BACKGROUND
chickpea  is one of the most important grain legume crops worldwide and a major source of protein for millions of families in developing countries.
despite considerable investment in breeding, average chickpea yield in major producer countries such as india stagnates at  <dig> – <dig>  mt hectare- <dig> since many years.
this low yield is far below the crop's potential of 3– <dig> mt hectar- <dig> under optimal conditions.
chickpea is mostly grown in low-input, rain-fed agriculture in mediterranean-type environments as inter-crop between cereals, and on residual moisture from monsoon rains on the indian subcontinent.
due to insufficient, untimely and erratic rainfall in these semi-arid and arid areas, the crop often suffers from drought at the end of the cropping season.
in future, global warming and soil erosion will even worsen conditions for many crops including chickpea.
thus, drought represents the major constraint to increase chickpea yield, and drought tolerance therefore is a major aim of chickpea breeding.
however, drought tolerance is a complex trait and hard to achieve by conventional breeding methods.
understanding of the molecular mechanisms underlying drought tolerance is therefore needed for successful, knowledge-based crop improvement  <cit> .
molecular genetics and genomics of stress-responses in model plants such as arabidopsis revealed that abiotic stresses such as drought, salinity and cold stress are characterized by ionic- and osmotic-disequilibrium components; eliciting general as well as specific responses and mechanisms of stress-protection  <cit> .
these studies underpinned the importance of early responses to the various stresses for the survival of the plants  <cit> .
much of our current understanding of stress-response mechanisms comes from genome-wide analysis of gene expression, facilitated by the availability of microarrays carrying a comprehensive set of genes.
in chickpea, like in many other under-researched crops, extensive microarray-based studies are not yet possible, because the necessary est data are not available.
for example, no more than  <dig>  chickpea ests are publicly available at the national centre of biotechnology information , as compared to at least  <dig> , <dig> ests available for arabidopsis.
in the absence of such resources, researchers often use less comprehensive approaches as e.g.
suppression subtractive hybridisation  libraries  <cit> , which do not per se allow the quantification of expression of differentially expressed genes.
ssh results are therefore often used for the generation of macro-arrays for subsequent analysis of gene expression  <cit> .
these authors used this cumbersome approach for the discovery of  <dig> dehydration-responsive transcripts in chickpea roots.
open-architecture, whole-genome transcription profiling technologies such as supersage  <cit> , however, provide a solution to the problem of lacking est and genomic data.
supersage is an improved version of the serial analysis of gene expression  technique  <cit> .
in the past years, it has demonstrated a high versatility due to its longer tag size   <cit> .
in principle, sage and all its variants rely on the assumption that a small, defined part of a cdna, a so-called "tag", characterizes this cdna, and that counting the number of times a particular tag occurs in the tag population faithfully reflects the abundance of the respective mrna in the transcriptome.
since  <dig>  to  <dig>  tags are sequenced in a single experiment, a comprehensive profile of the transcriptome is generated.
here we report on the stress responses of  <dig>  transcripts representing  <dig>  unique  <dig> bp tags  from roots of the drought-tolerant chickpea variety icc <dig> early after onset of desiccation.
we discuss the stress-regulated transcription of genes involved in signal perception and transduction, ros scavenging and metabolism, osmotic and ionic stress-related pathways, regulation of water and ion homeostasis, as well as several reported effector proteins.
to test the reliability of the present results we use microarrays carrying stress-responsive as well as constitutively expressed  <dig> bp tag sequences.
to further confirm the supersage results with a third method, we use sybrgreen and commercially available taqman assays produced from 3'-and 5'-race sequences from selected chickpea mrnas.
finally, we compare our transcription profiles from drought-stressed chickpea roots to results obtained from chickpea root ssh libraries  <cit>  and microarray experiments in medicago truncatula  <cit> , and discuss similarities and differences.
this study is the first of a series characterizing stress responses of chickpea on a molecular level as a prerequisite for production of expression markers and microrrays for high-throughput germplasm and expression qtl analysis at the onset of knowledge-based breeding for stress-tolerance in this important protein crop.
RESULTS
the combination of high-throughput  <dig> sequencing with supersage
drought libraries are part of a project, which aims at evaluating the transcriptional responses of chickpea upon diverse abiotic stresses, including several other treatments and various tissues .
for the sequencing of all the libraries, a single  <dig> plate divided into two sections was used, from which a total of  <dig>  reads were extracted.
after eliminating: i) incomplete reads, ii) twin-ditags, and iii) ditags without complete library-identification dna linkers, a total of  <dig>   <dig> bp tags were obtained for further analysis.
from these, about  <dig>  tags were singletons, that were excluded from analysis.
finally,  <dig>  tags remained for evaluation .
subsequently, sub-datasets were constructed for each experimental situation.
for the present study,  <dig>   <dig> bp tags from control and dehydrated roots were analyzed.
abundance of unitags and annotation to public databases
a total of  <dig>   <dig> bp tags from roots of the drought-tolerant variety icc <dig> either subjected to  <dig> h desiccation  or from well-watered controls  were sequenced, and represented  <dig>  unique transcripts, so called unitags.
less than 1% percent of these occurred in very high copy numbers , whereas 23% and 75% of the transcripts were present between  <dig> to  <dig>  and less than  <dig> copies.million- <dig>  respectively .
unitags from control and stress libraries were deposited in the gene expression omnibus  public domain under accessions gsm <dig> and gsm <dig>  respectively.
* ratio  values indicating up- or down-regulation calculated with the  <dig> h desiccation library as reference.
annotation of the  <dig>  unitags matched  <dig>   to well characterized sequences from the fabaceae family available in public databases.
of these, 53% matched to sequences from medicago truncatula, 29% to cicer arietinum, 6% to pisum sativum, 3% to glycine sp., 2% to medicago sativa, and 7% to other legume genera.
in many cases, tigr gene index annotations from legumes bridge automatically to characterized tentative consensus  sequences mostly from arabidopsis, rice, and maize.
of the remaining  <dig>   non-assignable  <dig> bp tags,  <dig>  were significantly homologous to anonymous est or dna sequences, whereas  <dig>  found no match at all.
a summary of the primary data is given in table  <dig>  annotation of the  <dig> bp tags and respective expression ratio values are deposited in the main data matrix .
annotation of virtual tags generated from chickpea ests deposited in public domains
in order to test the validity of the annotation of chickpea  <dig> pb tags through sequence homology with other legumes, virtual tags generated from chickpea est sequences deposited in the ncbi data bank were extracted, and their direct annotation was compared with the annotation of longer homologous ests from the model legume m. truncatula.
after retrieving the complete set of chickpea sequences deposited in the ncbi est database , a total of  <dig>  different in silico-generated  <dig> bp tags were selected to be directly blasted against the nr ncbi  nucleotide database .
from these, a total of  <dig> tags revealed high homology hits.
after exclusion of anonymous entries,  <dig> tags were linked to uniprot entries or to characterized non-protein coding rnas .
in parallel, the same  <dig>  tags were blasted against the plant est ncbi  and tigr  databases separately, where  <dig>  and  <dig> sequences, respectively, found at least one high homology hit.
then, the complete target sequences from each blast were retrieved and re-blasted against the nr ncbi  database.
a total of  <dig>  and  <dig>  sequences, each representing a different  <dig> bp tag, revealed high homologies with nr ncbi entries.
from these,  <dig> and  <dig>  respectively, were non anonymous .
the results from the direct blast were compared with each of the m. truncatula est-briged homology searches for commonly annotated sequences, either by direct id matching, protein name, or sequence description.
for m. truncatula ests from ncbi,  <dig> out of  <dig>  annotations were not congruent with the annotation of the corresponding chickpea in silico tag.
however, only  <dig> out of the  <dig> annotations belonged to uniprot-linked accessions .
for m. truncatula ests from tigr,  <dig> out of  <dig>  common annotations were not congruent.
from them,  <dig> belonged to uniprot-linked accessions .
the present results reveal that in more than 90% of the cases, the assignment of tags to uniprot-linked accessions is congruent with the annotation of longer ests from other legumes.
however, databases overloaded with anonymous entries considerably reduce the amount of biologically interpretable data.
differential gene expression in response to drought stress of chickpea roots and assignment of  <dig> bp tags to gene ontology  functional categories
we calculated the natural logarithm of expression ratios  of the  <dig> bp tags from control versus stressed roots as well as significance levels  according to audic and claverie  <cit>  of up- and down-regulation for each transcript using the software package discoveryspace  <dig>  .
differences in abundance of tags in control and stressed roots were considered relevant at r >  <dig> .
at this threshold,  <dig>   tags were significantly differentially expressed in stressed as compared to control roots.
of these,  <dig>  were up-, and  <dig>  down-regulated.
a total of  <dig> transcripts  showed more than 8-fold difference in expression  >  <dig> , p <  <dig> ).
of these,  <dig> were up-, and  <dig> down-regulated under stress.
as depicted in the venn diagram , a considerable number of tags occurred exclusively in either the control tissue or under stress.
the  <dig> most significantly up- or down-regulated transcripts matching well characterized genes in public databases are listed in table  <dig> and table  <dig>  respectively.
however, many transcripts could not be annotated, because they either matched to anonymous ests, or did not hit any entry in the database.
therefore, the annotatable transcripts coding for extensin , nadp-dependent isocitrate dehydrogenase i , s-receptor kinase-like protein  <dig> , chalcone isomerase , udp-glucose-pyrophosphorylase , beta-glucosidase , specific tissue protein  <dig> , s-adenosyl-l-methionine synthetase , and cysteine synthase  were the most up-regulated interpretable transcripts under stress though not the most up-regulated in the data set.
correlation of r to defined standard functional gene categories  in the gene ontology  database revealed that the majority of the most up-regulated transcripts are assigned to the go: biological process "metabolism" .
as depicted in figure  <dig>  stress perception and signalling , small gtpase-mediated signal transduction , rna metabolism , and cellular carbohydrate metabolism  were the most represented go: functional categories in desiccation-stressed roots.
further, over-representation of transcripts involved in transport , proteolysis , oxidative phosphorylation , and stress response  indicate the mechanisms by which the roots adapt to the stress.
over-representation of transcripts involved in go: functional category "oxygen and reactive oxygen species metabolism"  suggests that ros play an important role as side stress, but also as signalling molecules.
tags from transcripts assigned to the go: biological process "protein biosynthesis" , such as 40s ribosomal proteins s <dig>  and s <dig> , and 60s ribosomal proteins l <dig> , l <dig> , and l <dig> , all were significantly down-regulated suggesting a repression of de novo protein biosynthesis under drought stress in roots.
additionally, other go: biological processes like photosynthesis and light reaction , chromatin assembly , chromosome organization and biogenesis , biopolymer metabolism , dna replication  were also under-represented .
regarding go: cellular component categories, "unlocalized protein complexes" were most over-represented under drought stress , followed by golgi apparatus , endoplasmic reticulum , membrane integral genes  and external encapsulating structure .
go: cellular components such as "ubiquitin ligase complex" , mitochondrial and inner membrane , and ribosome  were amongst the most under-represented.
the transcription of genes coding for proteins of the serine/threonine phosphatase complex , cytoplasm , cytosol , cytoskeleton , thylakoid , microtubule cytoskeleton , cell-wall structure and modification as well as cell surface protein was constitutive .
members of a gene family assigned to over- or under-represented go: categories, respectively, could be up- as well as down-regulated to different extents.
for example, whereas unitagstca- <dig>  annotated to the putative extensin , was one of the most down-regulated transcripts, unitag stca- <dig> representing another extensin isoform  was the most up-regulated tag in our data set.
however, both isoforms are grouped in the same go:category.
differential expression of drought stress-related sub-transcriptomes in chickpea roots
since the present genome-wide expression analysis revealed a plethora of differentially expressed  <dig> bp tags with and without match to genes of known function, it is impossible to display or discuss all of them in the frame of this paper.
instead, in figure  <dig> and figure  <dig> we elaborate in more detail on the expression of genes and gene families belonging to the go: biological processes "signal transduction", "stress sensing", "regulation of transcription", "transport", "post-transcriptional regulation" and "pathway inhibitors", all involved in early responses to stress.
in these categories, the gene itself or members of its family have known functions in stress-perception, stress-signalling and stress-responsive regulation of transcription and chromatin structure.
further, we closely look at transcripts encoding proteins involved in reactive oxygen species  scavenging, and transcripts related to ros-mediated signal transduction cascades .
as examples for the regulation of effector genes down-stream of the signalling cascades, we will detail the differential expression of genes coding for water-channel proteins, so called aquaporins, and of genes coding for proteins involved in osmolyte metabolism .
confirmation of supersage results by qrt-pcr
our genome-wide expression analysis results were exemplarily confirmed by quantitative real-time  pcr using taqman probes xptm-ca- <dig>  and xptm-ca- <dig>  available from genxpro gmbh, as well as sybr green® assays.
oligonucleotides for sybr green assays were deduced from 5'- and 3'-race sequences generated with  <dig> bp tags as primers.
the following transcripts were targeted: st-ca <dig> , st-ca <dig> , st-ca <dig> , st-ca <dig> , st-ca <dig> , and st-ca <dig> .
for sybr green® as well as taqman assays, the sequence for either the forward or the reverse pcr primer was derived from the  <dig> bp tag, and the complementary primers from 3'- or 5'-race sequences, respectively.
confirming the supersage expression levels, amplifications from sybr green® assay st-ca <dig> and taqman probe xptm-ca <dig> revealed constitutive levels of expression  .
amplifications for sybr green® assays with st-ca <dig>  st-ca <dig>  st-ca <dig> as well as taqman probe xptm-ca- <dig> revealed up-regulation of the respective transcripts under stress , as already indicated by the differential expression analysis.
stress-induced down-regulation of  <dig> bp tags was corroborated by sybr green® assays for st-ca <dig> and st-ca <dig> .
however, for st-ca <dig>  amplification profiles as well as post-qrt-pcr amplicon melting curves suggested partially unspecific priming.
again, in agreement with the supersage results, the transcript chosen as invariable control indeed displayed almost completely similar expression in control and stressed roots.
these experiments confirm our present results and suggest  <dig> bp tags as reliable sequence information source for other expression profiling techniques.
profiles confirmation via microarray hybridization of spotted supersage-derived oligos
to characterize the chickpea transcriptome under drought stress, sequence information from supersage profiles was used to design an agilent  <dig> k microarray, onto which  <dig>  selected  <dig> pb tags were spotted for a comparison of both profiling techniques.
on the microarray, the majority of the oligonucleotides with original tag sequences were spotted twice .
additionally, oligonucleotides with different mismatch numbers from each original tag as well as a small sub-set of longer race-derived sequences were also included.
after statistical treatment of the different internal twin-replicas, normalization, and mismatch background correction, reproducible signals from different hybridization rounds  were selected for cluster analyses together with expression ratios of the  <dig> bp tags.
a total of  <dig> % probes on the micro-array shared the same tendency of expression with the respective tag when hybridized with crnas prepared from drought-treated roots from icc <dig>  from a total of  <dig>  spots showing congruent results among different replicas and dye-swap experiments,  <dig> and  <dig> were up- and down-regulated in both techniques, respectively, whereas  <dig> spots showed in-congruent results .
unitag expression ratios from both techniques are deposited in the main data matrix .
*regulation tendencies for salt-stressed roots in both techniques are included for comparative purposes
comparison of different transcript profiling techniques: supersage versus ssh macroarrays and microarrays
we tested, whether our genome-wide transcription patterns are typical for roots of chickpea and other legumes, even if the profiles were obtained with other methods.
therefore we compared the present results to two studies addressing similar questions.
in chickpea, boominathan and co-workers  <cit>  investigated whether pre-exposure to a dehydration shock improved adaptive responses of the roots during subsequent dehydration treatment.
these authors identified  <dig> dehydration-inducible transcripts by repetitive rounds of cdna subtraction, differential dna-array hybridization, and northern-blot analysis.
additionally, responses to exogenously applied abscisic acid  were also monitored.
since one analyzed time point was set  <dig> h after onset of drought, the results of this experiment should be at least partially comparable to our study in which rna was isolated from roots  <dig> h after onset of drought stress.
however, the results were not 100% congruent.
it is important to note, that micro- or macro-arrays do not reliably differentiate between different transcript isoforms from gene families.
hybridization signals may integrate the hybridization intensities over all closely related transcripts, whereas supersage generates absolute numbers for each transcript variant .
due to the much deeper coverage of the transcriptome by this technique, almost all differentially expressed transcripts represented in the ssh libraries have at least one, but usually more counterparts in our  <dig> bp tags libraries.
a cluster analysis of transcription profiles obtained by macroarray hybridization and the differential expression of  <dig> bp tags from drought-stressed roots  <dig> h or  <dig> h after onset of the stress, respectively, is shown in figure  <dig>
similarly up-regulated  and differentially regulated transcripts  under both conditions are listed in table  <dig>
comparison between supersage expression profiles and macroarray-generated data .
three main categories from the cluster analysis in figure  <dig> are detailed below.
section a: constitutive tags versus upregulated transcripts on the macroarray.
section b, d: non-corresponding supersage and macroarray transcript profiles.
section c: up-regulated tags and up-regulated transcripts on the macroarray.
investigating regulatory and protective mechanisms leading to desiccation tolerance  in medicago truncatula seeds, buitink and co-workers  <cit>  published another, at least partially comparable study.
these authors used the  <dig> k medicago microarray to monitor changes in the transcriptome of desiccation-sensitive 3-mm-long radicles at different time points during incubation in a polyethylene glycol  solution mimicking the effects of desiccation.
these experiments identified several specific expression profiles at different time scales.
a cluster analysis comparing the results from desiccation-stressed medicago radicles and drought-stressed chickpea roots is depicted in figure  <dig>  transcripts up-regulated in both species , regulated in opposite directions in the two species , and transcripts down-regulated in chickpea and medicago as a reaction to stress  are listed in table  <dig>
comparison between supersage expression profiles and 16k-microarray-generated expression data .
four main categories from the cluster analysis in figure  <dig> are detailed below.
section a: down-regulated tags and down-regulated transcripts on the microarray.
section b: contrasting supersage and microarray expression profiles
section c: contrasting supersage and microarray expression profiles
section d: up-regulated or constitutive tags and 16k-microarray transcripts, respectively
differential expression of natural antisense transcripts
as expected from an open-architecture technique, tags synthesized on coding strands of template cdnas and, in addition, tags originating from the opposite  strands were discovered.
here, at least  <dig> tags matching to the reverse sequences of ests in the public databases were detected.
these tags could represent potential natural antisense transcripts .
in lotus japonicus, sage tags representing nats were induced during nodulation  <cit> , and in arabidopsis endogenous sirnas derived from a pair of natural cis-antisense transcripts regulate salt tolerance  <cit> .
thus, it is possible that nats are also involved in stress tolerance in chickpea.
however, galante and co-authors  <cit>  demonstrated that a considerable portion of such nats found in the databases are artefacts.
in drought-stressed chickpea roots, we found – inter alias – up- and down-regulation of nats for transcripts of several members of the aquaporin gene family and differential expression of nats for phosphatase transcripts.
however, since we did not exclude genomic priming by dnase digestion of the rna prior to cdna synthesis, confirmation of the identity of these potential nats will be subject to further studies.
discussion
we analysed the expression of approximately  <dig>  transcripts from un-stressed control and drought-stressed chickpea roots, respectively.
it was previously estimated that the total number of average-sized transcripts per cell in higher plants ranges from  <dig>  to  <dig>   <cit> .
thus,  <dig>   <dig> bp tags provide ~1-fold coverage for transcripts present at a minimum of  <dig>  copies per cell.
therefore our study, which detected >  <dig>  unique transcripts, is not comprehensive.
however, even for the model legume medicago truncatula only  <dig>  unique ests/tcs are deposited in public databases.
therefore, our single study demonstrates that supersage is suited to overcome the problem of lacking resources in non-model organisms and under-researched crops.
since, as we have shown, 75% of transcripts are present in less than  <dig> copies million- <dig>  and a small portion of transcripts is represented by more than  <dig> copies million- <dig>  we probably missed a number of transcripts with less than 10– <dig> copies per cell.
these may include transcripts for highly interesting pleiotropic proteins such as transcription factors possibly present at only  <dig>  copies per cell  <cit> .
additionally, sampling larger organ sections with millions of cells obscures the specific transcript profiles of component cells.
as a consequence, transcripts found in high copy numbers in just a few specialized cells are under-represented.
as elegantly demonstrated for maize  <cit>  and arabidopsis  <cit> , there are strong differences in transcription profiles between adjacent tissues.
therefore, the detection of rare transcripts should be improved by increasing the number of sequenced transcripts  in combination with a more selective sampling technique such as laser-capture microdissection  <cit> .
also, annotation of 55% of the  <dig> bp tags to the databases was straightforward.
considering that roughly  <dig>  chickpea est sequences are publicly available , most tags matched to sequences from the related model legume medicago truncatula rather than to chickpea ests.
on the other hand, a large number of significant hits represented fully uncharacterized database accessions, a fact that handicaps functional interpretation of the present chickpea transcription profiles more than the size of the  <dig> bp tags.
additionally, a relevant problem arose from the interpretation of the assignment of different distinct 26bp tags to single unigenes or tentative consensus sequences .
we decided to classify such tags as isoforms of transcripts from members of gene families rather than assigning them to a particular gene, even if the expression patterns suggested functional homology.
in addition, we could not differentiate whether these tags with similar annotation came from alternatively spliced transcripts of the same gene.
since in arabidopsis and rice at least 21% of genes produce alternatively spliced transcripts  <cit> , evidence beyond sequence homology of the supersage tags  is needed to decide between these two possibilities.
differential expression of members of large gene families
one major advantage of the differential expression of 26bp tags as compared to macro- and micro-arrays is the very good differentiation between transcripts from different members of large genes families, which comprise the majority of all genes.
gene family members have more or less conserved sequences, and similar or different functions.
for example, the cytochrome p <dig>  gene superfamily comprises more than  <dig> members grouped in  <dig> families in arabidopsis thaliana, and more than  <dig> members in rice  <cit> .
in the present study,  <dig> 26bp tags revealed high-homology hits with members of the cyp <dig> superfamily, from which  <dig> could be assigned to their respective family through blasting against the arabidopsis-cyp database .
the involvement of cyp superfamily members in numerous catalytic reactions on a spectrum of substrates suggests that the transcription profiles of its members reflect this diversity.
in accordance with the expectation,  <dig>  of the  <dig> cyp-annotated 26bp tags changed their expression profiles at least  <dig> -fold with r  >  <dig> , whereas  <dig>  showed only slight differences or constitutive levels.
among the 26bp tags assigned to drought stress-related cyps, hits to cyp707a family  which harbours aba 8'-hydroxylases , revealed a very slight up-regulation , indicating that the turnover of aba is already active under our experimental condition and time-points.
unitag stca- <dig>  assigned to a cyp81f <dig> member, stands out for its extreme down-regulation .
although its function is not known, cyp81f <dig> members generally change their expression profiles dynamically after induction of water stress  <cit> .
considering the differential expression and diverse functions of individual members of gene families, whole-genome transcription profiling is only useful, if it differentiates between the different genes and their transcript isoforms in such families.
faithful discrimination between, and individual quantification of expression of these isoforms is therefore one advantage of the longer  <dig> bp tags.
however, also methodological inconsistencies may result in the observation of several different tags from a single gene  <cit> .
to prevent methodological artefacts and assure the validity of the detected transcript variants, confirmative procedures such as double nlaiii digestion were standard in all libraries.
additionally, in silico routines for the exclusion of artefacts were applied .
in the following sections, we discuss the expression of only a subset of drought-regulated genes and gene family members.
we focussed on genes known to be involved in stress-perception, signalling and transcription initiation, because at least some of them are well characterized, and often clear-cut evidence is available for their role in stress-responses  <cit> .
as has been shown for tolerance to salt-stress in rice, these genes or their products regulate early events in drought responses, differentiating between stress-tolerant and -susceptible genotypes  <cit> .
however, genes involved in stress perception and signalling are not necessarily most up- or down-regulated.
stress perception and signalling in drought-stressed chickpea roots
dehydration-related stresses such as drought and salinity have ionic as well as osmotic attributes that elicit signal transduction cascades resulting in activation of effector genes to adapt the metabolism of the plant to the stress.
in the model arising from research in arabidopsis or rice, the first step of signalling is the perception of the stress through g-protein coupled receptors , inositol polyphosphates, or receptor-like kinases .
in drought-stressed chickpea roots,  <dig> rlk transcript variants were detected.
one of these increased in abundance more than 20-fold, whereas fourteen were  <dig> to 8-fold up-regulated under stress, indicating a potential role of these transcripts in stress perception.
calcium transients are major signalling events in plants  <cit> .
thus, entry points and interconnecting links in major stress-related signalling cascades involve ca2+ sensors and proteins regulated by ca2+-concentration gradients between apoplast and cytoplasm  <cit> .
in drought-stressed chickpea roots, supersage revealed moderate to significant changes in expression levels of ca2+-responsive genes.
transcript isoforms encoding ca2+ sensors , ca2+-channel proteins, and inositol-3-phosphate -gated ca2+-release , were generally up-regulated .
in contrast, transcript isoforms transcribed from genes involved in downstream events like fine-tuning of, and interconnecting between, signalling cascades were up- as well as down-regulated in reaction to the stress.
these included transcripts encoding a wide range of kinases such as ca2+-dependent protein kinases , calcineurin-b-interacting protein kinases ., and protein phosphatases class 2c.
since the interplay between kinases and phosphatases balances activation and inactivation of proteins and with it the cross-talk between signaling cascades and metabolic pathways  <cit> , measurement of transcriptional activity of kinase-encoding genes is important for understanding drought-response homoeostasis.
mitogen-activated protein kinases , however, do not seem to interact much with early drought-stress signalling in chickpea roots, since of  <dig> mapk transcripts detected,  <dig> were down-regulated more than 8-fold, and  <dig> similarly expressed as in control roots.
in addition, the only mpkk detected was constitutively expressed, whereas from three  <dig> bp tags annotated to mpkkks, two were down-regulated at least 4-fold , and one transcript was up-regulated at least 6-fold .
regulation of 14-3- <dig> transcripts
signal transduction and regulation of metabolism achieved via phosphorylation-mediated transition of protein states require that phosphorylated proteins physically interact with specialized adapter proteins to fulfil their regulatory role.
an example for such adapters are the phosphoserine/threonine-binding 14-3- <dig> proteins  <cit> .
for example, 14-3- <dig> proteins are phosphorylation targets for snrk  <dig> , a member of the sucrose non-fermenting-related kinase family, that is down-regulated in plants deprived of nutrients and with reduced growth  <cit> .
plants have large 14-3- <dig> gene families, and various 14-3- <dig> isoforms have varying affinities to target proteins.
in arabidopsis, at least  <dig> expressed members of the 14-3- <dig> gene family exist and exhibit high cell- and tissue-specificity as well as diverse expression levels  <cit> .
in rice, at least four 14-3- <dig> transcript isoforms are induced by drought and salt-stress  <cit> .
in drought-stressed chickpea roots, we detected  <dig> isoforms of 14-3- <dig> protein transcripts.
however, contrary to results from drought-stressed rice, only three of these were up-regulated more than 2-fold, whereas ten were down-regulated more than 4-fold .
similar differential expression of 14-3- <dig> transcript isoforms was detected in young tomato roots under normal growth conditions as well as in response to salt stress and potassium and iron deficiencies, suggesting that especially one isoform  may mediate cross-talk between the salt stress and potassium and iron-deficiency signalling pathways, respectively  <cit> .
transcription factors and involvement of aba in early drought stress responses in chickpea roots
another large and complex class of genes encode transcription factors .
we could identify  <dig> unitags from tf transcripts classified into  <dig> tf families , whereas  <dig> remained un-classified.
the majority of tf-unitags annotated to the bzip tf family , followed by unitags matching to the hdz , hmg , and wrky  tf families.
as described for the bzip type tf family in figure  <dig>  expression profiles of transcription factors-encoding transcripts may display diverse regulation tendencies.
this is also true for unitags derived from transcripts encoding myb family members, though these were reportedly involved in signal transduction under water-deficit  <cit> .
this observation may be related to the constitutive and even slight down-regulation of transcripts for proteins involved in aba synthesis such as 9-cis-epoxy-carotenoid dioxygenase  <cit> , though boominathan and co-authors  <cit>  observed a strong up-regulation of a particular mrna for this enzyme under drought stress, that probably escaped our detection.
the difference in expression of genes involved in aba-dependent signalling such as the isoforms of myb tfs  <cit>  may suggest that, as in maize, aba as signal may be restricted to very specific regions of the root  <cit> .
from six myb transcription factors detected, two were up-regulated, and  <dig> were down-regulated.
of  <dig> members of the bzip tf family  to which areb factors belong  <cit> , four were up-regulated more than 3-, whereas three were down-regulated more than 4-fold.
in contrast, aba-independent signalling seems to prevail: two out of five drought-responsive-element-binding  tf isoforms thought to be regulated independently from aba  <cit>  were at least 3-fold up-regulated .
additionally, two further dreb transcript variants revealed constitutive levels, whereas one was 3-fold down-regulated .
besides dreb <dig> tfs, wrky tfs also seem to be involved in drought-stress responses in chickpea, because of the  <dig> detected family members, two were at least 2-fold , and two 4-fold up-regulated .
also, unitag stca- <dig>  homologous to an alfin-1-like tf transcript from alfalfa, belonged to the  <dig> most up-regulated transcripts under drought stress in chickpea .
in alfalfa, transgenic over-expression of a tf belonging to this class enhances expression of the endogenous msprp <dig> gene and improves salinity tolerance  <cit> .
thus, up-regulation of stca- <dig> indicates the involvement of alfin-1-like tfs in salt- as well as in drought-stress responses.
though already highly informative, our analysis of tf gene expression in drought-stressed chickpea roots certainly tapped the tip of an iceberg only, since we could assign only  <dig> unitags to this class of genes.
in fact, there are more than  <dig> tf genes in the arabidopsis genome  <cit> .
considering that we could assign only 22% of the  <dig> bp tags to well characterized entries in the public data bases, the number of tfs in our data set should be approx.
<dig> to  <dig> times as high as the one we could assign, i.e.
approx.
<dig>  this estimate is in good agreement with results from sugarcane, where  <dig>  ests contained  <dig> tf sequences  <cit> .
ros scavenging and ros-triggered signalling-related genes
plants generate singlet oxygen-, superoxide-, peroxide-, and hydroxyl-radicals  that trigger a wide range of partly genetically fixed responses.
ros are released by either nadph oxidases or peroxidases, that may exist alone or in combination in different plant species.
plant cells perceive changes in the concentrations of ros as second messengers, and transform them into signals that change the transcription of genes  <cit> .
on the other hand, disturbances in metabolism and photosynthesis by environmental stresses lead to ros accumulation, which, if not controlled, can rapidly reach toxic levels in the plant cell  <cit> .
major ros scavenging enzymes include superoxide dismutase , ascorbate peroxidase , mono-dehydroascorbate reductase , dehydroascorbate reductase  <cit> , glutathione-s-transferase   <cit> , glutathione peroxidase , glutathione reductase , and catalase   <cit> .
the balance between the activities of these enzymes could be crucial for determining the steady-state level of ros.
our data reflect the complexity of ros signalling and scavenging.
for example,  <dig> out of  <dig> peroxidase isoforms were significantly up-regulated , but only one nadph oxidase was detected and moderately up-regulated.
however, one transcript each for a hypersensitive-induced response protein and radical-induced cell death 1- <dig> protein were detected, and both were significantly up-regulated.
of the ros scavengers, six out of seven sod transcripts, and one out of two transcripts coding for each catalase and dehydroascorbate reductase were more than 2-fold up-regulated, whereas two of the three ascorbate peroxidase  transcripts were moderately or even significantly down-regulated.
since apx catalyses the h2o2-dependent oxidation of l-ascorbate , this potent ros scavenging mechanism seems to be under-used in early responses of chickpea roots to drought.
considering that stca- <dig> representing an nadp-dependent isocitrate dehydrogenase transcript belonged to the most up-regulated transcripts in our study   <dig> , table 4), scavenging of ros by gluthatione and recycling of oxydized gluthatione by this enzyme seems to prevail instead.
this assumption is supported by the strong up-regulation   <dig> , table 4) of cysteine synthase -encoding unitag stca- <dig>  in rice, as a consequence of cs up-regulation, both the total glutathione pool and reduced glutathione concentration were significantly increased in response to aluminium stress  <cit> .
still another stress-responsive gene family encodes glutathione-s-transferases , key defence enzymes against xenobiotic toxicity, and has at least  <dig> members in rice  <cit> .
from  <dig> chickpea  <dig> bp tags annotated to gsts, four isoforms  were at least 2-fold down-regulated, and three isoforms revealed up-regulation .
apart from ros-scavenging, gsts may also function in stress tolerance through signalling  <cit> .
considering the importance of these proteins for managing ros-related stress, the gst transcript variants strongly up-regulated in chickpea roots under drought stress are potential targets for molecular breeding for drought tolerance.
regulation of aquaporin gene activity under drought stress
despite considerable progress in understanding fundamental stress responses in model plants, we know little about the molecular basis of differences between stress-tolerant and susceptible genotypes of crops.
only recently, investigations into drought responses of upland and lowland rice, and genotypic variation for water status under different water regimes in a population of recombinant inbred lines  of sunflower  <cit>  demonstrated a possible involvement of certain aquaporin genes in differing dehydration-stress response phenotypes of these crops.
for example, the arabidopsis genome harbours at least  <dig> genes coding for different aquaporins, that are differentially expressed under different stress conditions and, whereas one family member is up-, the other may well be down-regulated.
in chickpea roots, we detected at least  <dig> different  <dig> bp tags from aquaporin transcripts, representing the three classes .
interestingly, the over-expression of a certain aquaporin isoform in transgenic arabidopsis resulted in altered expression patterns of other aquaporin isoforms with consequences for seed germination, seedling growth, and stress responses of the plants under various stress conditions  <cit> .
these results suggest a concerted transcriptional regulation of at least a subset of aquaporin genes.
like in arabidopsis, drought also elicited differential responses in the different members of the aquaporin gene family also in chickpea.
it will be interesting to learn, whether the differentially expressed putative nats corresponding to several of the sense aquaporin isoforms detected in chickpea are involved in the regulation of other gene family members.
also, whether differences exist in the expression of aquaporin isoforms between drought-tolerant and -susceptible genotypes of chickpea needs to be determined.
changes in transcription of genes regulating compatible osmolyte accumulation
beyond the broad repertoire of signalling cascades and signalling interactions that plants have on their defence lines against drought stress, the accumulation of sugars, sugar alcohols, amino acids, and polyamines, acting as compatible osmolytes against the osmotic disequilibrium is one of the most widespread strategies of plants to enhance their tolerance against drought stress  <cit> .
several genes involved in biosynthesis, transport as well as intermediate and catabolic pathways related to this strategy have been genetically and functionally characterized during the past years  <cit> .
after screening the c. arietinum unitags database for the behaviour of genes related to the above processes, transcription profiles and number of isoforms from at least  <dig> relevant genes were investigated in detail .
related to sugar accumulation, one unitag annotated to trehalose-6-phosphate synthase , and three  <dig> bp tags annotated to trehalose-6-phosphate phosphatase  were observed.
trehalose plays an important role as compatible osmolyte and signalling molecule under drought stress  <cit> .
however, since we do not observe strong up-regulation of genes encoding threhalose-6-phosphate synthase, we are hesitant to conclude that threhalose accumulated to high concentrations under our assay conditions.
additionally, one significantly up-regulated unitag stca- <dig> representing a galactinol synthase gene as well as at least three tags representing transcripts related to sucrose metabolism and transport with more than 2-fold expression changes  revealed, that the dynamics of sugar metabolism, transport and accumulation could be altered as a response to drought stress in chickpea.
the positive role of galactinol synthase in stress tolerance has already been reported elsewhere  <cit> .
several  <dig> bp tags representing amino acid transport- and accumulation-related genes were detected.
for proline, a compatible osmolyte  <cit> , one 6-fold up-regulated unitag with homology to a proline/betain transporter  as well as moderate down- and up-regulation of two  <dig> bp tags representing a negative regulator for proline accumulation  were revealed.
this suggests that prolin accumulation may occur to some extent under drought stress in chickpea.
the transcript levels for betaine aldehyde dehydrogenase unitag stca- <dig>  the key enzyme for glycine betaine synthesis  <cit> , did not markedly change upon drought stress, indicating that this osmolyte, otherwise reported to accumulate under water stress  <cit> , is not important for an early stress response.
since the accumulation of polyamines as compatible osmolytes is discussed as protection against stress, we paid special attention to transcripts encoding arginine decarboxylase and spermidine synthase, that share important roles in putrescine and spermidine accumulation  <cit> .
the detected transcripts were 2-fold  and 3-fold  up-regulated.
our results indicate a differential influence of drought stress on mechanisms for compatible osmolyte accumulation as an early stress response in chickpea.
comparison of supersage versus macro- and micro-arrays
though drought-stress responses from roots of adult chickpea plants were compared to the roots of much younger seedlings from medicago , the similar expression of several genes  suggests similar reactions of the roots of both legumes to drought stress, independently of the developmental stage of the roots.
genes similarly up-regulated in both species inter alias include a certain extensin isoform, actually the most up-regulated transcript in stressed chickpea roots .
extensins are hydroxyproline-rich proteins strengthening cell walls, and are often activated by mechanical stress  <cit> .
like other proline-rich cell-wall proteins, extensins require hydroxylation of prolines to 4-hydroxyprolines to form the cell wall matrix  <cit> .
consistent with extensive hydroxylation of extensin, unitag stca- <dig> representing a prolyl 4-hydroxylase alpha subunit-like protein belonged to the most up-regulated transcripts in drought-stressed chickpea roots   <dig> ).
at the same time, stca- <dig>  encoding an expansin-like protein, was most down-regulated  - <dig> ) in stressed chickpea roots.
expansins weaken cell walls  <cit> , and thus down-regulation of the expansin gene also would contribute to an increased strength of the cell walls of drought-stressed roots.
in addition, unitag stca- <dig>  representing a gibberellin 2-beta- hydroxylase transcript, belonged to the most up-regulated transcripts in stressed chickpea roots   <dig> , table 4).
the encoded enzyme inactivates gibberellin through β-hydroxylation  <cit>  and thus, its up-regulation should result in significantly deceased levels of bioactive hormone and a reduction of cell divisions and extensions.
we conclude that in chickpea and medicago, drought impairs division and extension of cells and results in growth-retarded roots with strengthened cell walls.
other transcripts similarly up-regulated in both chickpea and medicago inter alias code for dehydrin, dreb, 2c protein phosphatase, udp-glucose phosphorylase and blue copper protein which are discussed elsewhere in this paper.
taken together, a comparison of drought stress responses in chickpea and medicago reveals many inter-species similarities and suggests to exploit the huge resources available for medicago to test the functions of differentially expressed transcripts in chickpea.
CONCLUSIONS
applying supersage to the analysis of abiotic stress responses in chickpea for the first time, our study presents the most comprehensive transcriptome profile of this crop available to date.
it increases the number of chickpea ests from approximately  <dig>  to more than  <dig> , and the number of unique transcripts to more than  <dig> .
the study identified major drought-stress signalling cascades resulting in differential expression of effector genes, and hints to the importance of ros and n starvation as side stresses resulting from drought.
our study revealed, that  genes involved in photosynthesis and energy metabolism were down-regulated,  many genes involved in early responses to biotic and abiotic stresses were up-regulated, while  many other stress-responsive genes were down-regulated, and  regulatory genes encoding e.g.
transcription factors or signal transduction proteins were both up- and down-regulated.
we conclude that follow-up transcription profiling studies of responses to drought in chickpea must take into account the potentially deleterious effect of the stress on snf and thus, on n supply to the plant in order to prevent mixing up responses to different stresses.
one important fact arising from our study is the unexpectedly high number of differentially expressed isoforms of members of large gene families, that was also observed in sage libraries from lotus japonicus, where different levels of transcription induction among leghemoglobin gene paralogs were found  <cit> .
these findings highlight the efficiency of tag-based techniques to discriminate different gene family members.
at the same time, they underpin the necessity to – experimentally and linguistically – distinguish between certain transcript isoforms  rather than summarizing them under a common term.
with this work, we aimed at identifying candidate genes as targets for molecular breeding for drought tolerance in chickpea.
numerous studies confirmed the polygenic nature of drought tolerance, for which single qtls have only little individual effect.
considering the large number of genes located at qtls for drought tolerance and related traits in cereals, comparing our transcription profiles to genes mapped to drought qtls in these crops may help to decide whether supersage has identified such potential breeding targets.

 chickpea is one of the most important grain legume crops worldwide and a major source of protein for millions of families in developing countries. despite considerable investment in breeding, average chickpea yield in major producer countries such as india stagnates at – mt hectare- since many years. this low yield is far below the crop's potential of 3– mt hectar- under optimal conditions. chickpea is mostly grown in low-input, rain-fed agriculture in mediterranean-type environments as inter-crop between cereals, and on residual moisture from monsoon rains on the indian subcontinent. due to insufficient, untimely and erratic rainfall in these semi-arid and arid areas, the crop often suffers from drought at the end of the cropping season. in future, global warming and soil erosion will even worsen conditions for many crops including chickpea. thus, drought represents the major constraint to increase chickpea yield, and drought tolerance therefore is a major aim of chickpea breeding. however, drought tolerance is a complex trait and hard to achieve by conventional breeding methods. understanding of the molecular mechanisms underlying drought tolerance is therefore needed for successful, knowledge-based crop improvement. molecular genetics and genomics of stress-responses in model plants such as arabidopsis revealed that abiotic stresses such as drought, salinity and cold stress are characterized by ionic- and osmotic-disequilibrium components; eliciting general as well as specific responses and mechanisms of stress-protection. these studies underpinned the importance of early responses to the various stresses for the survival of the plants. much of our current understanding of stress-response mechanisms comes from genome-wide analysis of gene expression, facilitated by the availability of microarrays carrying a comprehensive set of genes. in chickpea, like in many other under-researched crops, extensive microarray-based studies are not yet possible, because the necessary est data are not available. for example, no more than chickpea ests are publicly available at the national centre of biotechnology information, as compared to at least, ests available for arabidopsis. in the absence of such resources, researchers often use less comprehensive approaches as e.g. suppression subtractive hybridisation libraries, which do not per se allow the quantification of expression of differentially expressed genes. ssh results are therefore often used for the generation of macro-arrays for subsequent analysis of gene expression. these authors used this cumbersome approach for the discovery of dehydration-responsive transcripts in chickpea roots. open-architecture, whole-genome transcription profiling technologies such as supersage, however, provide a solution to the problem of lacking est and genomic data. supersage is an improved version of the serial analysis of gene expression technique. in the past years, it has demonstrated a high versatility due to its longer tag size. in principle, sage and all its variants rely on the assumption that a small, defined part of a cdna, a so-called "tag", characterizes this cdna, and that counting the number of times a particular tag occurs in the tag population faithfully reflects the abundance of the respective mrna in the transcriptome. since to tags are sequenced in a single experiment, a comprehensive profile of the transcriptome is generated. here we report on the stress responses of transcripts representing unique bp tags from roots of the drought-tolerant chickpea variety icc early after onset of desiccation. we discuss the stress-regulated transcription of genes involved in signal perception and transduction, ros scavenging and metabolism, osmotic and ionic stress-related pathways, regulation of water and ion homeostasis, as well as several reported effector proteins. to test the reliability of the present results we use microarrays carrying stress-responsive as well as constitutively expressed bp tag sequences. to further confirm the supersage results with a third method, we use sybrgreen and commercially available taqman assays produced from 3'-and 5'-race sequences from selected chickpea mrnas. finally, we compare our transcription profiles from drought-stressed chickpea roots to results obtained from chickpea root ssh libraries and microarray experiments in medicago truncatula, and discuss similarities and differences. this study is the first of a series characterizing stress responses of chickpea on a molecular level as a prerequisite for production of expression markers and microrrays for high-throughput germplasm and expression qtl analysis at the onset of knowledge-based breeding for stress-tolerance in this important protein crop.  the combination of high-throughput sequencing with supersage drought libraries are part of a project, which aims at evaluating the transcriptional responses of chickpea upon diverse abiotic stresses, including several other treatments and various tissues. for the sequencing of all the libraries, a single plate divided into two sections was used, from which a total of reads were extracted. after eliminating: i) incomplete reads, ii) twin-ditags, and iii) ditags without complete library-identification dna linkers, a total of bp tags were obtained for further analysis. from these, about tags were singletons, that were excluded from analysis. finally, tags remained for evaluation. subsequently, sub-datasets were constructed for each experimental situation. for the present study, bp tags from control and dehydrated roots were analyzed. abundance of unitags and annotation to public databases a total of bp tags from roots of the drought-tolerant variety icc either subjected to h desiccation or from well-watered controls were sequenced, and represented unique transcripts, so called unitags. less than 1% percent of these occurred in very high copy numbers, whereas 23% and 75% of the transcripts were present between to and less than copies.million- respectively. unitags from control and stress libraries were deposited in the gene expression omnibus public domain under accessions gsm and gsm respectively. * ratio values indicating up- or down-regulation calculated with the h desiccation library as reference. annotation of the unitags matched to well characterized sequences from the fabaceae family available in public databases. of these, 53% matched to sequences from medicago truncatula, 29% to cicer arietinum, 6% to pisum sativum, 3% to glycine sp., 2% to medicago sativa, and 7% to other legume genera. in many cases, tigr gene index annotations from legumes bridge automatically to characterized tentative consensus sequences mostly from arabidopsis, rice, and maize. of the remaining non-assignable bp tags, were significantly homologous to anonymous est or dna sequences, whereas found no match at all. a summary of the primary data is given in table annotation of the bp tags and respective expression ratio values are deposited in the main data matrix. annotation of virtual tags generated from chickpea ests deposited in public domains in order to test the validity of the annotation of chickpea pb tags through sequence homology with other legumes, virtual tags generated from chickpea est sequences deposited in the ncbi data bank were extracted, and their direct annotation was compared with the annotation of longer homologous ests from the model legume m. truncatula. after retrieving the complete set of chickpea sequences deposited in the ncbi est database, a total of different in silico-generated bp tags were selected to be directly blasted against the nr ncbi nucleotide database. from these, a total of tags revealed high homology hits. after exclusion of anonymous entries, tags were linked to uniprot entries or to characterized non-protein coding rnas. in parallel, the same tags were blasted against the plant est ncbi and tigr databases separately, where and sequences, respectively, found at least one high homology hit. then, the complete target sequences from each blast were retrieved and re-blasted against the nr ncbi database. a total of and sequences, each representing a different bp tag, revealed high homologies with nr ncbi entries. from these, and respectively, were non anonymous. the results from the direct blast were compared with each of the m. truncatula est-briged homology searches for commonly annotated sequences, either by direct id matching, protein name, or sequence description. for m. truncatula ests from ncbi, out of annotations were not congruent with the annotation of the corresponding chickpea in silico tag. however, only out of the annotations belonged to uniprot-linked accessions. for m. truncatula ests from tigr, out of common annotations were not congruent. from them, belonged to uniprot-linked accessions. the present results reveal that in more than 90% of the cases, the assignment of tags to uniprot-linked accessions is congruent with the annotation of longer ests from other legumes. however, databases overloaded with anonymous entries considerably reduce the amount of biologically interpretable data. differential gene expression in response to drought stress of chickpea roots and assignment of bp tags to gene ontology functional categories we calculated the natural logarithm of expression ratios of the bp tags from control versus stressed roots as well as significance levels according to audic and claverie of up- and down-regulation for each transcript using the software package discoveryspace. differences in abundance of tags in control and stressed roots were considered relevant at r >. at this threshold, tags were significantly differentially expressed in stressed as compared to control roots. of these, were up-, and down-regulated. a total of transcripts showed more than 8-fold difference in expression >, p < ). of these, were up-, and down-regulated under stress. as depicted in the venn diagram, a considerable number of tags occurred exclusively in either the control tissue or under stress. the most significantly up- or down-regulated transcripts matching well characterized genes in public databases are listed in table and table respectively. however, many transcripts could not be annotated, because they either matched to anonymous ests, or did not hit any entry in the database. therefore, the annotatable transcripts coding for extensin, nadp-dependent isocitrate dehydrogenase i, s-receptor kinase-like protein, chalcone isomerase, udp-glucose-pyrophosphorylase, beta-glucosidase, specific tissue protein, s-adenosyl-l-methionine synthetase, and cysteine synthase were the most up-regulated interpretable transcripts under stress though not the most up-regulated in the data set. correlation of r to defined standard functional gene categories in the gene ontology database revealed that the majority of the most up-regulated transcripts are assigned to the go: biological process "metabolism". as depicted in figure stress perception and signalling, small gtpase-mediated signal transduction, rna metabolism, and cellular carbohydrate metabolism were the most represented go: functional categories in desiccation-stressed roots. further, over-representation of transcripts involved in transport, proteolysis, oxidative phosphorylation, and stress response indicate the mechanisms by which the roots adapt to the stress. over-representation of transcripts involved in go: functional category "oxygen and reactive oxygen species metabolism" suggests that ros play an important role as side stress, but also as signalling molecules. tags from transcripts assigned to the go: biological process "protein biosynthesis", such as 40s ribosomal proteins s and s, and 60s ribosomal proteins l, l, and l, all were significantly down-regulated suggesting a repression of de novo protein biosynthesis under drought stress in roots. additionally, other go: biological processes like photosynthesis and light reaction, chromatin assembly, chromosome organization and biogenesis, biopolymer metabolism, dna replication were also under-represented. regarding go: cellular component categories, "unlocalized protein complexes" were most over-represented under drought stress, followed by golgi apparatus, endoplasmic reticulum, membrane integral genes and external encapsulating structure. go: cellular components such as "ubiquitin ligase complex", mitochondrial and inner membrane, and ribosome were amongst the most under-represented. the transcription of genes coding for proteins of the serine/threonine phosphatase complex, cytoplasm, cytosol, cytoskeleton, thylakoid, microtubule cytoskeleton, cell-wall structure and modification as well as cell surface protein was constitutive. members of a gene family assigned to over- or under-represented go: categories, respectively, could be up- as well as down-regulated to different extents. for example, whereas unitagstca- annotated to the putative extensin, was one of the most down-regulated transcripts, unitag stca- representing another extensin isoform was the most up-regulated tag in our data set. however, both isoforms are grouped in the same go:category. differential expression of drought stress-related sub-transcriptomes in chickpea roots since the present genome-wide expression analysis revealed a plethora of differentially expressed bp tags with and without match to genes of known function, it is impossible to display or discuss all of them in the frame of this paper. instead, in figure and figure we elaborate in more detail on the expression of genes and gene families belonging to the go: biological processes "signal transduction", "stress sensing", "regulation of transcription", "transport", "post-transcriptional regulation" and "pathway inhibitors", all involved in early responses to stress. in these categories, the gene itself or members of its family have known functions in stress-perception, stress-signalling and stress-responsive regulation of transcription and chromatin structure. further, we closely look at transcripts encoding proteins involved in reactive oxygen species scavenging, and transcripts related to ros-mediated signal transduction cascades. as examples for the regulation of effector genes down-stream of the signalling cascades, we will detail the differential expression of genes coding for water-channel proteins, so called aquaporins, and of genes coding for proteins involved in osmolyte metabolism. confirmation of supersage results by qrt-pcr our genome-wide expression analysis results were exemplarily confirmed by quantitative real-time pcr using taqman probes xptm-ca- and xptm-ca- available from genxpro gmbh, as well as sybr green® assays. oligonucleotides for sybr green assays were deduced from 5'- and 3'-race sequences generated with bp tags as primers. the following transcripts were targeted: st-ca, st-ca, st-ca, st-ca, st-ca, and st-ca. for sybr green® as well as taqman assays, the sequence for either the forward or the reverse pcr primer was derived from the bp tag, and the complementary primers from 3'- or 5'-race sequences, respectively. confirming the supersage expression levels, amplifications from sybr green® assay st-ca and taqman probe xptm-ca revealed constitutive levels of expression. amplifications for sybr green® assays with st-ca st-ca st-ca as well as taqman probe xptm-ca- revealed up-regulation of the respective transcripts under stress, as already indicated by the differential expression analysis. stress-induced down-regulation of bp tags was corroborated by sybr green® assays for st-ca and st-ca. however, for st-ca amplification profiles as well as post-qrt-pcr amplicon melting curves suggested partially unspecific priming. again, in agreement with the supersage results, the transcript chosen as invariable control indeed displayed almost completely similar expression in control and stressed roots. these experiments confirm our present results and suggest bp tags as reliable sequence information source for other expression profiling techniques. profiles confirmation via microarray hybridization of spotted supersage-derived oligos to characterize the chickpea transcriptome under drought stress, sequence information from supersage profiles was used to design an agilent k microarray, onto which selected pb tags were spotted for a comparison of both profiling techniques. on the microarray, the majority of the oligonucleotides with original tag sequences were spotted twice. additionally, oligonucleotides with different mismatch numbers from each original tag as well as a small sub-set of longer race-derived sequences were also included. after statistical treatment of the different internal twin-replicas, normalization, and mismatch background correction, reproducible signals from different hybridization rounds were selected for cluster analyses together with expression ratios of the bp tags. a total of % probes on the micro-array shared the same tendency of expression with the respective tag when hybridized with crnas prepared from drought-treated roots from icc from a total of spots showing congruent results among different replicas and dye-swap experiments, and were up- and down-regulated in both techniques, respectively, whereas spots showed in-congruent results. unitag expression ratios from both techniques are deposited in the main data matrix. *regulation tendencies for salt-stressed roots in both techniques are included for comparative purposes comparison of different transcript profiling techniques: supersage versus ssh macroarrays and microarrays we tested, whether our genome-wide transcription patterns are typical for roots of chickpea and other legumes, even if the profiles were obtained with other methods. therefore we compared the present results to two studies addressing similar questions. in chickpea, boominathan and co-workers investigated whether pre-exposure to a dehydration shock improved adaptive responses of the roots during subsequent dehydration treatment. these authors identified dehydration-inducible transcripts by repetitive rounds of cdna subtraction, differential dna-array hybridization, and northern-blot analysis. additionally, responses to exogenously applied abscisic acid were also monitored. since one analyzed time point was set h after onset of drought, the results of this experiment should be at least partially comparable to our study in which rna was isolated from roots h after onset of drought stress. however, the results were not 100% congruent. it is important to note, that micro- or macro-arrays do not reliably differentiate between different transcript isoforms from gene families. hybridization signals may integrate the hybridization intensities over all closely related transcripts, whereas supersage generates absolute numbers for each transcript variant. due to the much deeper coverage of the transcriptome by this technique, almost all differentially expressed transcripts represented in the ssh libraries have at least one, but usually more counterparts in our bp tags libraries. a cluster analysis of transcription profiles obtained by macroarray hybridization and the differential expression of bp tags from drought-stressed roots h or h after onset of the stress, respectively, is shown in figure similarly up-regulated and differentially regulated transcripts under both conditions are listed in table comparison between supersage expression profiles and macroarray-generated data. three main categories from the cluster analysis in figure are detailed below. section a: constitutive tags versus upregulated transcripts on the macroarray. section b, d: non-corresponding supersage and macroarray transcript profiles. section c: up-regulated tags and up-regulated transcripts on the macroarray. investigating regulatory and protective mechanisms leading to desiccation tolerance in medicago truncatula seeds, buitink and co-workers published another, at least partially comparable study. these authors used the k medicago microarray to monitor changes in the transcriptome of desiccation-sensitive 3-mm-long radicles at different time points during incubation in a polyethylene glycol solution mimicking the effects of desiccation. these experiments identified several specific expression profiles at different time scales. a cluster analysis comparing the results from desiccation-stressed medicago radicles and drought-stressed chickpea roots is depicted in figure transcripts up-regulated in both species, regulated in opposite directions in the two species, and transcripts down-regulated in chickpea and medicago as a reaction to stress are listed in table comparison between supersage expression profiles and 16k-microarray-generated expression data. four main categories from the cluster analysis in figure are detailed below. section a: down-regulated tags and down-regulated transcripts on the microarray. section b: contrasting supersage and microarray expression profiles section c: contrasting supersage and microarray expression profiles section d: up-regulated or constitutive tags and 16k-microarray transcripts, respectively differential expression of natural antisense transcripts as expected from an open-architecture technique, tags synthesized on coding strands of template cdnas and, in addition, tags originating from the opposite strands were discovered. here, at least tags matching to the reverse sequences of ests in the public databases were detected. these tags could represent potential natural antisense transcripts. in lotus japonicus, sage tags representing nats were induced during nodulation, and in arabidopsis endogenous sirnas derived from a pair of natural cis-antisense transcripts regulate salt tolerance. thus, it is possible that nats are also involved in stress tolerance in chickpea. however, galante and co-authors demonstrated that a considerable portion of such nats found in the databases are artefacts. in drought-stressed chickpea roots, we found – inter alias – up- and down-regulation of nats for transcripts of several members of the aquaporin gene family and differential expression of nats for phosphatase transcripts. however, since we did not exclude genomic priming by dnase digestion of the rna prior to cdna synthesis, confirmation of the identity of these potential nats will be subject to further studies. discussion we analysed the expression of approximately transcripts from un-stressed control and drought-stressed chickpea roots, respectively. it was previously estimated that the total number of average-sized transcripts per cell in higher plants ranges from to. thus, bp tags provide ~1-fold coverage for transcripts present at a minimum of copies per cell. therefore our study, which detected > unique transcripts, is not comprehensive. however, even for the model legume medicago truncatula only unique ests/tcs are deposited in public databases. therefore, our single study demonstrates that supersage is suited to overcome the problem of lacking resources in non-model organisms and under-researched crops. since, as we have shown, 75% of transcripts are present in less than copies million- and a small portion of transcripts is represented by more than copies million- we probably missed a number of transcripts with less than 10– copies per cell. these may include transcripts for highly interesting pleiotropic proteins such as transcription factors possibly present at only copies per cell. additionally, sampling larger organ sections with millions of cells obscures the specific transcript profiles of component cells. as a consequence, transcripts found in high copy numbers in just a few specialized cells are under-represented. as elegantly demonstrated for maize and arabidopsis, there are strong differences in transcription profiles between adjacent tissues. therefore, the detection of rare transcripts should be improved by increasing the number of sequenced transcripts in combination with a more selective sampling technique such as laser-capture microdissection. also, annotation of 55% of the bp tags to the databases was straightforward. considering that roughly chickpea est sequences are publicly available, most tags matched to sequences from the related model legume medicago truncatula rather than to chickpea ests. on the other hand, a large number of significant hits represented fully uncharacterized database accessions, a fact that handicaps functional interpretation of the present chickpea transcription profiles more than the size of the bp tags. additionally, a relevant problem arose from the interpretation of the assignment of different distinct 26bp tags to single unigenes or tentative consensus sequences. we decided to classify such tags as isoforms of transcripts from members of gene families rather than assigning them to a particular gene, even if the expression patterns suggested functional homology. in addition, we could not differentiate whether these tags with similar annotation came from alternatively spliced transcripts of the same gene. since in arabidopsis and rice at least 21% of genes produce alternatively spliced transcripts, evidence beyond sequence homology of the supersage tags is needed to decide between these two possibilities. differential expression of members of large gene families one major advantage of the differential expression of 26bp tags as compared to macro- and micro-arrays is the very good differentiation between transcripts from different members of large genes families, which comprise the majority of all genes. gene family members have more or less conserved sequences, and similar or different functions. for example, the cytochrome p gene superfamily comprises more than members grouped in families in arabidopsis thaliana, and more than members in rice. in the present study, 26bp tags revealed high-homology hits with members of the cyp superfamily, from which could be assigned to their respective family through blasting against the arabidopsis-cyp database. the involvement of cyp superfamily members in numerous catalytic reactions on a spectrum of substrates suggests that the transcription profiles of its members reflect this diversity. in accordance with the expectation, of the cyp-annotated 26bp tags changed their expression profiles at least -fold with r >, whereas showed only slight differences or constitutive levels. among the 26bp tags assigned to drought stress-related cyps, hits to cyp707a family which harbours aba 8'-hydroxylases, revealed a very slight up-regulation, indicating that the turnover of aba is already active under our experimental condition and time-points. unitag stca- assigned to a cyp81f member, stands out for its extreme down-regulation. although its function is not known, cyp81f members generally change their expression profiles dynamically after induction of water stress. considering the differential expression and diverse functions of individual members of gene families, whole-genome transcription profiling is only useful, if it differentiates between the different genes and their transcript isoforms in such families. faithful discrimination between, and individual quantification of expression of these isoforms is therefore one advantage of the longer bp tags. however, also methodological inconsistencies may result in the observation of several different tags from a single gene. to prevent methodological artefacts and assure the validity of the detected transcript variants, confirmative procedures such as double nlaiii digestion were standard in all libraries. additionally, in silico routines for the exclusion of artefacts were applied. in the following sections, we discuss the expression of only a subset of drought-regulated genes and gene family members. we focussed on genes known to be involved in stress-perception, signalling and transcription initiation, because at least some of them are well characterized, and often clear-cut evidence is available for their role in stress-responses. as has been shown for tolerance to salt-stress in rice, these genes or their products regulate early events in drought responses, differentiating between stress-tolerant and -susceptible genotypes. however, genes involved in stress perception and signalling are not necessarily most up- or down-regulated. stress perception and signalling in drought-stressed chickpea roots dehydration-related stresses such as drought and salinity have ionic as well as osmotic attributes that elicit signal transduction cascades resulting in activation of effector genes to adapt the metabolism of the plant to the stress. in the model arising from research in arabidopsis or rice, the first step of signalling is the perception of the stress through g-protein coupled receptors, inositol polyphosphates, or receptor-like kinases. in drought-stressed chickpea roots, rlk transcript variants were detected. one of these increased in abundance more than 20-fold, whereas fourteen were to 8-fold up-regulated under stress, indicating a potential role of these transcripts in stress perception. calcium transients are major signalling events in plants. thus, entry points and interconnecting links in major stress-related signalling cascades involve ca2+ sensors and proteins regulated by ca2+-concentration gradients between apoplast and cytoplasm. in drought-stressed chickpea roots, supersage revealed moderate to significant changes in expression levels of ca2+-responsive genes. transcript isoforms encoding ca2+ sensors, ca2+-channel proteins, and inositol-3-phosphate -gated ca2+-release, were generally up-regulated. in contrast, transcript isoforms transcribed from genes involved in downstream events like fine-tuning of, and interconnecting between, signalling cascades were up- as well as down-regulated in reaction to the stress. these included transcripts encoding a wide range of kinases such as ca2+-dependent protein kinases, calcineurin-b-interacting protein kinases., and protein phosphatases class 2c. since the interplay between kinases and phosphatases balances activation and inactivation of proteins and with it the cross-talk between signaling cascades and metabolic pathways, measurement of transcriptional activity of kinase-encoding genes is important for understanding drought-response homoeostasis. mitogen-activated protein kinases, however, do not seem to interact much with early drought-stress signalling in chickpea roots, since of mapk transcripts detected, were down-regulated more than 8-fold, and similarly expressed as in control roots. in addition, the only mpkk detected was constitutively expressed, whereas from three bp tags annotated to mpkkks, two were down-regulated at least 4-fold, and one transcript was up-regulated at least 6-fold. regulation of 14-3- transcripts signal transduction and regulation of metabolism achieved via phosphorylation-mediated transition of protein states require that phosphorylated proteins physically interact with specialized adapter proteins to fulfil their regulatory role. an example for such adapters are the phosphoserine/threonine-binding 14-3- proteins. for example, 14-3- proteins are phosphorylation targets for snrk, a member of the sucrose non-fermenting-related kinase family, that is down-regulated in plants deprived of nutrients and with reduced growth. plants have large 14-3- gene families, and various 14-3- isoforms have varying affinities to target proteins. in arabidopsis, at least expressed members of the 14-3- gene family exist and exhibit high cell- and tissue-specificity as well as diverse expression levels. in rice, at least four 14-3- transcript isoforms are induced by drought and salt-stress. in drought-stressed chickpea roots, we detected isoforms of 14-3- protein transcripts. however, contrary to results from drought-stressed rice, only three of these were up-regulated more than 2-fold, whereas ten were down-regulated more than 4-fold. similar differential expression of 14-3- transcript isoforms was detected in young tomato roots under normal growth conditions as well as in response to salt stress and potassium and iron deficiencies, suggesting that especially one isoform may mediate cross-talk between the salt stress and potassium and iron-deficiency signalling pathways, respectively. transcription factors and involvement of aba in early drought stress responses in chickpea roots another large and complex class of genes encode transcription factors. we could identify unitags from tf transcripts classified into tf families, whereas remained un-classified. the majority of tf-unitags annotated to the bzip tf family, followed by unitags matching to the hdz, hmg, and wrky tf families. as described for the bzip type tf family in figure expression profiles of transcription factors-encoding transcripts may display diverse regulation tendencies. this is also true for unitags derived from transcripts encoding myb family members, though these were reportedly involved in signal transduction under water-deficit. this observation may be related to the constitutive and even slight down-regulation of transcripts for proteins involved in aba synthesis such as 9-cis-epoxy-carotenoid dioxygenase, though boominathan and co-authors observed a strong up-regulation of a particular mrna for this enzyme under drought stress, that probably escaped our detection. the difference in expression of genes involved in aba-dependent signalling such as the isoforms of myb tfs may suggest that, as in maize, aba as signal may be restricted to very specific regions of the root. from six myb transcription factors detected, two were up-regulated, and were down-regulated. of members of the bzip tf family to which areb factors belong, four were up-regulated more than 3-, whereas three were down-regulated more than 4-fold. in contrast, aba-independent signalling seems to prevail: two out of five drought-responsive-element-binding tf isoforms thought to be regulated independently from aba were at least 3-fold up-regulated. additionally, two further dreb transcript variants revealed constitutive levels, whereas one was 3-fold down-regulated. besides dreb tfs, wrky tfs also seem to be involved in drought-stress responses in chickpea, because of the detected family members, two were at least 2-fold, and two 4-fold up-regulated. also, unitag stca- homologous to an alfin-1-like tf transcript from alfalfa, belonged to the most up-regulated transcripts under drought stress in chickpea. in alfalfa, transgenic over-expression of a tf belonging to this class enhances expression of the endogenous msprp gene and improves salinity tolerance. thus, up-regulation of stca- indicates the involvement of alfin-1-like tfs in salt- as well as in drought-stress responses. though already highly informative, our analysis of tf gene expression in drought-stressed chickpea roots certainly tapped the tip of an iceberg only, since we could assign only unitags to this class of genes. in fact, there are more than tf genes in the arabidopsis genome. considering that we could assign only 22% of the bp tags to well characterized entries in the public data bases, the number of tfs in our data set should be approx. to times as high as the one we could assign, i.e. approx. this estimate is in good agreement with results from sugarcane, where ests contained tf sequences. ros scavenging and ros-triggered signalling-related genes plants generate singlet oxygen-, superoxide-, peroxide-, and hydroxyl-radicals that trigger a wide range of partly genetically fixed responses. ros are released by either nadph oxidases or peroxidases, that may exist alone or in combination in different plant species. plant cells perceive changes in the concentrations of ros as second messengers, and transform them into signals that change the transcription of genes. on the other hand, disturbances in metabolism and photosynthesis by environmental stresses lead to ros accumulation, which, if not controlled, can rapidly reach toxic levels in the plant cell. major ros scavenging enzymes include superoxide dismutase, ascorbate peroxidase, mono-dehydroascorbate reductase, dehydroascorbate reductase, glutathione-s-transferase, glutathione peroxidase, glutathione reductase, and catalase. the balance between the activities of these enzymes could be crucial for determining the steady-state level of ros. our data reflect the complexity of ros signalling and scavenging. for example, out of peroxidase isoforms were significantly up-regulated, but only one nadph oxidase was detected and moderately up-regulated. however, one transcript each for a hypersensitive-induced response protein and radical-induced cell death 1- protein were detected, and both were significantly up-regulated. of the ros scavengers, six out of seven sod transcripts, and one out of two transcripts coding for each catalase and dehydroascorbate reductase were more than 2-fold up-regulated, whereas two of the three ascorbate peroxidase transcripts were moderately or even significantly down-regulated. since apx catalyses the h2o2-dependent oxidation of l-ascorbate, this potent ros scavenging mechanism seems to be under-used in early responses of chickpea roots to drought. considering that stca- representing an nadp-dependent isocitrate dehydrogenase transcript belonged to the most up-regulated transcripts in our study, table 4), scavenging of ros by gluthatione and recycling of oxydized gluthatione by this enzyme seems to prevail instead. this assumption is supported by the strong up-regulation, table 4) of cysteine synthase -encoding unitag stca- in rice, as a consequence of cs up-regulation, both the total glutathione pool and reduced glutathione concentration were significantly increased in response to aluminium stress. still another stress-responsive gene family encodes glutathione-s-transferases, key defence enzymes against xenobiotic toxicity, and has at least members in rice. from chickpea bp tags annotated to gsts, four isoforms were at least 2-fold down-regulated, and three isoforms revealed up-regulation. apart from ros-scavenging, gsts may also function in stress tolerance through signalling. considering the importance of these proteins for managing ros-related stress, the gst transcript variants strongly up-regulated in chickpea roots under drought stress are potential targets for molecular breeding for drought tolerance. regulation of aquaporin gene activity under drought stress despite considerable progress in understanding fundamental stress responses in model plants, we know little about the molecular basis of differences between stress-tolerant and susceptible genotypes of crops. only recently, investigations into drought responses of upland and lowland rice, and genotypic variation for water status under different water regimes in a population of recombinant inbred lines of sunflower demonstrated a possible involvement of certain aquaporin genes in differing dehydration-stress response phenotypes of these crops. for example, the arabidopsis genome harbours at least genes coding for different aquaporins, that are differentially expressed under different stress conditions and, whereas one family member is up-, the other may well be down-regulated. in chickpea roots, we detected at least different bp tags from aquaporin transcripts, representing the three classes. interestingly, the over-expression of a certain aquaporin isoform in transgenic arabidopsis resulted in altered expression patterns of other aquaporin isoforms with consequences for seed germination, seedling growth, and stress responses of the plants under various stress conditions. these results suggest a concerted transcriptional regulation of at least a subset of aquaporin genes. like in arabidopsis, drought also elicited differential responses in the different members of the aquaporin gene family also in chickpea. it will be interesting to learn, whether the differentially expressed putative nats corresponding to several of the sense aquaporin isoforms detected in chickpea are involved in the regulation of other gene family members. also, whether differences exist in the expression of aquaporin isoforms between drought-tolerant and -susceptible genotypes of chickpea needs to be determined. changes in transcription of genes regulating compatible osmolyte accumulation beyond the broad repertoire of signalling cascades and signalling interactions that plants have on their defence lines against drought stress, the accumulation of sugars, sugar alcohols, amino acids, and polyamines, acting as compatible osmolytes against the osmotic disequilibrium is one of the most widespread strategies of plants to enhance their tolerance against drought stress. several genes involved in biosynthesis, transport as well as intermediate and catabolic pathways related to this strategy have been genetically and functionally characterized during the past years. after screening the c. arietinum unitags database for the behaviour of genes related to the above processes, transcription profiles and number of isoforms from at least relevant genes were investigated in detail. related to sugar accumulation, one unitag annotated to trehalose-6-phosphate synthase, and three bp tags annotated to trehalose-6-phosphate phosphatase were observed. trehalose plays an important role as compatible osmolyte and signalling molecule under drought stress. however, since we do not observe strong up-regulation of genes encoding threhalose-6-phosphate synthase, we are hesitant to conclude that threhalose accumulated to high concentrations under our assay conditions. additionally, one significantly up-regulated unitag stca- representing a galactinol synthase gene as well as at least three tags representing transcripts related to sucrose metabolism and transport with more than 2-fold expression changes revealed, that the dynamics of sugar metabolism, transport and accumulation could be altered as a response to drought stress in chickpea. the positive role of galactinol synthase in stress tolerance has already been reported elsewhere. several bp tags representing amino acid transport- and accumulation-related genes were detected. for proline, a compatible osmolyte, one 6-fold up-regulated unitag with homology to a proline/betain transporter as well as moderate down- and up-regulation of two bp tags representing a negative regulator for proline accumulation were revealed. this suggests that prolin accumulation may occur to some extent under drought stress in chickpea. the transcript levels for betaine aldehyde dehydrogenase unitag stca- the key enzyme for glycine betaine synthesis, did not markedly change upon drought stress, indicating that this osmolyte, otherwise reported to accumulate under water stress, is not important for an early stress response. since the accumulation of polyamines as compatible osmolytes is discussed as protection against stress, we paid special attention to transcripts encoding arginine decarboxylase and spermidine synthase, that share important roles in putrescine and spermidine accumulation. the detected transcripts were 2-fold and 3-fold up-regulated. our results indicate a differential influence of drought stress on mechanisms for compatible osmolyte accumulation as an early stress response in chickpea. comparison of supersage versus macro- and micro-arrays though drought-stress responses from roots of adult chickpea plants were compared to the roots of much younger seedlings from medicago, the similar expression of several genes suggests similar reactions of the roots of both legumes to drought stress, independently of the developmental stage of the roots. genes similarly up-regulated in both species inter alias include a certain extensin isoform, actually the most up-regulated transcript in stressed chickpea roots. extensins are hydroxyproline-rich proteins strengthening cell walls, and are often activated by mechanical stress. like other proline-rich cell-wall proteins, extensins require hydroxylation of prolines to 4-hydroxyprolines to form the cell wall matrix. consistent with extensive hydroxylation of extensin, unitag stca- representing a prolyl 4-hydroxylase alpha subunit-like protein belonged to the most up-regulated transcripts in drought-stressed chickpea roots ). at the same time, stca- encoding an expansin-like protein, was most down-regulated - ) in stressed chickpea roots. expansins weaken cell walls, and thus down-regulation of the expansin gene also would contribute to an increased strength of the cell walls of drought-stressed roots. in addition, unitag stca- representing a gibberellin 2-beta- hydroxylase transcript, belonged to the most up-regulated transcripts in stressed chickpea roots, table 4). the encoded enzyme inactivates gibberellin through β-hydroxylation and thus, its up-regulation should result in significantly deceased levels of bioactive hormone and a reduction of cell divisions and extensions. we conclude that in chickpea and medicago, drought impairs division and extension of cells and results in growth-retarded roots with strengthened cell walls. other transcripts similarly up-regulated in both chickpea and medicago inter alias code for dehydrin, dreb, 2c protein phosphatase, udp-glucose phosphorylase and blue copper protein which are discussed elsewhere in this paper. taken together, a comparison of drought stress responses in chickpea and medicago reveals many inter-species similarities and suggests to exploit the huge resources available for medicago to test the functions of differentially expressed transcripts in chickpea.  applying supersage to the analysis of abiotic stress responses in chickpea for the first time, our study presents the most comprehensive transcriptome profile of this crop available to date. it increases the number of chickpea ests from approximately to more than, and the number of unique transcripts to more than. the study identified major drought-stress signalling cascades resulting in differential expression of effector genes, and hints to the importance of ros and n starvation as side stresses resulting from drought. our study revealed, that genes involved in photosynthesis and energy metabolism were down-regulated, many genes involved in early responses to biotic and abiotic stresses were up-regulated, while many other stress-responsive genes were down-regulated, and regulatory genes encoding e.g. transcription factors or signal transduction proteins were both up- and down-regulated. we conclude that follow-up transcription profiling studies of responses to drought in chickpea must take into account the potentially deleterious effect of the stress on snf and thus, on n supply to the plant in order to prevent mixing up responses to different stresses. one important fact arising from our study is the unexpectedly high number of differentially expressed isoforms of members of large gene families, that was also observed in sage libraries from lotus japonicus, where different levels of transcription induction among leghemoglobin gene paralogs were found. these findings highlight the efficiency of tag-based techniques to discriminate different gene family members. at the same time, they underpin the necessity to – experimentally and linguistically – distinguish between certain transcript isoforms rather than summarizing them under a common term. with this work, we aimed at identifying candidate genes as targets for molecular breeding for drought tolerance in chickpea. numerous studies confirmed the polygenic nature of drought tolerance, for which single qtls have only little individual effect. considering the large number of genes located at qtls for drought tolerance and related traits in cereals, comparing our transcription profiles to genes mapped to drought qtls in these crops may help to decide whether supersage has identified such potential breeding targets. 
certain transcript isoforms characterizing these processes are potential targets for breeding for drought tolerance. the chip data confirmed 79% of the tag-based results, whereas rt-pcr confirmed the supersage data in all cases. it demonstrates that – inter alias – signal transduction, transcription regulation, osmolyte accumulation, and ros scavenging undergo strong transcriptional remodelling in chickpea roots already h after drought stress. further, supersage tags may be immediately used to produce microarrays and probes for real-time-pcr, thereby overcoming the lack of genomic tools in non-model organisms. supersage, an improved version of the serial analysis of gene expression technique, generating genome-wide, high-quality transcription profiles from any eukaryote, has been employed in the present study. to this end, we sequenced bp tags representing unique transcripts from drought-stressed and non-stressed control roots. we applied supersage to the analysis of gene expression in chickpea roots in response to drought. their large size enabled the unambiguous annotation of unitags to genes or proteins in public data bases and thus to stress-response processes. improving drought tolerance is therefore of outmost importance for breeding. drought is the major constraint to increase yield in chickpea.
 drought is the major constraint to increase yield in chickpea. improving drought tolerance is therefore of outmost importance for breeding. however, the complexity of the trait allowed only marginal progress. a solution to the current stagnation is expected from innovative molecular tools such as transcriptome analyses providing insight into stress-related gene activity, which combined with molecular markers and expression qtl mapping, may accelerate knowledge-based breeding. supersage, an improved version of the serial analysis of gene expression technique, generating genome-wide, high-quality transcription profiles from any eukaryote, has been employed in the present study. the method produces bp long fragments from defined positions in cdnas, providing sufficient sequence information to unambiguously characterize the mrnas. further, supersage tags may be immediately used to produce microarrays and probes for real-time-pcr, thereby overcoming the lack of genomic tools in non-model organisms.  we applied supersage to the analysis of gene expression in chickpea roots in response to drought. to this end, we sequenced bp tags representing unique transcripts from drought-stressed and non-stressed control roots. a total of unitags were more than -fold differentially expressed, and were regulated more than 8-fold upon stress. their large size enabled the unambiguous annotation of unitags to genes or proteins in public data bases and thus to stress-response processes. we designed a microarray carrying of these bp tags. the chip data confirmed 79% of the tag-based results, whereas rt-pcr confirmed the supersage data in all cases.  this study represents the most comprehensive analysis of the drought-response transcriptome of chickpea available to date. it demonstrates that – inter alias – signal transduction, transcription regulation, osmolyte accumulation, and ros scavenging undergo strong transcriptional remodelling in chickpea roots already h after drought stress. certain transcript isoforms characterizing these processes are potential targets for breeding for drought tolerance. we demonstrate that these can be easily accessed by micro-arrays and rt-pcr assays readily produced downstream of supersage. our study proves that supersage owns potential for molecular breeding also in non-model crops. 