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
2007-07-24 | GSE8554 | GEO
Palak Chaturvedi 
