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Gene expression profiling of chickpea responses to cold stress

ABSTRACT: ‘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 accessions tolerant and susceptible to cold stress. Two groups of a tolerant and susceptible accession were challenged with cold stress. 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 leaves and flowers/buds/early pods 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 and flower tissues separate) 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. The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress. Globally, the level of 221 transcripts was affected in response to cold stress in all the genotypes and tissue types studied. The DE transcripts in response to cold stress fell into various functional categories, indicating a broad response. Sixteen out of the 221 DE transcripts were consistently expressed in cold tolerant/susceptible genotypes. All these transcripts were repressed and none was found to be consistently induced in response to cold-stress. Most of the putative genes were identified in leaves of tolerant genotypes, and included a beta-galactosidase (DY475141) transcript that was possibly indicative of disaccharide (e. g. sucrose) retention with the effect of protecting cell membranes during cold stress. Several protein synthesis/modification and energy/metabolism transcripts were also repressed (e.g. DY475282, DY396371 and DY475555), which was likely due to the impairment of photosynthesis and respiration at low temperature. Other consistently repressed transcripts in tolerant genotypes included putative signalling (DY396262, DY475384 and DY396307) and defence-related proteins (CV793589 and DY396343), which may be involved in the repression of cell death mechanisms that are absent in tolerant genotypes. In susceptible genotypes, a putative superoxide dismutase precursor protein (DY475397) and sorting nexin protein (DY475523) were the only known transcripts to be consistently repressed. Keywords: Chickpea, Cold stress, Tolerant, Susceptible, cDNA microarray

ORGANISM(S): Lathyrus sativus Cicer arietinum Lens culinaris

PROVIDER: GSE7417 | GEO | 2007/04/03

SECONDARY ACCESSION(S): PRJNA105515

REPOSITORIES: GEO

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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 accessions tolerant and susceptible to high-salinity stress. Two groups of a tolerant and susceptible accession were challenged with high-salinity stress. 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 and root tissues were collected at 24 and 48 h post-treatment (hpt) 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 (shoot and root tissues separate for each time point) 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. The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress. Globally, the level of 409 transcripts was affected in response to high-salinity stress in all the genotypes and tissue types studied. Of the transcripts consistently expressed in tolerant genotypes in response to high-salinity stress, the annotation of transcripts at 24 hpt suggest a reduction in energy production in shoots and roots by repression of putative genes including P700 chlorophyll a-apoprotein (DY475501) and NADH-plastoquinone oxidoreductase subunit I (DY475287), cytosolic fructose 1,6-bisphosphatase (DY475548) and splicing factor-like protein (DY396290). The ATHP3 (DY396300) and protein kinase (DY475077) that are potentially involved in signalling cascades responsible for sensing and relaying osmotic stress signals, were consistently repressed in tolerant genotypes in response to high-salinity. Additionally a glycine rich protein (DY396342) that is associated with lignification of cell walls in response to wounding and pathogen attack was repressed in tolerant genotypes. In tolerant genotypes at 48 hpt, two energy and metabolic-related transcripts were consistently repressed in roots, including a carbonic anhydrase (DY475403) and putative thiazole biosynthetic enzyme (DY475242). In shoots, only a pathogenesis-related protein (DY396301) was consistently repressed at 48 hpt, but a similar transcript (DY396281) was induced in roots of tolerant genotypes at the same time. Only four transcripts were DE in susceptible genotypes at 24 hpt, all occurring in root tissue. Two of these were unknown/unclear, but the others included a proline oxidase transcript (DY475225) and a nuclear transport factor 2 (DY396436). At 48 hpi, a putative splicing factor-like protein (DY396290) and polyubiquitin (DY396328) were repressed in roots of susceptible genotypes. Interstingly, a putative xylosidase (DY475408) was induced in susceptible roots at 48 hpi. Finally, several unknown/unclear transcripts were DE in both tolerant and susceptible genotypes. Of interest were two unknowns (DY475293 and DY475521) that were consistently expressed in tolerant genotypes and may be important for high-salinity tolerance. Keywords: Chickpea, High-salinity stress, tolerant, susceptible, cDNA microarray

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 accessions tolerant and susceptible to drought stress. Two groups of a tolerant and susceptible accession were challenged with drought stress. 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 and flower/bud 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 and flower tissues separate) 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. The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress. Globally, the level of 114 transcripts was affected in response to drought stress in all the genotypes and tissue types studied. The DE transcripts in response to drought stress coded for various functional and regulatory proteins, most of which were repressed. Protein and other solute transport was repressed in susceptible-1 flowers but induced in tolerant-2 flowers, as evidenced by the induction of a protein-transport facilitation protein (DY475074) in flowers of tolerant-2 and repression of a aquaporin-like membrane channel protein (DY396334) and DNA-J like protein involved in intracellular protein transport (DY475488) in flowers of susceptible-1. Two putative auxin-repressed proteins (DY396289, DY396359) were highly repressed in flowers and leaves of tolerant-2, whilst being induced in flowers and leaves of susceptible-1. Defence-related genes including pathogenesis-related proteins (DY396305 and DY396343), nematode-resistance protein (CV793603), Cf-9 gene cluster (DY396352) and disease resistance response proteins (DY396265 and DY396276) were repressed in flowers of tolerant and susceptible genotypes. A Pea (pi230) disease resistance response protein (DY396390) and a multi-resistance protein ABC transporter (CV793605) were induced in flowers of both tolerant genotypes. Some genes involved in energy metabolism (DY396279 and DY475316) were repressed in leaves and flowers of tolerant and susceptible genotypes. Additionally, some transcripts related to senescence, including auxin responsive protein IAA9 (DY396315), senescence-associated protein DIN 1 (DY396338), and dehydration stress-induced protein (DY396321) were repressed in leaves and flowers of the tolerant genotypes. Of the 114 DE transcripts expressed in drought tolerant and susceptible genotypes, only two were consistently expressed. These included a cytosolic fructose 1,6-bisphosphatase (DY475548) and a gene with unknown function, which were repressed in flowers of both susceptible genotypes. Keywords: Chickpea, Drought stress, Tolerant, Susceptible, cDNA microarray

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

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Gene expression profiling of chickpea responses to cold stress

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