Abstract
Cool season crops face intermittent drought. Exposure to drought and other abiotic stresses is known to increase tolerance of the plants against subsequent exposure to such stresses. Storage of environmental signals is also proposed. Preexposure to a dehydration shock improved adaptive response during subsequent dehydration treatment in a cool season crop chickpea (Cicer arietinum). We have identified 101 dehydration-inducible transcripts of chickpea by repetitive rounds of cDNA subtraction; differential DNA-array hybridization followed by northern-blot analysis and analyzed their responses to exogenous application of abscisic acid (ABA). Steady-state expression levels of the dehydration-induced transcripts were monitored during the recovery period between 2 consecutive dehydration stresses. Seven of them maintained more than 3-fold of expression after 24 h and more than 2-fold of expression level even at 72 h after the removal of stress. Noticeably, all of them were inducible by exogenous ABA treatment. When the seedlings were subjected to recover similarly after an exposure to exogenous ABA, the steady-state abundances of 6 of them followed totally different kinetics returning to basal level expression within 24 h. This observation indicated a correlation between the longer period of abundance of those transcripts in the recovery period and improved adaptation of the plants to subsequent dehydration stress and suggested that both ABA-dependent and -independent mechanisms are involved in the maintenance of the messages from the previous stress experience.
Abstract
Plants are often exposed to various environmental stresses when grown in field and within a physiological tolerance limit. A mild abiotic stress induces an adaptive response in the plant, allowing it to grow with a greater tolerance to the same or different stresses (Siminovitch and Cloutier, 1982; Lang et al., 1994; Mantyla et al., 1995; Knight et al., 1998) . Pretreatment with thermal or chemical shock induced a substantial chilling tolerance in germinated cucumber seeds (Jennings and Saltveit, 1994) . Wilted excised cabbage leaves recovered turgor in absence of water uptake when allowed to lose water at a slow rate (Levitt, 1986) . Plants express a number of genes in response to water deficit. At the cellular level, a part of this response results from cell damage, whereas the others correspond to adaptive processes. Adaptation to water deficit brings about changes in the metabolic processes and perhaps in the structure of the cell that allows the cells to continue metabolism at low water potential (Ingram and Bartels, 1996) . Dehydration and other stresses cause rapid elevation in the cytosolic free calcium ion ([Ca 12 ] cyt ) concentration (Knight et al., 1991) . As an adaptive response, the subsequent stresses show altered magnitude and kinetics of [Ca 12 ] cyt , depending on the nature and intensity of the previous stress even after a 48-h deacclimation period, indicating existence of a signal storage mechanism. Different stress-exposure alters cytosolic calcium-signature and calcium-regulated gene expression differently in the following stresses (Knight et al., 1996 (Knight et al., , 1998 , suggesting that plants are able to discriminate different stimuli and then store the impression of individual stimuli in a unique way.
Abstract
Accumulation of abscisic acid (ABA) in the plant organs corresponds to many physiological and metabolic changes that occur during dehydration stress (Lee et al., 1993; Moons et al., 1995) . Most of the genes that respond to dehydration are also responsive to exogenous application of ABA (Seki et al., 2002a) . The involvement of ABA in dehydration signaling and hence in tolerance was illustrated using ABA-deficient plants (Xiong et al., 2002; Zhu, 2002) . Arabidopsis mutants deficient in or insensitive to ABA are impaired in dehydration-induced freezing tolerance and demonstrated the role of ABA in stress-adaptation (Mantyla et al., 1995) . However, there are cases of evidence suggesting existence of additional mechanisms (Trejo and Davies, 1991; Griffith and Bray, 1996).
Abstract
1 This work was supported by the Department of Biotechnology, Govt. of India and by the support fund of the National Centre for Plant Genome Research.
Abstract
* Corresponding author; e-mail wrightrhonda@example.com. co.in; fax 91-11-26167394. Article, publication date, and citation information can be wrightrhonda@example.com/cgi

Plants are often exposed to various environmental stresses when grown in field and within a physiological tolerance limit. A mild abiotic stress induces an adaptive response in the plant, allowing it to grow with a greater tolerance to the same or different stresses (Siminovitch and Cloutier, 1982; Lang et al., 1994; Mantyla et al., 1995; Knight et al., 1998) . Pretreatment with thermal or chemical shock induced a substantial chilling tolerance in germinated cucumber seeds (Jennings and Saltveit, 1994) . Wilted excised cabbage leaves recovered turgor in absence of water uptake when allowed to lose water at a slow rate (Levitt, 1986) . Plants express a number of genes in response to water deficit. At the cellular level, a part of this response results from cell damage, whereas the others correspond to adaptive processes. Adaptation to water deficit brings about changes in the metabolic processes and perhaps in the structure of the cell that allows the cells to continue metabolism at low water potential (Ingram and Bartels, 1996) . Dehydration and other stresses cause rapid elevation in the cytosolic free calcium ion ([Ca 12 ] cyt ) concentration (Knight et al., 1991) . As an adaptive response, the subsequent stresses show altered magnitude and kinetics of [Ca 12 ] cyt , depending on the nature and intensity of the previous stress even after a 48-h deacclimation period, indicating existence of a signal storage mechanism. Different stress-exposure alters cytosolic calcium-signature and calcium-regulated gene expression differently in the following stresses (Knight et al., 1996 (Knight et al., , 1998 , suggesting that plants are able to discriminate different stimuli and then store the impression of individual stimuli in a unique way.

Accumulation of abscisic acid (ABA) in the plant organs corresponds to many physiological and metabolic changes that occur during dehydration stress (Lee et al., 1993; Moons et al., 1995) . Most of the genes that respond to dehydration are also responsive to exogenous application of ABA (Seki et al., 2002a) . The involvement of ABA in dehydration signaling and hence in tolerance was illustrated using ABA-deficient plants (Xiong et al., 2002; Zhu, 2002) . Arabidopsis mutants deficient in or insensitive to ABA are impaired in dehydration-induced freezing tolerance and demonstrated the role of ABA in stress-adaptation (Mantyla et al., 1995) . However, there are cases of evidence suggesting existence of additional mechanisms (Trejo and Davies, 1991; Griffith and Bray, 1996) . Drought-induced accumulation of endogenous ABA does not correspond to drought-tolerance in sunflower (Helianthus annuus) or drought-induced development of freezing tolerance in Arabidopsis (Lang et al., 1994; Cellier et al., 1998) .

We have observed an improved dehydrationtolerance in chickpea (Cicer arietinum) seedlings preexposed to dehydration shock, even when a recovery phase was allowed between two subsequent stresses. This observation was in accordance with the hypothesis that experiences of previous encounters with stress define the response to the following stresses (Knight et al., 1998) . Studies with RNA synthesis inhibitors have suggested an important role of transcription regulatory mechanisms in the long-term memory consolidation in animal systems (Andrews, 1980; Korzus, 2003) . We have identified 101 dehydrationinducible genes in chickpea and analyzed their responses to exogenous application of ABA. In order to detect any long-term impression of a stress on gene expression after the removal of the stress, we followed the steady-state expression of those transcripts at 2 different time points during the recovery period using nylon filter arrays. We have detected that some of the transcripts maintained their expression levels more than 2-fold compared to unstressed condition even after 24 h of recovery phase. We have carefully checked, by northern analysis, the expression level of some of the selected transcripts that maintain high expression during recovery after the dehydration shock. In this paper we report a correlation between the long term abundance of some of the dehydrationinducible transcripts after the removal of stress and adaptation to subsequent stress in chickpea seedlings.
RESULTS

Dehydration Stress and Relative Water Content
For dehydration-shock experiments, stressed and control seedlings were sampled at the same time to avoid diurnal variation. The control seedlings were removed similarly from the soil and immediately planted in the same pot and kept under the same condition as the stressed samples to normalize the shock due to uprooting and changes in the environment. During this treatment relative water content (RWC) of the stressed leaves decreased from about 80% to about 55% after 5 h of dehydration and wilted, whereas the same for the unstressed samples did not change. RWC of the samples treated with ABA did not change appreciably in the period of treatment (not shown). To test their tolerance to the indicated condition, some of the plants were replanted in watered soil after 5 h of stress, returned to normal growth condition, and observed for a week. All of them recovered turgor within 4 h and resumed growth with the development of new leaves (not shown). RWC in this period returned to about 74% (average) after 5 h and became about 81% (average) after 24 h of replantation. For recovery, all the samples (control, dehydrated, and ABA treated) were replanted in the same pot containing water-saturated soil to maintain equal soil moisture content. After 24 h of recovery, all of them were subjected to a second dehydration as before. Again RWC of the leaf samples were determined at different time points as a measure of stress-adaptation as it (RWC) accounts for osmotic adjustment, which is considered as one of the most important mechanisms of plants for adaptation in water-limited environment (Turner, 1986; Munns, 1988) . The control samples, as expected, continued to lose water reaching to 61.02% (61.41%) after 3 h and to 55.95% (61.92%) after 5 h of dehydration and wilted. The rate of decrease in RWC for the ABA pretreated plants was much slower (67.5% [61.65%]) up to 3 h and then quickly reached 59.73% (62.02%) after 5 h, showing some degree of tolerance. Some of the lower positioned, matured leaves showed wilting after 5 h. In contrast, the plants, which wilted after 5 h during the first exposure to dehydration, retained turgor throughout the treatment and showed much higher RWC, 69.24% (61.2%) and 64.71 (61.53%), after 3 and 5 h of dehydration, respectively, compared to the control or ABA pretreated samples ( Fig. 1, top) . Figure 1 . Top, Effect of preexposure to dehydration or exogenous ABA on the RWC during the subsequent dehydration treatment to chickpea seedlings. RWC of leaves in response to dehydration stress (described in ''Materials and Methods'') at different time points applied 24 h after the end of no treatment (control); pretreatment with dehydration shock (5 h) or 100 mM of ABA (5 h). The traces shown are the averages of four replicates (20 fully expanded leaves each) with SEM. Bottom, Accumulation of Pro in response to dehydration in control (as above; white bar) and dehydration pretreated (as above; gray bar) chickpea seedlings.
Dehydration Stress and Relative Water Content
Stress-dependent accumulation of Pro has been observed in many plants (Yoshiba et al., 1997) . However, the importance of Pro in stress-adaptation is contradictory; its accumulation is considered as one of the indicators of adaptive response (Delauney and Verma, 1993) . We have monitored the accumulation of Pro in control and in dehydration pretreated chickpea seedlings during subsequent dehydration treatment. In the control seedlings, Pro started accumulating within 3 h and increased more than 15-fold within 5 h of the treatment. In contrast, the pretreated seedlings showed high constitutive level of Pro (8-fold of the unstressed) from the beginning of subsequent stress and maintained the level throughout the period of stress (Fig. 1, bottom) .
Identification of Dehydration-Inducible Transcripts
Transcripts accumulated during dehydration stress were identified by a combination of approaches. Sixday-old chickpea seedlings were subjected to dehydration treatment with proper control as mentioned above and sampled at the same time. A subtracted cDNA library was constructed with poly(A 1 ) RNA isolated from dehydrated and control seedlings by repetitive rounds of subtractive hybridizations as described in ''Materials and Methods.'' The length of the subtracted cDNA products visibly ranged from 200 bp to 1,200 bp (not shown). The whole library was represented by about 3,000 clones. A population of expression sequence tags (EST) representing dehydration-induced transcripts was identified by screening 1,000 randomly picked clones. PCR amplified cDNAs were spotted in duplicate on nylon membranes in 96-format. Drought-inducible ESTs were identified by differential hybridization with radiolabeled first strand cDNA probes prepared using poly(A 1 ) RNA isolated from control and stressed samples (Fig. 2) . Each DNA element was tested at least three times with different sets of cDNA probes from three independent dehydration experiments to verify reproducibility. Expression ratio was calculated according to Seki et al. (2001) . Normalized signal intensity of each spot was determined after subtracting the local background intensity. Signals, with at least five times more intensity than that of the negative control (NPTII) in the dehydrated samples, were included in the analysis (about 97%). Effective signal intensities of the spots were calculated by subtracting the normalized intensity of the negative control. Fold induction was presented as the expression ratio (dehydration to unstressed) of each EST to that of actin. Actin transcript level was shown to be unchanged by waterstress and ABA treatment in Arabidopsis (Xiong et al., 1999) and in chickpea ( Fig. 3 and not shown).
Fold Induction5
Effective signal intensity in dehydration Effective signal intensity in control ‚
Intensity of Actin in dehydration Intensity of Actin in control
ESTs (total 377 spots) showing at least 2-fold of induction (average of 3 independent experiments) were selected for sequencing and annotated by BLASTX homology search. There were a number of redundant clones, indicating their abundances in dehydrated sample. Among the most notable are b-amylase (21 clones), myoinositol-1-phosphate synthase (MIPS; 17 clones), albumin (14 clones), leuco anthocyanidine dioxygenase (8 clones), polygalacturonase inhibiting protein (8 clones), 9-cis-epoxycarotenoid dioxygenase (NCED; 6 clones), etc. Different sequences showing highest homology to the same accession number were also registered. Excluding the redundant clones, a list of 101 unique transcripts along with their annotations, average fold inductions, e-values, and SDs are presented in Table I . Most of the induced transcripts were subjected to northernblot analysis to validate the differential dot-blot hybridization results and all of them have shown more than 2-fold of induction. RNA gel analysis of 10 selected ESTs confirming dot-blot data is shown in Figure 3 .
Intensity of Actin in dehydration Intensity of Actin in control
Response of Dehydration-Inducible Transcripts to Exogenous Application of ABA Dehydration stress resulted in accumulation of ABA in chickpea seedlings within 2 h and continued to accumulate up to 5 h, when the last sample was assayed (1.087 mg/g dry weight in control to 13.99 mg/g dry weight at 5 h). Similar kinetics and magnitude of ABA accumulation in response to dehydration was reported in another legume crop, cowpea (Vigna unguilata; Iuchi et al., 2000) . Majority of the droughtinducible genes are induced by exogenous application of ABA in Arabidopsis (Seki et al., 2002a) . Labeled cDNA probes prepared from control and ABA-treated samples were used for differential hybridization. Drought-inducible genes (61 among 101) showed more than 2-fold of induction by exogenous ABA treatment (Table I) . We compared our results to a similar report in Arabidopsis (Seki et al., 2002a) . Previously reported drought-inducible transcripts totally corroborated ABA-mediated gene expression in Arabidopsis, which indicates that probably similar mechanisms for exogenous ABA mediated response operate in Arabidopsis and chickpea. We have revalidated our results by RNA blots with selected ESTs (not shown).
Steady-State Abundances of Dehydration-Inducible Transcripts upon Rehydration
A short-term dehydration shock improved tolerance of chickpea seedlings to the subsequent dehydration treatment when 24 h of recovery period was allowed ( Fig. 1) . We monitored steady-state expression levels of the dehydration-induced transcripts in the recovery period to detect any long-term impression of the previous stress. Nylon filter arrays were used to determine fold expression at 5 and 24 h time points after replantation of the dehydrated plants in watered soil. As controls, we kept untreated plants (same as the control for dehydration) harvested at the same time point. The average expression ratios from two independent experiments are presented as scatter plots (Fig. 4) . Accumulation of most of the transcripts was reduced down to less than 2-fold within 5 h of replantation; however, 15 out of 101 transcripts tested showed more than 2-fold of expression in comparison to the corresponding control after 24 h (Table II) .
Steady-State Abundances of Dehydration-Inducible Transcripts upon Rehydration
Northern-blot analysis of 6 selected transcripts showed total compliance with the dot-blot data (Table  II) . Expression of 3 unrelated transcripts, corresponding to chlorophyll a/b-binding protein, Rubisco small subunit, and actin during this period, was unchanged showing that the experimental condition did not cause a global change in transcript abundance (not shown).
Steady-State Abundances of Dehydration-Inducible Transcripts upon Rehydration
We have analyzed by northern blot the expression of 7 transcripts showing more than 3-fold of expression at 24 h time point (Table II) . Their expressions were monitored up to 72 h during recovery phase (Fig. 5 ). Other than CD051303 (myoinositol-1-phosphate synthase) and CD051297 (Dehydrin1), steady-state abundances of all the transcripts gradually decreased in this period. The kinetics of CD051303 were surprising. Its constitutive expression in the control sample was almost undetectable, however, accumulated more than 20-fold upon dehydration (Fig. 3) . Upon rehydration, its expression totally diminished to basal level within 5 h, however, again started accumulating and maintained above the basal level up to 72 h (Fig. 5 ). Two explanations can be given for this unique expression pattern. The rate of degradation of the accumulated transcripts upon rehydration was more than the rate of expression early in the recovery phase and/or expression of a new factor(s) was required for reaccumulation in the latter part. The transcript corresponding to Dehydrin1 (CD051297) continued to express at high level as the dehydrated sample up to 24 h of replantation (Fig. 5) . Five other transcripts were accumulated more than 3-fold over the control after 24 h as observed in the nylon filter hybridization (Table II; Fig. 5) . Expression levels of 5 of the 7 high expressing transcripts were maintained at above 3-fold even after 72 h when the last sample was harvested (Fig. 5 ). Longer periods of abundance of these transcripts over the others can be explained by higher intrinsic stability of these mRNAs. Alternatively, preexposure to dehydration may have selectively increased their stabilities or extended the duration of their expression.
Steady-State Abundances of Dehydration-Inducible Transcripts upon Rehydration
Expression of all the seven transcripts mentioned above was inducible by exogenous application of ABA (Table I) . To verify whether the characteristic longterm abundance of these transcripts is specific to dehydration or general for any stress, the chickpea seedlings were exposed to 100 mM of ABA for 5 h and were allowed to recover as before. Steady-state accumulation of those transcripts in this period was monitored similarly by northern analysis up to 24 h. As expected, all the seven transcripts were accumulated upon exposure to ABA, in some cases accumulated more than that in dehydration treatment (Fig. 5 , dotted lines). The transcript CD051303 (MIPS) followed the same kinetics as before. Its steady-state amount decreased abruptly at 5 h and reaccumulated at 24 h time point indicating that an ABA-dependent or a general stress-regulated mechanism is responsible for this unique pattern. Abundance of the rest of the transcripts decreased sharply to reach the basal expression level within 24 h; in some cases within 5 h Figure 3 . RNA-blot analysis of selected ESTs confirmed increased accumulation of the transcripts as determined by nylon filter array analysis. Twenty micrograms of total RNA isolated from unstressed (Con) and stressed (Deh) chickpea seedlings were analyzed by northern hybridization with a 32 P-dCTP labeled probes corresponding to indicated EST clones. A PCR product of chickpea actin cDNA (see ''Materials and Methods'') was used as internal control and 25S ribosomal RNA was shown as loading control. Annotations assigned by BLASTX homology search were mentioned. of replantation (Fig. 5) , suggesting that long-term abundance of these transcripts after removal of dehydration stress was not due to higher intrinsic stability of mRNAs.
Steady-State Abundances of Dehydration-Inducible Transcripts upon Rehydration
Accumulation of myoinositol in Actinidia seedlings in response to salt stress has been reported and its (myoinositol) level was shown to be maintained above the control level after removal of stress (Klages et al., 1999) . In our study, 1 of the 7 transcripts (CD051303) maintaining higher expression level after removal of stress encodes for MIPS, a key enzyme for synthesis of myoinositol. We have assayed myoinositol content in the chickpea seedlings during dehydration treatment and subsequent recovery period. Myoinositol accumulated more than 4-fold from the control after 5 h of dehydration (6.48 6 0.64-28.5 6 2.03 mg/g dry wt); however, it declined to a level 2-fold of the control after 24 h of recovery (13.15 6 0.97 mg/g dry wt) and maintained up to 72 h (11.8 6 1.15 mg/g dry wt).
DISCUSSION

Dehydration-Inducible Genes in Chickpea
We have selected chickpea, the third most consumed legume crop (Graham and Vance, 2003) for this study because it requires less irrigation and adapts well in a water-limited environment (Turner et al., 2001) . As a part of the chickpea genomics program, in this paper we have reported an EST database of dehydrationinducible transcripts. Temperate grain legumes such as pea, fava bean, lentil, chickpea, and others share similar gene arrangements (VandenBosch and Stacey, 2003) . Therefore, we expect that our database will benefit the study of other legume plants by comparing the expression patterns of the transcripts.
Dehydration-Inducible Genes in Chickpea
Small sugar molecules are believed critical to maintain osmotic balance of cells in stress (Bray et al., 2000; Lee et al., 2003) . Dehydration in chickpea induced a number of transcripts for the enzymes associated with degradation of starch such as b-amylase, Fru-1,6-biphosphate aldolase, phosphoglucomutase, and synthesis of small sugars such as trehalose-6-phosphate synthase and myoinositol-1-phosphate synthase (MIPS), enzymes that promote synthesis of trehalose and pinitol, respectively, (Nelson et al., 1998; Lee et al., 2003) . Pinitol accumulates in the leaves of legumes and halophytes in salt and drought stress (Pattanagul and Madore, 1999) . In Mesenbryanthemum crystallinum, salinity, but not drought or exogenous ABA, causes accumulation of MIPS transcript (Vernon et al., 1993) , whereas Arabidopsis does not show induction in MIPS gene expression in response to excess salt (Ishitani et al., 1996) . However, in our study, both dehydration and exogenous ABA caused accumulation of MIPS transcripts (Table I; Fig. 3 ). In several leguminous plants role of apyrase, an enzyme involved in nucleic acid metabolism, has been implicated in legume-rhizobia symbiosis. However, in Medicago truncatula, expression of apyrase is not regulated symbiotically, rather it is induced by wounding or stress associated with harvesting in an ethyleneindependent manner (Navarro- Gochicoa et al., 2003) . Expression of apyrase in chickpea in response to dehydration indicates either that the dehydration treatment mimicked the stress associated with harvesting or that dehydration regulates expression of apyrase through a different pathway.
Dehydration-Inducible Genes in Chickpea
The CBF/DREB family of transcription factors are an integral part of early regulation of water stressmediated response (Liu et al., 1998) . CBF regulon comprises a part of total genes expressed in cold stress (Jaglo et al., 2001; Fowler and Thomashow, 2002) . A number of genes expressed by overexpression of CBF homologs do not contain DRE/C-repeat in their promoters. It has been postulated that RAP2.1, a CBFinducible, AP2-family protein in turn may activate transcription of the CBF target genes in cold-response pathway (Fowler and Thomashow, 2002) . Expression of transcripts encoding DREB2, RAP2.4, and RAP2.6 indicates that a similar subregulon may function in dehydration-stress response. Simultaneous upregulation of DREB2, RAP2.4, and RAP2.6 transcripts during mechanical wounding in Arabidopsis (Cheong et al., 2002) reinforces this concept. Transcripts encoding ribosomal proteins and translation elongation factor are expressed during the initial phase of salt stress in a salt tolerant rice variety but not in a salt-sensitive one. It was speculated that part of the salt-tolerance may be conferred by the ability to translate the components of the response circuit early in the stress that lacked in the sensitive variety (Kawasaki et al., 2001) . In chickpea, we have observed induction of transcripts encoding a number of ribosomal structural proteins and the proteins involved in translational initiation, elongation, and termination. Generation of reactive oxygen molecules accompanied by protein degradation are the consequences of dehydration stress (Ingram and Bartels, 1996) . Mitochondrial uncoupling protein and metallothionin-like proteins may decrease and detoxify such species. A number of transcripts encoding protein-degrading enzymes were also identified (Table I) .
Long-Term Expression of Some Dehydration-Inducible Genes during Recovery
All the genes expressing more than 2-fold at 24 h after removal of stress were inducible by ABA. However, this cannot be explained by residual high ABA concentration, because at that time point endogenous ABA level had returned to normal level (1.136 mg/g dry weight). Other ABA-inducible genes also returned to basal level of expression at this time point (Fig. 4) . In another legume crop, cowpea, expression of the gene for 9-cis-epoxycarotenoid dioxygenase, the rate limiting enzyme for ABA biosynthesis, was also shown reduced down to basal level within 10 h of rehydration (Iuchi et al., 2000) .
Long-Term Expression of Some Dehydration-Inducible Genes during Recovery
Two of the transcripts expressing in the recovery period code for myoinositol-1-phosphate synthase and trehalose phosphate synthase are involved in synthesis of pinitol and trehalose, respectively. Concentration of pinitol was reported to be higher in the halophytic plants and the plants that are adapted to drought (Vernon and Bohnert, 1992) . We also have observed a high constitutive level of myoinositol in the dehydration pretreated chickpea seedlings. Interestingly, myoinositol concentration in Actinidia leaves increased more than 2-fold at harvest time when a short water-stress was given in early summer (Miller et al., 1998) .
Long-Term Expression of Some Dehydration-Inducible Genes during Recovery
Late-embryogenesis abundant (LEA) groups of proteins are thought to provide protection to the cellular macromolecular structures against desiccationmediated damages. A transcript (CD051297) encoding dehydrin1 (LEA D11) continued to express at the same level as in the dehydrated sample up to 24 h after the removal of dehydration stress. Accumulation of dehydrin (both dhn1 and dhn2) transcripts was shown to correlate drought-adaptive response in a relatively tolerant variety of sunflower; however, accumulation of ABA was not responsible for the varietal difference in drought tolerance (Cellier et al., 1998) . Alteration in the kinetics of dehydrin transcript accumulation during recovery from dehydration from that after ABA-exposure supports the observation in sunflower and suggests that additional factors other than ABA might be involved in the expression of dehydrin during stress-adaptation. In Arabidopsis, expression of rab18 gene in response to low temperature was shown to be ABA dependent. However, a small, but reproducible accumulation of rab18 mRNA persisted during low temperature adaptation in Arabidopsis even after endogenous ABA level decreased to the basal level (Lang et al., 1994) . RAB18 protein expression was maintained almost comparable to the induced level up to 3 d of deacclimation (Mantyla et al., 1995) .
Long-Term Expression of Some Dehydration-Inducible Genes during Recovery
Two transcripts (CD051343 and CF340748) encoding 2 putative Ser/Thr kinases that maintained higher expression for 72 h after stress-removal resemble closely CBL-interacting protein kinases (CIPK) 6 and 16, respectively, of Arabidopsis. CBL-interacting protein kinases are cytosolic calcium-regulated autophosphorylating enzymes (Halfter et al., 2002; Hardie, 1999) . Calcium was shown to be involved not only in the early events of signal transduction (Knight et al., 1991; Bush, 1995) , but also in the signal storage and retrieval (Verdus et al., 1997) . A signal storage mechanism involving an autophosphorylating kinase has been proposed (Lisman, 1985) and involvement of calcium-dependent protein kinases in long-term maintenance of seed embryo dormancy was suggested (Trewavas, 1986) . The transcript CD051312 possesses high homology to ABI1, a protein phosphatase. ABI1 locus regulates a wide spectrum of Abscisic acidmediated responses. ABI1 protein contains a Ca 21 binding site in its N-terminal region and interacts strongly with one of the CIPKs (PKS18), indicating a possibility that this protein may connect ABA and Ca
21
-mediated signaling pathways (Leung and Giraudat, 1998; Ohta et al., 2003) . Cytosolic calcium also regulates plasma membrane H 1 -ATPase (CD051280; Kinosita et al., 1995; Qui et al., 2002) . All these cases of evidence indicate that ABA and cytosolic calcium probably play major role in the maintenance of the transcript messages from the previous stress. Possible involvement of cell wall has been implicated as a reservoir of stored signal concerning the fate of the cell during development (Brownlee and Berger, 1995) . Changes in the cell wall structure were also proposed in drought acclimation (Levitt, 1986) . Cinnamoyl coA reductase, encoded by CD051301, is one of the enzymes involved in lignin biosynthesis for cell wall (Ruelland et al., 2003) .
21
Prolonged gene expression can be explained by two mechanisms: increased mRNA stability and/or slow mRNA synthesis for longer period. Salt-regulated increased mRNA stability has been demonstrated for the transcript accumulation of a cell wall targeted alfalfa protein MsPRP2, which contain a sequence for mRNA destabilization at the 3#-untranslated region (Deutch and Winicov, 1995) . It will be interesting to check whether these transcripts have such a common sequence at their 3#-UTR. Unlike dehydration, exposure to ABA had no appreciable effect on RWC of the Table II . A list of ESTs and their fold expressions with different treatments (as described) determined by nylon filter hybridization. Averages of the ratios from two independent experiments (described in Fig. 3 leaves; and in contrast to dehydration, preexposure to ABA did not prolong the expression of six transcripts during recovery. Therefore, it is possible that some dehydration-regulated, but ABA-independent factor(s) is responsible for the increased stability of those mRNAs. Alternatively, unlike ABA-exposure, water-limitation may cause a permanent alteration in the cell structure and in the signaling pathways, resulting in sustained expression of those transcripts. Preexposure to ABA, though it did not prolong the expression of 6 transcripts in the recovery phase as dehydration pre-exposure, improved water retention ability in the chickpea leaves to some extent in the following dehydration treatment (Fig. 1) . ABA pretreatment maintains the expression levels of MIPS and dehydrin1 transcript more than 2-fold up to 24 h during recovery. However, this may not be the only reason. We have analyzed the expressions of only 7 genes after ABA pretreatment. Detail expression analysis of more genes in the recovery period after ABA exposure is required to address this issue. Finally, it cannot be excluded that storage of previous stress signals does not result only from the maintenance of some gene expression; rather it is the manifestation of cumulative changes in the structural and functional relationships of multiple pathways involved in stress metabolism. 
MATERIALS AND METHODS

Plant Materials and Stress Treatment
Seeds of chickpea (Cicer arietinum L. cv BGD72), provided by IARI (New Delhi, India), were germinated, sown in composite soil (peat compost to vermiculite, 1:1), and grown for 6 d after germination at 18°C to 22°C day/ 10°C to 15°C night/50% relative humidity with a photoperiod of 10 h with appropriate watering. For dehydration treatment, seedlings were carefully removed from the pot and subjected to dehydration for 5 h on 3 MM paper (Whatman, Clifton, NJ) at room temperature under dim light. For control, plants were removed from the soil and immediately replanted in the same pot and kept under the same condition for the same period. For stress-recovery, seedlings exposed to dehydration for 5 h were replanted in water-saturated soil and kept for the indicated period in normal growth condition. As controls for stress recovery, seedlings were removed from the soil and replanted immediately as earlier and kept for the same period of time to avoid diurnal variation. For ABA treatment, plants were removed from the soil as before, and the roots were dipped into aerated deionized water with or without 100 mM of ABA for 5 h. For the recovery from ABA-stress, seedlings were similarly treated as in the case of dehydration. RWCs of the leaf samples were measured as described (Levitt, 1986) . Four samples each of 20 fully expanded leaves were taken for each of the time points.
RNA Isolation and Construction of Subtracted cDNA Library
Total RNA was isolated from whole seedling by using TRIzol Reagent (Life Technologies, Rockville, MD), and polyA 1 RNA was purified by mRNA isolation kit (Roche Applied Science, Manheim, Germany). Subtracted cDNA library was constructed by using CLONTECH PCR-Select cDNA subtraction kit (CLONTECH Laboratories, Palo Alto, CA) following manufacturer's protocol. In brief, tester (dehydration) and driver (control) double stranded cDNAs were prepared from 2 poly(A 1 ) RNA (2 mg each) samples. Tester and Driver cDNAs were separately digested with RsaI to obtain shorter bluntended molecules. Two tester populations were created by ligating two aliquots of diluted tester cDNA with two different adaptors (adaptors 1 and 2R) separately. First hybridization was performed by the following procedure. Each tester population was mixed with an excess of digested driver cDNA. The samples were heat denatured and allowed to anneal at 68°C for 8 h. The two samples from the first hybridization reaction were mixed together, and more denatured driver cDNA was added for further hybridization to enrich differentially expressed sequences. Differentially expressed cDNAs, with different adaptor sequences at two ends, were selectively amplified by PCR and a second PCR was done with nested primers to further reduce the background. The subtracted and enriched DNA fragments were directly cloned into T/A cloning vector (pT-Adv; CLONTECH Laboratories). Competent cells of Escherichia coli DH5a were transformed with the ligation mix and plated on Luria-agar plate containing ampicillin, isopropylthio-bgalactoside, and X-gal for blue-white selection (Sambrook and Russell, 2001) .
Library Amplification and Preparation of DNA Arrays
Individual clones of the subtracted cDNA library were amplified in a 96-well PCR reaction plate using M13 forward and reverse primers in a 50-mL reaction at an annealing temperature of 60°C for 30 cycles. The products were analyzed in agarose gel to confirm the insert size, quality, and quantity. Purified PCR products were denatured by adding an equal volume of 0.6 M sodium hydroxide. Equal volume of each denatured PCR product (about 100 ng) of $300 bp of size was spotted on two Hybond N membranes (Amersham Pharmacia Biotech, Uppsala) using dot-blot apparatus (Life Technologies, Bethesda, MD) in 96 format to make two identical arrays. In addition, a PCR product of chickpea actin cDNA (GenBank accession no. AJ012685) using primer sequences (5#-CCACGAGACAACATTTAACTC-3# and 5#-TATTCTGCCTTTGCAATCCAC-3#) was spotted as internal control to normalize the signals of two different blots corresponding to stressed and unstressed samples. A PCR product of Neomycin phosphotransferase (NPTII) gene from the vector pCAMBIA 1305.1 (GenBank accession no. AF354045) using primer sequences (5#-TTTTCTCCCAATCAGGCTTG-3# and 5#-TCA-GGCTCTTTCACTCCATC-3#) was also spotted as a negative control to subtract the background noise. The membranes were neutralized with neutralization buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl) for 3 min, washed with 2 3 SSC, and cross linked using UV cross linker (Stratagene, La Jolla, CA).
Probe Preparation, Hybridization, and Data Analysis
Control and stress mRNAs were labeled with a 32 P-dCTP by first-strand reverse transcription. One microgram of mRNA was labeled in a 20-mL reaction volume containing 1 3 reaction buffer, 2 mg of 5#-(dT) 30 VN-3# (V 5 A/G/C and N 5 A/G/C/T) primer, 2.5 mM dATP, dTTP, dGTP, 0.02 mM dCTP, 5 mL of a 32 P-dCTP (10 mCi/mL; 3,000 Ci/mmol), and 200 units of reverse transcriptase (Superscript II, Life Technologies, Grand Islands, NY). After incubation at 42°C for 1 h, RNA was removed by incubating with RNase H at 37°C for 20 min. Radio-labeled cDNAs were cleaned by Sephadex G-25 (Amersham-Pharmacia Biotech) and suspended in hybridization buffer (7% SDS, 0.3 M Sod-phosphate pH 7.4, 1 mM EDTA, 10 mg of sonicated salmon sperm DNA). Nylon membranes were prehybridized with the same buffer for 3 h at 65°C and hybridized with denatured control and experimental (drought-treated) cDNA probes at the same condition for 24 h. The membranes were washed three times with washing buffer (1 3 SSC, 1% SDS, 30 min each, 65°C). Images of the membranes were scanned in phosphorimager and signal intensities were analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The program allows normalization of the signal against background. Sequencing was done using Big Dye Terminator kit version 3.0 (Applied Biosystems, Foster City, CA) and analyzed with the 3,700 ABI Prizm 96 capillary sequence analyzer. The sequences were submitted to the EST data bank of NCBI for accession numbers.
Northern Hybridization
Twenty micrograms of total RNA from control and treated samples were analyzed in 1.2% agarose gel containing formaldehyde and transferred to Hybond N membrane (Amersham Biosciences, Buckinghamshire, UK) following the method mentioned in Sambrook and Russell (2001) . PCR-amplified individual cDNA fragment (with primers corresponding to adaptor 1 and 2R) was purified from agarose gel. Primers for the actin cDNA are mentioned above. Probes were labeled with a 32 P-dCTP using Megaprime DNA labeling system (Amersham Biosciences) and purified through Sephadex G-25. Northern hybridization was performed and band-intensity was calculated following the procedure described above for nylon membrane array.
Northern Hybridization
Assay of Abscisic Acid, Pro, and Myoinositol ABA content of chickpea seedlings with or without stress was measured according to Setter et al. (2001) . Lyophilized seedlings were crushed in chilled 80% methanol. The extracts were fractionated by C18 reverse-phase chromatography, and the ABA contents were assayed by enzyme linked immunosorbant assay (ELISA). The ABA-content is expressed as microgram of ABA per gram of dry weight. Free Pro content was measured from three different lyophilized samples for each time point according to Bates et al. (1973) . Myoinositol was measured according to Miller et al. (1998) . Briefly, total soluble sugar was extracted from the lyophilized samples with methanol to chloroform to trichloroacetic acid to water (12:5:1:2). Water to methanol (1:1) was added for phase separation. The aqueous phase was lyophilized to complete dryness. The sample was derivatized with Tri-Sil Z (Pierce, Rockland, Il) and run through gas-liquid chromatography coupled with flame ionization detector with nitrogen as carrier gas. Quantitation was made against similar run with standard myoinositol (Sigma-Aldrich, St. Louis). Samples from two independent seedlings were taken for each time point.

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