Growing economy in many ways has led to climatic change by increasing global temperature, thereby rise in sea level resulting in increased salinity of groundwater and arid land . Also, increase in global temperature has increased frequency of drought  posing a big challenge for agriculture . Drought and salinity affected soils contain high concentration of ions such as chloride, sodium, calcium and magnesium in soil  or are nutrient deficient. Thereby, forcing the use of chemical fertilizers leading to increased cost of crop production environmental pollution. Breeding techniques have been beneficial in creating new cultivars resistant to drought and salinity. Also, understanding the tolerant genotypes response towards stress gives insights into improving the tolerance of crops.
Plant response to drought and salinity have been studied in terms of alterations in metabolism , detoxification , ABA induced response , proteome , transcriptome . Transcriptome networks  and differential gene expression studies with model plants under abiotic stress   have provide candidate genes for improving tolerance and productivity. Plant hormones play a role in inducing osmotic effects eliciting overlapping responses under drought and salinity . Transcription factor families like NAC , WRKY , DERB  , are reported to be differentially regulated under stress. Specific protein like defensins, SpPKE1 , cysteine proteases , RING finger protein  an E3 ubiquitin ligase  and genes modified using RNAi mediated gene silencing  are known to increase tolerance towards abiotic stress.
Among food crops, legumes form a major source of protein and contribute to enhancement of soil quality through their nitrogen fixing ability. A Fabaceae member, Lablab purpureus L. (Hyacinth Bean) HA4 variety is extensively grown in south India for its pods, and previous reports have indicated its tolerance to drought  and salinity . Transcriptomic variations, in root tissue, under drought stress  have given insight into the expression pattern. A comparative account of leaf specific transcripts expressed under drought and salinity stress in Lablab purpureus L. (Hyacinth Bean) variety HA4, are described herein with the objective of delineating the mechanism of stress response in terms of differentially expressed ESTs.
2. Material and Methods
2.1. Plant Growth, Stress Induction and Total RNA Isolation
The seeds of L. purpureus (cv. HA-4) were obtained from National Seed Project, University of Agricultural Sciences, GKVK, Bangalore, India. Seeds were surface sterilized with 0.1% (w/v) mercuric chloride for 30 s, rinsed immediately with distilled water several times and imbibed overnight in distilled water. The overnight-soaked seeds were sown in plastic trays containing vermiculite and acid-washed sand (1:1 w/w) and irrigated daily with distilled water.
The germination was carried out under natural greenhouse conditions; day/night temperature and relative humidity were 30/25˚C, and 75/70%, respectively. The average photoperiod was 12 h light/12 h dark. Plants were grown for 10 days before inducing stress. Drought stress was induced by withdrawing water for 6 Days whereas control plants were watered. Salt stress was induced using half strength Hoagland media with 300 mM NaCl, and 150 mM CaCl2 for 48 h. Control plants were grown in half strength Hoagland media without 300 mM NaCl. After stress exposure leaves were collected from both control and stress plants. The samples were immediately ground to a fine powder in liquid nitrogen and used for total RNA isolation. Total RNA was isolated using Tri-Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. The quality of total RNA was observed by electrophoresis on 1.5% Formaldehyde-MOPS gel. The purity was assessed by reading the A260/A280 ratios and A260/A230 ratios using Biomate 3S UV-Visible spectrophotometer (Thermo Scientific).
2.2. Differential Display Reverse Transcription Polymerase Chain Reaction
Differential display was performed according to the method of Liang and Parde  with minor modifications. Total RNA was treated with RNase free DNase-I (0.1 U per mg RNA for 30 min) to remove the contaminating genomic DNA. In brief, first strand cDNAs was synthesized using 2 μg of Total RNA and 10 mM oligo dT primer. Initial incubation was at 70˚C for 5 min followed by quick chilling on ice. To this, 40 units of Superscript™ II RNase H RT (Thermo Scientific), 20 units RNasin (Thermo Scientific), 0.5 mM dNTPs (Thermo Scientific) and 1X reverse transcriptase reaction buffer (250 mM Tris-HCl, pH 8.3, 250 mM KCl, 20 mM MgCl2 and 50 mM DTT) were added and made up to final volume of 20 µL.
The reaction was performed in Eppendorf master cycler by incubating at 40˚C for 60 min, and terminated by heating at 70˚C for 10 min. All cDNA samples were stored at −80˚C until further use. A total of three anchor primer and 16 arbitrary primer (Table 1) were used for DDRT-PCR. Amplification was carried out in a final reaction volume of 50 µL containing 1 µL of the cDNA as template, 0.5 U Taq DNA polymerase (Thermo Scientific), 12.5 mM each of specific arbitrary primer and anchor primer with restriction sites of BamHI and HindIII, respectively. 0.1 mM dNTPs and 1x Taq DNA polymerase reaction buffer (10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl2, 50 mM KCl and 0.01% gelatin). The reaction conditions were: an initial denaturation step of 95˚C/4min, followed by 35 cycles of 94˚C/60s (denaturation), 50˚C to 55˚C (according to primer combination)/60s
Table 1. List of primer used in DDRT-PCR.
for annealing and 72˚C/60s for extension and 72˚C for 15 min for final extension. Each reaction was run in triplicate, and products obtained were subjected to denaturing 6% urea gel electrophoresis at a voltage of 0.45 V/cm2 for 12 h at room temperature. DNA was visualized by staining with ethidium bromide and documented using Alpha Imager gel doc. Differential bands were excised and cloned into pGEM-T-easy vector (Promega) according to manufacturer’s instructions. Plasmid expressing the band of interest was sequenced on an automated sequencer (ABI PRISM 3700) using Bio Rad Taq cycle sequencing kit (Amersham Pharmacia Biotech,). Sequence analysis was performed using BLASTn (http://www.ncbi.nlm.nih.gov/blast). All of these ESTs were submitted to the dbEST database at NCBI (http://www.ebi.ac.uk).
2.3. Quantitative Real Time PCR
qRT-PCR was carried out using iQ SYBR green supermix (Biorad, India) on Biorad iQ5 Multicolor Real Time PCR Detection System. The reaction mix contained 0.5 µL cDNA, 10 µL 2X SYBR green mix, 0.5 µL of 1 µM forward primer and 0.5 µL of 1 µM reverse primer in 20 µL reaction volume. The cycling conditions were: initial denaturation of 95˚C/3 min followed by 40 repetitive cycles at 95˚C for 20 s for denaturation, 60˚C for 30 s for annealing (according to primer combination) and 72˚C for 30 s for extension. Melt curve analysis was performed at 55˚C - 95˚C. β-Actin was used as the endogenous control and analysis was carried out in three replicates. The Cq values were converted to relative quantities using the formula: 2−ΔΔCt .
DDRT-PCR was performed using 48 combinations of primers. Bands with clear differential expression were excised from polyacrylamide gels, amplified and sequenced. All obtained ESTs were submitted to NCBI EST database. After removing the redundant sequences, 31 differentially expressed ESTs were obtained under drought stress and 12 under salinity stress. All the sequenced fragments were of the expected length as ascertained from their positions in the gel and ranged between 98 - 423 bp for ESTs under drought, and 137 - 311 bp for ESTs under salinity stress. Sequence homology of the expressed fragments expressed under stress ranged between 73% - 93% similarity to sequence in Reference mRNA database. The ESTs showed similarity to sequences from legume family. Quantitative real time PCR was carried out to ascertain expression pattern (Figure 1 and Figure 2). The ESTs from salinity stress showed homology to genes encoding sugar transporter, ion transporter, chromatin remodeling components and RNA splicing components. Expression profile of salinity induced ESTs indicated that 3 ESTs were up regulated and 9 were down regulated. Eight EST corresponded to genes with putative functions (Table 2) and 4 were hypothetical
Figure 1. Differential expression of 31 ESTs under drought stress. Actin gene used as control. The ESTs expression below zero are down regulated, above zero are up regulated. Changes in gene expression are calculated using ΔΔCt method. The plotted values are average of triplicates. (On the X-axis are the EST (Number assigned and their corresponding Genbank accession number are; 1-JZ515981, 2-JZ515982, 3-JZ168400, 4-JZ168401, 5-JZ168402, 6-JZ168403, 7-JZ168404, 8-JZ168405, 9-JZ168406, 10-JZ168407, 11-JZ168408, 12-JZ168409, 13-JZ515983, 14-JZ515984, 15-JZ515985, 16-JZ515986, 17-JZ515987, 18-JZ515988, 19-JZ515989, 20-JZ515990, 21-JZ546398, 22-JZ546399, 23-JZ546400, 24-JZ546401, 25-JZ546402, 26-JZ546404, 27-JZ546405, 28-JZ546406, 29-JZ546407, 30--JZ546408 and 31-JZ546409).
Figure 2. Differential expression of 12 ESTs transcripts under salinity. Actin gene is used as control. The ESTs with fold expression below zero are down regulated, above zero are up regulated. Changes in gene expression are calculated using ΔΔCt method. The plotted values are average of triplicates. On the X-axis are the EST (Number assigned and their corresponding Genbank accession number are 1-JZ515991, 2-JZ515992, 3-JZ515993, 4-JZ515995, 5-JZ515996, 6-JZ515997, 7-JZ515998, 8-JZ515999, 9-JZ516000, 10-JZ516001, 11-JZ516002 and 12-JZ516003).
Table 2. List of ESTs differentially regulated under drought and salinity stress.
proteins. Thirty-one ESTs characterized under drought stress exhibited homology to transcription factors, chlorophyll, ribosome and RNA polymerase associated proteins, enzymes involved in protein degradation and general metabolism. Expression profile of ESTs indicated that 19 ESTs were up regulated and 12 down regulated. While 21 of the EST corresponded to genes with putative functions (Table 2) 10 of them corresponded to hypothetical proteins.
Lablab purpureus has been reported to exhibit quantitative and qualitative variations in biochemical markers of stress responses under salinity and drought stress  . These variations indicate distinctive set of players involved in stress perception and response biochemistry. This is also corroborated by distinct set of differentially expressed ESTs characterized under drought and salinity stress using DDRT-PCR, employing a common set of 48 primers. The distinction was also clear from the fact that 31 ESTs were characterized under drought stress, while only 12 ESTs were characterized under salinity stress relative to their respective controls. Annotation of ESTs, suggested different functionalities involved in drought and salt stress response in Lablab purpureus. Even among 12 ESTs differentially expressed under salinity stress, none of them were observed to be similar to those identified under drought stress (Table 2). Broadly the annotated ESTs, from both drought and salt stress, were associated with metabolism, photosynthesis, energy production and post transcriptional regulation, such as alteration in mRNA levels.
Metabolic adjustment is crucial to cope with abiotic stress. These changes may involve selective regulation of genes associated with the maintenance of metabolite levels. While synthesis and storage of starch in plastids takes place during the day, under applied drought, the increase in demand for energy and reduced photosynthetic ability induces the breakdown of starch . The breakdown product, Glucose-6-phosphate, is transported across the plastidic membrane via the Glucose-6-phosphate transporter . Upregulation of EST homologous to Plastidic Glucose Transporter suggested breakdown of starch under salinity stress. A homologue of this transporter has been reported to be upregulated in Arabidopsis thaliana during increased sink demand   .
Transcript abundance of several genes encoding mitochondrial proteins are known to be upregulated in response to stress . An EST homologous to cytochrome-c oxidase (COX) was differentially regulated under drought stress with 1.17-fold over expression. COX expression has been shown to be associated with thermotolerance as observed in Arabidopsis thaliana   and monocots such as maize . Therefore, upregulated EST suggested may also contribute to drought tolerance of Lablab purpureus, also Lablab is also known for thermotolerance which has been shown through biochemical studies  COX may also play a role. Another EST showing homology to F1 complex assembly factor 2-like of F0F1 ATP synthase known to help in the stabilization of ATPase complex and facilitate the formation of ATP  , was upregulated under drought, is in conformity with increased demand for ATP during stress.
Catabolite repression is a general mechanism utilized by prokaryotes and lower eukaryotes to regulate carbon and nitrogen metabolism. Plant homologs are involved in catabolite regulation of various metabolites  . Upregulation of EST homologues to CCRP suggested a vital role for its mRNA turnover during drought stress . Plants respond to environmental cues by altering rigidity of the cell wall. Cell wall hydrolases have been shown to be crucial in cell wall dynamics  . While an EST homologue to cell wall hydrolase was upregulated under drought, another homologue was down regulated under salinity stress. While upregulation of hydrolases has been reported to have protective effects under drought stress  their down regulation has been found to be effective under salinity stress .
Cell volume is modulated by alteration in plasma membrane size which is due to dynamics of endocytic vesicles. Endocytosis in plants can be mediated by formation of clathrin-dependent vesicles or clathrin-independent vesicles. EPSIN (Eps15 interactor) has been identified as a key protein under clathrin-mediated endocytosis in flowering plants . In Lablab purpureus, the downregulation of EPSIN homologue indicated the role of clathrin independent endocytosis under drought stress. Another vesicle associated EST showed homology to VAMP727, which is associated with ARA6 a QSNARE   , was differentially expressed under drought stress. Overexpression under drought stress but not under salt stress, suggested different set of inducers under drought and salinity in Lablab. Dehydration leads to protein aggregation and damage  . One of the key enzymes in protein turnover, ubiquitin carboxy-terminal hydrolase, catalyses hydrolysis of ubiquitin polymers to monomers and release of ubiquitin from tagged protein . An EST showing homology to this gene was upregulated under drought as observed in Brassica napus . E3 ligase is a key protein which transfers the ubiquitin onto target protein, an EST corresponding to PUB22 U-box-containing E3 ligase is down-regulated under drought. First identified in Arabidopsis , PUB22 expression was shown to have negative influence on water stress response . The increased activity of hydrolase and reduced E3 ligase indicated the reduction in protein turnover under drought in L. purpureus.
Survival of plants under stress is critically dependent on storage and utilization of assimilated elements such as Sulphur and Nitrogen . EST homologous to Adenosine-5’-phosphosulfate-kinase (APSK), involved in sulphur metabolism, and nitrate transporter (NRT 1.5) 1.5 like were down regulated under salinity stress. Reduced APSK would result in reduced 3'-phosphoadenosine 5’-phosphosulfate (PAPS), a principal sulphur donor in metabolism of sulphur containing compounds  . Reduction in NRT 1.5 like would cause non mobilization of nitrate stores, with reduction in plant growth . Similar downregulation of NRT homologue has been reported for Arabidopsis thaliana  under cadmium stress, suggesting the possibility of similar molecular signals prevailing under salinity and heavy metal stress.
Stability and/or turnover of mRNA is one of the factors influencing gene expression under normal and stressed conditions . Differential expression of a homologue of poly A binding protein RBP45 under drought stress suggested its role in stability of mRNA under drought . But a similar response was not observed under salt stress when identical set of primers were employed for DDRT-PCR, although physiological response to dehydration by drought and salinity have been reported to be similar.
Transcription factors play key role in regulation of gene expression . ESTs corresponding to MYB was upregulated under drought stress. MYB transcription factors are known to be involved in cold tolerance , stress response , ABA induced drought tolerance mechanism . Another EST homologue to NF-Y A7 like has shown to be important in embryonic development, plastid biogenesis, abiotic stress tolerance  . Upregulation of MYB and NF-Y A7 like members strongly indicate priming of transcription regulation under drought stress in Lablab purpureus.
Another EST homologous to transcriptional activator CAMTA3, a major regulator in biotic defence   and regulator of rapid stress response element  was differentially expressed under drought. Considering the fact that CAMTA3 mutants accumulate greater DNA damage , its upregulation in Lablab purpureus suggested its protective role under drought stress. As most of the gene regulation by transcription factors are mediated by mediator complexes, up and down regulation of mediators     would have an impact on the extent of regulation by a given transcription factor . Upregulation of homologue of Med26 under drought suggested its positive effect in drought tolerance.
Post transcriptional regulation via siRNA and miRNA have been shown to contribute to stress response either positively, or negatively  . Down regulation of a homologue of Maturase K under drought stress, which is known to be involved in splicing group II introns, in relation to plant development indicated non processing of the transcripts   and has protective role in Lablab purpureus. As part of post transcriptional regulation of gene expression, modification of mRNA also plays a key role. Pentatricopeptide repeat proteins (PPR) are known to be associated with mRNA splicing and processing components of organelles, and overexpression of a PPR gene in A. thaliana has been shown to contribute to tolerance to salinity stress   . Down regulation of an EST homologous to pentapeptide repeat protein family (PPRP family) and pre-mRNA-splicing factor (CWC22) under salinity, indicate reduced stability of mRNAs under salt stress.
Abiotic stress responses are also known to be epigenetically regulated . A homologue of SSRP1, which is known to associate with FACT (Facilitates chromatin transcription complex) complex  , required for transcription elongation by RNA polymerase II, was down regulated under salinity. This indicated significant role for FACT mediated epigenetic regulation under salt stress.
Environmental stress has been shown to influence ribosomal synthesis . Two EST homologous of ribosomal subunits, chloroplast encoded S7 and the nucleus encoded L22, were upregulated suggesting the dynamics of ribosome under drought stress. A similar observation with upregulation of S7 has been observed in Tomato . An EST homologous to rRNA-processing protein EBP2 mRNA   showed down regulation under drought. In Arabidopsis this protein expression is altered in salt hypersensitive mutant 9 (sahy9) mutant under salinity stress . The down regulation of predicted EBP2 in Lablab purpureusmight slowdown the progression of mitosis and effect salinity induced ribosomal protein alteration in biogenesis.
Drought and salinity stress are known to affect the photosynthetic capacity, which is linked to chlorophyll content . Two ESTs one homologous to chlorophyll a/b binding protein, and another showing homology to Photosystem II CP47, a component of Light harvesting complex component was down regulated under drought stress, suggesting reduced photosynthetic capacity in Lablab purpureus. Declined photosynthetic activity due to down regulation of LHCB protein family has been shown in Arabidopsis  and pepper .
Downregulation of salt stress specific differentially expressed EST, homologous to chlorophyll reductase in Lablab, which indicated delay in senescence   under salt stress as observed in stay green phenotypes of Oryza sativa . Another EST homologous to TIC32, a short chain dehydrogenase, which is part of Toc-Tic complex involved in regulation of protein import  in response to calcium sensing was down regulated under drought. A similar down regulation has been observed in O. sativa under drought stress . Apart from these known homologs, there were ESTs matching the unknown function giving a scope for identification of new roles.
Previous studies have shown that Lablab purpureus exhibits more or less similar pattern of biochemical parameters such as antioxidants and antioxidant enzymes under drought and salt stress. However, the foregoing discussion shows a contrasting response in terms of relative abundance of transcripts under these two conditions. Further, the transcripts identified under these two conditions were altogether different despite identical set of primers employed in DDRT-PCR method. Compared to other study on root transcriptome, only one EST (MYB) showed similarity. Therefore, it can be concluded that, at transcription level, distinct signalling molecules and elicitors are employed by the plants under drought and salinity stress. Nevertheless, the observed levels of transcripts may not always indicate the expected levels of translation products, as the stability of mRNAs and translational regulation further add a layer of regulation to stress. A clear picture may require exhaustive transcript profiling and study of epigenetic regulation to compare the molecular events that distinguish response of the plant to drought and salt stress.
The authors are grateful to Department of Science and Technology, New Delhi, for providing financial assistance (SR/SO/BB-72/2007, dated 14.01.2009). Authors are also grateful to GKVK, Bengaluru, for providing seeds and the Department of Biological Science, IISc Bangalore, for their generosity to allow us use central facility.
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