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Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea.

ABSTRACT: Drought and salinity are the major factors that limit chickpea production worldwide. We performed whole transcriptome analyses of chickpea genotypes to investigate the molecular basis of drought and salinity stress response/adaptation. Phenotypic analyses confirmed the contrasting responses of the chickpea genotypes to drought or salinity stress. RNA-seq of the roots of drought and salinity related genotypes was carried out under control and stress conditions at vegetative and/or reproductive stages. Comparative analysis of the transcriptomes revealed divergent gene expression in the chickpea genotypes at different developmental stages. We identified a total of 4954 and 5545 genes exclusively regulated in drought-tolerant and salinity-tolerant genotypes, respectively. A significant fraction (~47%) of the transcription factor encoding genes showed differential expression under stress. The key enzymes involved in metabolic pathways, such as carbohydrate metabolism, photosynthesis, lipid metabolism, generation of precursor metabolites/energy, protein modification, redox homeostasis and cell wall component biogenesis, were affected by drought and/or salinity stresses. Interestingly, transcript isoforms showed expression specificity across the chickpea genotypes and/or developmental stages as illustrated by the AP2-EREBP family members. Our findings provide insights into the transcriptome dynamics and components of regulatory network associated with drought and salinity stress responses in chickpea.

SUBMITTER: Garg R

PROVIDER: S-EPMC4725360 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea.

Garg Rohini R Shankar Rama R Thakkar Bijal B Kudapa Himabindu H Krishnamurthy Lakshmanan L Mantri Nitin N Varshney Rajeev K RK Bhatia Sabhyata S Jain Mukesh M

Scientific reports 20160113

Drought and salinity are the major factors that limit chickpea production worldwide. We performed whole transcriptome analyses of chickpea genotypes to investigate the molecular basis of drought and salinity stress response/adaptation. Phenotypic analyses confirmed the contrasting responses of the chickpea genotypes to drought or salinity stress. RNA-seq of the roots of drought and salinity related genotypes was carried out under control and stress conditions at vegetative and/or reproductive st ...[more]

PMID: 26759178

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Project description:‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea genotype ICC 3996 to drought, cold and high-salinity stresses. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaf/shoot, flower/pod and/or root tissues were collected and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (leaf/flower/root tissues pooled separately) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1. Overall, 46, 54 and 266 ESTs were identified as DE under drought, cold, and high-salinity stresses, respectively. The important ones DE in response to drought stress include induction of transcripts associated with dehydrin-cognate, lipid-transfer protein precursor, and glutamate-hydrolysing asparagine synthetase, whilst repression of transcripts associated with senescence, photosynthesis/energy metabolism, auxin-repressed protein, starch metabolism, AP2/EREBP1 DNA binding domain, putative ARF1 GTPase activating protein, and histidine-containing phosphotransfer protein ATHP3. The interesting transcripts DE in response to cold-stress included induction of phosphate-induced protein, cationic peroxidase, UDP-glucose 4-epimerase, Avr9/Cf9 rapidly elicited protein, DNA-J like protein involved in intracellular protein transport, and protein kinase, whist repression of transcripts associated with a hypothetical transmembrane protein, membrane related protein CP5, lipid-transfer protein precursor, WD repeat protein and several transcripts associated with cellular metabolism, cell cycle and DNA processing, protein synthesis and photosynthesis/energy metabolism. Under high-salinity stress, several transcripts were DE only in roots at 24 hpt but DE in shoots or both, shoots and roots at 48 hpt, relating to the theory of upward movement of salt to shoots at later stages when roots fail to restrict it (Munns et al., 2002). The important transcripts DE in response to high-salinity stress included induction of aluminum-induced protein, auxin-repressed proteins, metallothionein-like protein, glutamate dehydrogenase, sucrose synthase, UDP-glucose 4-epimerase, xylose isomerase, class 10 pathogenesis related protein, disease resistance protein, SNAKIN 2 antimicrobial peptide, and DNA-J like protein involved in intra-cellular protein transport. Whilst high-salinity stress caused the repression of transcripts associated with cell rescue/death, cell cycle/DNA processing, cellular metabolism, photosynthesis/energy metabolism, and transport facilitation. Several of the above transcripts have been previously implicated to be associated with abiotic stress response in other crops. Hence, the study of more genotypes and transcriptional changes at several time points may provide a better picture of involvement of the genes being interrogated here in drought tolerance/susceptibility. Subsequently, the functionality of candidate tolerance genes detected through this approach could be validated by overexpressing the genes through transgenics or silencing them using knockout-mutants/antisense/RNAi. Keywords: abiotic stress response, drought, cold, high-salinity, chickpea

Project description:BackgroundDrought is the major constraint to increase yield in chickpea (Cicer arietinum). 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 (e)QTL mapping, may accelerate knowledge-based breeding. SuperSAGE, an improved version of the serial analysis of gene expression (SAGE) technique, generating genome-wide, high-quality transcription profiles from any eukaryote, has been employed in the present study. The method produces 26 bp long fragments (26 bp tags) 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.ResultsWe applied SuperSAGE to the analysis of gene expression in chickpea roots in response to drought. To this end, we sequenced 80,238 26 bp tags representing 17,493 unique transcripts (UniTags) from drought-stressed and non-stressed control roots. A total of 7,532 (43%) UniTags were more than 2.7-fold differentially expressed, and 880 (5.0%) were regulated more than 8-fold upon stress. Their large size enabled the unambiguous annotation of 3,858 (22%) UniTags to genes or proteins in public data bases and thus to stress-response processes. We designed a microarray carrying 3,000 of these 26 bp tags. The chip data confirmed 79% of the tag-based results, whereas RT-PCR confirmed the SuperSAGE data in all cases.ConclusionThis 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 6 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.

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Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea.

Publications

Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea.

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