Review Articles

Models and Tools for Studying Drought StressResponses in Peas

Katalin Magyar-Tabori, Nora Mendler-Drienyovszki, and Judit Dobranszki

Abstract

The pea (Pisum sativum L.) is an important pulse crop but the growing area is limited because of its relatively lowyield stability. In many parts of the world the most important abiotic factor limiting the survival and yield ofplants is the restricted water supply, and the crop productivity can only be increased by improving droughttolerance. Development of pea cultivars well adapted to dry conditions has been one of the major tasks inbreeding programs. Conventional breeding of new cultivars for dry conditions required extensive selection andtesting for yield performance over diverse environments using various biometrical approaches. Several mor-phological and biochemical traits have been proven to be related to drought resistance, and methods based onphysiological attributes can also be used in development of better varieties. Osmoregulation plays a role in themaintenance of turgor pressure under water stress conditions, and information on the behaviour of genotypesunder osmotic stress can help selection for drought resistance. Biotechnological approaches including in vitrotest, genetic transformation, and the use of molecular markers and mutants could be useful tools in breeding ofpea. In this minireview we summarized the present status of different approaches related to drought stressimprovement in the pea.

Introduction

The pea (Pisum sativum L., family Fabaceae), is a multi-purpose crop cultivated all over the world (Mendler-

Drienyovszki and Dobranszki, 2011; Nisar et al., 2008). Peasare the third most important pulse crop following soybeansand beans, and grown in over 6 million hectares in the world(FAO, 2009; www.faostat.fao.org). The pea crop is consumedas dried seed (mainly for animal feed) or in the immature statefor human consumption as fresh, canned, or frozen pea (McPhee, 2007).

The water requirements of the pea is relatively high duringgrowing season; the critical stages are the initial development(germination) and the flowering. After flowering, during thepod-filling phase the sensitivity of peas to drought stress is muchless (Neumann and Aremu, 1991). Drought stress appearingduring flowering resulted in yield losses mainly due to the lowernumber of pods per stalk (Neumann and Aremu, 1991).

There are large climatic variations between pea croppingareas, between years, and even within a cropping year (An-nicchiarico and Iannucci, 2008). Drought is one of the mostlimiting factors in pea cropping in many parts of the world,affecting both quality and quantity of the yield (Ali et al., 1994;Boyer, 1982). According to estimations, about 90% of arablelands suffer from one or more environmental stresses, and the

prediction is that water deficits will be the major abiotic factor(Dita et al., 2006).

Although precipitation during flowering has been provento be the most important factor in seed yield in Narits andKeppart’s experiments (2010), peas can suffer from droughtduring both the vegetative phase or during reproductive de-velopment. Both early (Ali et al., 1994) and terminal droughtmay be severe depending on cropping environments (An-nicchiarico and Iannucci, 2008), thus, occurrence and distri-bution of rainfall can also affect the degree of losses (Stoddardet al., 2006).

Development of new varieties with wide adaptation abilityincluding drought tolerance is the primary aim of peabreeding works (Abd-El Moneim et al., 1990), and it is nec-essary to increase competitiveness of legumes (Dar andGowda, 2010). Tolerance to drought is a complex phenome-non in which different adaptations are involved (Sanchezet al., 2001); thus, it is very important to unravel mechanismsleading to drought tolerance and improvement of crop plantperformance under drought circumstances.

Models

Plants can perceive environmental challenges includingbiotic and abiotic stresses and response to stress by an

Research Institute of Nyıregyhaza, Research Institutes and Study Farm, Center for Agricultural and Applied Economic Sciences,University of Debrecen, Nyıregyhaza, Hungary.

OMICS A Journal of Integrative BiologyVolume 15, Number 12, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/omi.2011.0090

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appropriate complex defense mechanism including alteredmetabolism, growth, and development (Bartels and Sunkar,2005; Bornberg-Bauer, 2009).

When mild soil water deficit occurs, plants can maintainphotosynthesis and turgor for a short period (Boyer, 1970;Morison et al., 2008.). However, with extended periods ofnegative soil water potential, the growth of plants is inhibited,the uptake and transport of mineral nutrition in plants aredisturbed, and concurrently, the photosynthetic activity de-creases (Boyer, 1970; Jewell et al., 2010). Simultaneously, mor-phological and physiological changes occur to decrease theevaporation of leaves; finally, some leaves will downfall andthe remainder can produce wax on their surfaces (Toldi andJenes, 2000). Drought stress can be accompanied by oxidativedamage, resulting in changes in chlorophyll fluorescence,membrane stability, and peroxidase levels. Morphological,physiological, and biochemical responses of plants are re-viewed by Anjum et al. (2011).

Although the plant height and leaf area were not influencedsignificantly, drought stress decreased the fresh and dryweight of the pea and especially the relative leaf water con-tent. The decrease in relative leaf water content was the mainfactor in reduced growth in drought-treated plants (Alexievaet al., 2001).

Moreover, in the case of pulse crops the nitrogen-fixingbacteria can also be affected by drought stress. Symbiotic N2

fixation decreased under drought stress due to severalphysiological changes accompanying water stress: sucrosesynthase activity of nodule decreased in addition to thedecreasing levels of UDP-glucose, glucose-1-phosphate,glucose-6-phosphate, and fructose-6-phosphate and adenylatecontent in the nodule. Activity of NADP( + )-dependent iso-citrate dehydrogenase increased as a result of water stress maybe compensating for a possible C/N imbalance and/orsupplying NADPH in circumstances where the pentosephosphate pathway was impaired (Galvez et al., 2005).Moreover, Frechilla et al. (2000) compared the effect ofwater stress on pea plants differing in their nitrogen nu-trition (nitrogen fixation or nitrate assimilation), and theyfound that nodulated plants were less sensitive to droughtconsidering the inhibiton of the growth, stomatal conduc-tance, and internal CO2 concentration. However, glycolateoxidase (a key enzyme in the photorespiratory cycle) de-clined by 50% only in nitrogen-fixing plants. They con-cluded that nitrogen source was the major factor affectingpea responses to water stress but the difference in sensi-tivity was related to complex interactions with photo-respiratory flux and stomatal conductance and not to thenitrogen assimilation (Frechilla et al., 2000). Physiologicalresponses of legume nodules to drought are summarized byArrese-Igor et al. (2011). Moreover, inoculation of peas withrhizobacteria containing 1-aminocyclopropane-1-carbox-ylic acid (ACC)-deaminase could help in eliminating theinhibitory effects of water stress on the growth of peas(Zahir et al., 2008). Ethylene synthesis in plants is increasedby several biotic and abiotic stresses, and its production isoften related with reduced growth and premature senes-cence and may be an indicator of plant susceptibility tostresses (Morgan and Drew, 1997). The ACC deaminaseenzyme catalize the reaction, in which lowering of theethylene levels occurs by hydrolysis of ACC, the immediateprecursor of ethylene in plants (Shah et al., 1998).

When water deficit appears, availability of water to cellsdecrease; this reduced availability of water could be quanti-fied as a decrease in water potencial (Verslues et al., 2006).Physiological processes of cells take place in aqueous ambi-ence; thus, when water deficit occurs and the water poten-tial falls (Cw) the cells start to accumulate inorganic ionsand to synthetize special molecules (proteins, proline, man-nitol, sorbitol, etc.) to withold water within the cells. Theprocess, called osmotic adjustment, leads to reduction of os-motic potential of cells (Cs), which in turn, attracts water intothe cell for maintenance turgor pressure (Sanchez et al., 2004).Reduction of turgor pressure induced by drought signifi-cantly inhibits the cell elongation; thus, cell growth is one ofthe most drought-sensitive physiological processes (Anjumet al., 2011). Accumulated solutes (known as compatible sol-utes or osmolyts) may play a role in protection of macro-molecules in dehydrating cells beside turgor maintenance(Smirnoff, 1998).

Drought stress often results in increased transport ofpolyols (sugar alcohols), which are the reduced form ofaldose and ketose sugars such as mannitol and sorbitol. Theyare osmotically active solutes in response to abiotic stressand they can accumulate in high concentration in the cell tocompensate for reduced cell water potential. Their hydroxylgroups could replace water in establishing hydrogen bondsin the case of water deficit; thus, they play a role in theprotection of enzyme activities and membranes (Noiraudet al., 2001). Accumulation of soluble sugars in pea epicotylswas the main factor in osmotic adjustment (34–46%), whilecontribution of increment of free proline content to osmoticadjustment proved to be much less (3–5%) (Sanchez et al.,2004). Sucrose content of seeds was also increased bydrought (Sorensen et al., 2003). Relationship between yieldperformance under drought stress and osmoregulationcapability of pea genotypes has often be proven (Neumannand Aremu, 1991; Rodriguez-Maribona et al., 1992). Corre-lation between growth and osmotic adjustment and turgormaintenance was observed on pea seedlings under waterstress induced by 46 mM polyethylene glycol (PEG) 6000(Sanchez et al., 2004). Measurements of turgor maintenancemade at the early stages of development could be used toidentify drought-tolerant genotypes (Sanchez et al., 2004).

Drought can result in changes in growth, yield, membraneintegrity, pigment content, osmotic adjustment water rela-tions, and photosynthetic activity in plants (Anjum et al.,2011). When pea plants suffer from drought they showedthe greatest reductions in the rate of photosynthesis, transpi-ration, and glycolate oxidase activity (78, 83, and 44%, re-spectively) (Iturbe-Ormaetxe et al., 1998; Moran et al.,1994). Water stress also inhibited the activities of catalase,dehydroascorbate reductase, and glutathione reductase (72–85%) but increased the activities of nonspecific peroxidaseand superoxide dismutase (32–42%) (Moran et al., 1994), andIturbe-Ormaetxe et al. (1998) found that mild water stresscaused an increase in zeaxanthin, malondialdehyde, oxidizedproteins, and mitochondrial, cytosolic, and chloroplastic su-peroxide dismutase activities. Less than 20% reductions werefound in the contents of chlorophyll a, carotenoids, and sol-uble protein (Moran et al., 1994.). According to Iturbe-Ormaetxe et al. (1998), severe water deficit almost completelyinhibited photosynthesis and damaged the photosyntheticapparatus.

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Traits Related to Drought Tolerance

Morphological and biochemical traits

The shoot-to-root ratio of drought-resistant cultivars(‘‘Gobo,’’ ‘‘Solara’’) were significantly smaller than that ofsensitive plants in both control and drought treatments(Grzesiak et al., 1997). Dwarf type pea plants (‘‘Progress’’ No.9) were more drought resistant compared to tall phenotype(‘‘Alaska’’) in Iwaya-Inoue et al. (2003) experiments. Water ofdwarf control and no-elongation zone of GA1-treated ‘‘Pro-gress’’ No. 9 grown under light condition was more sustainedduring 3-h air drying compared to those of ‘‘Alaska’’ (Iwaya-Inoue et al., 2003).

Several experiments were conducted to compare thedrought resistance of conventional leaf and semileafless culti-vars. The leafy cultivar ‘‘Bohatyr’’ produces significantly moreleaf area than semileafless ‘‘Grafila’’ (Semere and Froud-Williams, 2001). Even though in their experiments the leafy pea‘‘Bohatyr’’ showed greater root and shoot competitive abilitiesthan semileafless ‘‘Grafila,’’ the latter types (leaflets are re-placed by tendrils) are reputed as more tolerant to water deficitthan conventional leafed varieties, and it was supposed that thereduced leaf area of the semileafless varieties is the main factor.Gonzalez et al. (2001) studied the background of phenomenaand they revealed that total leaf area and transpiration rate perplant were not significantly different. Osmolarity also did notdiffer among different leaf structure at tissue level, whereas atthe epidermal vacuole level tendrils of the semileafless had ahigher osmolarity than those of conventional pea. On thesemileafless plants the tendrils are about 40% of the total leaf;thus, its more efficient osmotic adjustment may be involved inwater use efficiency under water deficits (Gonzalez et al., 2001).However, under water deficit only stipules of semileafless peaplants showed significantly better ability to increase osmolarityby accumulation of potassium, magnesium, and chloride inmore than other leaf structures (Gonzalez et al., 2002).

Epicuticular wax also plays important role in control theloss of water from the cuticle. Sanchez et al. (2001) measuredthe leaf epicuticular wax load in 20 pea cultivars and they didnot found differences between semileafless and conventionalleafy cultivars. It is varied between 0.19 and 0.41 g m - 2 anddepended more on crop year than cultivars, whereas thesemileafless types showed a greater residual transpirationrate than conventional leafy cultivars. Finally, there was nocorrelation between residual transpiration rate and wax load.However, under drought conditions the wax load of cultivarsincreased significantly and it was accompanied by increasedresidual transpiration rate.

Under drought stress the roots of field pea (‘‘Profi’’) growndeeper in the soil than those under irrigated conditions: about34% of the total pea roots were deeper than 0.23 m in the drysoil, whereas only about 20% of roots rooted in this depth ofsoil profile under irrigated conditions (Benjamin and Nielsen,2006). However, osmotic stress induced by PEG 6000 resultedin shortening of primary root and increase of lateral rootnumber (Kolbert et al., 2008).

Drought stress reduced the seed number in an intensity-dependent manner and the distribution of seeds was also af-fected: more seeds developed on the basal phytomers ofdrought-stressed pea plants than on control plants (Guilioniet al., 2003). Water deficit begun 1 week after forming the firstpods resulted in 79% fewer seeds than in the controls (de

Sousa-Majer et al., 2004). Net photosynthesis (Pn) was alsodecreased by water deficit only during the stress period. Re-lationship was revealed between final seed number and plantgrowth rate during critical period for seed set suggesting thatpea can adjust the number of reproductive sinks in a balancewith assimilate availability in the plant (Gulioni et al., 2003).Although yield was reduced when drought stress existsduring flowering and pod filling, the size distribution of seedswas not affected consistently (Sorensen et al., 2003).

Crop-growing areas with low rainfall representing theshort season environment, and field pea can performs wellunder these conditions if the crop flowers early, then filling ofpods occurs when plant water status is still adequate (droughtescape mechanism); thus, development of genotypes withvigorous early growth, flowering, and pod set are necessary.However, the yield performance of early-flowering genotypescan be low (Khan et al., 1996).

The chlorophyll content slightly increased, while theamounts of anthocyanins were not affected in drought-stressedpea plants. The soluble phenols in leaves increased markedlyunder drought stress (Alexieva et al., 2001). Drought stress ledto full disruption of the chiral macroaggregates of the light-harvesting chlorophyll a/b pigment–protein complexes(LHCIIs) measured by circularly polarized chlorophyll lumi-nescence (CPL) in detached pea leaves (Gussakovsky et al.,2002).

Water stress is often modeled by osmotic stress induced byPEG or mannitol. Generezova et al. (2009) studied the effect ofosmotic stress induced by mannitol (0.6 M) on mitochrondialmetabolic activity in etiolated pea seedlings and found thatthe growth of epicotyl and the water content of tissue weredecreased. At the level of mitochondria the oxidation rates ofmalate and other respiratory substrates were also decreased.The greatest inhibition was found in the rate of proline oxi-dation (by 70%).

Osmotic adjustment

Morgan (1983) first selected for superior osmotic adjustmentunder dehydrating conditions and observed improved yieldsin wheat. Even though Khan et al. (1996) found only a littlecorrelation between osmotic adjustment ability of pea geno-types and their yield performance under water-limited condi-tions, the most successful cultivar (‘‘Dundale’’) showed verygood osmotic adjustment. Moreover, osmotic adjustmentcould also be a useful trait by extending the period of favorablewater relations during pod fill (Khan et al., 1996).

Osmotic stress tolerance of different pea genotypes can beevaluated in in vitro experiments at the tissue level. Magyar-Tabori et al. (2009) tested eight field pea genotypes on mediawith 2.5, 5.0, 7.5, and 10.0% PEG, and they observed the rel-ative growth rate of shoots and the multiplication rate ofinitial explant. All observed parameters were inhibited byPEG, although the PEG concentrations of 5.0 and 7.5% werethe best treatments to classify genotypes into different toler-ance groups (Fig. 1). Osmotic tolerance of these genotypeswas also tested in other in vitro experiments at the cell level:the growth of callus cultures of the same genotypes weretested on media with osmoticum (mannitol in concentrationof 0.2, 0.6, and 1.0 M). The highest concentration was provento be the most efficient to distinguish genotype according totheir osmotic stress tolerance.

STUDYING DROUGHT STRESS RESPONSES IN PEAS 831

Oxidative damages and antioxidant capacity

Drought stress as other environmental stress can be ac-companied by oxidative damage including accumulation ofactivated oxygen forms (single oxygen, O2

1; superoxide rad-ical, O2

- ; hydrogen peroxide, H2O2, and hydroxyl radical,HO - ) in high concentration, resulting in changes in chloro-phyll fluorescence, membrane stability, and peroxidase levels.Although reactive oxygen species (ROS) control many pro-cesses in plants and also participate in signaling events, theyare toxic molecules and can injure cells (Mittler, 2002; Mittleret al., 2004). Producing, scavenging, and avoiding reactiveoxygen intermediates have been summarized by Mittler (2002).Among mechanisms that might reduce reactive oxygen inter-mediates the antioxidants (catalase, ascorbate, superoxidedismutase, etc.) play a significant role in elimination of reactiveoxygen molecules. In drought-stressed peas the specific activ-ities of antioxidant enzymes content increased (Alexieva et al.,2001). Catalase and SOD (superoxide dismutase) activitieswere inhibited, whereas peroxidase activity was stimulated,while hydrogen peroxide level increased in drought-stressedpea plants (Alexieva et al., 2001). Gluthatione synthesis is alsoinduced by oxidative stress (Smirnoff, 1998).

Mitochondria are the significant source of cellular ROS andoxidative damage of organelles disturbs the energy supply ofcells required for repair mechanism (Taylor et al., 2005).Taylor et al. (2005) studied the effect of environmental stresseson the pea (‘‘Green Feast’’) mitohocondrial proteome (entireset of proteins expressed by the genome). The drought re-gimes they used (plants were not watered for 7 days) did not

cause accumulation of lipid peroxidation end products sig-nificantly above controls, but drought treatment clearly af-fected leaf metabolism (the rates of dark respiration andespecially net photosynthesis were significantly decreased)and caused oxidative modification of mitochondrial proteins.However, in mitochondria isolated from stressed pea leavesthe assessment of lipoic acid moieties on mitochondrial en-zymes showed that drought treatment decreased significantlythe lipoic acid moieties apparent on the H protein of gly-cine decarboxylase (GDC-H), while lipoic acid moieties onboth the pyruvate dehydrogenase complex (PDC) and the2-oxoglutarate dehydrogenase complex were less affected.

Changes in phytohormone levels

Activity of cytokinins decreases in response to drought(Hare et al., 1997), while level of abscisic acid (ABA) increases(Morgan, 1990). Cytokinin oxidase/dehydrogenase enzymesmaybe is responsible for the changes in the cytokinin poolunder adverse environmental conditions (Vaseva-Gemishevaet al., 2005). Vaseva-Gemisheva et al. (2005) measured theexpression of two putative cytokinin oxidase/dehydrogenase(CKX) genes (PsCKX1, PsCKX2) in response to different stressin the leaf and root tissue of ‘‘Manuela’’ pea plants by real-time RT-PCR. They observed an increased PsCKX1 mRNAexpression in leaves of drought-stressed plants; however, themeasured CKX activity in drought-stressed plants was in-hibited in leaves and was above the control in roots. BecauseCKX enzyme is substrate-inducible it was supposed that de-creased cytokinin content of water-deprived plants could be

FIG. 1. Growth inhibition induced by increasing PEG levels in different laboratory experiments. (A) Young seedlings of‘Baccara’ grown in PEG 600 solutions; (B) seedlings of ‘Baccara’ germinated on filter paper soaked by PEG 600 solutions(concentration of PEG solutions was increasing from left to right from 0% up to 20%); (C) in vitro shoot culture of different peagenotypes developed on media with PEG 600 (PEG concentration of media was increasing from left to right from 0% up to10%). 190 · 253mm (200 · 200 DPI).

832 MAGYAR-TABORI ET AL.

the cause of the contrary results. Moreover, measurementswere made after a short period of water stress (4 days).

The phytohormone ABA is also involved in the response toenvironmental stresses such as drought, salt, and cold (Buskand Pages, 1998). The main functions of ABA are the regula-tion of plant water balance and osmotic stress tolerance. Therole of ABA in cellular dehydration tolerance including in-duction of genes that encodes dehydration tolerance proteinsin cells (Zhu, 2002). ABA is also essential for stomatal closure,stress-responsive gene expression, and metabolic changes(Seki et al., 2007). ABA accumulation induced by osmoticstress is a result of both activation of synthesis and inhibitionof degradation (Zhu, 2002). ABA aldehyde oxidase catalyzesthe last step of ABA biosynthesis. Three isoforms of aldehydeoxidase (AO) were detected in pea plants; among them, onlyPAO-3 seemed to be responsible for stress-induced ABA pro-duction (Zdunek-Zastocka et al., 2004). In drought-stressedpea plants seedlings especially showed increased PsSNF5(a member of SNF5 family of chromatin remodeling factors)expression, and it was assumed from the results that this geneinvolved in ABA response (Rıos et al., 2007). Although thestress-induced pea DNA helicase 47 (PDH47) was proven toplay role in both the ABA-dependent and ABA-independentpathways in abiotic stress, its transcription was not inducedunder drought stress (Vashisht et al., 2005).

Because exogenously applied brassinosteroids (BRs) canincrease the resistance of plants to water stress, Jager et al.(2008) studied BR mutants in the pea to determine whetherchanges in endogenous BR levels are involved in drought stressresponses. They observed that in wild-type plants the waterstress did not result in altered BR levels; moreover, the ABAlevels in response to water stress were also not affected by BRdeficiency. They concluded that in pea the changes in endog-enous BR levels did not play role in response to drought.

Osmotic stress induced by PEG 6000 led to a significantincrease of nitric oxide (NO) generation in pea roots. It wassupposed that the initial phase of NO generation may play arole in the osmotic stress-induced signalization process lead-ing to the modification of root morphology (Kolbert et al.,2008). The role of NO in stress responses in plants includingNO reactions, signaling pathways, NO plant hormone inter-actions, and NO-induced and -mediated signalization underosmotic stress are detailed by Erdei and Kolbert (2008) in theirreview.

Tools

Conventional methods

Improvement of abiotic stress tolerance of crops by tradi-tional breeding methods is limited by the multigenic nature ofthe trait (Bartels and Sunkar, 2005). However, stress tolerantcrops have been bred mostly by introducing traits from stress-adopted wild relatives (Bartels and Sunkar, 2005). Fieldscreening of genotypes for stress-response often involvesstudying them in contrasting conditions and estimating theirsusceptibility from their relative yield in different environ-ments. The inhibition of relative growth rate (including chan-ges in dry matter of both the above-ground parts and roots andlength of shoots and roots of seedling) was smaller in droughtresistant than in sensitive cultivar (Grzesiak et al., 1997).

Because the seriousness of the drought stress is unpre-dictable in field experiments and field selection for wide ad-

aptation requires long time and much cost, they areincreasingly supplemented with experiments in a controlledenvironment, and focus on methods could be applied in earlyselection stages (Annicchiarico and Iannucci, 2008). Thesekinds of experiments include mostly evaluation of germi-nation and growth of seedling or whole plant under os-motic stress induced by molecules with osmotic effect (PEG,mannitol).

Sanchez et al. (1998) tested 49 pea genotypes both in a fieldcondition and growth chamber for drought tolerance. In thefield experiment the grain yield was 25% less in nonirrigatedconditions than in the control. Genotypes with better turgormaintenance had higher yield but the trend of biomass pro-duction was opposite. Drought-tolerant pea genotypes hadbetter turgor maintenance, which was significantly related toosmotic adjustment (Sanchez et al., 1998).

Sanchez et al. (1998) studied the soluble sugar and prolineaccumulation measured in the leaves of pea plants subjectedto a period of dehydration. They found that accumulation ofsoluble sugars was significantly correlated to osmotic ad-justment and their concentrations were significantly higher ingenotypes with conventional leaf type than in the semileaflessgenotypes in both control and stress conditions. Moreover,the amount of carbohydrates increased proportionally to os-motic adjustment in all genotypes when exposed to stress.Sugars can also stabilize proteins and membranes, as osmo-protectors (Crow et al., 1992). The level of free proline wasalmost constant at high water potential and significantly in-creased when water potential decreased. However, its con-tribution to osmotic potential of leaf was only 0.5–1.8%(Sanchez et al., 1998).

Significantly increased levels of the metabolites indrought-stressed plants were detected including proline,valine, threonine, homoserine, myoinositol, c-aminobuty-rate, and trigonelline (Charlton et al., 2008).

Because drought is manifested primarly as osmotic stress,resulting in the disruption of homeostasis and ion distributionin the cell (Serrano et al., 1999), several experiments have beenconducted by induction of osmotic stress to study responsesof plants to stress. The high molecular weight PEG is com-monly used to induce water deficits and to study the ability ofdifferent genotypes to tolerate stress in other species (Foitoet al., 2009; Kie1kowska and Adamus, 2009; Orlikowska et al.,2009; Rakosy-Tican and Maior, 2009), as well as in peas(Sanchez et al., 2004; Singh et al., 1990; Singh & Singh, 1992)because of its ability to induce water stress.

Seed germination is a very important stage of development,determining successful crop production, and the early ger-mination stage is very sensitive to osmotic stress (Almansouriet al., 2001; Dobranszki et al., 2006); thus, several studies wereconducted in this phase.

Osmotic stress induced by PEG inhibited the germinationand seedling growth in all cultivars and under each osmoticpotential (from - 2 to - 8 bars) tested, but the rate of inhibitionvaried between genotypes (Okcu et al., 2005). The growth ofradicle was the most inhibited by water stress, although thelength of coleoptyle and the rate of germination were alsoaffected. The best screening method for drought tolerance ofgenotypes was the evaluation of differences in the length ofradicle at the level of - 6 bar of water potential (Marjani et al.,2006). The same level of mannitol ( - 6 bar or - 0.6 MPa) wasfound to be appropriate to detect differences in the seed

STUDYING DROUGHT STRESS RESPONSES IN PEAS 833

germination parameters of dry peas with different droughttolerance (Grzesiak et al. 1997).

Brosowska and Weidner (2011) studied the effect of os-motic stress on the formation of a population of polysomesand their stability in pea seeds. They found that the long-termosmotic stress inhibited the germination of seeds and thegrowth of embryos and seedlings proportionally to its inten-sity. Under osmotic stress the free polysomes were presentdominantly, whereas under intensive stress the share themembrane-bound and cytoskeleton-bound and cytoskeleton-membrane polysomes among total fraction of polysomes in-creased significantly. In early selection of pea breeding linesthe chlorophyll a fluorescence signals analysed with the self-organizing map (SOM) can be used as a routine tool for themonitoring their degree of resistance against drought stress(Maldonado-Rodriguez et al., 2003).

Biotechnological Tools

The yield performance of several crops can not be furtherincreased by traditional breeding methods; consequently, it isnecessary to increase the efficiency of breeding work bycombining all available tools including biotechnological pos-sibilities (Metzlaff, 2009). Application of biotechnologicaltools requires both biological knowledge of target species andthe mechanisms of stress tolerance (Dita et. al., 2006).

Molecular approaches

During molecular control of abiotic stress tolerance specificstress-related genes are activated and regulated; moreover,the activation of cascades of molecular networks is included(Vinocur and Altman, 2005; Wang et al., 2003). The role ofbiotechnological methods becomes more and more importantas genes involved in stress resistance are cloned and theirmode of action unravelled (Smirnoff, 1998). Novel breedingmethods includes the investigations of the cellular bases ofstress resistance and improvement of stress resistance bymaking transgenic plants with increased stress tolerance(Toldi et al., 2010). Investigation of mutants and transgenicplants with altered expression of genes involved in droughttolerance can significantly enhance to understand the molec-ular background of stress resistance (Smirnoff, 1998). Weigeltet al. (2009) studied the specific transcriptional and metabolicchanges of carbon–nitrogen metabolism in ADP–glucosepyrophosphorylase-deficient pea embryos (IAGP-3). Theyfound that IAGP-3 seeds displayed upregulation of severalgene expressions related to stress responses and osmoticstress signaling. Regulation of water evaporation is controlledby stomata, and Ghasemi et al. (2010) reported that lip1 geneis involved in regulation of stomata aperture.

Genetic composition of the pea is about 4,800 Mbp spreadacross 2n = 2x = 14 chromosomes (Mc Phee, 2007). Recently,genes for several morphological traits have been mapped (McPhee, 2007) and advancement in biotechnology approaches toovercome biotic and abiotic stress in legumes have been re-ported (Dita et al., 2006). Relatively few quantitative traitlocus (QTL) analyses in pea have been reported so far, andthey associated mainly to biotic stress (72 QTL for 11 traits)and none of them related to abiotic stress (Dita et al., 2006; McPhee, 2007).

The use of molecular markers for the indirect selection ofbreeding lines shortens the time required for selection process

compared to direct screening under greenhouse and fieldconditions (Dita et al., 2006). However, several factors shouldbe considered such as level of polymorphism between pa-rental lines, unclear expression of markers, false positivemarkers, discrepancy between the presence of the marker andtarget gene, and the presence of multiple genes scattered overseveral linkage groups (Dita et al., 2006).

Transgenic approaches

Up to the present the aim of genetic transformation in thepea were principally to prove the potential for transformationand establish a functional system for genetic transformation.During these experiments mainly antibiotic and herbicideresistance genes (npt II, hpt, bar, and all) were incorporatedinto the pea. Although difficulties can occurs associated withregeneration during organogenesis, several successful genetransformation with agronomic importance have been re-ported including incorporation of genes of virus coat proteins,a-amilase inhibitor (Mc Phee, 2008). Because the geneticallymodified pea has a relatively low risk to the environment (lowlevels of outcrossing < 1%), the gene transformation could bea useful tool in pea breeding for drought tolerance (Mc Phee,2008). Moreover, the genetic engineering would be the onlyoption when genes responsible for drought tolerance originatefrom species, by which sexual hybridization would be im-possible (distant relatives, nonplant sources, etc.) (Bhatnagar-Mathur et al., 2008). However, very few gene transformationexperiments were conducted to improve the drought toler-ance in the pea maybe due to the complexity of plant drought-resistant mechanisms at the whole plant, cellular, metabolic,and genetic levels ( Jewell et al., 2010).

Several stress-induced proteins exist with known function(enzymes for osmolyte biosynthesis, detoxification enzymes,and others); after genetic characterization these metabolictraits seemed to be suitable for manipulation to improve stresstolerance but the abiotic stress tolerance can involve manygenes at a time. However, expression of the cDNA encodingDREB1A (transcription factor) from the stress induciblerd29A promoter in transgenic Arabidopsis plants activatedthe expression of many stress-tolerant genes and resultedin improved tolerance to drought, salt loading, and freez-ing (Kasuga et al., 1999). In this way, by manipulation of asingle gene encoding stress-inducible factor many genesinvolved in stress response could simultaneously be regulated(Bhatnagar-Mathur et al., 2008).

Genes responsible for production of osmoprotectants(mt1D, P5CR, P5CS) were cloned in many cases and one ofthem (mt1D, mannitol phosphate dehydrogenase enzyme)was introduced using by GENEBOOSTERTM particle accel-erating device into the genotype ‘‘Akt’’ in order to improve itsdrought tolerance. A total of 36 putative transgenic plantswere regenerated on selective medium. Evaluation of re-generants is in process (Molnar, 2008).

Conclusions

Improvement of pea genotypes for water stress tolerancehas been facing difficulties due to the complex characteristicof drought tolerance. Factors in breeding for plant tolerance todrought can be manifested either as stress tolerance or asstress avoidance. Genetic engineering of specific genes canresult in tolerance, whereas avoidance strategies, such as

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plant morphological, anatomical, and physiological changes,can be reached by using conventional breeding methods, in-cluding QTL analysis (Vinocur and Altman, 2005).

The screening methods should be nondestructive andbased on simple selection criteria including traits that allow arapid and accurate selection when compared to field perfor-mance (Saxena et al., 1994; Serraj et al., 2003; Wery et al., 1994).Traditional breeding methods incorporate selection for traitssuch as morphological and developmental characters andmore frequently are based on osmotic adjustment ability.Recently, the newly revealed physiological changes inducedby osmotic stress have also been used as selection criteria. Themajority of physiological stresses result in disturbance inplant metabolism and cause oxidative damages by enhancingthe production of ROS. Resistance of plants to abiotic stressis related to their antioxidant capacity; thus, the increasedlevels of the antioxidant molecules can help in prevention ofstress damage (Monk et al., 1989).

Gradual acclimation of sensitive plants can result in in-creased tolerance, suggesting that genes responsible for tol-erance are also present in nontolerant plants, and duringgradual adaptation enhanced expression of these genesoccurs. Moreover, it is supposed that stress-relevant genes arepresent in the whole plant kingdom (Bartels and Sunkar,2005).

Clearing up the genetic background of the changes inphysiological processes related to stress resistance can effi-ciently be utilized in pea breeding work, and possible epige-netic modifications of gene expression should also be takeninto consideration (Metzlaff, 2009). However, the literature onthe biochemical nature of the responses is still very few. Somephysiological parameters especially traits for osmotic adjust-ment ability have been proven to be suitable for screeninggenotypes for abiotic stress.

Even though biotechnological methods are known aspromising tools for improvement of stress resistance of plantsvery few advancements in the pea have been reported to date.

Author Disclosure Statement

The authors declare that no conflicting financial interestsexist.

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Address correspondence to:Katalin Magyar-Tabori

Research Institute of NyıregyhazaResearch Institutes and Study Farm

Center for Agricultural and Applied Economic SciencesUniversity of Debrecen

P.O. Box 12H-4400, Nyıregyhaza, Hungary

E-mail: mtaborik@freemail.hu

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