Molecular Physiology Department, Agricultural Biotechnology Research Institute of Iran, PO Box 31535-1897, Karaj, IranBioinformatics Department, Agricultural Biotechnology Research Institute of Iran, Iran
a r t i c l e i n f o
rticle history:eceived 8 December 2012eceived in revised form 10 April 2013ccepted 10 April 2013vailable online xxx
Barley (Hordeum vulgare) is one of the most important cereals in many developing countries wheredrought stress considerably diminishes agricultural production. Glutathione S-transferases (GSTs EC2.5.1.18) are multifunctional enzymes which play a crucial role in cellular detoxification and oxida-tive stress tolerance. In this study, 84 GST genes were identified in barley by a comprehensive in silicoapproach. Sequence alignment and phylogenetic analysis grouped these HvGST proteins in eight classes.The largest numbers of the HvGST genes (50) were included in the Tau class followed by 21 genes in Phi,five in Zeta, two in DHAR, two in EF1G, two in Lambda, and one each in TCHQD and Theta classes. Phy-logenetic analysis of the putative GSTs from Arabidopsis, rice, and barley indicated that major functionaldiversification within the GST family predated the monocot/dicot divergence. However, intra-speciousduplication seems to be common. Expression patterns of five GST genes from Phi and Tau classes wereinvestigated in three barley genotypes (Yusof [drought-tolerant], Moroc9-75 [drought-sensitive], andHS1 [wild ecotype]) under control and drought-stressed conditions, during the vegetative stage. Allinvestigated genes were up-regulated significantly under drought stress and/or showed a higher level of
transcripts in the tolerant cultivar. Additionally, GST enzyme activity was superior in Yusof and inducedin the extreme-drought-treated leaves, while it was not changed in Moroc9-75 under drought condi-tions. Moreover, the lowest and highest levels of lipid peroxidation were observed in the Yusof andMoroc9-75 cultivars, respectively. Based on the achieved results, detoxification and antioxidant activityof GSTs might be considered an important factor in the drought tolerance of barley genotypes for further investigations.
ntroduction
The glutathione S-transferases (GSTs, EC 2.5.1.18) are a super-amily of multifunctional enzymes involved in the detoxificationf xenobiotic and endobiotic compounds through conjugatingripeptide (y-Glu-Cys-Gly) glutathione (GSH) to hydrophobic sub-trates. In plants, GSTs comprise approximately 2% of solubleroteins (Scalla and Roulet, 2002). Based on sequence relatedness,
mmunological cross reactiveness, kinetic properties, and genome
Please cite this article in press as: Rezaei MK, et al. Glutathione S-transferaand gene expression pattern. J Plant Physiol (2013), http://dx.doi.org/10.10
rganization, plant soluble GSTs have been grouped into differ-nt classes, which include Phi, Tau, Lambda, dehydroascorbateeductase (DHAR), Theta, Zeta, elongation factor 1 gamma (EF1G),
Abbreviations: AOS, activated oxygen species; CDNB, 1-chloro-2,4-initrobenzene; GST, glutathione S-transferase; MDA, malondialdehyde; RWC,elative water content; WHC, water holding capacity.∗ Corresponding author at: Agricultural Biotechnology Research Institute of Iran
and tetrachlorohydroquinone dehalogenase (TCHQD). The first fourclasses are plant specific (Edwards and Dixon, 2005).
Plant GSTs have been the center of attention because of theirrole in herbicide detoxification. Study of different plant tissue typesshowed the presence of these enzymes at every stage of plantdevelopment from early embryogenesis to senescence (Sari-Gorlaet al., 1993; McGonigle et al., 2000; Soranzo et al., 2004). Bioticand abiotic stresses, plant hormones such as auxins, ethylene,cytokinins and ABA, heavy metals, GSH, and hydrogen peroxidaseare considered to be inductive factors that regulate GST activ-ity differentially (Marrs, 1996). Plant GSTs are also involved inendogenous metabolism, including functioning as GSH-dependentisomerases (Dixon et al., 2000; Thom et al., 2001), noncatalyticallyacting as flavonoid-binding proteins, stress signaling proteins, andregulators of apoptosis (Dixon et al., 2003). Some GST isoforms actas glutathione peroxidases (GPOX) and protect membrane integrityby reducing toxic lipid peroxidation products (Bartling et al., 1993;
se (GST) family in barley: Identification of members, enzyme activity,16/j.jplph.2013.04.005
Cummins et al., 1999). Not only do they reduce lipid peroxidesdirectly, but also they may act to remove lipid peroxidation of endproducts such as alkenals, 4-hydroxynonenal, ethacrynic acid (EA),and other �,�-unsaturated aldehydes (Dalton et al., 2009).
Drought is one of the main environmental threats to agricul-ural production world-wide (Bray, 1993). During drought stresshe imbalance between antioxidant defense and the amount of acti-ated oxygen species (AOS) causes devastating effects on plants.lthough AOS play an important role in inter- and intracellularignaling, they are harmful in high concentrations (Asada, 1999;reusegem et al., 2001). One key role of GSTs in plants is referredo as defense mechanism against the toxicity of AOS (Galle et al.,009).
Barley is one of the most important cereal commodities inany developing countries, where drought stress and arid cli-ates significantly affect agricultural production (Ceccarelli, 1994;
eccarelli et al., 2007). It is ranked as the fourth most abundantereal in both area and tonnage harvested (http://faostat.fao.org).ecause of its relative adaption to diverse environmental condi-ions, barley remains a major food source in poor countries (Grandond Macpherson, 2002; Nevo et al., 2012) and due to its high solubleietary fiber, it is considered a functional food in many developedountries (Collins et al., 2010).
Because barley is a kind of self-pollinated cereal with a diploid2n = 14) genome, it is considered a genetic model of the Trit-ceae tribe within the Poaceae. The large genome size of thispecies (greater than 5 Gb, approximately 12 times that of rice)as limited vast investigation of barley genomics and moleculariology (Varshney et al., 2007; Matsumoto et al., 2011). Severalembers of the GST genes have been identified and divided into
ifferent classes in maize, soybean (McGonigle et al., 2000), Ara-idopsis (Wagner et al., 2002), wheat (Galle et al., 2009), and riceJain et al., 2010). Although conspicuous research activities wereonstructed to the identification and functional analyses of GSTenes in plants, there are few studies on barley in this gene familyMohsenzadeh et al., 2009).
We are interested in exploring the molecular aspects of barleyenotypes that may be involved in their various levels of toleranceo drought. In the present study, we have tried to identify and clas-ify different members of the GST family – that play a key rolen oxidative stress tolerance – in barley by an in silico approach.
oreover, GST enzyme activity and expression patterns of someembers as well as malondialdehyde (MDA) content were studied
n drought-tolerant and sensitive barley genotypes under differentevels of water stress to detect the likely involvement of these genesn drought tolerance.
aterials and methods
equence collection
The sequences of glutathione S-transferases (GSTs) were down-oaded from the National Center for Biotechnology InformationNCBI), the full-length Barley cDNA Database (http://barleyflc.na.affrc.go.jp/hvdb/index.html), (Matsumoto et al., 2011) andhole-genome shotgun assembly database at IPK Gatersleben
http://webblast.ipk-gatersleben.de/barley/) (Mayer et al., 2012).
n silico approach for GST gene identification
One protein sequence of each Arabidopsis thaliana GST classas used as the query to perform tBLASTn searches against trans-
ated nucleotide databases. A minimum cut-off E value (≤e−20)as applied to select significant matches. A threshold of at least
5% nucleotide sequence identity was employed to discrimi-
Please cite this article in press as: Rezaei MK, et al. Glutathione S-transferaand gene expression pattern. J Plant Physiol (2013), http://dx.doi.org/10.10
ate between duplicated genes (Soranzo et al., 2004). The cDNAequences with no reported protein were subjected to translationnd analyzed by the PROSITE program (http://prosite.expasy.org/)o confirm the presence of the GST domain. In addition, the presence
PRESShysiology xxx (2013) xxx– xxx
of a GST-N domain in each individual protein was reconfirmed bySMART analysis (http://smart.embl-heidelberg.de/).
Phylogenetic tree reconstruction
Conserved domains of eight GST classes were determinedbased on the protein sequence alignment of each class mem-ber from barley (Hordeum vulgare L.), rice (Oryza sativa L.), andArabidopsis. The graphic view of protein alignment was achievedusing BioEdit software (Hall, 1999). The online MEME program(http://meme.sdsc.edu) was used for motif prediction. Classifica-tion of GST proteins was performed according to the conserveddomain of GST proteins using the genes were already assignedto GST classes (Dixon et al., 2002). Sequences were aligned byClustalW (Larkin et al., 2007), using the MEGA4 program (Tamuraet al., 2007). Phylogenetic trees were drawn and formatted inMEGA4. The neighbor-joining method with p-distance was used forthe construction of the trees, and bootstrap analysis was performedwith 1000 replicates.
Plant material and drought stress treatments
Three barley genotypes (Yusof, HS1, Moroc9-75) were used forphysiological and gene expression experiments. Yusof is one ofthe drought tolerant varieties of barley that is cultivated in thearid regions of Iran; HS1 is a wild barley (Hordeum vulgare ssp.spantaneum) ecotype from the National Plant Gene Bank of Iran(accession no. 02TN37) that is well-known for its drought toler-ance; and Moroc9-75 as a drought sensitive cultivar was selectedfor comparative analysis (Ceccarelli, 1994; Ceccarelli et al., 2007).
A pot experiment was carried out based on complete randomdesign with three treatments (well-watered, moderate, and severedrought stress) and three replications (4 pots/replication) undercontrolled conditions in a greenhouse at the Agricultural Biotech-nology Research Institute of Iran (ABRII).
Pots contained perlite and peat moss (1:3, v/v). Differentwater regimes, including 70% water holding capacity (WHC)as well-watered, 30% and 10% WHC as moderate and extremedrought-stressed conditions, respectively (Doorenbos and Pruit,1977; Guo et al., 2009) were performed on the three barley geno-types. Drought treatment was induced at the two–+leaf stage bywithholding water until soil pots reached the desired WHC.
Relative water content (RWC) and dry matter
Complete spanned leaves were immediately weighed to obtainfresh weight (FW), then floated on distilled water for 16 h andweighed again for turgid weight (TW). Leaves were dried at 70 ◦Cfor 24 h to measure dry weight (DW). RWC was computed as(FW − DW)/(TW − DW) × 100 (Dhanda and Sethi, 1998; Galle et al.,2009).
To determine dry matter content, the harvested plants weredried at 70 ◦C for 72 h then weighed using a sensitive digital balance.
Malondialdehyde (MDA) content
The assay of MDA was performed using a thiobarbituric acidmethod as described by Ederli et al. (1997). The MDA con-centration was calculated according to the following formula:[(A532 − A600)/157,000] × 106 (Du and Bramlage, 1992).
Measurement of the GST activity
se (GST) family in barley: Identification of members, enzyme activity,16/j.jplph.2013.04.005
Crude protein was extracted from 0.2 to 0.5 g of homoge-nized leaves using an extraction buffer (0.1 M phosphate bufferpH 7.0, containing 1 mmol L−1 phenylmethylsufonylfluoride and
% polyvinyl-polypirrolidone). After centrifugation at 10,000 × gor 15 min, the supernatant was decanted. Enzyme activity wasetermined spectrophotometrically using standard substrate 1-hloro-2,4-dinitrobenzene (CDNB) (Habig et al., 1974). The reactionnitiated by the addition of CDNB to the leaf extract and the rate ofncrease in absorbance was measured at 340 nm. The amount ofnzyme that produced 1 �mol conjugated product in 1 min wasetermined as 1 U, ε340 = 9.6 mM−1 cm−1. The enzyme activity wasxpressed in terms of specific activity (Ug−1 FW−1).
NA isolation and real-time PCR experiment
Total RNA was isolated from leaf tissues using Trizol reagentLife Technology, Invitrogen, USA). Treatment with RNase–freeNase (Promega, USA) was applied to RNA samples to guarantee
he removal of DNA. For first cDNA strand synthesis, RNA sam-les were reverse-transcripted using iScript cDNA synthesis kitBIO-RAD, USA). The proper primers were designed (Table S1) andynthesized at the MWG Company (Germany). The relative quanti-ative PCR was performed in an iCycler iQ thermocycler (BIO-RAD,SA) using the iQ Syber Green Supermix kit (BIO-RAD, USA). Theousekeeping gene HvActin was selected as an internal control forormalizing real-time PCR data (Guo et al., 2009). The 2(−��Ct)
ethod (Livak and Schmittgen, 2001) was used to calculate GSTxpression levels of the genotypes under drought-stressed condi-ions which were normalized to the control Ct value of Yusof as arought tolerant cultivar.
tatistical analysis
The mean of each measurement was computed from the dataf at least three independent biological replicates. Analysis of vari-nce (ANOVA) for comparing treatment means was determined byuncan’s multiple range tests, available in the SAS package (Afifit al., 2004).
esults
dentification and classification of GST genes in barley
Comprehensive searches of the nucleotide collection of NCBI,he full-length Barley cDNA Database (Matsumoto et al., 2011), andhole-genome shotgun assembly data (Mayer et al., 2012) led to
he identification of 84 putative members of the GST gene family inarley (Table S2). The conserved pattern of N terminal domain ofhese HvGST proteins was determined through multiple sequencelignment (Fig. S2).
Using the protein sequence alignment and homology-basedree, barley GSTs were assigned to 8 classes. Tau class containedhe largest number of GST genes (50) followed by 21 members inhi, five in Zeta, two each in DHAR, EF1G and LAMBDA, and oneach in Theta and TCHQD classes (Table S2 and Fig. S3). The con-erved domains of different GST classes were determined based onhe protein sequence alignment of different class members fromarley, rice, and Arabidopsis. As is postulated, the sequence similar-
ty between GST members of barley and rice was higher than theimilarity between Arabidopsis and either barley or rice GST mem-ers (Fig. S1). The phylogenetic tree of corresponding GST genesf barley along with different GST members of rice (Table S3) andrabidopsis (Table S4) was also created (Fig. 1).
ry matter, RWC, and MDA content of the barley genotypes with
Please cite this article in press as: Rezaei MK, et al. Glutathione S-transferaand gene expression pattern. J Plant Physiol (2013), http://dx.doi.org/10.10
ifferent drought susceptibility
Statistical analysis showed that the effect of drought treat-ent on dry matter content was significant (p < 0.01), and its level
PRESShysiology xxx (2013) xxx– xxx 3
decreased significantly under extreme drought stress. The mostdry matter content was observed in Yusof and HS1 varieties underextreme drought-stressed conditions, and the least was observedin Moroc9-75 under both levels of drought treatment (Fig. 2).
Although drought stress reduced RWC in the three cultivars,there were no significant differences (p < 0.05) in RWC of differentwater regimes in the Yusof cultivar. The most reduction of RWCwas referred to Moroc9-75 under extreme water-stressed condi-tions. The pattern of RWC variation in wild ecotype HS1 was similarto Moroc9-75, but its level was higher compared to the droughtsensitive variety (Fig. 2).
Lipid peroxidation was estimated and quantified by measur-ing its specific end-products; MDA. This data (Fig. 2) showed anincrease in lipid peroxidation in all three barley genotypes underwater stress, but the increase was greatest in Moroc9-75.
GST activity under control and drought stress conditions
GST enzyme activity was higher in Yusof and was induced inextreme drought-treated leaves, yet no significant change wasobserved in the GST activity of the Moroc9-75 and HS1 cultivarsduring either control or stress conditions (Fig. 2).
Expression patterns of the candidate barley GST genes
Expression patterns of five GST genes from the Phi and Tauclasses and one member of the exostosin family were evalu-ated under control and drought stress conditions in the threecontrasting genotypes. In the tolerant cultivar, all the investi-gated genes were up-regulated significantly under drought stress(HvGSTF13, HvGSTF7, HvGSTU1 and HvEX1) and/or had the highestlevel of transcripts under control conditions (HvGSTF13, HvGSTF16,HvGSTF7 and HvGSTU32) compared to the other genotypes (Fig. 3).The highest amount of transcription of HvGSTF13 in the Yusof cul-tivar was detected in the least WHC level. A similar result wasdetected in Moroc9-75 with a lower transcription level, and theopposite expression pattern was observed in HS1 ecotype. TheHvGSTF16 was not up-regulated significantly by the drought stressin any of the investigated genotypes; however, it showed the mostamount of the transcripts in Yusof. Transcript level of HvGSTF7 wasincreased by the extreme stress in both Yusof and Moroc9-75 culti-vars, but it was not responsive to any applied stress in HS1. At Yusof,the expression of HvGSTU1 increased about threefold and fivefoldunder mild and severe drought stress respectively, while only slightor no significant elevations were observed in the other genotypes.The highest level of HvGSTU32 transcription was referred to theYusof cultivar under control conditions, although it was decreasedunder drought condition. The HvEX1 was significantly up-regulatedunder mild and severe drought-stressed conditions by about 5-and 17-fold in the Yusof cultivar while no significant change wasobserved at its expression in the other cultivars (Fig. 3).
Discussion
The complete genome sequences of Arabidopsis and rice notonly facilitate bioinformatics and molecular studies of these modelplants, but they also assist the exploration of other important crops,such as barley (Cai et al., 2011). In this research, based on theinformation of GST proteins from Arabidopsis and rice, an in silicoapproach was exploited to identify putative members of GST genefamily in barley. The conserved domain of GST proteins was rec-ognized by the alignment of protein sequences from Arabidopsis
se (GST) family in barley: Identification of members, enzyme activity,16/j.jplph.2013.04.005
and rice that were already assigned to GST classes. GST-N domain(domain I) is highly conserved among different GSTs and providesspecific residues for GSH binding and catalytic activity (Reinemeret al., 1996; Neuefeind et al., 1997a,b). A total of 84 sequences from
4 M.K. Rezaei et al. / Journal of Plant Physiology xxx (2013) xxx– xxx
F rabidof oining
btbt
befmsIdecstht2ac
piattahD
ig. 1. Phylogenetic tree and classification of GST proteins from barley, rice, and Aull-length protein sequences using ClustalW in MEGA4 software by the neighbor-j
arley were predicted to contain GST-N domain, which is more thanhe 79 and 54 members reported in rice (Jain et al., 2010) and Ara-idopsis (Wagner et al., 2002), respectively, as expected based onhe their genome size.
Phylogenetic analyses with GST proteins from barley, Ara-idopsis, and rice were performed to assign GST proteins usingvolutionary distances. Based on the resultant tree, majorunctional diversification within the GST family predated the
onocot/dicot divergence, and eight different classes of the plantoluble GSTs are present in all the above-mentioned plants.ntra-specious duplication, however, seems to be common. Geneuplication plays a key role in the GST family evolution of differ-nt plants (Soranzo et al., 2004; Duarte et al., 2006). Studies onarnation, Arabidopsis, and wheat all revealed that GSTs from theame subclass are grouped on chromosomes as tandem duplica-ions (Edwards et al., 2000). Evolution of the rice GST gene familyas occurred by gene duplication followed by retention due tohe sub- or neo-functionalization of duplicated genes (Jain et al.,010). According to the obtained results, several examples of thenalogous gene duplication can be proposed in barley based onhromosome location and sequence similarity (Table S5).
Tau and Phi GSTs were dominant classes in barley just as in otherlants. The rapid evolutionary rate and large number of members
n these two plant specific GST classes compared with the Zetand Theta classes might have arisen as an evolutionary strategyo counteract several environmental challenges over a period of
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ime (Wolfe et al., 1989; Soranzo et al., 2004). They are dimericnd can catalyze the conjugation of a diverse range of xenobioticsaving key roles in detoxifying selective herbicides (Edwards andixon, 2005). GSTs are related to other GSH- and cysteine-binding
psis. The unrooted tree was constructed based on multiple sequence alignment of method. Bootstrap values (≥500) from 1000 replicates are considered in the tree.
proteins, with which they share a thioredoxin-like fold, as well asto stress-related proteins in a wide range of organisms. Based onthis evidence, it has been proposed that primordial stress proteinsmay be the ultimate ancestors of GSTs (Sheehan et al., 2001). Manyresearchers have examined the responsibility of GSTs for oxidativestress tolerance in different plants such as rice (Jain et al., 2010),barley (Ozturk et al., 2002; Guo et al., 2009), wheat (Galle et al.,2009), and potato (Seppänen et al., 2000). The large phi and tauclasses are often highly stress inducible and regularly crop up inproteomic and transcriptomic studies (Dixon and Edwards, 2010).
For expression analysis, five GST genes from the Phi and Tauclasses were selected based on previous reports implying theirinvolvement in drought stress response (Michalek et al., 2002;Ozturk et al., 2002; Kunieda et al., 2005; Guo et al., 2009; Satoet al., 2009). The gene HvEX1 has also been introduced as a droughtresponsive GST (Maraschin et al., 2005), but our further analysisrevealed that it should be categorized in the exostosin family. Infact, it encodes a galactosyltransferase that has a main role in thesynthesis of xyloglucan, the principle glycans that interlace cellu-lose microfibrils in most flowering plants (Madson et al., 2003). Theexostosin family is responsible for the polysaccharide synthesis ofplant cell walls (Tesfaye et al., 2009). Wall synthesis during plantdevelopment and growth could be influenced by biotic or abioticstresses (Humphery et al., 2007). Interestingly, the expression ofHvEX1 was significantly up-regulated by drought stress only in thedrought tolerant genotype.
se (GST) family in barley: Identification of members, enzyme activity,16/j.jplph.2013.04.005
Expression patterns of the selected HvGST genes were inves-tigated in three barley genotypes, Yusof (drought-tolerant),Moroc9-75 (drought-sensitive) and HS1 (wild ecotype) under con-trol and drought-stressed conditions during the vegetative stage.
M.K. Rezaei et al. / Journal of Plant Physiology xxx (2013) xxx– xxx 5
Fig. 2. Dry matter, relative water content, malondialdehyde concentration, and GST enzyme activity analysis in Yusof (drought tolerant), Moroc9-75 (drought sensitive), andH (70%n w sta
Ads(ctMw
gtaottig
eMep2
ei
S1 (wild ecotype) varieties under three levels of water holding capacity (WHC) as Cot significantly different (p < 0.05, Duncan test). The error bars on the columns sho
ll the investigated genes were up-regulated significantly underrought stress (HvGSTF13, HvGSTF7, HvGSTU1 and HvEX1) and/orhowed the highest level of transcripts under control conditionsHvGSTF13, HvGSTF16, HvGSTF7 and HvGSTU32) in the tolerantultivar, compared to the other genotypes. The expression pat-erns of all selected HvGSTs in the reproductive stage of Yusof and
oroc9-75 cultivars were similar, although the expression levelas higher compared to the vegetative stage (data not shown).
GST has also been previously reported as one of the thirty-fourenes which were highly transcribed only in the two drought-olerant genotypes, not in the drought-sensitive one. Therefore, theuthors suggested that these genes may be involved in the processf drought tolerance in barley (Guo et al., 2009). Based on func-ional classification, the aforementioned genes were divided intowo groups including regulators and functional genes that directlymprove drought tolerance. GSTs were considered in the secondroup by playing a role in the scavenging ROS for detoxification.
GST enzyme activity was higher in Yusof and induced in thextreme drought-treated leaves, however it was not changed inoroc9-75 during drought conditions. Higher levels of GST gene
xpression and enzymatic activity were also observed in tolerantotato genotypes under cold and osmotic stresses (Seppänen et al.,
Please cite this article in press as: Rezaei MK, et al. Glutathione S-transferaand gene expression pattern. J Plant Physiol (2013), http://dx.doi.org/10.10
000).In spite of similar trends between enzyme activity and gene
xpression, there is not an absolute association, because GSTsoenzymes show different responses toward CDNB (Meyer et al.,
WHC), T1 (30% WHC), and T2 (10% WHC). Means denoted by the same letters werendard deviation (SD) plotted by Excel 2007.
1991; Hussey and Hayes, 1992; Dixon et al., 2009; Galle et al.,2009). As some GST isoenzymes have very little activity towardCDNB but very high activity toward other substrates, the absenceof any detectable CDNB activity does not provide proof of theabsence of GSTs (Meyer et al., 1991). Both Phi and Tau GSTs,however, have a broad range of conjugating activity towardxenobiotics, and their existence can be readily determined usingCDNB and 4-nitrobenzylchloride (Edwards and Dixon, 2005).Moreover, the lowest and the highest levels of MDA were observedin the Yusof and Moroc9-75 cultivars, respectively. The increase ofMDA content is reported in soybean (Liang et al., 2003; Shan andLiang, 2010; Anjum et al., 2011), barley (Liang et al., 2003; Zlatevet al., 2006), and wheat (Khan and Panda, 2008) during waterstress. MDA elevation is also considered a symptom of senescence(Yang et al., 2001). The negative correlation between antioxidantactivity and MDA content has been reported (Esfandiari et al.,2007, 2008). Rapid increase of MDA content in drought sensitivecultivars could be referred to the inadequate activity of antioxidantenzymes.
Conclusively, the putative barley GST genes were identified,revealing the presence of all the GST classes in barley. The datacan be useful for the further study of their functions in bar-
se (GST) family in barley: Identification of members, enzyme activity,16/j.jplph.2013.04.005
ley and their evolution. Based on the achieved molecular andphysiological data, GST enzyme activity was superior in the tol-erant cultivar and induced under severe drought, yet this wasnot the case in the sensitive genotype. Furthermore, the lowest
6 M.K. Rezaei et al. / Journal of Plant Physiology xxx (2013) xxx– xxx
Fig. 3. Expression pattern of five GSTs and one exostosin gene in Yusof (drought tolerant), Moroc9-75 (drought sensitive), and HS1 (wild ecotype) varieties under three levelso C). Met el 200s
atidelcaoab
f water holding capacity (WHC) as C (70% WHC), T1 (30% WHC), and T2 (10% WHest). The error bars on the columns show standard deviation (SD) plotted by Excample).
nd highest levels of lipid peroxidation were observed in theolerant and sensitive cultivars, respectively. Therefore, GST antiox-dant activity might be considered an important factor in therought tolerance of barley genotypes. On the other hand, thexpression analysis showed either drought inducibility or higherevels of transcripts for the investigated genes in the tolerantultivar, suggesting their participation in barley stress toler-
Please cite this article in press as: Rezaei MK, et al. Glutathione S-transferaand gene expression pattern. J Plant Physiol (2013), http://dx.doi.org/10.10
nce. Characterization of these genes would not only improveur understanding of the mechanisms of stress tolerance, butlso provide tools to improve the grain yield and quality ofarley.
ans denoted by the same letters were not significantly different (p < 0.05, Duncan7. It is considered 0 for Yusof cultivar in the control conditions (as the reference
Acknowledgments
This work was supported by the Agricultural BiotechnologyResearch Institute of Iran (ABRII). The authors are grateful to Rei-haneh Pishkam Rad for her technical assistance.
Appendix A. Supplementary data
se (GST) family in barley: Identification of members, enzyme activity,16/j.jplph.2013.04.005
Supplementary data associated with this article canbe found, in the online version, at http://dx.doi.org/10.1016/j.jplph.2013.04.005.
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