10

Plant Adaptation to Abiotic and Genotoxic Stress:

Relevance to Climate Change and Evolution

Brahma B. Panda, V. Mohan M. Achary, Srikrishna Mahanty, and Kamal K. Panda

Abstract

The global climate scenario has been changing alarmingly, attributed to theunabated emission of greenhouse gases into the Earth’s atmosphere. Some of theconsequences of climate change include a rise of surface temperature and sealevels, and abnormal precipitation across the world. The resulting abiotic stressesfrom drought, extreme temperature, salinity, radiation, or environmental pollutionare likely to impact plant genome integrity and function. This, in turn, would affectplant diversity and productivity adversely, having far-reaching consequences in theecosystems in various parts of the world. The problem can be mitigated consi-derably through the development of strategies for plant adaptation to abiotic stressthat nevertheless warrants better understanding. Excess generation of reactiveoxygen species (ROS) is the general adaptive syndrome that plants manifest inresponse to stress. The activation of the antioxidative machinery consisting ofantioxidant molecules and enzymes is crucial for the maintenance of redoxhomeostasis that is necessary for normal cellular metabolism and survival understress. Stress-induced perturbation of this delicate redox balance prompts ROS totrigger signal transduction networks that through gene expression and adaptiveresponses confer genome protection against environmental stresses. The presentchapter highlights the state of the art of transgenic vis-�a-vis epigenetic approaches toaccelerate adaptive evolution of plant tolerance to stress.

10.1

Introduction

Climate change and global warming have been the consequence of the phenom-enal increase in greenhouse gases in the atmosphere, which include carbon dioxide(CO2), methane and nitrous oxide, chlorofluorocarbons, aerosols such as sulfate,organic carbon, black carbon, nitrate, and dust, and solar radiation that togethercontribute to the land surface properties [1]. The global increase of CO2

concentration has been primarily due to the burning of fossil fuel, while the

251

Climate Change and Plant Abiotic Stress Tolerance, First Edition. Edited by Narendra Tuteja and Sarvajeet S. Gill.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

increases of methane and nitrous oxide are primarily due to agriculture. Recenttimes have witnessed a sharp acceleration in CO2 emission since 2000 to morethan 3% increase per year from 1.1% per year during the 1990s. The second aspectof climate change is global warming, as is now evident from the increase of theglobal average air and ocean temperatures, widespread melting of snow and ice,and rising global average sea level. The increase in global temperature has been ona linear trend at 0.13� 0.03 �C per decade during the last 50 years, nearly twice thatof the past 100 years. The Intergovernmental Panel on Climate Change (IPCC) [1]further predicted that, if not regulated, the average surface temperature increasewould range from 1.1 to 6.4 �C during the twenty-first century. Global mean sealevel has been rising at an average rate of 1.7� 0.5mm per year over the last 100years, which is significantly higher than that of last 1000�2000 years. The thirdaspect of climate change is stratospheric ozone depletion, resulting in increasedbiologically active UV-B radiation reaching the Earth’s surface [2]. Significantchanges in the frequency and nature of precipitation and storms are also predictedas the surface of the Earth warms up [3]. These are some of the challenges posed asa result of climate change that are likely to impact on the biota, human health,agriculture, and the overall ecosystem function [4,5]. Agricultural yield, especiallythat of wheat, rice, maize, and soybean, has been predicted to decline according tothe climate change scenarios developed by applying the HadCM3 global climatemodel [6]. The IPCC Special Report on Emissions Scenarios elucidated the complexregional patterns of projected climate variables, CO2 effects, and agriculturalsystems that indicated decreasing and increasing trends in crop yield in developingand developed countries, respectively [1,7]. The projected world scenario of climatechange emphasizes the urgent need for concerted efforts to protect plant diversityand to sustain crop productivity under stressful environments. Stress, bydefinition, is any external factor that exerts a disadvantageous influence on theplant. It is, therefore, imperative to understand the basics of plant responses tovarious abiotic stresses associated with climate change. Such knowledge is vital formodulating stress tolerance in crop plants so as to sustain productivity in the faceof climate change.

10.2

Plant Responses to Abiotic Stress

Cosmic and solar radiation (light, UV-B), temperature (cold, heat), water (drought,flooding), salinity, nutrient (deficiency, over supply, osmolarity), CO2, and oxygen(hypoxia, anoxia) represent some of the sources of abiotic stresses associated withclimate change (Figure 10.1).Plants, being sedentary, are incapable of escaping from stress and often display

unique adaptive responses to overcome stress. Oxidative burst, a rapid transientproduction of huge amounts of reactive oxygen species (ROS) (O2

��, H2O2, �OH,and 1O2), has been the most common feature associated with plant responses tostress [8�13] A hypothesis termed the “general adaptation syndrome (GAS)

252 10 Plant Adaptation to Abiotic and Genotoxic Stress

response” has been proposed, according to which different types of stress evoke asimilar adaptive response and implicating the role of ROS in the underlyingadaptive response mechanism [14,15]. The sites of oxidative burst are the cell walland the plasma membrane, involving the enzymes NADH peroxidase (NADH-PX)and NADPH oxidase (NADPH-OX or RBOH (respiratory burst oxidase homolog)),respectively [16�18]. ROS generated in response to environmental stress arenecessary for cell function, regulation, and development [19�21]. For instance,plant cells use ROS for polymerization of lignin during cell wall formation. Severalother enzymes associated with the cell wall also contribute to the generation ofROS including lipoxygenase, oxalate oxidase, xanthine oxidase, amine oxidases, andperoxidases [22�24]. Plant cells generate H2O2 during normal metabolism via theMehler reaction in chloroplasts, electron transport in mitochondria, and photo-respiration in peroxisomes (Figure 10.2). ROS generated by chloroplasts asbyproducts of photosynthesis include singlet oxygen (1O2) and the O2

��, whereasthe main species generated by peroxisomes are O2

�� and H2O2 [25,26]. In the dark,most of ROS are generated in mitochondria, which mainly form O2

�� by over-reduction of the electron transport chain [27]. O2

�� can be converted into H2O2 in areaction catalyzed by superoxide dismutase (SOD) and H2O2 serves as an inertdiffusible species that can give rise to reactive �OH through the catalysis by freetransition metal ions [28]. H2O2 diffuses freely, facilitated by movement through

WaterDrought

TemperatureHeat

Cold (chilling and frost)

WaterDrought

TemperatureHeat

Cold (chilling and frost)

WaterDrought

WaterDroughtDrought

TemperatureHeat

Cold (chilling and frost)TemperatureHeat

Cold (chilling and frost)TemperatureHeat

Cold (chilling and frost)

HeatCold (chilling and frost)

Flooding (hypoxia)

RadiationLight

UV

radiation

Flooding (hypoxia)

RadiationLight

UV

radiation

Flooding (hypoxia)Flooding (hypoxia)Flooding (hypoxia)

RadiationLight

UV

radiation

RadiationLight

UV

radiation

RadiationLight

UV

radiation

RadiationLight

UV

Light

UV

Light

UV

LightUV

Clim

ate

chan

ge

Abi

otic

str

ess

fact

ors

Chemicalstress

Mineral salts (Deficiency or over-supply)

Pollutants (metals, pesticides)

Ionizing radiation

Gaseous toxins

Mechanicalstress

WindSoil movementSubmergence

Signal transduction

Signal Perception

Signal transduction

Signal Perception

Signal transduction

Signal Perception

Signal transduction

Signal Perception

Signal Perception transduction

Redox-perturbation

Perception

Stress

transduction

Redox-perturbation

Perception

Stress

transduction

Redox-perturbation

Perception

Stress

transduction

Redox-perturbation

Perception

Stress

Redox-perturbation

Perception

Stress

ROS

responsive gene-

expression

Antioxidativeresponse

Signal transducers

Death Adaptation and survival

Figure10.1 Plantperceptionand response toabioticstress factorsassociatedwithclimatechange.

The resulting increase in ROS or redox perturbation perceived by plant cells triggers signal

transduction leading to gene expression that determines plant adaptation or death.

10.2 Plant Responses to Abiotic Stress 253

peroxiporin membrane channels. Although H2O2 is relatively stable, present inplant cells in the micromolar concentration range, the remaining ROS have veryshort half-lives [29]. Of the primary ROS, �OH is the most reactive and is capable ofoxidizing all known biomolecules at diffusion-limited rates of reaction (Fig-ure 10.3). It has been estimated that the average diffusion distance before �OH

Figure 10.2 Apoplastic and symplastic sources of H2O2 in the plant cell. For enzymatic reactions,

see Table 10.1.

Figure 10.3 Free radical generation and

scavenging by SOD, CAT, and GPX. GPX

catalyzes reduction of H2O2 to water and

simultaneously oxidizes glutathione (GSH) to

GSSG. Glutathione reductase (GR) in the

presence of NADPH reduces GSSG back to

GSH. For enzymatic reactions, see Table 10.1.

254 10 Plant Adaptation to Abiotic and Genotoxic Stress

reacts with a cellular component is only 3 nm (i.e., approximately the averagediameter of a typical protein) [30]. The extent of cytotoxic damage induced by ROSultimately depends on the redox homeostasis or the balance between ROSdetoxification and ROS production mechanisms in the cell. An antioxidative systemconsisting of antioxidant molecules and enzymes is in place in different compart-ments of plant cells that scavenges or detoxifies the ROS and maintains cellularredox homeostasis. The antioxidant enzymes include SOD, catalase (CAT),glutathione peroxidase (GPX) and the ascorbate�glutathione cycle (Halliwell�Asadapathway) enzymes: ascorbate peroxidase (APX), glutathione reductase, monodehy-droascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) [31,32].Whereas in animals GPXs function as key enzymes that scavenge H2O2, in plantsthis function mainly belongs to CAT and the enzymes of the ascorbate�glutathionecycle. However, studies have indicated that GPXs or GST/GPXs become the mainH2O2-scavenging enzymes under extreme or persistent stress conditions [33].Gueta-Dahan et al. [34] have reported a decrease of APX and corresponding increaseof GPX activities in response to salt stress in salt-sensitive citrus cells as a functionof time. In addition to these antioxidant enzymes that scavenge ROS (Table 10.1),plants synthesize antioxidant molecules that include L-ascorbic acid (vitaminC),glutathione, a-tocopherol (vitamin E), and carotenoids [31,35,36].Under physiological steady-state conditions, ROS are scavenged by different

antioxidants. However, the balance between production and scavenging of ROS isupset by a number of adverse environmental stress factors, resulting in a rapid

Table 10.1 ROS scavenging and detoxifying enzymes.

Antioxidative enzymes EC number Function Cellular

Compartments

Superoxide dismutase(SOD)

1.15.1.1 2O2��þ 2H!H2O2þO2" Chl, Mit, Cyt

Ascorbate peroxidase (APX) 1.11.1.11 2ASCþH2O2! 2MDHAþ 2H2O

Chl, Mit, Per,Cyt

Monodehydroascorbatereductase (MDHAR)

1.6.5.4 MDHAþNAD(P)H!NAD(P)þþASC

Chl, Mit, Per,Cyt

Dehydroascorbate reductase(DHAR)

1.8.5.1 2GSHþDHA!GSSGþASC

Chl, Mit, Per,Cyt

Glutathione reductase (GR) 1.6.4.2 2NADPHþGSSG!2NADPHþ 2GSH

Chl, Mit, Per,Cyt

Glutathione peroxidase(GPX)

1.11.1.9 2GSHþH2O2!GSSGþ 2H2O

Chl, Mit, Cyt

Glutathione S-transferase(GST)

2.5.1.18 RXþGSH!HXþR-S-GSH

Cyt, Nu

Catalase (CAT) 1.11.1.6 2H2O2! 2H2OþO2" Per

Peroxiredoxin (Prx) 1.11.1.15 H2O2þ 2R-SH!R-S-S-Rþ 2H2O

Chl, Mit, Per,Cyt

Chl: chloroplast, Cyt: cytosol, Mit: mitochondria, Nu: nucleus, Per: peroxisome.

10.2 Plant Responses to Abiotic Stress 255

increase of intracellular ROS levels. Although high concentrations of ROS can causeirreversible damage to the macromolecules leading to cell death, at the same timethey can also influence signaling and gene expression, indicating that cells haveevolved strategies to utilize ROS to their advantage in various cellular programs andfunctions [37]. The role of ROS is increasingly implicated in cell signaling processesinvolving the induction of stress-related genes regulated by a network oftranscription factors. Transcription factors are proteins that act together with othertranscriptional regulators, including chromatin-modifying proteins, for binding orobstruction of RNA polymerases with the DNA template [38]. Plant genomes assignapproximately 7% of their coding sequence to transcription factors [39]. The AP2(apetala 2)/ethylene-response element-binding protein (ERF) constitutes a largefamily of plant-specific transcription factors that share a well-conserved DNA-binding domain. This transcription factor family includes DRE-binding proteins(DREBs), which activate the expression of abiotic stress-responsive genes viaspecific binding to the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element in their promoters [40]. Stress-responsive WRKY [41] and NAC [42]proteins represent yet the other groups of plant-specific transcription factors.

10.3

ROS Induce Genotoxic Stress

The “genome” is the total genetic content consisting of both coding DNA and non-coding DNA contained in a haploid set of chromosomes in all eukaryotes. ROSgenerated either endogenously from different sources within the cells orexogenously in response to abiotic stress can attack almost all the cell componentsthat include sugars, lipids, proteins, and nucleic acids [43,44]. Genotoxic stressresults from agents (genotoxins or mutagens) that are capable of damaging thenuclear and extranuclear genetic material of cells (i.e., they are “toxic to thegenome”). Thus, the unifying characteristic of genotoxins is the ability to damageDNA. Environmental agents may directly or indirectly (through generation of ROS)damage DNA or induce mutations causing genomic perturbations [45,46]. NeitherH2O2 nor O2

�� are capable of inducing DNA damage, but their reactive product�OH generated via Fenton or Haber�Weiss reactions induces DNA damage [47].Oxidative attack to DNA generates both altered bases and damaged sugar residuesthat undergo fragmentation and lead to strand breaks. Oxidative attack to DNAbases generally involves �OH addition to double bonds, while sugar damage mainlyresults from hydrogen abstraction from deoxyribose [48]. Attack on either DNA orassociated proteins by �OH may result in DNA�protein cross-links [44]. Besidesdirect oxidation, DNA bases may also be indirectly damaged through reaction withreactive products generated by ROS attack to other macromolecules. One of themajor sources of such indirect oxidative damage is lipid peroxidation, caused byattack of oxygen radicals to the polyunsaturated fatty acid (PUFA) residues ofmembrane phospholipids [49]. Major reactive products of lipid peroxidation aremalondialdehyde (MDA), acrolein, and crotonaldehyde. MDA reacts with

256 10 Plant Adaptation to Abiotic and Genotoxic Stress

G residues in DNA to form the pyrimidopurinone adduct called M1G [50]. Acroleinand crotonaldehyde generate etheno modifications of DNA bases, such as etheno-Aand etheno-C [51]. An additional major source of oxidative damage is that ROSattack deoxyribose leading to single-strand breaks (SSBs). SSBs occurs frequentlythrough removal of the hydrogen atom from the C40 position of deoxyribose, whichgives rise to a deoxyribose radical that further reacts to produce DNA strandbreakage [52]. Endogenous ROS predominantly induce SSBs [53]. The generationof oxidized bases in DNA may have serious consequences. The failure to repair adamaged base before it is encountered by the replication fork may cause blockingof the DNA polymerase.The biological consequences arising from any damage to the genome depend

upon the chemical nature of the alteration caused to the structure of the DNA,which varies from the innocuous to the highly mutagenic events leading to achange in gene expression, senescence, with far-reaching consequences such asdecrease in biodiversity and crop yield. Invariably, the DNA damage is repaired.However, mis-repair commonly found under stressful conditions results inaccumulation of mutations, resulting in genomic perturbation leading togenetic instability, depletion of the gene pool, or genetic erosion that culminatesin the loss of biodiversity. ROS, on the one hand, can damage a number ofcellular targets, including DNA, resulting in genotoxic stress [54], and, on theother, are involved in plant defense or immunity [55], growth and development[56], programmed cell death or apoptosis [57], and signal transduction [58]. ROSare key components of cell signaling capable of affecting the activity ofantioxidant enzymes, the mitogen-activated protein kinases (MAPKs), calciumsignaling, DNA damage, and repair or cell death (Figure 10.4), which areelaborated further in the following sections.

10.4

Adaptive Responses to Oxidative Stress

Being sedentary, plants are equipped with innate protective mechanisms thathelp them to acclimatize and withstand environmental changes. Several studieshave shown that abiotic stresses, such as salinity, drought, excess light, oroxidative agents, activate the antioxidative system in plants that can be monitoredor quantified using a plethora of physiological, biochemical, or molecular end-points [11,59,60]. ROS, more particularly H2O2, play versatile roles in plantadaptation to stress [61]. The adaptive response could be considered as a non-specific phenomenon in which exposure to minimal stress could result inincreased resistance to higher levels of the same or to other types of stress somehours or days later [62]. Modulation studies have shown that conditioning ofseeds or seedlings with low concentrations of H2O2, salicylic acid, cinnamic acid,or mild abiotic stress makes them more tolerant against further exposure toabiotic stress like drought, salinity, temperature, metals, and so on (Table 10.2).An adaptive response of antioxidant defenses occurs in cells exposed to mild

10.4 Adaptive Responses to Oxidative Stress 257

oxidative stress that confers stress tolerance in plants. For example, drought-acclimated wheat seedlings modulated growth by maintaining favorable turgorpotential and relative water content, and were able to minimize H2O2 accumula-tion and membrane damage as compared with non-acclimated plants duringsevere water stress conditions [70]. This was due to systematic upregulation ofH2O2-metabolizing enzymes, especially APX, and by maintaining the ascorba-te�glutathione redox pool in acclimated plants. Hernandez et al. [11] reportedshort salt (NaCl) treatments drastically decreased CAT, APX, and POX activitiesin Brassica oleracea roots. However, the activities of CAT and POX recovered whenthe salt treatment was prolonged. The increase in ascorbate levels and themaintenance of the redox state was critical for root growth and developmentunder salt stress [11]. The involvement of H2O2 in acclimation of Cistus albidus, aMediterranean shrub, to summer drought has been demonstrated [76]. In a 2-year long-term climate chamber experiment, it was possible to induce frosthardening in 4-year-old Scots pine (Pinus silvestris), used as a model plant, byapplying a short period (6 h) of mild frost [77]. In response to enhanced UV-Bradiation, plants Abies balsamea, Anemone nemorosa, Fagus sylvatica, Kalmialatifolia, Picea abies, Picea rubens, Rhododendron maximum, R. periclymenoides,

ROS

macromolecular damage

DNA Protein Lipid

stress stress

Activation of repair systems

Activation of transcription factors

Activation of kinases

Gene activation/inactivation

Antioxidant response Genotoxic response

Genome adaptation

Cell death

Figure 10.4 Role of ROS in cell signaling

processes. ROS can interact with DNA,

proteins, or lipids by modifying their

structure or function, leading to the

activation or inactivation of stress-responsive

genes. These changes can be part of

antioxidant or genotoxic responses that may

either counter stress though adaptation or

lead to abnormalities that affect cell or tissue

functions, leading to cell dysfunction and

death.

258 10 Plant Adaptation to Abiotic and Genotoxic Stress

Pinus sylvestris, Pinus cembra, Senecio fuchsia, Vaccinium myrtillus, and so on,manifest smaller and thicker leaves through thickening of the epidermis andby an increase in the concentration of UV-B-absorbing compounds on the leafsurface layers as well as by activation of the antioxidant defense system. Theabove high-altitude alpine timberline plants that show better adaptation to UV-Bradiation also exhibit cross-tolerance to other harsh stress conditions comparedto their low-altitude counterparts [78]. The occurrence of cross-tolerance suggeststhat ROS through signal transduction play a common mediator role in theadaptation to different types of abiotic stresses. Abiotic stress conditioning ofseeds or seedlings of a number of plants through up- or downregulation ofantioxidant enzymes CAT, SOD, APX, GPX, POX, glutathione reductase, or GSTalleviates plant tolerance to stress, which underscores the role of redoxhomeostasis in the underlying mechanism (Table 10.2).

Table 10.2 Prior conditioning of seed or seedlings enhances plant tolerance to abiotic stress.

Plant name Conditioning treatment

(type/duration)

Alleviation of tolerance

to abiotic stress

Reference

Zea mays L. Drought (by withholdingwater for 7 d)

Chilling (5 �C for 5 d) [63]

Oryza sativa L. H2O2 or sodium nitro-prusside 1�1000mM/2 d

Salt (NaCl, 100mM/8 d) [64]

Lycopersicon esculentumMill.

Salicylic acid, 0.1mM,40ml (root drenching/2 d)

Salt (NaCl, 150, 200mMfor 3 d)

[65]

Festuca arundinacea cv.Barlexas; Lolium perennecv. Accent

Heat at 30 �C for 3 d Heat stress levels(38, 42, 46 �C)

[66]

Triticum aestivum L. H2O2, 1�120mM, 8 h Salt (NaCl, 150mM for15 d

[67]

Lycopersicon esculentum L. Salicylic acid, 1mM(foliar spray)

Salt (NaCl, 100mM) for14 d)

[68]

Triticum aestivum L. H2O2, 0.05�5mM, 24 h H2O2, 150mM, 6 h [69]

Triticum aestivum L. Mild drought (by with-holding water)/9 d

Severe drought (by with-holding water) 11 d

[70]

Cucumis sativus cv.Jinchun

H2O2, 1.5mM, 12 h Low light intensity of100mmolm�2 s�1 for144h

[71]

Artemisia annua L. Salicylic acid, 1mM(foliar spray)

Salt (NaCl, 50�200mM) [72]

Cucumis sativus L. Cinnamic acid, 50 mM,2 days

Chilling (15/8 �C) for 1 d) [73]

Cucumis sativus L. Cinnamic acid, 50 mM,2 days

Heat (42/38 �C for 3 d) [74]

Pisum sativum L. NaCl, 10, 25mM andPEG 6000, 2.5%/7 d

Salt (NaCl, 80mM) for14 d)

[75]

10.4 Adaptive Responses to Oxidative Stress 259

10.5

Transgenic Adaptation to Oxidative Stress

Fortification of the antioxidative machinery using genetic engineering to ameliorateoxidative stress tolerance has been one of the many approaches for themaintenance of plant productivity under environmental stress conditions [79].Transformation of plants with a single transgene, encoding one of the antioxidantenzymes, has been carried out yielding contrasting results. Studies that addressedthe overproduction of Cu/Zn-SOD [80,81], Mn-SOD [82,83], or Fe-SOD [84] in thechloroplasts resulted in enhanced tolerance to oxidative stress. In addition,transgenic plants expressing gene constructs for either cytosolic or a chloroplast-targeted cytosolic APX have increased tolerance against various abiotic stresses,including water stress [85,86]. Faiz et al. [87] demonstrated the involvement ofcytosolic APX and Cu/Zn-SOD in transgenic tobacco showing improved toleranceagainst drought stress. However, several other reports indicated no improvementsof tolerance to oxidative or environmental stress [88�91]. These contradictoryresults have usually been attributed to the complexity of the scavenging pathways,because modification of one enzyme may not affect the antioxidative system as awhole. To overcome this problem, pyramiding or stacking of antioxidant genes hasbeen attempted (for a review, see [92]). Therefore, instead of trying with any singlegene, a group of antioxidant genes are stacked to develop stress-tolerant transgenicplants [93,94]. While efforts are still ongoing to develop stress-tolerant transgeniccrops by cloning and overexpressing antioxidant enzymes involved in theascorbate�glutathione cycle and other ROS detoxification mechanisms(Table 10.3), the success achieved so far has been rather limited. To date, onlyabout 10 different stress-tolerant transgenic plant species against drought, salinity,or temperature have been developed. Information on the performance of suchtransgenic stress-tolerant crops under abiotic stress under field conditions is,however, not readily available for assessment, which is essential for the assessmentof stress tolerance vis-�a-vis productivity or yield.

10.6

Adaptive Response to Genotoxic Stress

Adaptive response precisely to the genotoxic stress, also termed genotoxicadaptation, is a phenomenon where a cell or organism, when subjected to“priming” or “conditioning” treatment with a mild non-toxic dose of an abiotic/genotoxic (oxidative and alkylating agents), develops increased tolerance to thegenotoxic challenge imposed later (Figure 10.5; for reviews, see [62,140]). The basisof the low-dose conditioning conferring genomic protection against the subsequenthigh-dose genotoxicity apparently involves an adaptive machinery comprising DNArepair, cell cycle checkpoints, apoptosis, and antioxidant enzyme systems [141].Quite often it has been observed that agents capable of causing genotoxic effects ata high dose, paradoxically, stimulated growth and cell division at a low dose. A

260 10 Plant Adaptation to Abiotic and Genotoxic Stress

generalized terminology, “conditioning hormesis,” has therefore been recom-mended to describe this phenomenon of low-dose protection from the subsequenthigh-dose genotoxicity [142]. Genotoxic adaptation, first discovered in Escherichiacoli [143] and later shown in mammalian cells [144], has been reported in almost alleukaryotes, including fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe,Aspergillus niger) [145�147], algae (Chlorella pyrenoidosa, Chlamydomonas reinhardtii,Euglena gracilis) [148�151], and higher plants (Vicia faba, Allium cepa, Hordeumvulagre,Helianthus annuus) [152�155]. In these studies, genotoxic adaptations wereevaluated on the basis of induction of cells with mitotic or chromosomeaberrations, micronuclei, cell death, DNA strand breaks, DNA damage (Cometassay), or homologous recombination [140,153,156]. Recently, employing thealkaline Comet assay and ROS scavengers, it has been established that ROSmediated the adaptive response induced by low non-toxic doses of Al3þ, rosebengal, and H2O2 against genotoxic stress of ethyl methanesulfonate (EMS) in rootcells in A. cepa (Figures 10.6 and 10.7) [157]. The inhibitor of protein synthesisblocks adaptive responses, suggesting that adaptive responses require de novoprotein synthesis [152,158]. Plants maintain genome integrity using different DNArepair pathways, such as direct repair (photoreactivation), base excision repair(BER), nucleotide excision repair (NER) or mismatch repair (MMR), and double-strand break (DSB) repair. DSBs are the most threatening type of DNA damage inliving cells [54]. There are two major DSB repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR) [46,159�162]. NHEJ repair is

Figure 10.5 Phenomenon of adaptive

response in plants. Seeds or seedlings

conditioned with a mild abiotic stress can

confer adaptation to subsequent high-level

exposure to oxidative or genotoxic stress.

Unconditioned seeds or seedlings fail to

show any adaptation to genotoxic stress.

10.6 Adaptive Response to Genotoxic Stress 263

error-prone and represents the predominant repair pathway during G1 to early Sphase of the cell cycle [163]. HR is important as an error-free repair pathway duringlate S to G2 phase of the cell cycle, when the homologous sequence from a sisterchromatid can be utilized for accurate repair (for review, see [164]). Mis-repairedlesions lead to biological effects, such as cell killing, chromosome aberrations,mutations, transformation, and so on [165]. These DNA repair pathways activatedupon exposure to DNA-damaging agents are involved in genotoxic adaptation [166].

Comet assay

6

bb

b

4

5

6

d

dd

b

d

1

2

3

Oliv

e ta

il m

omen

t (µm

)

00

RB 10µm

2O2 2.5mM

EMS 10mM

RB /EMS

H2O2 /EMS 0

SA 10µM

EMS 5mM

SA /EMS 0

Al 10µM

Al 800µM

l 10 /800µM

H ATreatments

Figure 10.7 Prior conditioning by RB 10 mM,

H2O2 2.5mM, SA 10 mM or Al3þ 10 mMconfers protection from DNA damage

induced by EMS 5 or 10mM or Al3þ 800 mM in

root meristems of A. cepa. Increase

significant compared to control (0) at

p� 0.01b; decrease significant compared to

EMS- or Al-challenge at p� 0.1d.

Figure 10.6 Comet assay in root cells of A. cepa showing genotoxic adaptation induced

by 10 mM salicylic acid (SA)-conditioning conferring protection from DNA-damage imposed by

5mM EMS (an alkylating mutagen).

264 10 Plant Adaptation to Abiotic and Genotoxic Stress

The DNA lesions induced by ROS are repaired via the BER pathway, in which thedamaged bases are removed by specific DNA glycosylases, leaving abasic sites thatare subsequently cleaved by apurinic/apyrimidinic endonucleases (APEs; EC4.2.99.18) that are activated by sublethal levels of ROS [167]. Angelis et al. [158],using the alkaline/neutral Comet assay in root meristem cells of V. faba, providedindirect evidence that glycosylase and APE are involved in the alkylating DNAdamage response. Furthermore, genotoxic stress induces b-polymerase and 3-methyl-adenine DNA glycosylase (AAG) that are involved in BER, and O6-ethylguanine-DNA methyltransferase [141,168]. Inhibitors of the DNA repair-related protein poly(ADP-ribose) polymerase (PARP) can also block adaptiveresponse implicating the repair processes [154].Abiotic and genotoxic stresses induce specific genes to express proteins that are

assigned to different functions of the DNA repair pathways (Table 10.4). InArabidopsis, DNA damage repair proteins SNI1, SSN2, and RAD51D are involved inhomologous DNA recombination during the defense response [186,187]. The Rad50protein is involved in the cellular response to DSBs, including the detection ofdamage, activation of cell cycle checkpoints, and DSB repair via recombination [172].

Table 10.4 Examples of plant genes and proteins induced by abiotic or genotoxic stress

conferring genome protection.

Phenotype Protein Abiotic/genotoxic

stress agent

Function References

Atuvc66 � UV-C, MMS, MC,salinity, ABA

Enhance stress toler-ance

[169]

ARS27A ARS27A MMS (100 ppm), UV-C(254 nm, 1 kJ cm�2)

Regulate RNA stability [170]

AtPARP-1 PARP-I c-rays BER (failed to respondto abiotic stress)

[171]

AtPARP-II PARP-II Drought (dehydration),metal (50mM CdCl2)

Implicated in addi-tional signalingresponses independentof DNA damage

AtRAD50 RAD50 MMS (0.0033�0.0167% v/v)

Activates G1 cell cyclecheckpoint and DSBrecombination repair

[172]

AtMKP1 MAPK1 UV-C (254 nm, 0.5�5 kJ cm�2), MMS(100�140 ppm), NaCl(150�225mM)

MAPK signaling andcross-talk betweengenotoxic and salinitystress

[173,174]

AtKU7,KU80

Ku70,Ku80

c-ray (10�150Gy),MMS (12.5�100 ppm)

Telomere lengthhomeostasis

[175]

Atku80 Ku80 MMS (25�150 ppm) Telomere homeostasis,DSB/NHEJ repair

[176]

(continued)

10.6 Adaptive Response to Genotoxic Stress 265

Table 10.4 (Continued)

Phenotype Protein Abiotic/genotoxic

stress agent

Function References

AtATR ATR Hydroxyurea, aphidico-lin, UV-B

Activated by SSB regu-lates G2 cell cyclecheckpoint

[177]

AtCEN2 CERTIN2 UV-C NER pathway [178]

AT SNM1 SNM1 Bleomycin (10�9 to10�4M), H2O2

(0.3�5mM))

HR repair [178]

AtRad51,Rad54,Rad51C,RecA

� UV-C (254 nm, 6 kergcm�2), bleomycin(10�6M)

HR repair [179]

PDH45 PDH45 NaCl (50�300mM) DNA�RNA unwindingpathways

[180]

AtRad51B,AtRad51C,AtXrcc2

AtRad51B,AtRad51C,AtXrcc2

Mitomycin C Somatic recombina-tion, DNA repair, andchromosome stability

[161]

AtATM,ATR

CYCB1;1 c-rays Activated by DSB, upre-gulates cyclinCYCB1;1, exact func-tion not known

[181]

NtGPDL GDPL Metal (100M AlCl3),osmotic (300mMNaCl), low temperature(4� 1 �C)

Demethylation andtranscriptional activa-tion

[182]

hpAtPARP2 PARP2 Hormones and abioticstress

Abiotic stress protec-tion via ABA signalingpathways

[183]

AtBRCA2 BRCA2 Cisplatin, c-ray DSB/HR repair [162]

AtNUDX7 AtNUDX7 Oxidative stress(2�3mM paraquat),salinity (250mMNaCl), high light(1600mEm�2 s�1),drought (dehydration)

Modulates PARylationby supplying ATP vianucleotide recyclingfrom free ADP-ribosemolecules and regu-lates the defensemechanisms againstoxidative DNA damage

[184]

AtTANMEI/ALT2 andATR

TANMEI/ALT2 andATR

Al3þ (0.75, 150mM,pH 4.2) for 7 d

Detect damage arisingfrom DNA cross-link-ing agents and Al-dependent alterationsin DNA topology, withthese changes trigger-ing cell cycle arrest

[185]

266 10 Plant Adaptation to Abiotic and Genotoxic Stress

The Cd-induced genotoxic stress response demonstrated in tobacco BY-2 cellsprovides evidence suggesting that telomerase together with DNA polymerases areinvolved in the re-establishment of genome integrity [188]. The role of Ku, aheterodimeric complex of 70- and 80-kDa subunits, has been shown to be essentialfor maintaining telomere homeostasis through the NHEJ pathway. Ku binds withhigh affinity to broken DNA ends to prevent degradation and facilitate alignment forsubsequent ligation by DNA ligase IV, and plays a major role in genotoxic stressresponse [175,176,189]. Abiotic stresses imposed by Al3þ, paraquat, salt, and lowtemperature induce glycerophosphodiesterase-like protein (NtGPDL), NtGPDLtranscripts, and demethylation of the corresponding coding region at CCGG loci inthe genome of the tobacco plant. Enhanced transcription has been linked to alteredchromatin structure through demethylation and histone modification [182]. Cellularresponses to genotoxic treatments include activation of DNA repair, temporal cellcycle arrest, and induction of stress signaling that modulates gene expression.Whereas the mechanism underlying genotoxic adaptation warrants further elucida-tion, the emerging new insights point to the roles of signal transduction, DNAdamage responses, and epigenetic mechanisms involving DNA methylation andsmall RNAs that are discussed in the following sections [53,190�192].

10.7

Role of MAPK and Calcium Signaling in Genotoxic Adaptation

Perception by sensors of external stress stimuli, transduction of the signal acrossthe cell wall, plasma membrane, cytoplasm to nucleus, and cellular responsethrough gene activation or expression comprise the components of plant signaltransduction. The mechanisms by which plants sense and transduce signals inresponse to environmental stresses are mostly unknown. Recent findings, however,suggested that, like Drosophila and mammals, plants have evolved TLR (Toll-likereceptors) and LRR (leucine-rich repeat) proteins that are involved in stressperception and downstream signaling through cytoplasmic protein kinasesfacilitating stress responses [193]. The MAPK cascade is one of the major pathwaysby which extracellular stimuli are transduced into the intracellular responses inyeast [194] and mammalian cells [195]. The basic assembly of the MAPK cascade isa three-kinase module conserved in all eukaryotes. A MAPK cascade minimallyconsists of a MAPKKK-MAPKK-MAPK module that is linked in various ways toupstream receptors and downstream targets. Receptor-mediated activation of aMAPKKK can occur through physical interaction and/or phosphorylation by thereceptor itself, intermediate bridging factors, or interlinking MAPKKKs.MAPKKKs are Ser/Thr kinases that activate MAPKKs through phosphorylation ontwo Ser/Thr residues in a conserved (S/T)X3�5(S/T) motif [196,197]. In contrast,MAPKKs are dual-specificity kinases that phosphorylate MAPKs on threonine andtyrosine residues in the TXYmotif. MAPKs are promiscuous Ser/Thr kinases thatphosphorylate a variety of substrates, including transcription factors, proteinkinases, and cytoskeleton-associated proteins [197,198]. The MAPKs are the

10.7 Role of MAPK and Calcium Signaling in Genotoxic Adaptation 267

terminal components in this three-kinase cascade. In mammals, the MAPK familywas subdivided into three subfamilies: the ERK (extracellular signal-regulatedkinase), the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase),and the p38 subfamilies that were activated in response to various signals,including UV and ionizing radiation, hyperosmolarity, oxidative stress, and so on[199]. An increasing body of evidence suggests that MAPKs play similar roles inplants responding to a multitude of biotic and abiotic stresses [200�202]. MAPKsare the intracellular mediators of information that shuttle between the cytoplasmand the nucleus, and among their targets are several classes of transcription factors[203]. The tobacco MAPKs, SIPK (salicylic acid-induced protein kinase) and WIPK(wound-induced protein kinase) are activated upon infection and elicitor treatment[204]. ROS do activate MAPKs [205,206]. In tobacco, SIPK and WIPK are activatedby ROS [207]. Heavy metals (Cd2þ, Cu2þ, Pb2þ, Zn2þ) also activate signaltransduction, apparently through ROS-mediated MAPK pathways [208,209]. H2O2,the most long-lived and, therefore, major ROS of the oxidative burst, initiatesMAPK cascades [210,211]. Being small molecules and able to diffuse over shortdistances, ROS are ideally suited to act as signaling molecules. Among differentROS, however, only H2O2 can cross plant membranes and can therefore directlyfunction in cell-to-cell signaling. Several reports have demonstrated the activationof MAPKs and induction of stress-responsive genes by H2O2 in Arabidopsis andother plants [212,213]. The fact that pretreatment of protein kinase inhibitorsprevented Al-induced genotoxic adaptation to EMS pointed to the involvement of aMAPK pathway in the underlying mechanism (Figure 10.8).

Comet assay

bbd

bbd

bbdb

d3

4

5

6

0

1

2

0

2AP 10

µM

Oliv

e ta

il m

omen

t (µm

)

Al 10µ

M

LY 1µ

M

LY 2µ

M

PD 2.5µ

M

PD 5µM

2AP 20

µM

EMS 5mM

Al /EMS

LY 1µ

M /Al /E

MS

LY 2µ

M /Al /E

MS

PD 2.5µ

M /Al /E

MS

PD 5µM/Al /E

MS

2AP 1

0µM /A

l /EMS

2AP 2

0µM /A

l /EMS

Treatments

Figure 10.8 Pretreatments of protein kinase

inhibitors LY (LY-294002) 1�2 mM, PD (PD

98059) 2.5�5 mM, and 2-AP (2-aminopurine)

10�20 mM prevented Al-induced adaptive

response to DNA damage induced by EMS

5mM in root meristems of A. cepa. Increase

significant compared to control (0) at

p� 0.01b; decrease significant compared to

EMS-challenge at p� 0.01d.

268 10 Plant Adaptation to Abiotic and Genotoxic Stress

Several phosphatases are able to dephosphorylate and thus inactivate variouscomponents of MAPK cascades. However, the direct inactivation of MAPKs isachieved only by phosphoSer/Thr phosphatase PP2A [214], phosphoTyr phospha-tases [215], and MAPK phosphatases (MKPs), all belonging to the family of theVH1-like dual-specificity phosphatases. Dephosphorylation of MAPKs by specificphosphatases plays a critical role in their inactivation. MKPs dephosphorylate bothtyrosine and Ser/Thr residues, exhibiting high specificity for MAPKs [216,217].Based on the fully sequenced Arabidopsis genome, 20 MAPKs, 10 MAPKKs, and 60MAPKKKs were identified, and a unified nomenclature for ArabidopsisMAPKs andMAPKKs was proposed [218].Calcium acts as an intracellular second messenger that couples extracellular

stimuli to intracellular and whole-plant responses. The changes in intracellularCa2þ are further translated into downstream actions through various Ca2þ sensorproteins [219,220]. These include calmodulins (CaM), CaM-binding proteins,calcium-dependent protein kinases (CDPKs), other EF-hand motif-containingCa2þ-binding proteins, and Ca2þ-binding proteins without EF-hands. CaM isconsidered as a versatile transducer in plants [221,222]. A growing number ofstudies revealed their role in signal transduction in plant responses to biotic andabiotic stresses [223,224]. Several CDPKs in Arabidopsis are involved in abscisic acid(ABA) signaling, having a role in plant resistance to drought or salt stress [225,226].Furthermore, Ca2þ is associated with ROS production in plants. A potato NADPH-OX is phosphorylated by two CDPKs in a Ca2þ-dependent manner, which in turnelevates its ability to produce ROS [227]. Plant CDPKs are responsive toenvironmental stresses [228,224]. Stress due to cold, salinity, and drought increasesCDPK transcripts in Arabidopsis [229]. Oxidative stress is often linked withactivation of Ca2þ channels [230]. Research over the past years has revealed thatplant CDPK/MAPK cascades constitute central elements in complex signalingnetworks that regulate plant responses to a multitude of abiotic and biotic stimuli.Recently, Achary et al. [231] have demonstrated that Ca2þ channel chelator/blockerscould block Al-induced genotoxic adaptation to genotoxic stress imposed by EMS inroot cells of A. cepa, suggesting a role for Ca2þ channels in abiotic stress-inducedgenotoxic adaptation (Figure 10.9).

10.8

Role of DNA Damage Response in Genotoxic Adaptation

Genotoxic stress in cells leads to a temporary arrest of cell division. This pausein the cell cycle progress allows time for repair of damaged cellular DNA beforethe onset of DNA replication. Critical molecules involved in cell cyclecheckpoints are cyclins and their associated kinases (cyclin-dependent kinases(CDKs)). Negative regulators of cyclins and CDKs can act as a brakingmechanism and pause the cell in the G1, S, or G2 phase of the cell cycle. The G1

delay allows for repair of damage before it is replicated. The S-phase completioncheckpoint, activated by stalled replication machinery and DNA damage, and the

10.8 Role of DNA Damage Response in Genotoxic Adaptation 269

G2 checkpoint have a protective effect by allowing additional time for repair ofDNA damage prior to entry into mitosis [232]. Genetic and biochemical analyseshave considerably advanced our understanding of DNA repair processes andtheir involvement in genotoxic signaling in plants [46,54,233]. Cells respond toDNA damage through regulation of the MAPK pathway by MKPs, the potentinactivators of MAPKs [173]. MKPs are considered important regulators ofMAPK signaling leading to genotoxic stress relief. These pathways initiate aseries of appropriate measures that include delaying the cell cycle progress, thusallowing time for DNA repair, and activation of genes required for repair andcellular protection. Although the identity of the sensors that directly recognizeDNA damage is not accurately known in most cases, in yeast and mammaliancells two related and conserved protein kinases of the phosphoinositide-3-OH-kinase-related (PI3K) family: ATM (ataxia telangiectasia-mutated) and ATR(ATM-Rad3-related) are considered central to DNA damage response [234�237].Downstream of these proteins are two families of checkpoint kinases (CHKs),CHK1 and CHK2, and their homologs. These kinases carry out subsets of theDNA damage response in mammals, and are the targets of regulation by ATMand ATR kinases. Below this level of signal transduction are the effectors thatexecute the functions of the DNA damage response. These include substrates ofboth PI3K and CHK kinases, and proteins involved in DNA repair, transcriptionregulation, and cell cycle control, such as BRCA1, Nbs1, p53, and Cdc25C [238].Activation of ATM and ATR kinases by two different types of lesions involvingindependent signal pathways has also been demonstrated in Arabidopsis thaliana[177,181,239]. ATM is activated by DNA damage, such as DSBs induced by

Comet assay

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7

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d

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5

d

0

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Oliv

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)

Al 10µM

EGTA 50µM

La 50

µM

VPL 25µM

W7 10µM

EMS 5mM

Al /EMS

EGTA/Al /EMS

La /A

l /EMS

VPL/A

l /EMS

W7 /Al /E

MS

Treatments

Figure 10.9 Pretreatments of Ca2þ channel

chelator/blocker EGTA 50 mM, La (LaCl3)

50 mM, VPL (verapamil) 25 mM, and

calmodulin-inhibitor W7 10 mM prevented Al-

induced adaptive response to DNA damage

induced by EMS 5mM in root meristems of A.

cepa. Increase significant compared to

control (0) at p� 0.01b; decrease significant

compared to EMS-challenge at p� 0.01d.

270 10 Plant Adaptation to Abiotic and Genotoxic Stress

ionizing radiation (c-rays), monofunctional alkylating agent methyl methanesulfonate (MMS) as well as oxidative stress [177,240]. ATM, in turn, activatesdownstream signaling pathways leading to the repair of DNA, transient arrest ofthe cell cycle, and inhibition of DNA replication [241,242]. While DSBs havebeen regarded as the major signal to activate ATM, there is also evidenceshowing that ATM can be activated by signals other than DSBs under certainconditions. For example, Cr6þ, which cannot induce DSBs, can activate ATM[243]. ATM is responsible for the signaling of DSBs that are not associated withthe replication machinery. ATR, on the other hand, is activated by various formsof DNA damage, including DSBs, arising at stalled replication forks or“replication stress” [244]. ATR regulates the slowing of the cell cycle during Sphase and the G2/M progression [245]. Typically, ATR is activated by non-ionizing radiation (UV), hyperoxia, and replication inhibitors like aphidicolinand hydroxyurea [181,242]. The ATR knockout A. thaliana obviously is severelysensitive to clastogenic and genotoxic stresses, likely because of failure to initiatethe necessary repair program for correcting the consequent DNA damage. ATMis also required for protection of short telomeres. ATR, by contrast, is requiredfor maintenance of telomeric DNA. ATM and ATR make essential and distinctcontributions to chromosome end protection and telomere maintenance inhigher eukaryotes [246]. Recently, the involvement of AtTANMEI/ALT2 and ATRin genotoxic adaptation resulting in arrest of the cell cycle and inhibition of rootgrowth in response to Al3þ has been also demonstrated [185]. However, sincelow doses of Al3þ stimulate root growth and cell division in A. cepa, the observedAl-induced genotoxic adaptation has been suggested to be independent of aDNA damage response [231].The role of poly(ADP-ribose) (PAR) involving PARPs, poly(ADP-ribose)

glycohydrolases (PARGs), and adenine dinucleotide (NADþ) is consideredcrucial in DNA damage response, genomic integrity, and cell survival[247�249]. As a DNA nick-activated enzyme, PARP is one of the firstresponders to the sites of DNA breaks. PARP acts as a DNA break sensor and aDNA repair signaling molecule, with a caretaker role that can lead cells eithertowards repair or towards programmed cell death, depending on the severity ofthe damage and amplitude of PARP activation [249]. Poly(ADP-ribosyl)ation(PARylation) is a unique post-translational protein modification mediated by thePARP enzyme that tags long-branched PAR polymers to nuclear target proteinsusing NADþ as substrate [247,248]. PARP detects DNA strand breaks andconverts the damage into intracellular signals that can activate DNA repairprograms or cell death, according to the severity of the injury, via thePARylation of nuclear proteins involved in chromatin architecture and DNAmetabolism, and interacts with the X-ray repair cross-complementing factor 1(XRCC1), an adapter protein that also has two interfaces with two importantSSB repair/BER enzymes: DNA ligase and DNA polymerase b [250�252]. DNApolymerase b fills the single nucleotide gap, preparing the strand for ligation bya complex of DNA ligase III and XRCC1 [253,254]. The enzyme ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) modulates PARylation [184]. The

10.8 Role of DNA Damage Response in Genotoxic Adaptation 271

catabolism of PAR is mediated primarily by PARG, an enzyme with both exo-and endoglycosidase activities that hydrolyzes the glycosidic linkages betweenthe ADP-ribose units of PAR producing free ADP-ribose [247]. Abiotic stressessuch as drought, high light, and heat activate PARP, causing NADþ breakdownand ATP consumption. When PARP activity is reduced by means of chemicalinhibitors or gene silencing, cell death is inhibited and plants become tolerantto a broad range of abiotic stresses like high light, drought, and heat. Plantlines with low PAR activity maintain their energy homeostasis under stressconditions by reducing NADþ breakdown and consequently energy consump-tion. The higher energy-use efficiency avoids the need for a too intensemitochondrial respiration and consequently reduces the formation of ROS[255]. This outperforming stress tolerance was initially attributed solely to amaintained energy homeostasis due to reduced NADþ consumption. However,genome-wide transcript analysis of stressed PARP2-deficient transgenic Arabi-dopsis (hpAtPARP2) revealed the induction of specific ABA signaling pathwaysthat might be steered through increased levels of the cyclic nucleotide cyclicADP-ribose, thereby attributing a signaling role for NADþ [183]. In addition togenotoxic stress tolerance, PARP also plays important roles in DNA repair,programmed cell death, transcription, and cell cycle control in plants [256]. Theright nuclear balance between unmodified and PARylated (PARP-1), whichdepends on the dynamics of PARPs/PARG activity, has been also considered askey to maintaining genomic methylation patterns via regulation of DNAmethyltransferase 1 activity [257].Current knowledge on DNA damage response emerging from plants also

suggested that strand breaks trigger a DNA damage response by inducing theexpression of molecular markers associated with DNA damage repair, such asPARP, RAD51, and breast cancer (BRCA) family members. DNA damage activatesATM and ATR signaling kinases in plant cells, and leads, via the WEE1 Ser/Thrkinase, to a transient cell cycle arrest that allows cells to repair DNA beforeproceeding into mitosis [249,258]. A generalized scheme involving CaM/CDPK orMAPK cascades in the cytoplasm leading to phosphorylation of transcriptionfactors giving rise to expression of specific genes of DNA repair proteins conferringgenome protection is proposed (Figure 10.10).

10.9

Epigenetics of Genotoxic Stress Tolerance

The chances of sustenance, survival, and fitness of plants largely depend on planttolerance or adaptation to abiotic stress due to impending climate change. Thecontemporary evolutionary concepts suggest that there is more to heredity thangenes, that some hereditary variations are non-random in origin, that someacquired information is inherited, and that evolutionary change can result frominstruction as well as selection [259,260]. For that matter, stress tolerance in plantsessentially is a manifestation of adaptive evolution resulting from the integration of

272 10 Plant Adaptation to Abiotic and Genotoxic Stress

evolution with development as influenced by the environment [261]. Adaptiveevolution is usually assumed to be directed by selective processes, whereasdevelopment is usually assumed to be directed by instructive processes; evolutioninvolves random genetic changes, development involves induced epigeneticchanges [262]. Epigenetics refers to heritable changes in gene expression or thecellular phenotype without changes in the DNA sequence. Evidence is over-whelming that upholds epigenetic inheritance of environmentally inducedhypomethylation or hypermethylation of DNA for several generations in plants[263]. Epigenetic regulation of gene expression is accomplished by DNA methyla-tion, histone modifications, histone variants, and chromatin remodeling, and mayinvolve small RNAs [264].A stressful environment results in a reduction of fitness of populations. The

ultimate success of plants to withstand environmental stress depends on the ability

Figure 10.10 Simplified scheme of signal

transduction from abiotic stress perception

to gene activation facilitating adaptive

response to genotoxic stress in plant cells.

Mild abiotic stress induced apoblastic

oxidative burst mediated by NADH-PX or

NADPH-OX bound to the cell wall or plasma

membrane, respectively, generating H2O2

that triggers the adaptive response, which

may involve CaM/CDPK or MAPK cascades in

the cytoplasm leading to phosphorylation of

transcription factors giving rise to expression

of specific genes of DNA repair enzymes

ensuring genome protection.

10.9 Epigenetics of Genotoxic Stress Tolerance 273

of individuals or populations to pass through the three phases of evolution:physiological elasticity, ecological plasticity, and genetic or epigenetic tolerancefacilitating microevolution. Physiological elasticity represents transient physiologi-cal acclimatization to stress as evident by an adaptive response that is reversible,whereas ecological plasticity favored through selection is irreversible [265,266].Developmental plasticity differentiates into ecotypes, affecting evolutionarydynamics by influencing the rate and direction of phenotypic change [267,268]. Itis based on regulatory changes in gene expression and gene products, which arepartially controlled by epigenetic mechanisms. Plasticity involves not justepigenetic changes in somatic cells and tissues, but also passes on to the progenypredominantly through the female rather than the male germline [269]. Germlineepigenetic plasticity increases the capacity to generate heritable, selectable,phenotypic variations, including variations that lead to novel functions or traits[268]. Terminologies such as “hard inheritance” and “soft inheritance” have beenused to describe Mendelian and epigenetic inheritance, respectively [270]. Plasticecotypes that are favored through selection pressure emerge over the course of timeas stress-tolerant genotypes [271,272]. The transition from metal-tolerant ecotypesto genotypes is well known in the evolution of metal tolerance in plants [273].Phenotypic variation in plants under stress is classically attributed to DNAsequence variants. Our present knowledge, however, suggests that phenotypes canbe influenced by epigenetic modifications that include DNA methylation,chromatin or histone modification, and small RNA-based mechanisms, which cancontribute separately or together to the phenotypes by regulating gene expressionin response to the stress effect [274�277]. Interestingly, Schwartz [278] observedthat evolution is not necessarily gradual, but often a sudden, dramatic expression ofchange that begins at the cellular level because of radical environmental stressors �like extreme heat, cold, or crowding � years earlier that underlines the greater rolefor epigenetics in evolution. The occurrence of “adaptive” mutations, also variouslytermed as “Cairnsian mutation” or “directed mutation,” was discovered first in E.coli in 1988 by Cairns that challenged the randomness of bacterial mutations[279,280]. The emerging hypothesis has been that environmental stress directsadaptive evolution, and in the process organisms evolve that acquire adaptive genesand lose redundant genes [281�283].

10.10

Transgenerational Inheritance and Adaptive Evolution Driven by the Environment

The aforementioned concept of adaptive evolution (Figure 10.11) gained supportfrom the studies in the aftermath of the Chernobyl nuclear disaster thatoccurred in the Ukraine in April 1986 [284]. There was extensive radioactivefallout contaminating large tracts of agricultural land in Belarus, Ukraine, andRussia, and scattered areas beyond those limits. Experiments on radiation-exposed P. silvestris plants in the aftermath the Chernobyl accident provide thefirst evidence that genome hypermethylation, an epigenetic mechanism, was the

274 10 Plant Adaptation to Abiotic and Genotoxic Stress

basis of the plant adaptation to ionizing radiation [285]. Subsequent studies onthe plant populations of A. thaliana that were exposed to radiation providedfresh insights into adaptive evolution directed by nuclear pollution. Seeds of A.thaliana that survived high levels of exposure to ionizing radiation producedprogeny that were more resistant to genotoxic and oxidative agents than theircounterparts obtained from A. thaliana exposed to no or low radiation levels.The foregone adaptive evolution underlined the involvement of DNA methyla-tion in the non-Mendelian epigenetic inheritance of radio-adaptation in plants[286]. Inheritance of such transgeneration memory of tolerance to stressconditions such as excessive or inadequate light, excess water or drought,salinity, extreme temperatures, oxidative or radiomimetic agents, UV orresistance to pathogens has been correlated with somatic homologous recombi-nation (SHR) [287�289]. Molinier et al. [287] have shown that a single exposureof A. thaliana plants to stress (UV-C) leads to an increased frequency of SHR inat least four consecutive non-stressed generations. In contrast, Boyko et al. [290]provided experimental evidence that the increased frequency of SHR isrestricted to the immediate progeny only and does not appear to persist acrosssuccessive generations of untreated plants. Maintenance of the response overmultiple generations requires repeated exposure to the same stress. Experimen-tally, it was shown that the increased SHR that indicates genome rearrangement

Climate change

Ecosystem evolution

Global warming, precipitation change, sea level change

Adaptive development

Adaptive evolution

ROS:

Abiotic stress

Plant Cell Oxidative adaptation

Oxidative stress

Genotoxicadaptation

Genotoxicstress

Oxidativedamage Genome

Figure 10.11 Response of the plant cell to abiotic stress leading to adaptive evolution of stress

tolerance in plants counteracting climate change.

10.10 Transgenerational Inheritance and Adaptive Evolution Driven by the Environment 275

is a dominant trait that can be equally transmitted via maternal and paternalgametes. The most prominent transgenerational increase of genome rearrange-ments, evident by a high frequency of SHR upon exposure to 25mM NaCl,whereas exposure to 100mM had no effect [290], was reminiscent of low-doseinduction of adaptive response to genotoxic stress discussed above. It ispertinent to note here that the NaCl-induced genotoxic stress has been to Cl�

ions and not to ROS [290]. The high frequency of SHR common to all stressresponses is the result of genomic perturbation associated with instability oftransposons in maize and other plant species that again are related to DNAmethylation [291�295]. Inheritance of transgenerational memory of heattolerance was evident in A. thaliana F3 progeny that were derived from ancestralplants grown under elevated temperatures over two consecutive generations Pand F1 generations earlier [296]. The increased frequency of SHR observed inthe untreated progeny of stressed plants has been shown to be mediatedepigenetically and considered to be a dominant trait equally transmitted viamaternal and paternal gametes [287]. Persistence of stress memory for shortdurations was evident from adaptive metal-induced responses to genotoxic stress[152,154]. The stress memory can persist for only short durations if the memorydepends on the half-life of stress-induced proteins, RNAs, and metabolites [297],while the memory can last longer if it involves reprogramming in the phenologyand morphology of plants [298]. Furthermore, epigenetic effects can contributeto phenotypic plasticity within generations (i.e., within individual genomes) andcould contribute to heritable variation across generations (i.e., parental tooffspring), and influence evolution in wild populations through their effects onphenotypic traits and fitness, suggesting that evolution in natural populationscould have significant epigenetic influences [299]. A number of studies haveshown that DNA methylation and histone modifications play a key role in geneexpression and plant development under stress. Most of these stress-inducedmodifications are reset to the basal level once the stress is relieved, while someof the modifications may be stable (i.e., may be carried forward as “stressmemory” and may be inherited across mitotic or even meiotic cell divisions). Inplants, the state of the chromatin can be modified rapidly and reversibly throughthe insertion of methyl groups in cytosine by DNA methyltransferases,acetylation and methylation of N-terminal histone tails for chromatin remodel-ing, linker histone H1 and components of chromatin complex, and themechanisms of small RNAs that influence gene regulation in stress responses[300]. Stresses can induce changes in gene expression through hypomethylationor hypermethylation of DNA. In maize roots, cold stress-induced expression ofZmMI1 was correlated with a reduction in methylation in the DNA of thenucleosome core. Even after 7 days of recovery, cold-induced hypomethylationwas not restored to the basal level [301]. In tobacco, aluminum, paraquat, salt,and cold stress-induced DNA demethylation in the coding sequence of theNtGPDL (a glycerophosphodiesterase-like protein) gene correlated with NtGDPLgene expression [182]. In rice, saline and heat stresses caused extensivedecondensation of 45S rDNA chromatin and also an increase in the distance

276 10 Plant Adaptation to Abiotic and Genotoxic Stress

between the two homologous 5S rDNA loci that was attributed to hypomethyla-tion [302]. Kovarik et al. [303], on the other hand, reported hypermethylation oftwo heterochromatic loci, defined with repetitive DNA sequences HRS60 andGRS, in tobacco BY-2 cells in response to osmotic stress. In plants, Arabidopsismutants defective in CG methylation exhibit stable inheritance of numeroushypomethylated loci for at least eight generations after out-crossing of themutant alleles [304,305]. Notably, such induced DNA hypomethylation appearsto revert over several generations in an RNA interference (RNAi)-dependentmanner [305,306]. This kind of epigenetic resetting resembles the progressiveinactivation of transposable elements in maize and the silencing of transgenicloci in many plants [307,308]. In plants, the major classes of small non-codingRNAs are microRNAs (miRNAs), small interfering RNAs (siRNAs), trans-actingsiRNAs (ta-siRNAs), natural cis-antisense transcript-derived siRNAs (nat-siR-NAs), and heterochromatic siRNAs (hc-siRNAs), which differ in biogenesis, andcontrol gene expression and epigenetic regulation in response to stress[309�312]. One key component of plant small RNA biogenesis is a family ofRNase III enzymes called Dicer-like (DCL) proteins [313,314]. These enzymesfunction to cut or “dice” specific stem�loop structures of single-strand RNAprecursors into miRNA, or double RNA into siRNA duplexes, respectively.miRNAs play important roles in plant post-transcriptional gene regulation bytargeting mRNAs for cleavage or repressing translation, signal transduction, andprotein degradation in response to environmental stress and pathogen invasion;they regulate their own biogenesis and expression of many important genes, anda majority of these genes are transcriptional factors [315,316]. siRNA directscytosine methylation of the target DNA sequences complementary to itself in theprocess of RNA-directed DNA methylation in the nucleus [317,318]. Furtherevidence for the involvement of small non-coding RNAs in memorizingepigenetic changes comes from the analysis of Arabidopsis plants deficient forDCL2 and DCL3, as such mutants were not able to properly establishtransgenerational stress effects on the frequency of somatic HR [290,308,319].Comparative genomics of moss Physcomitrella patens vis-�a-vis several angiosperms

(A. thaliana, Oryza sativa, and Populus trichocarpa) and aquatic unicellular greenalgae (Ostreococcus tauri, Ostreococcus lucimarinus, and Chlamydomonas reinhardtii)provided further evidence pertaining to long-term evolution or macroevolution thatupheld adaptive evolution that underscores changes in plant genomes concomitantwith their transition from aquatic to land ecosystems [320]. The genome evolutionis marked by loss of genes associated with aquatic environments (e.g., flagellarcomponents for gametic motility) and vegetative dehydration tolerance; andacquisition of genes for tolerance to abiotic stresses, such as drought, radiation,and extreme temperature, genes for auxin and ABA signaling pathwaysthat coordinating multicellular growth and morphogenesis, and morecomplex photoreception, and an overall increase in gene family complexity. Thecomparative functional genomics, therefore, vindicated environmental stress-drivenevolution of the genome in land plants. Primary adaptations included enhancedosmoregulation and osmoprotection, desiccation and freezing tolerance, heat

10.10 Transgenerational Inheritance and Adaptive Evolution Driven by the Environment 277

resistance, synthesis and accumulation of protective sunscreen molecules, aminoacids, polypeptides, and cell cycle control and DNA repair mechanisms [320].

10.11

Concluding Remarks

This chapter reinforces the view that rapid climate change imposes strong selectionpressures on traits important for fitness and promotes microevolution of stresstolerance in plants [321]. Genomic protection of plants from the possible impact ofclimate-related abiotic stress is fundamental for the conservation of plant diversity,ensuring agricultural sustainability and ecological security. Plants, being sessile,are inherently equipped with well-orchestrated antioxidative defense systems tocounter or adapt to the impending abiotic stress emanating from climate change.With the unraveling of the plants’ defense network, comprising antioxidantresponses, signal transduction, and DNA damage responses operating at multiplelayers, plant perception and response to stress is now better understood. Attemptsare currently underway to fortify the antioxidative mechanisms using geneticengineering, but have so far met with little success. Stress tolerance can beachieved through genomic tweaking of seeds or seedlings, which can be achievedby mild stress-conditioning conferring tolerance to the subsequent genotoxicchallenge. From the available evidence, it appears that perturbation of the genometriggers somatic as well as transgenerational epigenetic modifications: DNAmethylation-, chromatin-, and small RNA-based mechanisms that coordinatelycontribute to the adaptive evolution of stress tolerance. This natural phenomenonthat enables plants to cope with climate change can be hastened through artificialepigenetic interference so as to sustain plant productivity under the environmentalstress posed by climate change that is looming large day by day.

Acknowledgments

The authors are thankful to the authorities of Berhampur University, Berhampurand ICGEB, New Delhi for extending infrastructural support. V.M.M.A. and K.K.P.acknowledge the award of postdoctoral fellowships received, respectively, fromDBTand UGC, New Delhi.

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