Chapter 2 Review of Literature - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/39814/4/08... · 2018. 7. 2. · Chapter 2 Review of Literature Department of Biotechnology,

Chapter 2 Review of Literature

Department of Biotechnology, Jamia Hamdard 5

2. REVIEW OF LITERATURE

Plants are exposed to various biotic and abiotic stresses in their natural environment that

result in water insufficiency, sodium toxicity, ion deficiency, and photo-inhibition. The

growth and productivity of crop plants depends largely on their vulnerability to

environmental stresses. High salinity, water deficit, and temperature stress are the major

constraints that limit agricultural production (Cherian et al. 2006). Salinity, which has an

adverse effect on the growth and the productivity of crops, has emerged into a global

threat among the agricultural community by affecting approximately 20% of the globally

irrigated agricultural land (Flowers and Yeo 1995; Joseph et al. 2010). During the course

of evolution, plants have developed intricate mechanisms to perceive external signals,

allowing optimal response to environmental conditions to survive under such conditions.

An array of biochemical and physiological adaptations leading to renovation of cellular

homeostasis, toxin detoxification, and recovery of growth, by modulation of expression of

stress-related genes, is the essence of these adaptations (Xiong and Zhu 2002). Hence, any

attempt to improve the stress tolerance requires a better understanding of the underlying

physiological, biochemical and molecular events (Cherian and Reddy 2003).

A common adaptation in plants to overcome stress is the production of compatible solutes

and they accumulate to high levels without disturbing intracellular biochemistry (Bohnert

and Jensen 1996). Typically, compatible solutes are hydrophilic giving rise to the view

that they could replace water at the surface of proteins, protein complexes, or membranes

and thus, have the capacity to persevere the activity of enzymes and membranes in hostile

environments. These compounds have minimal effect on pH or charge balance of the

cytosol or luminal compartments of organelles. Compatible solutes include low molecular

weight sugars, organic acids, polyols, and nitrogen containing compounds such as amino

acids, amides, imino acids, proteins and quaternary ammonium compounds (Ashraf and

Harris 2004). Accumulation of low molecular weight osmolytes such as proline, betaines

and sugar alcohol is an important mechanism underlying adaptation to such stress factors.

In addition, detoxification enzymes, transport proteins and stress induced proteins such as

water channel proteins and osmotin also contribute to stress tolerance.



2.1 Osmotin

Singh et al. (1985) isolated osmotin and its gene from tobacco var. Wisconsin 38 that was

cultured for several generations on the culture medium with high NaCl concentration. The

name osmotin was applied because of the positive correlation between the accumulation

of the protein and the cell osmotic potential. However, this nomenclature is deceptive

because the specific function of the protein still has not been established (Singh et al.

1989). Osmotin is synthesized, as a pre-protein with a molecular weight of 26,380 Da and

the mature protein is a basic protein with molecular weight of 23,984 Da (Singh et al.

1989). The size of osmotin predicted by nucleotide sequence is smaller than the size

observed by electrophoresis. This difference may represent an undetected posttranslational

addition of some moiety, which may also explain the isoform of osmotin observed (Singh

et al. 1989).

Osmotin strongly resembles the sweet protein thaumatin (THN), a sweet-tasting protein

that was first isolated from the fruit of the West African shrub (Thaumatococcus daniellii

Benth.), in its molecular weight, amino acid composition, N- terminal sequence, and the

presence of a signal peptide on the precursor protein (Cornelissen et al. 1986; van der Wel

and Loeve 1972). Osmotin solution however, could not be detected as sweet as thaumatin,

at a concentration atleast 100 times that of the latter (Singh et al. 1987). Also thaumatin

does not cross react with anti-osmotin. Osmotin and thaumatin however, belongs to the

pathogenesis-related group 5 (PR5) proteins, also called thaumatin-like proteins (TLPs)

(Cornelissen et al. 1986; Singh et al. 1987; van der Wel and Loeve 1972). Many of PR5

plant proteins including thaumatin and osmotin have antimicrobial activity. Taking

advantage of recently completed plant genome data and discovery of multiple insect PR5-

L sequences, Shatters et al. (2006) performed phylogenetic comparisons among plants and

animals. Their analyses showed that the PR5 family is paraphyletic in plants, likely arising

from 10 genes in a common ancestor to monocots and eudicots. Structure and sequence

conservation among PR5/PR5-Ls suggests an important and conserved role throughout the

evolutionary divergence of the diverse organisms from which they reside (Shatters et al.

2006). PR-5 proteins have a conserved topology, consisting of a central lectin-like

domain of antiparallel strands surrounded by one or two disulfide-stabilized domains.



Osmotin proteins have peptide sequences similar to the bifunctional trypsin/α-amylase

inhibitor from maize, the tomato protein NP 24, the tobacco anti viral protein gp 22,

potato PR protein C, tobacco, and barley thaumatin-like proteins (Larosa et al. 1992).

Osmotin is a cationic protein that exists in at least two forms: an aqueous soluble form

(osmotin-I) and a detergent soluble form (osmotin-II): one with pI of 7.8 and other with a

pI of >8.2 and differing slightly in molecular weight (Singh et al. 1987). The N-terminal

amino acid sequences of osmotins I and II are identical through position 22. Immuno-

cytochemical detection of osmotin reveals that osmotin gets concentrated in dense

inclusion bodies within the vacuole. Min et al. (2004) have reported that the osmotin

crystals have a non crystallographic dimer in the asymmetric unit. The two monomers

have slightly different tertiary structures. Osmotin is composed of three domains and

shows a similar fold with thaumatin and other PR proteins. Domain I consists of an eleven

strand, flattened -sandwich that forms the compact core of molecule. Domain II consists

of several loops extending from domain-I. It is stabilized by four disulphide bonds.

Domain III consists of a small loop with two disulphide bonds. Osmotin has a pronounced

cleft formed by domains I and II. The tertiary structure of osmotin is homologous to those

of thaumatin and other antifungal PR-5 proteins. Most of the differences among them are

in domain II and distribution of surface charges. The cleft between domains I and II may

have an acidic, neutral or basic nature for binding different ligands/receptors. In all plant

PR5 proteins with known antifungal activity, this cleft is acidic because of five highly

conserved amino acids (arginine, glutamic acid, and three aspartic acid residues) (Figure

1). This acidic cleft is assumed to be relevant to their specific receptor binding for an

antifungal activity (Batalia et al. 1996; Koiwa et al. 1999; Min et al. 2004).

So far, crystal structures for seven plant TLPs have been determined, including thaumatin

(Ogata et al. 1992), zeamatin (Batalia et al. 1996), tobacco PR-5d (Koiwa et al. 1999),

osmotin (Min et al. 2004), cherry allergen Pru Av2 (Dall'Antonia et al. 2005), banana

allergen Ba-TLP (Leone et al. 2006) and tomato NP24-I (Ghosh and Chakrabarti 2008).

All show similar 3 dimensional (3D) structures with three domains and a cleft structure

between domains I and II (Ghosh and Chakrabarti 2008). In the 3D structures of these

plant TLPs, domain I is a lectin-like β-barrel that forms the compact core of a TLP



molecule. Each domain is stabilized by at least one disulfide bridge linked by up to 16

cysteine residues with a conserved spatial distribution throughout the protein (Min et al.

2004) (Figure 1).

Figure 1: (A) Ribbon diagram of osmotin viewing along the cleft. Red (domain I),

Blue (domain II), and Yellow (domain III). N- and C-termini are labeled. Residues

(Glu84, Asp97, Asp102, and Asp185) around the acidic cleft are drawn in green stick

models. The figure was drawn using MOLSCRIPT. (B) Stereo diagram of the

superposition of the Cα traces of osmotin (red), PR-5d (magenta), zeamatin (green),

and thaumatin (blue) in the same orientation with (A). An arrow indicates the

thaumatin loop in domain I. [Adapted from (Min et al. 2004)]

2.2 Osmotin/thaumatin-like protein superfamily

Osmotin belongs to Osmotin/thaumatin-like protein superfamily. Multigene/Super family

is a group of similar, but not necessarily identical sequences, each sequence representing a



gene, so that the gene is present in multiple copies. Zhu et al. (1995) have characterized

three cDNAs encoding osmotin-like proteins from Solanum commersonii cell cultures

having extensive nucleotide identity in the coding regions, but low homology in the 3’

non-coding sequences. Osmotin is encoded in Nicotiana tabaccum by a pair of genes, each

derived from one of the parental species, N. sylvestris and N. tomentosiformis (Nelson et

al. 1992). Osmotin like proteins are encoded by atleast six members of a multigenic family

in Solanum commersonii (Pierpoint et al. 1990; Zhu et al. 1995), two of which contain no

introns and are structurally similar to the tobacco osmotin (Nelson et al. 1992). A cDNA

clone encoding osmotin has been isolated from petal protoplast cultures of Petunia

hybrida and its protein sequence has revealed high identity with tobacco osmotin and

tobacco osmotin-like proteins (OLPs) (Kim et al. 2002). The SniOLP protein encoding

gene from S. nigrum represents a small multigene family and displays organ-specific

expression (Campos et al. 2008; Jami et al. 2007). Similarly, strawberry osmotin-like

protein gene (FaOLP2) belongs to a multigene family (Zhang and Shih 2007). Apart from

these, osmotin-like proteins (OLPs) have also been found in many other plant species such

as maize (Roberts and Selitrennikoff 1990; Vigers et al. 1991), tomato (Rep et al. 2002),

Arabidopsis (Capelli et al. 1997; Hu and Reddy 1997), rice (Reimmann and Dudler 1993),

flax (Anzlovar et al. 1998), apple (Oh et al. 2000), black nightshade (Campos et al. 2002)

and petunia (Kim et al. 2002). In addition to plants, thaumatin-like proteins have also been

discovered in the nematode Caenorhabditis elegans (Kitajima and Sato 1999) and the

desert locust Schistocerca gregaria (Brandazza et al. 2004).

2.3 Osmotin gene expression

Several studies have shown that osmotin gene expression can be induced by several

different hormonal and environmental signals. Singh et al. (1989) reported that abscisic

acid induces mRNA encoding osmotin. Levels of osmotin mRNA in NaCl adapted

tobacco cells are approximately 15-fold higher than in unadapted cells. Message for

osmotin is present at constant levels through the growth cycle of adapted cells, while in

unadapted cells, the level decreases during exponential phase of growth and increases

again when the cells approach stationary phase. While abscisic acid (ABA) induces the

message for osmotin, a low water potential environment appears to be required for



accumulation of the protein. Thus, it was hypothesized that after ABA induces or

stabilizes osmotin mRNA, a lower water potential environment is required for preferential

translation of osmotin mRNA or to reduce osmotin turnover (Singh et al. 1989).

Moreover, the message for osmotin may be preferentially translated over the messages in

an environment of high NaCl concentration. Besides its induction by ABA, osmotin

expression is controlled by a host of complex developmental, tissue specific and various

hormonal signals indicating substantial transcriptional and post-transcriptional regulation

of osmotin mRNA (Kononowicz et al. 1992; Larosa et al. 1992; Raghothama et al. 1993).

Grillo et al. (1995) showed that in tomato, both salt- and water stress caused increase in

the levels of osmotin transcripts and protein. The enhanced transcript levels were

accompanied by an increase in endogenous ABA. Interestingly, osmotin transcripts were

not induced by salinity or osmotic stress in an ABA deficient mutant of tomato suggesting

involvement of ABA in osmotin mRNA accumulation (Grillo et al. 1995).

In an investigation involving osmotin-promoter characterization, Kononowicz et al. (1992)

found high-level promoter activity in the mature pollen grains and in the pericarp tissue at

the desiccating stage of fruit development. The authors speculated that osmotin may be

involved in the protection of membranes during dehydration. Earlier reports on osmotin

from this group had implied that the intracellular accumulation of osmotin protein by cells

under salt- or drought stress conditions somehow enhances their ability to withstand

stresses (Larosa et al. 1989; Singh et al. 1987). However, support for the tolerance,

because of apoplastically secreted osmotin induced in response to abiotic stresses, comes

from results on soybean GmOLPa (Onishi et al. 2006). In Gossypium hirsutum, two

osmotin genes have been identified using probe of osmotin gene from tobacco (Wilkinson

et al. 2005). Based on the putative N-terminal 24 amino acid-long signal sequence of the

preprotein, the mature forms of the proteins are believed to be targeted for extracellular

secretion (Wilkinson et al. 2005). Thus, it is possible that both intracellular and

extracellular osmotin may be involved in plant response to abiotic stresses.

2.4 Osmotin and accumulation of compatible solutes

Osmotin was detected during characterization of salt-induced proteins in tobacco (Singh

et al. 1987). However, it also accumulates in response to cold, drought, waterlogging, and



at high as well as low levels of mineral nutrients (Barthakur et al. 2001; Das et al. 2011;

Goel et al. 2010; Kishor et al. 1995; Zhang and Blumwald 2001). Osmotin could be

involved in osmotic adjustment of cells by facilitating the accumulation or

compartmentation of solutes (Barthakur et al. 2001). Osmotin is also known to protect the

native structure of proteins during stress and involved in repairing the denatured proteins

(Singh et al. 1987). It has also been hypothesized that the synthesis of osmotin protein

could induce synthesis and accumulation of certain other compatible solutes or could be

involved in metabolic or structural changes (Singh et al. 1987). This function of osmotin is

linked to its action as compatible osmolyte that enhances osmotic potential of the cells.

The exact mechanism is however, not yet clear.

2.5 Osmotin and stress (biotic and abiotic) tolerance

Confirmation of the protective abilities of osmotin against abiotic stresses comes from

some overexpression studies published relatively recently. Transformation with the

osmotin gene has been reported to increase tolerance to different biotic and abiotic

stresses, in many plants (Grover et al. 2001).

Osmotin has been classified as a plant defense related protein under the PR5 protein

family. The production of pathogenesis-related (PR) proteins is a well characterized

response mechanism in tobacco plants against pathogen attack (Alexander et al. 1993).

The accumulation of osmotin and other PR-proteins is believed to play a role in the plant

disease defense response by limiting pathogen infection. Osmotin is shown to exhibit

antifungal activity against a broad range of fungal pathogens (Abad et al. 1996; Yun et al.

1998) in addition to its role as osmoprotectant. It is induced in response to viral and fungal

pathogen infections in tobacco and tomato (Stintzi et al. 1991; Woloshuk et al. 1991).

Osmotin proteins from both species were found to cause lysis of sporangia and growth

inhibition of Phytophthora infestans (Woloshuk et al. 1991). Tobacco osmotin was also

shown to inhibit the growth of Candida albicans, Neurospora crassa, and Trichoderma

reesei (Vigers et al. 1991). In a large-scale in vitro study, Abad et al. (1996) demonstrated

antifungal properties of osmotin against a wide range of fungal pathogens. Overexpression

of the osmotin gene in transgenic potato plants (Solanum tuberosum and Solanum

commersonii) confers enhanced tolerance to the oomycete pathogen P. Infestans (Liu et al.



1994). Overexpression of tobacco osmotin or a potato osmotin-1 protein was found to

delay the development of fungal disease symptoms in potato (Liu et al. 1994; Zhu et al.

1995) and provide Fusarium wilt resistance to transformed tomato (Ouyang et al. 2005).

In a subsequent study, Liu et al. (1994) showed that C-terminal truncated tobacco osmotin

expressed in potato was secreted to the extracellular matrix and the transgenic potato plant

showed resistance to P. infestans. Fungal challenge undertaken with three fungal species

known to cause serious losses to mulberry cultivation, namely Fusarium pallidoroseum,

Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic

plants with osmotin showed resistance against these pathogens (Das et al. 2011).

Constitutive expression of an osmotin gene in transgenic tobacco improved their tolerance

to salinity and drought stress (Barthakur et al. 2001). Transgenic strawberry expressing the

tobacco-osmotin showed improved germination of seeds and higher growth rates of plants

under high salinity (Husaini and Abdin 2008). Overexpression of a tobacco osmotin gene

in olive tree demonstrated its involvement in cold acclimation-related programmed cell

death, blocking cold-induced calcium signaling, and cold-induced cytoskeleton alterations.

However, the underlying mechanism of the protection provided by osmotin to tolerate

various abiotic stresses is still unclear (D'Angeli and Altamura 2007). Tomato transgenic

plants overexpressing osmotin gene has the potential to offset the adverse effects of such

environmental stresses as salinity and drought (Goel et al. 2010). Transformation of tea

somatic embryos showed normal embryo maturation, including accumulation of storage

reserves and acquisition of desiccation tolerance (Bhattacharya et al. 2006). Wheat plants

transformed with osmotin gene showed enhanced ability to produce roots at high NaCl

concentration (Noori and Sokhansanj 2008). The transformation of mulberry with the

osmotin gene confers tolerance against drought and salinity stress (Das et al. 2011).

Transgenic plants, when subjected to simulated salinity and drought stress conditions

showed better cellular membrane stability and photosynthetic yield than non-transgenic

plants under similar conditions. Overexpression of apoplast-targeted tobacco osmotin in

transgenic cotton conferred tolerance to water stress (Parkhi et al. 2009). The

transformants showed a slower rate of wilting during drought and faster recovery

following the termination of dry conditions in a greenhouse setting.



2.6 Osmotin and cell signalling

The recent research shows osmotin to be important signaling molecule. Osmotin is able to

induce programmed cell death in the Saccharomyes cerevisiae by weakening defensive

cell wall barriers. The genetic and biochemical studies showed that it mediate its effect

through the activation of signal transduction pathway that shares common elements with

the signal cascades for mating, invasive growth, and pseudohyphal development in yeast

(Yun et al. 1998). It was further suggested that programmed cell death in the S. cerevisiae

is induced by signaling suppression of cellular stress responses via RAS2/cAMP. The

resulting apoptosis occurs by intracellular accumulation of reactive oxygen species

(Narasimhan et al. 2001). Osmotin has recently been shown to specifically bind to

PHO36, a seven transmembrane domain receptor-like polypeptide, in yeast (Narasimhan

et al. 2005). It is required for full sensitivity to osmotin and controls osmotin-induced cell

death via a RAS2 signaling pathway.

Further, vertebrate taste receptors for thaumatin, a structural homolog of osmotin, have

been shown to be heterotrimeric G protein - coupled serpentine transmembrane proteins

(Abe et al. 1993). Therefore, it has been suggested that the ability to activate a G protein

coupled / seven transmembrane domain- serpentine receptor is a conserved characteristic

of PR-5 proteins and imply that osmotin may induce the MAPK pathway through an

unknown serpentine receptor in plants.

It has been shown that osmotin mimic the adiponectin in mammalian C2C12 myocytes

cell culture (Narasimhan et al. 2005). Adiponectin is an antidiabetic and

antiatherosclerotic protein hormone in mammals that conditions sensing of energy status,

fatty acid oxidation, and glucose transport upon interaction with adiponectin receptors

(Diez and Iglesias 2003; Yamauchi et al. 2003). It is a 30 kDa protein with an N-terminal

domain that is collagen-like and a C-terminal globular domain that is complement 1q-like

(Scherer et al. 1995). The C-terminal globular domain (17 kDa) is often referred to as

globular adiponectin. Osmotin is also a globular protein, but has no significant sequence

homology (≤10%) with the sequence of full-length or globular adiponectin (Narasimhan et

al. 2005). X-ray crystallographic studies however, have shown that both globular

adiponectin and osmotin consist of antiparallel β strands arranged in the shape of a β



barrel (Min et al. 2004; Shapiro and Scherer 1998). The domain I (lectin-like domain) of

osmotin can be overlapped with adiponectin with a rmsd of 3.1 Å for 121 Cα atoms,

suggesting that the two proteins share this particular domain (Narasimhan et al. 2005).

The binding of hormones and other proteins to receptors activates specific responses.

When bound to its receptor, adiponectin regulates sugar uptake and, in mouse models,

prevents the development of diabetes and atherosclerosis, or the hardening of the arteries

associated with heart disease. Two adiponectin receptors (AdipoR1 and AdipoR2) have

been found in humans and rats (Yamauchi et al. 2003). Binding of adiponectin to

adiponectin receptors has been shown to activate AMP-activated protein kinase (AMPK)

by phosphorylation. Adiponectin and osmotin are able to induce phosphorylation of AMP

kinase (Narasimhan et al. 2005). Thus, osmotin is able to propagate its effect through a

cascade of reactions. The exact mechanism of osmotin action, however, needs to be

explored.

2.7 Regulation of metabolic pathways by proteins

Biological functioning of a living cell involves coordinated activity of its metabolic and

regulatory networks. In a given environment, plants orchestrate its response by the

transcription regulatory network through up- or down-regulation of enzyme levels.

Proteins, an important component of this network, play key role in many biological

functions within a cell and many cellular processes and biochemical events are ultimately

achieved by a group of proteins interacting with one another. Thus, it is possible to deduce

functions of a protein through the functions of its interaction partners. Some of these

regulatory functions of proteins are described below:

2.7.1 Regulator of metabolic networks as transcription factors

Transcriptional regulation of the genes in metabolic pathways is a highly successful

strategy, which is virtually universal in all organisms. External and/or internal signals

appear to modulate the levels and activities of these regulators of biosynthetic genes,

leading to appropriate responses to stress or development. Transcription factors (TFs) are

sequence specific DNA-binding proteins that interact with the promoter regions of target

genes, and modulate the rate of initiation of mRNA synthesis by RNA polymerase II

(Riechmann and Ratcliffe 2000). These regions are responsible for tethering TFs to



specific cis-regulatory DNA elements, generally 6–8-bp long, but sometimes even longer.

The control of specific sets of genes is accomplished by the combinatorial interaction

among TFs, between TFs and non-DNA-binding proteins, and between TFs and cis-

regulatory elements. Of all the proteins encoding plant genes, 5–7% corresponds to TFs.

Moreover, some specific families of TFs have dramatically expanded in the plants,

compared to other kingdoms (Shiu et al. 2005). The cellular processes controlled by TFs

in other plants are rapidly being established in Arabidopsis as part of various functional

genomics initiatives. The work is also being extrapolated to other crops such as maize and

rice (Alonso-Blanco et al. 2005). Mutations in TF genes controlling developmental

processes with phenotypic alterations are easier to study than those affecting metabolic

pathways, as they can be visualized.

Transcription factor can act as activators or repressors of gene expression, mediating

either an increase or a decrease in the accumulation of messenger RNA (Latchmann

2003). Early work on pathway transcription factors in plants began with the discovery of

the maize flavonoid pathway regulators, COLORLESS1 (C1) and RED (R) (Latchmann

2003). Within a few years of their discovery, C1 and R were shown to induce flavonoid

gene expression and anthocyanin accumulation in transgenic plants (Goff et al. 1990).

These pioneering studies demonstrated the potential of transcription factors for the

manipulation of complex metabolic pathways in plants. Fuelled by the advent of

genomics, most is known with regards to transcriptional control of flavonoid biosynthesis

in the plant (Quattrocchio et al. 2006) and providing an example that similar results might

be expected for the regulation of other metabolic pathways. Flavonoids encompass a very

large family of phenolic compounds that include the anthocyanin, proanthocyanidin, and

phlobaphene pigments (Lepiniec et al. 2006), as well as the flavonol, flavone, and

isoflavone subfamilies that play specific functions in the plants in which they accumulate

(Subramanian et al. 2007; Taylor and Grotewold 2005). While the biosyntheses of most of

these compounds share some common metabolic enzymes and precursors, each branch of

the pathway can be under the control of a specific set of regulators. For example, in

Arabidopsis, PAP1/PAP2 (R2R3-MYB) control anthocyanin biosynthesis (Borevitz et al.

2000), AtMYB12/AtMYB11/AtMYB111 control flavonol accumulation (Mehrtens et al.



2005; Stracke et al. 2007) and TT2 (R2R3-MYB) and TT8 (bHLH) cooperate in the

control of seed coat proanthocyanidins (Nesi et al. 2000; Nesi et al. 2001). In maize, P1

(R2R3-MYB) controls phlobaphene accumulation (Grotewold et al. 1994), while C1/PL1

(R2R3-MYB) and R/B (bHLH) cooperate in the control of anthocyanins (Hernandez et al.

2004).

Plant transcription factors can also effectively reduce the flux by downregulation of

pathway activators by cis or trans activity and ultimately decrease the expression of the

genes they regulate. In fact, most of the transcriptional activators known to date were

identified genetically through mutants that are impaired in the production of specific

metabolites (Schmidt et al. 1990). Overexpression of AmMYB308 from Antirrhinum, for

example, represses the expression of lignin pathway genes and lignin biosynthesis in

tobacco plants (Tamagnone et al. 1998). Conversely, Arabidopsis plants that are

homozygous for a null allele of AtMYB4, a proposed orthologue of AmMYB308, express

higher levels of the gene that encodes cinnamate 4-hydroxylase, an early lignin pathway

enzyme, and accumulate high levels of sinapate esters (Jin et al. 2000). Strawberry

FaMYB1 down-regulates late flavonoid pathway gene expression when overexpressed in

transgenic tobacco plant (Aharoni et al. 2001).

2.7.2 Regulator of metabolic networks as cell signaling molecules

Cell signaling is one of the important processes required for the normal growth and

development of cell. It is the basic process that helps the cells to sustain through various

environmental cues and develop tolerance against stress conditions (Mahajan and Tuteja

2005; Redhead and Palme 1996; Tuteja and Mahajan 2007). The basic cell signaling

machinery involves a receptor molecule that perceives the signal. The signal or primary

stimulus could be light, hormone, odorant, antigen, neurotransmitter or the surface of

another cell, which transport into the cell via membrane receptor, through signal

transduction triad (receptor/transducer/effectors) (Trewavas and Malho 1997). The

intracellular signalling is carried out by second messenger that could be Ca2+ (for ion

channels), cAMP and cGMP (for adenylyl and guanlyl cyclases), inositol-1, 4,5-

triphosphate (IP3), diacyl glycerol (DAG) and arachidonic acid (for phospholipases)

(Mahajan and Tuteja 2005). The triad is responsible for converting the signal from first to



second messenger, which could be further regulated by protein kinases or phosphatases in

the cytoplasm. The target of the signal may be enzymes, intracellular receptors, special

transport vehicles and finally transcription factors (Hucho and Buchner 1997), which

ultimately control the gene expression. There are several signaling mechanisms present in

the cell to carry out normal functions and a number of metabolic messengers that acts

from within the cell to initiate a variety of signaling pathways (Figure 2).

Figure 2: The basic principle of a cell signaling pathway; Stimulus acts on cell-surface

receptors and relay information through intracellular signaling pathways that can have a

number of components. They usually begin with the activation of transducers that use

amplifiers to generate internal messengers that either act locally or can diffuse throughout

the cell. These messengers then engage sensors that are coupled to the effectors that are

responsible for activating cellular responses. The green and red arrows indicate that cell

signaling is a dynamic process consisting of ‘ SWITCH ON’ mechanisms (green arrows)

during which information flows down the pathway, opposed by the ‘SWITCH OFF’

mechanisms (red arrows) that stop the different steps of the signaling pathway. (Adapted

from Cell Signaling Biology, 2012).

Many proteins are involved in the process of cell signaling and signal transduction. In

animals, the widest varieties of signaling molecules are peptides, ranging in size from only



a few to more than a hundred amino acids. This group of signaling molecules includes

peptide hormones, neuropeptides, and a diverse array of polypeptide growth factors. Well-

known examples of peptide hormones include insulin, glucagon, and the hormones

produced by the pituitary gland (growth hormone, follicle-stimulating hormone, prolactin,

and others). Although all these molecules act as ligands that bind to receptors expressed

by their target cells, there is considerable variation in the structure and function of the

different types of molecules that serve as signal transmitters.

Protein ligands can bind to very diverse receptors. These may be peripheral membrane

proteins, transmembrane proteins or soluble globular proteins. Peripheral membrane

protein receptors adhere only temporarily to the biological membrane with which they are

associated. These molecules attach to integral membrane proteins, or penetrate the

peripheral regions of the lipid bilayer and usually forms regulatory subunits of many ion

channels and transmembrane receptors. The reversible attachment of proteins to biological

membranes has shown to regulate cell signaling and many other important cellular events,

through a variety of mechanisms (Cafiso 2005). Membrane binding promotes

rearrangement, dissociation, or conformational changes within structural domains of such

receptors, resulting in an activation of their biological activity (Johnson and Cornell 1999;

Thuduppathy et al. 2006). Additionally, the positioning of many receptor proteins are

localized to either the inner or outer surfaces or leaflets of their resident membrane

(Takida and Wedegaertner 2004). This facilitates the assembly of multi-protein

complexes by increasing the probability of any appropriate protein–protein interactions.

Transmembrane proteins broadly are classified as follows:

a) G protein-coupled receptors that are composed of seven transmembrane alpha helices,

the loops that are connecting the alpha helices form extracellular and intracellular

domains, the binding site for larger peptidic ligand that is usually located in the

extracellular domain, whereas the binding site for smaller non-peptidic ligands is often

located between the seven alpha helices and one extracellular loop (Congreve and

Marshall 2010).

b) Ligand-gated ion channels that have a heteropentameric structure. Each subunit of

ligand-gated ion channel consists of the extracellular ligand-binding domain and a



transmembrane domain, where the transmembrane domain in turn includes four

transmembrane alpha helixes (Cascio 2004). The ligand binding cavities are located at

the interface between the subunits.

c) Receptor tyrosine kinases that are homodimer functional receptors. Each monomer

possesses a single transmembrane alpha helix and an extracellular domain containing

the ligand binding cavity and an intracellular domain with catalytic activity.

d) Soluble globular proteins that are nuclear receptors composed of a C-terminal DNA-

binding domain (DBD) and a N-terminal ligand-binding domain (LBD). The LBD is

composed of twelve alpha helices and an antiparallel beta sheet. The ligand binding

cavity is buried within the interior of the LBD (Kumar and Thompson 1999).

Protein-protein interactions are fundamental to molecular understanding of all biological

processes including signalling, which describes the cells recognition of extracellular

stimuli and generation of a response. Extracellular signaling molecules (usually hormones,

neurotransmitters, cytokines, growth factors or cell recognition molecules) attach to the

receptor, triggering changes in the function of the cell. Protein-protein interactions recruit

cytoplasmic polypeptides to activated receptors, direct their assembly into larger

complexes, target them to defined subcellular locations, and determine the specificity with

which enzymes interact with their substrates. In this way the receptors play a unique and

important role in cellular communications and signal transduction on the intracellular side

of the membrane. Intracellular signaling networks control many different aspects of

cellular behavior, and are assembled through the interactions of proteins with one another,

and with lipids, nucleic acids and small molecules.

2.8 Cellular and molecular responses of plants to Stress

The growth and productivity of crop plants depend largely on their vulnerability to

environmental stresses. High salinity, water deficit, and temperature stress are the major

constraints that limit agricultural production by adversely affecting the plant growth and

yield (Blumwald E 2006; Boyer 1982; Cherian 2006). Plants respond to these conditions

with an array of biochemical and physiological adaptations, which involve the function of

many stress-related genes. The cellular and molecular responses of plants to

environmental stress have been studied intensively (Hasegawa et al. 2000; Thomashow



1999). A number of abiotic stress responsive genes have been identified in plants,

including rice and Arabidopsis by using molecular techniques such as microarray analysis

(Fowler and Thomashow 2002; Nakashima et al. 2009; Rabbani et al. 2003; Shinozaki and

Yamaguchi-Shinozaki 2006). Stress induced genes not only function to protect cells from

abiotic stress through the production of important enzymes and metabolic proteins

(functional proteins), but they also regulate signal transduction and gene expression in the

stress response (regulatory proteins).

Metabolic acclimation via the accumulation of compatible solutes is often regarded as a

basic strategy for the protection and survival of plants under abiotic stress (Bohnert and

Jensen 1996; Cuin and Shabala 2007; Hanson and Hitz 1982; Sakamoto and Murata

2000). This class of small molecules includes certain amino acids (notably proline),

quaternary ammonium compounds (glycinebetaine, prolinebetaine, β-alaninebetaine, and

choline-O-sulfate), and the tertiary sulfonium compound, 3-dimethylsulfoniopropionate

(DMSP). The quaternary ammonium compounds and DMSP are derived from amino acid

precursors. These compounds share the property of being uncharged at neutral pH, and

have high solubility in water (Ballantyne JS 1994). Moreover, at high concentrations they

have little or no perturbing effect on macromolecule-solvent interactions (Somero 1986;

Timasheff 1993). The class of compatible solutes that accumulate in plant cells in

response to high salinity includes glycine betaine, proline, and polyols (Bohnert et al.

1995; Di Martino et al. 2003; Rhodes and Hanson 1993). In addition to the conventional

role of these compatible solutes in cell osmotic adjustment, they are also suggested to act

as low-molecular-weight chaperones, stabilizing the photosystem II complex, protecting

the structure of enzymes and proteins, maintaining membrane integrity and scavenging

ROS (Bohnert et al. 1995; Hare et al. 1998; Mccue and Hanson 1990; Noiraud et al. 2001;

Robinson and Jones 1986; Santoro et al. 1992; Smirnoff and Cumbes 1989). Recently, it

was also shown that some of these compatible solutes are very efficient in reducing the

extent of K+ loss in response to both salinity (Cuin and Shabala 2005; 2007) and oxidative

stress (Cuin and Shabala 2007) in barley and Arabidopsis roots. Also, the introduction of

genes involved in the synthesis of proline, betaines, and polyols into plants contributes to

abiotic stress tolerance (Chen and Murata 2002; Rathinasabapathi 2000) and numerous



genetic engineering attempts have been made to manipulate the biosynthesis pathways of

compatible solutes in order to enhance salt tolerance (Chen and Murata 2002;

Rathinasabapathi 2000; Sakamoto and Murata 2000).

Although many genes that encode signal transduction proteins that are potentially

important to osmotic stress adaptation in plants have been identified, but still little is

known about how these gene products function in a signal pathway. The knowledge about

stress signal transduction is also vital for continued development of strategies to improve

stress tolerance in crops. The stress responses of plants are regulated by multiple

signalling pathways (Glazebrook 2001; Knight and Knight 2001), and there is significant

overlap between the patterns of gene expression that are induced in plants in response to

different stresses (Chen et al. 2002; Durrant et al. 2000; Schenk et al. 2000; Seki et al.

2001). Stress gene induction occurs primarily at the level of transcription factors which

then regulate the temporal and spatial expression patterns of specific genes (Rushton and

Somssich 1998). Plants devote a large portion of their genome capacity to transcription

factors, with the Arabidopsis genome coding in excess of 1500 transcription factors

(Riechmann and Ratcliffe 2000). These transcription factors often belong to large gene

families, which in some cases are unique to plants. There has been major progress in the

past years linking specific members of these families with plant stress responses. Members

of the MYB plant transcription factors have been linked to plant stress responses, such as

responses to ultra-violet (UV) light, wounding, anaerobic stress and pathogens. MYB

proteins have been the subject of several recent reviews (Hemm et al. 2001; Jin and

Martin 1999; Rushton and Somssich 1998; Stracke et al. 2001). Other transcription factors

that have been implicated in the expression of plant stress genes include salicylic acid

(SA)-inducible DOF (DNA binding with one finger) proteins that interact with and

stimulate the DNA-binding of stress-responsive bZIP proteins (Kang and Singh 2000),

and novel single-stranded-DNA-binding proteins that modulate the expression of the

potato pathogenesis-related-10a (PR-10a) gene (Boyle and Brisson 2001; Desveaux et al.

2000). Thus, these proteins regulate the responses of plant to abiotic stresses and are

important for stress tolerance. However, the understanding of exact mechanisms by which



plants perceive environmental signals and transmit the signals to cellular machinery to

activate adaptive responses is still an area of continued research.

2.9 Plant signaling proteins in response to stress

Recent development in the state of art had led to the identification of determinants of

stress tolerance in plants. These include effectors, viz. mitogen-activated protein

kinases (MAPKs), ion homeostasis proteins, osmolytes, toxic radical scavenging and

water transport proteins and signaling molecules, viz. growth hormones and

transcription factors regulating the signal transduction pathway components.

Transcription factors are identified based on interaction with promoters of stress

responsive genes. They participate in the activation of these genes, and presumably

lead to plant adaptation to the stressed environment (Abe et al. 1997; Shinozaki and

Yamaguchi-Shinozaki 2006). The promoters controlled by these transcription factors

however, are responsive to several environmental signals. It is not clear therefore,

whether any single transcription factor is specific for a particular environment. Basic

leucinezipper motif (bZIP), MYB, MYC, Ca2C-dependent protein kinases (CDPKs),

and dehydration response element (DRE) binding proteins (Liu and Zhu 1998;

Stockinger et al. 2001) are some well studied factors in plants. The first genetic

evidence of importance of bZIP proteins in stress tolerance was provided by over

expressed ABRE binding factor/ABA responsive element binding protein of bZIP

family in transgenic Arabidopsis thaliana (Kang et al. 2002). DRE motifs are involved

in regulation of ABA independent gene expression under drought, salinity and cold

stress. The ERF gene imparts tolerance to multiple stresses such as drought, salinity

and cold (Kasuga et al. 1999; Park et al. 2001).

Many genes encoding signal transduction proteins that are potentially important to

osmotic stress adaptation in plants have been identified. However, little is known

about the function of these gene products in a signal pathway in response to various

stresses. Cell differentiation and spatial location of these gene products within plant

tissues and the divergence between species affect the adaptation by the plants to the

environmental stresses and posses problems in cell signaling studies. So far, much of

genes encoding plant signaling proteins controlling responses to osmotic stress have



been found by mining gene sequence databases (Hasegawa et al. 2000) and still need

experimental validation.

2.10 Proline and stress tolerance

In addition to its role in protein synthesis, there is a growing consensus that proline is of

special importance in imparting stress tolerance to the plants under stress. It is a

proteinogenic amino acid with an exceptional conformational rigidity, and is essential for

primary metabolism. Since the first report on proline accumulation in wilting perennial rye

grass (Lolium perenne) (Kemble and Macpherson 1954), numerous studies have shown

that the proline content in higher plants increases under different environmental stresses.

Proline accumulation has been well documented during biotic and abiotic stresses such as

drought (Choudhary et al. 2005), salinity (Yoshiba et al. 1995), high light intensity and

UV irradiation (Saradhi et al. 1995), heavy-metals (Schat et al. 1997), oxidative stress

(Yang et al. 2009) and pathogenic distress (Fabro et al. 2004; Haudecoeur et al. 2009). An

osmoprotective function of proline was discovered first in bacteria, where a causal

relationship between proline accumulation and salt tolerance has been demonstrated

(Csonka et al. 1988). The large accumulation of proline that occurs during drought may be

explained in part by its basic chemical properties; it is the most water soluble among the

amino acids and exists much of the time in a zwitterionic state having both weak negative

and positive charges at the carboxylic acid and nitrogen groups, respectively. Proline

shares this property with other compounds collectively referred to as “compatible solutes”

that are accumulated in a wide range of organisms to adjust cellular osmolarity (Yancey

2005). Apart from this, proline may serve for C and N reserve for growth after stress

relief, detoxification of excess ammonia, stabilization of proteins and/or membranes and a

scavenger of free radicals (Fabro et al. 2004; Haudecoeur et al. 2009; Lehmann et al.

2010). Several comprehensive studies using transgenic plants or mutants demonstrate that

proline metabolism has a complex effect on the development and stress responses, and

that proline accumulation is important for the tolerance of certain adverse environmental

conditions (Hong et al. 2000; Mattioli et al. 2008; Székely et al. 2008).



2.11 Proline Metabolism

Plant under stress conditions accumulate low molecular weight compounds (compatible

solutes), such as proline, betaine and sugar alcohols (Munns 2005). Among these

osmoprotectants, proline has been studied extensively in recent years. Transgenic

approaches revealed that proline plays a role as a regulator of osmotic adjustment (Kishor

et al. 1995; Hmida-Sayari et al. 2005). The in vitro analyses provided the potential

function of proline as a destabilizer of DNA helices (Rajendrakumar et al. 1997), a radical

scavenger (Smirnoff and Cumbes 1989), a suppressor of ribulose-1,5-bisphosphate

carboxylase/oxygenase activity (Sivakumar et al. 1998) or a protector of complex II of the

mitochondrial electron transport system (Hamilton and Heckathorn 2001).

2.11.1 Proline biosynthesis

Proline accumulation under stress is found in both prokaryotic and eukaryotic systems.

Accumulation of proline in plant tissues has been suggested to result from; i) a decrease in

proline degradation, ii) an increase in proline biosynthesis, iii) a decrease in protein

synthesis or proline utilization and iv) hydrolysis of proteins (Charest and Ton Phan

1990). In prokaryotes such as Escherichia coli, Pseudomonas aeruginosa and Salmonella

typhimurium, proline is synthesized by phosphorylation of glutamate, which gets

converted to g-glutamyl phosphate and then to glutamic-g-semialdehyde (GSA) by the

enzymes g-glutamyl kinase and glutamic-g- semialdehyde dehydrogenase respectively.

GSA gets converted to pyrroline 5-carboxylate (P5C) by spontaneous cyclization (Figure

3).

Synthesis of proline occurs in the cytosol and in the plastids (like chloroplasts in green

tissues) in plants. It involves the sequential action of pyrroline-5-carboxylate synthetase

(P5CS) and pyrroline-5-carboxylate reductase (P5CR), which convert glutamate to

pyrroline-5-carboxylate (P5C) and P5C to proline, respectively (Delauney and Verma

1990; Verbruggen et al. 1993) (Figure 3). P5CS was first isolated from Mothbean (Vigna

aconitifolia) by functional complementation technique using E. coli mutants (Delauney et

al. 1993). Initially, single P5CS gene was cloned and sequenced from Arabidopsis

(Savoure et al. 1995). AtP5CS encoded a protein of 717 amino acids showing high identity

with the P5CS of Vigna. Later, transcriptional regulation and study of knockout mutants



showed two Arabidopsis P5CS genes, having clearly distinct functions. P5CS1 is

expressed most highly in shoot tissue but not in dividing cells and transcription of P5CS1

is strongly induced under high salinity, dehydration and cold (Hong et al. 2000; Hu et al.

1992; Yoshiba et al. 1995; Yoshiba et al. 1997). Thus, the transcriptional up-regulation of

At-P5CS1 is well correlated with proline accumulation in short-term dehydration

experiments (Yoshiba et al. 1995). It is however, still unclear whether this correlation

exist under longer-term stress or other types of stresses also (Kaplan et al. 2007).

Consistent with the upregulation of AtP5CS1, p5cs1 knockout mutants have greatly

reduced proline levels during salt or low water potential stress (Sharma and Verslues

2010; Székely et al. 2008). p5cs1 mutants were also reported to have reduced growth and

altered reactive oxygen levels, suggesting that they are hypersensitive to salt, osmotic

stress and low water potential (Nanjo et al. 1999; Székely et al. 2008). On the other hand,

AtP5CS2 is expressed in dividing cell cultures and its induction has little or no

transcriptional up-regulation under stress (Strizhov et al. 1997; Székely et al. 2008; Zhang

et al. 1997).

Promoter of an Arabidopsis P5CS gene was isolated and analysed using β-glucuronidase

(GUS) reporter gene in transgenic plants (Zhang et al. 1997). While GUS expression was

rapid and was only within 3 h period under NaCl stress, it was noticed for 24 h under

dehydration stress. Abscisic acid (ABA) is known to increase during dehydration stress,

but it also failed to induce the P5CS gene expression. Further, glutamine, proline and cold

also showed no induction or repression of P5CS gene expression (Zhang et al. 1997). It is

reported that the P5CS is feedback regulated by proline (Zhang et al. 1995) but its

inability to induce P5CS gene expression is a big bottleneck in understanding the

regulation of proline metabolism. Several efforts have been made in different species to

engineer a deregulated P5CS, with the aim of obtaining plants that are able to grow in the

desert or salty land areas (Hmida-Sayari et al. 2005; Verbruggen and Hermans 2008). The

identification of the proline binding site in P5CS and the mechanism responsible for its

inhibition however, is still an area of active research.

The second enzyme of proline biosynthesis, P5CR (pyrroline-5-carboxylate reductase),

reduces P5C to proline(Figure 3). The gene encoding this enzyme was the first gene to be



cloned in the proline pathway by functional complementation of a proC mutation in E.

coli with an expression library of soybean root nodule cDNA, and was found to be

osmoregulated (Delauney and Verma 1990). Genomic analysis showed that there are 2–3

copies of the P5CR gene in the genome of soybean. P5CR gene was later isolated from

Pisum sativum (Williamson and Slocum 1992), A. thaliana (Verbruggen et al. 1993) and

kiwifruit (Walton et al. 1998). The two isoforms of P5CR were biochemically identified in

pea and spinach, although it is currently unclear whether the two isoforms originate from

one or two genes (Murahama et al. 2001; Williamson and Slocum 1992). The P5CR is

encoded by only one gene in the Arabidopsis genome, but the enzyme seems to be active

in chloroplasts and cytosol (Rayapati et al. 1989; Szoke et al. 1992; Verbruggen et al.

1993). It is generally considered as a rate-limiting enzyme in proline biosynthesis based

on the studies showing lack of its transcriptional regulation by stress (Delauney and

Verma 1993; Hua et al. 1997; Sharma and Verslues 2010; Szoke et al. 1992; Verbruggen

et al. 1993). The accumulation and developmental regulation of transcript encoding P5C

reductase in Arabidopsis, however, suggest that it plays an important role in proline

synthesis in rapidly dividing cells and/or in cells undergoing changes in osmotic potential

(Hare and Cress 1996). Expression of AtP5CR is high in cells or tissues that experience

developmentally programmed osmotic adjustment, such as guard cells, hydathodes, pollen

grains and developing seeds. Its expression is also high in apical meristems, root

meristems and lateral root primordia (Hua et al. 1997). In plants, P5CR activity has been

detected in the cytosol and in plastids of several plant species. The location of this enzyme

in plastids suggests that it may assist in counteracting photoinhibitory damage of RuBisco

enzyme under adverse conditions.

While P5CS utilizes both ATP and NADPH, P5CR-1 and P5CR-2 isoenzymes of

Spinacea oleracea (spinach) leaf showed strong preference for NADPH rather than

NADH as the electron donor and also showed inhibition by ATP and Mg2+ (Murahama et

al. 2001). Hua et al. (1997) concluded that P5CR activity might be involved in the

metabolic regulation of cellular redox potential by affecting the level of reduction of

NADPH. Also the preference for NADPH is likely to be significant in linking proline

metabolism to the pentose phosphate pathway



In plants, a second pathway for proline synthesis from ornithine had also been postulated,

(Adams and Frank 1980; Hu et al. 1992). Ornithine is converted to GSA by the enzyme

ornithine aminotransferase (OAT), which is then converted to proline. The glutamate

pathway is however, predominant in plants under the osmotic stress and nitrogen

starvation. It was supported by the observations that ornithine aminotransferase (OAT)

was decreased, while pyrroline-5-carboxylate synthetase (P5CS) transcripts levels were

increased in salt-stressed and nitrogen starved moth bean plants (Delauney et al. 1993).

Recently, it has been shown that OAT feeds P5C exclusively into the catabolic branch of

proline metabolism, which yields glutamate as an end product and that the conversion of

ornithine to glutamate is an essential route for recovery of nitrogen stored or transported

as arginine (Funck et al. 2008). Further, proline biosynthesis occurs predominantly or

exclusively via the glutamate pathway in Arabidopsis and does not depend on glutamate

produced by arginine and ornithine catabolism (Funck et al. 2008).

2.11.2 Proline catabolism

For degradation, proline is imported into the mitochondria, where it is converted back to

glutamate by the tightly coupled activities of proline degrading enzymes. Proline is

oxidized in a two-step process involving sequential action of proline dehydrogenase

(PDH), which converts proline to P5C, and D1 -pyrroline-5-carboxylate dehydrogenase

(P5CDH), which converts P5C into glutamate (Deuschle et al. 2001; Elthon and Stewart

1981; Kiyosue et al. 1996; Verbruggen et al. 1996) (Figure 3). Proline catabolism is

repressed under osmotic stress (Delauney and Verma 1993; Kiyosue et al. 1996; Peng et

al. 1996), but once the stress is withdrawn, proline is oxidized to glutamate.

Proline dehydrogenase (PDH) is a flavoprotein that catalyzes the first and rate-limiting

step of two reactions converting proline into glutamate at the mitochondria. This enzyme

is located at the matrix side of the inner mitochondrial membrane, where it oxidizes

proline into Δ1-pyrroline-5-carboxylate (P5C) and transfers electrons to the primary

mitochondrial electron transport chain (Boggess et al. 1978; Elthon and Stewart 1981;

Huang and Cavalieri 1979). PDH is sensitive to environmental stresses. Dehydration,

extreme temperatures, and other abiotic injuries repress PDH and stimulate proline

synthesis, thus leading to net proline accumulation. Once the stress is relieved, PDH



becomes activated to consume the stored proline (Kiyosue et al. 1996; Peng et al. 1996;

Szabados and Savouré 2010; Verbruggen and Hermans 2008; Verbruggen et al. 1996).

In Arabidopsis, two PDH (PDH1 and PDH2) have been characterised at the molecular

level and are localized in mitochondria (Deuschle et al. 2001; Funck et al. 2010).

Expression analysis of PDH1 indicated ubiquitous expression in all organs, with the

highest expression levels detected in pollen grains and in the stigma of the carpel. No

PDH2 expression was detected stigma and in pollen while strong expression was detected

in roots carpel (Nakashima et al. 1998).

Pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyzes the second step of proline

oxidation in mitochondria and converts the intermediate P5C to glutamate. An alternative

source of substrate for the P5CDH enzyme can be derived from the conversion of arginine

to ornithine and subsequent catabolism to P5C by ornithine aminotransferase (Delauney

and Verma 1993). P5CDH expression is also repressed by osmotic stress and up-regulated

by proline. However, the changes are more pronounced for PDH (Verbruggen and

Hermans 2008). P5CDH is upregulated in pollen while it was weakly expressed in roots

(Deuschle et al. 2004a). Potentially, arginine catabolism, which is the second task of

P5CDH, is less prominent in roots.

Arabidopsis P5CDH was identified by functional complementation of a yeast P5CDH

(ScPUT2) deletion strain unable to utilize proline as a sole nitrogen source (Deuschle et al.

2001). P5CDH is encoded by a single copy gene in Arabidopsis and the enzyme is located

in mitochondria (Deuschle et al. 2001). P5CDH purified from potato appeared to be a

tetramer with 60kD subunits. It has Km of 0.11 and 0.46 mM for NAD+ and P5C,

respectively. It can however, also use either NAD+ or NADP+ as an electron acceptor

(Forlani et al. 1997). Data on the physiological role of P5CDH have come mainly from the

study of P5CDH mutants in Arabidopsis. P5CDH mutants had only slightly higher than

wild type or unchanged proline content under salt stress (Borsani et al. 2005; Deuschle et

al. 2004b). These mutants however, did maintain higher proline content after stress release

or removal of exogenous proline, which is consistent with a requirement for P5CDH

during rapid proline catabolism. When grown in presence of proline, arginine or ornithine,

which can all be metabolized to produce P5C, P5CDH mutants had tissue damage that is



likely related to ROS accumulation (Deuschle et al. 2004b). It is thought that this damage

was primarily caused by increased P5C, which may elicit ROS production in the

mitochondria.

Figure 3 : Pro biosynthesis pathway in bacteria and plants indicating the genes and

enzymes participating in the pathway.Abbreviations: AcGSA, N-actyl glutamyl

semialdehyde; GSA, γ-glutamyl-semialdehyde (or glutamate gamma semialdehyde);

GP, glutamyl phosphate; P5C, δ-1-pyrroline-5-carboxylate. Enzymes: γGK, γ-

glutamyl kinase; GSD, glutamic-γ-semialdehyde dehydrogenase (also known as GPR,

GP reductase); P5CS, P5C synthetase; P5CR, P5C reductase; ProDH, proline

dehydrogenase; P5CDH, P5C dehydrogenase. [Adapted from Plant Science, 2011]

2.11.3 Proline transport

While proline synthesis and catabolism pathways are well characterized in plants, the

uptake and its transport is not at all understood. It was reported that in alfalfa, proline

transport processes play an important role in adaptation to osmotic stress (Girousse et al.

1996). In plants, the transport of amino acids is regulated not only by endogenous but also

by environmental signals. The water deficit like drought and salt stress, affects long-

distance transport and results in massive changes in partitioning of carbon and nitrogen.



Developing tissues, reproductive organs and meristems, usually import amino acids to

support growth and development. Genes encoding transporters for the compatible solutes

proline and glycine betaine were isolated from a number of plant species, including

Arabidopsis (Rentsch et al. 1996), tomato (Schwacke et al. 1999), rice (Igarashi et al.

2000), barley (Ueda et al. 2001) and mangrove (Waditee et al. 2002). Initially these

transporters were described as specific transporters (Rentsch et al. 1996). However, as the

studies followed some were shown to be general transporters for the compatible solutes

proline and glycine betaine (Schwacke et al., 1999;Waditee et al., 2002). Still a limited

information on the significance of transport of compatible solutes is available. The

Arabidopsis genome encodes three proline transporters (AtProT1, AtProT2 and AtProT3).

Though ProT1 was expressed in all organs of the plants, highest levels were found in the

floral stalk phloem that enters the carpels and were down-regulated after fertilization. This

is consistent with the evidence that proline synthesis and degradation play an important

role in flowering and seed set. In contrast, mRNA levels of ProT2 were observed

throughout the plant, but their expression was strongly induced by water or salt stress.

This suggests that the gene may be distributing nitrogen during water stress unlike the

members of amino acid permease gene family, the expressions of which are generally

suppressed under similar conditions.

2.12 Signal transduction events of proline biosynthetic pathway genes

Although the importance of proline accumulation conferring hyperosmotic stress tolerance

has been demonstrated well, the regulatory molecules as well as the molecular signals

involved in the expression of proline biosynthetic genes are not understood. Less and

Galili (2008) analyzed publically available microarray data and found that, for many

amino acids, abiotic stress altered transcription of the catabolism genes but had little effect

on expression of genes encoding the biosynthetic enzymes. Notable exceptions to this

were the connected pathways of proline and arginine metabolism, where abiotic stress

caused extensive transcriptional regulation of the biosynthetic enzymes. In addition to

specific regulation of proline biosynthesis and catabolism pathways, the compartmentation

of its metabolism between cytoplasm, mitochondria and chloroplast is also of critical

importance. Proline transport (both intra- and intercellular), tissue specific differences in



proline metabolism, and the connections of proline metabolism to other metabolic

pathways are all important, both to understanding proline metabolism in a basic sense and

for plant improvement in an applied sense. Therefore, the ccomprehensive studies are

required at physiological, molecular and genetic levels to explore the signal transduction

events of proline biosynthesis and degradation.

The transcriptional upregulation of proline biosynthesis from glutamate and

downregulation of proline catabolism during stress are thought to control proline levels,

although exceptions to this pattern have been observed (Stines et al. 1999). Much data are

consistent with this straightforward model of proline metabolism being mainly regulated

by transcription of genes encoding the key enzymes. This is, however, unlikely to be the

whole story. The post-translational regulation of these enzymes has been little explored.

Based on the current knowledge of proline metabolism, regulation of P5CS and PDH

transcripts represents the rate-limiting step in proline biosynthesis and degradation,

respectively. The signals during salt stress appear to be mediated by ABA that can bring

about the expression of stress-related genes and subsequently the synthesis of organic

osmolytes. Proline accumulation appears to be mediated by both ABA-dependant and

ABA-independent signaling pathways (Ober and Sharp 1994; Savoure et al. 1997; Sharma

and Verslues 2010; Verslues and Bray 2006). In some plants like Arabidopsis causal link

between ABA and proline accumulation was suggested and it was shown that exogenous

application of ABA increases the level of AtP5CS1 and AtP5CS2 transcripts. Analysis of

the promoter elements in the proline biosynthetic pathway genes such as AtP5CS1,

AtP5CS2 and AtP5CR indicated that a cis-acting ABA responsive element sequence is

found in AtP5CS2 upstream region. Recently, quantitative comparison of P5CS1

expression to known ABA-induced genes found largely ABA-independent regulation of

P5CS1, implying that the underlying mechanisms controlling P5CS1 expression are

distinct from that of many commonly studied stress marker genes (Sharma and Verslues

2010). P5CS1 expression has also been found to be stimulated by light (Hayashi et al.

2000) and nitric oxide (Zhao et al. 2009), while its expression was decreased by

brassinosteroid application (Abraham et al. 2003). Other than effects of plant hormones,

the signal transduction controlling P5CS1 is not well known. In contrast to the P5CS1,



gene expression of P5CS2 has little or no transcriptional up-regulation under stress and all

indications are that it has only a minor role in proline accumulation induced by abiotic

stress (Abraham et al. 2003; Strizhov et al. 1997; Székely et al. 2008). P5CS2 can be

induced by pathogen response (Fabro et al. 2004) and may be more actively involved in

plant-pathogen interaction. Further post translational regulation is also unknown for either

P5CS1 or P5CS2. Proteomics studies showed phosphorylation of P5CS1 (Benschop et al.

2007; Reiland et al. 2009), but its significance in regulating P5CS1 activity is not known.

Conversion of P5C to proline is not a rate-limiting step in proline biosynthesis, yet the

control of P5CR activity implies a complex regulation of transcription, which was shown

to be under developmental and osmotic regulation (Verbruggen et al. 1993). Promoter

analysis of Arabidopsis P5CR identified a 69-bp promoter region that is responsible for

tissue-specific expression (Hua et al. 1999). However, trans-acting factors that can bind to

this promoter region have not yet been identified.

Downregulation of PDH expression during stress is widely accepted as one control point

that can promote proline accumulation under stress (Sharma and Verslues 2010;

Verbruggen et al. 1996; Verslues et al. 2007). PDH transcription is activated by

rehydration and proline, but repressed by dehydration; thus, preventing proline

degradation during abiotic stress (Kiyosue et al. 1996). Promoter analysis of PDH1

identified the proline and hypo-osmolarity-responsive element (PRE) motif ACTCAT,

which is necessary for the activation of the PDH gene (Satoh et al. 2002). Basic leucine

zipper protein (bZIP) transcription factors (AtbZIP-2, -11, -44, -53) have been identified

as candidates for binding to this motif (Satoh et al. 2004).

P5CDH is transcriptionally regulated by both environmental signals and developmental

factors. Developmentally, P5CDH is expressed at a basal level in all tissues except

flowers, where it has higher expression (Deuschle et al. 2001). The P5CDH gene is

expressed at a low basal level in all Arabidopsis tissues, and can be upregulated by proline

(Deuschle et al. 2001). Stress regulation of P5CDH is more varied. P5CDH was

upregulated by low water potential (Sharma and Verslues 2010) and was also found to be

upregulated in response to exogenous proline (Deuschle et al. 2004a). A short sequence

similar to the PRE motif has been identified on the promoters of P5CDH genes in



Arabidopsis and cereals (Ayliffe et al. 2005). However, under salt stress, P5CDH

expression was downregulated by siRNAs generated from the overlapping transcripts of

P5CDH and SRO5, a mitochondrial protein potentially important in ROS regulation

(Borsani et al. 2005).

The light and osmotic stresses upregulates proline biosynthesis whereas proline

catabolism is upregulated in the dark and during stress relief (Abraham et al. 2003;

Hayashi et al. 2000; Xue et al. 2009). Phospholipase D along with calcium was also

suggested to play an important role in the regulation of proline biosynthesis. Calcium

signals transmitted by a specific CaM4 calmodulin, interacts with the MYB2 transcription

factor and upregulates P5CS1 transcription (Yoo et al. 2005). Under non-stressed

conditions, phospholipase D (PLD) functions as a negative regulator of proline

accumulation (Thiery et al. 2004). Another phospholipase, phospholipase C (PLC), trigger

P5CS transcription and proline accumulation during salt stress (Knight et al. 1997; Parre

et al. 2007). Despite establishing the role of proline, the exact regulatory and biochemical

mechanisms responsible for the proline accumulation in a specific plant tissue or organ is

not known. The comprehensive studies at physiological, molecular and genetic levels are

required to explore the signal transduction events of proline synthesis and degradation.

2.13 Osmotin and proline

The over expression of osmotin confers tolerance against various biotic and abiotic

stresses. As discussed in the earlier sections, the plants over expressing osmotin gene were

found to accumulate higher levels of proline even when they were not exposed to stress.

Moreover, being a 26 KDa protein, osmotin is within the range of low molecular weight

transcription factors such as members of the GATA factor family of zinc finger

transcription factors, WRKY transcription factors family etc. It has also been shown to act

as a ligand for adiponectin receptor as well as PHO36, the yeast homologue of adiponectin

receptor, and activate the downstream signaling.

Keeping the above facts in view, the role of osmotin in modulating proline biosynthesis

and accumulation as cell signaling molecule and /or transcription factor involved in

regulating the expression of genes associated with proline metabolism has been evaluated

in the present study.

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Chapter 2 Review of Literature - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/39814/4/08... · 2018. 7. 2. · Chapter 2 Review of Literature Department of Biotechnology,