Abiotic Stress Related Genes and their Role in Conferring...

Indian Journal of Biotechnology Vol I, July 2002, pp 225-244

Abiotic Stress Related Genes and their Role in Conferring Resistance in Plants

M Z Abdin' *, R U Rehman', M Israr' , P S Srivastava' and K C Bansal2

'Centre for Biotechnology, Faculty of Science, Hamdard University, New Delhi 110 062, India 2National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012, India

Abiotic environmental stresses, which limit the plant distribution and productivity, include low and high temperature, salinity and water deficit. Over the last century human activities have increased the level of environmental stress in the form of pollutants such as ozone and heavy metals, levels of UV light reaching the biosphere and salinity in irrigated areas. Plants are sessile and have, therefore, developed mechanisms to survive under extreme environments sometimes in vegetative stages of their life cycle. The recent surge of information on regulation of gene expression under stress conditions as well as the biochemical function of individual proteins in conferring tolerance to stress will help in isolating the genes of interest to produce desired transgenics. The understanding of molecular basis of these survival mechanisms discussed in this review may ultimately help enhance plant productivity in current marginal areas. The review deals with effect of drought and salt stress on plants and the regulatory mechanisms.

Keywords: Arabidopsis, abiotic stress, stress inducible genes, stress resistance, transgenics

Introduction Abiotic stresses such as drought, temperature, and

salinity are location specific, exhibiting internal variation in occurrence, intensity and duration, and generally cause reduced crop productivity. There is a serious concern for food security in developing countries including India for the following reasons: (i) increasing food demand for the rapidly burgeoning population; (ii) stagnating or declining productivity in high productivity regions, often described as 'Green Revolution' fatigue; and (iii) increasing vulnerability of agriculture to potential climate change. Of all the parameters, abiotic environmental stresses contribute most significantly to the reduction in potential yield (Flowers & Yeo, 1995). There is, therefore, a conscious effort to improve production by extending cultivation of tolerant crops to areas commonly exposed to abiotic stresses such as drought, temperature, salinity, alkalinity, water logging and nutrition (Renu & Suresh, 1998). Despite the fact that a large number of genes have to contribute to the overall phenotypes, investigations on plant responses to environmental stresses have revealed relatively small number of major quantitative trait loci (QTL) (Yano & Saski, 1997). The prospects of changing the phenotype through genetic manipulations or conventional hreeding become much greater if one or

* Author for correspondence : Tel: + 91-6084685, 6089688/231 ,236; Fax: + 91-11-6088874 E-mail: Illzabdin @yailoo.colll.root@hamduni .rcn .nic.in

a few defined regions of chromosomes are of crucial importance. The identification of QTLs has, therefore, practical importance in attempts to enhance stress tolerance (Koyama et ai, 200 1).

Drought Stress Drought and salinity stresses constitute a

permanent and increasing agronomical problem in arid midwest/western growing areas. Plants respond and adapt to a variety of environmental stresses in order to survive; drought is one of the most adverse factors of plant growth and crop production. Drought stress induces variolls biochemical and physiological responses. A variety of genes have been described that respond to drought at the transcriptional level and their gene products are thought to function during stress tolerance and response (Table 1). Plant breeding has not produced varieties suitable for use in stressful environments. The complexity of salinity stress responses and assigned failure of breeding strategies to lack of mechanistic understanding has been reviewed (Flowers & Yeo, 1995). Indeed, breeding amounts to merging two multi genic, quantitative traits: tolerance and productivity. The limited knowledge available about the genetic basis of stress responses to salinity has been derived mainly from the analysis of a s ingle or few genes, expression of corresponding transcripts, and the proteins. The functional mechanism served by different gene products, however, may remain the same. For

226 INDIAN J BIOTECHNOL. JULY 2002

Table I---Genes involved in abiotic stress

Gene Gene ac tion Species

Genes encoding enzymes that sy nthesize osmoprotectants

Adc

AtHAL3a

BADH-I BetA

CodA

CodA

CodA GS2

IMTI

MtD

MtD

OtsA

OtsB

P5cs

P5cs

Pdc l

Arginine decarboxybse

Phosphoprotei n phosphatase

Betaine aldehyde dehydogenase Choline dehydrogenase (glyc inebetaine) Choline oxidase (glycine betaine sy nthesis)

Chloroplast glutamine synthetase

Myo- inositol o-methyltransferase (D-ononito l sy nthesis) Manni tol-phosphate dehydrogenase (mannito l synthesis)

Trehalose-6-phosphate synthase (trehalose synthesis)

Trehalose-6-phosphate sy nthase (trehalose synthesis)

Pyrroline carboxyl ate syntetase (proline synthesis)

Pyruvate decarboxylase

Late embryogenesis abundant (LEA)-related genes

CORI5a HVAI

HVAI

Regulatory genes

ABI3 ACC

AtGluR2

ATHB6

CBF I

DREB

Cold induced gene Group 3 LEA protein gene

Transcription factor ACC deaminase

Transcription factor

Transcription fac tor



Rice

Arabidopsis

Tomato Tobacco

Arabidopsis

Rice

Arabidopsis Rice

Tobacco

Tobacco

Arabidopsis

Tobacco

Tobacco

Tobacco

Rice

Rice

Arabidopsis Rice

Wheat

Arabidopsis Tobacco

Arabidopsis

Arabidopsis

Arabidopsis

Arabidopsis

Phenotypic expression

Reduced chlorophyll loss under drought stress

Reference

Capell e! al ( 1998)

Regul ate salin ity and osmotic tolerance and plant growth Maintenance of osmotic potential Increased tolerance to salinity stress

Espinosa-Ruiz et al ( 1999) Moghaieb et al (2000) Lili us et al (1 996)

Seedling tolerant to salini ty stress and increased germination under cold

Hayashi et al (1997). Alia et al ( 1998)

Increased tolerance to salinity and Sakamoto et al ( 1998) cold Increased stress to lerance Increased salinity resistance and chill ing tolerance Performed better under drought and salinity stress

Huang et al (2000) Hoshida et al (2000)

Sheveleva et al (1997)

Increased plant height and fresh weight under salinity stress

Tarczynski et al ( 1993)

Increased germination under salinity Thomas el al (1 995) stress Increased dry weight and Pilon-S mits et al (1 998) photosynthetic acti vity under drought Increased dry weight and Pilon-S mits et al (1998) photosynthetic acti vity under drought Increased biomass production and Kavi et al ( 1995) enhance flower development under salinity stress Increased biomass production under Zhu e ( al ( 1998) drought and salinity stress Increased submergence tolerance Quimio et al (2000)

Increased freezing tolerance Increased tolerance to drought and salinity stress Increased biomass and WUE under stress

Steponkus et al ( 1998) Xu et a (1996)

Sivarnani et al (2000)

Enhanced freezing tolerance Increased fl ooding (water logging)

Tamminen et al (200 I) Grichko & Glick (200 I)

tolerance Calcium utilization under ionic Ki m et al (200 I) stress Cell division and/or diffe rentiation Soderman et al (1999) in developing organs Increased cold to lerance

Increased tolerance to cold. drought and salinity

Jaglo-Ottosen el

( 1998) Kasuga et al ( 1999)

al

COlltd.

ABDIN et af : ABIOTIC STRESS RESISTANCE IN PLANTS 227

Table I---Genes involved in abiotic stress----Contd.

Gene Gene action Species

MsPRP2 Transcription factor Alfalfa

OsCDPK7 Transcription factor Rice

SCOF-I Transcription factor Arabidopsis, Tobacco

Tsil Transcription factor Tobacco

example, accumulation of mannitol in celery, pinitol in ice plant (Mesembryanthemum crystallinum), glycerol in yeast (Saccharomyces cerevisiae) and Dunaliella, and ectoine in halobacteria, all seem to assure osmotic adjustments. Though the most important of the osmotic adjustment is questionable, the compatible solute concept appears to hold. The accumulating solute seems to act in specific functions, such as protein solubilization (ectoine, glycinebetaine), and uncharged solutes (mannitol, pinitol) seem to act as scavengers of reactive oxygen species (ROS). Over-expression of the Fe-binding protein, ferritin, resulted in increased tolerance, is attributable to the removal of free iron from the cells. Protection by the removal of iron, which participates in the "Fenton reaction", and produces hydroxyl radicals, has already been shown (Shen et ai, 1996). Many reports suggest ROS scavenging as most essential for protection (Allen et aI, 1989; Roxas et ai, 1997). Nitric oxide (NO) is a very active molecule involved in many diverse biological pathways, where it has proved to be protective against damages provoked by oxidative stress conditions. Two NO donors, sodium nitroprusside (SNP) and S-nitroso-Nacetylpenicillamine (SNAP) when applied to wheat (Tri:ticum aestivum) showed 15% increase in the water retention than those pretreated with water (Carlos & Lorenzo, 2001 ).

In plants, the regulation of binding protein (BiP) gene expression has been examined primarily by the detection of BiP RNA and protein levels (Denecke, 1996). Elevated levels of B iP in the transgenic tobacco lines conferred tolerance to the glycosylation inhibitor, tunicamycin during germination and tolerance to water deficit during plant growth (AI vim et aI, 2001) . Infact, BiP expression has been correlated to a variety of abiotic and biotic stress conditions, such as water stress, fungus infestations, insect attack, nutritional stress, cold acclimation and elicitors of the plant pathogenesis response (Anderson et aI, 1994; Denecke et ai, 1995; Kalinski et ai, 1995 ;

Phenotypic expression Reference

Increased salinity tolerance Winicov & Bastola (1999)

Increased cold salinity and drought Saijo et af (2000) tolerance

Increased cold tolerance Kim et af (200Ia)

Increased osmotic stress tolerance Park et af (200 I)

Fontes et aI, 1996, 1999; Figueiredo et ai, 1997). In the endosperm of maize floury-mutant, the synthesis of a zein-like storage protein variant, containing uncleavable signal sequence is associated with increased accumulation of BiP (Boston et ai, 1991 ; Fontes et ai, 1991; Coleman et ai , 1995; Gillinkin et aI, 1997). Tunicamycin, a potent acti vator of unfolded protein response (UPR) pathway, effectively induces BiP expression at both, mRNA and protein level in several plant systems (Fontes et aI, 1991 ; D'amico et aI, 1992; Figueiredo et ai, 1997).

There are other biochemical mechanisms contributing to tolerance, such as reactions that lower the NADHINAD+ ratio, thereby decreasing reducing power. The balance, also between nitrogen assimilation and carbon proVISIOn requires adjustments. Uptake, transport and compartmentation of sodium ions require several genes and pathways while maintaining uptake of the essential potassium. Cellular integrity is maintained by changes in membrane fluidity and protein composition of membranes (Bohnert et ai, 1995). Accumulation of stress specific proteins such as the late embryogenesis abundant (LEA) proteins to high concentration within specific tissues is correlated with or causually related to improved tolerance to drought, cold, or salinity (Imai et aI, 1996; Close, 1996; Xu et ai, 1996), although the mechanistic basis of their effects remains elusive. Apparently, different species have solved the problem by strengthening unique biochemical pathways, but there seems to be a limited number of ways in which this can be achieved.

Substantial data from genetic transformation experiments have established that functionally analogous tolerance determinants exist in both unicellular organisms and plants. The DNA sequences involved in stress sensing, transduction of the signal and the regulation and function of the downstream gene induction and repression machinery are largely conserved (Serrano, 1996; Shinozaki & YamaguchiShinozaki, 1997; Zhu et aI, 1997; Ishitani eJ ai, 1997).

228 INDIAN J BIOTECHNOL, JULY 2002

Stress- inducible genes have been transferred to improve tolerance of plants. It is, therefore, not only important to analyze functions of stress-inducible genes for further understanding of molecular mechanisms of stress tolerance, but also for the improvement of stress tolerance of crops by gene manipulation (Shinozaki & Yamaguchi-Shinozaki , 1997).

The plant growth regulator, Abscisic acid (ABA) is produced under drought conditions and plays important roles in response and tolerance against dehydration . Most of the genes that have been studied are also induced by ABA. It appears that dehydration triggers the production of ABA, which in turn induces various genes. There are genes that are induced by dehydration but are not responsive to exogenous ABA treatments suggesting the existence of ABAindependent as well as ABA-dependent signal transduction cascades between the initial signal of drought stress and the expression of specific genes. To understand the molecular mechanisms of gene expression in response to drought stress, cis- and transacting elements that function in ABAindependent and ABA-responsive gene expression have been precisely analysed . A variety of transcription factors seem to be involved in stressresponsive gene expression that may further involve complex regulatory systems (Shinozaki & Yamaguchi-Shinozaki , 1997).

Functions of Drought Inducible Gelles A variety of genes are induced by drought stress,

and function s of their gene products have been predicted from sequence homology with known proteins. Genes induced during drought stress conditions are thought to protect cells against dehydration by the production of important metabolic proteins and also in the regulation of genes for signal transduction in the drought stress response (Shinozaki & Yamaguchi-Shinozaki, 1997). Thus, these gene products are classified into two groups: functional proteins and regul atory proteins (Fig. 1). Functional proteins function in the stress tolerance, whereas the regulatory proteins function in signal transduction and gene expression to stress response. Existence of a variety of drought inducible genes suggests complex responses of plants to drought stress. These gene products are involved in drought tolerance and stress response.

The expression patterns of genes induced by drought have been analysed by RNA gel blot analysis.

Results indicated broad variations in the timing of induction of these genes. All the drought inducible genes are induced by high salinity stress. Most of the drought inducible genes also respond to cold stress but some do not, and vice versa. Many genes respond to ABA, whereas, others do not. ABA-deficient mutants were used to analyse drought inducible genes that respond to ABA. Several genes were induced by exogenous ABA treatment, but were also induced by cold or drought in ABA-dependent (aba) or ABAinsenSitive (abi) Arabidopsis mutants. These observations indicate that these genes do not require an accumulation of endogenous ABA under cold or drought conditions, but do respond to ABA. There are ABA-independent as well as ABA-dependent regulatory systems of gene expression under drought stress (Shinozaki & Yamaguchi-S hinozaki , 1997). Analysis of the expression of ABA-inducible genes showed that several genes require protein biosynthesis for their induction by ABA, suggesting that at least two independent pathways exist between the production of endogenous ABA and gene expression under stress conditions.

Drought, salt and cold in aba or ab i Arabidopsis mutants induce a number of genes. Among these

Drought Stress Proteins

/\ Functional Proteins Regulatory Proteins

I. Prorcin"scs (Cytoplas m. Chloroplast) I. T ranscription Factors (MYC. MYB, bZlP

2. Membrane proteins (water channel 2. Prorein Kin"ses (MAPK. MPKKK , protein transporters) CDPK.S6K)

3. Osmoprot<ctants synthases (Proline, 3. PI turnover (Phospholipase C. PIP5K. Sugar) DGK. PAP)

4. Detoxification enzymes (GST. s. EH) 4. Protection faclors of macromolecules (Chaperones. LEA proteins).

Fig. I-Drought stress induci ble genes and their possible funct ion in stress tolerance and response. Gene products are classified into two groups. The first group inc ludes proteins that probably func tion in stress tolerance (Functional proteins), and the second group contains protein fac tors involved in further regulat ion of signal transduction and gene expression that probabl y function in stress response (Regul atory proteins) . CDPK, calcium-dependent prote in kinase; DGK, diacylglycerol ki nase; GST, g lutathione-Stransferase; LEA, late embryogenesis abundant; MAPK, mitogenac ti vated protein kinase; PA P, phosphatid ic ac id phosphatase; PIP5K. phosphatidyl inositol-4-phosphate-5-kinase; sEH, soluble ex poxide hydrolase; S6K, ribosomal protein S6kinase (Adapted from: Plant Response to Environmenta l Stress. Edited by M F Small wood, C M Calvert & D J Bow les BIOS Scientific Publishers Ltd, 1999).

ABDIN el a/: ABIOTIC STRESS RES ISTANCE IN PLANTS 229

genes, the expression of a drought-inducible gene for rd29AJlti78/cor78 was extensively analysed (Yamaguchi-Shinozaki & Shinozaki , 1994). At least two separate regulatory systems function in gene expression during cold and drought stress; one is ABA-independent pathway and the other is the ABAdependent pathway. A 9-bp conserved sequence, TACCGACAT, named the dehydration-responsive element (DRE) is essential for the regulation of the induction of rd29A under drought, low temperature and high salt stress conditions, but does not function as an ABA-responsive element. The rd29A promoter contains ABRE, which functions in ABA-responsive expression. DRE-related motifs have been reported in the promoter region of many cold and droughtinducible genes (Yamaguchi-Shinozaki & Shinozaki , 1994). These results suggest that the DRE-related motifs including C-repeat (CRT) and low temperature-responsive elements (LTRE), which contain a CCGAC core motif are involved in droughtand cold-responsive but ABA-independent gene expression (Fig. 2).

Protein factors, which specifically interact with the 9bp-DRE sequence, were detected in nuclear extracts prepared from either dehydrated or untreated Arabidopsis plants (Yamaguchi-Shinozaki & Shinozaki, 1994). Liu et ai (1998) have cloned five independent DRE/CRT-binding proteins using the yeast hybrid screening method. All DRE/CRTbinding proteins (DREBs and CBFs) have been shown to contain a conserved DNA-binding motif, also reported in EREBP and AP2 proteins (EREBP/AP2 motif) , that are involved in ethyleneresponsive gene expression and floral morphogenesi s, respectively (Liu et ai, 1998; Stockinger et ai, 1997) . These cDNA clones, which encode DRE/CRTbinding proteins, are classified into two groups, CBFlIDREB 1 and DREB2. Expression of the DREBIA gene and its two homologs (DREBIB=CBF1 , DREBIC) was induced by low temperature stress, whereas expression of the DREB2A gene and its single homologue (DREB2B) was induced by dehydration (Liu et ai, 1998 ; Shinwari et aI, 1998).

Over-expression of the DREBIA cDNA in transgenic Arabidopsis plants not only induced strong expression of the target genes under unstressed conditions but also caused dwarfed phenotypes in the transgenic plants. The DREB 1 A transgenic plants also revealed freezing and dehydration tolerance, which was also shown in the CBF 1 transgenics (Jaglo-

Ottosen et ai, 1998; Liu et ai, 1998). In contrast, bverexpression of the DREB2A cDNA induced weak expression of the target genes under unstressed conditions and caused only s light growth retardation of the transgenic plants (Liu et ai, 1998). These results indicate two independent families of DREB proteins, DREB 1 and DREB2, functioning as transacting factors in two separate signal transduction pathways, under low temperature and dehydration conditions, respectively (Liu et ai, 1998). Overproduction of the DREB 1 A and CBF lIDREB 1 B cDNAs driven by the 35S CaMV promoter in transgenic plants significantly improved stress tolerance to drought and freezing. However, the DREB lA transgenic plants revealed severe growth retardation under normal growth conditions. The

?tic~ (Drought, Salt) (Cold)

+ ~ / Sij percer~n ~ Protein synthesis

(MYC/MYB)

1 MY~'MYBR

EREPB/AP2

1 1 ~ . Dr

ABA biosynthesis pathway ABA Independent pathway

+ Ge ne Expression (rd22,rd29 A n,erdl )

Drought stress response and tolerance

Fig. 2-Signal transduction pathway between the perception of drought stress signal and gene expression. At leas t four-sign al transduction pathways ex ist (I-IV ): two are abscisic ac id (ABA)dependent (I and II ) and two are ABA-independent (Ill and IV ). Protein biosynthesis is required in one of the ABA-dependent pathways (I). In another ABA-dependent pathway, ABA responsive ele ment (A BRE) function as an ABA-responsive element and does not require protein biosynthesis ( II ). In one of the ABA-independent pathways, a drought-responsive element (DR E) is in volved in the regulation of genes not on ly by drought and sa lt but also by cold stress (IV). Another ABA-independent pathway is controlled by drought and salt , but not by cold (I ll ) (Adapted from: Plant Response to Env ironmental Stress. Edited by M F Small wood, C M Calvert & D J Bowles BIOS Scientific Publishers Ltd, 1999).


DREB I A cDNA driven by the stress inducible rd29A promoter was expressed at low level under unstressed controlled conditions and strongly induced by dehydration, salt and cold stresses . The rd29A promoter minimized negative effects on growth of plants, whereas the 35S CaMV promoter caused severe growth retardation under normal growth conditions. In addition, this stress-inducible promoter enhanced tolerance to drought, salt and freezing as compared to 35S CaMV promoter (Kasuga et ai, 1999).

Cold Stress During the warm growing season plants are

generally very sensitive to freezing. However, as the year progresses many plants in temperate regions sense changes in the environment that signal the coming winter and respond by increase in freezing tolerance. Freezing temperatures are a major factor determining the geographical locations suitable for growing crop and horticultural plants and periodically account for significant losses in productivity. Determining the mechanisms whereby plants sense low temperature and activate the cold acclimation response has a potential to provide new strategies for improving the freezing tolerance of agronomic plants. Such strategies would be highly significant as traditional plant breeding approaches have had limjted success so far. For instance, despite considerable efforts , the freezing tolerance of wheat varieties today is only marginally better than those developed in the early part of the century (Fowler & Gusta, 1979). Earlier classical genetic analyses had indicated that the ability of plants to cold acclimation is a quantitative trait largely involving the action of many genes, each with small additive effects (Thomashow, 1990). Until recently , however, there has been little progress in identifying specific genes with roles in freezing tolerance. Genetical approaches have been hampered by mUltiple factors including the quantitative nature of the trait as well as an accurate determination of freezing tolerance that requires the testing of plant populations. However, by using newly developed molecular marker technologies and methods of Quantitative Trait Loci (QTL) mapping, exciting new information on freezing tolerance loci has begun to emerge, particularly in wheat (Galiba et ai, 1995) and barley (Hayes et ai, 1993). In each of these species, a locus has been identified that has a major effect on freezing tolerance. These loci appear to be related. The locus, known as the Vrnl-Frl

interval in wheat, may encode regulatory gene(s) that controls the level of expression of cold-inducible genes (Limin et ai, 1997). The challenge now is to determine the identity and function of the genes at these freezing tolerance loci. This will not be an easy task, but the expanding efforts in cereal genomic research should greatly facilitate these efforts.

The changes in gene expression that occur during cold acclimation (Guy et ai, 1985), have quickly led to a new line of investigation, the isolation and characterization of genes that are induced during cold acclimation. The notion was that some genes, which are activated in response to low temperature probably have roles in metabolic adjustment to low temperatures, but that others might have specific roles in freezing tolerance. Indeed, recent studies with Arabidopsis have led to the identification of a freezing tolerance gene 'regulon' and a family of transcription factor genes that control their expression. Several cellular processes are associated with the reorganization of the actin cytoskeleton in plants. These include cell division and differentiation, stomatal movement, gravitropic tip growth, light induced plastid migration, wound repair, response to pathogen attack, pollen development, nuclear migration, cytoplasmic streaming, secretion, cell wall biosynthesis and transmembrane signalling. Actin filaments are tightly linked to the plasma membrane and believed to be involved in signal transduction events (Aon et ai, 1999). Disruption or reorganization of the cytoskeleton could thus impair or modify the activity of signalling molecules associated with cytoskeleton elements (Ouellet et ai, 2001).

Functions of Cold Inducible Genes Among the highly expressed cold-responsive genes

of Arabidopsis are the COR (cold regulated) genes, also designated L TI (low temperature induced), KIN (cold inducible), RD (responsive to desiccation) and ERD (early dehydration-inducible). The COR genes comprise four gene families, each of which is composed of two genes that are physically linked in the genome in tandem array. The COR78, CORI5 and COR6.6 gene pairs encode newly discovered polypeptides, while the COR47 gene pair encodes (late embryogenesis abundant) group II proteins, also known as LEA DB proteins, RAB proteins and dehydrins (Ingram & Bartels, 1996). Artus et al (1996) have provided direct evidence that the CORISa gene has a role in cold acclimation. This gene encodes a 15 kDa polypeptide, CORISa that is \

ABDIN el al: ABIOTIC STRESS RESISTANCE IN PLANTS 231

targeted to the stromal compartment of chloroplast. Ouring import, CORISa is processed to a 9.4 kOa polypeptide, designated CORISa. The polypeptide is hydrophilic, acidic (PH 4.S), rich in alanine, lysine, glutamic acid and aspartic acid residues, and is predicted to form an amphipathic a-helix. To determine whether CORISa might be a freezing tolerance gene, Artus et al (1996) made transgenic Arabidopsis plants that constitutively expressed CORISa and compared the in vivo freezing tolerance of the chloroplasts in non-acclimated transgenic and wild-type plants (the level of COR ISa in the nonacclimated transgenic plants approximated that in cold-acclimated wild-type plants).

Chloroplast Jolerance was assessed by freezing the leaves at various sub-zero temperatures, and after thawing, estimating the damage to photosystem II by measuring room temperature chlorophyll fluorescence. The results indicated that the chloroplasts of the non-acclimated transgenic plants were I-2°C more freezing tolerant than the chloroplasts from the non-acclimated wild-type plants. In addition, the effects of CORISa were not limited to the chloroplast. In particular, protoplasts isolated from leaves of non-acclimated transgenic and the wild-type plants were frozen in vitro at various sub-zero temperatures, thawed, and the percentage survival was determined by staining with fluorescein diacetate. The results indicated that over the temperature range of -S to -8°C, the freezing tolerance of the protoplasts from transgenic plants was about 1°C greater than that of the wild-type plants. Constitutive CORISa expression increases the cryostability of the plasma membrane (Artus et ai, 1996). This followed from the fact that the protoplast survival was measured by fluorescein diacetate staining, a method that reports on retention of the semipermeable characteristics of the pl asma membrane. COR IS a ex press ion might decrease the propensity of membranes to form hexagonal II phase lipids. Indeed, the constitutive expression of CORISa decreases (S teponku s et ai, 1998) the incidence of freeze-induced lamell ar-to-hexagonal II phase transitions that occur in regions where the plasma membrane is brought in close apposition with the chloroplast envelope as a result of freeze-induced dehydration . In addition, purified preparations of CORISa increase the lamellar-to-hexagonal II phase transition temperature of dioleoylphosphatidylethanolamine and promote formation of the lamellar phase in a lipid mixture composed of the major lipid

species that comprise the chloroplast envelope. CORISa polypeptide (Steponkus et ai, 1998) acts to defer freeze-induced formation of the hexagonal II phase to lower temperatures by altering the intrinsic curvature of the inner membrane of the chloroplast envelope.

Jaglo-Ottosen et al (1998) created transgenic Arabidopsis plants that constitutively express the entire battery of COR genes and compared the freezing tolerance of these plants to those that expressed CORISa alone. Expression of the COR gene regulon was accomplished by over-expressing the Arabidopsis transcriptional activator CBFl (CRT/ORE binding factor 1) (Stockinger et ai , 1997 ). This factor binds to CRT (C-repeat)/ORE (drought responsive element) ONA regulatory element present in the promoter of the COR genes and activates their expression without a low temperature stimulus. Using an electrolyte leakage test to assess the freezing tolerance of detached leaves from non-acclimated plants, Jaglo-Ottosen et al (1998) were unable to detect a statistically significant enhancement of freezing tolerance by expressing CORISa alone. In sharp contrast, they detected a 3°C increase in freezing tolerance in plants that over-expressed CBFI and consequently, the CRTIDRE COR gene regulon. In addition, expression of the CRT/ORE COR gene regulon resulted in an increase in whole plant freezing survival, whereas expression of CORISa alone did not. Taken together, these results implicate additional COR genes in freezing tolerance.

Liu et al (1998) have independently shown that expression of the CRT/ORE-regulated COR genes increases the freezing tolerance of Arabidopsis plants. They activated COR gene expression by overexpressing a homologue of CBFl designated OREBIA. These results indicate that expression of the CRT/ORE-containing COR genes not only affect freezing tolerance, but also increase tolerance to drought. One important difference between the results of Jaglo-Ottosen et al (1998) and Liu et at (1998) is that the latter invest igators found that over-expression of OREBIA resulted in the transgenic pl ants having a dwarf phenotype. However, it was not observed by Jaglo-Ottosen et at ( 1998) in plants that overexpressed CBFI.

Gene fusion studies have demonstrated that the promoters of the Arabidopsis COR ISa, COR 6.6 and COR 78 genes are induced in response to low temperature (Jaglo-Ottosen et ai, 1998). Yamaguchi-


Shinozaki & Shinozaki (1994) first identified the cold regulatory element that appears to be primarily responsible for this regulation in COR78 promoter. It is a 9bp element referred to as the ORE. The ORE with Sbp core sequence of CCGAC, designated as CRT (Baker et at, 1994), stimulates gene expression in response to low temperature, drought and high salinity, but not in response to exogenous application of ABA (Yamaguchi-S hinozaki & Shinozaki, 1994). The element, which is also referred to as LTRE (low temperature regulatory e lement) has been shown to impart cold-regulated gene expression in Brassica napus (Jiang et at, 1996).

It was established that CBFl protein binds to CRT/ORE sequence and activates expression of reporter genes in yeast (Saccharamyces cerevisiae) carrying the CRT/ORE as an upstream regulatory sequence. The first cONA for a protein that binds to the CRT/ORE sequence was thus isolated. This CBFI protein has a mass of 24 kOa, an acidic region that potentially serves as an activation domain and a putative bipartite nuclear localization sequence. It also has an AP2 domain, a 60 amino acid motif that exists in a large number of plant proteins including Arabidapsis APET ALA2, AINTEGUMENT A and TINY; the tobacco EREBPs (ethylene response element binding proteins); and numerous other plant proteins of unknown function (Riechmann & Meyerowitz, 1998). AP2 domain includes a ONAbinding region (Ohme-Takagi & Shinshi , 1995). These results indicated that CBFl is a transcriptional activator that can activate CRT/ORE-containing genes and thus, was a probable regulator of COR gene expression in Arabidapsis. Subsequently, 1agloOttosen et at (1998) showed that constitutive overexpression of CBFI in transgenic Arabidapsis results in expression of the COR genes without a low temperature stimulus. Thus, CBF I appears to be an important regulator of the cold acclimated response, controlling the level of COR gene expression, which in turn promotes freezing tolerance. It was further established that CBF 1 is a member of a small gene family comprised of three closely related transcri ptional acti vators referred to as CB F I, CB F2 and CBF3 (Gilmour et at, 1998) and OREB IB, OREBIC and OREBIA, respectively (Liu et at, 1998). The three CBF genes are physically linked in direct repeat on chromosome 4 near molecular markers PG II and m600 (72 .8 cM) but are unlinked to their target genes COR6.6, CORISa, COR47 and COR78, which are located on chromosomes S, 2, 1

and S, respectively (Gilmour et at, 1998). Like CBFl, both CBF2 and CBF3 can activate expression of reporter genes in yeast that contains the CRT/ORE as an upstream activator sequence indicating that these two family members are also transcriptional activators. Moreover, the over-expression of OREB 1 A (CBF3) in transgenic Arabidapsis results in constitutive expression of COR78 and, as described above, enhances both the freezing and drought tolerance of the transgenic plants (Liu et at, 1998). In wheat, the cold-regu lated TaAOF cDNA (T. aestivul1l actin-depolymerizing factor) shows a high homology to plants, animals and yeast actin-binding proteins (Danyluk et at, 1996). These AOFs (Cofilin group, a fami ly of small proteins; IS-22 kOa) include cofilin , destrin, depactin and actophorin (Staiger et at, 1997; Lappalainen et at, 1998). The members of this family are stimulus-responsive modulators of the cell actin cytoskeleton dynamics and show in vitro actin monomer binding, actin fi lament binding/severing, and nucleotide/monomer dissociation-inhibiting activ ities (Lappalainen et at, 1997; McGough & Chiu, 1999). Over-expression of cofilin in sl ime mould and bacterium, Listeria manacytagenes showed development of thick actin cables, dramatic membrane ruffling and increased motility. AOFls were shown to increase the turnover rate of actin fi laments in Arabidapsis (Aizawa et at, 1996; Carlier et at, 1997). Isolation of TaADF cDNA in wheat encodes AOF, which is correlated with freezing tolerance (Ouellet et at, 2001).

The expression of lipid transfer protein (LTP) genes in several plants is regulated by environmental factors such as cold (Hughes et at, 1992; White el at, 1994; Molina el at, 1996). L TPs unspecifically transfer all phospholipids and galactolipids and show activity with phosphatidyl choline (Kader, 1996). The Gal-binding seed, leaf Iectins, and a class 1-~-I, 3-glucanase (Hincha et ai, 1993, 1997a, 1997b) has cryoprotective actlVlty for isolated thylakoid membranes. Cryoprotective protein, cryoprotectin, has been purified from the leaves of cold acclimated cabbage (Brassica aleracea) . It is a small boil-stable protein that could not be purified from the leaves of non-acclimated plants. This indicates. that it may be cold induced (Hincha et at, 2001). RC12A and RCI2B, two homologous rare cold inducible genes, constitute a new small family of low temperature regulated genes in Arabidapsis (Capel el ai , 1997). The expression of these genes is transiently regulated during cold acclimation and is also induced by ABA

ABDIN et al: ABIOTIC STRESS RESISTANCE IN PLANTS 233

and water stress. Furthermore, RC12A and RC12B expression is up-regulated by low temperature in aba and abi mutants of Arabidopsis, suggesting that both pathways regulate their low temperature responsiveness. RC12A and RC12B encode small (54 residues), highly hydrophobic proteins with two potential transmembrane domains. The absence of signals for organelle targetting suggests that both proteins could be localized in the plasma membrane, considered to be a primary site of injury during freezing (Lyons, 1973). RC12A and RC12B were isolated and characterized. The promoter regions were fused to the uidA (GUS) reporter gene and the levels of GUS activity were analysed quantitatively in Arabidopsis transgenic plants during development under both stressed and . unstressed conditions. Transgenic plants with either RC12A or RC12B promoter showed strong GUS expression during the first stage of seed development and germination, in vascular bundles, pollen and guard cells (Medina et aI, 2001).

Salt Stress Plants vary widely in their ability to withstand

saline environments. The optimal salt concentration for the growth of halophytes is about 0.5 M NaCI (Flowers et aI, 1977), which is roughly equivalent of salt concentrations in seawater. Most crop plants, however, are glycophytes (salt sensitive plants). Plants, such as Medicago sativa are killed at 0.1 M NaCI concentration (Smith & McComb, 1981). The level of diversity in salt resistance, which often exists between cultivars of anyone glycophytic species, indicate that stable genetic mechanisms exist even in glycophytic plants that provide incremental levels of protection against salt stress. Because of the increasing salinization of agricultural land the world over, it is of utmost importance to identify genes encoding physiological traits that can confer improved salt tolerance to glycophytic plants. Plant breeding for tolerance to salt stress has been difficult, because a variety of physiological parameters appear to contribute to tolerance, salt tolerance has been considered to be a quantitative trait (Norlyn & Epstein, 1984; Dvorak et aI, 1985, 1988). The primary consequences of salt stress (Levitt, 1980) usually consist of toxic effects (Kingsbury & Epstein, 1986) and metabolic disturbances, which also include loss of chloroplast activity (Shqrp & Boyer, 1985). At the cellular level, there is increased growth coupled with maintenance costs (Handa et ai, 1983; Stavarek

& Rains, 1984, Rhodes et aI, 1986). It is, therefore, presumed that most of the enhanced gene expression observed under conditions of salt stress is to circumvent the deleterious effects of osmotic stress, increased energy requirements, and changes in membrane functions in order to protect the growth and survival of the acutely stressed plqnts. At the molecular level, most studies have focussed on identification of those genes expressed under conditions of salt stress.

The ability to adapt to osmotic stress is ubiquitous in all living systems with minor modifications from bacteria to higher plants. A number of these systems have been used as models for investigating genetic changes at the molecular level , to understand potential mechanisms for salt tolerance in higher plants. Not surprisingly, common molecular mechanisms for salt tolerance are emerging across the physiological spectrum. In Escherichia coli, the adaptation mechanism includes alterations in envelope protein (Barrons et aI, 1986), biosynthesis or transport functions for osmotically active compounds and a number of uncharacterized genes (Csonka, 1989). In bacteria, it is possible to identify single mutations that lead to tolerance, usually due to overproduction of the product of a physiologically important pathway for salt tolerance. Another type of single mutation that can lead to tolerance in bacteria and yeast is the osmoremedial mutation, in which slight alterations in the amino acid sequence of highly osmo-Iabile proteins can influence the tolerance of the organism (Csonka, 1989). Cyanobacteria are photosynthetic bacteria, which can survive high salinity. The cloning of salinity stress-induced genes from Anabaena torulosa has shown that a large portion of its genome is responsive to salt (Apte & Haselkorn, 1990). It is interesting to note that NaCI increases CO2 fixation, chlorophyll level and Rubisco content in the salt tolerant cyanobacterium, Aphanothece . stagnina (Takabe et ai, 1988; Rai , 1990). Salinity also increases the photosynthetic capacity of Synechococcus (Blumwald & Tel-Or, 1984) and affects at least three primary photosynthetic processes in red algae (Satoh et aI, 1983). These results indicate that carbon metabolism through the photosynthetic systems is very sensitive to environmental salt and organisms respond by maintenance of photosynthesis in presence of salt. Gaxiola et al (1992) by using functional gene complimentation cloned a novel gene HALl in S. cerevisiae, which improves growth under salt stress by over-expressing this gene. The HAL I


protein, most ly present in the cytosol of the yeast cells, is specific for NaCI and its sequence appears to be conserved in corn and Arabidopsis as detected by DNA hybridi zation and western analysis. The effect of water stress, salt stress and ABA on the steady state levels of tomato Asrl mRNA and protein have been studied. Application of polyethylene glycol (PEG), NaCI or ABA to the growth medium resulted in an elevation of the steady-state levels of both Asrl mRNA and protein (Amitai-Zeigerson et ai, 1995). This increase was transient reaching a maximum approximately 24 hrs after the application of the stress. The extent of the increase was correlated with the severity of the stress. The similarity between the response of mRNA and protein to the stress suggests that mainly RNA transcription or RNA stability regulates the Asrl gene. Anti-ASR 1 antiserum was used to screen the cellular location of the ASRI protein . ASRI protein was detected in the nuclear fraction. Treatment of the nuclei with Triton-X-IOO did not solubilize the protein, suggesting that the protein is bound to chromatin (Gilad et ai, 1997) . ABA is an important intermediate in transducing signals of osmotic stresses since the ABA levels are often elevated upon stress and most, if not all , of the stress-responsive genes are also induced by external ABA treatment (Skriver & Mundy, 1990). ABAinsensItIve mutants, abi, display a range of phenotypes including reduced seed dormancy , wilted seedlings, increased sensitivity to desiccation and diminished induction of normally ABA-inducible genes (Wei et aZ, 2001). ABII and ABI2 encode protein phosphatases indicating that protein phosphorylation plays an important role in other stress responsive pathways (Meyer et ai, 1994). Protein kinases are recogni zed as having a central role in the control of signal transduction . A number of stress-or ABA regul ated protein kinase genes from Arabidopsis, ATCDPK I and ATCDP K I a act ivate the promoter of the barley stress-inducible gene Hva I when transiently expressed in maize leaf protoplasts (Sheen, 1996). Protein kinases play key roles in detecting and relaying developmental and env ironmental signals for the regu lation of specific genes and thus mediate cellular response to those signals. Protein kinase gene (Esi 47) can be induced by ABA in Lophopyrum elongatul11 roots (Galvez et aI, 1993) and it was shown to mediate between certai n processes re lated to ABA and its antagoni st GA in aleurone ce ll s of barley (Wei ef ai , 2001). Transient gene expression assays in the barley

aleurone tissue showed that Esi 47 suppresses induction of barley low-pI a-amylase gene promoter, thus providing evidence for the role of this protein kinase gene in plant hormone sig nalling. Esi 47 contains a small upstream open reading frame in the S'-untranslated region of its transcript that is implicated in mediating the repression of the basal level of the gene expression and in regulating the ABA inducibility of the gene (Wei et ai, 2001 ).

Varietal, species and even genus diversi ty with different levels of salt tolerance have complicated the study of salt stress in glycophytic plants. The molecular responses to salt stress in different plant vanetles, therefore, have been compared against different genetic backgrounds, which add another level of complexity to an already multi-faceted problem of salt stress imposed on gene expression regulated by development, ti ssue specificity and diurnal rhythm. To circumvent these problems and to focus on the primary differences in gene expression due to environmental changes in salt or drought, several laboratories have utilized the ice plant as a model system to study genes expressed for adaptation to salinity. The ice plant is a facultative halophyte, able to adjust rapidly to growth in O.S M NaCI. Exposure to salt, drought or cold leads Mesembryanthemum plants to change their mode of carbon assimilation from C3 to the CAM pathway (Ting, 1985). The differential gene expression elicited by salt has been thoroughly reviewed (Bohnert et ai, 1988) .

The CAM pathway induction by salt requires the coordinate acti vation of the genes encoding enzymes of the CAM pathway, including phosphoenolpyruvate carboxylase (PEPCase), pyruvate orthophosphate dikinase and NADP malic enzyme (Cushman et ai, 1990). Transcriptional increase by salt stress has been demonstrated for PEPCase isogene Ppc 1 (Cushman el ai, 1989) with additional regulation occurring at the post-transcriptional level. The role of transcriptiona l regul ation in Ppc I expression in salt stress was further strengthened by identification of specific DNAbinding factor interact ions with this promoter that were modi fied by salt stress (Cushman & Bohnert, 1992). Salt stress leads to increased expression of a number of other genes involved in carbon metabolism such as NAD: glyceraldehyde-3-phosphate dehydrogenase as well as two unidentified genes, McUB4 and McUB5 (Ostrem er ai, 1990). At the same time other gene products are down regulated , such as phosphoribulokinase. However, the


expression of this gene is resumed to pre-stress levels after the plant has undergone salt adaptation (Michalowski & Bohnert, 1992). It is interesting to note that the Mesembryanthemum transcript levels for photosynthesis related proteins in general recover after the initial stress, indicating that this physiological process remains protected under conditions requiring salt tolerance. Photosynthetic organisms, including cyanobacteria are endowed with several kinds of mechanisms that allow them to acclimate to salt stress; for example, the inducible synthesis of compatible solutes (Allakhverdiev et ai, 2001); of sucrose in salt-sensitive strains of Cyanobacteria, such as Synechococcus (Mackay et ai, 1984; Hagemann & Erdmann, 1997); of glucosylglycerol in strains with intermediary tolerance such as, Synechocystis (Hagemann & Erdmann, 1997); and of glycinebetaine in salt tolerant strains of Synechococcus sp (Mackay et ai, 1984; Hagemann & Erdmann, 1997). Direct evidence of these compatible solutes to protect the cyanobacterial cell has been provided from the studies of transgenic systems (Deshnium et ai, 1995, 1997; Ishitani et ai, 1995; Nakamuru et ai, 1997). Also, several reports suggest that lipids might be involved in the protection against salt stress (Huflejt et ai, 1990; Khamutov et ai, 1990; Ritter & Yopp, 1990) that is borne by the fact that when photosynthetic organisms are exposed to salt stress, the fatty acids of membrane lipids are desaturated (Allakhverdiev et ai, 2001).

Functions of Salt Inducible Genes Direct screening of a genomic library of

Mesembryanthemum C for genes expressed at enhanced levels under saline growth conditions detected an estimated 100 or more genes (Meyer et ai, 1990) indicating that a large number of genes respond to salinity signal. These results are somewhat complicated and will require sorting out of the developmentally and saline stress controlled interactions/responses, since it has been shown that competence for the switch to CAM IS developmentally determined as well as induced by saline environmental conditions (Cushman et ai, 1990).

The isolation of a cDNA clone, lmtl , that encodes myo-inositol methyltransferase in Mesembryanthemum (Vernon & Bohnert, 1992), provides evidence that there may be several signals or pathways that regulate salinity tolerance in this plant. While CAM enzymes respond to salinity stress In a

developmentally controlled manner, the Imtl gene product, an enzyme involved in the biosynthesis of pinitol, which is a likely osmoprotectant, does not depend on developmental triggers for induction. Although, ABA appears to participate in stressinduced expression of the CAM pathway (Chu et ai, 1990), cytokinins and NaCl have been implicated to act as independent InItIators for ABA in Mesembryanthemum CAM specific genes (Schmitt & Piepenbrock, 1992; Thomas et ai, 1992).

The molecular basis for salt tolerance in glycophytic plants is not well-understood. Several attempts to identify genes that are differentially expressed in response to salt have compared the effect of NaCI on polypeptide patterns from different plant tissues. Similar comparisons have been made with drought-stressed plants (Bray, 1991).

Investigators have characterized differences in gene expression due to salt stress by analysing the distribution of cellular polypeptides and the influence of salt stress on this distribution. Comparisons of barley polypeptides from plants exposed to NaCI showed a large number of differences (Hurkman et ai, 1988) with increases in some protein concentrations and decrease in others, demonstrating global changes in protein concentrations. Subsequent analysis of polypeptide changes induced significantly by salt stress in barley roots and shoots as measured in in vitro translation showed that it was specific for genotype and tissues. Ramgopal (1987a,b) has detected preferential accumulation of unique mRNAs in roots of salt-tolerant and in shoots of the saltsensitive genotypes of barley. A similar comparison of two barley cultivars differing in salt tolerance was carried out by computer assisted two-dimensional PAGE polypeptide analysis (Hurkman et ai, 1989). The result from experiments with in vivo labelled polypeptides and in vitro translation products showed relatively minor quantitative changes. Transient shortterm changes in polypeptides were also shown in barley root plasma membrane and tonoplast during K+ deprivation coupled with replacement by Na+ (Fernando et ai , 1992). Gulick & Dvorak (1987 ) compared in vitro translation products from isolated mRNA from salt tolerant amphiploid before and after acclimation to NaCI. The amphiploid was derived from cross between salt-sensitive T. aestivum cv. Chinese Spring and salt-tolerant Elylrigia elongata. Many mRNAs showed differential accumulation primarily in the roots of the two cultivars, which is consistent with previous observations (Storey et ai,

236 INDIAN J BIOTECHNOL. JULY 2002

1985) that these plants exclude salt from the shoots. These results also indicate coordinate regul ation of many genes.

Salt increased the activity and gene expression of peroxidases, gl utathione-S-transferase (GST) and aldose reductase. Tobacco plants transformed with active GST/PHGPX showed increased tolerance to salinity, whereas catalase-deficient transgenic tobacco showed higher sensiti vi ty to the stress (Willekens et at, 1997). One of the most extensively studied proteins that are accumul ated in response to salt adaptation is osmotin (Singh et at, 1985), which was first identified in salt adapted tobacco cells. The analogous NP 24 gene was cloned from tomato (King et ai, 1988) and osmotin was cloned from tobacco (Singh el at, 1989). Osmotin is preferentially expressed in roots, but is also found in basal leaves and floral organs (Neale el at, 1990). At the cellular level, it accumulates in the vacuole of salt stressed cells (Singh et at, 1985). Osmotin is regulated in cells at the transcriptional level by ABA application (Singh et at, 1989), but post-transcriptional regulation has been shown to have significant control over osmotin protein accumulation (LaRosa et ai, 1992). Overexpression of osmotin induces increased proline accumulation and conferred tolerance to osmotic stress in transgenic tobacco (Barthakur et at, 2001). The BnD22 gene (Downing et at, 1992), expressed in Brassica napus leaves in response to salt and drought, interestingly also contains the signature motif of soybean Kunitz trypsi n inhibitor. Studies on salt adapted cell walls have identified several specific polypeptides that are secreted in the growth medium. A thaumatin-like protein is secreted from NaCI adapted tobacco cells (lraki et at, 1989a,b). A novel, hydroxyproline rich protein, which resembles the structural protein in cell walls of higher pl ants, is excreted from salt adapted winged bean cells (Esaka et at, 1992). A protein oxalate oxidase homologous to germin was identified from a salt induced clone of barley roots (Hurkman & Tanaka, 1996). Activity of another protein, cytochrome C oxidase increased in salt treated Vigna plants (Hernandez et at, 1993). In addition to photosynthesis related genes, a number of other genes are induced in salt tolerant alfalfa cell s, leading to specific mRNA accumulation (Winicov et at, 1989). Winicov & Seemann (1990) and Winicov (1992) have cloned several cDN As by differenti al screening of a cDN A library, constructed from Poly A + RNA isol ated from salt tolerant cells grown on 171 mMNaCI.

High soi l salinity results in ion toxIcity and hyperosmotic stress leading to numerous pathologies including generation of reactive oxygen species (ROS) and programmed cell death (Niu et at, 1995; Zhu et at, 1997; Hasegawa et at, 2000b). Enzymes that catalyze rate-limiting steps in the biosy nthesis of compatible solutes or osmoprotectants e.g. sugar alcohols, quarternary ammonium and terti ary sulfonium compounds are categorical examples of osmotic stress tolerance effectors (Hanson et ai, 1994; Hasegawa et at, 2000b). Other effectors are proteins that protect membrane integrity , control water or ion homeostasis and ROS scavenging (Bray, 1994; Ingram & Bartels, 1996; Hasegawa et at, 2000b). Determinant function of some effectors has been confirmed because expression in transgenic plants enhances salt tolerance efficiency. Regulatory determinants include transcription factors and signal pathway intermediates that post-transcriptionally activate effectors (Hasegawa et at, 2000b). Basic ion zipper motif, MYB and MYC and zinc finger transcription factors, including rd22BPI (MYC), AtMYB2 (MYB), DREBIA and DREB2A (AP2 domain) and ALFIN 1 (Zinc Finger), interact with promoters of osmotic-regulated genes (Abe et at, 1997; Liu et at, 1998; Hasegawa et at, 2000b). The osmotic stress tolerance function of DREB 1 A in Arabidopsis (Kasuga et at, 1999) and ALFIN in alfalfa (Winicov, 2000) has been confirmed by ectopic expression in transgenic plants. Regulatory intermediates that modulate plant stress responses include SOS3 (Ca2+-binding protein), SOS2 (Suc nonfermenting like) kinase, Ca2+-dependent protein kinases and mitogen-activated protein kinases (Sheen, 1996; Halfter et ai, 2000; Kovtun et at, 2000). Additional signal intermediates have been implicated in the plant response to salt (Hwang & Gooman, 1995; Mizoguchi et at, 1996; Mikami et at, 1998; Piao et at, 1999; Hasegawa et at, 2000b) . Salt tolerant determinants have been identified in Arabidopsis (Hasegawa et at, 2000a; Sanders, 2000; Zhu , 2000). Genetic and physiological data indicate that SOS3, SOS2 and SOS I are components of a signal pathway that regulate ion homeostasis and salt tolerance and their functions are calcium dependent. SOS I encodes a putative plasma membrane Na+/H+ anti porter, SOS2 encodes a Suc non- fe rmenting like (SNF) kinase and SOS3 encodes a Ca2+-binding proteins with sequence similarity to the regulatory subunit of calcineurin and neuronal Ca2+ sensors (Liu & Zhu, 1998; Liu et ai, 2000; Shi et at, 2000). Molecular interactions and

ABDIN el af: ABIOTIC STRESS RESISTANCE IN PLANTS 237

complementation analysis indicate that SOS3 is required for activation of SOS2 that regulate SOS 1 transcription (Halfter et ai, 2000; Shi et ai, 2000) , confirming that the order of the signal pathway is SOS3-S0S2-S0S 1 (Hasegawa et ai, 2000a; Sanders, 2000; Zhu, 2000).

Of the three families of plant potassium channels known, AKT/KAT constitute the subfamilies (Zimmermann & Sentenac, 1999). All potassium channels show high specificity for K+ over other alkali cations, making them unlikely candidates for significant inadvartent sodium intrusion even at high Na+ to K+ ratios (Maathuis et ai, 1997; Amtmann & Sanders, 1999). At high abundance, AKTl is predominantly expressed in Arabidopsis roots (Cao et ai, 1995; Lagarde et ai, 1996). Mutant plants grow poorly on media with potassium concentrations in the micromolar range in comparison to wild type (Hirsch et ai, 1998; Spalding et ai, 1999), suggesting that AKTl-type channels can function in the high-affinity range (Su et ai, 2001). Expression of AKT2 has been located most strongly in the leaves (Cao et ai, 1995) and AKT3 in leaf phloem (Marten et ai, 1999). KA Tl expressed in guard cells that constitutes a path for potassium influx during stomatal opening (Su et ai, 2001). KATI from Arabidopsis and KST from potato are activated by extracellular acidification (MuellerRoeber et ai, 1995 ; Very et ai, 1995). KATl when expressed in Arabidopsis guard cells or yeast mediates K+ uptake (Bruggemann et ai, 1999). KA T2 has been detected in Arabidopsis leaf mesophyll cells (Butt et ai, 1997). Transcripts for three potassium channel homologs Mktl , Mkt2 and Kmtl from the common ice plant (M. crystallinum) have been characterized for their expression during salt stress. Mktl is root specific ; Mkt2 is found in leaves, flower and seed capsules. After salt stress, Mktl (transcript and protein) is drastically down regulated, Mkt2 transcript do not change and Kmt is strongly and transiently up regulated in leaves and stems (Su et ai, 2001).

Wheat protein LCT 1 is known to be permeable for a wide range of cations when expressed in S. cerevisiae (Amtmann et ai, 200J). G19 cells of S. cerevisiae transformed with LCT were found to be hypersensitive to NaCI and it was shown that LCT expression results in a strong decrease of intracellular K+/Na+ ratio in G19 cells due to the combined effect of enhanced Na+ accumulation and loss ' of K+. Na+ uptake through LCT 1 was inhibited by K+ and Ca2+ at high concentrations and the addition of these ions

rescued growth of LCT 1 transformed G 19 on saline medium. LCT also mediated in the uptake of Li+ and Cs+. Expression of two mutant LCT cDNAs with Nterminal truncations resulted in decreased Ca2+ uptake and increased Na+ tolerance as compared to expression of the full length LCTl (Amtmann et ai , 2001), suggesting that LCTl represents a link between Ca2+ and Na+ uptake into plant cells.

The salt responsive genes VSP2 (Vegetative Storage Protein), CCRI (Cold Circadian RhythmRNA binding 1), ST2 (Salt Tolerance Zinc Finger), SAMT (S-adenosyl-L-Met: Salicylic acid carbonyl methyl transferase) and COR6.6/KIN2 (cold regulated/cold inducible) are differentially regulated. The transcript abundance is slightly elevated in Arabidopsis SOS3, but the steady state mRNA level s were hyperIinked by salt treatment. Only the abundance of CCRI gene transcript was reduced by salt treatment (Gong et ai, 2001). Expressed Sequence Tags (ESTs) corresponding to salt regul ated genes were identified by screening a subtracted cDNA library prepared from wild type (Col-O gIl) seedlings treated with 160 mM NaCI. Differential dot blot hybridization identified unique ESTs (Salt regul ated ESTs [SREs]) that detected salt regulated transcripts. The SREs have been further used to define the transcriptional response of plants to salt stress. These results identify genes whose expression is most likely controlled by transcriptional activation, although other factors such as salt stress-dependent mRNA stability might contribute to steady state transcription abundance (Gong et ai, 2001). Database comparisons of SREs using blast programmes determined that the corresponding encoded proteins included those involved in primary metabolism, cell wall synthesis or degradation, other cellular functions, transport or nutrient assimilation , signalling and defensive responses. Several identified salt responsive genes encode components of octadecanoid signalling through jasmonic acid (Gong et ai, 2001).

VSP 2 is a member of a two-gene family (87% nucleotide sequence identity over the coding region) that codes a protein with similarity to soybean VSPs (Berger et ai, 1995; Utsugiet et ai, 1998). These are vacuolarized glycoproteins with acid phosphatase activity . These proteins are presumed to be amino ac id sinks during water deficit , but are important reduced mitogen source after stress relief (Mason & Mullet, 1990). CCRI encodes a Gly-rich RNAbinding protein implicated in post-transcriptional regul ation. CCRI and CCR2 comprise a two-gene


family and their expression is regulated by diurnal circadian clock (Carpenter et ai, 1994; Heintzen et ai, 1997). Their expression negatively regulates either gene and this feedback loop presumably facilitates diurnal oscillations controlled by the master circadian clock (Heintzen et ai, 1997). CCR1 and CCR2 steady state m-RNA levels are regulated by ABA, whereas CCR2 expressIOn IS induced by dehydration (Carpenter et ai, 1994).

Targeted mutagenesis has been used to alter genes for fatty acid desaturases (desA) in Synechocystis sp. PCC6803, and in the strains with decreased levels of unsaturated fatty acids in their membrane lipids as well as decreased tolerance to salt. Wild type cells synthesized saturated and mono-unsaturated fatty acids, whereas des A + cell s transformed with des A gene for the ill 2 acyl-lipid desaturase also synthesized di-unsaturated fatty acids. Whereas des A cells were more tolerant to salt stress and osmotic stress than the wild-type cells and the extent of the recovery of the various photosynthetic activities from the effect of NaCI was much greater in desA + cells than wild type cells. The photosystem II activity of thylakoid membranes of desA + cells was more resistant to NaCI than that of membranes of wild type cells (Allakhverdiev et ai, 2001).

Future Prospects Although, there is a surge of information on plant

responses to abiotic stress, the understanding of the mechanisms involved is still enigmatic. One of the strategies could be the use of saturation mutagenesis in Arabidopsis by T-DNA insertions to identify mutations in a particular gene (Krysan et ai , 1996). Other approaches can also be exploited to isolate and functionally characterize knockout mutants in addition to functional assays, direct gene discovery strategies (EST collections and databases) with corresponding developments of specialized bioinformatics resources. These strategies will bring us much closer to comprehensive characterization of the different osmomes (Xerome)-the set of transcripts that are stress specific-in higher plant models, where genomic sequencing remains incomplete. EST collections need to be tried to identify stress-induced genes of known and unknown function solely on the basis of their expression profiles using microarray hybridization technology . This will help assess gene family redundancy and complexity, which will facilitate targeted gene disruption strategies in

Arabidopsis and other plants. The most rewarding will be to monitor and compare stress-induced gene expression profiles among different related stresses and in specific mutants. This will allow capturing individual "transcriptome profiles" or stressing fingerprints and discern interrelationships or connections relating stresses or mutants not immediately obvious through the study of one or few genes. Studies on the developmental regulation of genes whose expression is induced by stresses may not only help reveal how environmental conditions interact with developmental processes, but may also provide clues to their function by uncovering where and when they are required, moreover, if these genes are going to be expressed in heterologous pl ants to increase the stress tolerance, their developmental expression must be fully characteri zed. Isoforms of gene families of plant potassi um channels may function in a tissue or cell specific manner. Changes in combination or isoform abundance along the plant axis may playa crucial role in K+ homeostas is which otherwise is impossible to gauge by measuring the activity of individual proteins (Su et ai, 2001). LCn is a potentially important pathway for Na+ and Cs+ uptake in wheat and thus a rel iable target for molecular engineering of salt tolerance or exclusion of radioactive caesium. Molecular identity of Ca2+ and Na+ transport pathway could explain the limited success in the breeding of salt tolerant varieties (Amtmann et ai, 2001). More work needs to be done regarding the potential ability of nitric oxide (NO) to enhance plant fitness to withstand environmental constraints like drought. This could probably be through multiple factors that directly or indirectly result in better housekeeping to make use of the available water (Carlos & Lorenzo, 2001). Yeast and animal ADFs interact with actin and this activity is regulated by phosphorylation and binding to the potent secondary messenger PIP2 (Yonezawa et ai, 1990; Morgan et ai, 1993). It remains to be determined if this is also the case with TaADF. The stress sensing sequences, regulatory elements and stress-inducible genes imparting tolerance against abiotic stresses, once identified and cloned from different organisms, can be used to develop transgenic crops . These may be profitably cultivated in stress prone areas.

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Abiotic Stress Related Genes and their Role in Conferring...

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