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Journal of Plant Physiology 168 (2011) 671–677

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l homepage: www.e lsev ier .de / jp lph

roup 1 and 2 LEA protein expression correlates with a decrease in water stressnduced protein aggregation in horsegram during germination and seedlingrowth

ounipalli Veeranagamallaiaha, Jonnakuti Prasanthib, Kondreddy Eswaranarayana Reddyb,erum Pandurangaiahb, Owku Sudhakara Babub, Chinta Sudhakarb,∗

Department of Botany, Yogi Vemana University, Kadapa 516 003, IndiaDepartment of Botany, Sri Krishnadevaraya University, Anantapur 515 003, India

r t i c l e i n f o

rticle history:eceived 20 June 2010eceived in revised form4 September 2010ccepted 26 September 2010

eywords:orsegram

a b s t r a c t

Plants produce an array of proteins as a part of a global response to protect the cell metabolism when theygrow under environmental conditions such as drought and salinity that generate reduced water potential.The synthesis of hydrophilic proteins is a major part of the response to water deficit conditions. Anincreased expression of LEA proteins is thought to be one of the primary lines of defense to prevent the lossof intercellular water during adverse conditions. These LEA proteins are known to prevent aggregation ofa wide range of other proteins. In this study we report the water stress induced protein aggregation andits abrogation followed by expression of group 1 and group 2 LEA proteins of water soluble proteomes in

EA proteinsacrotyloma uniflorum

rotein aggregationater stress

horsegram. Water stress caused an increased protein aggregation with magnitude and duration of stressin horsegram seedlings. Tissue-specific expression of LEA 1 protein decreased in the embryonic axis whencompared to cotyledons in 24 h stressed seedlings. We found no cross reaction of LEA 1 with proteomeof 48 h stressed embryonic axis and 72 h stressed root and shoot samples. However, LEA 2 antibodieswere cross reacted with four polypeptides with different molecular mass in shoot tissue samples and

ot prregat

found no reaction with roin relation to protein agg

. Introduction

Plants are continuously exposed to various abiotic stresses suchs drought, salinity and low or high temperature, which causehe loss of intercellular water. The development of crop plantsble to grow in these adverse conditions assumes importance inhe emerging context of global warming. Proteins whose expres-ion increase during stress, include late embryogenesis abundantLEA) proteins, proteins responsive to abscisic acid, Osmotins, heathock proteins and pathogen related proteins, are the primaryine of defense to prevent the loss of intercellular water during

dverse conditions. Of these, LEA proteins have been known forany years to accumulate in maturing plant seeds as they acquire

esiccation tolerance (Cuming, 1999; Tunnacliffe and Wise, 2007)nd are largely unstructured in solution, because their extreme

Abbreviations: DM, dry mass; FW, fresh weight; HSPs, heat shock proteins; LEA,ate embryogenesis abundant; PEG, polyethylene glycol; PR, pathogenesis related;AB, responsive to abscisic acid; REC, relative water content; RH, relative humidity;M, turgid mass.∗ Corresponding author. Tel.: +91 8554255054; fax: +91 8554255054.

E-mail address: chintasudhakar@yahoo.com (C. Sudhakar).

176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.jplph.2010.09.007

oteome as evidenced from immuno-blot analysis. The role of LEA proteinsion during water stressed conditions was discussed.

© 2010 Elsevier GmbH. All rights reserved.

hydrophilicity favors association with water over intrachain inter-action. They can show increased folding when dried or whenassociated with phospholipids (Wolkers et al., 2001; Goyal et al.,2003; Tolleter et al., 2007).

LEA proteins constitute a very divergent family, which have beenidentified and cloned from many plants, classified at least into sixdifferent groups of LEA proteins on the basis of expression and pres-ence of certain sequence motifs; the major categories are groups1–3 (Bray, 1993; Cuming, 1999; Wise, 2003; Wise and Tunnacliffe,2004). Group 1 LEA proteins are only found in plants and are charac-terized by the presence of a hydrophilic 20-amino acid motif family.Members of group 2 LEA proteins, called dehydrins, are mainlyfound in plants and typically accumulated in dehydrating plant tis-sues in seeds and or in tissues subjected to environmental stressessuch as drought, salinity and low temperature. These group 2 LEAproteins contain three distinct motifs (Y, S and K), whereas group3 LEA proteins have multiple copies of an 11-mer sequence (Wiseand Tunnacliffe, 2004; Tunnacliffe and Wise, 2007).

LEA proteins are generally associated with desiccation stresstolerance in orthodox seeds, pollen and anhydrobiotic plants, butmany LEA proteins are induced by cold stress, osmotic stress and byexogenous application of ABA (Welin et al., 1994). Several molecu-lar functions have been ascribed to LEA proteins, such as protection

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72 G. Veeranagamallaiah et al. / Journa

f cellular or molecular structures from that of water loss. A num-er of putative mechanisms have been proposed, including ionequestration, membrane binding and stabilization, redox balances an antioxidant, buffering of hydrate water, and chaperone activ-ty (Bray, 1993; Cuming, 1999). LEA proteins are known to conferncreased resistance to osmotic or freeze stress when introducednto yeast (Zhang et al., 2000) and similarly barley LEA proteinmproves tolerance to water deficit in transgenic rice (Xu et al.,996) and wheat (Sivamani et al., 2000); furthermore, in vitro, anlgal LEA protein diminished freeze damage of the enzyme lac-ate dehydrogenase (Honjoh et al., 2000). Recently, an atypical LEArotein coding cDNA has been isolated and characterized from therought tolerant Prosopis juliflora (George et al., 2009). The authorshowed that this atypical protein differs from typical LEA in termsf hydropathy and found that unlike typical LEA, these atypical LEAossess alanine as most abundant amino acid followed by serine

ocated near the N-terminus.Although LEA proteins are widely held to protect cells against

ater stress, their precise role is not clear. Recently, Tunnacliffend Wise (2007) obtained evidence supporting the possibility thatome LEA proteins act to prevent other proteins aggregating dur-ng water loss. This is probably because, due to their hydrophilic,nsaturated nature, LEA proteins themselves are not susceptibleo aggregation on desiccation. This anti-aggregation activity could

ean they represent a novel form of dehydration-specific molec-lar chaperone. An alternative explanation is that analogous toolymer stabilization of colloidal suspension; LEA proteins behaves molecular shields that prevent the approach and interaction ofggregation-prone protein species by steric or electrostatic repul-ion (Tunnacliffe and Wise, 2007). Recently it has been noticedhat a nematode recombinant-LEA protein was able to abrogateesiccation-induced aggregation of the water-soluble proteomesrom nematodes and mammalian cells and afford protection dur-ng both dehydration and rehydration in vitro (Chakrabortee et al.,007). Further, when co-expressed in a human cell line, the LEArotein reduced the propensity of polyglutamine and polyalaninexpansion proteins associated with neurodegenerative disease toorm aggregates, demonstrating the in vivo function of a LEA proteins an anti-aggregant. Whether LEA proteins are molecular chaper-nes or molecular shields, we hypothesize that they have a physio-ogical role in protecting a wide range of proteins against aggrega-ion. We asked whether group 1 and group 2 LEA proteins are ableo prevent water stress induced aggregation of a complex mixturef proteins, represented by water soluble proteome of plants.

A pulse crop, horsegram member of Fabaceae, characteristic ofhe semi arid land is extensively cultivated in Rayalaseema regionf Andhra Pradesh, south Karnataka, some regions of Rajasthan andujarat. This crop has potential use as dry fodder and grain legume

eed for live-stock in semi-arid regions and as a cover crop for soilnd water conservation. Horsegram will germinate, grow and yieldarginally better than other pulse crops in receding soil moisture

evels and shows better tolerance to drought and salinity conditionsSreenivasulureddy et al., 1990). In the present study we report theccumulation of water-stress-induced proteins, protein aggrega-ion in vivo, expression of group 1 and 2 LEA proteins and theirmportance in anti-aggregation of other proteins during germina-ion and early seedling stages of horsegram subjected to differentevels of induced water stress conditions.

. Materials and methods

.1. Plant material and stress treatments

Healthy surface sterilized horsegram (Macrotyloma uniflorumLam.) Verde.) Cv. VZM1) seeds were germinated in Petri plates

nt Physiology 168 (2011) 671–677

lined with filter papers. Water stress was induced by usingpolyethylene glycol (PEG 6000) at different concentrations (0(control), 2.5, 5 and 10%) prepared with half strength Hoaglandmedium. The Petri plates were kept in a growth chamber (Conv-iron AR1000, Canada) at temperature of 28 ◦C, relative humidity75%, PAR 1500 �mol/m−2/s for three days. Samples were taken foranalysis at different time intervals i.e. at 12, 24, 48 and 72 h andthree day-old seedlings were separated into root and shoots, flashfrozen in liquid nitrogen and stored at −80 ◦C.

2.2. Relative water content

Fresh germinating horsegram seedlings subjected to differ-ent levels of water stress were weighed record fresh mass (FW).Then seedlings were immersed in distilled water and after 4 hthey were reweighed (turgid mass (TM)) after which they werekept at 80 ◦C for 48 h and dry mass (DM) was recorded. Relativewater content (RWC) was calculated according to Turner (1981):RWC = (FW − DM)/(TM − DM) × 100.

2.3. Protein extraction and estimation

The frozen samples were powdered by using liquid nitrogen andextracted in 50 mM Tris–HCl (pH 7.4) buffer contained 10 mM NaEDTA, 5% �-mercaptoethanol and 10% glycerol. The homogenatewas centrifuged at 5000 × g for 10 min in refrigerated high speedcentrifuge (SIGMA 3K18) to remove the cellular debris. The result-ing supernatant was centrifuged at 10,000 × g for 20 min to obtainclear supernatant. The concentration of proteins was estimated bythe method of Lowry et al. (1951) using bovine serum albumin(BSA) as the standard.

2.4. Protein aggregation

One gram of frozen samples were powdered using liquid nitro-gen and suspended in DTE buffer containing 56 mM dithiothretol,56 mM sodium carbonate, 2 mM sodium EDTA and 1% sucrose.The homogenate was centrifuged at 14,000 × g for 10 min and thesupernatant was collected. To the supernatant 10% trichloro aceticacid was added in 1:20 ratio and kept in an ice bath for 15 min. Theprecipitated proteins were centrifuged at 14000 × g for 5 min, thepellet collected and dissolved in 1 N NaOH.

Water-stress-induced protein aggregation assay was performedaccording to Goyal et al. (2005). All proteins were first dialyzedovernight with several changes of distilled water to remove buffersalts and protein aggregation was assessed by measuring appar-ent absorption at 340 nm in a spectrophotometer (Shimadzu 1601,Japan). Assays were performed in triplicates and the appropriatebuffer solution without proteins was used as blank.

For further quantification and SDS-PAGE analysis, aggregatedproteins were centrifuged at 15,000 × g for 30 min, at 4 ◦C andthe resulted pellet and supernatant containing soluble and insolu-ble proteins, respectively. Pellets were dissolved in buffer. Proteinquantification was performed in both fractions as described ear-lier (Lowry et al., 1951) and SDS-PAGE was carried out by loadingequal amount of protein from each fraction as described by Laemmli(1970).

2.5. Electrophoresis

For one-dimensional separation, 12.5% linear sodium dodecyl

sulphate (SDS)-polyacrylamide slab gels (1 mm thickness) over-layed with stacking gel was used. The electrophoresis was carriedout at 50 V for 1 h followed by 100 V for 2 h. Once tracking dyereached the anode, run was stopped and the gel was carefullyremoved and placed in five volumes of Coomassie briliant blue

G. Veeranagamallaiah et al. / Journal of Plant Physiology 168 (2011) 671–677 673

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Of the low molecular weight proteins, the amount of the ∼16and ∼17 kDa proteins gradually increased with increasing sever-ity and duration of stress. The most noticeable change was in theconcentration of ∼38 kDa protein, the expression of which was notmuch changed up to 5% stress but decreased at 10% stress during

ig. 1. Relative water content of horsegram subjected to different levels of watertress during different time intervals.

CBB) for 1 h or overnight. The gel was washed with the distilledater and destained using methanol, acetic acid and distilled water

n ratio 30:30:40.

.6. Protein gel blot analysis

The immunoblot was performed according to Towbin et al.1979). The proteins separated on SDS-PAGE were transferred to

PVDF membrane (Millipore, India) using semi-dry electroblot-ing apparatus. Anti-sera of LEA 1, raised against Eleusine coracanaGaertn) and LEA 2 raised against synthetic polypeptides corre-ponding to the conserved region of LEA-2 (EEKKGIMKIKEKLPG)ere used in 1:250 dilutions to check their cross-reaction with theroteins isolated from seedlings of horsegram.

Image of the gels and blots was scanned, analyzed and recordedsing Gel Documentation Image Analysis System (Alpha Innotech, Saneandro, USA) computer software.

. Results

.1. Relative water content

Relative water content (RWC) was measured in horsegrameedlings subjected to different levels of water stress during dif-erent time intervals (Fig. 1). RWC was decreased with increasingeverity of stress levels and decease was more significant by 10%tress. Further it was observed that RWC was gradually decreasedith the duration of stress treatment and 72 h water stressed sam-les showed half the RWC of control samples.

.2. Water stress induced protein aggregation

Water stress resulted in a significant aggregation of proteins inorsegram seedlings as measured by a light scattering assay (Fig. 2).ight scattering assay revealed that the protein aggregation wasore pronounced in stressed samples than controls. It was found

hat protein aggregation increased with stress intensity and dura-ion. The maximum increase was noticed at 10% stress condition.owever, the light scattering was decreased during 72 h when com-ared to 12, 24 and 48 h at all stress levels. Further, we observed thathe light scattering was more significant in root proteome whenompared to shoot proteome after 72 h (Fig. 3).

To determine whether a small subset or a broad range of proteins

as susceptible to aggregation upon subjection to water stress,e separated the insoluble, aggregated proteins from the soluble

raction by centrifugation; solubilised in DTE buffer and then ana-yzed both fractions by SDS-PAGE. Small fraction of the proteome

as apparent in the pellet of control samples, consistent with the

Fig. 2. Water stress induced protein aggregation during different time intervals inhorsegram as measured in light scattering assay.

light scattering results, but water stress of 5% and above resulted inpart of the protein sample becoming insoluble. However, a markeddifference was observed in the intensity of some protein bandsbetween soluble fractions and insoluble fractions, suggesting thatmost proteins form aggregates but that not all molecules of anyparticular protein do so (Fig. 4). Further, this water stress inducedaggregation of proteins was confirmed by quantifying the amountof proteins in each fraction (Fig. 5).

3.3. Accumulation of water stress induced proteins at differenttime intervals

In the present study a comparison of total protein profile of con-trol and different levels of water-stressed germinating seeds for12 h and 24 h revealed that water stress resulted in the synthesis ofa few polypeptides and enhanced or repressed that of others (Fig. 6).It was observed that four polypeptides with approximate molecularmass 51, 52, 67 and 95 kDa were increased in their concentrationwith increasing water stress levels. The ∼52 kDa protein accumu-lated more at 10% stress treatments compared to other stress levelsduring 12 h. Though this protein showed a gradual increase in itsexpression and reached maximum at 10% stress during 24 h, butwas lower when compared at all stress levels during 12 h (Fig. 6).Similarly, the four proteins ∼30, 51 67 and 95 kDa were also fol-lowed the same trend with ∼52 kDa protein with increasing stresslevels.

Fig. 3. Water stress induced protein aggregation in root and shoot tissues of horseg-ram during 72 h as measured in light scattering assay.

674 G. Veeranagamallaiah et al. / Journal of Plant Physiology 168 (2011) 671–677

Fig. 4. Coomassie blue-stained polyacrylaminde gel of soluble and insoluble frac-tions of horsegram proteome subjected to different levels of water stress during72 h.

Fig. 5. Total protein content in soluble and insoluble (pellet) fractions of horsegramsubjected o different levels of PEG induced water stress at 72 h.

Fig. 6. Total protein profile of horsegram subjected to different levels of PEG inducedwater stress for 12 h and 24 h during germination. Lane M. Low molecular weightmarker; Lanes 1, 2, 3 and 4 – control, 2.5, 5 and 10% PEG stress, respectively, during12 h; Lanes 5, 6, 7 and 8 – control, 2.5%, 5% and 10% PEG stress, respectively, during24 h.

Fig. 7. Total protein profile of horsegram subjected to different levels of water stress

during 48 h and 72 h. Lane 1. Low molecular weight marker; Lanes 1, 2, 3 and 4 – con-trol, 2.5%, 5% and 10% PEG stress, respectively, during 48 h respectively; Lanes 6, 7, 8and 9 – control, 2.5%, 5% and 10% PEG stress, respectively, during 72 h, respectively.

12 h. In contrary, this protein was not abundant at all stress levelsexcept at 10% stress during 24 h.

The comparison of total protein profile of 24 and 48 h(Figs. 6 and 7) revealed that ∼30 and ∼51 kDa proteins concentra-tion increased with the increase in the levels of stress at 24 and 48 h,but intensity were more at 48 h compared to 24 h. The low molecu-lar weight protein ∼17 kDa was increased at10% stress level whencompared to control, 2.5 and 5% stress levels.

The protein profile of control and stressed seedlings by 72 hwhen compared with 48 h revealed the existence of a significantdifference in the levels of polypeptides, as seedlings were differen-tiated morphologically into root and shoot by 72 h of germination.More specifically proteins with approximate mass of 51, 67 and95 kDa increased with increasing stress level during 48 and 72 h(Fig. 7). However, the intensity of protein bands was greater after48 h than 72 h. The synthesis of a ∼30 kDa protein decreased duringstress treatments at 72 h, although its intensity was high after 48 h.Similarly, others proteins such as ∼27, 28 and 37 kDa decreasedin their accumulation with increased levels of stress. Another pro-tein with molecular weight ∼25 kDa was accumulated more in 5%stress when compared to control and 2.5% and decreased in 10%stress only at 48 h.

3.4. Tissue specific accumulation of water stress induced proteins

In order to evaluate tissue specific accumulation of water-stress-induced proteins, germinating seeds at various time points (48 and72 h) were separated into cotyledons and embryonic axis, root andshoot parts.

Separation of total proteins, isolated from cotyledons andembryonic axis separately, revealed that ∼37, 51, 52, 74 and 95 kDapolypeptides were decreased with increase in the levels of waterstress in cotyledons (Fig. 8). Similarly, a low molecular weight∼19 kDa protein decreased with increase in stress level. Moreover,the ∼21 kDa polypeptide was decreased in 2.5 and 5% stress lev-els when compared to control but was increased in 10% stress incotyledons.

Total proteins in the embryonic axis were decreased in all levelsof stress when compared to cotyledonary proteins during 48 h. Fourpolypeptides including ∼51, 52, 74 and 95 kDa were decreased inembryonic axis with increased stress level compared to the sameproteins in cotyledons. Similarly, proteins with approximate mass∼27, 28 and 37 kDa were also decreased with increasing levels

of stress compared to cotyledonary proteins. The most significantchange observed in the ∼30 kDa protein, which was absent in con-trol and 2.5% stress, expressed at 5% stress, however disappearedat severe stress.

G. Veeranagamallaiah et al. / Journal of Plant Physiology 168 (2011) 671–677 675

Fig. 8. Total protein profile of cotyledons and embryonic axis in horsegram sub-jected to different levels of PEG induced water stress during 48 h. Lane 1. Lowmolecular weight marker; Lanes 1, 2, 3 and 4 – control, 2.5%, 5% and 10% PEG stress,respectively, in cotyledons; Lanes 5, 6, 7 and 8 – control, 2.5%, 5% and 10% PEG stress,respectively, in embryonic axis.

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Fig. 11. Immunoblot showing the tissue specific expression of LEA 1 antibodies inhorsegram subjected to control and 10% PEG stress at different time intervals. Lanes1, 3, 5, 7, 9, and 11 – control samples; Lanes 2, 4, 6, 8, 10 and 12 stressed samples.

ig. 9. Total protein profile of root and shoot in horsegram subjected to differentevels of water stress during 72 h. Lane 1. Low molecular weight marker; Lanes 1, 2,and 4 – control, 2.5%, 5% and 10% PEG stress, respectively, in root; Lanes 5, 6, 7 and– control, 2.5%, 5% and 10% PEG stress, respectively, in shoot.

The seedlings grown in control and stressed conditions for 72 here separated into root and shoot and isolated proteins were

esolved on SDS-PAGE. From Fig. 9, it was observed that the over-ll intensity of proteins was decreased in root when compared tohoot. The most noticeable observation is in ∼30 kDa and ∼52 kDaolypeptides of shoot, which were not changed with 2.5% stress butecreased with 5% stress and again increased in 10% stress.

.5. Expression of group 1 LEA proteins

Gel blot analysis indicated that antibodies raised against LEA 1ross-reacted with the ∼27 kDa protein in both control and 10%tress treated seedlings of 12, 24, 48 and 72 h. The expression ofhis protein was higher in stress treatments than control. Further-

ore, it was observed that the expression of this LEA-like proteinecreased in controlled conditions by 72 h (Fig. 10).

Group 1 LEA antibodies were also differentially cross-reactedith ∼27 kDa protein isolated from different tissues of control and

0% water stressed seedlings at different time intervals (Fig. 11).t was observed that the expression of this LEA protein decreasedn the embryonic axis when compared to cotyledons at 24 h. LEA 1

ig. 10. Immunoblot showing the cross-reacted of LEA 1 antibodies with horsegrameedling proteins subjected to control and 10% PEG stress at different time intervals.ane M. Low molecular weight marker; Lanes 1, 3, 5 and 7 – control during 12, 24,8 and 72 h, respectively; Lanes 2, 4, 6 and 8, 10% PEG stress for 12, 24, 48 and 72 h,espectively.

Fig. 12. Immunoblot showing the tissue specific expression of LEA 2 antibodies inhorsegram subjected to different levels of PEG treatment during 72 h. Lane 1. Lowmolecular weight marker; Lanes 1 and 5 – control; 2 and 6–2.5% stress; 3 and 7–5%stress; 4 and 8–10% stress.

antibodies did not show cross-reaction with proteins of the embry-onic axis at 48 h and root and shoot at 72 h.

3.6. Expression of group 2 LEA proteins

Expression of group 2 LEA protein analysis (Fig. 12) revealedthat antibodies raised against LEA 2 differentially cross-reactedwith proteins of shoot tissues subjected to control and stress condi-tions for 72 h. Four polypeptides of approximate molecular weight33, 51, 52 and 66 kDa were found to cross-react with LEA 2 anti-bodies in shoot tissue in control and at 2.5%, 5% and 10% levels ofstresses. However, the cross reaction with the ∼66 kDa polypeptidewas found only in 10% stress level in shoot tissues.

The most noticeable difference was the LEA 2 antibodies showedno cross-reaction with proteins of root tissue in both control andstressed seedlings, indicating the presence of LEA and LEA like pro-teins only in shoot tissues.

4. Discussion

LEA proteins were first identified in cotton and wheat as pro-teins that accumulated to high levels during the maturation phaseof seeds (Dure et al., 1981; Grzelczak et al., 1982; Galau et al., 1986).The precise function of LEA proteins in plants has not been defined,although their expression is associated closely with acquisition ofdesiccation tolerance (Blackman et al., 1995). When introduced intoyeast; tomato, wheat and barley LEA proteins have been shown toconfer increased resistance to osmotic and freeze stress (Honjohet al., 1996; Imai et al., 1996; Swire-Clark and Marcotte, 1999;

Zhang et al., 2000) and a barley LEA protein improved the toler-ance to water deficit in transgenic rice (Xu et al., 1996) and wheat(Sivamani et al., 2000). LEA proteins, therefore, seem able to offerpartial protection to biological structures at the molecular and cel-

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76 G. Veeranagamallaiah et al. / Journa

ular level against the effects of water loss, but how this function isinked is unclear so far. With this context, this report provides annderstanding of water stress-induced protein aggregation, which

s reduced upon accumulation of certain LEA group proteins andheir tissue specific expression at different stages of seedling estab-ishment in horsegram as evidenced from immunoblot expressionnalysis.

In the present study we observed significant aggregation ofroteins with initial stress treatments during early periods ofeed germination. This aggregation gradually decreased with thencreasing time and severity of stress (Fig. 2), suggesting theresence of some possible operative mechanism for protectionf cellular proteins during decreased RWC (Fig. 1). This could, inrinciple, be operated by the accumulation of LEA like proteins,hich have a protective role against cellular aggregation. Previ-

usly, it was very clear that desiccation stress caused increase ofrotein aggregation was reduced by addition of Aav-LEA1 proteinrom nematode (Chakrabortee et al., 2007). Further, the increasef some protein bands upon electrophoresis revealed that mostroteins form aggregates, but not all molecules of any particu-

ar protein do so (Fig. 4). The recent findings of Tunnacliffe andise (2007) and Chakrabortee et al. (2007) are supporting the view

hat some LEA proteins act to prevent aggregation of other pro-eins during water loss. These results are consistent with Goyal etl. (2005) who showed antiaggregation of single protein species.g., citrate synthase in the presence of LEA protein from nema-ode upon desiccation. The enzymes citrate synthase and lactateehydrogenase form insoluble aggregates when dried or frozenut are prevented from doing so in the presence of Ava-LEA1, aroup 3 member from nematode Aphelenchus avenae (Goyal et al.,005) or the group 1 LEA protein, Em, from wheat (Goyal et al.,003).

In order to get an insight into the abrogation of water stressnduced aggregation of the water soluble proteomes from horseg-am is weather contributed by LEA proteins, we performedxpression studies using LEA group 1 and 2 antibodies with horseg-am seedling proteomes. The expression of LEA 1 was observed inoth control and 10% PEG induced stress seedlings at 12 and 24 h.oreover, expression of this LEA 1 decreased in embryonic axishen compared to cotyledons at 24 h and also completely absent in

mbryonic axis at 48 h and in root and shoot at 72 h suggesting theifferential regulation of LEA 1 under water stress in germinatingeedlings of horsegram. Similar results were reported previouslyy Arati et al. (2003) in ragi cultivars under osmotic stress.

Similarly, expression studies with LEA group 2 antibodiesevealed that these group 2 LEA proteins were expressed only inhoot tissues at 72 h. These results further support the light scat-ering assay, which revealed the pronounced protein aggregationn roots compared to shoot at 72 h. This tissue specific expression ofroup 2 LEA was previously observed in response to chilling stressn floral buds of woody perennial tree, blue berry (Muthalif andowland, 1994) and to water stress in roots and shoots of Populusremula L. (Pelah et al., 1997).

In summary, we presented data on the developmentally reg-lated tissue specific expression of LEA 1 and 2 proteins underifferent levels of water stress in horsegram. From these resultse hypothesize that the reduced protein aggregation and water

tress induced accumulation of proteins could perhaps be due toigh expression of LEA proteins particularly LEA 1 and 2 whichre thought to play an important role during water stress inducedesiccation. Further, it might be suggested that LEA proteins acts

s a special form of molecular chaperones to protect the for-ation of protein aggregation during water stress and further

esearch on behavior of proteins during extreme water loss wille required to elucidate the exact function of the LEA proteins inetail.

nt Physiology 168 (2011) 671–677

Acknowledgements

We acknowledge the DST (SR/SO/PS-10/06), DBT(BT/PR9609/AGR/02/451/2007) and UGC, New Delhi for thefinancial assistance in the form of Research grants. We thank Dr.M. Udayakumar University of Agricultural Sciences, Bangalore,for generous gift of LEA antibodies. We thank Prof. T.J. Flowers,University of Sussex for reading the manuscript and his valuablecomments.

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