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First published online July 9, 2004; 10.1104/pp.104.043141 Plant Physiology 135:1608-1620 (2004) © 2004 American Society of Plant Biologists Long Term Transcript Accumulation during the Development of Dehydration Adaptation in Cicer arietinum1National Centre for Plant Genome Research, JNU Campus, New Delhi 110067, India (P.B., R.S., A.K., P.K.V., D.C.); Department of Biotechnology, University of Calcutta, Kolkata 700019, India (D.M.); and Department of Biotechnology, CCS University, Meerut 250004, India (D.N.)
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.
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+2]cyt) concentration (Knight et al., 1991). As an adaptive response, the subsequent stresses show altered magnitude and kinetics of [Ca+2]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, 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
We have observed an improved dehydration-tolerance 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
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
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).
Transcripts accumulated during dehydration stress were identified by a combination of approaches. Six-day-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+) 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+) 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)
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  -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 northern-blot 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.
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 µg/g dry weight in control to 13.99 µg/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
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). 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).
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.
Expression of all the seven transcripts mentioned above was inducible by exogenous application of ABA (Table I). To verify whether the characteristic long-term abundance of these transcripts is specific to dehydration or general for any stress, the chickpea seedlings were exposed to 100 µM 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 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.
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
Dehydration-Inducible Genes in Chickpea
We have selected chickpea, the third most consumed legume crop (Graham and Vance, 2003
Small sugar molecules are believed critical to maintain osmotic balance of cells in stress (Bray et al., 2000
The CBF/DREB family of transcription factors are an integral part of early regulation of water stress-mediated response (Liu et al., 1998
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
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 µg/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
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
Late-embryogenesis abundant (LEA) groups of proteins are thought to provide protection to the cellular macromolecular structures against desiccation-mediated 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
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
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 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.
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 µM 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
Total RNA was isolated from whole seedling by using TRIzol Reagent (Life Technologies, Rockville, MD), and polyA+ 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+) RNA (2 µg each) samples. Tester and Driver cDNAs were separately digested with RsaI to obtain shorter blunt-ended 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 DH5
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-µL 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
Control and stress mRNAs were labeled with
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)
ABA content of chickpea seedlings with or without stress was measured according to Setter et al. (2001) Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AJ012685, AF354045, CD051266, CD051265, CD051303, CD051329, CD051345, CD051358, CD051350, CD051347, CD051311, CD051301, CD051310, CD051304, CD051352, CD051279, CD051285, CD051305, CD051278, CD051342, CD051357, CD051343, CF340748, CD051322, CD051324, CD051317, CD051274, CD051264, CD051312, CD051261, CD051323, CD347670, CD051268, CD051271, CD051272, CD051277, CD051290, CD051297, CD051339, CD051320, CD051326, CD051288, CD051295, CD051286, CD051292, CD051346, CD051338, CD051333, CD051300, CD051284, CD051267, CD051351, CD051287, CD051308, CD051355, CD051360, CD051361, CD051330, CD051282, CF074502, CD051293, CD051336, CD051341, CD051262, CD051273, CD051299, CD051315, CD051353, CD051270, CD051289, CD051291, CD051296, CD051321, CD051335, CD051276, CD051281, CD051307, CD051283, CD051309, CD051327, CD051325, CD051280, CD051269, CD051275, CD051298, CD051306, CD051313, CD051314, CD051316, CD051332, CD051334, CD051337, CD051344, CD051348, CD051349, CD051354, CD051340, CD051331, CD051294, CD051302, CD051263, CD038847, and CD051328.
The authors gratefully acknowledge Dr. S.S. Yadav (Indian Agricultural Research Institute, New Delhi, India) for providing the chickpea seeds. Also acknowledged are Dr. Sudip Chattopadhyay and Dr. Subhra Chakraborty (NCPGR, New Delhi) for providing cDNAs for CAB, Rubisco small subunit, and actin used in this study. B.P. acknowledges UGC and R.S. acknowledges CSIR for providing junior research fellowships. Dipak Manna and Divya Negi worked as summer interns. Received March 20, 2004; returned for revision April 18, 2004; accepted April 19, 2004.
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.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.043141. * Corresponding author; e-mail debasis_chattopadhyay{at}yahoo.co.in; fax 91–11–26167394.
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ABSTRACT
RESULTS
32P-dCTP labeled probes corresponding to indicated EST clones. A PCR product of chickpea actin cDNA (see "Materi als and Methods") was used as internal control and 25S ribosomal RNA was shown as loading control. Annotations assigned by BLASTX homology search were mentioned.
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. 
