucleosomes ladders (McConkey and Orrenius, 1994; Wang et al., 1996). The stimuli that activate apoptosis are similar in plant and animal cells (O'Brien et al., 1998). Mammalian and plant PCD processes share several morphological and biochemical features, including cytoplasm shrinkage, nuclear condensation, DNA laddering, expression of caspase-like proteolytic activity, and release of Cytochrome c from mitochondria (Balk et al.,

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1999; Sun et al., 1999; Kim et al., 2003). Although, it should be noted that not all of the events were demonstrated in the same plant system, taken together these results infer a common basic cell death process in plants and animals. Apoptotic-like PCD (AL-PCD) is one type of cell death that can be identified by a distinctive corpse morphology where the cytoplasm has condensed away from the cell wall and nuclear DNA has been fragmented. AL-PCD is a controlled process that is initiated by the cells themselves (McCabe et al., 1997; Danon et al., 2000; Reape et al., 2008) and is distinct from necrosis, an unorganized cell death that results from overwhelming stress and severe cell damage and does not result in cytoplasmic condensation. As condensed cell morphology and fragmented DNA are distinguishing features of AL-PCD in plants, they can be used as markers to determine levels of AL-PCD in plant cells (Danon et al., 2000; Reape and McCabe, 2008). The involvement of chloroplasts in plant Apoptotic-like PCD (AL-PCD) because the cells contain chloroplasts if grown in the light, but not if grown in the dark. They showed that light grown and dark-grown culture cells respond differently to a PCD-inducing stimulus, resulting in different levels of cell condensation and DNA fragmentation. Evidence that chloroplasts regulate plant Apoptotic-like PCD (AL-PCD) and that this regulation may involve chloroplast-produced ROS, is presented. The obtained data suggested a complex interplay between light, chloroplasts, ROS, and nuclear protein synthesis during AL-PCD. In addition to that chloroplasts may be an important factor in AL-PCD regulation in green tissue. Arabidopsis thaliana cell suspension cultures are model systems shown that their cells contain well-developed, functional chloroplasts when grown in the light, but not when grown in the dark (Doyle et al., 2010). They found that heat treatment at 550 C induced apoptotic-like (AL)-PCD in the cultures, but light-grown cultures responded with significantly less AL-PCD than dark-grown cultures. Chloroplast-free light-grown cultures were established using norflurazon, spectinomycin, and lincomycin and these cultures responded to heat treatment with increased apoptotic-like (AL)-PCD, demonstrating that chloroplasts affect apoptotic-like (AL)-PCD induction in light-grown cultures. Antioxidant treatment of light-grown cultures

also resulted in increased apoptotic-like (AL)-PCD induction, suggesting that chloroplast produced ROS may be involved in AL-PCD regulation (Doyle et al., 2010). Chen and Dickman (2004) showed that transgenically expressed animal anti-apoptotic proteins in plant cells localized to chloroplasts and suppressed AL-PCD induced by oxidative stress within chloroplasts, suggesting chloroplast involvement in AL-PCD. However, Danon et al. (2006) showed that while plant PCD does occur independently of chloroplasts, when functional chloroplasts are present, they tend to play a major role in determining the severity of and the number of cells undergoing AL-PCD and this effect seems to be driven by chloroplast ROS production. TdT is mediated deoxyuridine triphosphate nick end labelling (TUNEL), which may be an accurate marker of Apoptotic-like PCD (AL-PCD) in dark-grown or chloroplast-free cells but may significantly underestimate levels of AL-PCD in light-grown or chloroplast-containing cells. Consequently, further experiments used cell condensation as a more accurate marker for levels of AL-PCD in both light-grown and dark-grown cultures (Doyle et al., 2010). The presence of functional chloroplasts in light-grown but not dark-grown cultures and the difference in Apoptotic-like PCD (AL-PCD) levels between the two culture types suggested that chloroplasts might affect AL-PCD progression. Norflurazon is an herbicide that inhibits phytoenedesaturase, an early enzyme in the carotenoid biosynthesis pathway (Sandmann, 1994). As carotenoids protect thylakoid membranes from photo-oxidative damage, norflurazon treatment results in early arrest of plastid development in the light. Norflurazon treatment may also indirectly result in the downregulation of nuclear genes encoding chloroplast proteins (Susek and Chory, 1992; Gray et al., 2003), over 90% of which are encoded by the nucleus rather than the chloroplast itself. Although plastids were observed in norflurazon culture cells using transmission electron microscopy, the lack of chlorophyll in the cells and the low fluorescence yield measurements indicated that these plastids did not possess photosynthetic function. Under low light conditions, photo-oxidative stress is reduced and chloroplasts may form in the presence of norflurazon (Sagar et al., 1988). Although carotenoid formation is inhibited by norflurazon, carotenoids are not always strictly

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necessary for chloroplast biosynthesis, but are needed for the assembly of photosystem II (Susek and Chory, 1992). It appears likely therefore, that chloroplast biosynthesis took place but that the thylakoid membranes were photosynthetically impaired. Norflurazon cultures responded to heat treatment with significantly higher levels of AL-PCD than untreated light grown cultures, as well as more extreme cell condensation in the cells, suggesting that suppression of chloroplast function affects AL-PCD progression (Doyle et al., 2010). 3. F. Hormonal and Oxidant Roles in

Developmental Cell Apoptosis

3. F. I. General Concepts

In plants, as in animals, cell death is an essential process during development and the responses to many stresses. The term programmed cell death (PCD) defines any form of cell death involving a series of orderly processes mediated by intracellular death programs, regardless of the triggers or the hallmarks it exhibits (Van Breusegem and Dat, 2006). PCD is a tightly regulated process for ensuring the proper development and the appropriate defense and stress responses. It is likely that, in addition to the putative regulators of PCD conserved throughout the animal and plant kingdoms, there are plant-specific mediators of PCD. Based on accumulated evidence, it has been proposed that plant hormones are strong candidates (Hoeberichts and Woltering, 2002). Generally, the structure of most of the leaves is determined by differential cell and tissue growth, but in some genus for instance in Monstera a group of cells die at early stages of leaf development, resulting in the formation of holes in the mature leaf (Kaplan, 1984; Greenberg, 1996). Reactive oxygen species (ROS) are key players in the regulation of programmed cell death (PCD) (Lamb and Dixon, 1997; Jabs, 1999; Overmyer et al., 2003); H2O2 potentially acts as a regulatory signal in plants (Gechev and Hille, 2005). ROS generation is associated with several forms of plant PCD, including aleurone layer cells, leaf senescence and the hypersensitive response (Alvarez et al., 1998; Bethke and Jones, 2001; Del Rio et al. 2003, Palma and Kermode, 2003; Zapata et al., 2005).

3. F. II. Hormonal Regulations

3. F. II. 1. Salicylic Acid (SA)

In plants, Salicylic Acid (SA) is best known for its role in systematic acquired resistant (SAR) (Raskin, 1995; Hoeberichts and Woltering 2002) for which it seems to be essential. It is also required for the execution of HR-like cell death (Durner et al., 1997). The role of SA has mostly been demonstrated through experiments with transgenic, SA-degrading NahG plants and the SA-insensitive Arabidopsis mutant npr1. Ozone exposure and pathogen attack both induce Salicylic Acid (SA) synthesis within a few hours after the beginning of the exposure (Orvar et al. 1997; Overmyer et al. 2005). These studies have shown that O3-induced cell death is abolished in the absence of SA or its action (Orvar et al., 1997; Rao and Davis, 1999; Overmyer et al., 2005). The integral role for SA in cell death is also supported by experiments in which exogenously applied SA significantly increased the ozone sensitivity of otherwise tolerant genotypes (Rao et al., 2000; Mazel and Levine, 2001). Furthermore, double mutant analysis in Arabidopsis has shown that O3-sensitive accessions became tolerant when either the transgene NahG or the npr1 mutation was introduced to the sensitive background (Orvar et al., 1997; Rao et al., 2000; Overmyer et al., 2005). It was suggested that SA has a vital role in cell death and that without SA active PCD is not initiated in O3-exposed plants. However, Rao and Davis (1999) showed that inhibition of SA accumulation in O3-exposed plants resulted in cell death either by necrosis (as opposed to PCD), or by HR-like programmed cell death (PCD), depending on the duration of exposure to O3. In short term O3 exposures the SA-deficient NahG Arabidopsis plants were more tolerant to O3 than the wild-type plants, whereas in long-term exposures NahG plants developed necrotic lesions without the involvement of the active superoxide formation typical of the lesions in short-duration O3 exposures (Kangasjarvi et al., 2005). In plants, salicylic acid (SA) is best known for its role in SAR (Raskin, 1995; Hoeberichts and Woltering, 2002) for which it seems to be essential. It is also required for the execution of HR-like cell death (Durner et al., 1997). The role of SA has mostly been demonstrated through experiments with transgenic, SA-degrading

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NahG plants and the SA-insensitive Arabidopsis mutant npr1. These studies have shown that O3-induced cell death is abolished in the absence of SA or its action (Orvar et al., 1997; Rao and Davis, 1999; Overmyer et al., 2005). In the systemic leaves, cell death as a response to a transmissible signal from the cells in contact with the pathogen does not spread further from these clusters of cells. In contrast to the pathogen-infected primary leaves in which cells around the primary infection site die by PCD, because of unidentified signals from initiation death site of cell. The necrotic cell death was interpreted as result of a gradual depletion of antioxidative capacity, which led to a drastic shift in the cellular redox balance and eventually to cell death. Thus, SA may also have a role in the upregulation of antioxidative systems during lesion spread and have a protective function when the SA concentration increases during lesion formation (Kangasjarvi et al., 2005). 3. F. II. 2. Ethylene Role

Autocatalytic activation by increasing the stability of ACC synthase by phosphorylation is not, however, the only process by which O3 regulates ethylene synthesis, as greatly increased ethylene synthesis is apparent only in the sensitive accessions. Either other components are required together with autocatalysis, or the down-regulation of ethylene synthesis is slower or more inefficient in the O3-sensitive accessions (Kangasjarvi et al., 2005). The very early activation of ethylene biosynthesis before the activation of ACC synthase gene expression suggested to be regulated at the post-translational level. It has been shown that in tomato the stability of LE-ACS2 protein is regulated by phosphorylation of a Ser-460 residue in the C-terminal part of the enzyme (Tatsuki and Mori, 2001), which presumably affects the binding of the ETO1 protein to the ACS dimer and prevents the degradation of the active dimer by the 26S proteasome (Wang et al., 2004). Kim et al. (2003) suggested the involvement of salicylic acid-induced protein kinase SIPK (AtMPK6 homologue) in the activation of ethylene biosynthesis, and the downregulation of this kinase by the ethylene-dependent feedback loop in tobacco. Liu and Zhang (2004) demonstrated that Arabidopsis MPK6 directly phosphorylates AT-ACS6 protein, which prevents its proteolytic degradation and causes the upregulation of ethylene synthesis. This model is in accordance

with the prolonged AtMPK6 activity in O3-exposed ethylene-insensitive Arabidopsis etr1 mutant (Ahlfors et al., 2004b), with the faster activation of AtMPK6 (Overmyer et al., 2005) as well as higher ethylene biosynthesis and AT-ACS6 induction (Overmyer et al., 2000) in the O3-sensitive rcd1 mutant. Similarly, inhibition of protein kinase activity with the general kinase inhibitor K252a prevented the O3-induced increase in ACC synthase activity in tomato (Tuomainen et al., 1997) and also O3 damage in the O3-sensitive Arabidopsis rcd1 mutant (Overmyer et al., 2005). Ethylene has been shown to initiate cell death through the initiation of ROS generation and proteolytic enzyme activation (de Jong et al., 2002). HR forming in the ethylene insensitive ein2 mutant in Arabidopsis (Arabidopsis thaliana) has suggested that ethylene plays, at best, a minor role in this type of cell death (Bent et al., 1992; Ciardi et al., 2000). Host ethylene production is required for the full virulence of P. syringae pv (P. s. pv) glycinea, P. s. pv tomato, Xanthomonas campestris pv vesicatoria, Verticillium dahlia, and Cucumber mosaic virus on their hosts (Lund et al., 1998; Van Loon et al., 2006). Some strains of P. syringae and X. campestris pathovars derive their own ethylene to serve a virulence function. Within these pathogens, ethylene is not synthesized from ACC, but from 2-oxoglutarate, by an ethylene forming enzyme (EFE) (Weingartet al., 2001). Additionally, ethylene is a clear regulator of petal senescence in carnation, petunia, tobacco and orchids following contact between pollen and stigma surface, however, it appear to play little or no part in senescence of other species including lilies such as Hemerocallis and Alstroemeria (Wagstaff et al., 2005; Rogers, 2006). The abscission associated ethylene production by tulip petals coincided with production of ethylene in carnations resulting in wilting of the flower (Nukui et al., 2004; Van Doorn 2004; Rogers, 2006). Inhibition of protein phosphatase activity with calyculin A significantly increased ethylene evolution and ACC synthase activity in the absence of other external elicitation (Spanu et al. 1994; Tuomainen et al. 1997). This treatment also significantly increased cell death in the O3-sensitive rcd1 mutant (Overmyer et al. 2005). Without a counteracting regulatory system that limits the spread of the lesion once it is initiated, induction of spreading cell death would result in

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a progressive destruction of the whole organ. This is evident, for example, in the Arabidopsis spontaneous lesion mutant lsd1 (Jabs et al., 1996), where a ‘runaway cell death’ spreads throughout the whole leaf once the lesion is initiated. Ethylene itself can be responsible for the containment of (the ethylene-dependent) O3 lesion propagation since ethylene causes desensitization of the cells to its own action (Wang et al., 2002). The ethylene receptor acts as a suppressor of ethylene signaling when it is not in contact with the hormone and this suppression is released after ethylene binds to the receptor. It is thought that the ethylene-induced synthesis of new, unoccupied receptor molecules is responsible for the desensitization of ethylene signaling (Kangasjarvi et al., 2005). Up-regulation of genes encoding ethylene receptors have been seen in ozone-exposed tomato (Moeder et al., 2002). Subsequently, the O3-induced synthesis of new ethylene receptor proteins could in part lead to decreased ethylene sensitivity and downregulation of ethylene-dependent lesion spread. It is noknown, whether the upregulation of new receptor synthesis takes place in, or around, the developing lesion (Kangasjarvi et al., 2005). The mutation in RCD1 seems to affect mostly hormone-related processes, since in microarray analysis with 6400 genes the few genes that had changed basal expression level in clean-air grown rcd1 are involved in either ethylene-, ABA-, or sugar-related processes (Ahlfors et al., 2004a). Furthermore, rcd1 shows slight insensitivity to both ABA and ethylene in some specific responses to these hormones, which is reflected, for example, in higher stomata conductance in rcd1 than in Col-0 wild type. It was speculated that the function of RCD1 could relate to post-translational modification of its targets by ADP ribosylation. The rcd3 was isolated in the same mutant screen as the two other rcd mutants. Similar to rcd1, also rcd3 possessesahigher stomata conductance than the Col-0 wild type. Unlike rcd1 micro-array analysis with 6400 genes reveal no genes with different basal expression between clean air-grown rcd3 and Col-0, which suggests that the gene mutated might be involved in a process that could be specifically related to O3-responses and the effect of the mutation becomes visible only when the plants are challenged with oxidative stress (Kangasjarvi et al., 2005). The rcd3 mutant was isolated in the same mutant screen as the two other rcd

mutants. The mutant has severe foliar injury and higher ethylene emission than the Ws wild type under O3.

3. F. II. 3. Jasmonic Acid and Methyl Jasmonate

The future identification of RCD3 is thus expected to reveal a specific process related to acclimation to O3 or stomata regulation. oji1 (ozone-sensitive and jasmonate-insensitive 1) was identified in a screening of a T-DNA insertion mutation population in the ecotype Ws (Kanna et al., 2003). The mutant has severe foliar injury and higher ethylene emission than the Ws wild type under O3. The mutant was also insensitive to jasmonate inhibition of root elongation, and the expression of a commonly used JA marker gene, VSP1 (VEGETATIVE STORAGE PROTEIN 1) and the jasmonate inhibition of O3-damage were reduced in oji1 when compared to Ws. The ascorbate content of oji1 was similar to the wild type, thus the O3-sensitivity of the mutant is attributable to the increased ethylene evolution and/or decreased jasmonate sensitivity (Kangasjarvi et al., 2005). Tuominen et al. (2004) postulated that these two are not necessarily separate since they have a mutually antagonistic interaction with each other. However, oji1 is another example showing the significance and role of both ethylene and jasmonate in plant O3 sensitivity. The location of the T-DNA tag in oji1 was preliminarily localized with TAIL-PCR (Thermal Asymetric Interlaced Polymerase Chain Reaction) between At3g61810 and At3g61820 (Kanna et al. 2003). The former is a β-1, 3, glucanase and the latter is an aspartyl protease family protein with one predicted transmembrane domain and a putative targeting to endomembrane system. It remains to be verified; if the OJI1 gene encodes the aspartyl protease protein and what is its function in the jasmonate signaling or regulation of ethylene biosynthesis (Kangasjarvi et al., 2005). The identification of the mutated genes in the O3

sensitive mutants described above has identified new and verified already known processes important for plant O3 tolerance. These mutants strengthen the role of ascorbate and the still unclear role for chloroplasts in plant O3 responses is evident, as demonstrated by the lcd1/rcd2 mutant (Kangasjarvi et al., 2005). Rcd1 and oji1 mutants could verify the importance of hormonal regulation especially the role of ethylene in determining plant ozone sensitivity. Mutant analysis has facilitated the

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elucidation of the oxidative cell death cycle, which seems to have universal relevance in the regulation of the spread of cell death, not only as a response to ozone, but in other responses as well in which induced lesion formation takes place (Overmyer et al., 2003). The evidence on the hormonal control of plant O3 responses is strong and the balance between SA, ethylene and JA is very likely to be largely responsible for the processes involved (Overmyer et al., 2003; Tamaoki et al., 2003; Tuominen et al. 2004). The balance seems to be accomplished by mutual interactions. Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are the most studied of the linolenic acid-derived signaling molecules in plants that are collectively referred to as oxylipins or jasmonate (Farmer et al., 1998). Not all JA-insensitive mutants are, however, sensitive to O3. The jasmonate insensitive jin1 is tolerant to O3 (Nickstadt et al., 2004), which suggests that JA has at least two different roles in O3-responses. It was shown that the MYC type transcription factor (AtMYC2) encoded by JIN1 is required to discriminate between two different branches of jasmonate-responses (Lorenzo et al., 2004). Both these branches of JA signaling seem to be, however, involved in O3-related processes, one in lesion formation and the other in lesion containment, since lesion formation seems to require AtMYC2 (JIN1) and lesion containment requires JAR1 and COI1(Kangasjarvi et al., 2005). Methyl jasmonate (MeJa) is a well-known plant stress hormone where exposure to stress, MeJa is produced and causes activation of programmed cell death (PCD) and defense mechanisms in plants (Zhang and Xing, 2008). The early events and the signaling mechanisms of MeJa-induced cell death have yet to be fully elucidated. To obtain some insights into the early events of this cell death process, mitochondrial dynamics, chloroplast morphology and function, production and localization of reactive oxygen species (ROS) at the single-cell level as well as photosynthetic capacity at the whole-seedling level under MeJa stimulation should be investigated (Zhang and Xing, 2008). They demonstrated that MeJa induction of ROS production, which first occurred in mitochondria after 1 h of MeJa treatment and subsequently in chloroplasts by 3 h of treatment, caused a series of alterations in mitochondrial dynamics including the cessation of mitochondrial movement, the loss of mitochondrial transmembrane potential (MPT), and the

morphological transition and aberrant distribution of mitochondria. Thereafter, photochemical efficiency dramatically declined before obvious distortion in chloroplast morphology, which is prior to MeJa-induced cell death in protoplasts or intact seedlings. Originally Jasmonic acid (JA) and its methyl ester (methyl jasmonate, MeJa) were identified as a major component of fragrant oils. They were first demonstrated to promote senescence, essentially a type of PCD, in detached oat (Avena sativa) leaves (Ueda and Kato, 1980), and have subsequently been shown to be a class of plant hormone that plays many diverse roles in several other aspects of plant development (Schaller, 2001). MeJa acts with mitochondria and chloroplasts in Arabidopsis protoplasts through ROS production, providing new insights into the ROS and MeJa signaling networks that modulate the cell death process. Recently, the relationship between ROS formation and the alterations in mitochondrial dynamics as well as the cross-talk between mitochondria and chloroplasts during MeJa-induced plant cell death remain unknown and need to be addressed in future research (Zhang and Xing, 2008). The roles of jasmonate in hypersensitive response- (HR) associated cell death have also been suggested since jasmonate synthesis and accumulation commence prior to fresh weight and protein loss, Malondialdehyde accumulation and cell death in elicitor-treated suspension cell cultures and pathogenesis-infected tobacco leaves (Creelman and Mullet 1997; Kenton et al., 1999). A wealth of molecular genetic studies have provided further evidence for their involvement in the regulation of the complex and highly regulated cell death program (Asai et al. 2000; Woo et al. 2001; He et al. 2002). Previous studies demonstrated that sustained exposure of both leaves and suspension-cultured cells of grapevine (Vitis vinifera L. cv. Limberger) to exogenously applied MeJa provokes HR-associated cell death (Repka 2002; Repka et al., 2004). In animals, the mitochondrion integrates diverse cellular signals and initiates the death execution pathway. The application of pro-death stimuli to mammalian cells leads to loss of mitochondrial transmembrane potential (MTP), changes in mitochondrial morphology and the release of cytochrome c, which initiates several downstream processes (such as the activation

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of cell-degrading caspase proteases) that culminate in cell death (Liu et al., 1996; Zou et al., 1997; Wang, 2001). It has been proposed that MeJa provokes death in cancer cells, not by changes in cellular mRNA transcription, protein translation or p53 expression, but by acting directly and selectively on mitochondria (Rotem et al., 2005). Evidence provided has demonstrated that MeJa could induce mitochondrial swelling and mitochondrial membrane depolarization via abnormal opening of the mitochondrial permeability transition pore mitochondrial transmembrane potential (MPTP) complex channel and therefore provoke cytochrome c release from mitochondria to the cytosol and ATP depletion, leading ultimately to cancer cell death (Rotem et al. 2005). In plants, the production of H2O2 and other ROS is a common feature of cell death in response to exogenous stimuli such as environmental stresses and pathogen attack (Levine et al., 1994; Apel and Hirt, 2004; Vacca et al., 2004; Amirsadeghi et al., 2006; Vacca et al., 2006; Gao et al., 2008). It is well known that plant hormones affecting cell death in plants may act as mediators of this core pathway also mainly by modulating the intracellular ROS levels (Hoeberichts and Woltering, 2002). MeJa has received considerable attention for its ability to induce cell death and suppress cell proliferation in several human cancer cell lines (Fingrut and Flescher 2002; Ishii et al., 2004; Kim et al., 2004; Oh et al., 2005; Rotem et al., 2005; Flescher, 2007; Wasternack, 2007). The ROS levels undergo a dramatic change during plant cell death as well as during apoptosis in human cancer cell lines in response to MeJa exposure (Kim et al., 2004; Oh et al., 2005; Mur et al., 2006). It is thus suggested that the ROS signaling cascade is the shared mechanism by which Methyl Jasmonate induces cell death in plants and animals (Hoeberichts and Woltering, 2002). Many death stimuli, including biotoxins (Andi et al., 2001), cell death protein substrate analogs (Yao et al., 2004; Yao and Greenberg, 2006), ROS (Gao et al., 2008; Scott and Logan, 2008), senescence-induced PCD (Zottini et al., 2006) and heat treatment (Vacca et al., 2004; Vacca et al., 2006; Scott and Logan, 2008) are sensed either directly or indirectly by mitochondria. These can alter mitochondrial polymorphism and motility at an early stage of subsequent cell death. In fact, Methyl Jasmonate provoked cell

death by acting directly and selectively on mitochondria in cancer cells, leading to abnormalities in mitochondrial morphology (Rotem et al., 2005). Using the Arabidopsis transformants (43C5) expressing GFP in mitochondria, Zhang and Xiang (2008) found that mitochondria in the protoplasts treated with MeJa for 3–5 h showed an aberrant phenotype and distribution prior to cell death, including an increase in the areas of individual mitochondria (e.g. mitochondrial swelling), the arrangement into tight clusters and the formation of extensive clumps. Scott and Logan (2008) observed in heat stress-induced cell death, the evident transition of mitochondrial morphology (from elongated rods or filamentous structures to spherical or ovoid shapes) could be observed during the onset of cell death. Moreover, in vitro experiments using isolated mitochondria showed that MeJa resulted in the occurrence of obvious mitochondrial swelling indicated by the declined in absorbance at 540nm within the first 3 h of exposure to Methyl Jasmonate. Mitochondrial transmembrane potential (MPT) is induced by multiple independent pro-apoptotic signaling pathways and multiple different molecules such as Ca2+

, ROS, MeJa and sphingolipids (Yao et al., 2004; Rotem et al., 2005). The role of superoxide in cell death initiation by GRI-peptide is further supported by the increased sensitivity of gri to X/XO, as well as the increased resistance to a virulent bacterial pathogen. The superoxide produced upon pathogen infection triggers cell death, which is enhanced by the truncated GRI protein, subsequently leading to pathogen resistance. This makes GRI-peptide a potential candidate for being involved in ROS-induced cell death. This role of the N-terminal GRI-peptide could explain the phenotype of gri plants as well as the phenotypes of the GRI overexpression lines and the genomic complementation lines (Wrzaczek et al., 2009). The presence of a signal peptide for the secretory pathway and induction of cell death by infiltration of GRI-peptide into the extracellular space of Arabidopsis leaves points to a function of GRI in the extracellular space. This is supported by secretion of STIG1 into the stigmatic lipid exudate (Verhoeven et al., 2005) and binding of LeSTIG1 to the extracellular domain of RLKs (Tang et al., 2004). Because of the very weak expression of GRI in Arabidopsis leaves, analysis of the subcellular localization of GRI required overexpression with the 35S promoter. Microscopic analysis of GRI-YFP

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localization suggested both cytosolic and extracellular localization for GRI-YFP. Similarly, when fused to YFP-GRI (1–30)-YFP], the GRI signal peptide directed YFP to the cell wall (Wrzaczek et al., 2009). Localization of the GRI-YFP fusion protein to the cytoplasm might be due to mislocalization caused by the 35S promoter, or an additional stimulus or protein might be required for correct GRI localization. Furthermore, fluorescent proteins frequently become unstable in the extracellular environment, accounting for very low fluorescence levels (Berg and Beachy, 2008). Further experiments will be required for determining the precise subcellular localization under control and stress conditions using an inducible promoter system and a transgenic approach in Arabidopsis. Taken together, our results provide evidence that the GRI protein is secreted to the extracellular space and it can perceive signals there (Wrzaczek et al., 2009).

3. F. III. Hormonal Coalitions

The balance seems to be accomplished by mutual interactions. In many cases, JA implication in the signaling of cell death processes was not due to it acting alone but rather coordinated with the action of ET and SA with complex synergistic/antagonistic roles and also ROS production (Kenton et al., 1999; Mur et al., 2006). Exogenously applied MeJa could induce the production of ET and SA (Turner et al., 2002; Sasaki-Sekimoto et al., 2005; Mur et al., 2006; Qu et al., 2006; Wasternack, 2007). JA and MeJa not only can induce cell death synergistically with other plant hormones such as ethylene (ET) and salicylic acid (SA) in Arabidopsis and tobacco explants, but also can increase the fungal toxin fumonisin B1- (FB1) induced apoptosis-like PCD in Arabidopsis protoplasts (Asai et al. 2000; Mur et al., 2006). The evidence on the hormonal control of plant O3 responses is strong and the balance between SA, ethylene and JA is very likely to be largely responsible for the processes involved (Overmyer et al., 2003; Tamaoki et al., 2003; Tuominen et al. 2004). The RCD1 protein (At1g32230) contains a WWE domain predicted to be involved in protein–protein interactions (Aravind, 2001), nuclear localization sequences, a ‘PARP’ domain (NAD-binding catalytic core for ADP-ribosyl transferases), and an unidentified C terminal protein–protein interaction domain identified in

yeast two-hybrid analysis (Belles-Boix et al., 2000). The C-terminal interaction domain in RCD1/CEO1 was shown to interact with several transcription factors or DNA-binding proteins involved in dehydration or osmotic stress responses, such as DREB2A, which is a central transcription factor in the ABA-independent responses to osmotic stress (Liu et al., 1998). The mutation in RCD1 seems to affect mostly hormone-related processes, since in microarray analysis with 6400 genes the few genes that had changed basal expression level in clean-air grown rcd1 are involved in either ethylene-, ABA-, or sugar-related processes (Ahlfors et al., 2004a). Furthermore, rcd1 shows slight insensitivity to both ABA and ethylene in some specific responses to these hormones, which is reflected, for example, in higher stomata conductance in rcd1 than in Col-0 wild type. It was speculated that the function of RCD1 could relate to post-translational modification of its targets by ADP ribosylation, which remains to be determined. ROS production drives the SA-dependent (Orvar et al., 1997; Rao and Davis 1999) cell death, which continues until the third, JA-dependent component, antagonistic to lesion propagation, contains the enlargement of the lesion. MAP kinases are connected to both ROS and cell death (Kovtun et al. 2000; Ren et al. 2002) and MAPK involvement in ethylene, SA, and ABA signaling has been established in different contexts (Zhang and Klessig, 1997; Kim et al., 2003; Ouaked et al., 2003; Xiong and Yang, 2003). Salicylic acid is required for the programmed cell death, ethylene promotes endogenous ROS formation and lesion propagation, and jasmonic acid is involved in limiting the lesion spreading. Abscisic acid is most likely involved through the regulation of stomata and thus is expected to affect lesion initiation (Kangasjarvik et al., 2005). SA (Shirasu et al., 1997; Mur et al., 2000) influences the biphasic pattern of the oxidative burst. Different concentrations of SA were injected into tobacco leaves and stimulated rapid rise in ethylene production, which declined after approximately 2 h (Mur et al., 2008). To examine the effect of SA on ethylene production within a HR, Psph was inoculated into leaves of cauliflower mosaic virus 35S salicylate hydroxylase (SH) transgenic tobacco plants, which degrade SA to catechol (Mur et al., 2008). Ethylene production was perturbed in salicylate hydroxylase (SH) tobacco leaves with C2H4-I being reduced and C2H4-II

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somewhat Delayed and reduced in amplitude compared to wild-type plants (Mur et al., 2008). An Arabidopsis gene probe for ASC6 was used because this exhibits the highest homology to the stress responsive NtACS2 gene. SA can act by influencing the generation of ROS to which C2H4 could act to augment (de Jong et al., 2002) or increase production in response to oxidative stress, likely in a positive feedback loop. Injections of either 1 mM methyl viologen or Glc: Glc oxidase (G: GO) elevated ethylene production. Hence, the observed patterns of ethylene production are likely to be linked to the well-established H2O2-SA interaction during defense (Lamb and Dixon, 1997). It is worthy to mention that BAK1 and BAK1-LIKE1 possesses dual roles in cell death: positive and negative regulation of brassinosteroid-dependent and brassinosteroid-independent growth and cell death pathways, respectively, through an alternating interaction with BRI1 and the LRR receptor-like kinase FLS2 (Chinchilla et al., 2007; He et al., 2007; Heese et al., 2007; Kemmerling et al., 2007).

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4. Stresses Implicated In Growth Limiting

Factors

4. A. Oxidant Roles in Programmed Cell Death

4. A. I. Reactive Oxygen Species (ROS)

4. A. I. 1. Reactive Oxygen Species in Triggering

PCD

In vivo, a range of biotic and abiotic stresses can elevate AOS levels in plants due to perturbations of metabolism in organelles and the generation of AOS in defense responses through NADPH oxidases, cell wall peroxidases and amine oxidases (Dat et al., 1998; Van Camp et al., 1998). A wide range of different environmental conditions can induce stresses, which significantly alter plant metabolism, growth and development and, at their extremes, ultimately lead to plant death. These abiotic stresses include drought, high salinity, extremes of high and low temperatures, heavy metals, ultraviolet radiation, nutrient deprivation, high light stress, and hypoxia (Dat et al., 2000; Noctor and Foyer, 1998). It was observed that transgenic tobacco plants with a lowered capacity to metabolize H2O2, as result of antisense suppression of a gene encoding CAT, develop necrotic lesions, exhibit defense-related gene expression in the absence of elicitors and are hypersensitive to pathogen attack (Chamnongpol et al., 1996; Takahashi et al., 1997; Mittler et al. 1999). Apoptosis is energy-demanding cellular death, which is regulated in several ways (Steven, 1998). Mitochondria play a key role in the signaling pathway resulting in apoptosis. Mitochondria play a key role in the signaling pathway resulting in apoptosis (Cavalli and Liang, 1998). Recent progress has further implicated ROS as mediators of PCD in plants (Apel and Hirt, 2004; Mittler et al., 2004), including cell death associated with the

hypersensitive response (Lamb and Dixon, 1997), barley aleurone cell death (Bethke and Jones, 2001), leaf senescence (Pastori and Del Rio 1997) and camptothecin-induced death of omato suspension cells (de Jong et al., 2002). Among the ROS, H2O2 is the most stable and is a key player in stress responses and PCD (Gechev et al., 2002; Gechev and Hille, 2005). Sub-lethal doses of H2O2 can protect cells by activating antioxidant enzymes, whereas toxic concentrations can trigger PCD or cause necrosis (Alvarez et al., 1998; Yao et al., 2001; Gechev et al., 2002; Gechev and Hille, 2005). ROS are toxic and in many cases short-lived, the activity of these oxidases is tightly regulated both temporally and spatially (Nanda et al., 2010). Kacperska (2004) has suggested that the role of ROS and H2O2 in the mediation of stress responses may depend on the severity of the stressor. This implies that, rather than sensor type, the quantitative effects of the sensor-initiated modifications in the oxidant-antioxidant activities in different cell compartments may be responsible for the different effects of a particular stressor. This suggestion is in line with observations that small increases of H2O2 allow the general enhancement of stress tolerance; whereas large increases in H2O2 trigger local responses that unavoidably lead to programmed cell death (PCD). Mittler et al. (1999) using transgenic catalase/Prx-deficient tobacco plants showed that these were hyper-responsive to pathogen challenge, thus providing direct evidence for a role for H2O2 in HR cell death.

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Transgenic tobacco plants with a lowered capacity to metabolize H2O2, as result of antisense suppression of a gene encoding CAT, develop necrotic lesions, exhibit defense-related gene expression in the absence of elicitors and are hypersensitive to pathogen attack (Chamnongpol et al., 1996; Takahashi et al., 1997; Mittler et al. 1999). H2O2 has been shown to be a diffusible signal mediating localized PCD during hypersensitive Response (HR) (Levine et al., 1996), as well as being involved in a systemic signaling network (Alvarez et al., 1998). H2O2 induces program cell death (PCD) in Arabidopsis and other species (Solomon et al., 1999); consequently, the expression of potential PCD-related genes following H2O2 treatment might be expected. Plants expressing a bacterial pyruvate decarboxylase showed enhanced levels of cell death in response to pathogen challenge (Tadege et al., 1998), suggesting that sugar metabolism is a crucial activity during the hypersensitive response (HR) and other stresses. Myb genes represent a large gene family in Arabidopsis (Kranz et al., 1998) and a myb on co-gene homolog has been implicated as a critical regulator of the hypersensitive response (HR) cell death pathway (Daniel et al., 1999). Moreover, myb transcription factors possess conserved amino acid motifs that are redox sensitive (Myrset et al., 1993). Several genes encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase, ACC oxidase, and cysteine proteinase are up-regulated during petal wilting in senescing Dianthus caryophyllus (carnation) flowers (Sugawara et al., 2002). A type of cysteine endproteinase that may mediate petal PCD has been identified in senescent Hemerocallis (daylily) petals (Valpuesta et al., 1995). The biochemical changes and increases in hydrolytic enzymes and respiratory activity, associated with petal senescence that consists of the breakdown of macromolecules, can be used to predict PCD (Rubinstein, 2000). Pyruvate decarboxylase was induced during oxygen deprivation stress in rice (Oryza sativa) seedlings (Minhas and Grover, 1999). Pyruvate decarboxylase catalyzes the decarboxylation of pyruvate to acetaldehyde and CO2 during ethanolic fermentation as result of oxygen deprivation (Desikane et al., 2001). Coordinated expression of several genes in response to a specific stimulus can be achieved via the interaction of transcription factors with cis-elements common to the promoter regions of

those genes. For example, the WRKY binding site was identified in the promoter region of all 26 genes making up the “pathogen regulon” in Arabidopsis (Maleck et al., 2000). 4. A. I. 2. Ros Role in Initiation and Spread of PCD The necrotic cell death was interpreted as result of a gradual depletion of antioxidative capacity, which led to a drastic shift in the cellular redox balance and eventually to cell death. Thus, SA may have a role in the upregulation of antioxidative systems during lesion spread and It have a protective function when the SA concentration increases during lesion formation (Kangasjarvi et al., 2005). Reactive Oxygen Species (ROS), such as superoxide anion (●O2

−) and hydrogen peroxide (H2O2), are by-products constantly produced during normal metabolic processes, such as photosynthesis or glycolysis. ROS produced at high levels have first been described as lethal for the cell integrity. However, high ROS production is also necessary for plant defense (oxidative burst, necrosis) (Nandan et al., 2010). Without a counteracting regulatory system that limits the spread of the lesion once it is initiated, induction of spreading cell death would result in a progressive destruction of the whole organ. This is evident, for example, in the Arabidopsis spontaneous lesion mutant lsd1 (Jabs et al., 1996), where a runaway cell death spreads throughout the whole leaf once the lesion is initiated. The rcd1 was isolated in a screen for increased O3-sensitivity. In addition to O3-sensitivity, rcd1 has several other phenotypes related to growth, development and hormone biology (Overmyer et al., 2000; Ahlfors et al. 2004). A second allele of rcd1 was identified in a mutant screen for paraquat tolerance (Fujibe et al., 2004). Genetic evidence has proved that elevated ●O2

−, singlet oxygen, or H2O2 levels are able to induce cell death under certain conditions. Arabidopsis mutants namely lsd1 and rcd1 produce more ●O2

−. Thus, undergo a PCD spontaneously (Jabs et al., 1996; Overmyer et al., 2005). Thus, rcd1 is sensitive to conditions that lead to the formation of apoplast ROS, which induces a spreading cell death phenotype in the mutant, but it has increased tolerance to ROS formation in the chloroplast manifested as increased tolerance to the ROS generating herbicide paraquat (Ahlfors et al., 2004; Fujibe et al., 2004). rcd1 has also increased tolerance to UV-B (Fujibe et al. 2004).

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RCD1 has also been reported earlier under the name CEO1 (Clone Eighty One), identified as an Arabidopsis cDNA that complements a yeast mutation that renders yeast sensitive to oxidative stress (Belles-Boix et al., 2000), and ATP8, an Arabidopsis protein that interacts with the movement protein of turnip crinkle virus (Lin and Heaton, 2001). 4. A. II. Reactive Nitrogen Species (RNS) Triggering PCD Phosphotidic acid (PA) and correspondingly, xylanase were shown to induce reactive oxygen species (ROS) production. Therefore, scavenging of ●NO or inhibition of either the phospholipase C (PLC) or the diacylglycerol kinase (DGK) enzyme diminished xylanase-induced reactive oxygen species (ROS) production. It was found that xylanase induced phospholipase β1 (PLDβ1) and PR1mRNA levels decreased when ●NO or phosphatidic acid (PA) production were compromised. Xylanase treatment was found to induce NO and that NO is necessary for PA production suggested that NO and the subsequent Phosphotidic Acid (PA) generation via the PLC/DGK pathway, are required for xylanase-induced ROS production, gene expression, and cell death. Xylanase-induced NO activates PLC/DGK signaling during plant defense. Therefore, NO and phosphatidic acid (PA) are involved in the induction of cell death by xylanase (Laxalt et al., 2007). Treatment with NO scavenger cPTIO, phospholipase C (PLC) inhibitor U73122, or DGK inhibitor R59022 diminished xylanase induced cell death. PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense (Laxalt et al., 2007). They showed increased levels of Phosphotidic Acid (PA) in cells treated with different concentrations of xylanase and PA accumulation was inhibited when cells were incubated together with 1 mM cPTIO. These studies indicate that NO participates in the elicitation of PA generation in xylanase-treated tomato cells. Consequently, cPTIO, [2-(4-carboxyphenylalanine) 4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxidepotassium] could not inhibit the xylanase-induced PLD activation, because PLD activation during xylanase treatments is low, typically 1.25-fold after 30 min (Van der Luit et al., 2000). The first wave of ROS was attributed to the PLC/DGK pathway, the second to PLD; both

phospholipase pathways acted upstream of ROS formation (Anderson et al., 2006). PA accumulated rapidly via PLC/DGK in tobacco cells expressing the tomato Cf-4 resistance protein treated with Cladosporium fulvum Avr4 protein. PLC activity was required for the rapid ROS accumulation (de Jong et al., 2002). NO dependent, PLC/DGK-generated PA is involved in the induction of ROS production during the first peak of the oxidative burst in xylanase-treated cells. PLD activation during xylanase treatments could contribute to activation of NADPH oxidase, as suggested in rice cells, via an NO-independent pathway (Laxalt et al., 2007). PLC activation also results in the formation of IP3 that, either directly or indirectly via its metabolized form IP6, may result in the release of Ca2+ from internal stores (Meijer and Munnik, 2003). Inhibition of PLC does also slightly affect NO production, suggesting a possible feedback mediated by either PA or IP3. Exogenously applied S-nitroso-N-acetylpenicillamine (SNAP) induces PA formation in a dose-dependent manner, but the concentrations required are high. 1mM SNAP(S-nitroso-N-acetylpenicillamine) induces a 2–3 fold increase in PA formation via PLC/DGK within minutes, as was shown for xylanase previously (Van der Luit et al., 2000). Inhibition of SNAP-induced PA formation was shown upon preincubation with inhibitors of PLC and DGK. Accordingly, PLC and the DGK inhibitors reduced SNAP-induced cell death (Laxalt et al., 2007). The concentration of SNAP that is required for the induction of significant level of PA is high, which makes confirmation of the direct effect of NO is uncertain. Scavenging the SNAP-released NO by cPTIO is required to show unequivocally that exogenously applied NO activates PA formation. The concentration of cPTIO required for the effective scavenging of NO released by 1mMSNAP is 10–20 mM (Pagnussat et al., 2002), which is toxic to tomato cell suspensions. High doses of S-nitroso-N-acetylpenicillamine (SNAP) must be applied to mimic xylanase induced NO. On the other hand, such high concentration of S-nitroso-N-acetylpenicillamine (SNAP) exceeds the global concentration as induced by xylanase, which might very well induce other pleiotropic or indirect effects. These results suggested that NO solely induces the formation of PA, although the concentration of S-nitroso-N-acetylpenicillamine (SNAP) required could induce pleiotropic effects (Laxalt et al., 2007).

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The role of transit metal Fe2+ in culture medium for ROS and NO● interaction; and how H2O2, ●O2

− and NO● affect each other’s accumulation at enzyme levels; how much ONOO‑ generated for cell death induction or antilipid peroxidation; what are downstream targets of ROS and NO● interactions and intermediates for cell death induction (Zhao, 2007). All these issues are critical for understanding ROS and NO● signaling leading to PCD. Recent progress from animal and plant studies has provided many clues for reaction can cause modification of many metallo proteins, which have metal binding sites at active center interact with tobacco aconitase that catalyze the isomerisation of citrate to isocitrate. In animals, NO● interacted with guanylate cyclase (GC), which generates cGMP and induces Ca2+ fluctuation (Gow et al., 2004). Soluble guanylate cyclase (GC) directly binds to NO● at heme moiety of N‑terminus, followed by conformational change and activation of enzyme. NO● binds to the oxidized form of cytochrome c oxidase (Cyt C oxidase) at the binuclear heme‑copper center as a competitive inhibitor of oxygen binding and inactivates the enzyme (Gow et al., 2004). NO● interacts with heme‑containing enzymes catalase and peroxidases and inhibits their activities (Clark et al., 2000). This was also confirmed in C. lusitanica, cell cultures. All NO● or ROS, or ONOO‑ mediated protein modifications can be essential cellular processes for PCD induction, not only by activation or inactivation of RNS/ROS generating and metabolizing enzymes but also through direct regulation of signaling and cellular process of PCD such as Ca2+ fluctuation, procaspase‑3 activation (Benhar et al., 2005). A plenty of evidence suggest that balances of RNS (mainly NO●) and ROS production are important for directing physiological consequences, either better survival or cell death (Delldonne et al., 2001; de Pinto et al., 2006; Espey et al., 2000). Physiological and genetic studies have convincingly shown their essential roles. However, the details and mechanisms by which ROS and NO● interplay and induce PCD are not well understood (Zhao, 2007). They studied Cupressus lusitanica, culture cell death and revealed the elicitor-induced co-accumulation of ROS and NO● and interactions between NO● and H2O2 or ●O2

− in different ways to regulate cell death. Therefore, NO● and H2O2 reciprocally enhanced the production of each other whereas

NO● and ●O2− showed reciprocal suppression on

each other’s production. It was found that the interaction between NO● and ●O2

− but not between NO● and H2O2 that induced PCD, probably through peroxynitrite (ONOO−) (Zhao, 2007). It was found that NO● was generated upon elicitor treatment in parallel with ●O2

− and H2O2 accumulation, and NO● donors induced a pronounced C. lusitanica, cell death (Zhao, 2007). Using biosynthetic enzyme inhibitors or scavengers showed that NO● and ●O2

− production was necessary for ●O2

− or NO● induction of cell death, respectively. Measuring H2O2, NO● and ●O2

− production in various treatments indicated that H2O2 and NO● reciprocally stimulated the production of each other, whereas NO● and ●O2

− suppressed the accumulation of each other (Zhao, 2007). Since NO● readily reacts with ●O2

− and generate a more potent oxidant ONOO‑, which plays pivotal roles in animal cells under oxidative and nitrosative stress, we proposed that NO● and●O2

− induced cell death mainly through their interaction product ONOO‑ (Zhao, 2007). Early physiological studies have shown many controversial results about if ROS and NO●

either ●O2− or H2O2 or NO● , is necessary or

sufficient to induce plant cell death (Delledonne et al., 2001; de Pinto et al., 2002; Beligni et al., 2002; Tada et al., 2004). Transgenic plants deficient in H2O2‑scavenging enzymes such as ascorbate peroxidase (APX) and catalase have elevated ROS levels, and therefore are more susceptible to NO● treatment by showing a more dramatically augmented cell death than wild type plants (Tarantino et al., 2005; Zago et al., 2006). These data suggest that elevated ROS levels in these plants are necessary for cell death induction. However, it may be too early to conclude that these ROS alone are sufficient to induce PCD since whether NO● is involved in initiating cell death by ROS in these plants was not tested. On the other hand, it has been shown that NO● requires the well-balanced H2O2 levels to induce plant cell death.It is more likely that ROS and NO● interactions undergoing in these plants might be the real causes for cell death (Zhao, 2007).

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4. A. III. Coalition of ROS and RNS in Triggering PCD NO● and ROS often show some overlapping and synergistic functions, particularly in cell death induction through interactions, which are determined by their reactive natures. The production balances of and diverse interactions between ROS and NO● under different physiological environments form a complex signaling cellular network to determine if plant cells continue to survive or are directed to death. These redox signaling and complex cellular processes play essential roles in innate immune response and other defense systems of plants. NO● donor induced cell death thus can be attributable to interactions between NO● and ROS. Similarly, treatment with H2O2 or ●O2

− generation system may also evoke NO● generation (de Pinto, et al., 2006; Bright et al., 2006). ROS induced cell death thus may not be solely caused by H2O2 or ●O2

−, but involve NO●. Advanced studies on animal cells convincingly demonstrate that NO●

‑ induced cell death largely attribute to its interaction ●O2

− with and the interaction product ONOO‑, although the early study on animals showed that NO● hasten apoptosis in macrophages, thymocytes, and tumor cells.14 However, there is a few data available for ●O2

− interaction with NO● and ONOO‑ production and function in plant. The lack of convenient and accurate tools to precisely capture and measure ONOO‑ and other ROS‑RNS interaction chain products may partly contribute to this situation. Several early experiments indicated the inhibition of ●O2

− on NO● production and vice versa (Caro and Puntarulo, 1998; Vanin et al., 2004). Zhao (2007) clearly demonstrated such interaction in plant cells and their correlations with cell death. 4. B. Temperature Stresses 4. B. I. Reactive Oxygen Species Role in Temperature Stresses Plants are unable to escape exposure to environmental extremes and, therefore, have developed defense responses in order to survive. The signal transduction pathways that elicit these responses, or even the way in which plants perceive these environmental stresses, are not well-understood (Braam et al., 1997; Mittler, 2002). Thermo tolerance induced in mustard seedlings by both heat acclimation and

salicylic acid treatment lead to the same modulations in H2O2 and catalase levels (Dat et al., 1998). Trehalose, thought only to provide tolerance to plants exposed to drought by acting as a compatible solute, was recently found to confer tolerance to a range of other environmental stresses (Garg et al., 2002). In a recent survey of 7000 Arabidopsis genes in response to cold, drought and high salt treatments, a significant number of changes in expression observed were common to more than one stress (Seki et al., 2002). Kreps et al. (2002) exposed Arabidopsis seedlings to chilling, salt and osmotic stresses, and found that, of the 2409 genes induced over all treatments, many were stress-specific 3 h after exposure, and this proportion increased 27 h following exposure. In the microarray study by Seki et al. (2002) observed, many stress-specific responses were observed between drought, cold and high-salinity treatments. There are common elements in the response of plants to different stresses; specific responses may be as important, or more important, than the generalized responses in allowing plants to recover from stresses. The degrees of importance of specific and common responses in stresses are active oxygen species (AOS) accumulations. It is now widely accepted that most environmental stresses lead to the accumulation of AOS such as ●OH, H2O2,

●O2 and O−

2 in plant cells (Dat et al., 2000; Mittler, 2002; Noctor and Foyer, 1998). This accumulation has a number of implications for biological processes within the plant as a whole andwithin mitochondria. 4. B. II. Reactive Nitrogen Species Role in Temperature Stresses There are numerous changes in biochemical and physiological processes during cold acclimation, ranging from accumulation of osmolytes and cryoprotectants (Xin and Browse, 1998) to disruption of reactive oxygen species homeostasis (Suzuki and Mittler, 2006). NO is involved in cold acclimation and freezing tolerance in plants. NO is also involved in metal toxicity induced proline production in Chlamydomonas reinhardtii (Zhang et al., 2008). The cross talk between NO and Proline exists in cold acclimation and freezing tolerance remains unknown. However, there has been no detailed study to evaluate the role of NO in cold acclimation and freezing tolerance in plants. Zhao et al. (2009) demonstrate that cold

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acclimation induced a marked increase in endogenous NO level in Arabidopsis leaves resulting from up-regulation of NIA1 expression. The elevated NO may function as a signal to evoke Proline accumulation via enhanced synthesis and reduced degradation, conferring tolerance of Arabidopsis to freezing stress. Therefore, these findings highlight the involvement of NR-dependent NO production in cold acclimation-induced freezing tolerance in plants. In plants, Proline contents are maintained by transcriptional regulation of both biosynthesis and degradation (Hare et al., 1999). The rapid upregulation of expression of the proline accumulation under osmotic (P5CS1) gene and down regulation of the Proline dehydrogenase (ProDH) gene by cold acclimation indicate that both enhanced synthesis and reduced degradation are responsible for the enhanced Proline accumulation. Like wild-type plants, expression of P5CS1 was also upregulated in nitrate reductase mutant (nia1nia2) plants by cold acclimation. In contrast, expression of Proline dehydrogenase (Pro DH) was upregulated in nia1nia2 plants during cold acclimation. These findings suggest that the lower accumulation of Proline in nia1nia2 plants than in wild-type plants is due to less enhanced synthesis and greater degradation of Proline during cold acclimation. There have been several reports showing that ●NO can stimulate genes responsible for proline accumulation under osmotic (P5CS1)activity and upregulated the expression of P5CS1 genes in plants (Uchida et al., 2002; Ruan et al., 2004; Zhang et al., 2008). Similar findings, Ruan et al. (2004) found that sodium nitroprusside (SNP) stimulates the activity of P5CS1 in wheat (Triticum aestivum). The observations that the ●NO donor SNP can mimic cold acclimation to upregulated expression of the P5CS1 gene, and that this effect can be abolished by the ●NO scavenger cPTIO, highlight the critical role of ●NO in cold acclimation-induced Pro accumulation. Cold acclimation-induced Proline accumulation has been widely observed in plants and varying responses of expression of P5CS1 and ProDH genes to cold acclimation (Hare et al., 1999). The up-regulation of P5CS1 and down-regulation of ProDH expressions during cold acclimation were reported by Xin and Browse (1998).

The elevated Proline content in wild-type plants can act as a compatible osmolytes to protect plants from dehydration, thus enhancing freezing tolerance (Xin and Browse, 2000). However, we did not observe a significant increase in osmolality of both wild-type and nia1nia2 plants during cold acclimation, suggesting that the ameliorative effect of ●NO on freezing tolerance cannot be explained by osmoregulation of accumulated Proline content. It has also been shown that Proline can function as a molecular chaperone to stabilize the structures of proteins and play a role in regulation of the antioxidant system and cellular redox potential (Hare et al., 1999; Szekely et al., 2008). Further studies to elucidate the mechanism underlying ●NO-dependent Proline accumulation in freezing tolerance are warranted. In addition, a recent study revealed that ●NO can induce phosphatidic acid (PA) where PA accumulation by activation of phospholipase C and phospholipase D (PLD) (Distefano et al., 2008). In this context, Li et al. (2004) demonstrated that increased accumulation of phosphatidic acid (PA) by overexpressing plasma membrane-bound PLD leads to enhanced freezing tolerance in Arabidopsis. Therefore, it is plausible that the cold acclimation dependent ●NO production may contribute to freezing tolerance by up-regulation of PLD-dependent production of phosphatidic acid (PA). The capacity of Proline to scavenge free radicals resembles the function of NO, because NO can also serve as an antioxidant under conditions of various stresses (Beligni et al., 2002). Another important observation is that nia1nia2 plants had much lower Proline contents than wild-type plants before and after cold acclimation. The lower Pro contents in nia1nia2 plants than in wild type plants are likely to result from the disruption of Proline biosynthesis due to less expression of P5CS1 in control conditions. Moreover, the cold acclimation-dependent increase in proline contents in wild-type plants can be markedly reduced in the presence of Nitrate Reductase (NR) inhibitor and ●NO scavenger. Therefore, these findings are indicative that induction of Proline accumulation may be a downstream component of cold acclimation-induced ●NO production. LOW Temperature Accumulation of ROS in cat2 was suppressed by the ROS-generating NADPH oxidase inhibitor

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diphenylene iodonium (DPI), demonstrating that this process is a secondary event caused by the activation of a signaling pathway that involves ROS production via NADPH oxidase activity, rather than a primary cause of CAT2 deficiency. The cat2 was more sensitive to heat stress in both pretreated (acquired) and non-pretreated (basal) plants and the lack of APX1 was able to compensate for this sensitivity (Vanderauwera et al., 2011). In cucumber roots, H2O2 accumulated rapidly in the cell wall and along the plasma membrane within the short period of only15 min following exposure to low temperature (LT). This correlated with adverse effects of LT on Lpr and H+-ATPase. The presence of highest concentrations of H2O2, initially in cell walls and along the plasma membrane, suggested that both the cell wall and the plasma membrane might be the major source of H2O2, as suggested by Ranieri et al. (2003). Oxidative burst generating ROS is known to occur in response to both biotic (Bolwell et al., 2002) and abiotic stresses (Vacca et al., 2004). Another antioxidant enzyme susceptible to oxidative damage is peroxiredoxins, which can be inactivated by high concentrations of peroxides via a mechanism thought to be important in allowing the enzyme to distinguish between AOS signaling and oxidative stress situations (Rabilloud et al., 2002; Wood et al., 2003). These enzymes are known in plant mitochondria (Sweetlove et al., 2002; Horling et al., 2003), but detailed analysis of their functions have yet to be undertaken. 4. C. Water Relation and Drought Stress The radicals of active oxygen facilitated the effects of decomposition enzymes of proteins by changing the situation of amino acids in the protein branches and therefore one of the most important reasons for decreasing the content of protein in the plants exposed to drought stress is the production of oxygen free radical (Somogy, 1982). Drought stress was associated with decreasing the amount of protein, and this reduction was so great in the 2nd level than the 1st level and the evidence plants. In the plants which were kept under the mid drought stress, using the ascorbic and salicylic acids in different concentrations, in most cases, was associated with increasing the amount of protein. For explaining this issue, we could say that under the unsuitable environmental conditions such as drought stress, the active species of oxygen would produce and concentrate, and

consequently, the increasing H2O2 would lead to increase in the oxidation of proteins in several types of plant species. Drought stress is a factor used to decrease the Rubisco activity and its amount in the plant (Yazdanpanah et al., 2011).Drought stress lead to the reduction of protein composition in several types of plant species through decreasing the cellular polysomic, and it was determined that the drought stress caused the activation of the protective genes in the plants, and consequently, it increased the amount of HSP protein. On the other hand, it caused the formation of the important enzymes such as Rubisco, phosphor phroktokinas, invertase and sucrose synthetase in the aerial organs in drought stress conditions (Inze and Montago, 1995). Drought stress increased the amount of Malonildialdehyde (MDA) and the other aldehydes, there were many reports based on drought stress on the amount of Malonildialdehyde (MDA) and the other aldehydes. Sairam et al. (1998) reported that the amount of Malonildialdehyde (MDA) increased in three wheat genotypes under drought stress. It was proved that the amount of Malonildialdehyde (MDA)-productive during the drought stress between the different cultivars of maize, banana and rice seedlings is so different. The resistant species are capable to sweep H2O2 by increasing the antioxidant ability but by decreasing H2O2, the less amount of Malonildialdehyde (MDA) would be created. Thus, the amount of the sensitive MDA - productive is so great (Sharma and Shanker, 2005; Li et al., 1998). Loggini et al. (1998) declared that drought stress did not affect lipids per oxidation in wheat cultivars. Mid drought stress, the amount of MDA increased and the other aldehydes increased in two plants of the cold region, Poa pratensis and Festuca (Fu and Haung, 2001). Yazdanpanah et al. (2011) observed increases in Malonildialdehyde (MDA) and the other aldehydes in the dry conditions resulted from active oxygen species generations such as super oxide radical, peroxide, hydrogen and radical hydroxide, which is in the oxidative stress condition. Yazdanpanah et al. (2011) found that the greatest amount of sugar is related to the plants which are placed under severe drought stress (irrigation equal to 1/3 field capacity), with 1 mM salicylic acid (D2S0A1) and was performed

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using the 1st level of drought stress (1/3 field capacity) with 1 mM salicylic and ascorbic acids (D1S1A1). The obtained results indicated that drought stress was associated with the sugar amount, and if the level of drought went up, the amount of sugar decreased. This reduction was mainly due to the effect of drought stress on the amount of chlorophyll and photosynthesis. In some conditions where the plants are placed under the 1st level of drought stress (irrigation equal to 2/3 field capacity), in combination with ascorbic and salicylic acids, the concentrations could be associated with the increasing amount of sugar and this effect could lead to the improvement of plant resistance. In general, application of different concentrations of ascorbic and salicylic acids is associated with the increasing amount of sugar, but this increase was only with 1 mM salicylic acid (D1S1A0) and was so great with 1 mM salicylic and ascorbic acids (D1S1A1). In addition, this will increase the height of the figure to the evidence group. In the other conditions where the plants were exposed to severe drought stress (irrigation 1/3 field capacity), in combination with ascorbic and salicylic acids, in most cases it was associated with increasing the amount of sugar, while in other cases, it was associated with the decreasing sugar content which was only seen with 3 mM ascorbic and salicylic acids. 4. C. I. Oxidant Roles in Drought Stress It was confirmed that the increasing concentration of secondary plant products due to an increasing drought stress gives a most reliable and solid explanation for the conjuncture that economically valuable products derived from plants grown under the drought conditions are well known for quality than those derived from equivalent plants cultivated in moderate climatic conditions. Gain in quality by increasing the secondary plant products concentration by using of drought stress would be compensated by decreasing yields in biomass. Ontogeny and circadian clock-controlled gene expression are important features of plant secondary metabolism, as are master regulatory transcription factors. These regulators are attractive targets for engineering secondary metabolic pathways and open an exciting field for research in near future. Plants that are exposed to drought stress produce a greater amount of secondary plant products such as phenols, terpenes as well as N and S containing substances such as alkaloids, cyanogenic

glucosides or glucosinolates. There are no doubts that the application of drought stress enhances the concentration of secondary plant products (Khan et al., 2011). Selmar and Mohamed (2007) stated that the content of secondary plant metabolites indeed is higher in plants that suffer from drought and salt stresses than those cultivated under optimal conditions. Continuously, pungency increases as salt stress increases in onion (Chang and Randle, 2004). Under abiotic stress conditions, increased ROS levels are associated with both signaling and oxidative damage. In water-stressed maize (Zea mays), sustained cell elongation in theapical root region is correlated with increased apoplastic ROS levels (Zhu et al., 2007). Accordingly, salt stress induced inhibition of leaf expansion in maize is correlated with reduced apoplastic ROS production (Rodriguez et al., 2007). These data suggested that increased apoplastic ROS production has a positive effect on growth under water stress conditions and consolidates previous reports on ROS mediated cell wall loosening sustaining the capability of cell walls to expand during abiotic stress conditions. The importance of an integrative role of mitochondrial oxidative respiration in the abiotic stress response of plants has been established further with the identification of the pentatricopeptide repeat protein (PPR40) that is important for the correct ubiquinol cytochrome c oxidoreductase activity of complex III and the adaptation to adverse environmental conditions (Zsigmond et al., 2012). The plants produce various secondary plant products with an economic value in normal conditions but various kinds of abiotic stress factors can result in an enhanced production of the secondary plant products. Especially, a wide array of abiotic stimuli like salinity and drought are capable to trigger changes in the plant metabolism, results enhanced production of plant secondary products (Khan et al., 2011). Plants have several metabolic pathways leading to tens of thousands of secondary products capable of effectively responding to stress situations imposed by biotic and abiotic factors. These pathways, often recruited from essential primary metabolism pathways upon initial gene duplication, are frequently restricted to specific taxonomic groups and play a major role in the plant and environment interaction (Nascimento and Fett, 2010). Polyphenols suppress a number of Lipopolysaccharides (LPS)-induced

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signals and thus could be effective against gingivitis (Zdarilova et al., 2010). Two other major groups of flavonoids found in flowers are flavones and flavonols function to protect cells from UV-B radiation by accumulating in epidermal layers of leaves and stems, absorb light strongly in the UV-B region and letting visible (PAR) wavelengths pass throughout uninterrupted (Lake et al., 2009). Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants and play a critical role in plant development and defense response. Isoflavonoids are secreted by legumes; and play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia (Sreevidya et al., 2006). It seems that synthesis of these flavonoids is an effective strategy against ROS (Posmyk et al., 2009). It could result from the fact that phenolic compounds, which could be also substrate for different peroxidase, were the first line of defense against various environmental stresses like metal stress (Posmyk et al., 2009). It could be possible to enhance a wide variety of useful metabolites in plant through applying different environmental stresses (Cisnero-Zevallos, 2003). Some of these useful metabolites have light absorptive properties, harvest light for photosynthesis and protect the cells from damaging effects of high energy radiation, while other promote defensive action against herbivores and pathogens. Concentration of a number of secondary products are strongly depending in the prevailing growing conditions and, it is obvious that any kind of stress conditions have a major impact on the metabolic pathwaysresponsible for the synthesis and accumulation of the secondary plant products. Increase in light intensity mostly is entailed with elevated temperatures or lower water availability and drought conditions often are correlated with high salt concentrations in the soil (Khan et al. 2011). Improving the content of some active compounds such as Volatile Sulphur compounds, ascorbic acid, carbonyl compounds, vitamins and flavonoids are the most important pharmaceutical content and could be enhanced through different environmental stresses in some vegetables (Cisneros-Zevallos, 2003). Most of the enzymes [like Glutathione-S-transferase (GSTs) and Phenylalanine ammonium lyase (PAL)], are stress inducible and play an important role in the protection of plants from adverse effects of stresses (Marrs, 1996). PAL, along with

cinamates 4-hydroxylase constitute important group of enzymes in allocating significant amount of carbon from phenylalanine into the biosynthesis of several important secondary metabolites (Singh et al., 2009). Plants were grown at high CO2 concentrations, a treatment that abolishes ROS production in peroxisomes in order to identify the subcellular source of ROS that triggers the mechanisms responsible for HL tolerance (Willekens et al. 1997). Constant growth at high CO2 suppressed growth retardation in cat2 and apx1/cat2 plants and lesion formation in cat2 plants. In contrast to apx1/cat2 plants grown under ambient conditions, apx1/cat2 plants grown under high CO2 and transferred to ambient air developed lesions. This result identified peroxisomal H2O2

as the ROS primarily responsible for the activation of defense and/or acclimation mechanisms in apx1/cat2 plants and showed that growth in ambient air constitutively triggers the high light (HL) Acclimation pathway in apx1/cat2 mutants. Measurements of glutathione redox state demonstrating that cat2 plants grown in ambient air have an oxidized cellular redox state and that growth of these plants in high CO2 prevents this oxidation. In contrast, apx1/cat2 plants grown under ambient air or high CO2 have a reduced cellular redox state (Vanderauwera et al., 2011). 4. C. II. Plasma Membrane Hydrulic

Conductivity Under Low Temperature

4. C. II. 1. H+ ATPase Activity

Water transport across root systems of young cucumber (Cucumis sativus L.) seedlings was measured following exposure to low temperature (LT, 8–13oC) for varying periods. Following low temperature (LT) treatment, hydrogen peroxide was localized cytochemically in root tissue by the oxidation of cerium (III) chloride (Lee et al., 2004). The effects of hydrogen peroxide on the hydraulic conductivity of single cells (LP) in root tissues, and on the H+-ATPase activity of isolated root plasma membrane, have been worked out. Cytochemically evidence suggested that exposure of roots to low temperature (LT) stress caused a release of hydrogen peroxide in the millimolar range in the vicinity of plasma membranes (Lee et al., 2004). In response to a low root temperature (8oC), the hydraulic conductivity of the root (Lpr) decreased by a

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factor of 4, and the half-times of water exchange increased by a factor of 5–6. Decreasing root temperatures from 25–13oC increased the half times of water exchange in a cell by a factor of 6–9. The measurement of axial water transport with a heat-balance sap-flow gauge showed that only a small amount of water was transported when 8oC was imposed on cucumber roots (Lee et al., 2004). Hydraulic conductivity of single cells (LP) and the H+-ATPase activity of the isolated root plasma membrane were very sensitive to externally applied hydrogen peroxide at a concentration of 1–16 mM. These observations suggest that the accumulation of hydrogen peroxide appears to mediate decreases in water transport in cucumber roots under low temperature (Lee et al., 2004). The most common visible consequence of the exposure of root system to a low temperature (LT) is the dehydration of leaves, implying that the tolerance of plants to LT may well be related to their ability to absorb water. Measurements of root hydraulic conductivity (Lpr) in response to low root temperatures support this hypothesis (Fennel and Markhart, 1998). It has been proposed that major abiotic stresses result in water deficits (Holmberg and Bulow, 1998). However, only a few attempts have been made to relate the LT-tolerance of plants to their ability to take up water directly (Aroca et al., 2001; Vernieri et al., 2001). Lee et al. (2004a) have shown that root pressure (Pr) in an excised cucumber root system, as measured with the root pressure probe at 25oC of root temperature, was 0.15–0.2 MPa. This value rapidly dropped to zero MPa, when the root temperature was gradually lowered to 8oC for 15–20 min. The result implied that the cucumber root system was very sensitive to LT. Interestingly, leaf gourd (Cucurbita ficifolia Bouche), a species tolerant to LT, was able to maintain positive root pressure (Pr) at 8oC (Leeet al., 2002). In the absence of transpiration, root pressure (Pr) is given by (Steudle, 1994). Production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals, is an early response of plants to low temperature (LT). In cells, enzymes such as superoxide dismutase, peroxidases and catalase, as well as several metabolites (Noctor and Foyer, 1998) tightly control the levels of these toxic species. The sensitivity of the cortical cells of cucumber roots after only 15 min of

exposure to low temperature (LT) was seen ultrastructurally (Lee et al., 2002) and this rapid response upon exposure to LT implies involvement of ROS. It is, then, reasonable to assume that ROS generated in excess of normal physiological levels due to LT stress may affect the function(s) of membrane intrinsic proteins, including H+-ATPases and aquaporins (Lee et al., 2004). A reduction in H+-ATPase activity in cucumber roots due to exposure to low temperature (LT) has been demonstrated, and Steudle model helps to explain the observed close relationship between the LT-induced rapid drop in root pressure (Pr) in cucumber and the decline in H+-ATPase activity (Steudle, 1994; Ahn et al., 2000). However, physiological and/or biochemical mechanisms related to the LT-induced reduction of H+-ATPase activity in the plasma membrane are not known. It has been suggested that the formation of intermolecular disulphide bonds from thiol groups caused by LT may result in the aggregation of membrane proteins (Levitt, 1980). Conformational alteration of the H+-ATPase due to the LT-induced phase change in the plasma membrane is also possible (Lee et al., 2004). Water transport across root systems of cucumber was examined using the root pressure probe. Root pressure (Pr) and Lpr were measured following an exposure to low temperature (LT). In addition to overall measurements of hydraulic conductivity of root (Lpr), turgor pressure (P) and hydraulic conductivity of individual cells hydraulic conductivity (LP) were measured to work out the role of aquaporins on water conductivity under conditions of LT stress. The consequence of LT on water transport through the intact plant was assessed using the heat-balance sap-flow gauge (Steinberg et al., 1990). Hydrogen peroxide generated in root tissue was localized using CeCl3. This was based on the possibility that H2O2 produced in excess of normal physiological levels under LT stress may affect the activity of H+-ATPases and aquaporins present in the plasma membrane. Evidence provide that LT-induced rapid reduction in water transport in cucumber plants may be associated with the generation of high concentrations of H2O2 in root tissues (Lee et al., 2004). It was found that LT causes a significant reduction in Lp and in the activity of plasma membrane associated H+-ATPase in the root cells of cucumber seedlings. There appeared to be a relationship between this phenomenon and the

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elevated production of H2O2 as indicated by cerium perhydroxide deposits. Decrease in hydraulic conductivity of root (Lpr) in cucumber due to LT and H2O2 production. The cytochemical method employed here has been used to study the role of H2O2 in plant–microbe interactions (Bestwick et al., 1997), and during extension growth of plants (Rodriguez et al., 2002). Penetration of cerium chloride across a mass of tissues such as epidermal and cortical cells, as result of the reaction between CeCl3 and H2O2, is likely to take some time and, therefore, this method could not be used to study dynamic processes related to hydrogen-peroxide generation and gradient across the plasma membrane. However, it has been proved as a useful tool in assessing major concentration differences in hydrogen peroxide at one point in time (Bestwick et al., 1997). Prasad et al. (1994) showed that, when corn (Zea mays L.) plants were grown under favourable environmental conditions, the H2O2 concentration was kept low, ranging between 0.1 and 2 mM. However, during chilling stress it may rapidly increase up to 10 mM. Frahry and Schopfer (1998) showed that in the soybean (Glycine max L.) root H2O2 production could be stimulated a 10-fold by exogenous NADH or NADPH. By contrast, Chara corallina internodes have been shown to tolerate up to 350 mM of H2O2 for quite some time (Henzler and Steudle, 2000). Depending on the species, the resistance to tolerate toxic stresses of high H2O2 may be quite different. It appears that cucumber is quite sensitive, and this high sensitivity correlates with a high sensitivity to LT. H2O2 accumulation in cells is likely to be a complex event, and the mechanism underlying H2O2-mediated reduction in the Lp in cucumber remains to be clarified. The rapid reduction of cell Lp suggests that, in addition to the H+-ATPases, the water-channel proteins, aquaporins, may be affected. Reduction in the activity of the H+-ATPases may lower cytoplasmic pH, and in turn cause a decrease in LP. Zhang and Tyerman (1999) have shown that the hydraulic conductivity of root (Lpr) was not affected by K+-channel blocker tetraethyl ammonium at concentrations that normally block K+ channels. Hence, the parallel inhibition by H2O2 simply means that H2O2 treatment is less selective on different transporters. It was shown that the rapid drop in root pressure (Pr) in response to low temperature (LT) was largely

caused by a reduction in the activity of the plasma membrane H+-ATPase activity (Lee et al., 2004a). Preincubation of plasma membrane vesicles with 1 mM H2O2 for 30 min, reduced the H+-ATPase, suggesting that the reduced H+-ATPase due to low temperature (LT) may be mediated by H2O2 production in root tissues. It is interesting to note that, in cucumber, a similar concentration of H2O2 affected both hydrulic conductivity (LP) (2 mM) and H+-ATPase activity (1 mM). The ways in which short-term exposures of root system to LT inhibit H+-ATPase are not yet known. H2O2 may interfere with ATP hydrolysis (Feng and Forgac, 1994) and/or disulphides exchange of oxidized glutathione with the reactive cysteine in V-ATPase (Wang and Floor, 1998). Jack pine (Pinus banksiana Lamb.) seedlings has been shown that the inhibition of the plasma membrane H+-ATPase activity by direct freeze and thaw was caused by the thiol oxidation of plasma membrane proteins (Zhao and Blumwald, 1998). Reduced glutathione prevented lipid peroxidation through a glutathione-mediated free-radical scavenging system (Kumar et al., 2007). Plasmodesmata can also facilitate water movement between adjoining cells; LT-induced closure of Plasmodesmata may be another reason for the observed reduction in cell hydraulic conductivity (LP). A low (non-freezing) temperature has been shown to cause a rapid reversible closure of Plasmodesmata (Holdaway-Clark et al., 2000). LT treatment caused an inhibition of H+-ATPase activity and a rapid elevation in H2O2 concentration, particularly in the region of the cell wall plasma membrane interface, and there appeared to be a relationship between H2O2 concentration and hydraulic conductivity, and H+-ATPase activity. Higher concentration of H2O2 may react with a Fenton catalyst, such as iron ions, in the bathing medium, generating reactive hydroxyl radicals, which may affect aquaporins activity (Lee et al., 2004). 4. C. II. 2. Aquaporin

There are indications that acidic cytoplasmic pH inhibits aquaporin activity. The sensitivity of water-channel activity to environmental conditions may affect the overall water uptake by roots and the regulation of the water balance of plants (Javot and Maurel, 2002). The activity of aquaporins in response to low temperature (LT) has been studied with the cell pressure probe in algae and in the root cortical cell of

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several species (Hertel and Steudle, 1997; Zhang and Tyerman, 1999). Although it has been shown that changes in root hydraulic conductivity (Lpr) result from expression of aquaporins (Javot and Maurel, 2002), the very rapid response of cucumber roots to LT makes it unlikely that the response in cucumber was mediated by gene expression. Water transport is inhibited by mercurial agents such as HgCl2, which reacts with sulfhydryl groups in proteins. Therefore, the application of HgCl2 to roots at micromolar concentrations usually closes the channels reducing hydraulic conductivity (Wanet al., 2001). It may be that H2O2 at elevated levels interferes with the activity of both H+-ATPase and aquaporins. However, a direct relationship of the activity of aquaporins (open/closed state) to H2O2 remains to be experimentally demonstrated. Cochard et al. (2000) postulated that gradual decrease in root pressure (Pr) soon after lowering of root temperature from 25oC suggests that aquaporins may start to close below this temperature. However, since the growth of cucumber plants is not adversely affected by 20oC of root temperature (Ahn et al., 1999), it is unlikely that aquaporins close at this temperature. It is not yet known what the critical threshold temperature is for the closure of aquaporins. In addition, there may be a reduction in the hydraulic conductivity due to the increase in the viscosity of water with lowering of temperature, which may affect water uptake, but this is usually small (Q10 of 1.25). Wan et al. (2001) have shown that low temperature (LT) significantly increased the resistance to water flow through the roots of aspen (Populus tremuloides Michx.) seedlings, and this increased resistance to water flow could not be fully explained by the corresponding increase in the viscosity of water. Raising the temperature back to 25oC after 22 h of low temperature (LT) treatment failed to restore the original rate of water transport through the main stem. Lee et al. (2004a) have shown that the half times and hydraulic conductivity of root (Lpr) of cucumber did not recover fully even after short-term exposure of root system to low temperature (LT). Confirmation that LT affects the architecture and function of aquaporins will require further studies. As observed here, H2O2 caused a reduction in the activity of H+-ATPases in isolated plasma membranes. However, the relationship of this to water channel activity is yet unclear, although the extrusion of protons

from cells should be linked to the uptake of nutrient ions.

4. D. Salt Stress

NaCl induces the membrane lipid peroxidation in seeds of the three salt treated species. The diverse responses of antioxidant enzyme activities to NaCl stress in seeds of M. sativa, M. officinalis and A. adsurgens suggested that oxidative stress could be an influential component of environmental stresses on plant seeds (Wang et al., 2009). Among wheat cultivars, level of Malondialdehyde (MDA) was increased under salinity in AlV and the salt sensitive but it was constant in the Sardari, a resistant cultivar (Esfandiari et al., 2007). Salt treatments (50 and 100 mM) were found to increase electrolyte leakage and malonyldialdehyde content of rosemary; however, this increase was greater at 0 µM than 1.0 µM Cu2+ (Mehrizi et al., 2012). However, drought stress induced free radical causes lipid per-oxidation and membrane deterioration in plants and it, also leads to an imbalance between antioxidant defenses and the amount of ROS resulting in oxidative stress (Van Breusegem et al., 2001). Lipid peroxidation is the introduction of a functional group containing two catenated oxygen atoms into unsaturated fatty acids in a free radical reaction (Dauqan et al., 2011). Furthermore, another oxidative enzyme, phospholipidhydroperoxide glutathione peroxidase (PHGPx) is over expressed in plants under abiotic stress conditions that mediate oxidative stress (Faltin et al., 2010). 4. D. I. Oxidant Roles in Salt Stresses

Current estimates indicate that 10-35% of the world’s agricultural land is now affected, with very significant areas becoming unusable each year. As pointed out for the drought stress response, also the plants suffering from salt-stress have to cope with the production of toxic oxygen species (Yokoi et al., 2002; Parida and Das, 2005). Therefore, the free radical scavenging and detoxification reactions are typical metabolic events for these plants. Moreover, they have to adjust the osmotic potential, which generally is achieved by accumulation of compatible osmolytes and osmoprotectant (Hasegawa et al., 2000; Bohnert et al., 1995). During environmental stress, the plant can lose control over this balancing. Loss

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of control often involves a combination of increasing AOS production and limited energy resources to replenish defense mechanisms, such as reductants for antioxidants, leading to these defenses being overwhelmed and ultimately resulting in AOS accumulation. An understanding of the targets of damage and their significance as points of AOS damage or as AOS sinks, will be essential to allow the breeding and possible engineering of plants to make them more robust in surviving exposure to environmental stress (Taylor et al., 2004). Much of the injury at cellular level caused by salinity stress is associated with oxidative damage due to ROS. Plants appear to possess a wide array of defense strategies to protect them from oxidative damage because oxidatively induced ROS oxidize biological macromolecules (Sharma et al., 2010). Plants under stress conditions, a strong oversupply of reduction equivalents are generated. The amount of secondary plant products were apparent in plants severely affected or grown under abiotic stress conditions than plants cultivated under optimal conditions of growth and productivity (Khan et al., 2011). For instance, high salt concentration causes osmotic and ionic stress in plants (Zhu, 2002). Salt tolerance is a complex trait involving the coordinated action of many gene families that perform a variety of functions such as control of water loss through stomata, ion sequestration, metabolic adjustment, osmotic adjustment and antioxidative defense (Abogadallah, 2010). It limits growth and development of plants by affecting several key metabolic processes (Hasegawa et al., 2000; Marschner, 2002; Siddiqui et al., 2009a; Khan et al., 2010). Salinity alters the activities of many enzymes involve in nitrate and sulphate (SO4

2-) assimilation pathways in plants, which lowers their energy status and increase the demand for nitrogen (N) and sulphur (S) (Siddiqui et al., 2009b). This hypothesis is supported by the enhanced expression of Leghaemoglobin (Lb) before active N2 fixation. Chung et al. (2008) demonstrated that increased ROS levels enhance the stability of the salt induced sodium over sensitive (SOS1) mRNA. This plasma membrane Na+/H+ antiporters interacts via its cytoplasmic tail domain with runaway cell death 1, revealing a new function for SOS1 within the oxidative stress response (Katiyar-Agarwal et al., 2006). The plasma

membrane slow anion channel-associated 1, a distant homolog of fungal and bacterial dicarboxylate/malic acid transport proteins, has been found to be essential for stomata closure during abiotic stress, including ozone stress, and to act as an essential subunit for S-type anion channel function or regulation (Vahisalu et al., 2008; Negi et al., 2008). These findings further elucidate the intricate networks of cellular processes that are modulated by ROS during abiotic stress. The zinc finger protein ZAT10was demonstrated to be a regulator of the abiotic stress response. Transgenic with enhanced or suppressed ZAT10 levels were more tolerant to multiple stresses (Mittler et al., 2006; Rossel et al., 2007). The enhancement in pungency due to salt stress could be explained through total flavour precursors (Methyl cysteine sulfoxide) were increased significantly in response to salt stress. The increased levels of free proline and soluble sugars were observed in transgenic plants compared to wild type plants under salt stress. Since, ROS readily oxidize methionine (Met) residues in proteins/peptides to form Met-R-sulfoxide or Met-S-sulfoxide, causing inactivation or malfunction of the proteins (Oh et al., 2010). Excess concentrations of salt stress are known to cause cellular oxidative damage. The production of glutathione-S-transferases (GST) under salt stress provide additional defense against oxidative stress and keep the metabolic activities in onion tissue functional. It could be noticed that exogenous application of antioxidant compounds (α-tocopherol) caused an inhibition of free radical generation, quench preformed free radicals and continuously enhance the activity of antioxidant enzymes such as glutathione S-transferase (GST). The essential for ROS detoxification during normal metabolism and particularly during stress, are antioxidant enzymes defense system (Khan et al., 2011).

4. E. Light Stress

Vanderauwera et al. (2011) found that the apx1/cat2 mutant was more tolerant to oxidative stress imposed by the superoxide-generating herbicide paraquat than Wild type (WT) or single mutants. The signaling pathway activated in apx1/cat2 therefore was functional against oxidative stress generated by at least three different treatments heat, high light (HL) and paraquat application). When subjected to high

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light (HL) under ambient conditions, cat2 and apx1/cat2 plants were smaller than wild type (WT) and apx1 plants, but the double mutants did not develop lesions. Dark-induced senescence of detached leaves and intact plants progressed more rapidly in the mutant compared with the wild type. Hydrogen peroxide, superoxide anion, oxidized lipid, and oxidized protein levels were all higher in the mutant. These results demonstrate that NOS1is a mitochondrial NOS that reduces ROS levels, mitigates oxidative damage, and acts as an antisenescence agent (Guo and Crawford, 2005). Blue copper binding protein gene was also induced by wilting and UV, and its expression upregulated in senescent leaves. Genes encoding blue copper-binding proteins have been shown previously to respond to abiotic stresses such as drought and ozone (Cho, 1997). An NADPH oxidase, a ROP, and the Rop regulatory protein Rop GTPase-activating protein 4 are involved in the plant’s response to oxidative stress induced by flooding (Baxter-Burrell et al., 2002). Among the genes induced by H2O2 was one encoding a blue copper binding protein, such proteins might function to sequester copper, a potentially toxic element that is also an essential cellular catalyst for redox reactions. Expression increased during senescence, in which copper sequestration is an important event (Himelbau and Amasino, 2000). Genes Analysis of the 1.1-kb 5'-upstream region of all the oxidative stress induced genes did not reveal the presence of a known binding site common to them all. However, 5'-upstream regions were identified in the H2O2-induced genes that are potential binding sites for redox-sensitive transcription factors. These included binding sites for myb (Myrset et al., 1993), Ocs/AS-1-like elements (that are present in SA- and auxin-induced genes; Qin et al., 1994) and AP-1 (Abate et al., 1990). Such redox-sensitive motifs have previously been identified in H2O2-induced genes such as tcI7 (Etienne et al., 2000) and GST6 (Chen et al., 1996). However, the identification of potential binding sites for redox-sensitive transcription factors is yet merely an observation.

4. E. I. Darkness Roles on Tissue Generated Ros

The correlation of extended darkness transcripts expression to database is shown as a color-coded heat map of correlation values. The

extended darkness data (2, 3, and 5 days under darkness) were normalized either to non-darkened tissue (d2/d0, d3/d0 and d5/d0) or to the value of the previous day (d3/d2 and d5/d3). The former comparison emphasizes the transition from light to dark, while the latter comparison indicates continuing changes in ROS signals during extended darkness (Rosenwasser et al., 2011). Inspection of the results for d (2-5)/d0 comparisons showed that high correlation values were obtained to AOX-MLD mitochondrial profile (0.51, 0.51, and 0.45) and rotenone mitochondrial profile (0.33, 0.36, and 0.33) on the second, third, and fifth day of darkness, respectively. Similarly, high correlation values were obtained with the peroxisomal profiles of aminotriazole (AT) (0.30, 0.31, 0.25), and CAT2HP1 high light (0.21, 0.38, 0.29). In contrast, a negative correlation was found to the singlet oxygen generated in the flu mutant (30 min) signature (-0.35,-0.20,-0.16) (Rosenwasser et al., 2011). These results suggested an increase in ROS-related stress signals emanating particularly from the mitochondria and the peroxisomes and a decrease in signal-related singlet oxygen from the chloroplasts. The transcriptome analysis of ROS-related genes suggested the existence of specific subcellular signatures during the dark treatment. It was of interest to examine to what degree these signatures might be reflected in actual measurable redox changes in the cell organelles (Rosenwasser et al., 2011). Changes in the glutathione redox potential are useful real-time indicator of oxidative stress (Meyer, 2008).To determine the redox changes in specific cellular compartments during darkness, we measured the degree of oxidation of the sensitive probes (roGFP) in Arabidopsis transgenic lines which included probes localized in the mitochondria (mit-roGFP1, mit-roGFP2), plastids (pla-roGFP2), cytoplasm (cyt-GRX1-roGFP2) and peroxisomes (per-GRX1-roGFP2). Both roGFP1 and roGFP2 were found to be suitable to monitor the glutathione redox potential and display different degrees of oxidation at the steady state due to their different mid-point redox potentials (Schwarzlander et al., 2008; Rosenwasser et al., 2009). Quantitative assessment provided by direct fluorometric measurements was limited to 3 days of darkness treatment, as longer periods were found to induce high-levels of autofluorescence

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of unknown origin at 405 nm that obviates measurements (Rosenwasser et al., 2009). Sugar deprivation occurs rapidly during extended night (UsaDelet al., 2008). Since roGFP oxidation occurred as early as the first day of darkness, it was of interest to examine the effect of sucrose addition on mit-roGFP2 oxidation. Where, addition of sucrose to detach rosettes during the dark treatment resulted in a degree of oxidation that was similar to control levels prior to darkness. In contrast, the addition of equimolar concentration of sorbitol showed an increase in mitochondrial roGFP oxidation (Rosenwasser et al., 2011). Supplementing the media with sucrose alleviated the increase in mit-roGFP oxidation. Additional support for the role of sugar starvation in the increase of ROS-related events comes from the analysis of transcriptome data obtained from starch-accumulation defective Phosphoglucomutase (pgm) mutant. This mutant was shown to have lower sucrose levels during the night than wild type plants (Blasing et al., 2005). The expression of general oxidative stress transcripts was extracted from the data ofPhosphoglucomutase (pgm) mutant at the ‘end of night’ (12 h darkness). The majority of the general ROS induced transcripts were found to be upregulated at the end of night in Phosphoglucomutase (pgm) but not in the wild type (WT) plants. An increase in these specific transcripts also occurred in WT, but only following 48 h after darkness. These results suggested that oxidative stress could be related to sugar depletion in this mutant. Moreover, when vector correlation analysis was applied to the pgm data generated above, high correlations of 0.45 to 12 h rotenone and 0.5 to the AOX-MLD were found. Since the transcriptomes of both treatments originate from mitochondrial stress, the results are consistent with the pgm mutation having an impact on mitochondrial activity. It can be concluded that mitochondrial ROS stress early during darkness can be ascribed to the lack of an energy supply (Rosenwasser et al., 2011). An increase in H2O2 and ●O2 content has been demonstrated in isolated mitochondria from pea 11 days after the initiation of the dark treatment (Jimenez et al., 1998). However, these events occurred after deterioration events were already prominent. In contrast, the changes reported here occurred before

measurable cell death and as early as the first day of darkness. An increase in mitochondrial oxidation and in transcripts related to mitochondrial ROS signals early during darkness may result from insufficient antioxidant capacity, although this was not the case at least for the antioxidant related genes. However, the observed decrease in mitochondrial oxidation level caused by sucrose has linked sugar deprivation to dark-generated ROS (Rosenwasser et al., 2011). Similarly, reduced electron transfer through the cytochrome-c-oxidase step because of sugar deprivation resulted in higher production of ROS at the level of mitochondria (Couee et al., 2006). This scenario is also consistent with the analysis of the Phosphoglucomutase (pgm) mutant, in which depletion of sugars occurs as early as the ‘end of the night’. In this case, it was revealed that the general markers of oxidative stress were expressed in the mutant earlier than in Wild Type plants. 4. E. II. High Light Intensity

High light (HL) irradiance is also an elicitor of gene expression responses, and this system has been well characterized in terms of the associated metabolic changes, including those that involve redox-reactive compounds (Noctor and Foyer, 1998). The transcriptional activity of all genes investigated was increased during the HL treatment when compared with the GL controls. The observation of a global activation of transcription in response to HL is in line with a possible stress-related signaling within the chloroplast. The psbA and trnQ promoters, despite quantitative differences, PTK seems capable of affecting the transcription of more than a single class of plastid genes. This would be consistent with a global up-regulation of transcription under HL conditions in vivo, when PTK is inhibited by elevated GSH levels (Baena-Gonzalez et al., 2001). High light (HL) intensity accelerated turnover rates of photosynthetic reaction center proteins (Aro et al., 1993). Usage of specific inhibitors of photosynthetic electron transport such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea has provided further support for the notion that chloroplast gene expression is under redox control (Trebst, 1980). The transcription rate of chloroplast genes were affected by the spectral quality of light photosystem I versus photosystem II excitation (Deng et al., 1989; Pfannschmidt et

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al., 1999) and electron transfer inhibitors (Pfannschmidt et al., 1999). Mustard seedlings were exposed to 1,000 (HL) or only 50 growth light (GL) µMol photons m-2 s-1 for 3 h. Chloroplasts were then isolated, and the GSH concentrations and GSH to oxidized GSH (GSSG) ratios were determined. As judged from the equal chlorophyll (chl) content present in the HL and GL plastids on a similar plastid number basis, this treatment did not seem to result in appreciable photo-oxidative damage in the cotyledons. Exposure to high irradiance resulted in a significant change in the GSH/GSSG ratio in chloroplasts, which was more than 3-fold higher upon the HL treatment of plants than in the GL conditions (Baena-Gonzalez et al., 2001). The light intensity dependent changes in the plastid GSH/GSSG ratio were accompanied by changes in the transcriptional activity. Isolated chloroplasts from seedlings exposed for 3 h either to growth light (GL) or high light (HL) illumination and carried out in organelle run-on transcription in the presence of radio labeled UTP. Chloroplast isolation typically yielded up to 90% intact plastids. Labeled transcripts were isolated and hybridized against chloroplast genes. These data indicate a relative increase in transcriptional activity in HL- versus GL treated seedlings for all the genes that were tested (Baena-Gonzalez et al., 2001). It is interesting that HL effect did not appear to be specific for photosynthesis-related genes because a similar induction of transcription was observed also for the 16S rRNA gene and for tRNA genes (trnS and trnG). The polypeptide composition of the Plastid Transcriptional Kinase (PTK) preparations from High light (HL) as compared with Growth light (GL) seedlings was very similar, the only difference being the accumulation of an about 94-kD protein in HL-treated seedlings. Phosphorylation activity, however, could only be observed in the GL preparation with no radioactive signal in the HL sample, even in overexposed films (Baena-Gonzalez et al., 2001). In the growth light (GL) sample, a single band was phosphorylated by the endogenous kinase following incubation of the preparation with labeled ATP. The size range of this band at approximately 72 to 76 KD matches that of the polymerase subunits previously shown to be preferred PTK substrates in highly (glycerol gradient-) purified kinase-polymerase complexes (Baginsky et al., 1999). The apparent lack of

32P-incorporation into the high light (HL) sample suggested a lower or entirely lost PTK activity relative to that in the GL sample. However, an alternative explanation for this result was that the HL proteins might have existed e.g. in a more highly phosphorylated form prior to the labeling reaction and therefore were not accessible to in vitro phosphorylation by PTK. A labeled 72- to 76-kD band was generated in both, GL and HL samples following phosphorylation by the heterologous kinase. Although the intensity of this band was lower in the HL lane, its presence indicated that phosphorylation sites were accessible in the HL sample. Hence, the absence of labeled products in the HL auto phosphorylation assay seemed to reflect inactivation of PTK as result of the HL treatment of seedlings (Baena-Gonzalez et al., 2001). In addition to the band at (72 to 76 KD) several other labeled bands were visible following phosphorylation by heterologous CK2. A number of them had about equal intensity in both the HL and GL samples (at proximately 35 and 20 KD), whereas others revealed reduced intensity in either the GL (at 65 and 29 KD) or HL samples (at 62 and 30 KD). These additional bands indicate a more stringent substrate specificity of the endogenous PTK as compared with the heterologous CK2 under the experimental conditions used here. The (differentially labeled) extra bands generated by CK2 may represent additional targets for phosphorylation control in vivo. 4. E. III. Photorespiration

The stress induced increase in leaf membrane damage, reduced uptake of CO2 as result of stomata closure, decreased hydrolytic enzyme activity and increased lipid peroxidation level; it may stimulate formation of AOS such as superoxide, hydrogen peroxide, and hydroxyl radicals. Among AOS, superoxide is converted by SOD enzyme into H2O2, which is further scavenged by Catalase (CAT) and various peroxidases. Ascorbate Peroxidase (APOX) and Glutatione Rductase (GR) also play a key role by reducing H2O2 to water through the Halliwell-Asada pathway (Noctor and Foyer, 1998). Plants suffer from drought and salt stress lead to stomata closure and an enhanced CO2 deficiency that generate a high oversupply of reduction equivalents. In order to prevent damage by oxygen radicals (●OH−, ●O2

−, 1O2 and H2O2), a massive amounts of NADPH+ is reoxidized by photorespiration and viola-

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xanthine cycle or xanthophylls cycle under such stress conditions. However, the corresponding strong reduction power or the markedly higher concentration of reduction equivalent seems to enhance the stronger rate of synthesis of highly reduced compound, like isoprenoids, phenols (Flavonoids) or alkaloids (Khan et al., 2011). Consequently, the synthesis and accumulation of highly reduced secondary plant products (SPP) reveals a meaning within the metabolism to prevent too massive production of ROS and the corresponding damage by photo-inhibition. Taylor et al. (2004) suggested that a slowing of both photorespiration and nitrogen assimilation during environmental stress that will have immediate direct effects on photosynthetic rate and efficiency and longer-term implications for plant nutrition. Further, the replenishment of the cellular antioxidant machinery is driven by reductants in the form of NAD (P)H and the availability of amino acids for glutathione synthesis. Deficits in all of these areas are common features of the phenotype of environmentally stressed plants (Vierling and Kimpel, 1992; Bohnert and Sheveleva, 1998; Dat et al., 2000). In order to prevent damage by ROS, NADPH+H+ is reoxidized by photorespiration or violaxanthine cycle. Yet, the higher concentration of reduction equivalents also precedes to a stronger rate of synthesis of highly reduced secondary plant products i.e. isoflavones, isoprenoids, phenols or alkaloids and phytosterols (Khan et al., 2011). The generation of ROS in plants occurred under oxidative stress at different location of the plant cell (Mitochondria, Chloroplast, Peroxisome and Nucleus), causes injury and cell death (Mano, 2002). 4. E. IV. Chlorophyll Destructions

Reactive oxygen species (ROS) are inevitable by-products of metabolism in all aerobic organisms (Girard and Boiteux, 1997). Plants and algae are especially prone to photo-oxidative stress because of ROS generated during oxygenic photosynthesis. Several types of ROS are generated at various sites in the photosynthetic electron transport chain in chloroplasts, and their production is enhanced by such factors as excess or varying light intensities and extremes of temperature, drought, nutrient deficiencies, and herbicides. These ROS can damage many chloroplast constituents, including lipids, proteins, pigments,

and the multi copy genome. Plants have evolved numerous mechanisms to deal with photooxidative stress, including dissipation of excess light energy, synthesis of antioxidant molecules and scavenging enzymes, and targeted repair (Niyogi, 1999). In transgenic tobacco (Nicotiana tabacum) plants expressing alfalfa (Medicago sativa) hemoglobin (a NO-scavenging enzyme), ROS levels are higher than in control plants during bacterial infection, indicating that NO normally suppresses ROS accumulation (Seregelyes et al., 2003). NO donors counteract photooxidative damage during treatment with methyl viologen herbicides by reducing H2O2,

●O2, and ●OH radical levels and slowing ion leakage, protein and lipid oxidation, loss of chlorophyll, and protein degradation (Beligni and Lamattina, 2002). Inhibition and rapid turnover of the D1 protein in chloroplasts in response to a wide range of environmental stresses (Aro et al., 1993), and of glycine decarboxylase (GDC) to herbicide application, drought and low temperatures (Taylor et al., 2002), further suggested that different stresses can have similar effects on specific molecular targets in chloroplast. The identification of such common induction responses and common damage sites has been the focus of much research to date (Dat et al., 2000; Mittler, 2002; Pastori and Foyer, 2002). Decrease in the chlorophyll (a) content, which happens under salinity could be due to stimulation of proline biosynthesis. It is demonstrated that glutamate is chlorophyll precursor in higher plants and during salinity a part of glutamate converts to proline that cause to decline input of this compound to chlorophyll biosynthesis pathway (Heath et al., 1968). On the other hand, it seems that reduce in chlorophyll a and chlorophyll a/b ratio under salinity could be related to decreasing K+ concentration in the leaf. Alikhani et al., (2011) showed that by supplementing of the media with high concentration of potassium decreased negative effect of salinity stress on chlorophyll content. In this regard, it found that chlorophyll degradation was improved when plant exposed to extreme salinity treatments while when salt stress applied in gradual chlorophyll degradation did not accrued (Luna et al., 2002; Yildirim et al, 2008). Increased osmotic potential and Na+ access into organelles creates damage in respiratory and photosynthetic electron transport (Allakhverdiev et al., 1999). Subsequently, plants cannot use effectively from absorbed light

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energy under stress and it is suggested that reducing photosynthetic pigments is a stress adaptation response (Demmig et al., 1996; Poormohmmad Kiani et al., 2008). Oxidative stresses occurs when there is a serious imbalance in any cell compartment between production of reactive oxygen species (ROS) and antioxidant defense, leading to damage. Oxidative stress is a central factor in abiotic and biotic stress phenomena (Halliwell and Gutteridge, 1999). In some cases ROS are necessary for inter and intracellular signaling, however, at high concentration can cause damage at various levels of organization including chloroplast (Smirnoff, 1993). Apart from morphological structures contributing to drought stress tolerance, plants have evolved a variety of physiological and biochemical processes which act as components of drought tolerance (Ren et al., 2007; Wang et al., 2007). Consequently, plants react with partial stomata closure. As results of this compromise between maximization of photosynthesis and minimization of water loss, the imbalance between the Delivery of reduction equivalents of nicotinamide-adenine dinucleotide phosphate (NADPH+H+) by photosynthetic electron transport chain and their consumption by CO2

fixation via Calvin cycle (C3 cycle or Calvin-Malvin cycle) is ever higher than under normal CO2-limiting conditions (Khan et al., 2011). It was evident from the present results that the genotypes (Co85004 and C 92038) which possess highest total chlorophyll and carotenoids content under salinity conditions signifying lower pigment bleaching and favors better adaptation under saline condition (Gomathi and Rakkiyapan, 2011). 4. E. V. UV Cell Division

Although it is well established that plant seeds treated with high doses of gamma radiation, arrest development as seedlings, the cause of this arrest is unknown. The uvh1 mutant of Arabidopsis is defective in a homolog of the human repair endonuclease XPF, and uvh1 mutants are sensitive to both the toxic effects of UV and the cytostatic effects of gamma radiation (Preuss and Britt, 2003). They found that gamma irradiation of uvh1 plants specifically triggers a G2-phase cell cycle arrest. Mutants, termed suppressor of gamma (SOG), that suppress this radiation-induced arrest and proceed through the cell cycle unimpeded were

recovered in the uvh1 background; the resulting irradiated plants are genetically unstable. The SOG mutations fall into two complementation groups. They are second-site suppressors of the uvh1 mutant’s sensitivity to gamma radiation but do not affect the susceptibility of the plant to UV radiation. In addition to rendering the plants resistant to the growth inhibitory effects of gamma radiation, the sog1 mutation affects the proper development of the pollen tetrad, suggesting that SOG1 might also play a role in the regulation of cell cycle progression during meiosis (Preuss and Britt, 2003).

4. E. VI. Cadmium Stress

4. E. VI. 1. Ros Production by Cadmium

Red ●O2

− dependent fluorescence of dihydroethidium (DHE) was not visible in leaves from control plants, but in Cd-treated plants, red fluorescence was observed mainly in xylem vessels and adaxial sclerenchyma, epidermis, stomata, and mesophyll cells. The pre-incubation of leaf sections with 2, 2, 6, 6-tetramethylpiperidinooxy (TMP), ●O2

− scavenger, abolished completely the fluorescence, thus showing the specificity of the fluorescent probe dihydroethidium (DHE) for the ●O2

−. To investigate if the Cd-dependent ●O2

− production was related to the Ca deficiency previously observed, ●O2

− was also imaged in Ca-supplemented plants. Ca prevented the accumulation of superoxide radicals induced by Cd in mesophyll cells but not in epidermis and vascular tissues. In these tissues, ●O2

− -dependent fluorescence was even higher in control plants, which suggests a possible involvement of Ca in ROS production, especially in the xylem vessels (Rodriguze-Serrano et al., 2009). Cross sections of control and Cd-treated pea leaves incubated with 2', 7'-Dichlorofluorescein Diacetate (DCF-DA) showed a very bright green fluorescence due to peroxides, mainly to H2O2, in xylem vessels from vascular tissues and epidermis. However, in Cd-treated leaves, the fluorescence increased considerably in palisade mesophyll cells and, to a lesser extent, in sclerenchyma cells. When pea leaf sections were incubated with 1 mM ascorbate, a peroxide scavenger, the fluorescence was considerably reduced. Some of the fluorescence due to 2',7'-Dichlorofluorescein Diacetate (DCF-DA) and dihydroethidium (DHE) in mesophyll cells

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overlapped with the tissue auto fluorescence, which indicates that part of the ROS production observed in leaf sections could be associated with chloroplasts, although some fluorescence punctuates, different from chloroplasts, can also be observed in the cytoplasm. A higher magnification of the images shows fluorescence in chloroplasts and spherical organelles, which could be peroxisomes or mitochondria. In the sclerenchyma, the production of both ROS was mainly located in the apoplast, especially in the intercellular space connecting cells. The analysis of H2O2 by cytochemistry with CeCl3 and electron microscopy showed that in xylem vessels from control plants, the H2O2-dependent precipitates were found on the inner side of cell wall (Rodriguze-Serrano et al., 2009).

A Cd-dependent accumulation of peroxides has also been observed in alfalfa (Medicago sativa) roots (Ortega-Villasante et al., 2005) and pea roots (Rodriguez-Serrano et al., 2006) using 2',7'-Dichlorofluorescein Diacetate (DCF-DA) and CLSM, and overproduction of ●O2

− was also observed using DHE in Lupinus luteus roots (Kopyra and Gwozdz, 2003). The analysis of H2O2 in pea leaf sections by DCF-DA fluorescence showed an induction of this ROS production by Cd mainly in mesophyll cells, probably associated with chloroplasts, mitochondria, and peroxisomes, and in plasma membrane from epidermal cells. In a previous work, using CeCl3 cytochemistry, the Cd-dependent accumulation of H2O2 in peroxisomes, mitochondria, and plasma membrane was demonstrated, with NADPH oxidase being the main source of ROS in the plasma membrane (Romero-Puertas et al., 2004). An oxidative burst has also been associated with Cd toxicity in tobacco (Nicotiana tabacum) cell suspensions, with a NADPH oxidase being involved (Olmos et al., 2003; Garnier et al., 2006). The highest fluorescence detected was localized in the cell wall of the xylem vessels. Similar results have been observed in pea roots, where the highest ROS production was associated with the vascular tissue (Rodriguez-Serrano et al., 2006). In different plant species, ROS production has been associated with cell wall lignifications in the xylem (Ogawa et al., 1997; Ros-Barcelo, 1999). The production of ROS in vascular tissues could serve as a signal under stress condition, as has been proposed in wounding damage (Orozco-Cardenas and Ryan, 1999).

ROS overproduction was partially due to the Ca deficiency induced by Cd treatment to judge by the negative effect of Ca supply on ●O2

− production. Thus, exogenous Ca supply reversed the Cd-treated phenotype, getting similar results to those obtained in control plants grown in the presence of Ca, reducing considerably the Cd-dependent ●O2

− production in mesophyll cells. However, in control plants, Ca produced a slight increase of ●O2

−

accumulation in xylem vessels and epidermis, which can be explained by the NADPH oxidase activation by Ca. These results suggest that other sources different from NADPH oxidase can be involved in the Cd-dependent ROS production in mesophyll cells, such as peroxidases in the plasma membrane (Choi et al., 2007), the electron transport chain in mitochondria (Romero-Puertas et al., 2004; Garnier et al., 2006), glycolate oxidase in peroxisomes (Romero-Puertas et al., 2004), and chloroplasts. The protecting role of Ca can be also explained by the up-regulation of antioxidants such as CuZn-SOD (Azpilicueta et al., 2007). In Arabidopsis plants, however, JA regulates genes involved in glutathione and phytochelatins synthesis under Cd treatment (Xiang and Oliver, 1998). JA is a component of the signaling processes under biotic and abiotic stresses (Devoto and Turner, 2005). Wu and Bradford (2003) demonstrated that Ethylene (ET) and Jasmonic acid JA in tomato leaves regulate Chitinases. Additionally, Rakwal et al. (2004) reported a regulation by Ethylene (ET), Jasmonic acid (JA), and ROS in rice plants. PrP4A is a hevein-related protein that binds chitin, can inhibit the growth of fungus, and belongs to chitinase I and II classes (Broekaert et al., 1990). Chitinases catalyze the hydrolytic cleavage of the β-1,4-glycoside bond of GlcNAc and is considered a defense mechanism against pathogens (Kasprzewska, 2003). An induction of chitinase activity has been observed in pea plants by Cd (Metwally et al., 2003) and other heavy metals like lead and arsenic (Bekesiova et al., 2008) and also by osmotic stress (Tateishi et al., 2001), low temperature (Stressmann et al., 2004), and wounding (Wu and Bradford, 2003). Chitinases are probably components of the general defense response program of cells, although they can also play unknown specific roles in heavy metal stress. Thus, transgenic plants expressing fungal Chitinases showed enhanced tolerance to metals (Dana et al.,

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2006), and Chitinases isoforms are differentially modified by different metals (Bekesiova et al., 2008). The gene induced by Cd corresponds to the cytosolic heat shock protein, HSP71.2, which is known to be up-regulated under heat stress; its function is to act as a molecular chaperone facilitating protein transport into organelles (DeRocher and Vierling, 1995) or preventing protein aggregation (Ma et al., 2006). The induction of Heat shock Protein (HSPs) in pea plants under Cd treatment is apparently regulated by H2O2 overproduction, since ascorbate reversed the up-regulation induced by Cd; in addition to that, transcription factors involved in HSPs regulation can act as H2O2 sensors (Miller and Mittler, 2006). In Arabidopsis plants, the regulation of Heat Shock Potein (HSP71.2) by JA has also been described (Cheong et al., 2002). The inducible nature of peroxisomal metabolism is the light-induced transition of glyoxysomes, the specialized peroxisomes of oilseeds, to leaf type peroxisomes (Masters and Crane, 1995; Mullen and Trelease, 1996). Likewise, during plant senescence and under the effect of abiotic stress by cadmium the reverse process is observed, that is the metabolic conversion of leaf peroxisomes into glyoxysomes (De Bellis et al., 1990; Landolt and Matile, 1990; Nishimura et al., 1993). In these metabolic transitions of peroxisomes, endogenous proteases could be involved in the turnover of peroxisomal proteins. In plant peroxisomes, the presence of exo and endo proteolytic activity was reported for the first time in pea leaves, and an exo peptidase was characterized as a leucine amino peptidase, belonging to the family of the serine proteinases (Corpas et al., 1993). Total endo-proteolytic activity and in the number of endoproteases isoenzymes was found in peroxisomes purified from senescent pea leaves in comparison with peroxisomes from young leaves (Distefano et al., 1999). In pea plants grown with cadmium an increase in the activity of four endoproteases of leaf peroxisomes was recently found (McCarthy et al., 2001). In general, there are data that suggest the involvement of peroxisomal endoproteases in a regulated modification of proteins in this organelle (Distefano et al., 1999). In recent years, different experimental evidence has suggested the existence of cellular functions for leaf peroxisomes related to reactive oxygen species. These ROS-related roles add to the other well-established functions known for peroxisomes from plant cells. In this review, the

production of reactive oxygen species (ROS), the different antioxidant systems and the generation of nitric oxide in peroxisomes will be analyzed in the context of these new ROS mediated functions of plant peroxisomes (Del Rio et al., 2002).

4. E. VI. 2. Cadmium Induces Hormone

Biosynthesis

The stimulation of ethylene (ET) biosynthesis by Cd has been reported in different plant species, although the molecular relationship between ET biosynthesis and Cd stress has not been well-established (Sanitadi Toppi and Gabbrielli, 1999). Jasmonates (JA) is a component of the signaling processes under biotic and abiotic stresses (Devoto and Turner, 2005). Under Cd stress, an increase of two times in methyl jasmonate (MeJA) took place in pea leaves, and free JA was detected neither in control nor in Cd-treated plants. The analysis of SA content shows that free SA was the main form present in pea leaves. On the contrary, Cd treatment did not produce any statistically significant effect on the SA levels, although the contents of conjugated (methyl salicylate [MeSA]) and free SA were slightly reduced in Cd-treated plants. Analysis of ET by GC showed an increase of two times in leaves from pea plants grown with 50 µM CdCl2, and this increase was reversed by supplying Ca to the nutrient solution, although a slight increase of ET emission was also observed in control plants (Rodriguze-Serrano et al., 2009). Salicylic acid (SA) plays an important role in signal transduction pathways, being involved in the induction of the hypersensitive response (Alvarez, 2000). However, the results obtained in this work suggest that, under Cd toxicity, SA is not involved in the cellular response in leaves, although in pea roots, different results were obtained (Rodriguez-Serrano et al., 2006). SA alleviates Cd toxicity in barley (Hordeum vulgare) roots, but the mechanisms involved are not well known (Metwally et al., 2003). The analysis of peroxidase (PR) expression in pea plants under Cd stress showed the up-regulation of Chitinases, PrP4A, and HSP71.2, while Phenyl Amonia Lyase (PAL) did not change.

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4. E. VI. 3. Minerals Competing Cadmium

Absorption

Higher concentrations have been used to study transcriptome effects of H2O2, though most of the added H2O2 is metabolized by cells within a few minutes (Desikan et al., 2001). Longer-term maintenance of H2O2 concentrations in the millimolar range would require continuous generation and perhaps decreased activity of the antioxidative system. A concentration as high as 10 mM H2O2, distributed uniformly in the apoplast, would contribute around 1 µMol g-1 FW, assuming that the apoplastic free space is about 10% of total volume (Winter et al., 1993; Fleischer and Ehwald, 1995). The cellular production of reactive oxygen species (ROS) in leaves from pea (Pisum sativum) plants under Cd stress has been reported (Romero-Puertas et al., 2004). Cd was proved toproduce disturbances in the plant antioxidant defenses, producing an oxidative stress (Rodriguez-Serrano et al., 2006; Romero-Puertas et al., 2007). Cadmium (Cd) is a toxic element whose presence in the environment is mainly due to industrial processes and phosphate fertilizers and then is transferred to the food chain (Pinto et al., 2004). Cd is rapidly taken up by plant roots and can be loaded into the xylem for its transport into leaves. Most plants sensitive to low Cd concentrations reveal plant growth inhibitions. As consequence of alterations in the photosynthesis rate and the uptake and distribution of macronutrients and micronutrients (Lozano-Rodriguez et al., 1997; Sandalio et al., 2001; Benavides et al., 2005). The content of polyvalent cations can be affected by the presence of Cd through competition for binding sites of proteins or transporters (Gussarson et al., 1996). Therefore, Cd produced a decrease of calcium (Ca) content in different plant species (Sandalio et al., 2001). Kinraide et al. (2004) found that Cd induced a strong reduction in the Ca content of leaves (Sandalio et al., 2001). Ca is an important signaling component in biotic and abiotic stresses, and disturbances in its content have been associated with toxicity by Cd, zinc (Zn), copper (Cu), or aluminum (Al). Although the mechanisms involved are not well known. Growth with 50 µM CdCl2 produced a decrease in the contents of Ca, Cu, Fe, Mn, and Zn in pea leaves (Rodriguze-Serrano et al., 2009). On the contrary, sulfur was accumulated 3-fold in Cd-treated plants with respect to the control plants.

The induction of sulfur metabolism by Cd was previously described; and involves a coordinated transcriptional regulation of genes for sulfate uptake and its assimilation. Cd is well known to produce disturbances in both the uptake and distribution of elements in pea plants (Hernandez et al., 1998; Sandalio et al., 2001; Tsyganov et al., 2007) and other plant species (Gussarson et al., 1996; Rogers et al., 2000). Analytical studies by inductively coupled plasma optical emission spectrometry demonstrated that after 24 h treatment of rooted potato cuttings with 60µM Cd2+, the accumulation of the heavy metal is 9-fold higher in the roots than in the leaves. This lower accumulation may explain the absence of Cd2+ genotoxicity in leaves. The role of thiol-rich phytochelatins in binding Cd and accumulating it within vacuolar compartments of root cells is well known and perhaps responsible for the high Cd content in roots (Sanita di Toppi and Gabbrielli, 1999). Cd2+ generates various reactive oxygen species (ROS) in plant cells, including H2O2 (Olmos et al., 2003).

4. E. VI. 4. Gene Regulations

Studies of gene regulation under different stress conditions are important to get deeper insights into the regulation of defenses involved in each particular condition and the cross talk processes occurring in different stress conditions. Some PRs have been described to be upregulated under abiotic stress (Tateishi et al., 2001; Stressmann et al., 2004), which suggests the existence of common effectors with biotic stress. The analysis of the expression of PRs and HSP71.2 (for heat shock protein 71.2) in pea plants treated with Cd was carried out by semi quantitative RT-PCR. Cd treatment is upregulated chitinase, PrP4A, and HSP71.2, while the expression of PAL (Phe ammonia-lyase) transcripts did not change significantly with the treatment. The induction of PrP4A and HSP71.2 was reverted by the supply of ascorbate, a H2O2 scavenger, which suggests that both genes are at least partially regulated by ROS. However, Ca did not change the expression level of PrP4A in both control and Cd treated plants (Rodriguze-Serrano et al., 2009). FISH results showed an intense hybridization signal in pea leaves of Cd-treated plants, in contrast to control plants, where a very faint signal was observed. No hybridization signal was observed in control experiments with the sense probe. PrP4A expression was observed in

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mesophyll cells, especially in palisade cells from Cd-treated pea plants; the hybridization signal was localized in the cytoplasm, with the nuclei (visualized by DAPI with blue fluorescence) being free of signal. However, the signal was absent in epidermis and xylem tissue, although a weak/low FISH signal was observed in stomata and in parenchyma cells surrounding xylem vessels (Rodriguze-Serrano et al., 2009). The NO synthase-dependent NO production was strongly depressed by Cd, and treatment with Ca prevented this effect. Under these conditions, the pathogen-related proteins PrP4A and chitinase and the heat shock protein 71.2, were up-regulated, probably to protect cells against damages induced by Cd. The regulation of these proteins could be mediated by jasmonic acid and ethylene, whose contents increased by Cd treatment. A model is proposed for the cellular response to long-term Cd exposure consisting of cross talk between Ca, ROS, and NO. 4. F. Herbicides Impacts

Methylviologen compounds are normally used in agronomy as herbicides. They cause an overproduction of reactive oxygen species (ROS) within chloroplasts, subjecting the plant to a severe oxidative stress. An Arabidopsis gene probe for ASC6 was used because this exhibits the highest homology to the stress responsive NtACS2 gene (Lei et al., 2000). SA can act by influencing the generation of ROS to which C2H4 could act to augment (De Jong et al., 2002) or increase production in response to oxidative stress, likely in a positive feedback loop. Injections of either 1 mM methylviologen or Glc: Glc oxidase (G: GO) elevated ethylene production. Hence, the observed patterns of ethylene production are likely to be linked to the well-established H2O2-SA interaction during defense (Lamb and Dixon, 1997). Since, nitric oxide (NO) is a bioactive ROS scavenger, its effect over some toxic processes caused by the methylviologen diquat and paraquat in potato leaves (Solanum tuberosum L. cv. Pampeana). Three NO donors, (i) sodium nitroprusside (SNP), (ii) S-nitroso-N-acetylpenicillamine, and (iii) a mixed solution of ascorbic acid and NaNO2, were able to prevent chlorophyll loss. Residual products from NO generation and decomposition failed to prevent chlorophyll decline and a specific NO scavenger, carboxy-PTIO, arrested NO-mediated chlorophyll protection. Dichlorophenyldimethylurea, an

inhibitor of chloroplastic electron transport, mimicked NO-mediated chlorophyll protection. During oxidative stress, cell ion leakage to intercellular compartments occurs as an early step, leading to a special kind of programmed cell death. NO was proved to specifically decrease the extent of ion leakage originated by diquat, since the protection originated by 100 µM SNP was completely arrested by carboxy-PTIO. These results suggest that NO can strongly protect plants from methylviologen damage and strengthen the evidence in favor of NO as a potent antioxidant in some situations (Beligni and Lamattina, 1999).

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Wang X., G. Zhao and H. Gu (2009). Physiological and antioxidant responses of three leguminous species to saline environment during seed germination stage. African Journal of Biotechnology Vol. 8 (21), pp. 5773-5779. Wang Y. and E. Floor (1998). Hydrogen peroxide inhibits the vacuolar H+-ATPase in brain synaptic vesicles at micromolar concentrations. Journal of Neurochemistry 70, 646-652. Willekens H., S. Chamnongpol, M. Davey, M. Schraudner, C. Langebartels and M. Van Montagu et al. (1997). Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants. EMBO J. 16: 4806-4816. Winter H., G. Robinson and H. W. Heldt (1993). Subcellular volumes and metabolite concentrations in barley leaves. Planta 191, 180-190. Wood Z., E. Schröder, J. Robin Harris and L. Poole (2003). Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28, 1: 32–40. Wu C. T. and K. J. Bradford (2003) Class I chitinase and b-1-3-glucanase are differentially regulated by wounding, methyl jasmonate, ethylene, and gibberellin in tomato seeds and leaves. Plant Physiol 113: 263-273. Xin Z. and J. Browse (1998). eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proc Natl Acad. Sci. USA 95: 7799-7804. Xin Z. and J. Browse (2000). Cold comfort farm: the acclimation of plants to freezing temperature. Plant Cell Environ 23: 893-902. Yano H., J. H. Wong, M. J. Cho and B. B. Buchanan (2001). Redox changes accompanying the degradation of seed storage proteins in germinating rice. Yazdanpanah S., A. Baghizadeh and F. Abbassi (2011). The interaction between drought stress and salicylic and ascorbic acids on some biochemical characteristics of Satureja hortensis. African Journal of Agricultural Research. 6, 4: 798-807.

Yildirim E., M. Turan and I. Guvenc (2008). Effect of foliar salicylic acid applications on growth, chlorophyll and mineral content of cucumber (Cucumis sativus L.) grown under salt stress. Journal of Plant Nutrition, 31:593-612. Yokoi S., R. A. Bressan and P. M. Hasegawa (2002). Salt stress tolerance of plants. Working Rep/Jpn Intern. Res. Center. Agric. Sci., 25-33. Zago E., S. Morsa, J. F. Dat, P. Alard, A. Ferrarini, D. Inze, M. Delledonne and F. Van Breusegem (2006). Nitric oxide- and hydrogen peroxide-responsive gene regulation during cell death induction in tobacco. Plant Physiology 141, 404-411. Zdarilova A., A. Rajnochova-Svobodova, K. Chytilova, V. Simanek and J. Ulrichova (2010). Polyphenolic fraction of Lonicera caerulea L. fruits reduces oxidative stress and inflammatory markers induced by lipopolysaccharide in gingival fibroblasts. Food Chem. Toxicol., 48, 6: 1555-61. Zhang L. and D. Xing (2008). Methyl Jasmonate Induces Production of Reactive Oxygen Species and Alterations in Mitochondrial Dynamics that Precede Photosynthetic Dysfunction and Subsequent Cell Death. Plant Cell Physiol. 49 (7): 1092-1111. Zhang W. H. and S. D. Tyerman (1999). Inhibition of water channels by HgCl2 in intact wheat root cells. Plant Physiology 120, 849-857. Zhao J. (2007). Interplay Among Nitric Oxide and Reactive Oxygen Species A Complex Network Determining Cell Survival or Death. Plant Signaling & Behavior 2:6, 544-547. Zhao M. G., L. Chen, L. L. Zhang, and W. H. Zhang (2009). Nitric Reductase-Dependent Nitric Oxide Production Is Involved in Cold Acclimation and Freezing Tolerance in Arabidopsis. Plant Physiology, 151, 755-767. Zhao S. and E. Blumwald (1998). Changes in oxidation-reduction state and antioxidant enzymes in the roots of jack pine seedlings during cold acclimation. Physiologia Plantarum 104, 134-142. Zhu J. K. (2002). Salt and drought stress signal transduction in plants. Annual Review of Plant Biology 53: 247–273.

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Zhu J., S. Alvarez, E. L. Marsh, M. E. LeNoble, I. J. Cho, M. Sivaguru, S. Chen, H. T. Nguyen, Y. Wu, D. P. Schachtman et al (2007) Cell wall proteome in the maize primary root elongation zone. II. Region-specific changes in water soluble and lightly ionically bound proteins under water deficit. Plant Physiol 145: 1533-1548. Zsigmond L. A. Szepesi, I. Tari, G. Rigo, A. Király and L. Szabados (2012). Overexpression of the mitochondrial PPR40 gene improves salt tolerance in Arabidopsis. Plant Sci. 182: 87-93.

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5. Oxidant Roles in Signaling

5. A. Oxidant Signaling Advantages

The plant to serve as a stress signal to activate acclimation could also channel reactive oxygen species (ROS) generated due to metabolic imbalances during stress and defense mechanisms that would in turn counteract stress associated oxidative stress (Mittler et al., 2004). The two, somewhat opposing faces of ROS, that is, on the one hand, the damaging toxic molecule, and on the other hand, the beneficial signal transduction molecule, underscore the need to control the steady state level of ROS in cells during normal metabolism, as well as in response to different stresses. Elucidating the mechanisms that control ROS signaling in cells during drought and salt stresses could therefore provide a powerful strategy to enhance the tolerance of crops to these environmental stress conditions. ROS signals originating at different organelles have been shown to induce large transcriptional changes and cellular reprogramming that can either protect the plant cell or induce programmed cell death (Rhoads et al., 2006). These types of reprogramming suggest the involvement, at least in part, of organellar retrograde signaling in mediating ROS signals in the coordination of the stress response between ROS generating organelles to the nucleus, and perhaps directly between the organelles themselves. Retrograde signaling is largely divided into two categories: (1) developmental control of organelle biogenesis; and (2) operational control, that is, rapid adjustments in response to environmental and developmental constraints (Pogson et al., 2008). They also stated that ROS generated in these organelles are considered to be important signaling molecules that are involved in retrograde signaling under stress conditions. There is now no doubt that oxidative signaling is central to the mechanisms by which plants cells sense the environment and make

appropriate adjustments to gene expression, metabolism and physiology (Foyer and Noctor, 2005). Therefore, they suggested that oxidative stresses has outgrown its usefulness as a term to denote plant responses to environmental and metabolic fluctuations, and has become something of a misnomer that might be phased out of current nomenclature. Overproduction of alternative oxidase (AO) in transgenic tobacco (Nicotiana tabacum) led to increased ozone sensitivity. This suggested that within a narrow window specific level of mitochondria ROS might be necessary to trigger mitochondrial retrograde signaling events required to launch a proficient defense response (Pasqualini et al., 2007). An alternative mode by which H2O2 and probably other ROS mediate abiotic stress responses is stabilization of specific transcripts that encode stress related proteins. Moller et al. (2010) suggested that peptides deriving from proteolytic breakdown of oxidatively damaged proteins have the requisite specificity to act as secondary ROS messengers, regulate source-specific genes, and in this way contribute to retrograde ROS signaling during oxidative stress. Increasing evidence indicates that H2O2 functions as a signaling molecule in plants and H2O2 generation during the oxidative burst is one of the earliest cellular responses to potential pathogens and elicitor molecules (Lamb and Dixon, 1997). Generation of H2O2 occurs under a diverse range of conditions, and it appears likely that H2O2 accumulation in specific tissues, and in the appropriate quantities, is of benefit to plants and can mediate cross tolerance toward other stresses (Bolwell, 1999). A number of similarities can be seen in the cellular responses to stresses, suggesting that H2O2 could be a common factor regulating various signaling pathways (Neill et al., 1999). ROS play a vital

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role in intracellular redox signaling activating antioxidant resistance mechanisms. Thus, it is a surviving response for plants to control the concentration of ROS (Khan et al., 2011). Direct reactive oxygen species (ROS) signal transduction will ensue only if ROS escape destruction by antioxidants or are otherwise consumed in a ROS cascade. Thus, the major low molecular weight antioxidants determine the specificity of the signal. They either are also themselves signal transduction molecules that can signal independently or further transmit ROS signals (Foyer and Noctor, 2005). 1% to 2% of the genes represented on the array (taking into account redundancy) is affected by oxidative stress imposed by H2O2. 5. B. Signaling Mechanisms

Desikan et al. (2001) demonstrate that H2O2 can modulate the expression of a subset of genes within the Arabidopsis genome. Furthermore, other studies cleared that H2O2 can alter the activity of cellular proteins. It is possible that in some cases H2O2 can interact directly with target proteins; for example, by oxidizing Cysteine (Cys) residues and thereby altering protein conformation (Wu et al., 1998). Plant cellscontain redox sensors that detect and respond to signals such as H2O2. In this context, the induction of a gene encoding a potential hybrid Histidine kinase is of particular interest. His kinases and two component signal transduction systems are well represented in the Arabidopsis genome and have already been shown to modulate cellular responses to ethylene, cytokinin, and possibly osmotic stress (Inoue et al., 2001). His kinases are important sensory enzymes in yeast, in which the osmo-sensing SLN1-SSK1 system has been particularly well characterized (Maeda et al., 1994). His kinase signaling module is connected to a mono adenine phosphate kinase (MAPK) system, such that activation of the HOG1MAPK is regulated by osmotic stress. Recent work has shown that the SLN1 His kinase-HOG1 MAPK signaling system also functions as an H2O2 sensor in yeast (Singh, 2000). H2O2 activates the Arabidopsis MAPK ATMPK6 (Kovtun et al., 2000), a MAPK with high sequence homology to HOG1, coupled with the observation that H2O2 induces the expression of a His kinase, suggests strongly that this His kinase may also function as an H2O2 sensor in plants (Desikan et al., 2001).

The tapestry of mechanisms influenced by ephemeral singlet oxygen, superoxide (O2

●─), hydrogen peroxide (H2O2), hydroxyl radical (●OH), peroxynitrite (ONOO2), and nitric oxide (●NO) in plant cells are not completely understood.In general, previous studies on Nitric acid radical (●NO) revealed that this radical is a relatively stable paramagnetic free radical molecule involved in many physiological processes under normal and stress conditions in both animal and plant cells (Arasimowicz and Floryszak-Wieczorek, 2007; Corpas et al., 2008; Neill et al., 2008). Nitric acid radicals (●NO) serve as a synchronizing chemical messenger involved in cytotoxicity and program cell death (PCD) (Van Camp et al., 1998; Durner and Klessig, 1999). The molecular identity of plant nitric oxide synthase (NOS) is unknown. However, Guo et al. (2003) isolated an Arabidopsis (Arabidopsis thaliana) AtNOS1 gene that encoded a protein with sequence similarity to a protein that is involved in NO synthesis in the snail. Further studies have discounted the possibility that AtNOS1 per se is an Arg-dependent NOS enzyme (Zemojtel et al., 2006). Accordingly, AtNOS1 was renamed as AtNOA1 for NO Associated1 (Crawford et al., 2006). In plants, the possibility that NO might influence protein kinase activities have been poorly explored and most of the available data come from studies based on artificially generated ●NO. NO-dependent activation of protein kinases exhibiting, for instance, MAPK or cyclin dependent phosphokinase (CDPK) properties were reported in A. thaliana suspension cell cultures and roots (Clarke et al., 2000; Capone et al., 2004), cucumber explants (Pagnussat et al., 2004; Lanteri et al., 2006) and tobacco leaves and suspension cell cultures (Yamamoto et al., 2004). With the exception of Salicylic acid-Induced Protein Kinase (SIPK) (Klessig et al., 2000), none of these protein kinases has been identified. Number of mechanisms by which NO activates phospholipase C (PLC)/ Diacylglycerol kinase (DGK) can be envisaged. NO could act directly on PLC or Diacylglycerol kinase (DGK) enzymes by nitrosylation or nitration (Stamler et al., 2001) act indirectly, e.g. via a mitogen-activated protein kinase (MAPKs) signaling cascade (Kumar and Klessig 2000) or by increasing cytosolic Ca2+ levels (Garicia-Mata et al., 2003), and change the redox potential affecting the signaling state of plants (Mou et al., 2003). At this point, it is not clear which of the

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above mechanisms are involved in NO-mediated PLC/DGK activation. NO has been demonstrated to be associated with abiotic stresses, including drought (Mata and Lamattina, 2001), salt stress (Zhao et al., 2007), heat (Song et al., 2006), UV-B radiation stress (Shi et al., 2005), and metal toxicity (Rodriguez-Serrano et al., 2006; Tian et al., 2007). Moreover, the NO-dependent signaling network is often closely related to the plant hormones ABA (Desikan et al., 2004) and ethylene (Ederli et al., 2006) and other signaling molecules such as H2O2 (Neill, 2007), cytosolic Ca2+ (Lamotte et al.,2004), and phosphatidic acid (PA) (Distefano et al., 2008). Interestingly, those molecules are also involved in cold acclimation and freezing tolerance in plants: ABA (Gusta et al., 2005), ethylene (Yu et al., 2001), H2O2 (Prasad et al., 1994), cytosolic Ca2+ (Knight et al., 1996), and phosphatidic acid (PA) (Li et al., 2004). The analysis of the cross-talk operating between NO, protein kinases, and the second messengers Ca2+ and Cyclic Guanidine Mononphosphate (cGMP) provided a framework for understanding the molecular bases of major physiological processes, such as egg fertilization or modulation of neuronal excitability (Willmott et al., 1996; Ahern et al., 2002). In plants, however, synchronized changes in [Ca2+] cyt and NO levels were apparent during the transduction of biotic and abiotic signals (Garcia-Mata et al., 2003; Gould et al., 2003; Lamotte et al., 2004; Vandelle et al., 2006). NO signaling includes various messenger molecules, such as cGMP, cADP ribose, and Ca2+

(Durner et al., 1998; Wendehenne et al., 2001), which both directly and indirectly modulate the expression of specific genes (Polverari et al., 2003; Parani et al., 2004). It was reported that NO might affects transduction processes imply the regulation of key signaling proteins such as protein kinases and Ca2+-permeable channels as well as the mobilization of second messengers including Ca2+, cGMP, and cADPR. Additionally, Ca2 + may function not only as an inducer of the oxidative burst, but also as a signaling molecule downstream of the oxidative burst that causes various cellular responses, including defense system (Torres and Dangl, 2005). Previous studies contended that most eukaryotic signaling pathways involve spatially and temporally specific elevations in [Ca2+]cyt, either via release from intracellular stores and/or via influx from the extracellular space, likely both processes are closely related

(Lanteri et al., 2006). Furthermore, Ca2+-dependent protein kinase (CDPKs) are involved in signaling pathways that utilize changes in [Ca2+]cyt to couple cellular responses to extracellular stimuli (Roberts and Harmon, 1992). An increase in a 50 kDa Ca2+-dependent protein kinase (CDPK) activity was reported after 1 d exposure of cucumber explants to either the NO donor sodium nitroprusside (SNP) or the auxin IAA (Lanteri et al., 2006).The other major mechanism in cell signaling is the reversible protein phosphorylation, including protein kinases and phosphatases (Neill et al., 2003). It seems that these two signaling events are activated during the NO-induced Adventitious Root (AR) formation and may represent key components of the NO responses in plants. Mechanistically, Ca2+ might represent a signaling carrier of NO and/or H2O2-triggered cell death pathways or, as described in animal cells, cell death could also be related to the cellular Ca2+ overload or perturbation of intracellular compartmentalization resulting from NO and/or H2O2-induced Ca2+ fluxes (Orrenius et al., 2003). Therefore, understanding of the mechanisms underlying cell death should also include experiments designed to delineate the cross talk between Ca2+, NO, and H2O2 in further detail. The Ca2+ channels shown in the plasma membrane (pm) are representative of voltage-gated Ca2+ channels, cyclic-nucleotide-gated channels (CNGCs) and of the N-methyl D-aspartate (NMDA) receptor. NO, produced by the Ca2+/CaM-dependent constitutive NOSs (cNOSs), generates Ca2+ signals via several routes (not all pathways occur necessarily in the same cell). First, NO regulates plasma membrane and endomembrane Ca2+ channels through direct S-nitrosylation of critical cysteine, leading to activation (P/Q- and L-type voltage-gated channels, CNGCs, RYRs) or inhibition N-methyl D-aspartate (NMDA) receptor, L-type voltage-gated channels (Courtois et al., 2008). Nitric oxide (NO) is an important signaling molecule involved in many physiological processes in plants (Zhao et al., 2009). It acts as an intermediate in multiple signaling pathways in plants where, NO can directly influence the activity of plant proteins as well as the signaling cascade leading to gene expression (Lindermayr et al., 2006; Grun et al., 2006; Belenghi et al., 2007). In similarly to Ca2+ signaling, NO signaling might display spatial and temporal

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organization (Stamler et al., 2001). Subsequently, several NOsignaling components such as Ca2+-permeable channels, p21ras, sGC, and Mitogen-Activated Protein Kinases (MAPKs) are organized into macromolecular complexes in which NO signaling functions within highly localized environments (Kone et al., 2003).The complementary aspect of NO and Ca2+ signaling is reinforced by the occurrence of transduction networks in which Ca2+ acts both as a promoter and a sensor of NO signaling (Clementi, 1998). 5. C. Protein Nitrosylations

Evidence indicates that NO, and derived species such as peroxynitrite (ONOO─), exert part of their biological activities by chemical modification of protein targets (Bogdan, 2001). These include the nitrosylation, nitrosation, nitration, and oxidation of proteins. In particular, nitrosylation, that is the direct binding of an NO group to a transition metal or cysteine residues (Mannick and Schonhoff, 2002), is emerging as an important post-translational modification of proteins. More than 100 proteins have been found to undergo reversible regulation by nitrosylation in vitro and/or in vivo (Hanafy et al., 2001; Stamler et al., 2001). The broad spectrum of functions ascribed to proteins found to be nitrosylated affects essentially all major cellular activities, highlighting the multifunctional roles of NO (Stamler et al., 2001). Furthermore, nitrosylation is one of the molecular strategies used for signaling (Hess et al., 2001). NO promotes cGMP synthesis by nitrosylation of the soluble guanylate cyclase (sGC). In turn, cGMP activates cGMP-dependent protein kinases (PKGs) and cyclic nucleotide-gated ion channels (CNGCs) through its binding to cyclic-nucleotide binding sites. cGMP might also regulate L-, T- and N-type voltage-gated channels indirectly where, both activation and/or inhibition have been reported (Courtois et al., 2008). Depending on the cell type and the initial stimulus, PKGs inhibit in most frequent situation or activate the inositol (1, 4, 5)-triphosphate receptor (InsP3R) via phosphorylation. PKGs might also activate ADP-ribosyl cyclase (ADPRC) by phosphorylation. ADPRC catalyses the cyclization of NAD to cADPR, an endogenous ligand for ryanodine receptors (RYR). Finally, NO contributes to the overall cytosolic Ca2+ homeostasis through the positive regulation of Ca2+ transporters including Na+/Ca2+ exchanger (Na+/Ca2+ Ex) and plasma membrane and

sarcoplasmic endoplasmic reticulum Ca2+-ATPases (PMCA and SERCA, respectively). Phosphorylation, S-nitrosylation, the covalent attachment of an NO group to the thiolside chain of cysteine residues, has been established further as a component of stress signal transduction. Together with a proteomic survey of S-nitrosylation targets during the hypersensitive response (HR), new insights were gained into the regulation of individual proteins by S-nitrosylation (Belenghi et al., 2007; Romero-Puertas et al., 2008). Of particular interest is the S-nitrosylation of proteins related to the antioxidant system (germin-like protein, monodehydroascorbate reductase, and type II peroxiredoxins [PrxIIE]). S-nitrosylation of PrxIIE inhibits both its peroxidase and peroxynitrite reductase activities, suggesting that NO might regulate the effects of its own radicals through S-nitrosylation of crucial antioxidant systems components (Romero-Puertas et al., 2007). NO signaling pathways often include posttranslational modification of target proteins, such as NO-dependent Cys S-nitrosylation that can modulate the activity and function of different proteins (Sokolovski and Blatt, 2004; Feechan et al., 2005). Kinases proteins are related to primary metabolism for instance pyruvate kinase which was reported to be activated or inhibited by S-nitrosylation (Gao et al., 2004). Nitrosylation, NO was shown to regulate key signaling related proteins including the soluble guanylate cyclase (sGC), the small GTP-binding protein p21ras, and a number of Ca2+-permeable channels (Mannick and Schonhoff, 2002).These pathways control a diverse set of processes, including programmed cell death, stomata movements, auxin-induced lateral root formation, abiotic stress, and defense responses (Neill et al., 2003; Delledonne, 2005; Besson-Bard et al., 2008; Palavan-Unsal and Arisan, 2009). Additionally, studies manifested that low level of free NO could function in signaling processes in nodules, where it may participate in the low oxygen response of the fixing cells (Pauly et al., 2006). Lammote et al. (2004) indicated that NO is intimately involved in the signal transduction processes leading to cryptogein induced defense responses. NO modulates the activity of distinct classes of protein kinases that play a key role in signal transduction including mitogen activated protein

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kinase (MAPK) cascades, protein kinase C, and Janus kinases (Beck et al., 1999). NO-dependent regulation of protein kinase activities occurs by nitrosylation of the kinases themselves or by modulation of interacting/upstream factors such as cGMP, Ca2+, p21ras or protein phosphatases (Beck et al., 1999). NO regulates Ca2+ channel activities through different mechanisms: direct S-nitrosylation or indirect modulation via Cyclic Guanidine Mononphosphate (cGMP)-dependent cascades. S-nitrosylated Ca2+ channels include voltage gated Ca2+ channels (P/Q- and L-type), cyclic nucleotide-gated ion channel (CNGC), ryanodine receptors (RyR), and the N-methyl D-aspartate receptor (Clementi, 1998; Ahern et al., 2002). NO was reported to enhance the activity of type 1 Ryanodine receptor (RYR) by S-nitrosylation of a single cysteine (Sun et al., 2001). The NO-cGMP cascade modulates the activity of L-, T-, and N-type voltage-gated Ca2+ channels, cyclic nucleotide-gated ion channel (CNGC), inositol (1,4,5)-triphosphate receptor, and RyR (Clementi, 1998; Ahern et al., 2002). NO impacts on their activity directly through S-nitrosylation the reversible formation of a covalent bound between a cysteine residue and an NO group or indirectly (Stamler et al., 2001; Ahern et al., 2002). The indirect means involve cGMP, produced following the NO-induced activation of soluble guanylate cyclase, and/or cyclic ADP-ribose (cADPR) a Ca2+-mobilizing metabolite that is synthesized from NAD+ by ADP-ribosyl-cyclase (Willmott et al., 1996; Hanafy et al., 2001). They suggested that cADPR mediates Ca2+ release by activating the intracellular Ca2+ channels ryanodine receptors (RYR) in mammals but also in plants (Allen et al., 1995; Fliegert et al., 2007). It was postulated that hyperosmotic and salt stress-activated protein kinase (NtOSAK) activity might be upregulated through phosphorylation by an upstream NO-dependent protein kinase, by auto-phosphorylation, and/or through direct S-nitrosylation or nitration by NO-derived species. Preliminary experiments are not in favour of the last possibility. In animal systems, S-nitrosoglutathione (GSNO) has been reported to function as an intercellular and an intracellular NO● carrier, and in plants GSNO was found to be a powerful inducer of defense genes (Durner et al., 1998). As hypothesized previously (Durner and Klessig, 1999), S-nitrosoglutathione (GSNO) could

function as a long distance signal molecule, transporting glutathione-bound NO● throughout the plant. In this mechanism, leaf peroxisomes could participate through the endogenous production of S-nitrosoglutathione (GSNO) which could diffuse to the cytosol. Corpas et al. (2001) considered peroxisomes as cellular compartments with the capacity to generate and release into the cytosol important signal molecules such as H2O2, NO●, ●O2

─ and possibly S-nitrosoglutathione (GSNO), which can contribute to achieve a more integrated communication among cell compartments both under normal physiological conditions and in response to biotic and abiotic stresses. This signal producing function of plant peroxisomes is still more significant from a physiological viewpoint considering that the cellular population of these oxidative organelles can proliferate in plants during senescence and under different stress conditions. The study of the molecules that mediate the interorganellar communication within the cell is an important emerging area of plant organelle research, which can supply more information on how peroxisomal, mitochondrial, chloroplast and nuclear fractions are coupled throughout plant development and under different stress conditions by means of an elaborate signaling network (Mackenzie and McIntosh, 1999). Nitration of Tyrosine residues in plant proteins has also been observed in antisense nitrite reductase tobacco (Morot-Gaudry et al., 2002), and following administration of ONOO‑ in vitro. Tyrosine nitration causes conformation, structure or catalytic activity changes; it also could block protein phosphorylation, which may be one mechanism for activation of target proteins (Delledonne et al., 2001). In A. thaliana, nitrosylation of a specific cysteine residue of PrxIIE inhibits the capacity of the enzyme to detoxify peroxynitrite (Romero-Puertas et al., 2007). This post-translational modification of PrxIIE causes a dramatic increase in nitrotyrosine formation, modulating tyrosine kinase signaling pathways, and is biologically relevant. Thiol modification by ROS such as hydrogen peroxide is already recognized as a potential signaling mechanism in plants. Benhar et al. (2005) stated that S‑nitrosylation of plant proteins is also regarded as an important regulatory mechanism similar to that of protein

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phosphorylation. S‑Nitrosylation of glyceraldehyde 3‑phosphate dehydrogenase (GAPDH), hemoglobin and Met adenosyltransferases has been reported; more substrate proteins have been identified as potentially S‑nitrosylated targets in Arabidopsis (Lindermayr et al., 2006). H2O2 also modified and inhibited GAPDH.24 Tyrosine nitration is mediated by ONOO‑ and nitrogen dioxide (NO●) formed as interaction between RNS and ROS or transition metals. In animals, peroxynitrite mediates the inactivation of Mn‑superoxide dismutase (Mn‑SOD) through tyrosine nitration at Tyr34 into 3‑nitrotyrosine. This modification also activates cytochrome c (Cassina et al., 2000).

5. D. Annexins

5. D. I. Annexin Functions

Annexins have been identified as protein components of an M. truncatula plasma membrane lipid raft alongside signaling and redox proteins (Lefebvre et al., 2007). They could, in common with raft associated animal annexins (Babiychuk and Draeger, 2000), anchor rafts to the actin cytoskeleton (Konopka-Postupolska, 2007). Peroxide can induce the channel forming vertebrate AnxA5 to be inserted into membranes in vitro, and peroxide-induced Ca2+ influx in vivo in DT40 pre-B cells requires AnxA5 (Kubista et al., 1999). From this it follows that channel forming plant annexins such as AnxAt1 are candidates for the ROS-activated channels identified in several plant cells (Foreman et al., 2003).Alternatively, conceivably, raft-associated annexins could function as channels or peroxidases operating in a localized ROS signaling ‘hub’. AnxAt1 is present at the root hair apex which is thought to harbour lipid rafts (Jones et al., 2006) and as a peroxidase could help regulate the intracellular peroxide generated during polar growth (Foreman et al., 2003). Annexins could regulate peroxide generated as inter or intracellular messenger or relay/terminate a signal through peroxide dependent oxidation. A protective role can also be envisaged. Peroxidase activity of annexins associated with chloroplast RNA polymerase could protect newly synthesized transcripts from oxidation (Pfannschmidt et al., 2000).

Actin binding actin filaments help shape a cell, are essential for the development of certain plant cell types, and act in signaling (Drobak et al., 2004). Evidencefor binding of plant annexins to actin is mixed; and appears to be species specific. Tomato and mimosa annexins both undergo Ca2+-dependent F-actin binding in vitro (Calvert et al., 1996; Hoshino et al., 2004). Two plasma membrane associated annexins from zucchini bind to zucchini derived F-actin (Hu et al., 2002). Mimosa annexin organizes F-actin into thick bundles in the presence of 2 mM Ca2+ in vitro (Hoshino et al., 2004). Cotton, bell pepper, and maize annexins have all been extensively tested and show no affinity for actin, in either the presence or absence of calcium (Delmer and Potikha, 1997). However, the latter all possess the IRI motif (needed in F-actin binding to mysosin) implicated in actin binding for tomato annexin (Lim et al., 1998). This suggests that the structural requirement for actin binding is more complex. Plant annexin expressions and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signaling. Potentially, they could operate in signaling pathways involving cytosolic free calcium and reactive oxygen species. Plants sense and respond to a range of environmental, metabolic, and developmental signals. Operation and control of interacting signal transduction pathways will involve cell and endomembrane, and integral, peripheral and soluble proteins. Downstream responses may require remodeling of the cytoskeleton, changes to exocytosis machinery, and walls. One family of plant proteins appears to have the capacity to function at all of those levels—the annexins (Mortimer et al., 2008). Plant annexins can cause aggregation of liposomes and secretory vesicles, implicating them in membrane organization (Blackbourn and Battey, 1993). 5. D. II. Annexin Localizations

Medicago truncatula annexin1 (AnxMt1), localizes to the nuclear membrane (De Carvalho-Niebel et al., 1998), and M. sativa (the model legume) AnxMs2 has been shown to localize in the nucleolus under stress conditions even though the protein shows no typical nuclear localization signal (Kovacs et al., 1998).

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A nuclear localization has also been reported for pea annexin (Clark et al.,2000). A putative spinach annexin has been identified in chloroplast envelope membranes (Seigneurin-Berny et al., 2000). Arabidopsis AnxAt1 has been found in chloroplasts (Peltier et al., 2006; Renaut et al., 2006) but also as a tonoplast protein (Carter et al., 2004) and an integral plasma membrane protein ostensibly under non-stressed conditions (Alexandersson et al., 2004). Its membrane association is prompted by salinity stress (Lee et al., 2004). ArabidopsisAnxAt4 has also been identified as a plasma membrane protein (Alexandersson et al., 2004). An annexin from Bryonia diocia relocates from the cytoplasm to the plasma membrane following mechano-stimulation (Thonat et al., 1997). In wheat exposed to low temperatures, two wheat annexins accumulate in the plasma membrane (Breton et al., 2000). Moreover, they are integral membrane proteins, which cannot be released by addition of Ca2+ chelators (Breton et al., 2000). That annexin association with or insertion into membranes can be dynamic and responsive to environmental change is consistent with their involvement in signaling and adaptation (Mortimer et al., 2008). 5. E. Oxidant Signaling In Cell Organelles

5. E. I. Oxidant Signalling In Peroxisomes

Studies on the compartmentation of the photorespiratory pathway in peroxisomes have led to the proposal that, in these organelles, unlike mitochondria and chloroplasts, the compartmentation of peroxisomal metabolism is, in major part, not caused by the boundary membrane but by the specific structure of the protein matrix. The enzymes of the photorespiratory pathway are arranged in the peroxisomal matrix in the form of multi enzyme complexes that allow efficient metabolite channeling, and transfer of metabolites proceeds across the peroxisomal membrane by porin-like channels (Reumann, 2000; Corpas et al., 2000). A characteristic property of peroxisomes is their metabolic plasticity since their enzymatic content can vary depending on the organism, cell/tissue-type and environmental conditions (Van den Bosch et al., 1992; Mullen and Trelease, 1996). Plant cellular proliferation of peroxisomes has been reported under natural and abiotic stress conditions. The induction of peroxisome biogenesis genes (PEX) by H2O2 has been demonstrated in both plant and animal

cells, indicating that the signal molecule H2O2 is responsible for the proliferation of peroxisomes (Lopez-Huertas et al., 2000). In plant wounding and pathogen attack a proliferation of peroxisomes takes place and, probably, this also happens in other stress situations that lead to an increased generation of H2O2 (Lopez-Huertas et al 2000). However, it must be taken into account that quantitative evidence for the proliferation of the peroxisomal population. For instance, counting of the number of organelles per cell section of tissue, has only been reported in plants treated with isoproturon (De Felipe et al., 1988), clofibrate (Palma et al., 1991), cadmium (Romero-Puertas et al., 1999) and during senescence of pea leaves (Pastori and del Rio, 1994). The roles of ROS and RNS are not restricted to the stress response, but can encompass the whole lifespan of the plant, including normal growth and development stages. Moreover, ROS/RNS bioactivity is largely dependent upon their turnover, which not only includes ROS/RNS production, but also their detoxification. ROS/RNS detoxification relies on the antioxidant defense (AD), which involves enzymatic activities such as catalases, superoxide dismutase, peroxidases and antioxidant molecules such as glutathione, GSH, ascorbate (Pauly et al., 2006). They reported that Reactive oxygen and nitrogen species ROS/RNS production, Antioxidant defense (AD) is actively regulated in the response to environmental cues and plant development. Taken together, ROS/RNS and AD contribute to the redox balance, the modulation of which is probably crucial for physiological regulation. The recycling of NADPH from NADP+ can be carried out in peroxisomes by three dehydrogenases: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate. Therefore, it was suggested that the existence of cellular functions for peroxisomes related to reactive oxygen species (ROS). However, the recent demonstration of the presence of nitric oxide synthase (NOS) in plant peroxisomes implies that these organelles could also have a function in plant cells as a source of signal molecules like nitric oxide (NO●), superoxide radical, hydrogen peroxide, and possibly S-nitrosoglutathione (GSNO) (Del Rio et al., 2002). H2O2 has been described as a diffusible transduction signal in different physiological

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processes and plant stress, leading to the induction of genes encoding different cellular protectant. Some functions that have been reported for H2O2 as a signal molecule were found (De Rio et al., 2002). In Arabidopsis thaliana, a peroxisomal membrane protein (PMP22) was identified and characterized at the molecular level and had a 55% similarity with mammalian Mpv17 (Tugal et al., 1999). However, the generation of ROS by this Arabidopsis PMP has not been studied. Antioxidant systems in peroxisomes superoxide dismutases (SODs; EC 1.15.1.1) are a family of metalloenzymes that catalyze the disproportionation radicals into H2O2 and O2, and play an important role in protecting cells against the toxic effects of superoxide radicals produced in different cellular compartments (Elstner, 1991; Halliwell and Gutteridge, 2000). SODs are distributed in different cell loci, mainly chloroplasts, cytosol, and mitochondria (Bowler et al., 1994; Halliwell and Gutteridge, 2000), but the presence of SOD in peroxisomes was demonstrated for the first time in plant tissues (Del Rio et al., 1983; Sandalio et al., 1987). Since then, the occurrence of SODs in isolated plant peroxisomes has been reported in at least nine different plant species. In five of these plants the presence of SOD in peroxisomes has been confirmed by immuno gold electron microscopy using an antibody been proposed (Singh, 2000). In yeast cells, the gene of a human CuZn-SOD (SOD1) was expressed and immuno cryo electron microscopy of cells showed that human CuZn-SOD was accumulated in peroxisomes (Keller et al., 1998). In an ectomycorrhizal fungus, the gene encoding a Mn-SOD was cloned, and the protein was found to maintain a C-terminal peroxisomal localization peptide (PTS1) and lack an N-terminal mitochondrial transit peptide. This putative peroxisomal Mn-SOD appears to be involved in the cellular response of the fungus to cadmium stress (Jacob et al., 2001). The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied (Barros et al., 1999). They purified plant organelles by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca2+-dependent NOS activity of 5.61 nMol of L-[(3)H]citrulline mg-1 protein min-1 was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was found to be clearly inhibited (60-90%) by different well

characterized inhibitors of mammalian NO synthases. The immuno blot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immuno reactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms. 5. E. II. Oxidant Signalling In Chloroplasts EXECUTER1 was proposed earlier as a candidate for singlet oxygen perception within the chloroplasts (Van Breusegem et al., 2008). Lee et al. (2007) provided genetic evidence that EXECUTER1 acts in concert with the highly similar EXECUTER2 to transfer stress related signals from the plastid to the nucleus. The primary function of EXECUTER2 is that of a modulator that attenuates and controls the activity of EXECUTER1 dependent upon enzymatic lipid peroxidation events (Przybyla et al., 2008). Another key component of the chloroplast-to-nucleus signaling, GUN1, that mediates ROS and/or redox responses in Arabidopsis (Arabidopsis thaliana) also has been identified recently (Koussevitzky et al., 2007), and its role in the plant’s response to heat stress has been demonstrated (Miller et al., 2007). There is new information on the fate of oxidized proteins from investigations that link protein oxidation and autophagy and describe the role of protein oxidation in seed dormancy. It was found that protein oxidation is followed by controlled degradation and recycling and that these processes are essential for cellular viability, signaling, development, and recovery from oxidative stress (Bassham, 2007; Moller et al., 2007; Oracz et al., 2007). 5. F. Oxidant Signaling For Growth and

Developments

5. F. I. Nitrogen Fixation

An important case of modulation and signaling by NO and other RNS is closely related to the function of some Hbs. Three types are known and may coexist in plants: nonsymbiotic, symbiotic, and truncated Hbs. The first group is

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classified, in turn, into class 1 Hbs (with very high O2 affinity) and class 2 Hbs (with lower O2 affinity and a primary sequence more similar to those of symbiotic Hbs). Class 1 Hbs are expressed under hypoxia, cold, and osmotic stress, upon treatment with NO, and during rhizobial infection .In hypoxic conditions, these Hbs are part of an NO dioxygenase system, converting NO to nitrate. This system consumes NAD(P)H and maintains ATP concentrations, allowing plant survival (Igamberdiev and Hill, 2004). In L. japonicus, a class 1 Hb controls the plant’s defense response during the early stages of the rhizobial interaction, by modulating NO concentration, and overexpression of this protein enhances symbiotic N2 fixation (Shimoda et al., 2009). By contrast, very little is known about class 2 and truncated Hbs, albeit recent data suggest that at least some of them can also modulate NO concentrations and are expressed in nodules (Vieweg et al., 2005). Symbiotic Hbs include Lbs and Hbs from some actinorhizal plants. In addition to the role of Lbs in facilitating O2 diffusion to symbiosomes, these abundant proteins can form complexes with NO and thus modulate NO bioactivity. The nitrosyl complexes (LbNO) are very stable and can be detected in intact nodules by electron paramagnetic resonance (Mathieu et al., 1998; Meakin et al., 2007). During the first minutes of interaction between plants and microorganisms, a molecular dialogue involving several signal molecules, takes place in the rhizosphere and at the cell surface, leading to physical interaction. For example, in the case of the Legume – Rhizobia symbiotic interaction, flavonoids from the plant root exudates induce the synthesis of Nodulation Factor (NF) from Rhizobia. Both compounds are responsible for the setup of the early interaction steps and for the establishment of the new root organ, the nodule (Oldroyd and Downie, 2008). A similar dialogue is observed during mycorrhizal fungus and plant interaction leading to the production of plant strigolactons (Akiyama et al., 2005) and putative Myc factor by the fungus (Kosuta et al., 2003). 5. F. II. Organ Development

Bioinformatics analysis of ROS-linked stresses described by Gadjev et al. (2006) identified transcriptome footprints common to several ROS stresses or unique to a ROS signal emanating from a specific organelle. This approach has been used to analyze ROS-related events in a senescence-related mutant

(Jing et al., 2008). Hence, this analysis provides a tool to identify organelle-specific ROS signals (Rosenwasser et al., 2011). Some ROS are second messengers implicated in signaling pathways that are activated in the plants in response to developmental and environmental cues; ROS can modify gene expression in an ROS-specific manner; and the production of ROS is, in many cases, genetically programmed. This can be exemplified in the nodulation process. ROS can also serve as important signaling molecules that participate in a diverse range of plant processes. These are root hair development and elongation, leaf expansion, apical dominance, tracheary element maturation, trichomes development, senescence, and response to biotic and abiotic stress (Rodriguez et al., 2002; Foreman et al., 2003; Overmyer et al., 2003; Sagi et al., 2004; Gapper and Dolan, 2006; Gechev et al., 2006; Miller et al., 2008). 5. F. III. Oxidant Mediating Developmental

Signaling

ROS is considered as signal molecule during cellular growth, control of stomata closing (Pei et al., 2000), plant-pathogen interactions (Apel and Hirt, 2004), programmed cell death (Gechev and Hille, 2005) and stress responses have been demonstrated in plants (Laloi et al., 2007). The regulation and the involvement of ROS in Legume – Rhizobia symbiotic relations (Rubio et al., 2004) and during the establishment of both endo- and ectomycorrhizal have also been described (Fester and Hause, 2005; Baptista et al., 2007). Whatever the system involved during the different symbiotic interactions, it would be of interest to analyze the consequences of modifying plant ROS-producing activities on the symbiotic capacities. This could allow us to better understand the signaling role of ROS molecules and its consequences on the establishment of symbiosis (Nanda et al., 2010). Prevention of ROS toxicity and control of ROS signaling require a large gene network, the so called “ROS gene network” is composed of at least 150 genes in Arabidopsis (Mittler et al., 2004). A smaller network is also detected in microorganisms. Several families of proteins from plants and microorganisms are associated with the regulation of ROS levels. Among them, Catalases (Kat), detected in all kingdoms, and catalase-peroxidases (CP), present in some fungi and in the majority of bacteria, can both reduce H2O2 (Passardi et al., 2007). Other

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proteins detected in all kingdoms can generate ROS such as NADPH oxidases (NOx/RBOH), or scavenge ROS such as glutathione peroxidases (GPx) (Margis et al., 2008), peroxiredoxins (Rouhier and Jacquot, 2002) and thioredoxin (Alkhalfioui et al., 2008). The plant specific Class III peroxidases (Prx) are also members of the “ROS gene network” and have the interesting capacity to both scavenges and produces ROS (Passardi et al., 2004). Whatever the source of ROS, H2O2 acts as a signal molecule to induce an array of molecular, biochemical, and physiological responses in plant cells (Neill et al., 2002). H2O2 is produced in response to a wide variety of abiotic and biotic stimuli, it is likely that H2O2 mediates the cross-talk between signaling pathways and is a signaling molecule contributing to cross tolerance; that is, the exposure of plants to one stress confers protection towards others (Bowler and Fluhr, 2000). It may be that cellular responses to H2O2 differ according to their site of synthesis or perception (Neill et al., 2002). Kacperska (2004) has suggested that the role of ROS and H2O2 in the mediation of stress responses may depend on the severity of the stressor. This implies that, rather than sensor type, the quantitative effects of the sensor-initiated modifications in the oxidant-antioxidant activities in different cell compartments may be responsible for the different effects of a particular stressor. This suggestion is in line with observations that small increases of H2O2 allow the general enhancement of stress tolerance; whereas large increases in H2O2 trigger local responses that unavoidably lead to programmed cell death (PCD). Neill et al. (2002) using Arabidopsissuspension cell cultures to elucidate the role of H2O2 as a signaling molecule, showed that H2O2 is generated following elicitor and pathogen challenge and that this H2O2 acts as a signal to induce PCD and defense gene expression (Desikan et al., 2000). Through its relative stability and ability to diffuse through membranes, H2O2 already had been recognized as the most potent signaling ROS in plants. At present, specific aquaporins have been demonstrated to channel H2O2 actively across membranes (Bienert et al., 2007). New insights have been gained into the modes of action and regulation of previously identified molecular targets of ROS signaling.

5. G. Oxidant Signaling For Biotic Stress

Resistance

In a few stresses, ROS increase has been shown to serve as a signaling molecule. For example, pathogen attack was associated with oxidative burst in the apoplast, leading to hypersensitive response which culminates in localized cell death (Lamb and Dixon, 1997; Grant and Loake, 2000; Sagi and Fluhr, 2001). Reactive oxygen species (ROS) have earlier been considered merely as cytotoxic compounds, but clearly, their functions are far more diverse. ROS have important and tightly regulated roles as signaling molecules in disease resistance, stress adaptation, and development in many organisms (Kangasjarvi et al., 2005; Valko et al., 2007).The active defense responses, which require de novo protein synthesis, are regulated through a complex and interconnected network of signaling pathways that mainly involve three molecules, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), and which results in the synthesis of pathogenesis-related (PR) proteins (Almagro et al., 2009). Many proteins particularly, those containing thiol groups are known to react directly with H2O2, to date no complete ROS signal transduction pathways have been described (Mahalingam and Federoff, 2003). Two component circuits provide an interface between environmental cue sensing and a downstream kinase signaling cascades in eukaryotes (Hwang et al., 2002). Heterotrimeric G protein signaling to membrane bound NADPH oxidases has been implicated in the developmental of disease resistance and hyper sensitive response (HR) in the rice apoplast (Suharsono et al., 2002). An oxidative signal-induced kinase OXI1 has been shown to be upstream of two mitogen activated protein kinases (AtMPK3 and AtMPK6) in Arabidopsis (Mittler et al., 2004). Mitogen activated protein kinases (MAPK3 and MAPK6) have been reported to be activated in response to H2O2 and to be a potential integrating point of environmental and developmental signals that regulate stomata development (Wang et al., 2008). The activity of MAPK3 and MAPK6 can be stimulated by the nucleoside diphosphate kinase 2 (NDPK2). The extracellular matrix (ECM)-plasma membrane cytoskeleton continuum is considered to play important roles in the perception of environmental signals (Baluska et al., 2003). In animals, heterodimeric

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plasma membrane proteins known as integrin anchor the cytoskeleton to the extra cellular matrix (ECM). These trans-membrane linker proteins can also function as bi-directional signal transduction molecules in processes such as apoptosis (Hyun, 2002). NO is a signal molecule involved in triggering the defense response of cells against different stress conditions (Romero-Puertas and Delledonne, 2003; Neill et al., 2008). The imbalance of the ●O2

−/●NO ratio could favor oxidative conditions but could also interfere in the signal transduction pathways of the defense mechanism against stress (Delledonne et al., 2001). Studies indicate that nitric oxide (NO) is an endogenous signal in plants that mediates responses to several stimuli. Experimental evidence in support of such signaling roles for NO has been obtained via the application of NO, usually in the form of NO donors, via the measurement of endogenous NO, and through the manipulation of endogenous NO content by chemical and genetic means (Neill et al., 2008b). It is clear that ●NO and, in general, most of the reactive nitrogen species (RNS) (●NO, NO+, NO–, ●NO2, and ONOO–), are major signaling molecules in plants which can be synthesized during stress responses at the same time as H2O2 (Durner and Klessig, 1999). NO being part of the intracellular signaling cascade is activated in plant cells in response to pathogens or elicitors (Garcia-Brugger et al., 2006). At the transcriptional level, microarray and cDNA-AFLP data obtained from ●NO donor-treated Arabidopsis cells indicated that ●NO modulates related to the expression of several defense genes, including genes encoding pathogen related (PR) proteins and proteins related to secondary metabolism (Polverari et al., 2003; Parani et al., 2004). 5. H. Oxidant Signaling For Abiotic Stress

Resistance

ROS serve as signaling molecules that regulate stress responses, as well as growth and development (Foyer and Noctor, 2005). The oxidative stress that accompanies drought and salt stresses should not necessarily be viewed as a harmful event needed to be avoided or alleviated, but could also be viewed as a perquisite for the plant to adequately respond and induce proper acclimation mechanisms (Miller et al., 2010). ROS signaling was shown to be an integral part of the acclimation response

of plants to drought or salinity stresses. It is used to sense stress due to enhance ROS production caused by metabolic imbalances, as well as to actively send different signals via enhance production of ROS at the apoplast by different respiratory burst oxidase homolog (RBOH) proteins. ROS Signaling during drought and salinity stresses is highly integrated into many of the other Signaling networks that regulate plant acclimation, including calcium, hormone and protein phosphorylation (Miller et al., 2010). In light of this view of the integrated signaling network of plants, which is responsible for the timely activation of different acclimation pathways, it is easy to see why some changes in ROS metabolism were found to cause enhanced tolerance to stress, whereas other changes were found to cause enhanced sensitivity. It is also not easy to predict how different changes in ROS metabolism, engineered by different genetic manipulations, will affect crop tolerance to abiotic stress. H2O2 is intimately involved in plant defense responses, affecting both gene expression and the activation of proteins such as monoadenine phosphate kinase (MAPK), which in turn function as regulators of transcription. Protein Tyr phosphatase is important signaling enzymes that regulate protein phosphorylation events in all eukaryotes (Walton and Dixon, 1993), particularly the inactivation of monoadenine phosphate kinase (MAPK) cascades (Luan, 1998). Oxidative stress activates MAPK cascades not only in plants (Kovtun et al., 2000). Tyr phosphatase was identified as a primary target for H2O2 (Wu et al., 1998). Expression of the MAP kinase ATMPK3 is induced by oxidative stress, that oxidative stress also activates the Arabidopsis MAP kinases ATMPK3 and ATMPK6, and that such activation can itself mediate the induction of oxidative stress-responsive genes (Kovtun et al., 2000). Tyr phosphatase was identified as H2O2 inducible in wilting and UV irradiation circumstances. An Arabidopsis protein Tyr phosphatase has previously been identified that is transcriptionally regulated by environmental stresses such as cold and salt stress (Xu et al., 1998). Several key transcription factors involved in ROS signaling have been identified (Gadjev et al., 2006; Miller et al., 2008). The majority (39/44) of transcription factor expressions during dark treatment were found to be modified during, suggesting a role of these elements in

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modulating the plant response to darkness. Of these genes, 15 genes were downregulated following 2 days in darkness. This group includes the genes ZAT12 (At5g59820) and WRKY53 (At4g23810), whose expression was confirmed by Q-RT-PCR and is likely due to the influence of light/dark transition (Rosenwasser et al., 2011). The expression of 11/15 of these genes was increased when compared to end of night. The rest of the ROS-related transcription factors were upregulated on the second day following transition to darkness. Such an expression pattern was confirmed for WRKY6 (At1g62300) by Q-RT-PCR (Rosenwasser et al., 2011). Moreover, inspection of d3 and d5 fold changes showed that the induction continues to increase in the majority of transcripts (e.g. AtWRKY75 is 47, 92 and 198-fold induced on d2, d3 and d5, respectively). In addition, analysis of the expression data obtained by Usadel et al., (2008) of night extension, revealed that 30 out of 44 genes were upregulated following 48 hours, and none of them were downregulated. It is interesting to note that the expression of some of these transcription factors increased earlier than 48 h of extended night. Hence, our analyses support the notion that ROS-related transcription factors are involved in the early events of dark treatment (Rosenwasser et al., 2011). They suggested the involvement of ROS in the dark response, and it was of interest to see if the transcriptome analysis can be used to identify the compartmental source of ROS. Compartment/type-related ROS-induced genes have been compiled by Gadjev et al. (2006), where 5 groups of specific gene expression were identified. These groups were related to responses of singlet oxygen in plastids, superoxide in chloroplasts or mitochondria and hydrogen peroxide in cytoplasm and peroxisomes. The transcript levels of the genes within each of the groups were examined in our gene expression data and the relative portion of upregulated or downregulated transcripts during darkness was calculated. One group consists of 289 transcripts, which were stimulated specifically by singlet oxygen generated in the flu mutant. Of these transcripts, expression of 126, 128, and 140 genes were modified on the second, third, and fifth day of dark treatment, respectively. Most of the genes were decreased on day 2 and 3 (71% and 62%, respectively), whereas on day 5 the expression of an equal number of genes was decreased or increased. This suggests that signals emanating from (1O2) in chloroplasts decreased during early stages of

darkness, but were induced when darkness persisted (Rosenwasser et al., 2011). Cells decipher ROS signals with regard to type, localization, and timing of ROS produced (Overmyer et al., 2003), but mechanisms of ROS perception have remained mostly obscure. Only a few examples have linked the cellular machinery to perception and integration of ROS signals. Plant heat-shock transcription factors (HSFs) might function as H2O2 sensors (Miller and Mittler, 2006), and the salicylic acid (SA) signaling protein NPR1 confers redox regulation to the transcription factor TGA1 (Despres et al., 2003). Various components of the ROS detoxification machinery could be used to detect differences in the oxidative load (Davletova et al., 2005). However, the mechanisms and processes involved in the ROS perception and signaling in the extracellular space have remained almost completely elusive (Wrzaczek et al., 2009). Nitric oxide (NO) is emerging as an important signaling molecule with multiple biological functions in plants (Wilson et al., 2008). Several messenger molecules such as Abscisic acid ABA (Xiong et al., 2001), hydrogen peroxide (H2O2) (Prasad et al., 1994), and cytosolic Ca2+ activity (Tahtiharju et al., 1997) are involved in perception and transduction of low temperature signal to mediate cold acclimation dependent changes in physiological processes (Zhao et al., 2009). The dehydration and cold response gene family genes have been identified as key transcriptional activators to activate the associated downstream cold-regulated genes, conferring tolerance of plants to freezing (Thomashow, 1999; Iba, 2002). Identification and characterization of genes involved in cold acclimation have advanced our knowledge of the molecular mechanisms underlying cold acclimation and freezing tolerance in plants (Chinnusamy et al., 2006).ROS are also involved in signalingin different processes such as growth, development, and response to biotic and abiotic stresses, and a balance between ROS production and scavenging (Bailey-Serres and Mittler, 2006) controls this signaling process. The O3-perception and immediate downstream signaling support the notion that the action of O3 in plants is due to processes perceived, transduced, and induced by the network of regulatory mechanisms in the cells affected. This seems to involve at least three separate signaling cascades downstream of the

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perception of O3. Since, the involvement of the Ga subunit in the O3-lesion formation (Booker et al. 2004; Joo et al. 2005),Gb or Gbg Signaling to the chloroplast (Joo et al. 2005), and the activation of MAP kinases by O3 (Samuel and Ellis, 2002; Ahlfors et al., 2004b; Joo et al., 2005) represent three independent branches of events downstream of O3 perception. One of the major challenges of research on plant O3 responses will be the identification of the signaling processes involved and elucidation of their interactions that result in the acclimation of the plant to the external challenge by O3

(Kangasjarvi et al., 2005). It is plausible that plant cells challenged by osmotic stress might use NO as an early signaling compound acting upstream of [(Sucrose Non Fermenting) 1-related protein kinase type 2 (SnRK2)]-induced pathways (Courtois et al., 2008). This could be true for other plant responses in which both changes in osmotic pressure and NO production are observed, such as responses to pathogens or elicitors of defense responses (Gauthier et al., 2007). Together with ROS, RNS, and in particular nitric oxide (NO), are considered as major components of oxidative burst and redox state regulation. NO has been shown to be a ubiquitoussignalingmolecule in plants, controlling physiological processes as diverse as flowering, iron homeostasis, drought response, or resistance against pathogens (Neill et al., 2003). 5. I. Oxidant Signalling For Stomata Behaviour

5. I. I. Reactive Oxygen Specie Signalling For

Stomata Behaviour

ROS are essential signals mediating Abscisic acid (ABA) induced stomata closure and induced ROS synthesis in chloroplast. Stomata closure during drought stress is a major plant mechanism for reducing the loss of water through leaves. The opening and closure of stomata are mediated by endo membrane trafficking pathway, involves AtVAMP7C (a protein) in stomata movement and this protein play an important role in the localization of ROS, detected in nuclei, chloroplasts, and vacuoles and in the regulation of stomatal closure by ABA treatment (Leshem et al., 2010). Islam et al., (2010) examined stomata movements and mobilization of second messengers in the

attpc1-2 loss-of-function mutant in response to ABA, methyl jasmonate (MeJA) and Ca2+ to investigate whether Arabidopsis thaliana two pore channel 1 (AtTPC1) that encodes the slow vacuolar (SV) channel is involved in stomata closure. 5. I. II. Reactive Nitrogen Specie Signalling For

Stomata Behaviour Nitric oxide synthase (iNOS) constitutively binds calcium and calmodulin and is regulated primarily at the transcriptional level. eNOS and nNOS produce NO at much lower rates than iNOS and thus serve primarily to generate NO for signaling. iNOS makes large amounts of NO as a cytotoxic agent in immune responses. A mitochondrial NOS (mtNOS) activity has been characterized in mammals, but the identification of the protein(s) responsible for this activity has been controversial (Brookes, 2004; Lacza et al., 2004; Ghafourifar and Cadenas, 2005). All three isoforms of NOS have at one time or another been implicated as mtNOS. Nitric oxide (NO) is a central signaling molecule in plants. It was shown to serve as a signal in defense and programmed cell death (PCD), hormone responses, abiotic stress, root and xylem development, germination, iron homeostasis, and flowering (Lamattina et al., 2003; Neill et al., 2003; Wendehenne et al., 2004; Crawford and Guo, 2005; Delledonne, 2005; Lamotte et al., 2005; Simpson, 2005). NO mediates Abscisic acid–induced stomatal closing (Desikan et al., 2004) and auxin-induced lateral and adventitious root growth (Pagnussat et al., 2003). The role NO on other physiological aspects was mentioned by Neill et al. (2008b) as they found that stoma closure, initiated by Abscisic acid (ABA) is affected through a complex symphony of intracellular signaling in which NO appears to be one component. Where, exogenous NO induces stomata closure, ABA triggers NO generation, removal of NO by scavengers inhibits stomata closure in response to ABA, and ABA-induced stomata closure is reduced in mutants that are impaired in NO generation. Their data indicated that ABA-induced guard cell NO generation requires both nitric oxide synthase like activity and, in Arabidopsis, the NIA1 isoform of nitrate reductase (NR). Studies have also illustrated the major role of NO as a signalin controlling primaryand adventitious root organogenesis, a developmental process, which shares common

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features with nodule formation (Pagnussat et al., 2002; Correa-Aragunde et al., 2004). 5. J. Oxidant Signaling for Hormonal

Metabolisms and Functions

It was proposed on a hypothetical basis that free radicals might induce an endogenous response culminating in more effective adaptations, which protect against exogenous radicals, and possibly other toxic compounds (Tapia, 2006). Reactive oxygen species do have useful functions in cells, such as redox signaling, the function of antioxidant systems is not to remove oxidants entirely, but instead to keep them at an optimum level (Rhee, 2006). Many environmental and hormonal stimuli, such as anoxia, Abscisic acid (ABA), light, salt stress, pathogens, and elicitors, induce a rapid increase in NO levels. It has been reported that NO is involved in salt resistance as excess salt increases NO levels and salt stress can be alleviated by application of NO donors or be aggravated by lowering NO accumulation (Zhao et al., 2007; Corpas et al., 2009). Treating plants with NO donors can often elicit the same responses induced by these hormonal or environmental stimuli. It is thus important to elucidate the processes that control NO synthesis and accumulation; however, in most cases, these processes are poorly understood (Wilson et al., 2008; Gas et al., 2009; Leitner et al., 2009). The reduced accumulation of NO in response to H2O2 in the phb3-3mutant is that H2O2 signaling or accumulation is impaired so that the mutant has lower levels of or cannot respond to H2O2 (Wang et al., 2010). Plant prohibitins play a role in plant defense (Nadimpalli et al., 2000), root hair elongation (Wen et al., 2005), cell division, development, and senescence (Van Aken et al., 2007), oxidative stress (Ahn et al., 2006), signaling (Christians and Larsen, 2007). Similar to animals, plant prohibitins are targeted to mitochondria, form multimeric complexes, and function in mitochondria biogenesis and function (Ahn et al., 2006; Van Aken et al., 2007). In wild type Arabidopsis thaliana plants, NO levels increase eightfold in roots after H2O2 treatment for 30 min. A mutant defective in H2O2-induced NO accumulation was identified, and the corresponding mutation was mapped to the prohibitins gene PHB3, converting the highly conserved Glycine-37 to an Aspartic in the protein’s SPFH domain. This point mutant and a T-DNA insertion mutant were examined for other

NO related phenotypes. Both mutants were defective in Abscisic acid induced NO accumulation and stomata closure and in auxin-induced lateral root formation. Both mutants were less sensitive to salt stress, showing no increase in NO accumulation and less inhibition of primary root growth in response to NaCl treatment. In addition, light-induced NO accumulation was dramatically reduced in cotyledons (Wang et al. 2010). No evidence was observed for impaired H2O2 metabolism or signaling in the mutants as H2O2 levels and H2O2 induced gene expression were unaffected by the mutations. These findings identify a component of the NO homeostasis system in plants and expand the function of prohibitin genes to include regulation of NO accumulation and NO-mediated responses. Nitric oxide (NO) is a reactive nitrogen species that acts as an intermediate in multiple signaling pathways in plants. These pathways control a diverse set of processes, including programmed cell death, stomata movements, auxin-induced lateral root formation, abiotic stress, and defense responses (Neill et al., 2003; Delledonne, 2005; Besson-Bard et al., 2008; Palavan-Unsal and Arisan, 2009).

5. J. I. Oxidant Signaling For Auxin


The complex interaction of auxin and reactive oxygen and nitrogen species during plant development is not well known yet, but more and more data accumulate indicating a mutual interaction that may be based on auxin transport inhibition and auxin-mediated ROS generation and removal what finally may converge on chromatin remodeling (Pasternak et al., 2001). Auxin promotes the production of ROS in the outer epidermis of maize coleoptiles (Schopfer, 2001) and ROS themselves stimulate their elongation. In contrast, when auxin inhibits maize root growth, there is a decrease in ROS production (Liszkay et al., 2004). As they inhibit germination, Abscisic acid (ABA) and far-red light inhibit ROS production in the embryos and seed coats of germinating radish Raphanus sativus, while gibberellin relieves this inhibition (Schopfer et al., 2001). Although not strictly a growth response, the NADPH oxidases, AtrhbohD and AtrhbohF, are known to be necessary for ABA signaling for stomatal closure (Kwak et al., 2003). Alfalfa leaf cells and freshly prepared protoplasts constitute a reliably

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homogenous population of mesophyll cells, most of which can be considered to be at the G0-phase of the cell cycle. Up to the moment of protoplast isolation, these cells are specialized for photosynthesis, with RUBISCO (ribulose-1, 5-bisphosphate carboxylase/oxygenase) comprising more than 70% of their protein content (Feher et al., 2008). As a consequence, majority of nuclear genes are switched off in these cells. Therefore, mesophyll cells and their protoplasts have compact nuclei with a diameter of 3.8–4.4µm, containing a small nucleolus (diameter around 1µm), and are characterized by highly condensed chromatin. In the presence of appropriate growth regulators such as auxin and cytokinin, these cells start to divide; the remodeling of the chromatin and the de novo synthesis of ribosomes, in order to allow the reorganization of the gene expression pattern and protein synthesis, respectively precedes that. These changes are reflected in the morphology and size of the nucleus and the nucleolus and for dyes they can accumulate (Pasternak et al., 2000). Therefore, these parameters can be well used as cellular markers of G0-to-G1 transition in plant as well as in animal cells (Bottiroli et al., 2004). The specific NO-scavenger cPTIO delayed the Ca2+-dependent protein kinase (CDPK) activity in SNP-treated explants and diminished it in IAA-treated ones (Lanteri et al., 2006). The obtained results suggested that the 50 kDa Ca2+-dependent protein kinase (CDPK) detected by in-gel assays in both NO and IAA treatments is the same and requires the presence of NO for its maximal and sustained activity. The induction of Ca2+-dependent protein kinase (CDPK) gene expression in response to Auxin was previously reported in alfalfa (Medicago sativa) (Davletova et al., 2001). Auxin and NO are put together in the Ca2+-mediated signaling pathway that regulates Ca2+-dependent protein kinase (CDPK) activity leading to adventitious root (AR) formation (Lanteri et al., 2006). The rate of NO release from the NO-donor SNP displays a peak around the second day in aqueous solutions. This fact might explain the partial inactivation of the 50 kDa Ca2+-dependent protein kinase (CDPK) in SNP+cPTIO treatment at the second and third days of treatment. With regard to the auxin action, a transient increase in the endogenous NO level was reported to occur during the first day of IAA-treated cucumber explants (Pagnussat et al., 2002). Thus, the amount of cPTIO could be insufficient to

scavenge the NO entirely and therefore to inhibit the Ca2+-dependent protein kinase (CDPK) activity completely during the first and second days of IAA+cPTIO treatment. The different dynamics of Ca2+-dependent protein kinase (CDPK) activity measured by in vitro and in-gel assays could be explained by the nature of the experiments. While in the in vitro assays the total Ca2+-dependent protein kinase (CDPK) activity is measured, in the in-gel assays only the Ca2+-dependent protein kinase (CDPKs) that are able to re-nature under the experimental conditions are detected. In addition, syntide2 is used as substrate for in vitro assays and histone IIIS for in-gel analysis (Lanteri et al., 2006). The NO donor SNP was shown to increase the amount and histone H1 phosphorylating activity of the p34cdc2 cyclin-dependent kinase in auxin-treated alfalfa protoplasts (Otvos et al., 2005). The NO-induced activation of these kinases has been thought to be part of processes related to defense responses and/or cell death (Yamamoto et al., 2004) and to auxin-mediated adventitious root formation and cell division (Otvos et al., 2005; Lanteri et al., 2006). Several kinases including phosphoglycerate kinase, nucleoside diphosphate kinase, and adenosine kinase have been shown to be S-nitrosylated in vitro (Lindermayr et al., 2005). The first evidence that NO modulates the activation of a member of the plant SNF1-related protein kinase2 (SnRK2) subfamily has been reported recently (Lamotte et al., 2006). Plant Sucrose Non Fermenting (SNF) as 1-related protein kinase type 2 (SnRK2/SnRKs) are classified into three subfamilies: SnRK1, SnRK2, and SnRK3, the SnRK2 and SnRK3 subfamilies being specific to plants (Harmon, 2003). Members of the SnRK2 subfamily function in abiotic stress signaling and include the tobacco 42 kDa protein kinase NtOSAK (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase (Mikolajczyk et al., 2000). NtOSAK is activated very rapidly in response to osmotic stress through phosphorylation of two serine residues (154 and 158) located within the enzyme activation loop (Burza et al., 2006). Salicylic acid-Induced Protein Kinase (SIPK) remained fully active in response to NO, where NO was found to activates Salicylic acid-Induced Protein Kinase, Hyperosmotic and salt stress-activated protein kinase (NtOSAK) and Mitogen-Activated Protein Kinase (MAPKs) enrich our understanding of how NO exerts its effects. The assumed that Hyperosmotic and salt stress-

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activated protein kinase (NtOSAK) is an interesting candidate; the analysis of its implication in the changes of [Ca2+]cyt triggered by NO is under investigation (Besson-Brad et al., 2008). The auxin indole acetic acid (IAA) regulates many aspects of plant growth and development from seed germinations to fruit ripening, mediating cell division, expansion, and differentiation (Davies, 1995). Adventitious root (AR) formation involves the development of a meristematic tissue after removal of the primary root system. The auxin IAA promotes adventitious root (AR) formation by inducing the dedifferentiation of cells to develop new meristems. This process is essential for the propagation of woody species and for adaptation to particular environmental conditions. However, despite the critical role of auxin throughout the plant life cycle, the molecular mechanisms underlying their action are still poorly understood. Protein extracts from cucumber hypocotyls were assayed for Ca2+-dependent protein kinase (CDPK) activity by using histone IIIS or syntide 2 as substrates for in-gel or in vitro assays, respectively. The activity of a 50 kDa CDPK was detected after 1 d of either NO or IAA treatments and it extended up to the third day of treatment. This Ca2+-dependent protein kinase (CDPK) activity was affected in both extracts from NO and IAA-treated explants in the presence of the specific NO-scavenger 2-(4-carboxyphenylalanine) 4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium (cPTIO), suggesting that NO is required for its maximal and sustained activity. The in-gel and the in vitro CDPK activity, as well as the NO- or IAA induced adventitious root (AR) formation, were inhibited by Calmodulin antagonists. Furthermore, the induction of Ca2+-dependent protein kinase (CDPK) activity by NO and IAA was shown to be reliant on the activity of the enzyme guanylate cyclase (Lanteri et al., 2006). Auxin-induced lateral root formation is another well-studied, NO mediated process (Pagnussat et al., 2002; Correa-Aragunde et al., 2006). Root formation by auxin in the monocotyledonous plant Commelina communis suppressed by treatment with either (i) a GC inhibitor, (ii) a Ca2+chelators, (iii) an inhibitor of Ca2+ release from intracellular stores, or (iv) an inhibitor of cADPR synthesis (Cousson, 2004). These findings suggested that cGMP could be

implicated in Ca2+ mobilization by stimulating cADPR synthesis and subsequent Ca2+ release from cADPR/ryanodine-sensitive Ca2+ channels. Results indicate that this signaling pathway is also operative in a dicotyledonous plant during adventitious root (AR) formation and that it relies on NO. Although some other Ca2+ channels might be affected due to the low specificity of the inhibitors methoxyverapamil hydrochloride and ruthenium red, the results presented in this work collectively argue that cADPR/ryanodine-sensitive Ca2+ channels are involved in the signaling pathways triggered by IAA and NO to induce adventitious root (AR) formation. A similar process occurs in animals in which NO was shown to activate cADPR/ryanodine-sensitive Ca2+ channels indirectly via a cGMP/cADPR-dependent pathway (Willmott et al., 1996) or directly through S-nitrosylation (Xu et al., 1998). Besides the function of cGMP in mediating a cADPR/Ca2+-dependent pathway, cGMP has also been shown to activate plasma membrane (PM) Ca2+ channels. These channels are called cyclic nucleotide-gated ion channels (CNGCs) and were identified throughout the plant kingdom. Cyclic nucleotide-gated ion channels (CNGCs) are permeable to both monovalent and divalent cations (typically K+, Na+, and Ca2+) and are directly activated by cGMP and/or cAMP (Lemtiri-Chlieh et al., 2004; Bridges et al., 2005). It was reported that formation and elongation of adventitious roots in marigold Targetes erecta involves not only NO but also H2O2, which supports the linkage between tissue proliferation and PHB3 mediated NO accumulation (Liao et al., 2009). Nitric oxide (NO) and cGMP are also involved in the auxin response during adventitious root (AR) formation in cucumber (Cucumis sativus). More recently, a mitogen-activated protein kinase cascade was shown to be induced by IAA in a NO-dependent, but cGMP-independent, pathway. The involvement of Ca2+ and the regulation of Ca2+-dependent protein kinase (CDPK) activity during IAA- and NO-induced adventitious root (AR) formation were evaluated in cucumber explants. The effectiveness of several broad-spectrum Ca2+ channel inhibitors and Ca2+ chelators in affecting adventitious root (AR) formation induced by IAA or NO indicated that the explants response to IAA and NO depends on the availability of both intracellular and extracellular Ca2+ pools (Lanteri et al., 2006). It was analyzed whether the NO-induced and cGMP-mediated adventitious root

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(AR) formation (Pagnussat et al., 2003) involves a cADPR/Ca2+-dependent pathway. Data obtained from treating cucumber explants with compounds that are known to block cADPR/ryanodine-sensitive Ca2+ channels (methoxyverapamil hydrochloride and ruthenium red) or cADPR synthesis (nicotinamide) indicate that these inhibitors provoked a significant reduction in both IAA and NO-induced adventitious root (AR) formation. Pagnussat et al. (2002) demonstrated that nitric oxide (NO) is involved in the auxin response during adventitious root (AR) formation in cucumber. Notwithstanding, convergent and complex cGMP-dependent and independent signaling pathways are orchestrating the formation of a new root system when the primary root is removed (Pagnussat et al., 2004). Wang et al. (2010) treated mutant and wild-type seedlings with different concentrations of indole-3-acetic acid (IAA) for 3d, and then the number of emerged lateral roots was determined. They showed that lateral root number increased in a dose dependent manner in wild-type roots. However, lateral root number was unchanged in phb3-3 roots for all IAA treatments tested, indicating that IAA-induced lateral root formation is inhibited in phb3-3. Interestingly, without IAA treatment, phb3-3 had a similar number of lateral roots as wild-type seedlings, showing that phb3-3 was fully capable of producing lateral roots. They tested the induction of several IAA-responsive genes, including IAA1, IAA5, and IAA19. They found that all three genes were induced to the same extent in both wild-type and phb3-3 mutant roots after 0.5 and 1 h of 1 mM IAA treatment, indicating that the mutant was still able to sense IAA and support rapid induction of gene expression. One of the well-established pathway in which Ca2+ is released from intracellular stores is through inositol 1, 4, 5-trisphosphate (IP3)-regulated channels (Alexandre et al., 1991; Allen et al., 1995). These data indicated that inhibitors of inositol 1, 4, 5-trisphosphate (IP3)-regulated Ca2+ channels promoted a significant reduction in adventitious root (AR) formation in both NO- and IAA-treated cucumber explants. There is evidence that Auxin could induce an increase in inositol 1, 4, 5-trisphosphate (IP3) concentration (Ettlinger and Lehle, 1988; Zbell and Walter-Back, 1988). It was also proposed that inositol 1, 4, 5-trisphosphate (IP3)-regulated Ca2+ channels at the tonoplast might be involved in auxin

triggered increases in [Ca2+]cyt (Shishova and Lindberg, 1999). Recent results indicated that the plasma membrane (PM) Ca2+ channel blocker lanthanum chloride significantly compromised the IAA and NO-induced adventitious root (AR) formation (Lanteri et al., 2006), where NO can regulate the changes in [Ca2+]cyt through the control of Ca2+ influx across the plasma membrane (PM) in tobacco cells (Lamotte et al., 2004). In the same trends, auxin might activate Ca2+ transport from the extracellular space through plasma membrane (PM) Ca2+ channels as shown in wheat (Triticum aestivum) leaf protoplasts (Shishova and Lindberg, 1999). The requirement of extracellular Ca2+ to promote adventitious root (AR) formation in cucumber explants was corroborated via the use of the membrane-impermeable Ca2+chelators EGTA. Notwithstanding, the limitations of the pharmacological approach used in this study, these data collectively indicate that both intracellular and extracellular Ca2+ pools are required for the action of IAA and NO in triggering adventitious root (AR) formation. Thus, in addition to the function of Ca2+ as a mineral nutrient modulating the root growth (Druart, 1997; Bellamine et al., 1998), these results provide evidence to support the involvement of Ca2+ as a second messenger linking both auxin and NO to the activation of processes leading to adventitious root (AR) formation. Basipetal transport of auxin induces a NO burst in the basal region of the cucumber hypocotyl, where the adventitious roots (AR) primordial develop (Pagnussat et al., 2002). Then, NO triggers a bifurcated signaling pathway that includes: (i) increases in the levels of cGMP, cADPR, and IP3 that results in elevations in both [Ca2+]cyt and Ca2+-dependent protein kinase (CDPK) activity; and (ii) the induction of a cGMP independent MAPK cascade (Pagnussat et al., 2004). TFP, W-7, and CPZ compounds are not specific for inhibiting Ca2+-dependent protein kinase (CDPKs). These compounds also affect the activity of other Ca2+-binding proteins such as CaM and calcineurin B-like proteins (Anil and Rao, 2000). However, an inhibition of the Ca2+-dependent protein kinase (CDPK) activity by those compounds in both in vitro and in-gel protein kinase assays. Nevertheless, this does not preclude the possibility that inhibition of other proteins by TFP, W-7, and CPZ could also be affecting adventitious root (AR) formation.

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Therefore, the activation of all the pathways triggered by NO seems to be required for adventitious root (AR) formation since the stimulatory effect of auxin and NO is abolished when one of the pathways is compromised. It remains to be determined whether co-ordination and/or synchronization between MAPK- and Ca2+/CDPK-dependent signaling pathways occur during adventitious root (AR) formation (Lanteri et al., 2006). 5. J. II. Oxidant Signaling For Abscsic Acid


In guard cells, H2O2 participates in the activation of plasma membrane localized anion channels that lead to stomata closure (Schroeder et al., 2001a). The significance of H2O2 in stomata regulation has been questioned (Kohler et al., 2003). Two genes encoding subunits of an NADPH oxidase (AtrbohD and AtrbohF) appear to be involved in stomata closure, as well as in other Abscisic acid (ABA) responses such as seed dormancy. Double mutants in which both genes are inactivated show impaired closure of stomata, an effect which can be rescued by exogenous H2O2 (Kwak et al., 2003). H2O2-induced stomata closure was reversed by the application of exogenous ascorbate, presumably due to peroxidase-dependent H2O2 scavenging (Zhang et al., 2001). Moreover, high constitutive expression of dehydroascorbate reductase (DHAR) in transgenic plants increased the amount of ascorbate relative to DHA in leaves and guard cells and significantly affected guard cell signaling and stomata movement (Chen et al., 2003). The leaves of the dehydroascorbate reductase (DHAR) overexpresses contained less H2O2 in the guard cells and had a higher percentage of open stomata and increased stomata conductance (Chen and Gallie, 2004). H2O2 is synthesized in response to exogenous Abscisic acid (ABA), and that H2O2 mediates at least in part, ABA responses including stomata closure and gene expression (Pei et al., 2000). Recent experimental evidence strongly suggests that this is indeed the case, and that such induction of endogenous free radical production extends life span of a model organism. Most importantly, this induction of life span is prevented by antioxidants, providing direct evidence that toxic radicals may mitohormetically exert life extending and health promoting effects. A further interaction between ABA and ascorbate is that the latter is a cofactor

for 9-cisepoxycarotenoid dioxygenase (NCED), a key enzyme in ABA synthesis. The abundance of NCED mRNA is modulated by ascorbate, such that transcripts are increased when ascorbate is low and decreased when ascorbate is high (Pastori et al., 2003). Some of the H2O2 sensitive genes could also be involved in plant hormone signaling. For example, a gene encoding a syntaxin was identified as H2O2 responsive by both microarray and RNA-blot analyses. Syntaxins are docking proteins involved in vesicle trafficking, and a role in the hormonal control of guard cell ion channels has been demonstrated for an ABA-inducible syntaxin in tobacco (Leyman et al., 1999). Because both elicitors and ABA induce H2O2 production in guard cells (Lee et al., 1999). Induction of a syntaxin by H2O2 is involved in regulating guard cell functioning. In addition to pathogen challenge, other stimuli that induce H2O2 synthesis and oxidative stress include drought stress and ABA, itself synthesized following loss of turgor (Pei et al., 2000); low and high temperatures (Dat et al., 1998); excess excitation energy (Karpinski et al., 1999); UV irradiation (Mackerness et al., 2001); and ozone (Langebartels et al., 2000). The phytohormone accumulates in response to drought stress and induces a range of stress adaptation responses including stomata closure. Earlier work has shown that H2O2 induces stomata closure (McAinnsh et al., 1996), and that guard cells synthesize ROS in response to abiotic stress challenges (Allan et al., 1997; Lee et al., 1999). ABA-stimulated ROS accumulation induced stomata closure via activation of plasma membrane calcium channels (Orozco et al., 2001). Therefore, it can be hypothesized that ABA might signal via ROS during primary cell growth (Laloi et al., 2004). While there are clear indications that ROS and hormones interact, it is not clear whether this is in specific signaling pathways or in the growth mechanism itself. ROS networks of production and action are extremely complex, so there are many points at which ROS can be regulated or have their effect (Mittler et al., 2004). In general, the atmospheric CO2 concentration is limiting for process of photosynthesis. Yet, when this maximum photosynthetic rate need be achieved, CO2 influx must be maximum i.e. by opening of the stomata (Khan et al., 2011). The drought tolerant species control stomata function to allow some carbon fixation at stress, thus improving water use efficiency or open stomata rapidly

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when drought is relieved. Although stomata closure generally occurs when plants are exposed to drought, in some cases photosynthesis may be more controlled by the chloroplast capacity to fix CO2 than by the increased diffusive resistance (Faver et al., 1996; Herppich and Peckmann, 1997). This, however, due to the corresponding high evaporation rates results in a severe loss of water, which at least in all water limiting habitats, would lead to desiccation processes. As a rule, stomata responses are more closely linked to soil moisture content than to leaf water status. This suggests that stomata are responding to ABA (a chemical signal) produced by dehydrating roots (Davies and Zang, 1999). A clear time dependency in stomata responsiveness to air humidity and water status was also found (Franks et al., 1997), suggesting that some of diurnal changes in stomata function may result from metabolic processes targeted to production of secondary plant products with a circadian rhythm (Chaves et al., 2002). Changes in cell carbon metabolism are also likely to occur early in the dehydration process. With the onset of drought, ABA and H2O2 levels were 3-fold and 2-fold higher than those attained in well hydrated plants, respectively. However, the highest and most significant levels of ABA and H2O2 were recorded during hot dry month, showing a peak of 6-fold and 11-fold, respectively. These results showed that drought, ABA, and H2O2 accumulation were related. Indeed, ABA accumulation preceded that of H2O2 (Jubany-Mary et al., 2009). Water deficit triggers an increase of ABA in plants (Zhu, 2002; Nambara and Marion-Poll, 2005). This increase is important for physiological and molecular responses of plants to water deficit, with stomata closure and modulation of the expression of gene networks being the most studied responses. Furthermore, ABA induces the accumulation of H2O2. The relationship between ABA and H2O2 shown in Arabidopsis and Vicia faba guard cells (She et al., 2004; Ann et al., 2008). In addition, ABA-induced H2O2 accumulation has been demonstrated in detached maize leaves in which water stress was induced by polyethylene glycol (Jiang and Zhang, 2002). However, there is a lack of knowledge of what occurs in ABA induction of H2O2 in plants growing under natural climatic conditions and subjected to variations in environmental parameters, including summer drought. In studying the time course of H2O2 and

ABA content it is necessary to consider that both are very sensitive to brief changes in environmental conditions, and that the general trend of the time course is the most representative of their response to environmental conditions. Under drought It has been proposed that the primary H2O2 accumulation in mesophyll cells is apoplastic, increasing in the plasma membrane and organelles only under conditions associated with visible damage or enhanced lipid peroxidation rates (Ranieri et al., 2003; Cheeseman, 2006). Involvement of ABA signaling in O3-induced stomatal closure is demonstrated by a comparison of the wild-type Arabidopsis Col-0 to ABA insensitive mutants; O3-induced stomatal closure was significantly faster in the wild type than in the mutants (Ahlfors et al. 2004a). Eventually, however, O3 caused the closure of stomata in ABA insensitive mutants indicating that O3 affects stomata also by ABA-independent mechanisms. It has been shown that ABA-dependent closure of stomata is ROS-mediated (Pei et al., 2000; Murata et al., 2001; Zhang et al., 2001). Therefore also O3 or the ROS formed from the degradation of O3 in the apoplast – could have direct effect on stomata function. The activity of the Arabidopsis NADPH oxidases ATRBOH D and ATRBOH F (Arabidopsis thaliana Respiratory Burst Oxidase Homolog), which generate ROS during hypersensitive response, is required for elevation in [Ca2+] cyt in guard cells, which in turn triggers the closure of the stomata. The ROS formed from O3 degradation could affect stomata either by acting as an ‘artificial’ replacement for the function of the ABA-induced ROS, or by affecting (unidentified) components in the perimeter of the guard cells and eliciting downstream signaling responsible for the activation of the NADPH oxidase. ROS also affect two protein phosphatases, ABI1 and ABI2, which are negative regulators of ABA signaling; H2O2 inhibits the activity of these proteins (Meinhard and Grill 2001; Meinhard et al., 2002). Thus, this is one way in which ROS, and possibly also O3 affect (the ABA-dependent) plasma membrane Ca2+ influx. It has also been shown that O3 can affect the stomata opening directly by regulating the K+ fluxes in the guard cells (Torsethaugen et al., 1999). However, only a few reports have addressed the effect of O3

onion fluxes across the plasma membrane in general (Castillo and Heath 1990; McAinsh et al. 1996; Clayton et al. 1999) and only one in the

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stomata guard cells specifically (Torsethaugen et al. 1999), thus the mechanisms by which O3

affects the stomata function require further study. (ROS damage)The O3 concentration inside the leaf during ozone exposure is close to zero (Laisk et al., 1989), which means that it is rapidly degraded in the apoplast, and thus the apoplastic anti oxidative capacity is the next factor in determining the fate of the leaf cells. Ascorbate provides important protection from oxidative injury by removing the harmful ROS generated from ozone (Luwe et al., 1993). The role of ABA in the stomata regulation and O3 influx has been discussed above. Ethylene and salicylic acid are needed for the development of the visible O3-lesions and jasmonic acid acts to limit and contain the lesion spread (Kangasjarvi et al., 2005). The mutants and accessions of Arabidopsis and other species, which were described as O3-sensitive. They have turned out to be either partially JA insensitive (Arabidopsis mutants oji1, rcd1, ecotype Cvi-0, poplar clone NE-388), or ethylene over-producers (Arabidopsis mutants rcd1, oji1 and ecotypes Ws and Kas-1) (Koch et al. 2000; Overmyer et al., 2000; Rao et al., 2000b; Kanna et al., 2003; Tamaoki et al., 2003). ABA induces production of ROS and nitric oxide (NO), elevation of the cytosolic free calcium ion concentration ([Ca(2+)](cyt)) and cytosolic pH (pH (cyt) ), and activation of S-type anion channels in guard cells, causing stomatal closure (Khan et al., 2011). Ali et al. (2007) reported that the Ca2+-dependent NOS-like-mediated NO production occurring in response to Lipopolysaccharides (LPS) in A. thaliana guard cells is brought about by the activation of the plasma membrane cyclic nucleotide gated channel CNGC2. Supporting this conclusion, plants without functional CNGC2 (dnd1: defense no death 1 mutants) lack inward plasma membrane Ca2+ currents and failed to produce NO in response to Lipopolysaccharides (LPS) (Besson-Brad et al., 2008). Interestingly, the hypersensitive response (HR), which is normally reduced in the dnd1 plants inoculated with virulent pathogens, was partially restored by the NO donor sodium nitroprusside (SNP). These findings highlight a role for NO produced through a Ca2+-dependent process in mediating the HR, in agreement with previous pharmacological-based studies (Delledonne et al., 1998; Lamotte et al., 2004). In Abscisic acid signal transduction for stomata closure, NO and H2O2 induced by Abscisic acid individually inhibit stomata opening

specific to blue light (Bright et al., 2006; Zhang et al., 2007a). Phospholypase D (PLDβ1)-derived PA interacts with ABI1 phosphatase 2C and regulates Abscisic acid (ABA) signaling during stomata closure in Arabidopsis (Zhang et al., 2004; Mishra et al., 2006). NO was shown to act upstream of ABI1 in ABA-induced stomata closure (Neill et al., 2002). Subsequently exogenous NO can alsoprotect cells against oxidative stress which suggested an emerging model of stress responses in which ABA has several ameliorative functions. These include the rapid induction of stomata closure to reduce transpiration water loss and the activation of antioxidant defenses to combat oxidative stress. These are two processes that both involve NO as a key signaling intermediate (Neill et al., 2008b). ROS serve as second messengers in Abscisic acid (ABA) signaling in the regulation of the stomata aperture (Vahisalu et al., 2008), and are involved in root hair growth (Carol and Dolan, 2006). ABA enhances H2O2 production (Pei et al., 2000; Kwak et al., 2003), which stimulates guard cell NO accumulation and stomata closure (Bright et al., 2006; Neill et al., 2008b). Because phb3 mutants reduce H2O2-stimulated NO accumulation in roots, NO accumulation in guard cells and closure of stomates were examined in the mutant. Therefore, epidermal peels were prepared from wildtype and phb3-3 plants and treated with ABA, and then NO levels and stomata apertures were determined. NO levels in wild-type stomates increased 2.7-fold when treated with 10 mM ABA. No increase was observed for phb3-3 mutant stomates after similar ABA treatment. These data show that ABA-induced NO accumulation was severely inhibited in phb3-3 guard cells (Wang et al., 2010).They found no evidence of reduced H2O2 accumulation in response to ABA using the fluorescent indicator dye CM-H2DCFDA. Stomata closure was induced by ABA in wild-type stomates, which showed statistically significant reductions in aperture of 33 and 39% using 1 and 10 mM ABA, respectively, compared with control stomates. However, ABA-induced stomata closure was strongly inhibited in the phb3-3 mutant. Only 6 and 9% with 1 and 10 mM ABA treatments, respectively reduced mutant apertures, which was not significant compared with untreated stomates. Measurements of aperture ratios confirmed these results (Wang et al., 2010). Moreover, ABA treatments significantly decreased the

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ratios by 29 and 42% of control for 1 and 10 mM ABA treatments, respectively whereas, there were no significant differences in the ratios between ABA-treated and control stomates in phb3-3. These experiments demonstrate that PHB3 is required for ABA-induced NO accumulation and stomata closure. Roots of 7-d-old seedlings grown hydroponically were treated with 0.5 mM H2O2 for 0.5 h, and the mRNA levels in roots for these genes were determined by quantitative RT-PCR. The results showed that WRKY transcription factor gene WRKY6, the heat stress transcription factor gene HSFA4, the touch gene TCH3, and the glutathione S-transferase gene GSTU12 genes were induced by H2O2, and the induction levels for each anti NO accumulation gene (phb3-3) in roots were similar to those in the wild type (Wang et al., 2010). They measured fluorescence in both untreated roots and ABA treated stomata. No difference in fluorescence between wildtype and mutant roots or stomata was observed. From these data, they find no evidence that phb3-3 is defective in H2O2 sensing or accumulation and thus conclude that its phenotype is due to defects in NO accumulation. Stomata closure was induced by ABA in wild-type stomata, which showed statistically significant reductions in aperture of 33 and 39% using 1 and 10 mM ABA, respectively, compared with control stomata. However, ABA-induced stomata closure was strongly inhibited in the phb3-3 mutant. Only 6 and 9% with 1 and 10 mM ABA treatments, respectively reduced mutant apertures, which was not significant compared with untreated stomata. Measurements of aperture ratios confirmed these results. Moreover, ABA treatments significantly decreased the ratios by 29 and 42% of control for 1 and 10 mM ABA treatments, respectively, whereas there were no significant differences in the ratios between ABA-treated and control stomata in phb3-3. These experiments demonstrate that PHB3 is required for ABA-induced NO accumulation and stomata closure.

5. J. III. Oxidant Signaling For Ethylene


Nitric oxide (NO) is a widespread intracellular and intercellular messenger with a broad spectrum of regulatory functions in many physiological processes (Moncada et al., 1991;

Ignarro, 2002; Del Rio et al., 2004; Grun et al., 2006). NO was involved in ethylene (ET) emission (Leshem and Haramaty, 1996), response to drought (Leshem, 1996), disease resistance (Durner et al., 1998; Clark et al., 2000; Delledonne et al., 2001). Besides, growth and cell proliferation (Ribeiro et al., 1999), maturation and senescence (Leshem and Haramaty, 1996; Corpas et al., 2004), apoptosis/programmed cell death (Magalhaes et al., 1999; Clark et al., 2000; Pedroso and Durzan, 2000), and stomata closure (Garcia-Mata and Lamattina, 2002; Neill et al., 2002). There is a linkage between NO and ethylene where NO and ethylene levels are negatively correlated during fruit ripening (Leshem and Pinchasov, 2000). NO has been shown to inhibit ethylene synthesis (Zhu and Zhou, 2007; Cheng et al., 2009). This linkage provides clues on how phb3 mutations could produce the eer3 phenotypes, which include higher ethylene production (Christians and Larsen, 2007). Results from a transcriptome study reported for phb3 mutants are also consistent with above mentioned findings. Many of the phb3 mutant genes whose expression is altered by the phb3 mutation are significantly upregulated by oxidative or salt stress as well as other abiotic stresses (Van Aken et al., 2007). Addition of sodium nitroprusside (SNP) resulted in biphasic patterns of ethylene biosynthesis and, just as during the Hypersensitive Response (HR), the second rise in ethylene was perturbed in Salicylate hydroxylase (SH) transgenic tobacco plants. SA alone was required to initiate the biphasic pattern, and only a single burst of C2H4 biosynthesis was initiated (Mur et al., 2008). They suggested that NO/SA influences the transcription of 1-aminocyclopropane-1-carboxylic acid synthase (ACS). Hence, the biphasic switch could arise from defined transcriptional events. Rakwal et al. (2004) reported a regulation by Ethylene (ET), Jasmonic acid (JA), and ROS in rice plants. It was identified in a screen for ethylene hypersensitive mutants and shows reduced hypocotyl length in the dark. The mutant; eer3 is an ethylene overproducer in the dark, and has reduced induction of ethylene responsive genes. The eer3/phb3 mutation is epistatic to two ethylene insensitive mutations (ein2 and ein3), indicating that PHB3 functions downstream of these regulators (Christians and Larsen, 2007).Wu and Bradford (2003)

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demonstrated that Ethylene (ET) and Jasmonic acid JA in tomato leaves regulate Chitinases.

5. J. IV. Oxidant Signaling For Jasmonic Acid


Genes encoding myrosinase binding proteins and JA-inducible proteins were shown to be H2O2 responsive on the microarray. Myrosinase are enzymes involved in the degradation of Glucosinolates, and a myrosinase-binding protein was found to be induced by both wounding and dehydration (Reymond et al., 2000). It also found that wilting induced the expression of a gene encoding a myrosinase-binding protein. Levels of Jasmonic acid (JA) and ROS increase with water stress, which might lead to the induction of such genes. Various genes encoding transcription factors were induced by H2O2, suggesting that these transcription factors mediate further downstream H2O2 responses, and that several other genes are likely to be induced at later times (Desikan et al., 2001). Jasmonic acid (JA) and its methyl ester (methyl jasmonate, MeJa) are key signaling molecules well known for their roles during plant development as well as plant defense and stress responses (Hoeberichts and Woltering 2002; Turner et al., 2002; Qu et al., 2006; Farmer 2007; Wasternack 2007; Balbi and Devoto, 2008). Rererences

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6. Oxidants Role in Developmental Processes

6. A. Oxidant Initiate Gene Transcription and

Relative Protein

6. A. I. Annexins

Annexins are a multigenic, multifunctional family of soluble proteins with a broad taxonomic distribution. Over 200 unique annexin sequences have been described in >65 species covering plants, fungi, protists, higher vertebrates, and recently a prokaryote (Gerke and Moss, 2002; Morgan et al., 2006). They found in all monocot and dicot plants (Hofmann, 2004), including the model plant Arabidopsis (Clark et al., 2001) and the model legume Medicago (Kovacs et al., 1998). Plant annexins have a molecular weight in the region of 32–42 kDa and, although sharing a common evolutionary ancestor, differ structurally from their animal counterparts (Mortimer et al., 2008). Plant annexins, typically only the first and fourth repeated domains have the characteristic endonexin sequence and crystal structures have been described (Hofmann et al., 2003). Plant annexins have, on average, a larger surface area than mammalian annexins (Clark et al., 2001). This is due to extra grooves and clefts, perhaps suggesting a wide range of interaction partners and a broad range of roles within the cell. However, in contrast to their animal counterparts, the N-terminal region of all known plant annexins is short (10 amino acids). The crystal structure of bell pepper annexin (AnxCa32) revealed that the short N-terminal region interacts with the annexin core, suggested the conservation of some regulatory functions in this region of plant annexins (Hofmann et al., 2000). Annexin expression is dynamic even under normal growth conditions. Transcript levels of annexin genes in Arabidopsis vary depending on tissue type and age, suggesting specific purposes at different developmental stages in different tissue (Cantero et al., 2006). Annexin expression increases during fruit ripening and

gall ontogeny, implying hormonal control (Vandeputte et al., 2007). Nod factors induce M. truncatula annexin1 (AnxMt1) expression, and co-localization studies using AnxMt1–GFP (green fluorescent protein) have suggested that it may be involved in the early stages of cell division required for nodule formation (de Carvalho-Niebel et al., 2002). The in vitro ability to bind membranes, Ca2+, purine nucleotides, and actin predicts critical roles for both animal and plant annexins in membrane trafficking and signal transduction. The clearest demonstration to date of annexin function in planta has been the stimulation of Ca2+-dependent vesicle fusion to the plasma membrane of maize root cap protoplasts by AnxZm33 and 35 (Carroll et al., 1998). Plant annexins are abundant and underlie the plasma membrane in cells associated with high secretion rates. Annexins are prominent at the apex of cells undergoing polar elongation, such as root hairs, pollen tubes, and fern rhizoids (Blackbourn et al., 1993). Annexin expression has detected in the root elongation zone of maize (Bassani et al., 2004) and Arabidopsis. As well as possibly being involved in primary and root hair elongation growth, a specific annexin in Arabidopsis (AnxAt2) is implicated in lateral root development, implying upstream regulation by growth regulators (Clark et al., 2005a). Cotton annexin (AnxGh1) mRNA is upregulated during cotton fiber elongation, and the protein may be associated with Golgi-derived coated vesicles mediating fiber elongation (Shin and Brown, 1999; Bulak Arpat et al., 2004). Recent work on a Saprolegnia annexin has revealed an ability to stimulate (1–3)-β-D-glucan synthase activity, implying a role in the regulation of wall synthesis (Bouzenenza et al.,

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2006). This is in contrast to the inhibitory effect of cotton annexin AnxGh1 (Andrawis et al., 1993) and suggests that different annexins play distinct regulatory roles. Arabidopsis AnxAt7 expression is upregulated by oxylipin treatment that induces callose formation and causes wavy roots and lateral root inhibition (Vellosillo et al., 2007). In addition to roles in exocytosis and wall synthesis, individual annexins could be acting as Ca2+ or GTP sensors to co-ordinate growth. A role as a Ca2+ sensor has proposed for vacuole-associated annexins (Seals et al., 1994). Vacuolar biogenesis is a key component of cell expansion, and expression of the vacuole-associated tobacco annexin VCaB42 correlates with vacuolar biogenesis in expanding cells (Seals and Randall, 1997). The annexin is associated with a ROP GTPase a type of protein viewed as master regulators of growth (Lin et al., 2005). Addition of GTP inhibited annexin-mediated exocytosis in root cap protoplast suggesting that local GTP and Ca2+ levels (Carroll et al., 1998) coordinate annexin function. Mimosa annexin binds F-actin in vitro. Actin filaments in Mimosa believed to control pulvinus movement, in conjunction with altered osmotic pressures, through decreased tyrosine phosphorylation of the actin, leading to tissue bending. However, since Mimosa annexin distribution does not precisely follow actin distribution in vivo, the relationship between the two remains unclear (Kanzawa et al., 2006). Annexins are asymmetrically distributed in the direction of gravity at the periphery of cells just below the apical meristem of etiolated pea plumules (Clark et al., 2000).They found that annexins were found to redistribute within 15 min of a gravitational stimulus (and prior to the onset of plumule curvature), occupying a more evenly distributed peripheral position. They interpreted their finding in context of an annexin involvement in redirecting materials for growth. In gravistimulated Arabidopsis roots, the abundance of AnxAt1 increases in roots (Kamada et al., 2005) and predominates in epidermal cells that would undergo the greatest growth rate to bend the root. AnxAt1 distribution largely matched that of polysaccharide secretion, supporting a role for annexins in the gravistimulated growth response. In gravistimulated hypocotyls, AnxAt2 detected preferentially in the epidermis that would grow the fastest (Clark et al., 2005b). Determination of

how these distributions are caused and link to gravistimulated changes in [Ca2+]cyt, actin, and ROS is increasingly within the technical range of plant biologists. Overall, the ability to bind membrane, GTP, and actin suggests the involvement of annexins in (differential) growth and places them downstream of a wide range of signal transduction pathways (Mortimer et al., 2008).

6. A. I. 1. Annexins in Cell Membranes

Animal and plant annexins bind Ca2+ and, in the presence of (micromolar) Ca2+, will bind to negatively charged phospholipids including phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Binding could reverse by addition of Ca2+ cheater (Balasubramanian et al., 2001). An annexin may be membrane associated or even membrane inserted, depending on the [Ca2+]cyt, pH, lipid composition, and voltage (Gerke and Moss, 2002). Certain annexins such as AnxB12 from Hydra have the capacity to be soluble, peripheral, and integral proteins (Ladokhin and Haigler, 2005). Strict sequence conservation does not appear to be necessary for membrane-binding function. This supported by the observation that both plant and animal annexins bind to a range of negatively charged phospholipids in addition to phosphatidylserine, including phosphatidylinositol, phosphatidic acids, and Malonaldehyde conjugated lipids (Balasubramanian et al., 2001). In plants, hydrophobic interactions are also involved in membrane binding. AnxCa32 attachment to membranes involves the hydrogen bonding of several amino acid residues to the phospholipid head group and glycerol backbone (Dabitz et al., 2005). Site directed mutagenesis of recombinant tomato annexin p35 (AnxLe35) revealed that the fourth repeat of the core domain was critical to lipid binding (Lim et al., 1998). Although Ca2+ is required for membrane binding at neutral pH, at acidic pH (<pH 6.0) some animal annexins bind to membranes independently of Ca2+ (Langen et al., 1998; Rosengrath et al., 1998; Golczak et al., 2004). Plant annexins also appear capable of Ca2+-independent membrane binding. Recently, it has been reported that 20% of the annexin protein (AnxGh1 and AnxCa32) remains bound to lipid vesicles in the absence of Ca2+ at neutral pH, and the proteins can be released following addition of detergent (Dabitz et al.,

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2005). However, rather than Ca2+-independent membrane binding, may represent a proportion of the population undergoing membrane insertion, hence the requirement for detergent to release the protein. In addition to promoting Ca2+-independent binding, acidic pH can reduce the Ca2+ requirement for phosphatidylserine binding. The mechanism of plant annexin Ca2+-independent membrane binding is still unclear, although a pair of conserved tryptophans (W35/W107) is involved. This suggested that Ca2+-independent membrane binding serves as a platform for an annexin population whose membrane binding is Ca2+ dependent. Given that sequential and co-operative binding of annexins showed the two modes of membrane binding may be intimately linked (Dabitz et al., 2005). The majority of plant annexins tend to be found in the cytosol, they are also found bound to (or in some cases inserted into) both plasma membranes and endo-membranes (Thonat et al., 1997). Annexins can localize to the plant vacuolar membrane (Carter et al., 2004), the Golgi, and Golgi-derived vesicles (Clark et al., 1992). 6. A. I. 2. Environment Stimulate Annexins

Annexin expression and distribution can change in response to environmental stimuli. Light affects the expression of certain annexins in Arabidopsis. In hypocotyls, AnxAt5 expression found to increase by red light and this is reversible by application of far red light; in cotyledons, AnxAt6 has a similar red/far red response (Cantero et al., 2006). It seems that annexin expression being downstream of phytochrome A, and further dissection of this relationship is awaited. Phytochrome action has previously implicated in the regulation of polarized annexin distribution in fern rhizoids (Clark et al., 1995). Nyctinastic (nighttime) movement of the Mimosa pulvinus provides a beautiful example of temporal regulation of annexin abundance and positioning. The amount of annexin is more significant at night (when the leaf droops) and the protein is largely cytosolic, whilst in the morning (when the leaf held up) it has redistributed to the outermost periphery of the motor cells in the pulvinus. Annexin abundance positively regulated by ABA which, suggested that annexins link stress responses with nyctinastic and possibly seismonastic (touch-induced) movement (Hoshino et al., 2004). Both

types of movement involve Ca2+ influx from the apoplast, and may be involved in annexin relocation (Campbell and Thomson, 1977). The mechanical stress caused by wind results in short, bushy plants (thigmomorphogenesis). Annexins respond to mechanical stress in B. dioica internodes by redistributing from the cytosol to the plasma membrane in parenchyma cells sampled 30 min after the stimulus (Thonat et al., 1997). Mechanical stress is known to elevate [Ca2+]cyt and this could stimulate annexin–plasma membrane association. The significance of annexin relocation is not understood, but as regulators of growth, they may govern the radial expansion that results from mechanical stress or could be ‘conditioning’ the plasma membrane for further stress responses (Mortimer et al., 2008). Cold causes increased annexin expression in poplar leaves (Renault et al., 2006). In wheat, the cold-induced accumulation of annexins p39 and p22.5 and their insertion into the plasma membrane could be involved in sensing or transducing Ca2+ signals or in regulating [Ca2+]cyt during signaling or acclimation (Breton et al., 2000). Annexin expression, abundance, and cellular position can respond to osmotic stress, salinity, drought, and ABA (Watkinson et al., 2003; Lee et al., 2004; Buitnik et al., 2006; Vandeputte et al., 2007). 6. A. II. Oxidant Inductions Of Calmodulin

Genes

Evidence of interplay between the monomeric GTPase Rho-like GTPase of plants (ROP), Respiratory Burst Oxidase Homolog (RBOH), NADPH oxidases, cytosolic calcium transients, and ROS production were reported (Gapper and Dolan, 2006; Kwak et al., 2006). Several pieces of this remarkable tapestry were assembled. First, it was shown that activation of the plasma membrane localized RBOHs involves phosphorylation of two N-terminal Ser by a calcium-dependent protein kinase as well as interaction with ROP. Respiratory Burst Oxidase Homolog (RBOH) phosphorylation as well as binding to calcium synergizes its activation, raising the possibility that it may function as a calcium sensor (Ogasawara et al., 2008; Takeda et al., 2008). A calmodulin gene strongly induced by H2O2, which coded calcium-binding protein that may well have a pivotal role in stress tolerance. Intracellular calcium concentrations

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increase in response to oxidative stress (Price et al., 1994), and calcium influx is required for the activation of ROS generation (Harding et al., 1997). Furthermore, NADPH oxidase, a potential ROS generating enzyme, contains EF hand calcium binding motifs (Keller et al.,1998), and at least one of the NADPH oxidase genes is induced by H2O2 (Desikan et al., 1998). These observations suggest that H2O2 induction of a calmodulinmight be at least in part regulated the activity of this enzyme. Moreover, a calmodulin has shown to mediate between calcium and ROS generation in tobacco (Nicotiana tabacum) cells undergoing the hypersensitive response (HR). Calmodulin is a regulator of NAD kinase, which generates NADPH for NADPH oxidase activity (Harding et al., 1997). Thus, a significant amount of cross talk occurs between ROS and calcium, and both these signaling molecules mediate cross-tolerance to a variety of stresses (Bowler and Fluhr, 2000). It was found that exogenous H2O2 not only activated gene expression, but also repressed the expression of some genes. Oxidative stress represses several genes in animals and the expression of 62 genes was downregulated. Many of these encode proteins of unknown function (Morel and Barouki, 1999). It is interesting to note that genes encoding a receptor protein kinase and Cys proteases were repressed by H2O2.

6. A. III. Oxidant Inductions of Diamine Oxidase

The localization and activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) together with polyamine levels were investigated in developing grains of barley (Hordeum vulgare L.). DAO (pH 7.5) is present mainly in vascular tissue and its neighbouring cells, namely chalazal cells and nucellar projection, while PAO (pH 6.0) is mainly localized in the chlorenchymatous cells of the crease and at the base of the vascular tissue (Asthir et al., 2002). Activities of both these enzymes appear to be independently regulated, as DAO activity increased steadily throughout grain development while PAO activity was higher during the early stages of grain filling, declined thereafter and again increased towards maturity. The maximum activities of DAO coincided with the maximum content of putrescine while the levels of PAO did not seem to be directly correlated with spermidine or spermine contents. Isoelectric focusing (IEF) of DAO and PAO activities revealed the presence of bands at 30 and 45

DPA. The possible involvement of DAO and PAO in H2O2 supplement to peroxidase-catalyzed reactions, in the chalazal cells during grain filling is discussed (Asthir et al., 2002). The histochemical results show that Diamine oxidase (DAO) activity is mainly present in the vascular tissue (xylem and phloem) and its neighbouring cells, namely the chalazal cells and nucellar projection, while polyamine oxidase (PAO) activity is present mainly in chlorenchymatous cells of the crease region and the base of the vascular tissue (i.e. xylem/phloem). Diamine oxidase (DAO) activity gave intense staining at neutral pH (i.e. 7.5) while PAO gave more intense colour at acid pH (i.e. 6.0) indicating that DAO is more active at neutral pH while PAO is more active at acidic pH (Asthrin et al., 2002). The apparent absence of polyamine oxidase (PAO) activity from the cells of the chalazal region suggests that this enzyme might be not involved in the biosynthesis of lignin in these cells. That the observed activity is indeed due to DAO/PAO is evidenced by the fact that no, or very little, colour is produced in the absence of the substrates. Further support for the hypothesis that amine oxidases are active in the tissue of the crease comes from evidence that the substrates are present in the cells or cell walls where activity was observed. That is, using light microscopy. It was observed that some cut sections of the control (without Put or Spd), also developed slight orange coloration in the chalazal cells (Asthrin et al., 2002). This is sufficient to indicate the presence of endogenous Putrescine (Put) or spermidine (Spd). That these substrates are present in developing barley grains over the period from 11-45 DPA has confirmed in the present work following extraction and chromatographic separation. The spots identified as putrescine ran slightly behind the standard. The reason for this may be that the extracted putrescine has co-polymerized with other phenolics thus resulting in the observed lag. Another possibility is that these spots may be diamines other than authentic Putrescine (Put). Extractable diamine oxidase (DAO) activity increased steadily throughout grain development while polyamine oxidase (PAO) activity was higher during the early grain filling stages. The presence of DAO activity in chalazal cells over the period from 7-45 DPA suggests that it may be involved not only in lignin/suberin deposition (which appears to be restricted to the period 16-

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18 DPA) but also to the general provision of cell wall and subsequent separation of the apoplast from the symplast. Any role of polyamine oxidase (PAO) in the control of assimilate supply seems unlikely since no activity was detected in the chalazal cells. Peroxidase substrate in these cells occurs throughout grain development. It is noteworthy that activity could not be detected earlier than 30 days post anthesis (DPA) using isoelectric focusing (Asthrin et al., 2002). A possible correlation between Diamine oxidase (DAO) and polyamine oxidase (PAO) activities and the endogenous content of their preferential substrates has emerged. In developing barley grain, the highest DAO and PAO activities corresponded to the highest putrescine (Put) and spermdine (Spd) contents. DAO activity increased concomitantly with Put content (Scoccianti et al., 1990). This suggested that enzyme activity is not regulated by substrate supply. Very similar results were obtained previously at different physiological stages of Helianthus tuberosus tubers (Torrigiani et al., 1989) where it was shown that the increase in diamine oxidase (DAO) activity paralleled the accumulation of putrescine. It was suggested that a direct correlation between the biosynthesis and oxidation of putrescine and is associated with stages of intense metabolism such as, in the present case, organ formation. The proportion of wall bound diamine oxidase (DAO) and polyamine oxidase (PAO) activity was shown to vary from 2-24%. However, with the exception of PAO at 7 DPA, wall-bound activity was low (Asthrin et al., 2002). This suggested that there might be two forms of PAO activity present in developing barley grain, one soluble and the other cell wall bound. It seems likely that both enzymes exist in a single form, some of which may be in association with the cell wall. In Helianthus tuberosus tubers it was shown (Torrigiani et al., 1989) that a greater proportion (50%) of total recovered DAO activity was associated with the re-suspended pellet. It was suggested that the soluble and cell wall enzymes might play distinct roles. Thus, the apoplastic enzyme might have a direct role in wall stiffening and lignification and the cytoplasmic enzyme might have a role in polyamine regulation. Asthrin et al. (2002) demonstrated the presence of both Diamine oxidase (DAO) activity and its substrate in the chalazal cells of the crease

region at a time when lignin and suberin deposition is taking place. It seems likely then that the H2O2 required for the peroxidase activity, which may be involved in lignin/suberin synthesis is derived from DAO activity. Thus, it may be that DAO is involved in the regulation of the H2O2 supply in the cell walls/apoplast of chalazal cells. Extractable diamine oxidase (DAO) activity increased steadily throughout grain development while polyamine oxidase (PAO) activity was higher during the early grain filling stages. The presence of DAO activity in chalazal cells over the period from 7-45 days post anthesis (DPA) suggests that it may be involved not only in lignin/suberin deposition (which appears to be restricted to the period 16-18 DPA) but also to the general provision of cell wall and subsequent separation of the apoplast from the symplast. Any role of polyamine oxidase (PAO) in the control of assimilate supply seems unlikely since no activity was detected in the chalazal cells. Peroxidase substrate in these cells occurred throughout grain development. It is noteworthy that activity could not be detected earlier than 30 days post anthesis (DPA) using isoelectric focusing (Asthrin et al., 2002). A possible correlation between Diamine oxidase (DAO) and polyamine oxidase (PAO) activities and the endogenous content of their preferential substrates has emerged. In developing barley grain, the highest DAO and PAO activities corresponded to the highest putrescine (Put) and spermdine (Spd) contents.

6. A. IV. Nitric Acid and Nitrate Reductase

The two known substrates for NO synthesis in plants are nitrite and Argenine. It was known that nitrate reductase could reduce nitrite to NO (Dean and Harper, 1988; Klepper, 1990; Yamasaki and Sakihama, 2000; Rockel et al., 2002). It was shown that mitochondria also support nitrite-dependent NO synthesis (Tischner et al., 2004; Planchet et al., 2005). NO emission from wild-type plants or intact mitochondria is very weak in air and requires anaerobic conditions to detect strong signals. Other mechanisms of nitrite-dependent NO production include non-enzymatic reactions (Bethke et al., 2004) and a Ni–NO reductase activity detected in roots (Stohr et al., 2001). Arg-dependent nitric NOS characteristic of animal systems has also been reported in plants (Neill et al., 2003; Del Rio et al., 2004). Plant NOS activity can be inhibited by animal NOS

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inhibitors that act as Arg analogs, such as L-NAME and L-NMMA; however, no gene or protein with similarity to the full animal NOS proteins has been found. Recently, a gene (AtNOS1; referred to hereafter as NOS1) was identified that is needed for NO production in Arabidopsis thaliana (Guo et al., 2003). NOS1 has no sequence similarity to animal-type NOS isoforms yet catalyzes NO synthesis in vitro, indicating that NOS1 is a novel NOS. NOS1 was first identified by its similarity to a snail protein that was implicated in NO synthesis in an unknown way (Huang et al., 1997). In Arabidopsis, NOS1 is needed for efficient germination, root and shoot growth, seed fertility, and Abscisic acid–induced stomatal closure (Guo et al., 2003) and participates in the control of flower timing (He et al., 2004). NOS1 is needed for defense responses; nos1 mutants are more susceptible to a bacterial pathogen and show almost no response to lipopolysaccharide treatment by microarray analysis (Zeidler et al., 2004). Phosphatidic acid (PA) activates a calcium-dependent protein kinase (CDPK) and a wound-related mitogen activated protein kinase cascade (Lee et al., 2001; Farmer and Choi, 1999). NO activates a mitogen activated protein kinase and a calcium-dependent protein kinase (CDPK) during adventitious root formation (Pagnussat et al., 2004; Lanteri et al., 2006). The gaseous free radical nitric oxide (●NO) is a widespread intracellular and intercellular messenger with a broad spectrum of regulatory functions in many physiological processes (Wendehenne et al., 2001). The effects of nitric oxide on biological systems mentioned above are either regulatory or toxic (peroxy-nitrite formation), but a protective function for nitric oxide has also been proposed (Wink et al., 1996). ●NO are involved in many physiological processes, such as ethylene emission (Leshem and Haramaty, 1996), response to drought (Leshem, 1996), disease resistance (Delledonne et al., 2001; Clarke et al., 2000), growth and cell proliferation (Ribeiro et al., 1999), maturation and senescence (Leshem et al., 1996), apoptosis/programmed cell death (Zhang et al., 2003), and stomata closure (Garcia-Mata et al., 2003). It was proposed that peroxisomal ●NO could be involved in the process of senescence of pea leaves (Corpas et al., 2004). Specialized peroxisomes are root-nodule peroxisomes from certain tropical legumes, in which the synthesis of allantoin (Schubert, 1986).

6. A. V. Prohibitins

Arabidopsis has seven members of the PHB family, which can be divided into two groups: the PHB1 group (PHB3, 4, and 5) and the PHB2 group (PHB1, 2, 6, and 7) (Ahn et al., 2006; Van Aken et al., 2007).The highest similarity among these proteins is between PHB3 and PHB4, which show 89% identity. Alignment of the ArabidopsisPHB proteins along with two Saccharomyces cerevisiae PHBs shows extensive similarity among these proteins, especially in the region containing the SPFH/band 7 domain, which is a common feature shared by a super family of proteins that form multimeric complexes and interact with membranes and the cytoskeleton (Hoegg et al., 2009). The mutation in PHB3-3 is at the beginning of the SPFH/band 7 domain of PHB3. Of the Arabidopsis prohibitin genes, PHB3 is the most studied. It is expressed primarily in regions of active cell proliferation, including the root and shoot apices (Van Aken et al., 2007). Prohibitin was found to be induced by auxin and shows elevated expression in pericycle cells that give rise to lateral roots. PHB3 knockout mutants show severe growth defects and have decreased cell division and expansion in the root apex. PHB3 mutants also have larger, rounder mitochondria. Interestingly, phb4 single mutants display no phenotype (it is expressed at lower levels than PHB3), yet phb3/phb4 double knockout mutants are not viable, suggesting that these two genes have compensating functions, with PHB3 being the predominant gene. PHB3 also functions in ethylene signaling (Christians and Larsen, 2007). It was identified in a screen for ethylene hypersensitive mutants and it shows reduced hypocotyl length in the dark. The mutant eer3 is an ethylene overproducer in the dark, and has reduced induction of ethylene responsive genes. The eer3/phb3 mutation is epistatic to two ethylene insensitive mutations (ein2 and ein3), indicating that PHB3 functions downstream of these regulators (Christians and Larsen, 2007). Strong NO accumulation occurs when leaves are exposed to light (Gould et al., 2003). During our analysis of the phb3-3 mutants, we noticed that the normal increase in DAF-FM fluorescence observed during green light exposure in cotyledons was drastically reduced in the phb3-3 mutant compared with wild type. The cotyledons of 3-d-old wild type and 5-dold

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phb3-3 mutant plants (ages that have similar sizes) were stained with DAF-FM DA and then exposed to green light from the fluorescent microscope. Fluorescence increased rapidly in wild-type cotyledons during the light exposure, saturating after 1 min. By contrast, no detectable increase in fluorescence was observed in mutant cotyledons except in stomates during light exposure. This effect was specific to cotyledons, as true leaves show comparable increases in fluorescence in wild type and mutant plants (Wang et al., 2010). There is also linkage between NO and ethylene. NO and ethylene levels are negatively correlated during fruit ripening (Leshem and Pinchasov, 2000). However, PHB3 provide a potential explanation for the diverse phenotypes reported for phb3 mutants: PHB3 regulates the level of NO accumulation and thus affects diverse processes involving NO signaling. For example, PHB3 involvement in cell division and tissue proliferation (Van Aken et al., 2007) may be in part mediated via NO. For the case of auxin induced lateral root formation, NO induces the cell cycle gene CYCD3;1 and represses the inhibitory KRP2 gene. This response could be reduced in phb3 mutants due to lower levels of NO (Correa-Aragunde et al., 2006). It was recently reported that formation and elongation of adventitious roots in marigold Targetes erecta involves not only NO but also H2O2, which supports the linkage between tissue proliferation and PHB3-mediated NO accumulation (Liao et al., 2009). Many of the genes whose expression is altered by the phb3 mutation are significantly upregulated by oxidative or salt stress as well as other abiotic stresses (Van Aken et al., 2007). Prohibitins in general and PHB3 in particular, affect NO accumulation and NO-mediated processes. The phb3 mutations do not appear to affect H2O2 accumulation or signaling. Another article reported substantial increase in reactive oxygen species production and susceptibility in Nicotiana after suppressing PHB expression; however, this study used virus-induced gene silencing of Nicotiana PHBs so that it is difficult to compare findings of (Ahn et al., 2006). The result that phb3 mutations reduce the level of NO at lower but not higher levels of H2O2 suggests that a simple loss of NO synthesis cannot explain the mutant phenotype. A more complex mechanism that affects rates of synthesis or degradation resulting in reduced

NO accumulation in response to H2O2 is indicated. For example, disruption of mitochondrial function may result in decreased electron flux in the respiratory chain, resulting in less NO synthesis or more degradation. A model for this idea is the process of NO synthesis from nitrite via electron transfer, through the respiratory chain in root mitochondria during anoxia (Gupta et al., 2005; Benamar et al., 2008). PHB3 data provide further support for the involvement of NO signaling in developmental and abiotic stress responses in plants. NO accumulation in response to H2O2 is reduced in phb3 mutants, and, at the same time, ABA-induced stomata closure, auxin induced lateral root formation, and salt induced repression of primary root growth are also impaired (Wang et al., 2010). The roots of 7-d-old seedlings grown hydroponically in half-strength MS were treated with 1 mM IAA for 0.5 and 1 h. RNA was extracted from the roots and mRNA levels were determined by quantitative RT-PCR. The results revealed that gene ACT2 (ACTIN2; At3g18780) showed stable expression when treated with IAA and was used as an internal control and the three IAA genes have the same mRNA levels in wild-type and phb3-3 roots without IAA treatment. Fold induction values were determined as the ratio of treated versus control root mRNA levels with SD. Three-day-old wild-type and 5-d-old phb3-3 mutant seedlings grown in liquid media were treated with different concentrations of NaCl for 2 d. (A) NO induction in wild-type and mutant roots was tested after 10 min treatment with 200 mM NaCl using DAF-FM DA. Induction ratios were determined as the ratio of treated versus control root levels. (B) Increases in primary root length were calculated by subtracting primary root length at time zero from that measured after 2 d of the treatments (Wang et al., 2010). 6. A. VI. Ascorbate Peroxidase

Changes in H2O2 concentrations are important in developmental and hormone signaling (Kwak et al., 2003; Gapper and Dolan, 2006). H2O2 was first discovered in the early nineteenth century. The molecule is not a radical and is relatively unreactive with only weak oxidizing power. For example, the rate constant for uncanalyzed reaction of H2O2 with ascorbate is about 100 000 times slower than that of superoxide (Polle, 2001). It is evident that the overwhelming

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majority of the H2O2 that is produced must be metabolized since it has long been recognized that micromolar concentrations of H2O2 can inactivate thiol-regulated enzymes such as fructose-1,6-bisphosphatase and modulate the activities of metabolic pathways in isolated chloroplasts (Kaiser, 1979; Charles and Halliwell, 1980). It is unclear whether the H2O2 sensitivity of isolated enzymes or organelles is an accurate reflection of the sensitivity of photosynthesis in vivo, where reductants availability could be greater and H2O2 metabolism more powerful H2O2-metabolizing enzymes (Queval et al., 2008). Among the primary enzymes in H2O2 metabolism are ascorbate peroxidases (APX), whose existence was demonstrated in isolated thylakoids and chloroplasts by Groden and Beck (1979). Subsequently, the notion that catalase is an important enzyme in H2O2 metabolism inside chloroplasts became discounted and it is now accepted that APX functions alongside peroxiredoxins as a major H2O2-metabolizing enzymes in this organelle (Asada, 1999; Dietz, 2003). Leaf H2O2 contents in the range of 1 µmol g-1 FW or greater could reflect preferential accumulation of H2O2 concentrations in specific compartments. With a pKa value of 11.6 for deprotonation to HO−

2, H2O2 is the overwhelmingly predominant form at physiological pH and its movement across membranes is facilitated by specific aquaporins (Henzler and Steudle, 2000; Bienert et al., 2007). On the basis of data obtained with knockout mutants for a cytosol APX form, it has been suggested that some part of the H2O2 produced within the chloroplast could diffuse into the cytosol to be metabolized (Davletova et al., 2005). Ascorbate peroxidase (APX) has high affinity for H2O2 (Km around 20-50 µM: (Chen and Asada, 1989; Yoshimura et al., 1998). The Km H2O2 of a chloroplastic peroxiredoxins has been estimated at about 2 µM. The affinity of an enzyme for its substrate is not proof of the substrate’s concentration range in vivo, Km values that are close to or higher than typical substrate concentrations allow catalytic activity to accelerate rapidly in response to increasing availability of substrate. Such properties act both to drive metabolic flux and to damp increases in substrate pools, in response to increases in substrate production. They may be important in controlling the accumulation of molecules such

as H2O2, whose rate of production likely shows rapid changes in response to factors such as fluctuating irradiance (Queval et al., 2008). If substrate concentrations are higher than enzyme Km values, the enzyme activity has more limited sensitivity to increases in substrate. For chloroplast APX, this may well be the case for ascorbate, since Km values are within the range 0.2–0.4 mM, probably significantly below ascorbate concentrations in this compartment (Chen and Asada, 1989; Yoshimura et al., 1998). Ascorbate concentration has little influence over chloroplastic (APX) activity has come from analysis of ascorbate deficient mutants (Muller-Moule et al., 2002). The catalytic rate of ascorbate peroxidase (APX) is similarly insensitive to changes in H2O2

concentrations then other mechanisms such as increased enzyme abundance or post-translational modification would be necessary to allow flexibility in enzyme activity, in response to fluctuations in H2O2 production. Increases in extra chloroplastic ascorbate peroxidase (APX) capacities in ascorbate-deficient lines could reflect a compensatory response to limitation by cytosol ascorbate concentration (Veljovic-Jovanovic et al., 2001). Mitochondria contain an APX isoform encoded by the same gene as the stromal APX (Chew et al., 2003). In the cytosol, APX isoforms so far characterized have Km H2O2 values around 20 µM (Yoshimura et al., 1998). The primary physiological function of these enzymes is the maintenance of H2O2 homeostasis; their properties could be taken as circumstantial evidence for H2O2 concentrations in the micromolar range. Plant cells are more tolerant to H2O2 than other types of cell or most measured leaf contents provide information of little relevance to H2O2 concentrations in compartments such as the chloroplast and mitochondria. Pathways that generate H2O2 in bacteria and animal cells, plants produce H2O2 through specific metabolic pathways such as photosynthesis and photorespiration (Queval et al., 2008). However, contents higher than 2 µMol g-1 FW have been reported for dark-grown maize seedlings (Prasad et al., 1994). 6. A. VII. Metallothioneins Metallothioneins (MTs) are cysteine-rich, low molecular weight intracellular proteins that were initially shown to regulate the metabolism of

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metals such as zinc, copper, and cadmium, and play a role in heavy metal tolerance (Lanfranco et al., 2002; Palmiter, 1998). A number of investigations have demonstrated metallothioneins (MTs) as being efficient scavengers of ROS production in animals (Li et al., 2006; Dong et al., 2007; Peng et al., 2007). During oxidative stress, MTs protect against ROS-induced DNA degradation with higher molar efficiency than glutathione (Jourdan et al., 2004). Plants contain a multiple metallothioneins (MT) gene family in which different types may play distinct and overlapping biological roles by the regulation of gene expression or signaling networks. In Arabidopsis, all four types of metallothioneins (MTs) provided similar levels of Cu tolerance and accumulation to the yeast mutant Dcup1 (Lee et al., 2004; Guo et al., 2008). Cu2+, Ag+, Cd2+, Zn2+, and Ni2+ all induced significant levels of Arabidopsis MT2 gene expression; however, MT1 in Arabidopsis could not be induced by these ions except for Cu2+ in excised leaves (Zhou and Goldsbrough, 1994; Murphy and Taiz, 1995). Expression of LSC54, a rape metallothioneins (MT1) gene, was proven to be induced by ROS production and related to the misbalance of ROS during leaf senescence (Navabpour et al., 2003), and transgenic Arabidopsis plants overexpressing cgMT1 from beefwood (Casuarina glauca) reduced the accumulation of H2O2 (Obertello et al., 2007). In addition, OsMT2b may also function as a ROS scavenger involved in the response to bacterial blight and blast fungus infections in rice (Wong et al., 2004). A type 3 MT encoding cDNA, GhMT3a, was isolated from a NaCl induced cotton cotyledons cDNA library. The up-regulation of GhMT3a expression was observed in cotton seedlings treated not only with high salinity but also with drought and low temperature (Xue et al., 2009). Interestingly, the levels of GhMT3a in cotton seedlings were markedly increased by H2O2 and PQ treatment. The induced expression of GhMT3a by these abiotic stresses could be completely inhibited in the presence of 1500 lM NAC, an antioxidant. Just as in the case of GhMT3a, NAC also decreased the levels of H2O2 in cotton seedlings, indicating that there is a high correlation between the expression of GhMT3a and the misbalance of ROS production in cotton and GhMT3a may act as an antioxidant to minimize ROS toxicity, which was further

confirmed by overexpressing GhMT3a in tobaccos and yeast. Transgenic tobaccos displayed high tolerance against salt, drought, and low temperature stresses, and their H2O2 levels were only half of that in WT plants. Transgenic yeast overexpressing GhMT3a showed more tolerance to ROS toxicity than the control. The purified GhMT1 protein from E. coli exhibited antioxidative capacity in vitro when no other metals and other antioxidants were applied. A number of studies have proved that the cysteine ligands in proteins are remarkably reactive towards oxidizing agents (Chae et al., 1994; Haslekas et al., 2003; Maret, 2004; Hao and Maret, 2006), including MTs (Zhou et al., 2002; Maret, 2004; Hao and Maret, 2006). Therefore, it could be concluded that Gossypium hirsutum (GhMT1) acts as an endogenous antioxidant to respond to ROS stress in a direct manner. It was accepted that high levels of ROS lead to phytotoxicity, while relatively low levels can be signals inducing ROS scavengers and other protective mechanisms in plants (Couee et al., 2006; Gadjev et al., 2006; Miller et al., 2007). These results strongly support the idea that ROS signaling is indispensable for the regulation of GhMT3a expression during environmental stresses in plants. (Antioxidant) The fact that Gossypium hirsutum metallo gene(GhMT3a) had antioxidant ability in vitro indicated the function of GhMT3a as a ROS scavenger, revealing that plant metallothioneins play important roles as do their animal counterparts (Mattie and Freedman, 2004; Hao and Maret, 2006). A number of transgenic plants or mutants with higher ROS scavenging ability showed increased tolerance to environmental stresses (Avsian-Kretchmer et al., 2004; Moradi and Ismail, 2007), it is proposed that the higher tolerance against abiotic stresses in transgenic tobaccos might be due to the scavenging of ROS production by the overexpression of GhMT3a. In addition, previous studies demonstrated that ROS may act as second messengers in redox signal transduction and implicated in hormonal mediated events (Guan et al., 2000; Zhang et al., 2001). Thus, the ROS signal may also be the intermediate for the induced expression of GhMT3a by ABA and ethylene in our study. The effects of metal ions on the expression of plant MT genes vary with

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plant species, tissues, and types of metallothioneins (MTs) genes (Foley et al., 1997; Chang et al., 2004; Bellion et al., 2007). However, very little known about the mechanism for the regulation of plant metallothioneins (MTs) gene expression by metal ions. In this study, GhMT3a showed a high affinity to Zn2+ in vitro. The cysteine ligands in proteins are reactive towards oxidizing agents and release zinc (Maret, 2004; Hao and Maret, 2006). When released from metallothioneins (MTs), zinc may become available for the synthesis of antioxidant metal binding proteins, such as Cu, Zn-superoxide dismutase, and at the same time be part of a mechanism that conducts spatial regulation of the oxidoreductive environment in the cell (Liochev and Fridovich, 2004). There is evidence that metallothioneins (MTs) release bound metals during oxidative stress and trigger a Zn-mediated antioxidant response in mammals and fungi (Maret, 1994; Tucker et al., 2004). The zinc-released can function as a reducing agent because of its high content of cysteine or rebind zinc under reducing conditions. Therefore, metallothioneins (MTs) may interact with other metal proteins, through releasing zinc in cells, in response to spatial or temporary changes in the redox environment. This might be another function of metallothioneins (MTs) in plant. Taken together, the results indicate that the rapid accumulation of ROS in cotton plants after abiotic stresses (high salinity, drought, and low temperature) and the application of ABA or ethylene will induce the expression of GhMT3a. As a ROS scavenging, accumulation of GhMT3a during defense signaling would diminish ROS damage and then increase the tolerance of plants against abiotic stresses (Xue et al., 2009). 6. A. VIII. Catalases

Under highly repressive conditions for alkaloid synthesis, including normal culture conditions in the light, ROS scavengers like dimethyl thiourea (DMT) or catalase (CAT), both resulted in significant induction of Putrescine N-methyltransferase (PMT) promoter activity. Moreover, treatment of callus with catalase resulted in the upregulation of PMT promoter activity and alkaloid accumulation in this tissue. These results suggest that ROS affects the regulation of the alkaloid pathway in undifferentiated cells and have implications for

regulation of the pathway in other plant tissues (Sachan et al., 2010). In cell CAT interacts with non-receptor tyrosine kinases: c-Abl and Arg (Cao et al. 2003). When the H2O2 concentration reaches 0.25–1.0 mM, CAT is phosphorylated by c-Abl and Arg, resulting in an increase in its activity. In post-germinative white spruce megagametophyte cells, one CAT that reacted with antibodies specific for cotton seed CAT was phosphorylated on tyrosine residue(s), a modification that stimulates its activity. Furthermore, tyrosine phosphorylation was enhanced when the H2O2 level was elevated. Thus, at least in white spruce megagametophyte cells, tyrosine phosphorylation of CAT appears to be one aspect of ROS signaling. At present, we do not know the site(s) of tyrosine phosphorylation on the white spruce CAT (He and Kermode, 2010). Signal transduction by protein tyrosine phosphorylation was identified. It appears to be involved in ABA signaling (Ghelis et al., 2008), but this process is not well understood in plants. Mitogen-activated protein kinases (MAPKs) are subject to regulation by tyrosine phosphorylation (Gupta and Luan, 2003). 6. B. Oxidant Mediate Differenciation of

Reproductive Organs

Participation of ROS molecules in various biological processes has been demonstrated in growth, flowering, cell cycle progression, senescence and biotic and abiotic stress responses (Mittler et al., 2004; Foyer and Noctor, 2005; Moller et al., 2007; Miller et al., 2008; Wagner et al., 2004; Zimmermann and Zentgraf, 2005). Some of the spontaneous and rapid chain reactions of RNS and ROS may scavenge some toxic radicals and protect cells from oxidative or nitrosative damaging (Rubbo et al., 2000). Guo and Crawford (2005) showed that NO● production deficiency in AtNOS1 mutant resulted in unbalanced accumulation of ROS. Higher NO● production obviously inhibited lipid peroxidation (Zhao, 2007). This often observed concentration dependent function changes may reflect the importance of balance between RNS and ROS and their interactions. The different fates resulted from these reactive free radicals also depend on physiological environments that plant cells are in details and mechanisms for these aspects remain to be elusive (Delledonne et al., 2001; Espey et al.,

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2000). In angiosperm flowers, ROS are present in pollen and stigmas (Mclins et al., 2006) and regulate pollen tube growth (Potocky et al., 2007), but they might also have further functions including self-incompatibility and pathogen defense (Mclins et al., 2006). Wrzaczek et al. (2009) characterized a component involved in the regulation of stress-induced cell death in plants. The GRIM REAPER (GRI) protein is an Arabidopsis ortholog of the tobacco stigma-specific protein 1 (STIG1) (Goldman et al., 1994). STIG1 helps to regulate exudate secretion in the pistils of petunia and tobacco (Verhoeven et al., 2005). Tomato LeSTIG1 binds to the extracellular domain of pollen receptor kinases and promotes pollen tube growth in vitro (Tang et al., 2004). No roles for STIG1 have been described in vegetative tissues or in the regulation of cell death. The GRI protein is related to tobacco STIG1, which is predominantly present in tobacco stigma and required for stigmatic lipid exudate secretion (Verhoeven et al., 2005). The tomato ortholog, LeSTIG1, binds the extracellular domain of 2 pollen receptor kinases in vitro (Tang et al., 2004). Consistent with a role for STIG1 in flowers, GRI transcript abundance is very low in Arabidopsis leaves and significantly higher in flowers. Genetic and functional analyses of homeotic mutants with changed floral organ identities in the model dicot plants Arabidopsis thaliana and Antirrhinum majus led to the ABC model, which explains how the stamen is specified by the combinatorial action of class B, C, and E genes (Coen and Meyerowitz, 1991; Ditta et al., 2004). Rice B-class gene, SUPERWOMEN1 (SPW1 or MADS16), which is orthologous to the ArabidopsisAPETALA3 gene, has been shown to be crucial for stamen specification (Nagasawa et al., 2003). MADS box genes, such as SPOROCYTELESS/NOZZLE (SPL/NZZ) (Yang et al., 1999; Schiefthaler et al., 1999) and AG (Ito et al., 2007) from Arabidopsis and MADS2 from maize (Zea mays; Schreiber et al., 2004), have been implicated in regulating anther development. SPL/NZZ regulates the formation of anther walls and pollen mother cells, as the primary sporogenous cells cannot form pollen mother cells in spl anthers, thereby blocking early cell differentiation (Yang et al., 1999). spw1 mutants were found to show homeotic conversions of stamens to carpels and lodicules to palea/lemma-like structures. AG has been

shown to activate the expression of SPL/NZZ, suggesting that this gene is necessary for early stamen development (Ito et al., 2004). In Arabidopsis, the C-class gene AGAMOUS (AG) acts to specify stamen and carpel identities and floral meristem determinacy (Yanofsky et al., 1990; Bowman et al., 1991). Studies in rice identified two C-class MADS box genes, MADS3 and MADS58, that may have distinct functions in specifying stamen identity, with MADS3 playing a more important role (Yamaguchi et al., 2006). During later developmental stages, AG continues its expression in the anther and regulates anther dehiscence by directly regulating the expression of the gene that encodes a jasmonic acid (JA) synthetic enzyme, DEFECTIVE IN ANTHER DEHISCENCE1 (Ito et al., 2007). Maize MADS2 is required for anther dehiscence and pollen maturation, and knockdown of MADS2 resulted in abortion of anthers and defective pollen development (Schreiber et al., 2004). The cellular level of ROS is thus, tightly regulated by an efficient and elaborate system, which modulates the production and scavenging of ROS. However, plants regulate ROS levels according to cellular needs at different developmental stages, and within different cell types and organs, remains poorly understood (Hu et al., 2011). They showed that MADS3 has a critical role in regulating rice late anther development via modulating ROS homeostasis. MADS3 is expressed during late anther development, in the tapetum and microspores. The rice mads3-4 mutant is male sterile, contains aborted anther walls, and shows disrupted pollen development due to oxidative stress. The abnormal increase in ROS level, peroxisome-like organelles, superoxide dismutase, and peroxidase activities in the mads3-4 mutant during later anther development is likely the result of the altered expression of genes involved in maintaining ROS level. Hu et al. (2011) demonstrated that MADS3 is able to bind to the promoter region of a metallothioneins gene, MT-1-4b, which encodes a small Cys-rich and metal binding protein in a ROS-scavenging pathway. These findings together provide insight into the role of MADS3 in regulating male reproductive development and show that it acts, at least in part, through regulating ROS homeostasis. Previous investigations showed that the rice floral homeotic C-class gene, MADS3, is

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expressed in stamen and carpel primordial during early floral development, and that the protein it encodes is crucial for stamen specification (Kang et al., 1998; Yamaguchi et al., 2006). Hu et al. (2011) characterized a mutant allele of MADS3 (mads3-4), further analyzed the expression pattern of this gene, and revealed an important role for MADS3 in regulating postmeiotic anther development in rice. It was reported MADS3 alleles; both mads3-1 and mads3-2 are in the Nipponbare (a japonica cultivar) background and contain Tos17 retrotransposon insertions in the gene. mads3-1 is a weak allele carrying an insertion in the C-terminal region of the gene and showing no defective floral organs, whereas mads3-2 is an intermediate allele with a mutation at the C terminus of the K-domain and reduced expression of the gene, which resulted in mild transformation of stamens into lodicules (Yamaguchi et al., 2006). mads3-3 is a strong allele in the Dongjin (a japonica cultivar) background. It contains a T-DNA insertion in the second intron of the gene, which leads to no detectable MADS3 transcript and homeotic transformation of nearly all stamens in whorl 3 into lodicule-like organs (Yamaguchi et al., 2006). mads3-4, which results in a mutation in the middle region of the K-domain of MADS3, seems to be another intermediate mutant with reduced expression of MADS3. In contrast with mads3-3, the allele introduced is in the 9522 (a japonica cultivar) background and is male sterile. These phenotypic differences between the mads3 alleles may be caused by differences in genetic background and/or the nature of the mutations in MADS3. Moreover, the possibility that mads3-2 also has defective anther development because in the previous report on mads3-2, the authors did not perform a detailed observation of anther development (Yamaguchi et al., 2006). The finding that MADS3 is important for postmeiotic anther development in rice is consistent with the expression pattern of MADS3 in the tapetum and microspores from stage 9 to stage 12. Furthermore, transcriptome analyses demonstrated that MADS3 affects the expression of 1728 genes, many of which are associated with cellular processes and signaling, carbohydrate transport and metabolism, secondary metabolite biosynthesis, detoxification, and transcriptional regulation.

Among these 1728 genes, such as TDR (Li et al., 2006; Zhang et al., 2008), CYP703A3 (Aya et al., 2009), CYP704B2 (Li et al., 2010), C6 (Zhang et al., 2010a), MST8, and INV4 (Oliver et al., 2007), have been shown to be involved in anther development, suggesting that MADS3 may regulate anther development by affecting the expression of these genes. Unlike MADS3, the Arabidopsis C-class gene AG is expressed from anther initiation to anther maturation in sporogenous cells, the connective, anther walls and the filament, and no AG expression was detected in pollen grains (Bowman et al., 1991). The distinct spatial and temporal expression patterns of AG and MADS3 suggest that these two proteins have diversified functions in anther developmental stages. AG plays a key role in regulating early anther development by activating the expression of the MADS box gene SPL/NZZ (Ito et al., 2004) and in late anther development and anther dehiscence by regulating JA synthesis (Ito et al., 2007). Besides MADS3 and AG, there are other MADS box genes reported to be expressed in anther development in rice (Hobo et al., 2008), maize (Schreiber et al., 2004), Arabidopsis (Alvarez-Buylla et al., 2000), and Antirrhinum (Zachgo et al., 1997), indicating important roles for MADS box genes in plant anther development and pollen formation. The steady state ROS level, which is important for normal development processes, is determined by the interplay between ROS-producing and ROS-scavenging mechanisms (Gapper and Dolan, 2006; Gechev et al., 2006; Miller et al., 2008). In rice, however, functional aspects of ROS-related genes remain less understood. It was observed that expression changes in 51 genes with putative functions in the ROS network in mads3-4 anthers, suggesting that MADS3 may play a role in adjusting ROS homeostasis during anther development. Consistent with the gene expression profile analysis, mads3-4 has high ROS accumulation in the anther, starting from stage 9, when young microspores form. Furthermore, mad3-4 tapetal cells at stages 10 to 12 display features characteristic of those associated with oxidative stress, including less cytoplasmic condensation, fewer mitochondria, and more peroxisome-like organelles. Increased ROS might be associated with the decreased expression of ROS-scavenging genes, including those encoding MTs and peroxidase.

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The increased number of peroxisomes in mads3-4 tapetal cells and elevated peroxisome-associated enzyme activities, such as superoxide dismutase and peroxidase, may in part result from higher ROS levels during late anther development (Hu et al. 2011). They reported that in vivo and in vitro analyses suggested that MADS3 may directly regulate a ROS-removal gene, MT-1-4b. An LM-mediated microarray analysis revealed the expression of MT-1-4b in the tapetum and microspores during late anther development (Hobo et al., 2008), an expression pattern that is similar to that of MADS3 in rice anther development. The biological importance of the regulation of MT-1-4b by MADS3 is further supported by the biochemical activity of the recombinant MT-1-4b protein in removing the superoxide anion and hydroxyl radical. Moreover, knockdown of MT-1-4b led to reduced pollen fertility and abnormal accumulation of superoxide anion in the transgenic lines, which is similar to the phenotypes in mads3-4 during late anther development. Lastly, we found changes in the expression of a few other genes encoding proteins putatively involved in ROS removal in mads3-4, suggesting that these genes may also encode components of a ROS-scavenging network that modulates late anther development. Hu et al. (2011) have demonstrated a role of the floral homeotic C-class gene MADS3 in regulating late anther development and pollen formation. They have shown that this protein functions, at least in part, by regulating ROS homeostasis in anthers through the ROS-removal protein MT-1-4b. Although it remains to be seen whether MADS3 functions in late anther development through other cellular targets, our study sheds light on the molecular mechanisms underlying the genetic control of postmeiotic tapetum degeneration and development. 6. C. Role of Oxidants in Symbiosis

6. C. I. Nodulation Factor The symbiosis between legumes and Rhizobia is a complex process relying on finely tuned infectious and developmental events. The initial step of the symbiotic interaction is a chemical crosstalk between the plant and the bacterial partners leading to the production of bacterial nodulation factors (NF) upon sensing of the flavonoids present in root exudates. Nodulation

factors (NF) not only participate in bacterial infection, but also trigger the initiation of a specific developmental program ending in the formation of a new organ, the nodule (Oldroyd et al., 2005). The dedifferentiation and division of root cortical cells assure nodule formation and the bacteria released from the infection threads formed upon infection (Pauly et al., 2006) subsequently colonize nodules. During the first minutes of interaction between plants and microorganisms, a molecular dialogue involving several signal molecules, takes place in the rhizosphere and at the cell surface, leading to physical interaction. For example, in the case of the Legume – Rhizobia symbiotic interaction, flavonoids from the plant root exudates induce the synthesis of Nodulation Factor (NF) from Rhizobia. Both compounds are responsible for the setup of the early interaction steps and for the establishment of the new root organ, the nodule (Oldroyd and Downie, 2004). A similar dialogue is observed during mycorrhizal fungus and plant interaction leading to the production of plant strigolactons (Akiyama et al., 2005) and putative Myc factor by the fungus (Kosuta et al., 2003). The symbiosis between legumes and compatible Rhizobia takes place in a nitrogen limited environment where a molecular dialogue between them. Rhizobia secret Nod Factors (NFs) in response to plant root exudates containing flavonoids. The perception of these NFs by the plant triggers several responses such as ion changes, cytoplasmic alkalization, calcium oscillations and gene expression leading to the formation of an infection thread and a new organ, the root nodule, containing the nitrogen fixing rhizobia bacteroid (Cardenas et al., 2000; Oldroyd and Downie, 2004). During the last few years, significant progress were made in understanding nod factor transduction several nodulation factors (NF) receptor candidates have been identified (Spaink, 2004). Moreover, a series of genes involved in signaling processes acting downstream of Nodulation factors (NF) perception was characterized (Geurts et al., 2005; Stacey et al., 2006). However, elements integrate to form the complex signaling network regulating symbiotic interaction are still unknown. Hitherto, there is increasing evidence that ROS, RNS and/or GSH play an important role in legume rhizobia symbiosis (Herouart et al., 2002). As for plant pathogen interactions,

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they may be important actors in the initial interaction between the two partners, which leads to the recognition of the bacteria as a partner or as a foe. Indeed, 90% of the infection threads initially formed abort in Medicago. Such abortion is achieved by a hypersensitive-like response, well-known ROS and RNS regulated process during plant pathogenesis (Vasse et al., 1993; Penmetsa and Cook, 1997). Compatible interaction initiated by a molecular dialogue between the plant and bacterial partners, leads to the formation of a novel root organ capable of fixing atmospheric nitrogen under nitrogen limiting conditions. ROS/RNS detection during the symbiotic process highlights the similarity of the early response to infection by pathogenic and symbiotic bacteria, addressing the question as to which mechanism Rhizobia use to counteract the plant defense response. Moreover, there is increasing evidence that ROS are needed to establish the symbiosis fully (Pauly et al., 2006). On the other hand, reduced glutathione (GSH) synthesis appears to be essential for proper development of the root nodules during the symbiotic interaction. Elucidating the mechanisms that control ROS/RNS signaling during symbiosis could therefore contribute in defining a powerful strategy to enhance the efficiency of the symbiotic interaction. ROS production might also have a signaling role during symbiosis. Indeed, a S. meliloti mutant over expressing the constitutive catalase, KatB, which exhibits the highest affinity for H2O2 among the three catalases, was constructed in this laboratory (Ardissone et al., 2004). The katB++ resulting strain was able to degrade H2O2 very rapidly and displayed an intracellular H2O2 concentration below that of the wild-type strain. This clearly indicates the complexity of the process. Moreover, as ROS are produced by the plant partner, it would also be of interest to analyze the consequences of modifying plant ROS-scavenging or ROS-producing activities on the symbiotic capacities (Pauly et al., 2006). 6. C. II. Role of Nitrogen Reactive Species (RNS)

In Nitrogen Fixation

Mesa et al. (2003) showed that, in B. japonicum, ●NO could regulate specific transcription factors such as NnrR, which control denitrification gene expression. This activation would involve the FixLJ pathway, which was activated by low oxygen concentrations and can readily fix ●NO (David et al., 1988; McGongile et al., 2000). A

series of genes related to the plant response to hypoxia was recently isolated in a screen for M. truncatula genes induced upon ●NO treatment. As hypoxia related genes are also regulated in fixing L. japonicus nodules, this may extend the hypothesis of ●NO regulating the low oxygen response to both symbiotic partners (Colebatch et al., 2004). ●NOS-like activity has been detected in plant extracts during the interaction of Rhizobium-legumes (Cueto et al., 1996) and fungi plants (Ninnemann and Maier, 1996), in soybean cell suspensions and Arabidopsis (Delledonne et al., 1998), and in tobacco leaves (Durner et al., 1998). The presence of large amounts of the O2 carrier leghaemoglobin, which has a high affinity for ●NO, can act as a ●NO scavenger, which may modulate ●NO bioactivity (Herold and Puppo, 2005). ●NO scavenger may be a function of non-symbiotic leghaemoglobin, which scavenge ●NO in plants (Romero-Puertas et al., 2004). Therefore, this scavenging is rapidly induced upon symbiotic infection and accumulates in fixing nodules (Shimoda et al., 2005; Vieweg et al., 2005). It was shown that leghaemoglobin overexpression in alfalfa roots efficiently prevented the inhibition of aconitase, a ●NO sensitive enzyme, by exogenous and endogenous ●NO generation (Igamberdiev et al., 2005). The cell redox balance (Vernoux et al., 2000) tightly regulates the cell mitotic cycle. Therefore, nodule organogenesis may be deeply dependent on the cellular redox conditions. More generally, nodulation efficiency is highly dependent on plant fitness (Oldroyd et al., 2005), which may be modulated by ROS and RNS formation and the antioxidant content. This is important when considering that the active nodule metabolism involved in the nitrogen fixation may itself provoke the formation of ROS, which will impair nodule functioning (Puppo et al., 2005). ●NO may also be involved in legume–rhizobia symbiotic interactions where, several studies have reported direct or indirect evidence for the production of ●NO during plant symbionts interactions (Pauly et al., 2006). Shimoda et al. (2005) suggested that a rapid and transient ●NO production, detected with the permanents NO-sensitive probe 4,5 diamino fluorescein diacetate, occurs in Lotus japonicus roots inoculated with Mesorhizobium loti (Shimoda et al., 2005). Using the same approach, such production was not observed during M. truncatula–S. meliloti interaction. The ●NO production in L. japonicas may reflect a

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specificity of this symbiotic model. The presence of ●NO during the early stages of symbiosis remains puzzling, its production in mature nodules has been clearly shown. 6. C. III. Role of Reactive Oxygen Species in

Nitrogen Fixation The plant provides bacteria with energy and a micro-aerobic environment compatible with nitrogenase activity. In exchange, bacteria provide the plant with a nitrogen supply. Nodules represent therefore a unique model for the study of developmental processes, plant-microorganism and carbon/nitrogen/oxygen metabolism interactions (Nanda et al., 2010). In the very early stages of the symbiotic interaction, the production of H2O2 appears to be inhibited by the Nod factors (Shaw and Long, 2003); in the same way, a S. meliloti nod C mutant, defective in Nod factor biosynthesis, showed an increase in H2O2 accumulation (Bueno et al., 2001). Moreover, the compatible interaction between M. sativa and S. meliloti is linked, at least in part, with an increase in antioxidant defense particularly, catalase and lipoxygenase during the up to 12 hrs pre infection period (Bueno et al., 2001). Simultaneous treatment of an alfalfa suspension culture with yeast elicitors and S. meliloti lipopolysaccharides (LPS) was unable to induce an alkalinization of the culture medium or an oxidative burst that is systematically observed when the cells aretreated with yeast elicitors alone. Thus, S. meliloti lipopolysaccharides (LPS) released from the bacterial surface might function as a specific signal molecule, promoting the symbiotic interaction and suppressing a pathogenic response in the host plant, alfalfa (Albus et al., 2001). The involvement of ROS in the establishment of the legume Rhizobium symbiosis was underlined; supporting the hypothesis that symbiosis and pathogens are variations on a common theme (Baron and Zambryski, 1995). However, the situation differs according to the infection time course where, in the early stages of the symbiotic interaction, the oxidation of nitro blue tetrazolium (NBT) can be detected in infection threads, indicating that ●O2

─ is produced during the infection process (Santos et al., 2001; Ramu et al., 2002). Addtionally, this production was not observed when M. truncatula plants were inoculated with a S. meliloti nodD1ABC mutant that was unable to produce

Nod factors, suggesting that they have a role in the oxidative burst (Ramu et al., 2002). ROS accumulation is observed during the infection process and during the degeneration of the symbiotic association. It is, however, important to note that in the first hours of the infection, the production of ROSis inhibited. In the later stages of the nodulation process, however, ROS such as H2O2 and ●O2

─ can be observed in infected cells of young nodules, revealing the prolonged production of these radicals during nodule development. H2O2 production was detected in ultra-thin sections of mature 6-week-old nodules as an electron-dense precipitate stained with cerium chloride (Rubio et al., 2004). Therefore extensive cerium labeling was observed in the cell walls of infected cells and in some infection threads all around bacteria. In functioning nodules, leghaemoglobin autoxidation appears to be an important source of ROS. In planta, the mutant displayed a delayed nodulation phenotype, Nodd, associated with cytological modifications. These last results clearly indicate that the presence of H2O2 is essential for optimal symbiosis development. It still remains to define more precisely the role of H2O2 in the infection thread development including bacterial proliferation. An open question concerns the enzymes responsible for enhanced ROS formation during infection and nodule organogenesis, which have not been identified yet. The superoxide radicals are formed in the infection threads possibly by a membrane bound NADPH oxidase, as observed in activated neutrophils (Santos et al., 2001). Indeed, inhibition of the oxidative burst with DPI (a specific suicide inhibitor of the NADPH oxidase) corroborates this hypothesis (Shaw and Long, 2003; Rubio et al., 2004). Moreover, other possible sources for H2O2 are cell wall peroxidases, germin-like oxalate oxidases, and diamine oxidases (Wisniewski et al., 2000). Reactive oxygen species (ROS) production in the infection threads is dependent on the NFs production because no ROS was observed when Medicago truncatula plants were inoculated with bacteria unable to produce Nodulation factor (NFs) (Ramu et al. 2002). Jamet et al. (2007) showed that an S. meliloti mutant impaired in H2O2 steady state is affected in its ability to establish an optimal symbiosis. This clearly indicates the role of H2O2 in the early steps of the interaction. Moreover, the generation of ROS in the cortical cells of M. truncatula roots after inoculation with

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Sinorhizobium meliloti was observed in vivo, using a ROS fluorescent probe (Peleg-Grossman et al., 2007). A transient increase in intracellular ROS level at the tip of growing Phaseolus vulgaris root hairs has been shown within a few minutes after treatment with NFs (Cardenas et al., 2008). However, the extracellular ROS situation may be different in the very early steps of the symbiotic interaction, where the production of H2O2 appears to be inhibited by NFs treatment (Shaw and Long 2003; Lohar et al., 2007). The role of ROS in root hair deformationin the M. truncatula – S. meliloti symbiosis has been highlighted. Exogenous application of ROS as well as the inhibition of ROS production using diphenylene iodonium (DPI), a commonly used NADPH oxidase inhibitor, both prevented root hair swelling and branching normally induced following treatment with NFs. However, transient treatment of M. truncatula roots with diphenylene iodonium (DPI), mimicked Nodulation factors (NFs) treatment and resulted in root hair branching in the absence of NFs. Interestingly, the same transient diphenylene iodonium (DPI) treatment on non-legumes such as Arabidopsis thaliana and Lycopersicon esculentum did not induce root hair branching. These results suggest a role for the transient reduction of ROS accumulation in governing NF-induced root hair deformation in legumes (Lohar et al., 2007). The transient decrease of intracellular ROS accumulation in legume root hairs, in response to rhizobial secretion of Nodulation factors (NFs), seems to play a key role in a compatible Legume-Rhizobium interaction by actively promoting the root infection by bacteria. However, without recognition of the NFs or by using non host NFs, the plant seems to consider the bacteria as a pathogen and mobilizes its defense mechanisms (Nanda et al., 2010).

6. C. IV. Oxidant Role On Mycorrhizal

Most land plants can form mutuality symbiosis with mycorrhizal fungi, which can be divided into two categories: ectomycorrhizae (EM) with extracellular hyphal structures and endomycorrhizae or arbuscular mycorrhizae (AM) with intracellular hyphal structures (Bonfante and Anca, 2009). In AM symbioses, fungal hyphae form appressoria at the root surface, before intercellular invasion of epidermal and root cortical cells (Harrison,

2005). Intensive nutrient exchange takes place across membrane interfaces between the fungus and the plant during symbiosis. The fungus provides the plant with nutrients like phosphorus, which the plant can have difficulty extracting from the soil. In return, the plant delivers carbon and lipids to the fungal symbionts (Nanda et al., 2010). As observed during the other plant-microorganism interactions, ROS have also been evidenced in mycorrhizal symbiosis: in the M. truncatula – Glomus intraradices interaction, H2O2

accumulation observed in plant cells was hypothesized to be a consequence of activation of a plant plasma membrane NADPH oxidase (NOx) in response to the fungus (Salzer et al., 1999).H2O2 accumulation is mostly observed in arbuscule-containing cells, more precisely surrounding the arbuscular structures. This suggests that ROS play a role in the control of fungal proliferation within the plant (Fester and Hause, 2005). H2O2production was reported during the symbiosis between Gigaspora margarita and two legume species including M. truncatula and Lotus japonicus, mainly located in the intra-radical fungal structures (Lanfranco et al., 2005). Evidence for the participation of ROS and antioxidant systems in ectomycorrhizal symbiosis has been found between the fungus Pisolithus tinctorius and the plant Castanea sativa (Baptista et al. 2007). During the early stages of this symbiosis, three peaks of H2O2 production were detected in C. sativa 2 h, 5 h and 11 h post inoculation, the first two coinciding with ●O2

−bursts. It is noteworthy that no ●O2− was

detected by Nitro blue tetrazolium (NBT) staining in P. tinctorius hyphae. The first phase of production of seems ●O2

− to be extracellular which suggests that the early Ectomycorrhizae (EM) fungus-plant interaction takes place at the cell wall and plasma membrane surfaces (Baptista et al. 2007). This first phase is similar to pathogenic attack responses, where the main sources of ROS production have been identified as membrane bound NADPH oxidase (NOx) (Wojtaszek, 1997). During the second phase of ROS production, ●O2

− accumulates in micro-domains within cells. This could result from activation of the ROS-producing systems and down-regulation of the ROS-scavenging ones (Nanda et al., 2010). Furthermore, superoxide dismutases (SOD) appear to be upregulated and catalases (Kat) to

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be downregulated during these early stages. This could explain the H2O2 accumulation, as SOD convert ●O2

− to H2O2 and Kat convert H2O2to water. The combined action of these superoxide dismutase and catalase enzymes seems to play an important role during plant-EM fungus interactions (Baptista et al., 2007). Reactive oxygen species (ROS) seem to play an important role in the different symbiotic interactions. These results suggest a main role in the control of the fungus proliferation in the plant. However, knowledge about ROS during early symbiotic interactions between plants and fungi is very limited, due to the asynchronous nature of the mycorrhization process (Nanda et al., 2010). 6. C. V. Ros Homestasis for Optimal Symbiotic

Reactive oxygen species (ROS) are known to play major roles in various plant and microorganism developmental processes, such as cell elongation (root hairs, pollen tube or appressoria growth) and during biotic interactions. In order to avoid ROS accumulation leading to cell death, organisms have evolved enzymatic and non-enzymatic antioxidant mechanisms constantly generating and deteriorating ROS (Mittler et al., 2004). In plants, ROS are unavoidable by-products of biochemical pathways, such as glycolysis and photosynthesis. As a result, plants have evolved enzymatic and non-enzymatic antioxidant mechanisms to eliminate ROS and avoid oxidative destruction (Apel and Hirt, 2004). On the other hand, ROS production is necessary for cell elongation (root hairs, appressoria growth) and plant-microorganism interactions. It is therefore necessary for the plant to possess very complex and well-tuned ROS producing and scavenging systems capable of maintaining ROS homeostasis in the cells (Nanda et al., 2010). Considering the extracellular region, both plant and microorganism are capable of regulating the ROS level in this area during the early steps of the interaction. The symbiotic Rhizobia appear to have an efficient antioxidant defense. Indeed, although ROS were present in the infection threads they were not detected inside the bacteria progressing within the infection thread (Santos et al., 2001; Rubio et al., 2004). The large battery of Reactive oxygen species (ROS) scavenging enzymes included in the “ROS gene network” contains catalases (Kat), superoxide dismutases (SOD), ascorbate

peroxidases (APx, detected in plants) cytochrome C peroxidases (CcP, detected in fungi). They are present in several intracellular compartments as well as in the apoplast in order to regulate both intracellular and extracellular ROS accumulation (Mittler et al., 2004). S. meliloti possesses two superoxide dismutase (SOD) that convert ●O2

− to O2 and H2O2 (Santos et al., 2000; Herouart et al., 2002) and three heme b containing catalases, which are able to scavenge H2O2 (Herouart et al., 1996; Ardissone et al., 2004). Bacterial catalases appear to play an important role in the nodule formation process as the double katB/katC and katA/katC mutants of S. meliloti are strongly impaired in nodule formation (Jamet et al., 2003). The S. meliloti genome contains three thiol peroxidase encoding genes: the alkyl hydroperoxide reductase ahpC-like and two organic hydroperoxide resistance ohr-like genes. Both types of enzyme display biochemically equivalent functions and catalyze the reduction of organic peroxides to the corresponding less toxic organic alcohols (Nanda et al., 2010). The cell wall has an enormous capacity to retain proteins in normal growth conditions, most of the peroxidases for instance, which may be released following abiotic stress. The involvement of class III peroxidases during the symbiotic process has already been observed. For example, Rip1, encoding a peroxidase from Medicago is rapidly and transiently induced by Rhizobium meliloti or after Nodulation factors (NFs) treatment (Cook et al., 1995). Moreover, this gene is induced by H2O2 (Ramu et al. 2002). Class III peroxidase (Srprx1) has been shown to be crucial for the bacterial invasion of the tropical legume Sesbania rostrata. The expression of Srprx1 is strictly dependent on bacterial nodulation factors (NFs) and could be modulated by, a down H2O2 stream signal for crack entry invasion. Its expression was not induced after wounding or pathogen attack, indicating that the peroxidase is a symbiosis-specific isoform. More interestingly, lack of Srprx1 gene expression could cause an aberrant structure of the infection threads (Den Herder et al., 2007). OsPrx53, encoding a peroxidase from rice, is the strongest gene induced after Glomus infection.Peroxidasesseem important for the initiation of symbiosis but no direct evidence has demonstrated their implication for ROS production in the early steps of interaction and the development of the infection (Guimil et al., 2005). Other possible sources for in the H2O2

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Legume – Rhizobium symbiosis are germin-like oxalate oxidases or diamine oxidases (Wisniewski et al., 2000). A germin-like oxidase from Pisum sativum has been characterized (PsGER1). This protein has a superoxide dismutase activity, and is associated with nodules (Gucciardo et al., 2007). Interplay between localized ROS production and amelioration is fundamental to responses to biotic and abiotic cues as well as development (Mittler et al., 2004). In the early stages of infection, superoxide radicals and H2O2 are produced by the root cells in response to rhizobia, which suggests that the symbiotic bacteria are initially perceived as invaders (Santos et al., 2001). H2O2 accumulation has been detected in the invasion zone of alfalfa and pea nodules, in association with infection threads (Rubio et al., 2004). H2O2 is required for inter and intra molecular cross-linking of extensions and may be produced by CuZnSOD activity (Rubio et al., 2004), diamine oxidase activity using putrescine as a substrate (Wisniewski et al., 2000), and ⁄ or a germin-like protein with SOD activity (Gucciardo et al., 2007). The concentration of H2O2 in the infection threads may be also modulated by the catalase activity of bacteroids, as shownby experiments with S. meliloti mutants overexpressing KatB (Jamet et al., 2007). Controlled ROS production is essential for the onset of symbiosis. Therefore, Rhizobia mutant strains defective in exo-polysaccharides, lipopolysaccharides, or cyclic β-glucans are unable to infect root cells and activate defense reactions which are strong evidence for a signaling role of these complex carbohydrates during the symbiotic interaction (Mithofer, 2002). Experiments with incompatible rhizobia or with S. meliloti nodC- mutants have shown that Nod factors are implicated in suppressing the plant defense response (Bueno et al., 2001). Also, application of compatible Nod factors to M. truncatula slowed the rate of H2O2 efflux from excised root segments (Shaw and Long, 2003), and similar studies in bean showed a transient increase of ROS, within seconds, at the tip of actively growing root hair cells (Cardenas et al., 2008). Redox signaling can be also mediated by RNS, for example, via post translational modification of antioxidant proteins or transcription factors. Thus, RNS can cause nitrosylation (addition of ●NO group) or nitration (addition of an NO2 group) of cysteine or

tyrosine residues, respectively. For example, in nodules of M. truncatula, ●NO has been shown to activate two genes encoding proteins involved in H2O2 metabolism (a peroxidase and a germin like oxalate oxidase), suggesting a cross-talk between ROS and RNS signaling (Ferrarini et al., 2008). The ●NO bound to Lb may have originated in the host cells (Baudouin et al., 2006), in the bacteroids (Meakin et al., 2007), or in both nodule compartments. It can be argued that the presence of Lb-NO, decreasing O2 buffering in the cytoplasm, is potentially detrimental to nitrogenase. However, Lb-NO complexes are most abundant at the early stages of nodule development, which suggests a beneficial role of Lb as ●NO reservoir or as part of a mechanism to detoxify RNS or prevent rejection of symbiotic rhizobia. In fact, the important ROS accumulation during the second phase has been reported to precede the hypersensitive response (HR) cell death that often accompanies successful pathogen recognition leading to the incompatible interaction (Mehdy, 1994; Levine et al., 1996). The involvement of ROS during the Legume Rhizobium symbiosis has been highlighted during this last decade (Pauly et al. 2006). During the establishment of the symbiotic interaction, by using Nitro blue tetrazolium (NBT) that forms a dark blue precipitate with ●O2

─ (Bielski et al. 1980), can be detected in infection threads, indicating that ●O2

─ is produced during the infection process and could have a role in the control of the bacteria development (Santos et al. 2001; Ramu et al. 2002). S. meliloti nodC- mutant, defective in Nodulation factors (NFs) biosynthesis, triggers an important increase in H2O2 accumulation in Medicago sativa roots after inoculation. The compatible interaction between M. sativa and S. meliloti is linked, at least in part, with an increase of the antioxidant defense (particularly catalase and lipoxygenase) during the preinfection period (Bueno et al., 2001). The use of Diphenylene iodonium (DPI), that inhibits flavoproteins such as NADPH oxidase (NOx), and abolished ROS production, strongly supports the possible involvement of M. truncatula NOx homologues in ROS production. Moreover, a DPI treatment during the early stages of M. truncatula – S. meliloti interaction not only abolished ROS production but also suppressed root hair curling and infection thread formation. These results emphasize the involvement of M. truncatula NADPH oxidase

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homologues in the early steps of Rhizobium infection (Peleg-Grossman et al., 2007; Cardenas et al., 2008). The involvement of plant NADPH oxidase NOx in plant-microorganism interactions have clearly been shown (Torres and Dangl, 2005). The oxidative burst that often takes place at the very first stage of the interaction acts as the first line of plant defense. The microbe must therefore produce ROS scavenging enzymes in order to successfully infect the plant or down-regulate the plant ROS producing systems. This massive ROS production and scavenging takes place in the apoplast between the cell surfaces of the two organisms or in the rhizosphere. However, the oxidative burst seems to differ in intensity and length between plant-pathogen and plant-symbiote interactions. This difference could act as a specific signal predefining the host’s response to the microbe (Nanda et al., 2010). Moreover, the plant NADPH (NOx) has been found to play an important role in the oxidative burst during plant-pathogen interactions. However, no functional evidence of NOx involvement in ROS production during Legume – Rhizobium interactions has been found. This further supports the idea that different ROS regulating systems could be activated by interactions with a pathogen or a symbiote. In this line, class III peroxidases could be a good candidate for ROS regulation. Whatever the system involved during the different symbiotic interactions, it would be of interest to analyze the consequences of modifying plant ROS-producing activities on the symbiotic capacities. This could allow us to better understand the signaling role of ROS molecules and its consequences on the establishment of symbiosis (Nanda et al., 2010). Nodule natural senescence (aging) is a complex and programmed process, which shares some features with stress-induced senescence, such as a decrease of N2-fixing activity and Lb content and an increase of proteolytic activity and ROS production. In aging soybean nodules, Evans et al. (1999) found an increase of ROS (mainly organic peroxides), catalytic Fe, oxidized homo glutathione, and oxidatively modified proteins and DNA bases, but no changes in ascorbate or tocopherol, concluding that these nodules were suffering from oxidative stress. Lipid peroxidation was also found to be elevated in nodules of pigeon pea (Cajanus cajan) and bean with advancing age (Swaraj et al., 1995;

Loscos et al., 2008). The findings mentioned above clearly illustrate that ROS⁄RNS are produced in plants, and particularly in nodules, with useful purposes, one of the most important being redox signaling. Other studies also support the concept of ‘oxidative ⁄ nitrosative signaling’. For example, using proteomic analysis and detection with an antibody against nitrotyrosine, only 21 nitrated proteins were identified in sunflower (Helianthus annuus) hypocotyls (Chaki et al.,2009), suggesting that nitration is specifically targeted in cells rather than an indiscriminate phenomenon. To avoid any deleterious effect, nitrogen-fixing nodules are fitted with a very efficient antioxidant defense (Matamoros et al., 2003). Furthermore, a strong cerium precipitate can also be observed around peribacteroid and bacteroid membranes of senescent infected cells, strongly suggesting that H2O2 is involved in the senescence process (Aleasandrini et al., 2003).

6. D. Mescellenuous Roles Ofh2o2

The first description of peroxisomes was carried in 1954 in the course of electron microscopy studies in mouse kidney tubules and these organelles are designated as microbodies, a morphological name not implying any biochemical function. However, it was DeDuve at the beginning of the 1960s that carried out the biochemical characterization of peroxisomes from mammalian tissues, which led to their recognition as distinct cell organelles (De Duve et al., 1960). It was demonstrated that peroxisomes are involved in a range of important cellular functions in almost all eukaryotic cells (Reddy et al., 1996; Tabak et al., 1999). These organelles have an essentially oxidative type of metabolism and can carry out different metabolic pathways depending on their source. Another type of specialized peroxisomes is root-nodule peroxisomes from certain tropical legumes, in which the synthesis of allantoin, the major metabolite for nitrogen transport within these plants is carried out (Schubert, 1986). The main metabolic processes responsible for the generation of H2O2 in different types of peroxisomes are the photo respiratory glycolate oxidase reaction, the β-oxidation of fatty acids, the enzymatic reaction of flavin oxidases, and the disproportionate of superoxide radicals (Huang et al., 1983; Del Rio et al., 1996). Chloroplasts and mitochondria are often included among the major sites of intracellular

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H2O2 production. The densities of many leaves are close to 1 g ml-1 (Vile et al.,2005), and so if H2O2 sensitivity of metabolic enzymes and APX affinities are indicators of typical H2O2 concentrations, and these are uniform within the leaf, this would convert to about 20-50 nMol g-l fresh weight (FW). Such values are at the low end of those reported in the literature. It should be emphasized that even concentrations of 20-50 µM in chloroplasts, mitochondria, and the cytosol could be considered somewhat high (Queval et al., 2008). H2O2 and free radicals are important in cell wall metabolism and the apoplast compartments could be enriched in H2O2. Other compartments in which H2O2 could accumulate include the intra thylakoids space, mitochondrial inter-membrane space, endomembrane systems, and vacuole (Halliwell, 1978; Bolwell et al., 2002). H2O2 was reported to be preferentially concentrated in endosomes that are targeted to the vacuole during the response to salt stress (Leshem et al., 2006). Redox buffering in most of some cell compartments could be low compared with the chloroplast stroma and mitochondrial matrix (Foyer and Noctor, 2005). The extent to which inter-compartmental gradients in H2O2 concentration are established will depend on restriction of movement by membranes compared with the sink effect of the antioxidant system. The relative importance of these two factors were proposed to vary with H2O2 concentration (Henzler and Steudle, 2000). Potassium also plays several biochemical roles including: maintain integrity of photosynthetic structure, CO2 fixation, photosynthate transport, regulating of chlorophyll content and adjusting of turgour pressure (Maathuis and Sanders, 1996; Kochian and Luccas, 1988; Pier and Benkowitz, 1987; Zhao et al., 2001). References

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7. Oxidant Homeostasis Initiates the Defense

System

7. A. Oxidant Homeostasis

In order to detoxify of toxic oxygen species, during evolution, plants have acquired an effective detoxification system to maintain low ROS level and avoid the detrimental effects of excessively high ROS concentrations but, however, they cannot be eliminated completely because plants use ROS as second messengers in signal transduction cascades of diverse physiological processes (Ruciniska-Sobkowiak, 2010). Cross talk between distinct ROS such as singlet oxygen and H2O2 were explored (Laloi et al., 2007). These studies not only exposed redundancy of the ROS scavenging network but also suggested that different antioxidant enzymes and different ROS in the same or different compartments mediate signature signals that control chloroplast function and plant response to various environmental stimuli (Van Breusegem et al., 2008). The importance of peroxiredoxins, glutaredoxin, and thioredoxin as scavengers of ROS has gained significant support in recent years (Cheng et al., 2006; Dos Santos and Rey, 2006). Antioxidants are able to modulate ROS⁄RNS concentrations and thereby are likely to affect signaling transduction cascades. This has led to the concept of oxidative signaling, which emphasizes the multiple useful roles of ROS in plants, especially in redox signaling (Foyer and Noctor, 2005). The balance between ROS production and scavenging in the various organelles is disrupted under stress conditions leading to ROS increase which either serves as a signal or if uncontrolled, as toxic molecules (Mittler et al., 2004; Moller and Sweetlove, 2010). H2O2 can function as a potent signaling molecule in both stress and developmental processes implies that concentrations are likely to be under

homeostatic regulation. Proof of the importance of H2O2 homeostasis has come from the study of plants deficient in ascorbate peroxidase and catalase. These plants mimic stress induced redox perturbation, gene expression and/or phenotypes such as cell death (Davletova et al., 2005; Queval et al., 2007). They also mentioned that marked increases in leaf H2O2 contents have generally not been described. It is therefore unclear to what extent localized changes in H2O2 accumulation affect whole leaf contents, and whether measurement of these contents provides useful information on tissue redox state.

7. A. I. Escavenging Enzymes It has been accepted that antioxidant defense systems, including non-enzymatic antioxidants such as ascorbate, reduced glutathione, and tocopherol, and enzymatic antioxidants such as SOD and CAT, play a crucialrole in plants against various stresses. Previous studies demonstrated that the regulation of the concentrations of antioxidants and of the activities of antioxidant enzymes is an important mechanism for combating oxidative stress (Alscher et al., 2002; Blokhina et al., 2003; Heiber et al., 2007). However, because of the complexity and diversity of cell metabolism, other unknown antioxidant systems may exist in plant cells and need to be clarified. Leaf metabolism produces H2O2 at high rates, but current concepts suggest that the potent signaling effects of this oxidant require that a battery of antioxidative enzymes (Queval et al., 2008) control concentrations. ROS production also occurs in the course of major metabolic pathways, especially those in the peroxisomes, and ROS are used as a weapon against

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invading pathogens in the oxidative burst. Because ROS can cause damage to proteins, lipids, and DNA, ROS production and removal must be strictly controlled. All cell compartments should therefore have mechanisms for preventing and repairing damage caused by ROS (Moller, 2001). The extent to which H2O2 is allowed to accumulate remains unclear. There is little consensus on leaf H2O2 values in the literature and measured concentrations in unstressed conditions range from 50–5000 nmol.g-1 fresh weight, a difference that probably reflects technical inaccuracies as much as biological variability (Queval et al., 2008). Intense interest has focused on H2O2 as a potent signaling molecule whose production can occur though basal energy metabolism as well as through specific enzyme systems. Production of H2O2 through both routes can be affected by a variety of environmental factors such as light, cold, and biotic stress (Lamb and Dixon, 1997; Apel and Hirt, 2004). Among ROS, superoxide radicals (●O2) are most damaging to cellular structures. Whereas, superoxide dismutase (SOD) is key enzyme in cellular defense catalyzes the dismutation of superoxide radicals to H2O2 and O2 (Foyer and Noctor, 2000). To avoid any deleterious effect, nitrogen-fixing nodules are fitted with a very efficient antioxidant defense (Matamoros et al., 2003). Furthermore, a strong cerium precipitate can also be observed around peribacteroid and bacteroid membranes of senescent infected cells, strongly suggesting that H2O2 is involved in the senescence process. It was noted that ROS have not been detected in the microorganisms progressing within the threads, suggesting that the rhizobia have an efficient antioxidant defense (Santos et al., 2001; Rubio et al., 2004). Legume root nodules are characterized by an early senescence and during nodule senescence, GSH and hGSH are found to decline (Evans et al., 1999). It was mentioned that nodule ageing causes a 50% decrease in hGSH in soybean and pea nodules and an 82% decrease in GSH in pea nodules (Matamoros et al., 2003; Groten et al., 2005) with a concomitant accumulation of catalytic Fe and oxidation of thiols, lipids, proteins, and DNA (Evans et al., 1999). Recent invitro experiments have demonstrated that ferrous Leghaemoglobin (Lb) in the oxygenated form can scavenge NO and peroxynitrite, and that these RNS can reduce ferryl-Lb, an inactive form produced by oxidation of Lb with H2O2 (Herold and Puppo, 2005). Non-symbiotic Haemoglobin (Hbs) and Leghaemoglobin (Lb) are involved in

metabolism, transport, and signaling by RNS. Apart from their function in controlling ROS⁄RNS concentration, antioxidants themselves may act as signals, as can be illustrated with two examples. Studies with A. thaliana mutants with ascorbate deficiency (vtc1) have shown that ascorbate influences plant growth and development by modulating expression of genes involved in defense and Abscisic acid signaling (Pastori et al., 2003). Much less attention has been paid so far to the reactions of AOS and its products with cellular components, especially to sites of action outside the chloroplast. The exact mechanisms that facilitate AOS accumulation during environmental stress are not entirely clear (Taylor et al., 2004). However, AOS production through disruption or inhibition of the mitochondrial electron transfer chain (Hernandez et al., 1993; Kowaltowski and Vercesi, 1999; Lam et al., 2001; Skulachev, 1996) or photosynthetic apparatus (Aro et al., 1993; Noctor and Foyer, 1998) are major factors. Most research on AOS accumulation in plants hitherto has focused on the roles of antioxidant defense systems in alleviating the accumulation of AOS. Largely validating this focus, over expression of certain antioxidant enzymes has clearly been shown to enhance yield and survival under some environmental stresses (Alscher et al., 2002; McKersie et al., 1996; Van Camp et al., 1996). AOS produced at lower levels by cell wall NADPH oxidase, peroxidases, amino oxidases or flavin-containing oxidases, may form part of a defense strategy against invading pathogens or, when produced at very low levels, act as signaling molecules (Dat et al., 1998; Delledonne et al., 1998; Van Camp et al., 1998; Vera-Estrella et al., 1992). Subsequently, plants, like other organisms, have learnt to live with AOS and there is a delicate balance between AOS production and AOS scavenging.

7. A. II. Escavenging Soluble Phenolic

Compound The soluble phenolic compounds are the most widely distributed secondary metabolites in plant kingdom and could be enhanced as a powerful antioxidant in plant tissues under differe nt stresses (Dixon and Paiva, 1995). Phenolic compounds in onions are mainly formed by anthocyanins and flavonoids, these constituents

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may be involved in response of onion bulbs to abiotic stress (Benkeblia and Selselet-Attou, 1999), and considered as an important factor of the overall antioxidant activity (Hertog et al., 1993). Moreover, the increase of phenolic compounds in the tissue ameliorates the ionic effect of salt stress (Muthukumarasamy et al., 2000). Phenol accumulation could be a cellular adoptive mechanism for scavenging oxygen free radicals during stress and this free radical scavenger could be radical oxidized in the system of this tissue preventing sub-cellular damage. Generally, salt stress treatments were capable of acting as activities of flavonoid accumulation. In Hordeum vulgare, the significant higher concentration of flavonoids observed in salt-stressed plants (Ali and Abbas, 2003). Elevated ozone (mean 32.4ppb) increased the total phenolic content of leaves and had minor effects on the concentration of individual compound (Savirnata et al., 2010). One of the largest classes of plant phenolics, perform very different functions in plant system including pigmentation and defense (Kondo et al., 1992; Hahlbrock and Scheel, 1989). Phenolics extracted from berries of lonicera cearulea L. particularly, phenolic acids, flavonoids and anthocyanins have multiple biological activities in vitro and in vivo such as anti-adherence, antioxidant and anti-inflammatory. Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities beneficial to health such as protects mouse brain against D-galactose-induced oxidative damage and activates significantly AMP activated protein kinase (AMPK) via down regulation of protein phosphatase 2C (PP2C). Also, results suggest that AMPK activated by quercetin may be a potential target to enhance the resistance of neurons to age-related diseases (Lu et al., 2010). Polyphenols are known as pro-oxidant agents, one of the possible explanations of this deleterious behaviour comes from the high capacity of these compounds to lose H-atoms. This process originates free radical scavenging but also the formation of phenoxyl radicals stabilized by π-electron delocalization over the aromatic rings (Kosinova, 2011). Even if these radicals are relatively stable they may react by following different pathways including, a second H-atom abstraction (HAT) to form stable quinones or semiquinone, regeneration by HAT

from another antioxidant or solvent, so the polyphenol may, degradation into metabolites, adduct formation and dimerization with another phenoxyl radical. If two identical phenoxyl radicals meet together, a dimer may be formed. The dimerization process exists in plants. It is often only an initial step to form oligomers or polymers of polyphenols, e.g., tannins, which give characteristic colours and tastes to red wine or tea (Kosinova, 2011). 7. A. III. Thiol Escavnging of Sugar Produced

ROS Hruda et al. (2010) proved that high glucose concentrations add to the effect of tert-butylhydroperoxide (tBH). As it was prevented by N-Acetyl cysteine – an antioxidant – it is clearly related to oxidative stress. The explanation for this may be the metabolic linkage between glucose breakdown and antioxidant protection of the cell. Since tert-butylhydroperoxide (tBH) oxidizes the -SH groups it attenuates the cell’s ability to protect against oxidative stress rather than directly inducing it. The -SH group is a key structure of action in glutathione, one of the most important mechanisms of cellular antioxidant defense. Reduced glutathione is essential for the breakdown of hydrogen peroxide via glutathione peroxidase. Once glutathione is oxidized, glutathione forms dimers and can only be restored with the reduced form of NADP. The source of reduced NADP within the cell is the pentose phosphate shuttle, an alternate metabolic pathway of glucose sharing its first step with glycolysis (Wamelink et al., 2008). The next step of the pentose phosphate shuttle is catalyzed by specific enzyme glucose-6-phosphodehydrogenase, which is notably inhibited by high glucose concentrations (Leverve, 2003). This is why high concentrations of glucose lead to increased glycolysis, mitochondrial metabolic activity and subsequently ROS production but at the same time they block production of NADP, thus attenuating the antioxidant protection of the cell (Hruda et al., 2010). They suggested that in this way, high glucose concentration increases the toxic effect of an oxidizing agent. This finding is in agreement with other authors’ observations that high rate of substrate influx to mitochondria induces cell sensitivity to oxidant-induced apoptosis (Jeong et al., 2004). The DNA damage observed in cells at high glucose concentrations can be explained in the same

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way as the additive effect of glucose to tBH increased ROS production together with insufficient restoration of glutathione (Hollins et al., 2006). Fructose enters the glycolytic pathway down the flow at the level of triose-phosphates and therefore bypasses the first step of glycolysis, its most important regulatory mechanism (Mayes, 1993).The breakdown of fructose proceeds in a fast and rather uncontrolled manner and increases mitochondrial ROS production. This is probably not substantial under otherwise stable conditions but may become critical when the cell has to challenge further oxidative insults. Fructose proved to be toxic to cells only at high levels of tBH. Glutamine has been reported to have certain anti-oxidant and anti-apoptoticcapabilities (Chang et al., 2002). Hruda et al. (2010) found no protective effect of glutamine. The reason for this might be that glutamine was used here as the only available substrate for the cells and not just added as extra treatment. The concentration of glucose of 30 mM is quite high, and may already have considerable osmotic effect. To ensure that this effect did not falsify our results, Hruda conducted a limited series of experiments with media containing 5 mM D-glucose and 25 mM L-glucose, which is not metabolized by the cells (Buhler et al., 2001). 7. B. Oxidant Role in Gene Expressions and

Enzymes

7. B. I. Role of Reactive Oxygen Species (ROS) Desikan et al. (2000) utilized differential mRNA display to identify H2O2regulated genes in Arabidopsis suspension cultures. Therefore, they used cDNA microarray technology to carry out a transcriptome analysis of oxidative stress regulated genes in Arabidopsis. They identified H2O2 regulated genes and used RNA-blot analyses of some of the genes to demonstrate that their expression is modulated also by other stimuli that involve oxidative stress. A substantial proportion of these genes have predicted functions in cell rescue and defense responses, cell signaling, and transcription, implying that H2O2 does have multiple roles in plant responses to stress. It should be noted that the AFGC microarray used here is estimated to represent only about 30% of the Arabidopsis genome, depending on redundancy (Desikan et

al., 2001). Moreover, RNA was taken from undifferentiated suspension cultures as the hybridization probes. However, suspension cultures do represent excellent model systems (McCabe and Leaver, 2000), and many of the genes analyzed by RNA blots were found to be similarly H2O2 responsive in rosette leaves. Although this study was restricted to an analysis of ESTs that are responsive to H2O2, it does identify those genes necessary to form the basis of further studies using gene specific sequences to analyze the expression and function of the genes that are sensitive to H2O2. 175 genes were identified as being H2O2 responsive; most of them do not have an obvious direct role in oxidative stress. However, roles in other abiotic and biotic stresses and developmental processes that might be linked to oxidative stress could explain their sensitivity to H2O2. The genes that were sensitive to H2O2 have a range of potential functions based on their sequence homologies. Identification of genes and proteins regulated by H2O2 is thus an important step toward treatments that might confer tolerance of multiple stresses (Desikan et al., 2001). H2O2 can induce the expression of genes involved in antioxidant defense (Levine et al., 1994). Grant et al. (2000) reported that H2O2 induces the expression of defense-related genes such as GST, encoding glutathione S-transferase (GST), and PAL, encoding Phe ammonia lyase. H2O2 induces the expression of genes required for peroxisomebiogenesis where peroxisomes are organelles of direct importance for antioxidant defense (Lopez-Huertas et al., 2000). One of the genes identified via microarray analysis as being expressed at low levels but H2O2 responsive was that encoding a protein Tyr phosphatase. H2O2 is able to initiate the octadecanoid pathway leading to the biosynthesis of JA, JA-related compounds, and other oxylipins, which havebeen reported to function as inducers of plant secondary metabolites biosynthesis (Thomma et al., 2001). The H2O2 responsive transcriptome has been determined (Gadjev et al., 2006) and can be distinguished from the responses to other reactive oxygen species, such as singlet oxygen (Gadjev et al., 2006; Laloi et al., 2007). ROS produced by the NADPH oxidases function in defense, development and redox-dependent signaling. They share common structural features and are evolutionarily of ancient origin and thus

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ubiquitous in multicellular eukaryotes (Bedard and Krause, 2007; Bedard et al., 2007). In plants, NADPH oxidases form a small multigenic family and are involved in diverse events including innate immunity development. NADPH oxidases (NOx) are present in all the fungi forming fruit bodies where they seem to participate in sexual reproduction. The inactivation of Aspergillus nidulans NoxA gene resulted in a decrease of ROS production, inhibition of the formation of cleistothecia at early stages of development, stimulation of mycelium growth and suppression of asexual reproduction (Lara-Ortiz et al., 2003). In addition NOx from fungi areimportant during the infection process. Moreover in the symbiotic interaction between the fungus Epistle fistulae and the plant Folium perenne, a NOXA deficient fungus mutant is unable to undergo symbiosis and induces plant death. This shows that fungus produced ROS that also play a major role in the establishment of this symbiosis (Takemoto et al., 2006; Tanaka et al., 2006). Egan et al. (2007) used a genetic approach targeting two NOxgenes they showed that these genes are independently required for pathogenicity of M. grisea (inability to initiate appressorium-mediated cuticle penetration for the mutants) and are involved in ROS production. The deletion of a putative NOx from the ergot fungus of ryegrass, Claviceps purpurea, has an impact on germination of conidia and pathogenicity, although its involvement in focusing ROS production has not been shown (Giesbert et al., 2008). Other proteins, such as class III peroxidases, regulate for ROS homeostasis. Class III peroxidases are only detected in Viridiplantae and are present as large multigenic families in all land plants (Bakalovic et al., 2006). Released from the cell surface into the apoplast, peroxidases are an important class of enzymes responsible for the stress-induced formation and degradation of ROS (Bindschedler et al., 2006; Fecht-Christoffers et al., 2006). Hydrogen peroxide (H2O2) has been implicated, both in pathogen attack and in the defensive oxidative cross-linking of plant cell wall proteins, which renders the cell wall less digestible (Dey et al., 1997). Increasing evidence indicates that H2O2 functions as a signaling molecule in plants and H2O2 generation during the oxidative burst is one of the earliest cellular responses to potential pathogens and elicitor molecules (Lamb and Dixon, 1997). Generation of H2O2

occurs under a diverse range of conditions, and it appears likely that H2O2 accumulation in specific tissues, and in the appropriate quantities, is of benefit to plants and can mediate cross-tolerance toward other stresses (Bolwell, 1999). A number of similarities can be seen in the cellular responses to stresses, suggesting that H2O2 could be a common factor regulating various signaling pathways (Neill et al., 1999). It has been known for many years that ROS can be cytotoxic and contribute to disease and aging in animals (Ames et al., 1993; Hensley and Floyd, 2002) and defense responses in plants (Lamb and Dixon, 1997). It has been shown that ROS act as signals (Finkel, 2003; Laloi et al., 2004; Mittler et al., 2004). NO is intimately linked with ROS, and many processes that respond to one also respond to the other or both (Neill et al., 2003; Wendehenne et al., 2004). ROS can lead to successful defense responses against biotic factors, adaptation to abiotic factors in tolerant plant species, and can cause injury to susceptible plants (Prasad et al., 1994). Elevated level of H2O2 causes alterations in permeability properties of the plasma membrane, and its integral proteins and protein complexes may be affected. The H2O2-mediated decrease in hydraulic conductivity in wheat (Triticum aestivum L.) roots in relation to salt stress has recently been reportedand the results showing a close correlation between H2O2 level and Lp and H+-ATPase activity in isolated plasma membrane, support this view. Considerable reductions of Lpr occurred at concentrations of H2O2 as low as 2 mM. This concentration appears to be within the range that may occur in the apoplast. Environmental stresses have often been linked to AOS production and AOS-induced damage in plants (Dat et al., 2000). To understand this link, the mechanism of AOS accumulation and the degree to which stresses differ from one another must be considered, and it must be acknowledged that AOS targets can be, on the one hand, antioxidant defenses and, on the other, sensitive sites of damage. Although the physical nature of environmental stress conditions varies greatly, a number of common responses have been shown to exist and sites of damage are often the same. For example, mild heat treatment of tomato plants confers resistance to subsequent oxidative stresses, and vice versa, and this has been shown to be correlated with the induction of a series of heat

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shock proteins in both cases (Banzet et al., 1998). 7. B. II. Nitric Oxide (

●No)

In tobacco (Nicotiana tabacum) cells, Cyclic Adenosine Diphosphate Ribose (cADPR) was able to mimic the ●NO-mediated induction of defense gene expression. This cADPR effect was inhibited by ruthenium red, an inhibitor of Ca2+ release via cADPR/ryanodine-sensitive Ca2+ channels (Durner et al., 1998). Furthermore, 8-Br-cADPR, an antagonist of cADPR, blocked gene expression mediated by recombinant neuronal nitric oxide synthase in tobacco (Klessig et al., 2000). It was demonstrated that ●NO triggered an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) by promoting Ca2+ release from intracellular stores through the activation of cADPR/ryanodine-sensitive Ca2+ channels in fava bean (Vicia faba) and tobacco cells (Garcia-Mata et al., 2003; Lamotte et al., 2004). It seems plausible that interplays of ●NO and Ca2+ might be implicated in cell death. This idea is supported by the fact that both ●NO and Ca2+ are triggers and modulators of cell death (Lam, 2004; Delledonne, 2005). This hypothesis is further reinforced by the finding that H2O2, which acts in concert with ●NO in triggering cell death (Zago et al., 2006) which contributes to stimulus-induced [Ca2+]cyt changes (Garcia-Brugger et al., 2006; Lecourieux et al., 2006). The combination of ●NO and H2O2, but not ONOO─, takes part in the induction of defense responses (Delledonne et al., 2001). ●NO induced accumulation of the pathogenesis-related (PR)-1 transcripts in tobacco leaves was suppressed in the presence of the cyclic adenosine diphosphate ribose (cADPR)-selective antagonist 8-bromo-cADPR (Klessig et al., 2000). In agreement with these data, ryanodine receptor (RYR) inhibitors (Durner et al., 1998) suppressed the expression of the PR-1 gene. Naturally, these data do not conclusively prove that ●NO activates RYR-like receptor, but collateral evidence supports that cADPR has a dominant role in generating NO-dependent Ca2+ fluxes: the NO-mediated Ca2+ transient influx is reduced by almost 40% by 8-bromo-cADPR (Lamotte et al., 2006). Nitric oxide (●NO) scavengers and mammalian NOS inhibitors reduced the increase in [Ca2+]cyt triggered by hyperosmotic stress or elicitors of defense responses, including cryptogein and endo

polygalacturonase I from Botrytis cinerea (Gould et al., 2003; Lamotte et al. 2006; Vandelle et al., 2006). Therefore, treatment of transgenic Nicotiana plumbaginifolia cells expressing the Ca2+ reporter aequorin addressed in the cytosol with the ●NO donor diethylamine-NONOate (DEA/NO) was followed by a rapid and transient biphasic elevation in [Ca2+]cyt. Experiments based on the use of the NO scavenger cPTIO demonstrated that the first increase in [Ca2+]cyt was caused by the ●NO donor diethylamine-NONOate (DEA/NO) solubilization buffer whereas the second elevation was specifically due to ●NO (Lamotte et al., 2006). The second elevation reached 350–500 nM, where the transient aspect of ●NO-induced [Ca2+]cyt changes suggests that Ca2+-ATPase and other Ca2+ transporters as well as Ca2+-buffering compounds are active within the cell membranes after ●NO treatment. Interestingly, a second treatment with DEA/NO 30 min after a first treatment with the ●NO donor induced a delayed and reduced [Ca2+]cyt rise compared with the first one, indicating that the same cells are poorly responsive to repeated ●NO treatments (Besson-Bard et al., 2008). It is unlikely that the ineffectiveness of the second NO treatment was caused by a breakdown of ion gradient regulation by NO, since a subsequent exposition to hyper-osmotic stress led to a typical biphasic increase in [Ca2+]cyt (Gould et al., 2003; Lamotte et al., 2006). The inefficiency of the second NO treatment may reflect the inactive state of components of the pathway(s) mediating the NO-dependent [Ca2+]cyt increase. It should also be noted that treatments with DEA/NO did not elicit increases in nuclear free Ca2+ concentration in transgenic tobacco cells expressing aequorin addressed to the nucleus (Lecourieux et al., 2005). This latter finding highlights a specific role of ●NO in governing the cytosolic Ca2+ homeostasis. In various cell types cGMP-dependent protein kinases (PKGs) trigger the activation of Ryanodine Receptor (RYR) by promoting the synthesis of the nicotinamide adenine dinucleotide (NAD+) metabolite cyclic cADP ribose. Furthermore, NO also decreases [Ca2+]cyt by activating plasma membrane and endomembrane Ca2+ transporters (Clementi, 1998). The detailed mechanism of this action is still unclear and might involve both nitrosylation and cGMP-dependent processes (Yao and Huang, 2003). Cyclic GMP targets Ca2+ channels by virtue of their cyclic nucleotide

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binding sites, particularly in the case of cyclic-nucleotide-gated channels (CNGCs) or mediates its effects through serine/threonine cGMP-dependent protein kinases (PKGs). It is especially noteworthy that protein sequences from practically every type of ion channel contain PKG consensus phosphorylation sites (Ahern et al., 2002). The rise in [Ca2+]cyt was blocked by sGC and RYR inhibitors, establishing cGMP as a putative mediator for NO-induced activation of cADPR-dependent endomembrane Ca2+ channel. A similar situation was found by analyzing the effects of the non-thiol NO donor diethylamine (DEA-NONOate) in tobacco cell suspensions expressing the Ca2+ reporter apoaequorin in the cytosol (Lamotte et al., 2006). NO markedly enhanced the [Ca2+]cyt through a process sensitive to 8-bromo-cADPR, indicating that NO-induced Ca2+ mobilization operates predominantly via a cADPR-mediated Ca2+-release mechanism (Courtois et al., 2008). Nitric Oxide (●NO) is a second messenger related to development and abiotic stress responses in plants (Laxalt et al., 2007). One of the second messengers reported to participate in plant defense responses is nitric oxide (NO). NO treatments induce plant defense-related transcript accumulation (Delledone et al. 1998); Durner et al., 1998). ●NO has been shown to activate proteases that appear to contribute to a HR-type cell death (Clarke et al., 2000; Belenghi et al., 2003), most likely by interacting with ROS-associated signals (Delledonne et al., 2001) as well as inducing defense gene expression (Grun et al., 2006). Treatments with different inhibitors of ●NO accumulation compromise the hypersensitive response (HR), a form of programmed cell death induced during plant defense (Durner and Klessig 1998). Nitric Oxide (●NO) and reactive oxygen species (ROS) have been found to act together triggering apoptosis and executing invasive pathogens. However, in plants, a balanced production of ●NO and ROS was required for Hypersensitive Resistance (HR) (Delldonne et al., 2001 and De Pinto et al., 2002). NO and ROS are required for the induction of Hypersensitive Resistance (HR) (Delldonne et al., 2001). ROS and ●NO are believed to play important roles independently or coordinately in plant innate immunity. ROS generated on the plasma membrane are released to the apoplast, inducing oxidative crosslinking of glycoproteins, strengthening the cell wall against secondary infection (Bradley et al., 1992) and simultaneously activating the

Ca2+ channel to increase the level of cytosolic Ca2+

(Lecourieux et al., 2002). Several genes that affect ●NO accumulation have been identified through genetic analysis. The Arabidopsis thalianaNOA1 gene, which encodes a cGTPase (Moreau et al., 2008) and is needed for ●NOaccumulation during abiotic and biotic responses (Gas et al., 2009), was identified through reverse genetics (Guo et al., 2003). In the case of hypoxia or anoxia, it is clear that nitrite serves as a substrate and is reduced to ●NOby nitrate reductase, mitochondria, or acid-catalyzed reactions (Rockel et al., 2002; Gupta et al., 2005; Planchet et al., 2005). Under normoxic conditions, however, the mechanisms and genes have not been resolved, but chloroplasts (Gas et al., 2009) and peroxisomes play a role (Corpas et al., 2009). Hydrogen peroxide works synergistically with ●NO to stimulate or delay programmed cell death and assist in defense responses to pathogens (Wendehenne et al., 2004; Zaninotto et al., 2006; Asai and Yoshioka, 2009; Zhang et al., 2009). 7. C. Hydroxyproline Containing Proteins Extensions are the most studied family of hydroxy-proline (Hyp)-rich proteins (HRGPs). The polypeptide backbone of extensions contains many repeats of the structural Ser (Hyp) 4–6 motif. Short sequences rich in Tyr, Lys, Val, and His, often flank these structural motifs the Val-Tyr-Lys motifs being the sites for extension cross-linking (Fry, 2004). Theyrevealed that extensions are secreted into the apoplast as soluble monomers where the positively charged Lys and the protonated His residues interact ionically with the negatively charged uronic acids of pectins. The formation of the insoluble extension network is a well-characterized Prxs-mediated and H2O2-dependent process, which, it has been proposed, involves the coupling of extension Tyr residues to form isodityrosine linkages and larger Tyr oligomers such as di-isodityrosine or pulcherosine. The interaction of peroxidase (Prxs) with extensions also has a defense function since it makes the cell wall harder to penetrate. The common relationship between extensions and Prxs could be the pectin layer. Indeed, some Prxs can bind to calcium pectate complexes (Shah et al., 2004), and there is growing evidence that covalent bonds exist between pectins and extensins. Pectins would

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act as an anchor for Prxs, which would then crosslink extensins to create a dense and solid network in the host plant cell wall, with the aim of limiting pathogen colonization (Passardi et al., 2004). 7. D. Oxidant Regulates Defense Genes Studies of gene regulation under different stress conditions are important to get deeper insights into the regulation of defenses involved in each particular condition and the cross talk processes occurring in different stress conditions. Some PRs have been described to be up-regulated under abiotic stress (Tateishi et al., 2001; Stressmann et al., 2004), which suggests the existence of common effectors with biotic stress. The analysis of the expression of PRs and HSP71.2 (for heat shock protein 71.2) in pea plants treated with Cd was carried out by semi quantitative RT-PCR. Cd treatment tended to upregulate chitinase, pathogen-related proteins (PrP4A), and HSP71.2, while the expression of PAL (Phe ammonia-lyase) transcripts did not change significantly with the treatment. The induction of PrP4A and HSP71.2 was reverted by the supply of ascorbate, a H2O2 scavenger, which suggests that both genes are at least partially regulated by ROS. However, Ca did not change the expression level of PrP4A in both control and Cd treated plants (Redriguze-Serano et al., 2006). FISH results showed an intense hybridization signal in pea leaves of Cd-treated plants, in contrast to control plants, where a very faint signal was observed. No hybridization signal was observed in control experiments with the sense probe. Pathogen related proteins (PrP4A) expression was observed in mesophyll cells, especially in palisade cells from Cd-treated pea plants; the hybridization signal was localized in the cytoplasm, with the nuclei (visualized by DAPI with blue fluorescence) being free of signal. However, the signal was absent in epidermis and xylem tissue, although a weak/low FISH signal was observed in stomata and in parenchyma cells surrounding xylem vessels (Redriguze-Serano et al., 2006). Pathogen related proteins (PrP4A) is a hevein-related protein that binds chitin, can inhibit the growth of fungus, and belongs to chitinase I and II classes (Broekaert et al., 1990). An induction of PrP4A gene was reported under ozone, UV-B radiation, and pathogen attack. The regulation of PrP4A gene was also dependent on ET in Chinese

cabbage (Brassica campestris ssp. pekinensis) plants (Chung et al., 2005) and was sensitive to ET and JA in Arabidopsis (Thomma et al., 2001), and in both cases it was not dependent on SA, which could explain its up-regulation by Cd. It was demonstrated that PrP4A is also regulated by ROS, although exogenous Ca supply does not affect the up-regulation under Cd toxicity. In situ localization of PrP4A transcripts in pea leaves revealed an accumulation mainly in the cytoplasm of palisade mesophyll cells, suggesting that PrP4A gene products have specific functions in these cells. The absence of transcripts in epidermis is in contrast to the data obtained for chitinase, β-1-glucanase, teonin, and SAR8.2, which were accumulated in phloem and epidermal cells of different plant species under pathogen infection (Wubben et al., 1996; Lee and Hwang, 2003). 7. E. Oxidative Brust

7. E. I. Regulation of Oxidative Brust Asai et al (2008) investigated the roles of MEK2-WIPK/SIPK/NTF4 and MEK1-NTF6 cascades in the regulation of NO and oxidative bursts in N. benthamiana. Gain of function and loss of function analyses showed that the MEK2-SIPK/NTF4 cascade controls the NOA1-mediated NO burst and that MEK2-SIPK/NTF4 and MEK1-NTF6 cascades regulate the NADPH oxidase dependent oxidative burst. They also showed that the NO burst and the oxidative burst have distinct effects on resistance to Phytopthera infestans and Colletotrichum orbiculare (syn. C. lagenarium) in N. benthamiana. Whereas, Asai et al. (2008) showed that the MEK1-NTF6 cascade regulates the oxidative burst accompanied by induction of Respiratory Burst Oxidase Homolog B (RBOHB) expression. MEK1 has been shown to be an activator of NTF6 in cytokinesis (Soyano et al., 2003). The NTF6 activation profile by Nb MEK1DD was obtained only when NTF6 was over expressed in N. benthamiana leaves, suggesting that the amount of endogenous NTF6 was not sufficient to detect the activity by anti-NTF6 antibody. However, the possibility that another efficient MAPKK activating NTF6 might exist for INF1-induced oxidative burst. How NTF6 can bifurcates two distinct cellular functions, cytokinesis and innate immunity is unclear. It was found that during plant infection, NOx from M. grisea generate ROS. This oxidative burst is associated with the

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development of specialized infection structures called appressoria. Pharmacological scavenging of these oxygen radicals significantly delayed the development of appressoria and affected their morphology (Nanda et al., 2010). Desikan et al. (2001) analyzed cellular responses to H2O2 through undertaken a large-scale analysis of the Arabidopsis transcriptome during oxidative stress by using cDNA microarray technology. They identified 175 non-redundant expressed sequence tags that are regulated by H2O2. Of these, 113 are induced and 62 are repressed by H2O2. A substantial proportion of these expressed sequence tags have predicted functions in cell rescue and defense processes. RNA-blot analyses of selected genes were used to verify the microarray data and extend them to demonstrate that other stresses such as wilting, UV irradiation, and elicitor challenge also induce the expression of many of these genes, both independently of, and, in some cases, via H2O2. It is clear that there are overlapping spectra of genes induced by stresses such as ozone, UV, and pathogen challenge (Langebartels et al., 2000). Furthermore, the phenomenon of cross tolerance, in which exposure to one stress can induce tolerance to other stresses, is one in which H2O2 is likely to play a pivotal role (Bowler and Fluhr, 2000). It was demonstrated that H2O2 production is induced by a number of elicitors in plant cells, primarily cell wall-derived oligosaccharides, and pathogen a virulence proteins, which are recognized by specific receptors, in many cases represented by leucine rich repeat (LRR) kinases (Dievart and Clark, 2004). In this regard, it is worth recalling that in soybean suspension cell cultures, different elicitors share a common mitogen activated protein kinase (MAPK) intermediate in the signal transduction pathway leading to the oxidative burst (Taylor et al., 2001). 7. E. II. Oxidative Brust Significance Oxidative stress occurs as an essential response when plants are challenged with abiotic stresses. Oxidative stress results from the disturbance in balance between ROS production and scavenging such as hydrogen peroxidate, superoxidate anions, and hydroxyl radicals that damage or kill cells by destroying lipids, nucleic acids, and proteins (Apel and Hirt, 2004; Knight and Knight, 2001). To cope with different internal and external stresses, plants

have developed a variety of adaptive mechanisms for survival by activating cascades or network events starting with stress perception and ending with the expression of many effector genes (Mittler, 2002; Xiong et al., 2002). The double-antisense tobacco plants deficient in both APX1 and CAT1 were less sensitive to oxidative stress than single-antisense plants lacking APX1 or CAT1(Rizhsky et al., 2002). Vanderauwera et al. (2011) generated an Arabidopsis thaliana double mutant lacking APX1 and CAT2 (the equivalent of CAT1 in tobacco). In contrast to the single mutant cat2, theapx1/cat2 double mutant was able to grow under high light (HL) conditions with outaccumulating ROS to detectable levels. It was visualized withdiaminobenzidine (DAB) staining indicative of H2O2 accumulation, and had low levels of oxidized ribulose-1, 5-bisphosphate carboxylase, indicating that the mechanism(s) activated in double mutants lacking cytosolic and peroxisomal H2O2-scavenging mechanisms are conserved. The role of the protein phosphorylation status in the regulation of the burst activity has been demonstrated by pharmacological studies, where the kinase inhibitors staurosporine and K-252a have been shown to block progression of the oxidative burst (Chandra and Low, 1995). Accordingly, protein phosphatases inhibitors such as calyculin A, cantharidin, and okadaic acid can stimulate H2O2 synthesis in the absence of elicitors (Chandra and Low, 1995). Laxalt et al. (2007) demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. In plants a number of Phosphotidic acid (PA) targets have been identified, suggesting PA is involved in many processes, where PA has been shown to bind a protein kinase in Arabidopsis (PDK1) and to activate protein kinase AGC2-1 in a PDK-dependent manner (Anthony et al., 2004). PLD-generated PA in cell suspensions treated with xylanase (Anthony et al., 2006) specifically activates PDK1. AGC2-1 is identical to OXI1, a protein kinase implicated in oxidative burst-mediated signaling in Arabidopsis (Rentel et al., 2004). An increase in intracellular protein tyrosine phosphorylation is one of the earliest events of oxidative stress responses. Tyrosine phosphorylation is carried out by a protein tyrosine kinase; the kinase catalytic activity of this enzyme is regulated by a phosphatase

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(Yamamura, 2002). Mammalian non-receptor tyrosine kinase c-Abl and the product of the c-abl-related gene (Arg) respond to oxidative stress (Kharbanda et al., 1995; Cao et al., 2003a). c-Abl is activated and targeted to mitochondria in H2O2-treated cells and induces loss of the mitochondrial trans membrane potential, resulting in cytochrome c release and induction of apoptosis (Sun et al., 2000; Kumar et al., 2001; Cao et al., 2003a). In addition, c-Abl and Arg can stimulate CAT and glutathione peroxidase activities by tyrosine phosphorylation (Cao et al., 2003b). In Arabidopsis plants, salt stress induced the expression of three peroxisome-associated genes including thiolase(PED1), PEX10, and PEX1, and required components of the ethylene, jasmonate, and Abscisic acid signaling pathways (Charlton et al., 2005). Additionally, in animal cells an enhanced synthesis of nitric oxide was found to increase the peroxisomal H2O2-producing β-oxidation. Under any type of plant biotic and abiotic stress an induction of the peroxisomal production of ●O2

─ radicals takes place, this can lead to the inhibition of catalase and Ascorbate peroxidase (APX) activities and, possibly to an increase of the H2O2 level from the enhanced fatty acid β-oxidation. This break down of the peroxisomal antioxidant defenses would originate an overproduction of H2O2 in peroxisomes and a toxic situation to the plant cell. The rate of ROS and ●NO generation in plant cells can have an ambivalent effect. 7. E. III. Pathogen Infections Initiate Oxidative

Brust ROS are produced by all living organisms, either constitutively as by-products of several metabolic processes or in a more controlled manner during developmental processes as well as at the early stages of plant-microorganism interactions (Nanda et al., 2010). Reactive Oxygen Species (ROS) are continuously produced as a result of aerobic metabolism or in response to biotic and abiotic stresses. ROS are not only toxic by-products of aerobic metabolism, but are also signaling molecules involved in several developmental processes in all organisms. Previous studies have clearly shown that an oxidative burst often takes place at the site of attempted invasion during the early stages of most plant-pathogen interactions. Moreover, a second ROS production can be observed during certain types of plant-pathogen interactions, which triggers hypersensitive cell

death (HR). This second ROS wave seems absent during symbiotic interactions. This difference between these two responses is thought to play an important signaling role leading to the establishment of plant defense (Nanda et al., 2010). Therefore, in order to cope with the deleterious effects of ROS, plants are fitted large panel of enzymatic and non-enzymatic antioxidant mechanisms. Characterizations of ROS producing and scavenging systems from plants and from microorganisms during interactions should be made. In this review, we present the current knowledge on the ROS signals and their role during plant-microorganism interactions. Oxidative stress is a result of overproduction of reactive oxygen species (ROS) which are produced in aerobic organisms by electron transport chain of mitochondria. The ROS are highly reactive agents readily attacking macromolecules including nucleic acids. Under physiological conditions, the complex I of the electron transport chain seems to be the major site of ROS production (Leverve and Fontaine, 2001). Reactive Oxygen Species (ROS), which are formed in numerous cellular processes, were first described as deleterious, as they can provoke cellular damage (Halliwell and Gutteridge, 1986). It is now largely admitted that they can play a signaling role in various cellular mechanisms (Neill et al., 2002). It has been demonstrated that ROS are key players in the plant defense system against pathogens oxidative burst (Apel and Hirt, 2004), and also in fundamental processes such as cellular growth (Foreman et al., 2003), stomata closure (Pei et al., 2000), and in the regulation of gene expression (Vranova et al., 2002). Some strains of P. syringae and X. campestris pathovars derive their own ethylene to serve a virulence function. Within these pathogens, ethylene is not synthesized from ACC, but from 2-oxoglutarate, by an ethylene forming enzyme (EFE) (Weingart and Volksch, 1997). 7. E. IV. Developmental Oxidative Brust

7. E. IV. I. Reactive Oxygen Species The tip growth of pollen tubes parallels that of root hairs. As yet, ROS have not been shown to be involved in pollen tube growth. However, speculation that ROS are involved has led to the discovery that pollen grains have intrinsic

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NADPH oxidase activity (Carol and Dolan, 2006). Pollen from 10 weed species, 10 grasses, and 20 trees were tested, and all were found to have intrinsic NADPH oxidase activity (Boldogh et al., 2005). Of the sample tested in a H2DCF-DA fluorescence-based assay, redtop grass Agrostis alba had the highest redox activity and pine (Pinus sp.) the least. In ragweed Ambrosia artemisiifolia, NADPH oxidase was shown to localize to the plasma membrane of the pollen grain by using an antibody that cross-reacts with the mammalian NADPH oxidase involved in the phagocytic oxidative burst. Pollen-borne NADPH oxidase activity was thus shown to be a major cause of inflammation and mucin production in antigen-induced allergic response of lungs (Carol and Dolan, 2006). Photosynthetic tissues have a high capacity for H2O2 production through electron transport chain activity, and in C3 plants, through the photorespiratory enzyme, glycolate oxidase. Although it is very difficult to establish precise rates of H2O2 production linked to photosynthesis, approximate calculations can easily be performed by simple modeling (Noctor et al., 2002). Thus, it is possible to estimate likely rates of H2O2 generation by electron leakage to O2 followed by dismutation or reduction of superoxide. A typical net rate of photosynthesis under moderate light is 50 µmol O2 evolved mg-1 chlorophyll h-1, equivalent to more than 200 µmol electrons mg-1chlorophyll h-

1. Assuming that 1% of the total electrons from water reduces O2 (a fairly conservative estimate), that all superoxide is converted to H2O2, and that the chloroplast volume is 25-70µlmg-1 chlorophyll. The photosynthetic electron transport chain is theoretically capable of generating a stromal H2O2 concentration of 0.2–0.5 M within 12 h photosynthesis (Heldt, 1980; Winter et al., 1993). In C3 plants, equally or more rapid H2O2 production can simultaneously occur in the peroxisomes through photorespiration, and the mitochondrial electron transport chain is a further source of H2O2 production via superoxide (Foyer and Noctor, 2003). Guo and Crawford (2005) suggested that because nos1 mutant plants suffer from a heavier burden of ROS and oxidative damage, they are more vulnerable to dark-induced senescence. This view is consistent with the free radical theory of aging in animals, which states

that ROS and oxidative damage promote aging, cellular senescence, and PCD (Finkel and Holbrook, 2000; Hensley and Floyd, 2002; Balaban et al., 2005). For example, oxidation of proteins increases with age (Oliver et al., 1987; Stadtman, 2001) and is associated with increased protein degradation (Levine et al., 1994), cellular deterioration, and disease (Stadtman, 2001; Nystrom, 2005). Protein oxidation increases with age only during the vegetative phase and then declines dramatically during flowering (Johansson et al., 2004). Levels of protein oxidation were much higher in growing nos1 mutant plants but decreased dramatically when leaves were induced to senesce by dark treatment (Guo and Crawford, 2005). It is unlikely that the dark treatment alone induced this decline, because protein oxidation levels are not much affected by light conditions, as shown in a previous study (Johansson et al., 2004). An oxidative burst has also been associated with Cd toxicity in tobacco (Nicotiana tabacum) cell suspensions, with a NADPH oxidase being involved (Olmos et al., 2003; Garnier et al., 2006). Prooxidants such as H2O2 and paraquat did not induce expression, an observation suggests that heme oxygenase is not involved in antioxidant protection in nodules. By contrast, more recent studies have shown that, under oxidative conditions induced by Cd(Balestrasse et al., 2005) or salt stress (Zilli et al., 2008), there is a marked increase in heme oxygenase expression (mRNA and protein) in nodules, providing credence to an antioxidative role. Furthermore, both UV irradiation and application of exogenous H2O2 caused oxidative damage and up-regulation of heme oxygenase in soybean leaves (Yannarelli et al., 2006). These treatments also increased ascorbate peroxidase (Apx) and catalase activities, making it tempting to include heme oxygenase in the list of antioxidants. The second enzyme of the heme degradation pathway, biliverdin reductase, has been found in A. thaliana (Gisk et al., 2010). The fact that heme oxygenase is encoded by a small gene family, with four putative members in A. thaliana, argues further that the reactions of heme degradation have an importance in plant physiology that has not previously been appreciated. The participation of ROS is deleterious consequences of stress on N2 fixation. Indeed, drought induced the expression of several antioxidant genes and caused oxidative damage

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in alfalfa nodules (Naya et al., 2007). However, results were different in nodulated plants exposed to salt stress. In soybean or bean nodules exposed to high salinity, no symptoms of oxidative stress could be found, although antioxidant enzyme activities were induced (Comba et al., 1998; Loscos et al., 2008). The up-regulation of antioxidant enzymes, and particularly of SOD, was also seen in several other studies, suggesting that plants are perceived an increase in ROS production and that antioxidants contribute to salt tolerance (Tejera et al., 2004; Jebara et al., 2005; Nandwal et al., 2007). Plant response to antioxidants and oxidative damage, is dependent on the type of stress and the legume species. The complexity of the interaction between the two symbiotic partners, probably differing in stress tolerance, and the structural and biochemical differences between indeterminate and determinate nodules make it difficult, if not impossible, to establish a general model for stress-induced nodule senescence (Becana et al., 2010). Superoxide radicals are known to inhibit catalase activity (Kono and Fridovich, 1982) and it has been reported in tobacco plants that ●NO and peroxynitrite inhibit catalase and Ascorbate peroxidase (APX) activity, the two major H2O2-scavenging enzymes of plant peroxisomes (Clark et al., 2000). Ozone enters plant tissues through the stomata and induces the generation of reactive oxygen species (ROS) namely superoxide anion radicals and hydrogen peroxide, and triggers oxidative burst (Sharma et al., 1996; Rao and Davis, 1999; Kangasjarvi et al., 2005). The activities of specific phosphatases and kinases are also needed in the ABA signaling cascade responsible for stomatal closure (Murata et al., 2001; Mustilli et al., 2002). The activation of a MAPK cascade and transcription factors acting downstream of the LRR receptor has recently been proposed as a conserved mechanism involved in resistance responses to bacterial and fungal pathogens (Asai et al., 2002).

7. E. IV. II. Reactive Nitrogen Species The Arabidopsisnox1 mutant was identified in a screen for ●NO hypersensitive mutants and has a defective CUE1 gene (encodes a chloroplast phosphoenolpyruvate/phosphate translocator), resulting in higher levels of ●NO and in delayed flowering (He et al., 2004). Arginasenegative

mutants have increased ●NO accumulation and enhanced lateral root formation (Flores et al., 2008). Nitrate reductase mutants are defective in some ●NO-mediated processes, such as ABA induced stomata closure but are normal in others (Gas et al., 2009). Hydrogen peroxide is also a signal for stomata closure; ABA induces H2O2 synthesis, which, in turn, induces ●NO accumulation (Desikan et al., 2004; Bright et al., 2006; Neill et al., 2008). Several kinases in the mitogen-activated protein kinase cascade regulate ●NO bursts during defense responses in Nicotiana benthamiana (Asai et al., 2008; Asai and Yoshioka, 2009), and GPA1, a subunit of heterotrimeric G proteins, is necessary for NOA1-dependent ●NOaccumulation that is stimulated by external calmodulin in stomates (Li et al., 2009). The Arabidopsis thaliana protein nitric oxide synthase1 (NOS1) is needed for nitric oxide (●NO) synthesis and signaling during defense responses, hormonal signaling, and flowering. The cellular localization of NOS1 was examined because it is predicted to be a mitochondrial protein (Guo and Crawford, 2005). NOS1–green fluorescent protein fusions were localized by confocal microscopy to mitochondria in roots. Isolated mitochondria from leaves of wild-type plants supported Arg-stimulated ●NO synthesis that could be inhibited by NOS inhibitors and quenched by a ●NO scavenger; this NOS activity is absent in mitochondria isolated from nos1 mutant plants. Because mitochondria are a source of reactive oxygen species (ROS), which participate in senescence and programmed cell death, these parameters were examined in the nos1 mutant (Guo and Crawford, 2005). The main production of ROS and NO took place in the xylem, sclerenchyma, and epidermis (Rodriguez-Serrano et al., 2009). In cell wall lignifications of xylem elements, an oxidative burst is involved and a NO burst also participates in the programmed cell death associated with the differentiating vessels. Moreover, ROS and NO production could be involved in signal transduction pathways to activate the response to stress in other tissues (Gabaldon et al., 2005). Nitric acid synthase 1 (NOS1), which produces NO in mitochondria, reduces ROS accumulation and the resulting oxidative damage in entire leaves. Such protection is important for mitochondria because mitochondrial proteins are especially vulnerable

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to oxidative stress (Sweetlove et al., 2002; Bartoli et al., 2004).

7. E. V. Hypersensitive Response ROS H2O2 has been shown to be a diffusible signal mediating localized PCD during hypersensitive Response (HR) (Levine et al., 1996), as well as being involved in a systemic signaling network (Alvarez et al., 1998). Mittler et al. (1999) using transgenic catalase/Prx-deficient tobacco plants showed that these were hyper-responsive to pathogen challenge, thus providing direct evidence for a role for H2O2 in HR cell death. Hypersensitive Response (HR) cell death may require a fine balance between NO and ROS (Delledonne et al., 2001). NO or ROS are not essential for Hypersensitive Response (HR) in plants but induce apoptosis in adjacent cells during the defense response (Tada et al., 2004). The HR is initiated and regulated by calcium (Grant et al., 2000) and reactive oxygen species (ROS) mainly the superoxide anion ●O2

− and H2O2 (Lamb and Dixon, 1997) and nitric oxide (●NO) (Delledonne et al., 1998). In regulating gene expression, ●NO can induce the expression of Phenyl ammonia-lyase (PAL) and chalcone synthase independently of ROS, and induction by ●NO of defense-related genes, such as glutathione S-transferase, depends on H2O2 (Grun et al., 2006). G: GO was injected into the apoplast to generate H2O2 at levels that had previously shown to be an effective initiator of plant defense (Mur et al., 2005). G:GO only produced a single burst of ethylene production in marked contrast to ethylene production following injection of sodium nitroprusside (SNP), an NO+ donor that is a potent nitrosylating agent, but subsequently releases gaseous ●NO following electrophilic attack (Membrillo-Hernandez et al., 1998). It was found that ●NO appears to be involved in the pathways leading to the accumulation of transcripts encoding the heat shock protein TLHS-1, the ethylene forming enzyme cEFE-26, and cell death. In contrast, ●NO does not act upstream of the elicitor induced activation of mitogen activated protein kinase, the opening of anion channels, nor expression of GST, LOX-1, PAL, and PR-3 genes (Lammote et al., 2004). Mur et al. (2008) abstracted that tobacco leaves treated with the C2H4 biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), suggested that C2H4 influenced the kinetics of a Hypersensitive

Response (HR). Challenging salicylate hydroxylase expressing tobacco lines and tissues exhibiting systemic acquired resistance (SAR) suggested that C2H4 production was influenced by salicylic acid (SA). Subsequently, treating leaves to increase oxidative stress or injecting with SA initiated monophasic C2H4

generation, but the nitric oxide (●NO) donor sodium nitroprusside initiated biphasic rises. The first transient C2H4 rise appeared to be unaffected by NG-nitro-L-arginine methyl ester, but the second rise was reduced. These data suggest that ●NOand SA are required to generate the biphasic pattern of C2H4 production during the HR and may influence the kinetics of HR formation (Mur et al., 2008). Application of MeJa has been found to induce the ROS burst in suspension-cultured cells of parsley (Petroselinum crispum L.), Taxus (Taxus chinensis), Arabidopsis and tobacco Bright Yellow-2 (BY-2) (Nicotiana tabacum L.) (Kauss et al. 1994; Kauss and Jeblick 1995; Wang and Wu 2005). Mur et al. (2006) show that SA and MeJa co-potentiation of the ROS burst is a feature and mechanism of synergistic gene expression and cell death in Arabidopsis and tobacco explants. In fact, MeJa-induced ROS production has also been implicated as one of the mechanisms by which MeJa induces cancer cell death. For example, MeJA induces apoptosis in A549 human lung adenocarcinoma cells through induction of the expression of pro-apoptotic members of the Bcl-2, Bax and Bcl-XS protein families and the activation of caspase-3 via ROS production. These observations prompted the suggestion that H2O2 might sit at the key node of the MeJa signaling pathways (Kim et al. 2004; Oh et al. 2005).

7. F. Oxidants Induction of Tolerance Several ESTs encoding heat shock proteins were induced by H2O2. Heat stress stimulates H2O2 generation in plants (Dat et al., 1998). Moreover, heat shock proteins are involved in enhancing survival following oxidative stress in yeast, animals, and plants (Finkel and Holbrook, 2000). Thus, the induction of genes encoding heat shock proteins and a heat shock transcription factor by H2O2 may lead to increased tolerance of further oxidative stress, as in tomato (Lycopersicon esculentum) cells (Banzet et al., 1998), as well as contributing to tolerance of other stresses such as pathogen challenge (Vallelian-Bindschedler et al., 1998) or

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high temperatures (Dat et al., 1998). It is interesting that one of the heat shock protein genes was also induced independently by wilting, UV irradiation, and elicitation, demonstrating signaling cross talk (Desikan et al., 2001). Hernandez et al. (2001) reported that two pea cultivars with different degrees of salt tolerance exhibited changes in apoplastic ROS-scavenging activities, such as superoxide dismutase (SOD), which positively correlated with salt tolerance. Salt-induced ROS accumulation in endosomes of AtVAMP7C antisense plants gave rise to a cytosolic signal that enhanced salt tolerance (Leshem et al., 2007). Deficiency in either cytosol ascorbic acid peroxidase (APX1) or tylAPX in Arabidopsis resulted in ROS accumulation that in turn generated a signal that enhanced the plant’s tolerance to both osmotic and salt stresses (Miller et al., 2007). Reactive oxygen species (ROS) are also necessary for the development of symbiosis and nodule development. They showed that nodules will not form on roots if GSH synthesis is blocked by addition of BSO, suggesting that like the root meristems, the nodule meristems are unable to develop in the absence of GSH (Frendo et al. 2005). Co-regulation of these genes by various stresses supports the hypothesis that H2O2 mediates cross tolerance (Bowler and Fluhr, 2000). However, it is likely that the exact mechanism and levels of expression of individual genes is dependent on cell type and the specific stress stimulus. Plant cells produce ROS, particularly superoxide and H2O2, as second messengers in many processes associated with plant growth and development (Foreman et al., 2003). Moreover, one of the major ways in which plants transmit information concerning changes in the environment is via the production of bursts of superoxide at the plasma membrane (Doke et al., 1994). Situations, which provoke enhanced ROS production have in the past been categorized under the heading of oxidative stress, which in itself is a negative term implying a harmful process. In fact it is probably in many cases quite the opposite, enhanced oxidation being an essential component of the repertoire of signals that plants use to make appropriate adjustments of gene expression and cell structure in response to environmental and developmental cues. Rather than involving simple signaling cassettes, emerging concepts suggest that the

relationship between metabolism and redox state is complex and subtle (Foyer et al., 2005). References

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Wendehenne D., J. Durner and D. F. Klessig (2004). Nitric oxide: A new player in plant signalling and defense responses. Curr. Opin. Plant Biol. 7, 449-455. Winter H., G. Robinson and H. W. Heldt (1993). Subcellular volumes and metabolite concentrations in barley leaves. Planta 191, 180-190. Wubben J. P., C. B. Lawrence and P. J. G. M. de Wit PJGM (1996). Differential induction of chitinase and 1,3-b-glucanase gene expression in tomato by Cladosporium fulvum and its race-specific elicitors. Physiol Plant Pathol 48:105-116. Xiong L., K. S. Schumaker, J. K. Zhu (2002). Cell signalling for cold, drought, and salt stresses. The Plant Cell 14, S165–S183. Yamamura H. (2002). Redox control of protein tyrosine phosphorylation. Antiox. Redox Sig.4: 479-480. Yannarelli G. G., G. O. Noriega, A. Batlle and M. L. Tomaro (2006). Heme oxygenase up-regulation in ultraviolet-B irradiated soybean

plants involves reactive oxygen species. Planta 224: 1154-1162. Yao X., and Y. uang (2003). From nitric oxide to endothelial cytosolic Ca2+: a negative feedback control. Trends in Pharmacological Sciences 24, 262-266. Zaninotto F., S. La Camera, A. Polverari and M. Delledonne (2006). Cross talk between reactive nitrogen and oxygen species during the hypersensitive disease resistance response. Plant Physiol.141: 379-383. Zhang D. B. and Z. A. Wilson (2009). Stamen specification and anther development in rice. Chin. Sci. Bull. 54: 1-12. Zilli C. G., K. B. Balestrasse, G. G. Yannarelli, A. H. Polizio, D. M. Santacruz and M. L. Tomaro (2008). Heme oxygenase up-regulation under salt stress protects nitrogen metabolism in nodules of soybean plants. Environmental and Experimental Botany 64: 83-89.

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8. Oxidants Ameliorate the Adversity of

Stresses

8. A. Cadmium Detoxifications

Cadmium (Cd) induced an increase of JA and ET, which suggests that these molecules are involved in the cellular response to Cd toxicity. JA is an oxylipin that acts as a signaling compound in different defense situations; such as response to pathogens and herbivore attack (Wasternack and Parthier, 1997). However, responses mediated by JA can be also triggered by diverse abiotic stresses (Devoto and Turner, 2005). Jasmonic acid (JA) is obtained from linolenic acid, and its production is associated with lipid peroxidation and membrane damage (Rodriguez-Serrano et al., 2006). Sandalio et al. (2001) demonstrated that growth of pea plants with Cd induced lipid peroxidation in leaves which would explain the increase observed in this work in JA production. The activation of the pathogen-dependent JA receptor is linked to the ion channel stimulation and ROS production and these conditions come together under Cd stress (McDowell and Dangl, 2000; Garrido et al., 2003). In Arabidopsis plants, JA regulates genes involved in glutathione and phytochelatins synthesis under Cd treatment (Xiang and Oliver, 1998). The increase of JA could also contribute to metal toxicity through the activation of lipoxygenase activity, H2O2 production, and lipid peroxidation (Wang and Wu, 2005; Maksymiec et al., 2007). JA is a component of the signaling processes under biotic and abiotic stresses (Devoto and Turner, 2005). Under Cd stress, an increase of two times in methyl jasmonate (MeJA) took place in pea leaves, and free JA was detected neither in control nor in Cd-treated plants. The analysis of SA content shows that free SA was the main form present in pea leaves. On the contrary, Cd treatment did not produce any statistically significant effect on the SA levels, although the contents of conjugated

(methyl salicylate [MeSA]) and free SA were slightly reduced in Cd-treated plants. Analysis of ET by GC showed an increase of two times in leaves from pea plants grown with 50 µM CdCl2, and this increase was reversed by supplying Ca to the nutrient solution, although a slight increase of ET emission was also observed in control plants (Rodriguze-Serrano et al., 2009). Ethylene (ET) plays a pleiotropic role in plant growth and development and is involved in a number of processes, including germination, senescence, and fruit ripening, but it also participates in a variety of defense responses (Guo and Ecker, 2004). The stimulation of ethylene (ET) biosynthesis by Cd was reported in different plant species, although the molecular relationship between ET biosynthesis and Cd stress has not been well-established (Sanitadi Toppi and Gabbrielli, 1999). Cd can be detoxified by phytochelatins, whose synthesis is induced by Cd and other metals and is accompanied by a decrease in the concentration of glutathione (Zenk, 1996). Cd is well known to produce disturbances in both the uptake and distribution of elements in pea plants (Hernandez et al., 1998; Sandalio et al., 2001; Tsyganov et al., 2007) and other plant species (Gussarson et al., 1996; Rogers et al., 2000). Induction of phytochelatins is one of the main detoxification strategies against Cd, by chelating Cd ions and preventing its toxicity (Howarth et al., 2003; Nocito et al., 2006). Differential Expression of SODs by Cd dependent reduction of SOD activity has been reported in wheat (Milone et al., 2003), pea (Sandalio et al., 2001), and bean Phaseolus vulgaris. Although the opposite effect was observed in Alyssum plants (Schickler and

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Caspi, 1999), sunflower Helianthus annuusandcoffee Coffea arabica cells (Gomes-Junior et al., 2006), and radish roots (Vitoria et al., 2001). These discrepancies are due to differences in the metal concentration and also in the period of treatment used in each case, in addition to the plant tissue studied. Thus, in garlic (Allium sativum) plants, SOD increased under short Cd treatment but decreased after long-term exposure (Zang et al., 2005). Long-term exposure to high Cd concentrations produced in pea leaf the down-regulation of Mn-SOD and CuZn-SOD transcripts, which is correlated with the reduction of their activities observed previously (Sandalio et al., 2001; Romero-Puertas et al., 2007). The plastidic Fe-SOD, in turn, was up-regulated, although its activity was previously observed to be reduced by the metal (Sandalio et al., 2001). This suggests a possible posttranslational regulation of Fe-SOD by oxidation Fe-SODs are sensitive to H2O2 or by reduction of Fe availability (Del Rio et al., 1991). A similar effect was observed in pea roots under the same experimental conditions, except for the Mn-SOD, which was up-regulated by Cd at both transcript and activity levels (Rodriguez-Serrano et al., 2006). Down-regulation of CuZn-SOD by Cd was reverted by Ca supply, and the same results were observed in Arabidopsis plants, which suggests a role of this element in the regulation of CuZn-SOD at the transcriptional level, although the mechanism involved is unknown. CuZn-SOD activity was also recovered by Ca where Ca deficiency has also been associated with a reduction of SOD activity and oxidative stress in tomato Solanum lycopersicum plants (Schmitz-Eiberger et al., 2002). Sunkar et al. (2006) have demonstrated that micro RNA (miR398) regulates CuZn-SOD protein in Arabidopsis under oxidative stress. The reduction observed in SOD activity and other antioxidants such as CAT observed previously could be responsible for the overproduction of ROS detected by CLSM, which would produce oxidative damages at macromolecules, being responsible for the Cd toxicity (Sandalio et al., 2001; Romero-Puertas et al., 2007).

8. B. Calcium Mitigates Stresses Risk

8. B. I. Calcium Mitigates Cadmium Risk

Kinraide et al. (2004) found that Cd induced a strong reduction in the Ca content of leaves (Sandalio et al., 2001). Ca is an important signaling component in biotic and abiotic stresses, and disturbances in its content have been associated with toxicity by Cd, zinc (Zn), copper (Cu), or aluminum (Al). Although the mechanisms involved are not well known. Growth with 50 µM CdCl2 produced a decrease in the contents of Ca, Cu, Fe, Mn, and Zn in pea leaves (Rodriguze-Serrano et al., 2009). On the contrary, sulfur was accumulated 3-fold in Cd-treated plants with respect to the control plants. The induction of sulfur metabolism by Cd has been described previously and involves a coordinated transcriptional regulation of genes for sulfate uptake and its assimilation. Several studies have demonstrated that Cd can enter the cells by the same uptake systems used by cations such as Fe, Cu, Ca, and Zn. Excess Cd could compete with those elements for the transporters promoting a reduction in both uptake and accumulation of those cations (Clemens, 2006). The exogenous supply of Ca to the nutrient solution reduced the accumulation of Cd in the tissue, which demonstrates the competition between both elements for the same transporters. In contrast, the addition of Ca did not alter considerably the accumulation of the rest of the elements, except for Mn and Mg in control plants. It has been observed that Cd competes with Ca not only for the transporters but also for intracellular Ca-binding proteins (Rivetta et al., 1997) and plasma membrane (Kinraide, 1998). Tsyganov et al. (2007) observed a relationship between Cd tolerance and homeostasis of Ca in both roots and shoots in a Cd-tolerant pea mutant (SGECdt). Ca has also been reported to alleviate Cd toxicity in radish Raphanus sativus (Rivetta et al., 1997), rice (Oryza sativa) roots (Kim et al., 2002), and Arabidopsis Arabidopsis thaliana seedlings (Suzuki, 2005), by reducing Cd uptake. Ca also prevents Al-dependent growth inhibition in wheat Triticum aestivum (Kinraide and Parker, 1987). It was found that a reduction of Ca content could interfere with the expression of antioxidant enzymes like CuZn-SODor could inactivate Ca-CaM-dependent proteins (Rivetta et al., 1997).

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8. B. II.Calcium and Nitric Acid Mitigate

Cadmium Risk

Cross talk between ROS, NO, and Ca2+ in the regulation of the cellular response to longterm Cd exposure is proposed. Cd produces nutrient disturbances, with Ca2+ being one of the most negatively affected elements. Ca and calmodulin (CaM) are involved in the control of many physiological and biochemical processes, mainly through different signal transduction pathways (Rodriguez-Serrano et al., 2009). It was stated that the reduction of NO could affect the activity of proteins regulated by S-nitrosylation, such as Met adenosyltransferases (MAT-1) involved in Ethylene (ET) biosynthesis, and this could be the reason for the induction of ET emission in Cd treated plants. A decrease in the level of NO could, directly or indirectly, promote the accumulation of O2

●─ and induce oxidative stress. In turn, ROS accumulation can cause membrane damage that is involved in JA and ET production (Rodriguez-Serrano et al., 2009). Finally, the overproduction of JA, ET, and ROS could activate the cell response with the induction of PRs in order to protect proteins from damage associated with Cd toxicity. The increase of JA, ET, and ROS production, the up-regulation of PRs, and the NO reduction are common features of senescence (Obregon et al., 2001; Corpas et al., 2004; Rodriguez-Serrano et al., 2006), which suggests that Cd accelerates senescence processes in plants. In pea plants, Cd could generate a senescence response induced by pathogen attack, which is characterized by ROS overproduction, NO reduction, and PR up-regulation (Rodriguez-Serrano et al., 2009). However, unlike ozone or pathogen attack, Cd did not produce any visible symptoms of local necrosis (Sandalio et al., 2001), although the formation of micro lesions not visually detectable cannot be excluded. Rodriguez-Serrano et al. (2009) observed that Ca supply reversed the induction of ethylene (ET) by Cd, and this fact could be due to an indirect effect of Ca on ROS and NO production. ET and NO are antagonistic which would explain the reduction of NO and the rise of ET emission observed in this work. This fact is supported by the effect of Ca on the accumulation of NO and ET. Proposed that NO could inhibit 1-aminocyclopropane-1-carboxylic acid synthase or 1-aminocyclopropane-1-carboxylic acid

oxidase and so prevent ET formation (Leshem, 2000). Lindermayr et al. (2006) demonstrated the reversible inhibition of Met adenosyltransferases (MAT-1) by S-nitrosylation, which can originate a reduction of the S-adenosylmethionine pool and, therefore, a decrease of ET biosynthesis. Thus, the Cd-dependent reduction of the NO level in leaves, observed in this work, could alter MAT-1 regulation by NO and, therefore, increase ET biosynthesis. The growth of pea plants in full-nutrient solutions containing 50 µM CdCl2 for 15 d produced an accumulation of Cd in the leaves of about 13 µg g-1 dry weight. In these conditions, a reduction in the content of the following nutrients was observed: Ca (27%), Cu (30%), iron (Fe; 19%), manganese (Mn; 47%), magnesium (Mg; 20%) and (Zn; 41%). On the contrary, Cd produced a 3-fold increase in the sulfur content, while the sodium contents were not affected by the heavy metal treatment (Rodriguze-Serrano et al., 2009). They found that the addition of Ca (NO3)2 to the nutrient solution produced an increase in the content of this element in both control and Cd-treated plants. Besides 30% reduction in the Cd accumulation in the leaves of Cd-treated plants, without affecting the content of the remaining elements, except the Mn, which increased, and Mg, which decreased slightly, in control plants. Lettuce plants irrigated with 0, 100, 200 and 300mg.l-1cadmium polluted water, during the growing season plants were sprayed and irrigated twice by 2ml.l-1 humic + fulvic acid mixture, 200 mg.l-1 ascorbic acid and calcium chloride 100 mg.l-1. Lettuce manifested significantly higher cadmium content 3.35 fold and 3.186 fold for untreated and 300mg.l-1, respectively, itexceeded untreated in lead accumulation as compared to untreated (18.464 fold) and 100mg.l-1(7.847 fold). Cadmium treated lettuce tended to reduce dry matter percentage, chlorophyll percentage out of total pigments and nitrogen content. However, cadmium treated lettuce revealed substantial increases in terms of accumulated cadmium, accumulated lead and accumulated calcium in edible tissues. The most potent ameliorating agents was humic + fulvic mixture followed by ascorbic acid and then CaCl2 (Abdel, 2012). The constitutive L-Arg-dependent NOS activity previously described in pea plants is dependent on Ca and CaM (Corpas et al., 2004). The Cd dependent reduction of NO observed was due to

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a Ca deficiency, pea plants were supplemented with Ca (NO3)2 during the Cd treatment. The reduction of constitutive NO production by Cd was reversed by supplying Ca to the nutrient solution, reaching similar levels of NO to those observed in control leaves. This suggests that the NO decrease by Cd could be due in part to an inactivation of the NOS activity as a consequence of the Cd-induced Ca deficiency in leaves. A higher magnification of images from Ca-Cd-treated plants shows the production of NO associated with the apoplast in xylem vessels and also in sclerenchyma cells. A punctate pattern of fluorescence was also observed in mesophyll cells (Rodriguze-Serrano et al., 2009). Heat Shock Proteins (HSPs) are also upregulated in response to wounding, osmotic stress, light (Wang et al., 2004), and oxidative stress (Ma et al., 2006). The upregulation of genes involved in protein folding in response to Cd treatment has been observed in Arabidopsis plants (Suzuki et al., 2001), which demonstrates that Cd toxicity is in part due to the induction of protein denaturation, probably by oxidative modifications (Romero-Puertas et al., 2002). In contrast with the former PRs, the metal did not affect Phenyl Ammonia Lyase (PAL) expression. The regulation of Phenyl Ammonia Lyase (PAL) is dependent on NO (De Pinto et al., 2002; Wang and Wu, 2005), and the reduction of NO induced by the Cd treatment could explain the absence of changes in the PAL transcript levels.

8. B. III. ROS, NO And Ca Mitigate Cd Adversity

In plant cells, some of the proteins modulated by CaM include NAD kinases, Glu decarboxylase, HSPs (Lu and Harrington, 1994), and Catalase CAT (Yang and Poovaiah, 2002). Ca is involved in the regulation of plant cell metabolism and signal transduction (Yang and Poovaiah, 2002; Rentel and Knight, 2004) and modulates cellular processes by binding proteins such as calmodulin (CaM), which in turn regulates the activity of target proteins (Roberts and Harmon, 1993). NO is a free radical that can react with ●O2

− and, thus, regulate its accumulation in the tissue (Romero-Puertas and Delledonne, 2003). The reduction of NO under Cd treatment could favor ●O2

− accumulation, promoting oxidative damages. This fact is supported by the reduction of ●O2

− accumulation after the restoration of NO production induced by Ca treatment. The

involvement of NO in different biotic and abiotic stresses has been demonstrated (Gould et al., 2003). The NO synthase-dependent NO production was strongly depressed by Cd, and treatment with Ca prevented this effect. Under these conditions, the pathogen-related proteins PrP4A and chitinase and the heat shock protein 71.2, were up-regulated, probably to protect cells against damages induced by Cd. The regulation of these proteins could be mediated by jasmonic acid and ethylene, whose contents increased by Cd treatment. A model is proposed for the cellular response to long-term Cd exposure consisting of cross talk between Ca, ROS, and NO. In L. luteus roots, the supply of NO as SNP reduced the negative effects of Cd, NaCl, ET, and paraquat and reduced the ●O2

− production induced by Cd and lead (Kopyra and Gwozdz, 2003). A protective role of NO has also been observed in sunflower leaves under Cd toxicity. NO is a signal molecule involved in triggering the defense response of cells against different stress conditions (Romero-Puertas and Delledonne, 2003; Neill et al., 2008). The analysis of NO production by 4,5-diaminofluorescein diacetate (DAF-2DA) fluorescence microscopy showed that fluorescence of control leaves was mainly due to a NOS-like activity, to judge by its inhibition by amino guanidine (Corpas et al., 2004). However, production of NO was strongly reduced by Cd. The reduction of NO levels by Cd was also observed previously in pea roots and leaves under the same experimental conditions (Barroso et al., 2006; Rodriguez-Serrano et al., 2006). Al treatment also led to a reduction of NO production in roots from Hibiscus (Tian et al., 2007) and Arabidopsis plants (Illes et al., 2006). However, Bartha et al. (2005) observed a Cd-dependent increase of NO accumulation in roots from pea seedlings and soybean (Glycine max) cell suspensions, respectively, after short-term treatment with Cd. This discrepancy could be attributed to differences in the cell response to short and long periods of metal treatment. The constitutive NOS activity described in pea plants is dependent on Ca and CaM (Corpas et al., 2006). The Ca-dependent restoration of NO accumulation in Cd-treated plants shows that Ca could be a key point in Cd toxicity by reducing NOS activity and modulating NO production and, therefore, those proteins regulated by S-nitrosylation. This result suggests the existence

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of cross talk between NO, ROS, and Ca under Cd toxicity (Rodriguez-Serrano et al., 2009). In the presence of transition metals such as iron or metal complexes, H2O2 can be rapidly degraded through oxidation or reduction reactions. Such reactions are exploited in industrial processes where H2O2 is reductively cleaved to the highly oxidizing hydroxyl radical, the existence of which was first inferred by Fenton in 1876. The potential for conversion of H2O2 to more reactive species is controlled inside cells, first, by systems that regulate the availability of metals such as iron (Hell and Stephan, 2003) and, second, by the existence of numerous H2O2-metabolizing enzymes, many of which themselves depend on iron-dependent catalysis through haem groups (Mittler et al., 2004). In animal cells, most of the biological regulatory properties of ●NO have been explained on the basis of its capacity to act as an iron ligand in haemoproteins. Dual activating or inhibitory effects of ●NO on haemoproteins have been described, and the nature of the effect seems to depend to a great extent on the resting state (oxidation state) of the haemoproteins, which conditions the ligand properties and the electronic configuration of the haemiron (Tsai, 1994). However, independently of ONOO─, NO and O2

●─ real roles in plant cells, which remains to be clearly established, ONOO– may be regarded as a substrate of plant peroxidases (Prxs) (Gebicka and Gebicki, 2000). In fact, ONOO– reacts (k=3x106 M-1 S-1) with the resting form of the enzyme to lead to CII (Floris et al., 1993): [Fe3+ + ONOO─ → C11 +●NO2], yielding the nitrosylating species, ●NO2. Compound 11 (CII) is catalytically inactive towards ONOO–, and the decay of CII to the native enzyme, Fe3+, only takes place in the presence of an external electron donor, such as a phenol. In such a scenario, a role in the scavenging of ONOO– has been proposed for the chlorogenic acid/Prx system, which may be functional with any other phenol (Grace et al., 1998). The NO-derived 4, 5-diaminofluorescein diacetate (DAF-2DA) green fluorescence was found in xylem vessels, sclerenchyma, and epidermal cells of control plants. However, in contrast with ROS generation, Cd treatment produced a significant reduction of NO-dependent fluorescence observed in control leaves. The incubation of control leaves with amino guanidine or L-NAME, two well-known

inhibitors of animal NOS, also produced a strong reduction of 4,5-diaminofluorescein diacetate (DAF-2DA), fluorescence, which is indicative of the involvement of a NOS-like activity in the production of the NO detected. As a positive control, Cd-treated pea plants were incubated with 10 µM sodium nitroprusside (SNP), a NO donor, and the NO-dependent fluorescence was observed by confocal laser microscopy. In these conditions, a NO-dependent increase in 4,5-diaminofluorescein diacetate (DAF-2DA) fluorescence in the leaf tissue was observed, showing the specificity of DAF-2DA for NO (Ridriguze-Serrano et al., 2009). 8. B. IV. Calcium Binding Annexins

Ca2+ binding is a defining annexin characteristics, but studies on animal annexins show them to be capable of sensing and regulating free cytosolic calcium (Hawkins et al., 2000; Watson et al., 2004). Mammalian AnxA6 has been shown to act as a Ca2+ sensor, mediating membrane association of a GTPase-activating protein (GAP) that then regulates a monomeric GTPase (Grewal et al., 2005). In addition to controlling trafficking of ion transporters to their target membranes and regulating their activity (Gerke et al., 2002), animal annexins can also form Ca2+-permeable ion channels themselves. This ability was first demonstrated with bovine AnxA7 which forms a highly selective, voltage-gated. The ability of plant annexins to form or regulate Ca2+ channels in plasma and endo membranes would enable signal transduction and amplification (Kovacs et al., 1998; White et al., 2002). Salt bridges are quite well conserved in plant annexins. It has been proposed that plant annexins could act as the plasma membrane Ca2+-permeable channels that mediate Ca2+ entry into the cell at very negative (hyperpolarized) membrane voltage (White et al., 2002). These channels are strongly implicated in growth and signaling. To date, AnxCa32 has been shown to mediate passive Ca2+ flux in fura-2-loaded vesicles, supporting the general concept of channel function (Hofmann et al., 2000). Plant annexins not only bind purine nucleotides but also hydrolyze them in maize (McClung et al., 1994), tomato (Lim et al., 1998), cotton (Shin and Brown, 1999). Both maize and tomato annexins are able to hydrolyzes ATP and GTP at a similar rate, but GTP is the preferred substrate for cotton annexin AnxGh1. Tomato

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annexin GTPase activity still proceeds when the protein is bound to actin (Calvert et al., 1996), suggesting that cytoskeleton association may specifically locate the annexins GTPase function in the cell. Ca2+ has an inhibitory effect on cotton annexin GTPase activity but not the ATPase/GTPase activity of maize annexins AnxZm33/35. Alignments of the primary sequence of cotton annexin, AnxAt2, and AnxZm33/35 showed that GTP-binding motifs overlap the Ca2+-binding motif of the fourth endonexin domain (McClung et al., 1994; Shin and Brown, 1999). Ca2+ and GTP may therefore compete for binding. Ca2+-mediated phospholipid binding has been shown to inhibit hydrolytic activity of tomato annexin (Calvert et al., 1996). Mutagenesis of the Ca2+-binding sites does not impair GTPase activity of the soluble form demonstrating that membrane binding prevents GTP from reaching its catalytic site (Lim et al., 1998). Overall, modulation of enzyme activity by Ca2+ and membrane binding may afford spatiotemporal definition of annexin function, where tomato, maize, and cotton annexins have different requirements, despite catalyzing the same reaction, may reflect the diverse roles that plant annexins fulfill. Maize annexins AnxZm33/35 contain the putative salt bridges, and a partially purified preparation formed hyperpolarization activated Ca2+-permeable channels in planar lipid bilayers (Nichols, 2005). One gene has been verified as encoding a plant Ca2+ channel, TPC1 encoding a vacuolar channel, it will be of great interest to see whether plant annexins purified to homogeneity support Ca2+ channel activity. Of the eight Arabidopsis annexins, AnxAt1 has been found to form K+-permeable channels in bilayers, with channel formation favoured at low pH (Gorecka et al., 2007). AnxA5 and AnxB12, acidic pH increases the hydrophobicity of AnxAt1 and promotes the oligomerization thought necessary for transport activity. The Ca2+ permeability of the AnxAt1 channel remains to be determined, as does its in vivo function, but AnxAt1 illustrates clearly the multifunctional nature of annexins potentially a peroxidase and a channel in one.

8. C. Mechanism of Stressameliorations

8. C. I. Cytosolic Calcium

Increases in cytosolic free Ca2+ concentration ([Ca2+]cyt) have been detected in response to a wide range of environmental, developmental, and growth stimuli (Lecourieux et al., 2006). According to the ‘Ca2+ signature hypothesis’, each stimulus induces Ca2+ transient which have unique temporal and spatial arrangements determining the specificity of the physiological response (Scrase-Field and Knight, 2003).The data discussed above propose that NO triggers cellular events in plant cells by causing an increase in [Ca2+]cyt. This process has been shown to occur in ABA, hyper-osmotic and elicitor transduction pathways (Garcia-Mata et al., 2003; Gould et al., 2003; Lamotte et al., 2006; Vandelle et al., 2006). In tobacco, NO-sensor protein kinases include NtOSAK, a member of the SnRK2 subfamily activated in response to osmotic stress. Other NO sensors correspond to nitrosylated proteins. The cross-talks operating between NO, Ca2+, and protein kinases in plant cells exposed to biotic and abiotic stimuli (including auxin, ABA, osmotic stress, pathogens, and elicitors of defense responses) might have important functional implications such as the dynamic regulation of gene expression. As described in animal cells, NO might also display a dual role in controlling [Ca2+]cyt concentration since both NO-dependent activation and inhibition of extracellular Ca2+ uptakes are reported. PK: protein kinases (Courtois et al., 2008). Ca2+ is well established as a universal intracellular second messenger (Sanders et al., 2002; Petersen et al., 2005).The changes in [Ca2+]cyt are decoded and relayed through Ca2+ sensors such as calmodulin (CaMs), Ca2+-Dependent Protein Kinases (CDPKs) or annexins (Berridge et al., 2003). A connection among NO, cGMP, Ca2+, and calmodulin (CaM) pathways was suggested the participation of NO in light-mediated processes in plants (Beligni and Lamattina, 2000). It has been shown that NO contributes to [Ca2+]cyt increases in plant cells exposed to biotic and abiotic stresses including hyper-osmotic stresses and elicitors of defense responses (Gould et al., 2003). Using tobacco cells constitutively expressing the Ca2+ reporter apoaequorin in the cytosol, have shown that NO participates in the cryptogein mediated

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elevation of cytosolic free Ca2+ through the mobilization of Ca2+ from intracellular stores. The NO donor diethylamine NONOate promoted an increase in cytosolic free Ca2+ concentration, which was sensitive to intracellular Ca2+ channel inhibitors (Lammotte et al., 2004). 8. C. II. Calcium and Nitric Acid

It was demonstrated that the NO donor DEA/NONOate induced a fast and transient activation of NtOSAK, in tobacco suspension cell cultures. Additionally, NO might be a key component of the hyperosmotic stress-induced signaling cascade leading to NtOSAK activation. An attempt was also made to clarify the NO-dependent upstream pathway of NtOSAK activation. Initial data established that neither NO-mediated Ca2+ influx nor Ca2+ release from internal stores were required for NtOSAK activation (Lamotte et al., 2006). Courtois et al. (2008) illustrated that NO can directly influence the activity of target proteins through nitrosylation and has the capacity to act as a Ca2+-mobilizing intracellular messenger. The interplay between NO and Ca2+ has important functional implications, expanding and enriching the possibilities for modulating transduction processes. Furthermore, protein kinases regulated through NO-dependent mechanisms are being discovered, offering fresh perspective on processes such as stress tolerance.Cryptogein, an elicitor from oomycete Phytophthora cryptogea, triggers NO production that subsequently participates in the mobilization of Ca2+ from internal stores, thereby increasing Ca2+ cytosolic concentrations (Lamotte et al., 2004). 8. C. III. Cellular Membranes Modulations by

Oxidants and Calcium

NO possesses a role in modulating plasma membrane Ca2+-permeable channels. Indeed, NO releases by NO donor diethylamine (DEA-NONOate) was found to trigger a fast and transient influx of extracellular Ca2+ in tobacco cells. Because the NO evoked Ca2+ influx occurred concomitantly with a Ca2+-independent plasma membrane depolarization, it was assumed that NO may promote the opening of voltage gated Ca2+ channels subsequent to membrane depolarization (Lamotte et al., 2006). It was suggested that NO might modulate plasma membrane Ca2+-permeable channels by

showing that NO negatively regulates Ca2+ entry in grapevine cells challenged by the elicitor endo polygalacturonase 1 (Vandelle et al., 2006). These results expands the role of NO in plant signaling as a more general regulator of Ca2+ homeostasis, promoting both activation and/or inhibition of Ca2+ fluxes. As reported in endothelial cells or neurons, a negative feedback could serve to protect cells from the detrimental effects of excessive NO and Ca2+ (Yao and Huang, 2003). However, artificially generated NO were not evokes rises in nuclear free Ca2+ concentration in tobacco cell suspensions expressing apoaequorin in the nucleus (Lecourieux et al., 2005). Therefore, NO action on Ca2+ homeostasis might be restricted to specific cellular compartments. A specific role for NO in activating intracellular Ca2+ channels was assumed through pharmacological and biochemical approaches in tobacco and grapevine cells exposed to the elicitors cryptogein and endo polygalacturonase 1, respectively (Vandelle et al., 2006). Pharmacological experiments suggested that NO is active upstream of [Ca2+]cyt transients during the processes of ABA-induced stomata closure and auxin–induced adventitious root formation (Desikan et al., 2002; Lanteri et al., 2006). Pharmacological, biochemical and electrophysiological approaches have shown that NO modulates the activity of plasma membrane as well as intracellular Ca2+-permeable channels. Almost all types of Ca2+ channels appear to be regulated by NO (Clementi, 1998). The concept that NO also modulates Ca2+-permeable channels in plant cells was supported by the observation that the NO released by NO donors induced a transient rise in [Ca2+]cyt in Vicia faba guard cells and in tobacco cell suspensions (Garcia-Mata et al., 2003; Gould et al., 2003; Lamotte et al., 2006). Inhibitors of plasma membrane and intracellular Ca2+ permeable channels have both been found to inhibit NO induced increases in [Ca2+]cyt (Garcia-Mata et al., 2003; Vandelle et al., 2006). Thus, NO might promote an influx of Ca2+ from the extracellular space and/or mobilization of Ca2+ sequestered in intracellular Ca2+ stores. Although the identity of the Ca2+ permeable channels involved in that process remains unknown, all pharmacological-based studies unanimously point out Ryanodine Receptors (RYR)-like channels as main targets for NO (Besson-Bard et al., 2008).

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8. C. IV. Phosphorylations

Phosphorylation-dependent events in the mediation of NO-induced Ca2+ mobilizations are required (Sokolovski et al., 2005). It is likely that protein kinases might represent an important pathway by which NO dependent Ca2+ signals are decoded. A 50 kDa CDPK, the activity of which was induced by NO through a Ca2+-dependent process, was characterized in cucumber explants (Lanteri et al., 2006). The 50 kDa CDPK might contribute to NO-induced adventitious root formation. Likewise, in our laboratory, evidence was obtained that the activation of the tobacco MAPK SIPK (Salicylic acid-Induced Protein Kinase) by NO donors (Klessig et al., 2000) requires a transient influx of extracellular Ca2+ in tobacco cells (Courtois et al., 2008). Elevating [Ca2+]cyt, NO might influence indirectly the activity of proteins including protein kinases and Ca2+-sensitive K+ and Cl– channels as described in guard cells (Garcia-Mata et al., 2003). It was speculated that NO/Ca2+ pathways, as well as the combined action of NO and Ca2+, might modulate the transcriptional regulation of specific set of genes involved, for instance, in disease resistance or developmental process. NO production occurs through two enzymatic routes: a nitrite-dependent and L-arginine (L-arg) routes. The L-arginine-dependent pathway is up-regulated by upstream Ca2+ fluxes which might be partly mediated by cyclic nucleotide gated channels (CNGCs). The increases in [Ca2+]cyt caused by NO are due to extracellular Ca2+ uptakes and/or mobilization of intracellular Ca2+. Mechanism through which NO can mobilizes intracellular Ca2+ may involve cADPR, cGMP, and phosphorylation-dependent processes. The NO/Ca2+ information seem to be partly processed by CDPKs and MAPKs (Courtois et al., 2008). cADPR and increases in [Ca2+]cyt have been described as messengers in the cGMP-dependent signaling pathways induced by NO in both animals and plants (Lamattina et al., 2003; Neill et al., 2003). Together with cyclic ADP-ribose (cADPR) the involvement of protein kinases in mediating NO-induced changes in [Ca2+]cyt has become an interesting object of study (Besson-Bard et al., 2008). Inhibitors of protein kinases, such as K252a and staurosporine, reduced the [Ca2+]cyt rises triggered by NO in Vicia faba guard cells and tobacco cell suspensions, indicating that protein kinases might be downstream effectors

of NO action on [Ca2+]cyt (Sokolovski et al., 2005). Lamotte et al. (2006) identified these protein kinases by analyzing the protein kinase activities of protein extracts from N. plumbaginifolia cells exposed to the NO donor diethylamine-NONOate (DEA/NO) a treatment leading to an increase in [Ca2+]cyt. Treatment with NO resulted in the activation of a 42-kDa protein kinase within 5 min. Its activation was observed with diethylamine-NONOate (DEA/NO) concentrations as low as 50 µM (Lamotte et al., 2006). The NO-induced 42-kDa protein kinase was identified as NtOSAK (Nicotiana tabacum). Osmotic-Stress-Activated protein Kinase a member of the plant SNF (Sucrose Non Fermenting) 1-related protein kinase type 2 (SnRK2) family (Mikolajczyk et al., 2000; Kelner et al., 2004). The Ca2+ surrogate La3+, which blocks the NO-induced Ca2+ influx and therefore reduces the subsequent [Ca2+]cyt elevation (Lamotte et al., 2004), completely suppressed the activation of the 48-kDa kinase by NO, as revealed by Western blotting and in-gel kinase assay (Besson-Bard et al., 2008). By contrast, the ryanodine receptors (RYR) inhibitor did not affect the activation of the 48-kDa kinase. Thus, the activation of Salicylic acid-Induced Protein Kinase (SIPK) could be preceded by a rise in [Ca2+]cyt triggered by the NO dependent activation of plasma membrane Ca2+-permeable channels. The scenario is bound to be different for Hyperosmotic and salt stress-activated protein kinase (NtOSAK), whose activation is insensitive to La3+ and intracellular Ca2+-permeable channel inhibitors (Lamotte et al., 2006). The demonstration that artificially generated NO stimulated Mitogen-Activated Protein Kinase (MAPKs) including Salicylic acid-Induced Protein Kinase (SIPK) was previously reported (Clarke et al., 2000; Kumar and Klessig, 2000; Pagnussat et al., 2004). However, the key role of Ca2+ in that process has not been investigated so far. The observation that a convergence of Ca2+and NO-signaling pathways might occur at the Mitogen-Activated Protein Kinase (MAPK) level was similarly observed in neuronal cells by Lee et al. (2000). It should be noted that NO has been shown to activate Salicylic acid-Induced Protein Kinase (SIPK) through a salicylic acid (SA)-dependent pathway in tobacco leaves (Kumar and Klessig, 2000).

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8. C. V. Inositol Triphosphate (Ip3)

It has been reported that ●NO could activate the enzyme phospholipase C (PLC), which catalyzes the formation of inositol 1, 4, 5-trisphosphate (IP3). Thus, PLC activity has been proposed to be part of the NO-dependent pathway that control [Ca2+]cyt via inositol 1,4,5-trisphosphate (IP3) (Clementi et al., 1995). NO-dependent phospholipase C (PLC) activation in xylanase elicited tomato (Solanum lycopersicum) cells (Lanteri et al., 2006), suggested that auxin and ●NO effects might also be accomplished through IP3-regulated Ca2+ channels. It was shown that ●NO is required for the mobilization of Ca2+ from internal stores (Garcia- Mata et al., 2003). Downstream, Ca2+ might affect PLC, Diacylglycerol kinase (DGK), Phospholypase (PLD) (Munnik et al., 1998), and NADPH oxidase (McPhail et al., 1999), there with constituting a feedback toward PA generation via PLC/DGK regulation and PLD. Inhibition of Diacylglycerol kinase (DGK) or reduction of Phospholypase D (PLD)-produced PA does not affect ●NO production, suggesting Inositol 1, 4, 5-trisphosphate (IP3) and Ca2+ to be responsible for this putative feedback. Finally, it should be mentioned that the possibility that NO might also influence the activity of inositol 1, 4, 5-triphosphate receptors has been suggested (Vandelle et al., 2006). 8. C. VI. Calcium Chennel

Respiratory Burst Oxidase Homolog (RBOH)/ROP-GTP interaction is regulated by the binding of calcium to two EF-hand motifs at the oxidase N terminus (Wong et al., 2007). The consequence of Respiratory Burst Oxidase Homolog (RBOH) activation is localized production of ●O2

─, which is rapidly converted to H2O2, presumably in the apoplastic space. Tip localized activation of RBOHC promotes calcium channel activation and calcium influx, thereby stimulating RBOH activity and amplification of the initial signal (Takeda et al., 2008). Demidchik et al. (2007) indicates that H2O2 and ●OH might serve as distinct signals in the regulation of calcium influx in roots, due to the existence of calcium channels that are distinctly sensitive to specific intracellular or extracellular generated ROS. The calcium and ROS connection in the regulation of stomata aperture was studied further. It was shown previously that Abscisic acid promotes ROS production that results in

increases in cytosol calcium that lead to stomata closure (Kwak et al., 2006). 8. D. Induction of Salt Tolerance

Under salinity stress (100 mM NaCl), peroxisomes are required for NO accumulation in the cytosol, thereby participating in the generation of peroxynitrite (ONOO2) and in increasing protein tyrosine nitration, which is a marker of nitrosative stress (Corpas et al., 2009). Hyperosmotic and salt stress-activated protein kinase (NtOSAK) was identified as a hyperosmotic and salt stress-activated protein kinase, suggesting that it may play an important role in osmotic-stress signaling (Mikolajczyk et al., 2000). Accordingly, NO was shown to be required for NtOSAK activation in tobacco cell suspensions exposed to hyperosmotic stress (Lamotte et al., 2006). In addition to NtOSAK, NO induced the activation of a second protein kinase with a molecular mass of 48 kDa. Its activity peaked at 30 min before returning to the basal level and was detected only at high diethylamine-NONOate (DEA/NO) concentrations that is mM ranges (Besson-Bard et al., 2008). Salt effects on the phb3-3 mutant were tested by measuring NO accumulation and primary root lengths after treatment with NaCl. NO levels increased 2.7-fold in roots of wild-type seedlings after 0.5 h treatment with 200 mM NaCl. In contrast, no change was observed in NaCl-treated phb3-3 roots. When growth of primary roots was measured, mutant seedlings showed less inhibition from salt stress than wild-type seedlings. After 2 d of 50 and 100 mM NaCl, the growth of wild type roots was reduced by 45 and 69%, respectively, whereas root growth was reduced by only 33 and 46% in the mutant (Wang et al., 2010). The involvement of NO in salt stress in several plant species has been demonstrated using pharmacological agents to manipulate endogenous NO level (Zhao et al., 2004; Zhang et al., 2006) and using Atnoa1/rif1 mutant plants (Zhao et al., 2007). Similar to freezing tolerance, tolerance to salt stress is also positively correlated with endogenous NO level, such that treatments with NO donor and NO scavenger alleviate and exaggerate salt stress symptoms, respectively (Zhao et al., 2007; Zhang et al., 2006). However, in contrast to freezing stress, salt stress inhibits NOS activity, thus reducing endogenous NO level in roots (Zhao et al., 2007). The reduction of endogenous NO level under salt stress may facilitate accumulating more Na+ and fewer K+

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ions and enhancing associated oxidative stress, thus rendering plants more vulnerable to salt stress (Zhao et al., 2007). The mitigating effect of exogenous NO on plants under salt stress may lie in up-regulation of H+-ATPases in the plasma membranes and tonoplasts, minimizing Na+ accumulation and/or compartmentation into vacuoles (Zhao et al., 2004; Zhang et al., 2006). It remains unknown whether the NO-dependent increase in freezing tolerance is associated with plant H+-ATPases. To identify genes whose expression is correlated with salinity stress in cotton, a cDNA library was constructed by using mRNA isolated from salt-induced seedlings of a salt tolerant cotton cultivar, ZM3, and screened by differential hybridization cDNAs encoding specific proteins whose activity may contribute to salt tolerance. A cDNA clone, GhMT3a, which encodes a type 3 plant MT, was isolated and characterized. Northern blot analysis indicated that the expression of GhMT3a in cotton seedlings was induced by several abiotic stresses factors, including salinity, drought, and low temperature, and these induced expression patterns of GhMT3a could be inhibited in the presence of antioxidants. Recombinant GhMT3a protein showed an ability to bind metal ions and scavenge ROS in vitro. Transgenic tobacco and yeast that overexpress GhMT3a displayed increased tolerance to environmental stresses, indicating its role in response to abiotic stresses is by mediating the ROS balance as a ROS scavenger in plants (Xue et al., 2009). Bharali and Bates (2002) demonstrated accentuated loss of bisulfite from the incubation solution in P. schreberi and R. triquetrus following pretreatment with Fe3+. Analytical data showed that this treatment increased the total Fe concentration of the tissues, with probably most of the metal becoming exchangeably bound or otherwise adsorbed in the cell walls. There is little doubt that the Fe3+ catalysed extracellular oxidation of bisulfite as the pretreatment was accompanied by an increase in the sulfate concentration of the external solution (Bharali and Bates, 2006). High salinity, low temperature and drought are critical environmental factors that limit agricultural production worldwide, mainly by affecting plant growth and development. The cellular and molecular responses of plants to these stresses have been studied intensively (Hasegawa et al., 2000; Thomashow, 1999; Xiong et al., 2002).

8. E. Induction of Cold Acclimation Zhao et al. (2009) evaluated the role of NO in cold acclimation and freezing tolerance using Arabidopsis (Arabidopsis thaliana) wild type and mutant nia1nia2 (for nitrate reductase [NR]-defective double mutant) and Atnoa1/rif1 (for nitric oxide associated1/resistant to inhibition by fosmidomycin1) that exhibit defects in NR and reduced NO production, respectively. Cold acclimation induced an increase in endogenous NO production in wild-type and Atnoa1/rif1 leaves, while endogenous NO level in nia1nia2 leaves was lower than in wild-type ones and was little changed during cold acclimation. Cold acclimation stimulated Nitrate reductase (NR) activity and induced up-regulation of NIA1 gene expression. In contrast, cold acclimation reduced the quantity of NOA1/RIF1 protein and inhibited NO synthase (NOS) activity. These results indicate that up-regulation of NR-dependent NO synthesis underpins cold acclimation induced NO production. Seedlings of nia1nia2 were less tolerant to freezing than wild-type plants. Subsequently, pharmacological studies using NR inhibitor, NO scavenger, and NO donor showed that NR-dependent NO level was positively correlated with freezing tolerance. Furthermore, cold acclimation up and down regulated expression of gene responsible for Pro accumulation under osmotic stress (P5CS1)andproline Dehydrogenase (ProDH) gene resulting in enhanced accumulation of proline (Pro) in wild type plants. NR inhibitor and NO scavenger reduced the stimulation of Pro accumulation by cold acclimation, while the NOS inhibitor did not affect Pro accumulation by cold acclimation. In contrast to wild type plants, cold acclimation up-regulated ProDH gene expression in nia1nia2 plants, leading to less accumulation in nia1nia2 plants than in wild-type plants. These findings demonstrate that NR-dependent NO production plays an important role in cold acclimation-induced increase in freezing tolerance by modulating Pro accumulation in Arabidopsis. Many plants from temperate and cold climates have evolved a mechanism to enhance their freezing tolerance during exposure to low, nonfreezing temperatures in a process known as cold acclimation (Guy, 1990).

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8. F. Induction of Drought Tolerance

NO appears to be involved in plant developmental processes and participates in a number of physiological processes such as stomata closure, flowering, response to environmental stresses, and cell death (Lamattina et al., 2003; Wendehenne et al., 2004; Delledonne, 2005; Lamotte et al., 2005). The SnRK2 kinases present in guard cells, AAPK (ABA-Activated Protein Kinase) from Vicia faba and its Arabidopsis orthologue SnRK2.6/OST1/SRK2E play an important role in ABA signaling in response to drought and regulate stomata closure under low humidity stress (Mustilli et al., 2002). Osmolytes and some of them by ABA activate protein kinases of the (Sucrose Non Fermenting) 1-related protein kinase type2 SnRK2 subfamily as well, highlighting a role for these enzymes in a general response to osmotic stress (Kobayashi et al., 2005). It has been shown that Arabidopsis ABA dependent SnRK2 kinase, SRK2C/SnRK2.8, improves plant drought tolerance, probably by promoting the up regulation of stress-responsive genes expression, including DREB1A/CBF3 encoding a transcription factor that broadly regulates stress-responsive genes (Umezawa et al., 2004). SnRK2 kinases can also phosphorylate and activate transcription activators AREB1 and TRAB1 in Arabidopsis and rice, respectively (Kobayashi et al., 2005; Furihata et al., 2006). These data strongly suggested that SnRK2 protein kinases are involved in the regulation of expression of ABA-responsive genes. It has been shown that Arabidopsis OST1 kinase and NO production are required in the plant innate immunity against bacterial invasion (Melotto et al., 2006). A remarkable tolerance to drought stress has been reported recently in transgenic tobacco plants that express an isopentenyl transferase gene under the control of a drought stress-responsive promoter (Rivero et al., 2007). Tolerance to drought stress in these plants was accompanied by the enhanced ability to scavenge ROS supported by a battery of ROS scavenging mechanisms, implying a link between cytokinin accumulation and enhanced ROS scavenging capability under stress (Van Breusegem et al., 2008). In leaves treated with ABA, H2O2 was generated in the apoplast (Hung and Kao, 2004), and induced cell wall peroxidase activity in roots (Lin and Kao, 2001). Drought stress is responsible

for the increase in cell wall lignifications (Lee et al., 2007) in xylem vessels and sclerenchyma. However, the increase in lignin preceded summer drought, even though it was after the decrease in leaf water status registered in April, which coincided with a slight increase in H2O2, and a significant increase in ABA. The mechanisms of drought-induced lignifications could be mediated by H2O2, which is used by cell wall peroxidases to polymerize cinnamyl alcohols into lignin (Vreeburg and Fry, 2005). However, comparing the increase in lignin accumulation with H2O2 values, the highest values for lignin were observed from May to August (~27 mg.g-1 DW), which preceded the peak of H2O2 that occurred in July (10 µmol.g-1 DW). If H2O2 is a signal molecule for lignin increase, it acts at low levels. It is plausible that the slight rise in H2O2 observed in April, together with the significant increases in ABA levels and changes in environmental factors such as light (April PFD,1856 µmol m-2 s-1), functions as a signal for the onset of lignin accumulation (Jubany-Mari et al., 2009). Leaf plasticity is one of the most striking aspects of C. albidus, which showed significant differences in leaf dimensions throughout the experiment. A 60% reduction in leaf area was recorded from June to August in parallel with an increase in ABA and H2O2. This was not a result of increased lignin biosynthesis, which may be related to xylem and sclerenchyma differentiation but not to area reduction. The effect of drought on leaf area of several species of Cistus has been described (Nunez-Olivera et al., 1996), but here it is suggested that the decrease in leaf area preceded summer drought and that it was due to plant phenology and environmental conditions such as increased light and temperature. The leaves that were measured throughout summer were those that emerged after plant flowering. These leaves were smaller and did not grow during the summer. This reduction in leaf area contributes to subsequent acclimation of the plant to summer drought (Munne-Bosch et al., 2003) and complements the shedding of older leaves. After rainfall, the leaves resumed growth and large leaves were attained by autumn. Leaf plasticity contributes to acclimation of plants to environmental conditions to a large extent. Furthermore, the plasticity of C. albidus leaves has been described (Grant and Incoll, 2005), but no studies have addressed the mechanisms that

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regulate leaf reduction under drought conditions in this Mediterranean shrub. Jubany-Mari et al. (2009) showed that H2O2 is involved in the acclimation of C. albidus to summer drought. No studies have been carried out so far on the role of drought stress in inducing ABA and triggering H2O2accumulation in plants subjected to a combination of stresses. The drought stress–ABA–H2O2 interaction can induce an increase in ascorbic acid (AA), maintaining and even decreasing the ascorbate oxidative status under summer drought conditions, and thereby protecting plants from oxidative damage. Therefore, C. albidus plants tolerate high concentrations of H2O2 because of its localization in the apoplast of mesophyll cells, xylem vessels, and in differentiating sclerenchyma cells. Xylem and sclerenchyma lignifications and differentiation together with a reduction in leaf area contributed to drought acclimation. Leaf metabolism produces H2O2 at high rates, but current concepts suggest that the potent signaling effects of this oxidant require that a battery of antioxidative enzymes control concentrations. The extent to which H2O2 is allowed to accumulate remains unclear. There is little consensus on leaf H2O2 values in the literature and measured concentrations in unstressed conditions range from 50–5000 nmol.g-1 fresh weight, a difference that probably reflects technical inaccuracies as much as biological variability (Queval et al., 2008). 8. G. Induction of Ozone Tolerant

In plants, G-proteins are thought to be involved in interactions of plant hormones and plant defense responses to wounding and pathogen infection (Assmann, 2002). Booker et al. (2004) showed that insertion mutants of Arabidopsis canonical G-protein a subunit gene (GPA1) were more tolerant to low-level O3than the wild-type Col-0 in long-term O3-exposures. This suggests that these null mutants had altered perception of O3 or downstream signals. Similarly, it was shown that in short-term acute O3 exposures Arabidopsis plants with null mutations in the genes encoding α and β subunits of the single heterotrimeric G protein were less and more sensitive, respectively, to O3 damage than wild-type Col-O plants (Joo et al. 2005). G-proteins are involved in guard cell ABA signaling (Wang et al., 2001; Coursol et al., 2003; Pandey and Assmann 2004). Stomatal conductivity might be one of the factors affecting plant sensitivity to ozone. Knocking out the putative G-protein

coupled receptor GCR1, rendering plants hypersensitive to ABA in respect to stomatal responses, has been shown to interact with GPA1 (Wang et al. 2001). These plants were also more drought tolerant than the wild type, probably due to their lower transpiration rates. However, this was not reflected in the ozone sensitivity of the GCR1 null mutants, which did not differ from the wild type. This suggests that the role of G-proteins in O3-related processes is separate from their role in stomata regulation (Booker et al., 2004). 8. H. Light Acclimation

The role of the reactive oxygen species (ROS) in plants merits special attention because ROS can exert two opposite effects: They can activate pathways aimed at saving the cell from demise (Pastore et al., 2000; Di Cagno et al., 2001; Mittler, 2002; Vranova et al., 2002). In plants and other organisms, antioxidants prevent the potentially deleterious effects of ROS (oxidative stress) and RNS (nitrosative stress). However, these reactive molecules also perform critical functions at low controlled concentrations by acting in certain cellular locations, developmental stages, or stressful conditions (Becana et al., 2010). Under moderate light levels (150 µmol.m-2 s-1), the lcd1/rcd2 mutant displayed constant H2O2 accumulation around the veins in a pattern and manner similar to the H2O2 accumulationduring acclimation to high light (> 1000 µmol m-2 s-1) conditions. The processinvolves a redox signal-mediated communication between the chloroplast and the nucleus (Fryer et al., 2003).Very early changes in oxidation and in ROS-related transcriptome, suggest that these are part of physiological adaptations as cell death is detected only on the fifth day of darkness. Although the increase in redox state is observed within two days in mitochondria and peroxisomes, the cytoplasmic oxidation state remained constant despite the fact that H2O2 can readily diffuse through membranes via aquaporins (Bienert et al., 2007). References Abdel C. G. (2012). Irrigating lettuce (Lactuca sativa L.Var longiflia) with cadmium (Cd) polluted water: A comparative trail to detect the validity of consuming urban grown lettuce. The International Journal of the Environment and Water, Vol. 1, Issue, 253-269.

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Glossary and Idoms 5FU: 5-fluorouracil 8-OHdG: 8-hydroxydeoxyguanosine ACS: 1-aminocyclopropane-1-carboxylic acid synthase AD: Antioxidant defense ADPRC: ADP-ribosyl cyclase AL-PCD: Apoptotic-like PCD AnxMt1: Annexin1 AO: Alternative oxidase AOX: alternative oxidase AP: adenosine phosphate APOX: Ascorbate Peroxidase APX: Ascorbate peroxidases ATM: ataxia telangiectasia-mutated AtSNM1: Arabidopsis sensitive to nitrogen mustard protein BER: Base excision repair BLM: Bleomycin BQ: benzoquinone BT: benzenetriol bZIP: basic Leu zipper cADPR: Cyclic Adenosine Diphosphate Ribose CaM: Calmodulin cAPX: ascorbate peroxidase CAT: Catalase CCO: cytochrome c oxidase CcP: cytochrome C peroxidases CDPK: Ca2+-dependent protein kinase CDPK: Calcium-dependent protein kinase CDPK: Cyclin dependent phosphokinase cGMP: Cyclic Guanidine Mononphosphate CLP: caspase-like protease CLSM: Confocal Laser Scanning Microscopy CNGC: Cyclic nucleotide-gated ion channel CPDs: cyclobutane-pyrimidine dimers cPTIO: [2-(4-carboxyphenylalanine) 4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide potassium] Cyt c: Cytochrome c DAB: Diaminobenzidine DAF-2DA: 4,5-diaminofluorescein diacetate DAO: Diamine oxidase DAPI: 4′,6-diamidino-2-phenylindole dihydrochloride DCF-DA: Dichlorofluorescein Diacetate DDR: DNA damage response DEA-NONOate: NO donor diethylamine DFO: desferoxamine mesylate DGK: Diacylglycerol kinase DHE: Dihydroethidium DMT: Dimethyl thiourea Dox: doxorubicin DPI: Diphenylene iodonium DPI: NADPH oxidase inhibitor diphenylene iodonium

Ph ton 262

DSBs: DNA strand break ECM: Extracellular matrix EFE: Ethylene forming enzyme ELISA: Enzyme-linked immunosorbent assay EM: Ectomycorrhizae EMS: Ethylmethanesulphonate EPR: Electro Paramagnetic Resonance ET: Ethylene ETF: Electron transfer flavoproteins GC: Guanylate cyclase GDC: Glycine dehydrogenase GhMT1: Gossypium hirsutum Metallothioneins GPA1: G-protein a subunit gene GR: Glutatione Rductase GSNO: S-nitrosoglutathione GSSG: Oxidized GSH GST: Glutathione S-transferase HAT: H-atom abstraction Hbs: Haemoglobin HNE: 4-hydroxy-2-nonenal HPLC-MS/MS: High performance liquid chromatography-tandem mass spectrometry HQ: hydroquinone HR: Hyper sensitive response HRc: Homologous recombination HRF: Somatic homologous recombination HRGPs: Hydroxy-proline (Hyp)-rich proteins HSP71.2: Heat Shock Potein HSPs: Heat Shock Proteins ICLs: Interstrand and intrastrand crosslinks InsP3R: (1,4,5)-triphosphate receptor IP3: Inositol 1,4,5-trisphosphate JA: Jasmonic acid Lb: Leghaemoglobin LP: hydraulic conductivity Lpr: Hydraulic conductivity of the root LPS: lipopolysaccharides LRR: Leucine rich repeat LSD1: Lesions simulating disease resistance 1 MAPK: Monoadenine phosphate kinase MAPKs: Mitogen-activated protein kinase MAT-1: Met adenosyltransferases MDA: Malondialdehyde MED: Minimal erythema dose MeJA: Methyl jasmonate MMC: cisplatin and mitomycin C MMS: Methyl methane sulphonate MPT: Mitochondrial transmembrane potential MPTP Mitochondrial transmembrane potential MTs: Metallothioneins NBT: Nitro blue tetrazolium NBT: Nitro blue tetrazolium NDPK2: Nucleoside diphosphate kinase 2 Nei: endonuclease VIII NFs: Nodulation factor NHEJ: non-homologous end joining

Ph ton 263

nia1nia2: nitrate reductase mutant NMD: nonsense-mediated decay NMDA: N-methyl D-aspartate NOS: Nitric oxide synthase NOx: NADPH oxidase NR: Nitrate reductase Nth: endonuclease III NtOSAK: Salt stress-activated protein kinase OGDC: 2-oxoglutarate dehydrogenase OXI1: oxidative signal-inducible 1 P5CS1: proline accumulation under osmotic PAL: Phenylalanine ammonium lyase PAO: Polyamine oxidase PARP: poly (ADP-ribose) polymerase PDC: pyruvate dehydrogenase PDK1: phosphorinositide-dependent protein kinase 1 PEP: plastid-encoded polymerase PHB: Prohibitin is a protein family, which can be divided into two groups: the PHB1 group (PHB3, 4, and 5) and the PHB2 group (PHB1, 2, 6, and 7) PHGPx: Phospholipidhydroperoxide glutathione peroxidase PLC: phospholipase C PLC: phospholipase C PLDβ1: Phospholipase β1 PM: Plasma membrane PMP22: Peroxisomal membrane protein PMT: Putrescine N-methyltransferase Pn: net photosynthesis rate PNPase: polynucleotide phosphorylase protein PP2C: Protein phosphatase 2C PPR40: pentatricopeptide repeat protein ProDH: Proline dehydrogenase PrP4A: Pathogen-related proteins PrxIIE: Type II peroxiredoxins PTK: plastid transcription kinase PUFA: polyunsaturated fatty acids RBOH: Respiratory Burst Oxidase Homolog RIA: Radio immunoassay RUBISCO: Ribulose-1,5-bisphosphate carboxylase/oxygenase RYR: Ryanodine receptor SAGs: senescence-activated genes sGC: Soluble guanylate cyclase SIPK: Salicylic acid-Induced Protein Kinase SNAP: S-nitroso-N-acetylpenicillamine SNF: Sucrose Non Fermenting snoRNPs: small nucleolar ribonucleoproteins SNP: Sodium nitroprusside SOD: Superoxide dismutases SOSI: Sodium over sensitive Srprx1: Class III peroxidase SSB: single-strand break STIG1: Stigma-specific protein 1 tBH: tert-butylhydroperoxide TCA cycle: Tricarboxylic acid cycle TG: Thymine glycol TMP: 2,2,6,6-tetramethylpiperidinooxy

Ph ton 264

TUNEL-positive (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) UVA: (320–400 nm) UVB: Lower energy (290–320 nm) wavelengths. UVG: UV-induced mRNA granules WIPK: wound-induced protein kinase Xm1: 5′-3′ exo-ribonuclease YB-1: Y-box-binding protein

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