A Comparative Study of ESTs Induced under Drought and Salinity Stress in Hyacinth Bean ( Lablab purpureus )

Abiotic stressors like drought and salinity are major causes for loss of agricultural productivity. Comparison of expressed sesquence tags (ESTs) under different abiotic stresses provides insight into underlying mechanism of stress response, and candidate genes to improve tolerance to abiotic and biotic stresses via breading and transgenic methods. In order to identify and compare stress-specific ESTs from drought and salinity stressed Hyacinth Bean, ten days old seedlings were subjected to respective stresses and RNA was ex-tracted from control and stressed leaves for EST identification. 31 and 12 ESTs, respectively, were characterized from leaves of drought and salinity stressed seedlings of Hyacinth Bean, Lablab purpureus by differential display RT-PCR using identical combinations of 48 primers and validated using quantitative RT-PCR. Relative fold expression was higher under salt stress than drought stress. Whereas 19 EST overexpressed under drought, 12 EST were down regulated. Of the 12 EST under salinity, 9 EST were downregu-lated and 3 EST upregulated. Putative functions predicted from sequence homology indicated that 11 drought specific EST corresponded to metabolic functions, and 4 of them corresponded to transcription regulation. Under salinity, 4 and 2 EST, respectively, corresponded to metabolic and RNA associated functions. Under both stresses, there were ESTs associated with unknown functions, whose characterization may throw light on the regulatory mechanism. Differing number of ESTs differentially expressed under drought and salt stress, and their predicted functionalities, suggested distinct set of response genes involved under


Introduction
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 [1]. Also, increase in global temperature has increased frequency of drought [2] posing a big challenge for agriculture [3]. Drought and salinity affected soils contain high concentration of ions such as chloride, sodium, calcium and magnesium in soil [4] 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.
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 [22] and salinity [23]. Transcriptomic variations, in root tissue, under drought stress [24] 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.
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 CaCl 2 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).

Quantitative Real Time PCR
qRT-PCR was carried out using iQ SYBR green supermix (Biorad, India) on

Discussion
Lablab purpureus has been reported to exhibit quantitative and qualitative varia-  (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 Transcript abundance of several genes encoding mitochondrial proteins are known to be upregulated in response to stress [31]. 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 [32] [33] and monocots such as maize [34]. 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 [ [51]. 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 [52], PUB22 expression was shown to have negative influence on water stress response [52].  [55]. Reduction in NRT 1.5 like would cause non mobilization of nitrate stores, with reduction in plant growth [56]. Similar downregulation of NRT homologue has been reported for Arabidopsis thaliana [57] 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 [58]. Differential expression of a homologue of poly A binding protein RBP45 under drought stress suggested its role in stability of mRNA under drought [59]. 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.
Downregulation of salt stress specific differentially expressed EST, homologous to chlorophyll reductase in Lablab, which indicated delay in senescence [93] [94] under salt stress as observed in stay green phenotypes of Oryza sativa [95]. Another EST homologous to TIC32, a short chain dehydrogenase, which is part of Toc-Tic complex involved in regulation of protein import [96] in response to calcium sensing was down regulated under drought. A similar down regulation has been observed in O. sativa under drought stress [97]. Apart from these known homologs, there were ESTs matching the unknown function giving