UPLC HRMS based untargeted metabolic profiling reveals changes in chickpea (Cicer arietinum) metabolome following long term drought stress

Transcription

1 Received: 11 Octoer 2017 Revised: 8 Mrch 2018 Accepted: 9 Mrch 2018 DOI: /pce ORIGINAL ARTICLE UPLC HRMS sed untrgeted metolic profiling revels chnges in chickpe (Cicer rietinum) metolome following long term drought stress Neem Khn 1 Asghri Bno 1,2 Mohmmd Atikur Rhmn 3 Bl Rthinspthi 4 Md Ali Br 3 1 Deprtment of Plnt Sciences, Quid I Azm University, Islmd, Pkistn 2 Deprtment of Biosciences, University of Wh, Wh Cntonment, Pkistn 3 Deprtment of Agronomy, IFAS, University of Florid, Ginesville, FL, USA 4 Horticulture Science Deprtment, IFAS, University of Florid, Ginesville, FL, USA Correspondence Md Ali Br, Deprtment of Agronomy, IFAS, University of Florid, Ginesville, FL, USA. Emil: mr@ufl.edu Funding informtion Deprtment of Agronomy, University of Florid; Institute of Food nd Agriculturl Sciences, University of Florid; Higher Eduction Commission, Pkistn Astrct Genetic improvement for drought tolernce in chickpe requires solid understnding of iochemicl processes involved with different physiologicl mechnisms. The ojective of this study is to demonstrte genetic vritions in ltered metolic levels in chickpe vrieties (tolernt nd sensitive) grown under contrsting wter regimes through ultrhigh performnce liquid chromtogrphy/high resolution mss spectrometry sed untrgeted metolomic profiling. Chickpe plnts were exposed to drought stress t the 3 lef stge for 25 dys, nd the leves were hrvested t 14 nd 25 dys fter the imposition of drought stress. Stress produced significnt reduction in chlorophyll content, F v /F m, reltive wter content, nd shoot nd root dry weight. Twenty known metolites were identified s most importnt y 2 different methods including significnt nlysis of metolites nd prtil lest squres discriminnt nlysis. The most pronounced increse in ccumultion due to drought stress ws demonstrted for llntoin, L proline, L rginine, L histidine, L isoleucine, nd tryptophn. Metolites tht showed decresed level of ccumultion under drought conditions were choline, phenyllnine, gmm minoutyric cid, lnine, phenyllnine, tyrosine, glucosmine, gunine, nd sprtic cid. Aminocyl trna nd plnt secondry metolite iosynthesis nd mino cid metolism or synthesis pthwys were involved in producing genetic vrition under drought conditions. Metolic chnges in light of drought conditions highlighted pools of metolites tht ffect the metolic nd physiologicl djustment in chickpe tht reduced drought impcts. KEYWORDS drought, metolic pthwy, metolites, UPLC HRMS nlysis, wter stress 1 INTRODUCTION Chickpe (Cicer rietinum) is the fourth lrgest grown legume crop in the world, which is grown in n re of 10.2 million hectres with n verge production of 11 million metric tons. Chickpe significntly contriutes to world food security y providing clories nd dietry protein for millions of people (Vrshney et l., 2013). More thn 80% of chickpe is grown under rinfed conditions where unpredictle This is n open ccess rticle under the terms of the Cretive Commons Attriution NonCommercil NoDerivs License, which permits use nd distriution in ny medium, provided the originl work is properly cited, the use is non commercil nd no modifictions or dpttions re mde The Authors Plnt, Cell & Environment Pulished y John Wiley & Sons Ltd Plnt Cell Environ. 2019;42: wileyonlinelirry.com/journl/pce 115

2 116 KHAN ET AL. rinfll pttern or drought stress is the most common limiting fctor tht ffects its growth nd productivity. Moreover, drought conditions lso ffect symiotic ssocition of nodulting cteri tht contriute 45% of nitrogen needed for griculture (Blol, 2010). In ddition, the climte chnge scenrio, especilly progressive drought stress induced y either declining rinfll or higher wter stress llied with hot climte, will cuse significnt dmge to crop production in the future (Di, 2013; Loell et l., 2014; Rune et l., 2014). The mjor effects of drought include reduction in cell elongtion, cell division, stem enlrgement, nd decrese in root growth (Frooq, Whid, Koyshi, Fujit, & Bsr, 2009; Y. Li, Ye, Wng, & Yn, 2009). In ddition, drought stress interrupts photosynthesis nd increses trnsloction of crohydrtes, ccelertes phsic development, nd oosts senescence (Kuot & Kozi, 1992; Yng, Zhng, Hung, Zhu, & Wng, 2000; Yng, Zhng, Wng, Zhu, & Wng, 2001). Genetic improvement is the most efficient nd sustinle wy to reduce drought stress effect nd develop geneticlly superior germ plsm tht dpts to future climte chnge conditions. However, genetic improvement requires solid understnding of the iochemicl mechnisms controlling different trits. The nlyses of iologiclly importnt molecules re necessry to understnd which molecules re influencing stress tolernce mechnisms in chickpe plnts. Plnts produce n rry of iochemicl compounds including hundreds of metolites under stress conditions. Plnt metolites ply essentil roles in growth, cell integrity, energy storge, cell signlling, memrne formtion nd scffolding, cellulr replenishment, nd whole plnt resource lloction, s well s roles in plnt development nd stress responses (Wen et l., 2015). Plnts cn modify their physiology to dpt to different conditions through metolic chnges (Khn, Ali, Shhid, & Khrin Msouleh, 2017; Kim, Choi, & Verpoorte, 2011). Plnts hve vrious metolic dpttion mechnisms to defend ginst the negtive effects of stress, which cn ply crucil role in the dptive mechnisms in plnts. Chnges in the metolic level in n orgnism re likely to e correlted with the phenotype s metolites re the end products of the iologicl system. Empiricl evidence suggests tht metolic components re linked to high temperture stress tolernce in corn (Chen, 2010) nd in coolseson grss (Su, Bremer, Jennotte, Welti, & Yng, 2009) s well s to cold nd freezing tolernce in Aridopsis (Welti et l., 2002). Additionlly, recent studies hve demonstrted ltered levels of metolites etween drought stressed nd well wtered plnts (Bowne et l., 2012; Hong Bo et l., 2006; Ot et l., 2015; Witt et l., 2012). The incresed level of rnched chin mino cids (leucine, isoleucine, nd vline) ws reported under drought conditions in tolernt cultivrs of whet nd rley (Bowne et l., 2012; Krugmn et l., 2011; Rontein, Bsset, & Hnson, 2002). Differing metolite expression ws ssocited with high temperture stress tolernce in mize (Chen, 2010) nd in cool seson grsses (Su et l., 2009). Mize plnts under hightemperture stress showed increses in metolites such s tryptophn, serine, threonine, et lnine, proline, glutmte, myoinositol, nd ure (Ot et l., 2015). In ddition to this, certin metolites showed negtive correltion (threonine, vline, trehlose, nd glycerol) or positive correltion (fumrte, succinte, nd rffinose) with grin yield nd tht chnge in metolite level ws consistent etween greenhouse plnts nd field grown plnts (Ot et l., 2015). Approches to profile metolites re receiving incresing ttention from the plnt iologist community. Metolic quntittive trit locus identifiction reveled tht heterosis is minly under the control of epistsis in Aridopsis, wheres in tomto, the underlying genetic vrition ws explined minly y primry metolites (Lisec et l., 2009; Schuer et l., 2006). Riedelsheimer et l. (2012) found strong correltion etween metolites nd gronomic trits in field evluted hyrid progeny in corn. They uilt whole genome nd metolic prediction models with ccurcies rnging from 0.72 to 0.81 for single nucleotide polymorphisms nd from 0.60 to 0.80 for metolites. Little is known out the complex metolic regultion for drought stress tolernce in chickpe. Thus, using the metolic phenotype to study differentil expression of contrsting vrieties for complex trits might deepen our understnding of the metolic pthwys nd expedite genetic gins in chickpe productivity. Metolomics llows the prllel ssessment of lrge numer of metolites in iologicl smple. Recent metolomics studies hve een of gret vlue in phenotyping nd dignostics in crop reeding (Fernie & Schuer, 2009). Recent progress in mss spectrometry with dvnced dt processing technology llows simultneous mesurement of hundreds of chemiclly different metolite species in single smple of plnt tissue. These dvnces hve mde it possile to investigte more thoroughly the regultion of metolic networks nd to study their influence on complex trits. The pplicility of this technology hs een demonstrted in tomto, Aridopsis, nd potto through discovery of iomrkers ssocited with trits of interest (Lisec et l., 2009; Rowe, Hnsen, Hlkier, & Klieenstein, 2008; Schuer et l., 2006). Thus, metolomics represents n importnt nd exciting new ddition for genetic improvement of plnts. Metolomics could e powerful selection tool to estlish the ssocition etween phenotype nd genotype, leding to etter understnding of the genetic sis of plnt responses to stress. The ojective of this study is to demonstrte the differentil ccumultion of metolites in chickpe leves under drought stress conditions in different periods nd the involvement of those metolites in different pthwys in reltion to drought tolernce in chickpe. To chieve this ojective, we employed nontrgeted glol ultrhighperformnce liquid chromtogrphy/high resolution mss spectrometry (UPLC HRMS) for the first time to identify metolites from the lef tissue of irrigted nd drought stressed chickpe plnts. The UPLC HRMS technique is sensitive nd potentilly more roust thn nucler mgnetic resonnce nd gs chromtogrphy mss spectrometry (GC MS) technology s it cn identify metolites in low concentrtion nd hs etter cpcity to control the flse discovery rte. 2 MATERIALS AND METHODS 2.1 Plnt mterils nd growth conditions The experiment ws conducted t controlled greenhouse condition in the Deprtment of Agronomy, University of Florid, in Ginesville, Florid. Drought sensitive chickpe (C. rietinum) vriety Punj Noor 2009 (G1) nd drought tolernt vriety (G2) were used for the study (Khn & Bno, 2016). The seeds of those vrieties were otined from Ayu Agriculturl Reserch Institute Fisld,

3 KHAN ET AL. 117 Pkistn. Chickpe seeds were wshed in 100% distilled wter followed y surfce steriliztion with 95% ethnol for 2 3 min nd then soked in 10% Clorox with concomitnt shking. The seeds were susequently wshed in utoclved distilled wter (Khn & Bno, 2016). Seeds were grown in pots filled with 2,000 g of Metro Mix 360 soil mixture. The pots were well wtered (typiclly two times per week) throughout until drought stress ws pplied, nd ech pot contined five plnts. Wter ws pplied until the sustrte ws completely wet nd the wter strted to seep out through the holes t the ottom of the pot. A tespoon of Osmocote (15N 9P 12K) ws pplied one time fter germintion. Experiment ws lid out in completely rndomized lock design with six (iologicl) replictions. During the experiment, the greenhouse condition ws mintined t 26 nd 19 ± 1 C (dy nd night tempertures, respectively) with 70 ± 2% reltive humidity nd dy nd night lengths (11 nd 13 hr, respectively). Drought stress ws imposed on 25 dy old plnts (three lef stge) y withholding wter supply for 25 dys until the soil wter content reched 4%. A set of well wtered plnts served s control. Chlorophyll content, chlorophyll fluorescence (F v /F m rtio), shoot nd root dry weight, nd reltive wter content (RWC) were estimted fter 14 nd 25 dys of the drought condition. Lef tissues were hrvested for metolomics nlysis lso fter 14 nd 25 dys of the drought condition. 2.2 Physiologicl chrcteriztion Dt were collected on physiologicl trits known to e ffected y the drought condition. Chlorophyll fluorescence (the rtio of vrile, F v, to mximum fluorescence, F m ) nd SPAD chlorophyll content were used s n indirect method to ssess photosystem II efficiency (Kdir, Von Weihe, & Al Khti, 2007; Ristic, Bukovnik, & Prsd, 2007) nd chlorophyll dmges due to stress. Both trits were mesured t 14 nd 25 dys fter imposition of drought stress following the methods descried y Tlukder (2014). Chlorophyll fluorescence ws mesured on intct leves of the xil surfce (third lef) fter 30 min of drk dpttion with pulse modulr fluorometer (Model OS5 FL, Opti Sciences, Hudson, NH) in oth the control nd drought stressed leves. Estimtes of lef chlorophyll content were tken using SPAD chlorophyll meter (Model 502, Spectrum Technologies, Plinfield, IL) in oth the control nd drought stressed leves. Chlorophyll fluorescence nd chlorophyll content were mesured t three leflets in ech plnt nd five plnts per pot ( totl of 15 redings) nd verged. The verge vlue of 15 redings ws considered s single repliction, nd six replicted vlues/vriety were used for sttisticl nlysis nd comprison of tretment mens, nd significnt testing t P <.05 level. The two chickpe vrieties demonstrted differences from ech other for different morphologicl nd phenotypic trits under drought conditions (Khn & Bno, 2017). Lef RWC ws determined with fully expnded leves fter 14 nd 25 dys of drought stress imposition using the method descried y Brrs nd Wetherley (1962). Leves were clipped nd weighed (fresh weight, FW) nd plced in Ziploc g filled with distilled wter to ensure oth sides of leves fully sor wter. They were soked in wter for 24 hr t room temperture nd then weighed immeditely fter excess moisture ws removed with pper towels (turgid weight, TW). The leves were then dried in n oven t 60 C for 72 hr to determine dry weight (DW). Lef RWC ws clculted s (FW DW)/(TW DW) Mesures of shoot nd root dry weights Shoots of five plnts per repliction were cut t the se nd dried t 60 C for 72 hr, nd dry weight ws tken y using n electronic scle. The roots of the sme plnts with soil were isolted from the pots, wshed crefully to seprte roots, dried t 60 C for 72 hr, nd weighed using n electronic scle. The root nd shoot dry weights were mesured fter 25 dys of drought stress imposition. 2.4 Antioxidnt enzyme extrction Fresh leves (0.5 g) were ground in 5 ml of 50 mm phosphte uffer nd plced in n ice th. The superntnt ws tken fter centrifuging the homogente for 20 min t 18,928 g t 4 C, which ws used for ssys of enzyme ctivities s outlined y Verm nd Duey (2003). 2.5 Lef tissue collection nd smple preprtion Lef tissues were collected from control nd drought plnts t middy fter 14 nd 25 dys of drought stress. Lef ldes were collected from six individul pots (iologicl replictes) (Bis, Moon Quneck, Nikolu, & Dickerson, 2011), frozen in liquid nitrogen immeditely fter collection, nd stored t 80 C. Tissue smples were lyophilized for 72 hr nd ground using TissueLyser. Lyophilized powder (30 mg) ws used for nontrgeted glol UPLC HRMS sed metolite profiling. Freeze dried ground lef tissues (30 mg) were spiked with mixture of internl stndrds (20 μl) into clen Eppendorf tue. Methnol (750 μl) nd mmonium cette (10 mm, 750 μl) were dded to ech smple nd vortexed for 1 min t room temperture. Centrifugtion (t 17,000 g for 10 min) of ll smples ws done fter ultrsoniction for 20 min t room temperture. Superntnt (>1 ml) ws trnsferred to 1.5 ml tue, followed y 50 μl trnsfer of superntnt to n Eppendorf tue. The superntnt ws dried down nd ws reconstituted with 50 μl of injection stndrd solution. Smples were then vortexed (30 s) nd put t 4 C for 10 min nd centrifuged t 20,000 rpm for 10 min, nd the superntnt ws trnsferred into n LC vil. 2.6 UPLC HRMS nlysis Untrgeted metolomics profiling ws performed on UPLC HRMS (Model: Thermo Ultimte 3000 UPLC nd Thermo Q Exctive mss spectrometer). All smples were nlysed in positive nd negtive heted electrospry ioniztion with mss resolution of 70,000 t m/z 200 s seprte injections. Chromtogrphic seprtion ws ttined on n ACE Excel 2 C18 PFP mm, 2 μm prticle size column with Moile Phse A s 0.1% formic cid in wter nd Moile Phse B s cetonitrile, t flow rte of 350 μl/min with run time of 16.8 min, mss resolution of 35,000 t m/z 200, nd mss rnge of 70 1,000 m/z. Injection volume ws 4 μl for negtive ion mode nd 2 μl for positive ion mode. The totl run time per smple ws 20.5 min. Proe (HESI proe) temperture ws mintined t

4 118 KHAN ET AL. 350 C for oth positive nd negtive runs with spry voltge of 3,500 V nd cpillry temperture of 320 C. 2.7 Dt nlysis The rw files were converted to.mzxml using MSConvert (ProteoWizrd 3.0). MZmine 2.1 ws used to identify fetures, deisotope, lign fetures, nd perform gp filling to fill in ny fetures tht my hve een missed in the first lignment lgorithm. All dducts nd complexes were identified nd removed from the dt set. The dt were serched ginst n internl retention time metolite lirry. Dt from positive nd negtive ion modes were seprtely sujected to sttisticl nlyses. Dt tles with metolite peks (mz/rt) t two time points under oth drought nd control conditions for tolernt nd sensitive vrieties were formtted s commseprted vlues (.csv) files nd uploded to the MetoAnlyst 3.0 server ( Xi, Mndl, Sinelnikov, Brodhurst, & Wishrt, 2012). To shrink ny possile vrince nd to improve the performnce for downstrem sttisticl nlysis, metolite dt generted y UPLC HRMS were checked for dt integrity nd normlized using MetoAnlyst's normliztion protocols (selecting normliztion y sum, log trnsformtion, nd utoscling) for sttisticl nlysis. We pplied univrite nlysis (t test nd one wy ANOVA) to clculte the sttisticl significnce nd fold chnge of the metolites etween two group mens (drought over control). As the multivrite methods tke ll the vriles into considertion, we pplied multivrite methods for comprehensive dt nlysis, for exmple, supervised method (prtil lest squres discriminnt nlysis [PLS DA]) nd n unsupervised method (hierrchicl clustering with het mp). The supervised method, PLS DA, ws used to mximize the difference of metolic profiles etween control nd drought groups to enle the detection of metolites existing in the iologicl smples. The het mp ws generted sed on the Person distnce mesure nd the Wrd clustering lgorithm, showing top 19 metolites selected y PLS DA with vrile importnce in projection (VIP) score using significnce level of P.05, nd post hoc nlysis of Fisher's lest significnt difference. The smples were rrnged ccording to their smpling time points in oth control nd drought groups. The importnt metolites were identified y using two different methods seprtely: significnt nlysis of metolites (SAM) nd PLS DA. The pthwy nlysis ws performed using MetoAnlyst for the identified importnt metolites using Aridopsis thlin pthwy lirries. The Kyoto Encyclopedi of Genes nd Genomes pthwy dtse ( ws lso used for the metolites tht were not found in the rice nd Aridopsis pthwy lirries. For phenotypic dt nlysis, we used SAS version 9.1. An ANOVA ws performed to determine the effect of tretments nd error ssocited with the experiment with replictions nd tretments s rndom effects. To identify significnt differences mong tretments, men comprison ws crried out y using protected lest significnt difference (P <.05) test where error men squre ws used to estimte the stndrd error of differences etween mens. 3 RESULTS 3.1 Physiologicl responses Chlorophyll content nd photochemicl efficiency (F v /F m ) In generl, the chlorophyll content decresed significntly (P <.01) in oth the sensitive vriety (G1) nd tolernt vriety (G2) under the drought condition s compred with control (Figure 1) nd decresed with the increse in the durtion of drought stress. However, the percentge of decrese in the tolernt vriety (G2) ws less thn tht in the sensitive vriety (G1). Mximum decrese (64%) in chlorophyll content ws oserved in the sensitive vriety t 25 dys under the drought condition s compred with control. The drought tolernt vriety, on the other hnd, showed 7% nd 16% decrese of chlorophyll compred with control fter the 14 nd 25 dys of stress. The photochemicl efficiency of photosystem II (F v /F m ) showed vriility etween vrieties nd t two time points (14 nd 25 dys) of stress tretments (Tle 1). The F v /F m rtio ws decresed for oth vrieties t oth time points under the drought condition, ut the decrese ws more pronounced (50% nd 63%) for the sensitive vriety fter 14 nd 25 dys of stress imposition Reltive wter content (%) Drought stress cused significnt (P <.01) chnges in RWC in oth the vrieties (Figure 2). The RWC of the tolernt vriety (G2) decresed y 27% nd 33% under the drought condition compred with the control condition t 14 nd 25 dys, respectively, ut the decrese in RWC ws higher for the sensitive vriety (G1) t oth time points (40% Spd Chloro[hyll Content G1 G2 G1 G2 Tretments Control Drought 14 Dys 14 Dys 25 Dys 25 Dys FIGURE 1 Chlorophyll content (±SE) in leves of two chickpe vrieties under drought nd control conditions t 14 nd 25 dys fter wter tretment. G1: drought sensitive vriety (Punj Noor 2009); G2: drought tolernt vriety (93127). Error rs represent stndrd errors of the men (n = 6) t ech time point. Different letters indicte significnt differences (P <.05) mong tretments (drought vs. irrigtion) for genotype in prticulr time point

5 KHAN ET AL. 119 TABLE 1 imposition Chlorophyll florescence (F v /F m rtio) of two chickpe vrieties under control nd drought conditions t 14 nd 25 dys fter stress Chickpe vriety Dys fter stress imposition Chlorophyll florescence (F v /F m ) Control (M ± SE) Drought stress (M ± SE) Punj Noor 2009 (sensitive vriety, G1) ± ± (tolernt vriety, G2) ± ± Punj Noor 2009 (sensitive vriety, G1) ± ± (tolernt vriety,g2) ± ± Reltive Wter Content (%) G1 G2 G1 G2 Tretments Control Drought 14 Dys 14 Dys 25 Dys 25 Dys FIGURE 2 Reltive wter content (±SE) of chickpe under drought versus control conditions. G1: drought sensitive vriety (Punj Noor 2009); G2: drought tolernt vriety (93127). Error rs represent stndrd errors of the men (n = 6) t ech time point. Different letters indicte significnt differences (P <.05) mong tretments (drought vs. irrigtion) for genotype in prticulr time point Shoot nd Root Dry wt. (g) Control Drought G1 G2 G1 G2 Shoot DW Shoot DW Root DW Root DW Tretments FIGURE 3 Shoot nd root dry weights (±SE) of chickpe under drought versus control conditions fter 25 dys of drought stress imposition. G1: drought sensitive vriety (Punj Noor 2009); G2: drought tolernt vriety (93127). Error rs represent stndrd errors of the men (n = 6). Different letters indicte significnt differences (P <.05) mong tretments (drought vs. irrigtion) for genotype nd 65%, respectively) under the drought condition. This result demonstrtes tht the drought sensitive vriety ws more ffected due to drought stress Shoot nd root dry weights (g) Shoot nd root dry weights of oth sensitive nd tolernt vrieties were reduced significntly (P <.01) due to drought stress compred with control fter 25 dys of drought stress imposition (Figure 3). However, the reduction ws more for the sensitive vriety thn for the tolernt vriety. Sensitive nd tolernt vrieties showed 69% nd 24% reduction in shoot dry weight under the drought condition compred with control, respectively (Figure 3). Similrly, 77% nd 25% reductions in root dry weights were shown y the sensitive nd tolernt vrieties, respectively, under the drought condition s compred with control Antioxidnt enzyme ctivity The ntioxidnt enzyme ctivities were significntly enhnced in plnts under drought stress conditions (Figure 4). Mximum increse ws noted for superoxide dismutse ctivities in the leves of stressed plnts s compred with control plnts in the sensitive (45%) nd tolernt vrieties (62%), respectively. The tolernt vriety showed higher ntioxidnt ctivities thn the sensitive vriety. The increse ws significnt nd 25% higher in tolernt vriety s compred with the sensitive vriety under stress conditions. Similrly, the tolernt vriety lso showed significnt increse in ctlse (24%), scorte peroxidse (33%), nd peroxidse (27%) ctivities s compred with the sensitive vriety under stress conditions Metolite profiling The nonised UPLC HRMS glol metolomics pproch detected totl of 691 peks, of which 175 were identified s known metolites (178 including duplictions) nd the remining peks were unknown metolites. Metolites were highly reproducile mong the six nlysed iologicl replictions t the two different time points. The identified metolites included mino cids, orgnic cids, sugrs, polymines, nitrogenous compounds, nd polyphenols.

6 120 KHAN ET AL. Antioxidnt enzymes (units/g fw) * * Tretments Control * Drought FIGURE 4 Antioxidnt enzyme content (±SE) in the leves of drought sensitive vriety (G1) nd drought tolernt vriety (G2) fter 25 dys of drought tretment. Dt re mens of six replictes long with stndrd error rs. APOX = scorte peroxidse, POD = peroxidse, SOD = superoxide dismutse. Different letters indicte significnt differences (P <.05) mong tretments (drought vs. irrigtion) for vriety. Asterisk represents significnt difference etween sensitive (G1) nd tolernt (G2) vrieties under the drought condition The PLS DA ws performed for drought (nd control) conditions on oth drought tolernt nd drought sensitive vrieties together t two different time points. The first PLS component (PC1) explined 61.8% of totl vrition, wheres the second component (PC2) explined 8.9% vrition cross the dt set (Figure 5). The scores plot * etween PC1 nd PC2 reveled two distinct groups ssocited with the drought nd control smples t two different smpling points (Figure 5), suggesting cler distinction in the metolite ccumultion under two conditions. The sensitive vriety (G1) nd tolernt vriety (G2) smples were seprted from ech other under oth drought nd control conditions, with overlp t two time points, especilly under the drought condition. The effect of wter stress tretment on metolic ccumultion ecomes evident from hierrchicl clustering with het mp (Figure 6). Two mjor clusters were identified with distinct ptterns of ltered metolite undnces. Metolites tht were highly ccumulted in plnts grown under the drought condition compred with the control condition formed the first cluster including mino cids (e.g., proline, tryptophn, histidine, nd isoleucine) nd orgnic cids. On the other hnd, metolites tht showed decresed level of ccumultion under the drought condition included putrescine, choline, phenyllnine, gmm minoutyric cid (GABA), nd lphketoglutric cid, forming the second cluster. Choline, phenyllnine, nd putrescine were undntly found in the sensitive vriety, wheres tyrosine, GABA, nd lph ketoglutric cid were undntly present in the tolernt vriety under the control condition. The incresed level of proline nd tryptophn ws evident in oth the sensitive nd tolernt vrieties when plnts were exposed to the drought condition. The ptterns of metolite clustering clerly indicte the metolic chnges under different wter regimes. To identify the importnt metolites ssocited with the drought condition, two sttisticl models, nmely, SAM nd PLS DA, were crried out (Tle 2). The most significntly different compounds were identified y the SAM plot with delt vlue of 1.4, flse discovery rte of 0.003, nd flse positive of 0.4. Similrly, the PLS DA method identified the most importnt metolites sed on the VIP score using the five component model. The two methods showed quite similr results, identifying the sme metolites. The top most importnt 20 metolites (sed on fold chnge vlues) FIGURE 5 Prtil lest squre discriminnt nlysis nd 2D scores loding plot for the Chickpe Punj Noor 2009 (G1) nd (G2) under control (well wtered) nd drought tretments t two time points (14 nd 25 dys). Smples t control nd drought tretments did not overlp with ech other, indicting n ltered stte of metolite levels in the chickpe leves. G1, sensitive vriety; G2, tolernt vriety

7 KHAN ET AL. 121 FIGURE 6 Het mp illustrting levels of top metolites in the leves of two chickpe vrieties ccording to the prtil lest squre discriminnt nlysis vrile importnce in projection scores under control (well wtered) nd drought conditions t two time points (14 nd 25 dys). The het mp ws generted using Person nd Wrd for distnce mesure nd clustering lgorithm, respectively. G1: drought sensitive chickpe vriety (Punj Noor 2009); G2: drought tolernt chickpe vriety (93127) TABLE 2 Importnt metolites with their compound ID (Kyoto Encyclopedi of Genes nd Genomes ID or PuChem CID) nd moleculr formul, identified through PLS DA, rndom forest, nd SAM S. no. Importnt metolites Compound ID Moleculr formul SAM (d vlue) PLS DA VIP score (vrince for Component 1) 1 Alnine C00041 C 3 H 7 NO N Methyl D sprtic cid C00123 C 5 H 9 NO Choline C00114 C 5 H 13 NO BOC L tyrosine C1 4 H 19 NO BOC D phenyllnine C 14 H 19 NO Adenosine C00212 C 10 H 13 N 5 O Aminoutnote C00334 C 4 H 9 NO Gunine C00242 C 5 H 5 N 5 O Allntoin C01551 C 4 H 6 N 4 O N Methyl L glutmte C01046 C 6 H 11 NO L Arginine C00062 C 6 H 14 N 4 O L Histidine C00135 C 6 H 9 N 3 O L Proline C00148 C 5 H 9 NO N Acetyl putrescine C02714 C 6 H 14 N 2 O Glutthione disulphide C00127 C 20 H 32 N 6 O 12 S Alph ketoglutric cid C00026 C 5 H 6 O L Isoleucine C00407 C 6 H 13 NO Tryptophn C00078 C 11 H 12 N 2 O Isocitric cid C00311 C 6 H 8 O Glucosmine C00329 C 6 H 13 NO Note. PLS DA = prtil lest squre discriminnt nlysis; SAM = significnt nlysis of metolites; VIP = vrile importnce in projection. PuChem CID. were identified y two different methods nd included different mino cids, sugrs, orgnic cids/compounds, mines, ftty cids, nd other compounds (Tle 2). Among different groups of metolites, mny mino cids were significntly ccumulted in the leves of plnts grown in the drought condition. Proline, L rginine, L histidine, L isoleucine, llntoin,

8 122 KHAN ET AL. tyrosine, nd tryptophn showed incresed levels of ccumultion in the leves of drought stressed plnts (Tle 3). However, compounds such s GABA, denosine, lnine, lph ketoglutric cid, phenyllnine, choline, glucosmine, gunine, nd sprtic cid were highly ccumulted in the leves of plnts grown under the control condition ut decresed under the drought condition. Higher ccumultion of L rginine occurred in the sensitive vriety when plnts were exposed to the drought condition; however, the level of L rginine ws decresed in the tolernt vriety. On the contrry, llntoin nd tyrosine were highly ccumulted in the tolernt vriety compred with the sensitive vriety under the drought condition. Isoleucine ws ccumulted under the drought condition in the tolernt vriety t oth time points (14 nd 25 dys), ut ccumultion occurred only t 14 dys fter stress in the sensitive vriety. The levels of isocitric cid nd lphketoglutric cid were reduced in the tolernt vriety t 25 dys under the drought condition (Tle 3). It ws evident from Tle 2 tht tryptophn ws the most contriutory of metolites in the PLS DA model with VIP score of nd men decrese ccurcy of , followed y choline nd BOC L tyrosine (1.36), wheres N methyl D sprtic cid hd the lowest VIP score ( ) with men decrese ccurcy of , followed y 4 minoutnote (0.987) with men decrese ccurcy of The results reveled tht tryptophn, proline, nd histidine were highly ccumulted in the leves of plnts under the drought condition t two time points nd hence cn e considered s responding to the drought condition. Similrly, denosine, lnine, nd choline were significntly ccumulted in the leves of plnts grown under the control condition. The mximum increse (4.279) in TABLE 3 Fold chnges of importnt metolites identified y prtil lest squre discriminnt nlysis nd significnt nlysis of metolites S. no. Compounds Fold chnge P 1 Alnine E 19 2 N Methyl D sprtic cid Choline E 09 4 BOC L tyrosine E 09 5 BOC D phenyllnine E 09 6 Adenosine E Aminoutnote (gmm minoutyric cid) 8 Gunine Allntoin E N Methyl L glutmte E L Arginine L Histidine E L Proline E N Acetyl putrescine E Di tert utylenzldehyde E Alph ketoglutric cid E L Isoleucine E Tryptophn E Isocitric cid E Glucosmine fold chnge ws recorded in 3,5 di tert utylenzldehyde, followed y llntoin > N methyl L glutmte > L rginine > tryptophn (3.845, 3.613, , nd 3.395; Tle 3), wheres the mximum decrese in fold chnge ws recorded in gunine followed y rioflvin < denosine < N methyl D sprtic cid < lnine < lphketoglutric cid (Tles 3 nd S1). Figure 7 presents mz/rt pek vlues of the most importnt metolites for sensitive nd tolernt vrieties t 14 nd 25 dys fter drought stress initited. In generl, the pek vlues of choline, gunine, GABA, denosine, phenyllnine, sprtic cid, nd gunosine decresed due to drought stress in oth vrieties. This indictes tht the ccumultion of these metolites ws reduced over time due to drought stress. Glutthione disulphide ccumultion ws incresed fter drought stress imposition ut reduced due to longer period of drought stress. Arginine lso showed similr trend for tolernt lines; however, sensitive lines showed continuous ccumultion in long term drought stress (25 dys of drought stress). The pek vlues of llntoin, histidine, L proline, tyrosine, isoleucine, nd tryptophn incresed in oth the tolernt nd sensitive vrieties. However, the pek vlues were consistently higher in the tolernt vriety, which indictes the higher ccumultion of these metolites in tolernt vrieties compred with sensitive vrieties under the drought stress condition. The pek for isocitric cid showed n increse t oth time points for the sensitive vriety, wheres it showed n increse only t 25 dys fter drought stress for the tolernt vriety. The mz/rt pek vlues were significntly different etween tolernt nd sensitive vrieties under drought conditions for llntoin, histidine, L proline, tyrosine, isoleucine, tryptophn, rginine, phenyllnine, glutmine, nd glucosmine Metolic pthwy nlysis The pthwy nlysis of MetoAnlyst3 ws performed on significntly different known metolites using A. thlin s the pthwy lirries. Seventeen different pthwys were identified where these metolites re involved in different steps (Tle 4), of which five re iosynthetic pthwys (minocyl trna iosynthesis; indole lkloid iosynthesis; glucosinolte iosynthesis; phenyllnine, tyrosine, nd tryptophn iosynthesis; nd vline, leucine, nd isoleucine iosynthesis), nd the remining re involved with primry metolism. 4 DISCUSSION Drought dversely ffects plnt morphology nd physiology nd hence results in reduced growth. Drought cuses reduction in photosynthesis nd photochemicl efficiency, which ffects photosynthetic mchinery, reduces lef expnsion, enhnces senescence of new leves, nd results in decresed productivity (Frooq et l., 2009). To cope with the drought stress, the plnt must develop specific tolernce mechnism. Different vrieties hve shown different ilities to mintin growth nd productivity y cclimting to stress conditions through specific tolernce mechnisms. Genotypic vriility in different ttriutes relted to photosynthesis, RWC, nd root nd shoot dry weight re good indictors of wter stress or drought monitoring. Our results demonstrted tht in the sensitive vriety, even short term

9 KHAN ET AL. 123 FIGURE 7 Significntly different levels of selected metolites (ANOVA, P.05, Tukey's honestly significnt difference) in the leves of two chickpe vrieties under control nd drought conditions t two time points (14 nd 25 dys). G1: drought sensitive chickpe vriety (Punj Noor 2009); G2: drought tolernt chickpe vriety (93127). Error rs represent stndrd errors of the men (n = 6) t ech time point. C = control, D = drought. Different letters indicte significnt differences (P <.05) mong tretments (drought vs. irrigtion) for genotype for mz/ rt pek in prticulr time point. Asterisk represents significnt difference in mz/rt pek etween sensitive (G1) nd tolernt (G2) vrieties under the drought condition t specific time point of smpling exposure (14 dys) to the drought condition resulted in significnt decrese in the chlorophyll production ut the tolernt vriety sustined, nd lower decrese ws recorded. The photochemicl efficiency (F v /F m ) of oth vrieties ws significntly ffected y drought stress. However, the sensitive vriety showed greter F v /F m reduction thn the tolernt vriety. Some other studies lso demonstrted significnt reduction of photosynthetic cpcity nd chlorophyll content due to stress condition (Bsu, Rmegowd, Kumr, &

10 124 KHAN ET AL. FIGURE 7 Continued. Pereir, 2016; Cielnik, Filek, & Cielnik, 2006; J. Li et l., 2017; Luo, Zhng, & Zhng, 2016; Ullh, Sun, Yng, & Zhng, 2017; Zhou et l., 2017). Our results re ligned with other reported studies nd demonstrted tht the experimentl condition seprted the expression of these trits etween the tolernt nd sensitive vrieties. Drought induced reduction in plnt dry weight hd lso een studied previously in different crops (Boutr, Akhkh, Al Shoii, & Alhejeli, 2010; Munns, 2002; Slm, Khtun, Bhuiyn, Ysmin, & Khn, 2016). We hve found significnt reduction in chickpe dry weights when grown under the drought condition in oth the drought tolernt nd drought sensitive vrieties. It is worth noting tht the percentge of decrese in dry weights ws significntly higher for the sensitive vriety. Erlier studies demonstrted significnt reduction in shoot dry weights when grown under drought conditions in chickpe (Png et l., 2016) nd in corn (Anjum et l., 2017). Silvente, Soolev, nd Lr (2012) ttriuted the

11 KHAN ET AL. 125 FIGURE 7 Continued. decrese in dry weight under drought in the sensitive soyen vriety to the depletion of sucrose in the leves of this vriety. However, Reddy, Chitny, nd Viveknndn (2004) reported tht wter stress inhiited dry mtter production due to limittion of photosynthesis. Our dt lso demonstrted reduction of photosynthetic cpcity (demonstrted y reducing chlorophyll content nd F v /F m vlues), which might hve ultimtely reduced shoot dry weight in our study. Lef RWC is n indictor of wter sttus in plnts under the drought condition nd reflects the lnce etween wter supply to the lef tissue nd trnspirtion rte. RWC hs een used to identify stress tolernt vrieties in different crops (Rmpino, Ptleo, Gerrdi, & Perott, 2006; Soltys Klin, Plich, Strzelczyk Żyt, Śliwk, & Mrczewski, 2016). Significnt correltions etween RWC nd reltive growth rte, photosynthesis rte, chlorophyll, nd proline contents hve een reported in Aeluropus lgopoide (Mohsenzdeh, Mlooi,

12 126 KHAN ET AL. TABLE 4 Pthwy nmes, totl metolites involved in tht pthwys, metolites significntly ccumulted in present study (hits), nd flse discovery rte (FDR) Pthwy nme Totl Hits FDR Aminocyl trna iosynthesis Glycine, serine, nd threonine metolism Arginine nd proline metolism Indole lkloid iosynthesis Glucosinolte iosynthesis Purine metolism Histidine metolism Selenomino cid metolism Phenyllnine, tyrosine, nd tryptophn iosynthesis Cron fixtion in photosynthetic orgnisms Alnine, sprtte, nd glutmte metolism Glycerophospholipid metolism Vline, leucine, nd isoleucine iosynthesis Glutthione metolism Tryptophn metolism Vline, leucine, nd isoleucine degrdtion Amino sugr nd nucleotide sugr metolism Rzvi, Frrhi Aschtini, & Rzvi, 2006). Though RWC ws reduced due to drought stress in our study, reduction in the tolernt vriety (93127) ws significntly lower thn in the sensitive vriety (Punj Noor 2009). In ddition, the tolernt line showed less dmge to chlorophyll content nd photosynthetic cpcity nd lso reduced loss of dry weight nd incresed proline level compred with the sensitive vriety, which is in greement with the findings of Mohsenzdeh et l. (2006). The increse of ntioxidnt enzyme ctivities is known to occur under wter deficit, slinity, low temperture, hevy metl exposure, nd UV rditions (Shrm & Dietz, 2006). These enzymes form n ntioxidnt defence system in plnts to mitigte oxidtive dmge. Under drought stress, cells of plnts re suffered from the ccretion of rective oxygen species (ROS), which is responsile for oxidtive dmge nd cell deth. The ROS molecules re found in vrious sucellulr comprtments. The stility mong the cretion nd reclmtion of ROS is influenced y enhnced ntioxidnt enzyme ctivities (Cverzn, Csssol, & Brmmer, 2016). Both verities showed incresed level of ROS enzymes; however, the tolernt vriety demonstrted higher level thn the sensitive vriety. The oserved increse in ntioxidnt enzyme ctivities in the tolernt vriety enhnced ROS detoxifiction mechnism. Mtsumur, Tyshi, Kmgt, Soum, nd Sruym (2002) reported n increse in Ct gene expression under stress tht improved tolernce to stress in trnsgenic rice y reducing the dverse effects of hydrogen peroxide (H 2 O 2 ). Significnt increse in the ctivities of superoxide dismutse, peroxidse, scorte peroxidse nd ctlse ws lso reported previously in other plnt species under stress environments (Mfkheri, Siosemrdeh, Bhrmnejd, Struik, & Sohri, 2010; Pompelli et l., 2010; W. B. Wng et l., 2009). In order to understnd chnges in metolites in chickpe under the drought condition, we crried out metolomic profiling nlysis in two different chickpe vrieties (drought sensitive nd tolernt) s levels of metolite ccumultion during stress t different growth stges cn provide more specific nd ccurte indiction of stress tolernce. This is the first report on chickpe to demonstrte comprehensive differences in metolic profiling in tolernt nd sensitive chickpe vrieties over n extended period of drought condition. For this purpose, we used glol UPLC HRMS nlysis to clssify metolites linked with drought tolernce in the chickpe crop. The study demonstrted vritions in the metolite levels in chickpe leves of plnts grown under drought nd control conditions. Imposition of drought stress hd considerly incresed the ccumultion of mino cids including proline, tryptophn, tyrosine, histidine, nd isoleucine t oth time points. The incresed ccumultion of mino cids under drought stress ws reported previously in whet (Bowne et l., 2012; Rhmn et l., 2017), in soyen (Silvente et l., 2012), in grpe vine (Hocherg et l., 2013), nd in Phseolus vulgris (Sssi, Aydi, Hessini, Gonzlez, & Arrese Igor, 2010). The expnded level of mino cids is considered to enhnce stress resilience in plnts y influencing vrious physiologicl mechnisms, for exmple, djustment to osmotic chnges, ROS detoxifiction, nd regultion of the intrcellulr ph level (Krsensky & Jonk, 2012). Regrdless of the vriety (tolernt or sensitive), drought stress conditions enhnced ccumultion of those metolites, ut their reltive intensity ws higher in the tolernt vriety (Figure 7) t different time points. These results suggest the iochemicl pthwys involving those metolites potentilly relted to drought tolernce in chickpe. Drought stress leds to the reduction in protein synthesis nd n increse in hydrolysis of proteins, endorsing surge in solule nitrogen compounds such s free mino cids (Frooq et l., 2009; Krsensky & Jonk, 2012). Proline, highly ccumulted due to wter stress, not only cts s n osmolyte during drought ut lso cts s signlling molecule nd provides defence ginst oxidtive dmge (Cheng et l., 2015; Ggné Bourque, Bertrnd, Clessens, Aliferis, & Jji, 2016; Hyt et l., 2012). In cell cytoplsm, it not only stilizes protein structure ut lso helps in mintining cell ph nd redox sttus under severe stress condition y reducing the mount of singlet oxygen present, which cuses lipid peroxidtion of thylkoid memrnes (Ali & Mohnty, 1997; Hmmd & Ali, 2014; Hyt et l., 2012; Szdos & Svouré, 2010). As in our study, proline ws lso highly ccumulted in soyen under the drought stress condition (Ds, Rushton, & Rohil, 2017), which clerly shows the potentil for n dvnced tolernce response provided y proline during dverse environmentl conditions. Under the drought condition, high ccumultion of proline might function s sink for excess products tht re needed for the mintennce of photosynthetic nd respirtory processes (Kishor et l., 2005). Tripthi et l. (2016) lso found similr results for mino cid metolism where metolomic ltertions during drought stress were monitored in soyen. Both vrieties showed dmge to photosynthetic cpcity in the present study; however, the dmge ws significntly lower in the tolernt vriety (16%) s compred with the sensitive vriety (64%). This could potentilly e contriuted y the strong ccumultion of proline, which helped in protecting the photosystem nd mintining redox lnce in photosynthetic memrnes. Incresed level of romtic mino cids, such s L tryptophn nd tyrosine, ws exhiited in the current study, which ws lso found in

13 KHAN ET AL. 127 whet (Bowne et l., 2012; Rhmn et l., 2017) nd in soyen (Ds et l., 2017) under drought conditions. The highly undnt romtic mino cids potentilly cted s n lternte source of energy supply nd provided stress tolernce in chickpe. Under vrious stress conditions, the plnt metolic response is regulted y convergent signl trnsduction ssocited with energy production. These romtic mino cids re the precursors of different secondry metolites including indole cette, lipid precursor, nd lignin in the shikimte pthwy, which plys vitl role in stress tolernce (Med & Dudrev, 2012; Suguiym, Silv, Meirelles, Centeno, & Brg, 2014). In ddition of cting s lternte source for energy under stress conditions, L tryptophn hs een demonstrted to ply roles in trnsporttion of ions, opening of stomt, reducing ROS nd ct s n osmolyte (Ri, Ydv, & Gde, 2009; Ford, Cssin, & Bcic, 2011). Tryptophn nd ROS re synthesized in photosynthetic orgns like chloroplst, nd tryptophn is the trget of oxidtion. As tryptophn is trget for oxidtion, free proteins my provide uffer etween ROS nd protein (Bowne et l., 2012). Ford et l. (2011) demonstrted correltion etween incresed protein levels nd their involvement in ROS scvenging nd oxidtive stress metolism. Photosynthetic mchinery generlly genertes different ROS, such s singlet oxygen, superoxide, nd peroxide. However, under stress conditioning, the production of ROS could e incresed to such level tht cn inhiit the production of D1 protein, which is essentil for the repir of dmge in photosystem II (Allkhverdiev & Murt, 2004; Nishiym et l., 2001). In ddition, ROS cn oxidse different proteins including D1 protein (Silv et l., 2003). Higher tryptophn ccumultion in our study hs contriuted to ROS scvenging mechnism, which is evidenced y higher ROS scvenging enzyme ctivities in drought stress compred with control, thus ultimtely reducing the dmge to the photosystem nd the reduction in growth in chickpe plnts. The tryptophn histidine iosynthesis pthwy is integrted with numer of other metolic pthwys. In our study, drought tolernt vriety showed very strong ccumultion of histidine fter 25 dys of drought stress (sed on mz/rt pek). Zemnová, Pvlík, Pvlíková, nd Tlustoš (2014) demonstrted tht Nocce cerulescens ccumulted 10 fold higher histidine thn Aridopsis hlleri under cdmium stress. Their oservtions indicted tht histidine my e involved in Cd resistnce nd ccumultion y reducing oxidtive dmge. Histidine might hve plyed similr role in reducing oxidtive stress in our study, which helped in mintining memrne structure nd wter sttus in the plnt (demonstrted y RWC). The incresed level of tyrosine under drought stress nd its possile role in tolernce hve een studied y different uthors (Bowne et l., 2012; Rhmn et l., 2017; Yo, Wng, Cheng, & Wu, 2013). Our study highlights the increse of mino cid tyrosine (mz/rt pek) in the tolernt vriety under the drought condition. Since the lst decde, extensive reserch hs een crried out to study the role of protein tyrosine on plnt physiology nd on iotic nd iotic stress tolernce (Mt Pérez et l., 2016), nd it ws found to e ssocited with drought tolernce in plnts (Hmmd & Ali, 2014; Shnkr, Agrwl, Shrm, Pndey, & Pndey, 2015). Witt et l. (2012) reported n incresed level of tryptophn nd phenyllnine in corn under drought conditions. However, the phenyllnine level ws decresed under drought compred with the control condition in our study. An incresed level of rnched mino cids hs een reported in whet nd Aridopsis under drought conditions (Bowne et l., 2012; Joshi & Jnder, 2009; Rhmn et l., 2017) nd lso hs een regulted t the trnscriptionl level in Aridopsis (Urno et l., 2009). Our study lso demonstrted incresed level of isoleucine in the tolernt vriety under the drought condition, wheres the level of the sme metolite ws decresed in the sensitive vriety. Tylor, Hezlewood, Dy, nd Millr (2004) reported tht rnched chin mino cids re source of lternte energy when Aridopsis plnts were under sugr strvtion, which is common phenomenon under drought stress conditions. This supports the incresed level of isoleucine in the tolernt vriety over different time points in our study. This present study hs demonstrted enhnced ccumultion of rginine in the sensitive vriety under the drought condition. Arginine is vitl mino cid in plnts tht cts s precursor in the synthesis of other mino cids including some polymines nd provides medium for the trnsporttion of nitrogen in plnts under stressful environmentl conditions (Bruc et l., 2012; Flores et l., 2008). It hs een reported erlier tht the expression of genes involved in the iosynthesis of rginine incresed wheres those enzymes involved in the rekdown of rginine decresed during stress. Besides this, enzyme rginine decroxylse tht is involved in rginine metolism is known for its synergistic effect on polymine concentrtion nd thus enhnces plnt tolernce to vrious iotic stresses in Aridopsis, rice, tomto, nd tocco (Q. Wng et l., 2011; Wimlseker, Tertz, & Scherer, 2011). Peremrti, Bssie, Christou, nd Cpell (2009) showed tht overexpression of rginine decroxylse in trnsgenic rice plnts leds to incresed tolernce to wter stress. These results show tht the vritions in mino cid concentrtions induced y iotic stresses my e connected with the ltered expression of genes tht encode for enzymes intricte with plnt metolism (Krsensky & Jonk, 2012). For plnt development nd metolism, nucleotide metolism is one of the essentil components tht ffect mny metolic pthwys. Nucleotides re n essentil uilding lock for nucleic cid. For the synthesis of crohydrte, lipids, peptides, nd secondry metolites, nucleic cid provides nucleic cids tht serve s n ultimte energy source (Stsoll, Kthir, Thorpe, & Ashihr, 2003). Enhnced ccumultion of purine compounds such s llntoin ws lso recorded for the tolernt vriety, ut not for the sensitive vriety in our study. Allntoin, n intermedite in purine ctolism, ws reported to ccumulte in plnts during stress (Tkgi et l., 2016). Silvente et l. (2012) seprted sensitive nd tolernt soyen genotypes on the sis of ccumultion of llntoin, pinitol, nd citric cid. Allntoin plys vitl role in plnt survivl under stress condition nd hs significnt role in plnt metolism, plnt signlling, ioenergetics, nd heredity. Allntoin my function in stress defence y reducing ROS ccumultion nd deth of plnt cells (Brychkov, Alikulov, Fluhr, & Sgi, 2008). Besides its role in scvenging ROS, llntoin my lso trigger stress responses y incresing levels of the stress induced hormone scisic cid (ABA; Wtne et l., 2014). ABA hs essentil roles in developmentl nd dptive responses to numerous environmentl stresses, ecuse of its synergistic nd ntgonistic interctions with other plnt hormones, notly with slicylic cid, jsmonic cid, nd ethylene (De Vleesschuwer,