Water stress (WS) can affect the structural attributes of membrane lipids, nucleic acids and proteins (Vertucci and Leopold, 1987). WS also enhances accumulation of sugars and free amino acids and decreases leaf turgidity, chlorophyll, ascorbic acid and soluble proteins in leafy vegetables (Guehl et al., 1993; Irigoyen et al., 1992; Ishitani et al., 1995; Keller and Ludlow, 1993; Nguyen and Lamant, 1988). Plants subjected to water stress show changes in physiological and biochemical processes reflected in altered enzyme activities and exhibit enhanced activities of proteases, and increased concentration of low-molecular weight. nitrogenous compounds (Jharna et al., 2001; McCue and Hanson, 1990; Smirhoff and Colombe, 1989). Investigations of osmoregulation at the whole plant level indicated that free amino acids and proteins may play a significant role in response to water deficits and create a dynamic adjustment in nitrogen metabolism (Stewart and Larher, 1980) contributing to osmotic adjustment (Morris et al., 1990; Jacobson et al., 1992; Delauney and Verma, 1993). Metabolic changes in response to water stress include reduction in photosynthetic activity (Ritchie et al., 1990) and in protein synthesis (Mason et al., 1988) and accumulation of organic acids such as malate, citrate and lactate accompanied by accumulation of proline, sugars and betaine (Aspinall and Paleg, 1981; Bohnert et al., 1995; Shinozaki and Yamaguchi, 1996; Tabaeizadeh, 1998). Exposure of plants to low water potential often leads to loss of cell turgor, and plants undergo osmotic adjustments by the rapid accumulation of abscisic acid (ABA) and osmoprotectants (Bray, 1997; Grumet and Hanson, 1986; Skriver and Mundy, 1990). Dehydrins and Rab-related proteins are typically accumulated (Zhu, 2002) in response to low temperature, drought, salinity, or extra cellular freezing. These proteins are believed to be associated with desiccation tolerance, and their major function is to rescue the metabolic processes that are required for survival and regrowth under drought conditions. Our previous studies using dehydrin antibodies showed that peanut seed produces dehydrins following water stress, and the amount of dehydrins increase with increased stress period (Mazhar and Basha, 2002). Pre-harvest aflatoxin contamination is a major problem in semi-arid tropics where drought stress aggravates the Aspergillus infection and aflatoxin contamination in peanut (Cole et al., 1989; Sanders et al., 1993; Turner et al., 2000). Aspergillus thrive in extremely dry conditions and invade seeds under high temperatures causing severe damage in peanut seeds (Widstorm et al., 2003). Drought stress is known to predispose peanut to aflatoxin contamination (Blankenship et al., 1984; Cole et al., 1982 and 1985; Hill et al., 1983; Wilson and Stansell, 1983) possibly due to loss of the phytoalexin producing capacity of the peanut seed (Wooton and Strange, 1985; Dorner et al., 1989) and altered seed composition. Occurrence of aflatoxin contamination depends upon maturity status, condition of the seed and duration of stress. This would suggest that, susceptibility of peanuts to aflatoxin contamination is inconsistent and vary depending upon genotype, environment, maturity and severity of stress. Exposure of peanut plants to water stress for 5 to 20 d showed significant increases in leaf amino acids, soluble sugars, proteins and polypeptide composition (Ali-Ahmad and Basha, 1998; Amaregouda et al., 1994; Patil and Patil, 1993; Purushotham et al.,, 1998; Sharma et al.,, 1990). The genetic mechanisms leading to drought induced aflatoxin contamination are still unclear (Holbrook and Stalker, 2003). Depending upon the developmental stage or the external stimuli applied, the drought induced genes are classified as dehydrin (dehydration-induced), RAB (responsive to ABA), or LEA (late embryogenesis abundant) genes. Although genes induced by drought have been identified in a wide range of plant species (Iuchi et al., 1996; Oliver et al., 1998; Shinozaki and Yamaguchi, 1996), molecular basis of plant's tolerance to water stress remains far from being completely understood (Cellier et al., 1998; Ingram and Bartels, 1996). Proteomics has proved to be a powerful tool for identification of protein and mechanisms involved in drought response and tolerance (Hajhaeidri 2005; Kawasaki et al., 2000; Riccardi et al., 1998; Salekeh et al., 2002 a and b) and has been used to identify stress induced proteins in several crops that may play a role in food and nutrition (Haravarth et al., 2005). Several stress-induced genes have been isolated which play major role in understanding the molecular mechanism of drought. The ABA responsive gene expression modulated during seed development (Choi et al., 1987; Galau et al., 1987; Gomez et al., 1988; Heikkila et al., 1984) have been isolated in response to plant dehydration (Bray, 1997; Close et al., 1989; Guerrero and Mullet, 1986) or low temperature (Close, 1997; Shinozaki and Yamaguchi, 1996). Zegzouti et al. (1997) have isolated a novel ethylene-responsive cDNA, designated ER5 by differential display which also showed strong homology to LEA protein family. In peanut, several stress induced genes such as dehydration-induced cDNA encoding a putative 9 cis-epoxycartenoid dioxygenase (Wan and Li, 2005) and phospholipase D (Guo et al., 2006) have been isolated. All these genes have been found useful in germplasm screening for drought tolerance. Luo et al. (2005 a and b) have generated 1825 Expressed Sequence Tag (EST) libraries using leaf and seed tissue of two peanut lines, and found that 52.8% of the ESTs from leaf tissue and 78.6% of the ESTs from the pod tissue have homology to genes of known function. The major seed storage protein genes of arachin, conarachin and conglutin were sequenced and full-length cDNAs that encode isoforms of five arachin and six conglutin precursors were obtained (Yan et al., 2005). Earlier, we have identified the isoforms of conglutin as methionine rich protein subunits (Basha and Pancholy, 1981). Several peanut seed and leaf derived ESTs were used to validate their function through microarray and real-time PCR, however, their association with drought and disease tolerance traits are not yet confirmed (Cushman and Bohnert, 2000; Keon et al., 2003; Luo et al., 2003, 2005 a and b; Seki et al., 2001). Improvements in drought and disease tolerance characteristics are critical in peanut breeding programs aimed at reducing aflatoxin contamination. However, limited information is available on peanut seed components, especially the seed proteins and their role in maintaining drought tolerance in peanut. The primary objective of this research was to identify the proteins/polypeptides affected by drought stress for their possible use as markers for evaluating peanut germplasm to select drought-tolerant genotypes. Here, we report the seed polypeptides that are affected by water stress, and show that these polypeptides are differentially expressed among peanut genotypes under water stress. These proteins may be useful as potential markers for screening peanut genotypes to identify drought tolerant genotypes.

Qualitative and quantitative change in peanut seed proteins due to water stress was determined by comparing polypeptide profiles of control and stress treatments, and among peanut genotypes.

Water stress alters plant gene expression leading to the induction of specific gene/s (Romo et al., 2001) accompanied by increased transcription and thus an increase of the corresponding proteins. In this study we have evaluated peanut genotypes for water stress-regulated proteins in order to identify the seed proteins affected by WS, and to understand the mechanisms associated with peanut plant acclimation and adaptation to drought. Identification of drought-responsive proteins would be useful to distinguish DT and DS peanut genotypes, and to understand the biochemical and molecular events responsible for drought-induced aflatoxin contamination of peanut. Some of the peanut genotypes used in this study were developed (ANGR Agri. Univ., Kadiri; National Research Center for Groundnut and ICRISAT, India) for drought tolerance, and are cultivated under rain-fed conditions in the arid and semi-arid regions of India. For example, the genotypes viz. Vemana, CSMG-84-1, Kadiri-4, and TMV-2 have been developed and released as DT cultivars for Andhra Pradesh, India. In addition, these genotypes are also being widely used in several breeding programs at Kadiri, Tirupathi, and Hyderabad, India for identifying peanut genotypes with high water use efficiency. Inclusion of these genotypes with proven drought-tolerance characteristics was helpful in identifying drought-responsive proteins as well as demonstrating that WS had limited effect on their seed protein composition. The genotypes, M-13, JL-220, JL-24 and TKG-19A which are classified as DS showed significant changes in seed polypeptide composition following WS, reflecting their DS trait. Peanut seed proteins have been grouped into arachin, conarachin / non-arachin (conglutin) based on their solubility (Basha and Pancholy, 1981). Our results clearly revealed that seed polypeptide composition, specifically arachin and conglutin proteins of DT genotypes were least affected while that of DS genotypes, significantly altered due to WS. Our data showed that proteins # 1 and 13 to18 were significantly suppressed in DS while they were unaffected in DT during water stress. Based on amino acid sequence data, protein # 1 is identified as Ara h1, the arachin subunit (Kolarich and Altman, 2000). Earlier, Basha and Pancholy (1981 a and b) have isolated the conglutin subunits and found two of them (# 15) to be rich in methionine (>4%). The MRP (methionine-rich protein) is a 120 kDa protein and consists of six polypeptides with molecular weights ranging from 15.5 to 20 kDa and pI between 4.5 and 5.5 pH. Later, the gene sequences of these conglutin subunits were identified by Yan et al., (2005). Our results show that the storage proteins, particularly the MRP subunits were highly suppressed in DS genotypes compared to DT genotypes due to WS. This data would suggest that the observed differential responses in subset of arachin and conglutin proteins could be employed to distinguish DT and DS peanut genotypes. Previously, our studies using Differential Display Reverse Transcriptase technique has enabled us to identify peanut transcripts affected by water stress (Jain et al., 2001). The observations are consistent with our earlier findings (Jain et al., 2001) which showed prolonged gene expression in DT genotypes compared to DS genotypes upon imposition of various levels of WS. In our previous studies, we have found transcripts that are severely affected (up-regulated, down-regulated or newly produced) due to water stress and are termed as Peanut Transcripts Responsive to Drought (PTRD). The down-regulated transcripts were studied by Northern Dot-Blot hybridization in drought-tolerant and drought-susceptible peanut lines. Results showed differential expression of these transcripts in the drought-tolerant and drought-susceptible lines indicating that these three transcripts (PTRD-1, PTRD-2 and PTRD-16) can be used as molecular tools to screen peanut germplasm for identifying drought-tolerant lines. Once the genotypes are classified, it is necessary to work with a large number of genotypes differing in their drought tolerance level to isolate differentially expressed transcripts induced upon water stress to address various stress related problems in peanut. Such markers would be of great value in the breeding programs aimed at identifying DT genotypes for developing drought- and aflatoxin-tolerant peanut genotypes. Although several molecular markers are currently available in peanut (Burrow and Blake, 1998), these have limited polymorphism in peanut and proved inadequate for identifying the drought specific peanut genotypes. Therefore, identification of drought responsive proteins would be useful to determine drought tolerance characteristics of peanut genotypes. Further studies are needed to clone and characterize the genes expressed due to drought stress and use them as probe to study the expression levels across genotypes.