Genomics approaches for salt stress tolerance in chickpea (Cicer arietinum L.)
Creators: name: KAASHYAP, Mayank
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310299
subject: Bioinformatics and computational biology not elsewhere classified
classificationCode: 310803
subject: Plant cell and molecular biology

description: Salinity is a major constraint for the second most important grain legume, chickpea, because of intrinsic salt sensitivity. Chickpea exhibits large genetic variation with some improvement in salt tolerance over the years. However, it still needs to be improved further for sustainable crop production. RNA-Sequencing is a revolutionary tool which allows comprehensive transcriptome profiling to identify genes/alleles associated with stress tolerance/sensitivity. This study comprehensively analyzed gene expression in JG 11 and ICCV 2 genotypes that were identified as salt tolerant and salt sentitive, respectively upon extensive multilocation field trials. Physiological and transcriptome data was generated for root and flower tissues to identify genes responsible for salt tolerance/sensitivity. 
RNA-Sequencing of root tissues from the vegetative and flowering stages of tolerant and sensitive genotypes resulted in a total of ~480 million reads. Bioinformatic analysis led to the identification of 3,053 differentially expressed genes (DEGs) in response to salt stress. Reproductive stage shows a high number of DEGs suggesting a major transcriptional reorganization in response to salt to enable tolerance. Importantly, dirigent proteins (Ca12786; FC: 1024 â†‘), expansin (Ca27453; FC: 302.2 â†‘), cationic peroxidase (Ca09667; FC: 70.5 â†‘) and cation/H<sup>+</sup> exchanger (Ca18432; FC: 9.57 â†‘) genes were significantly induced in the tolerant genotype but repressed in the sensitive genotype. In addition to this, a suite of important genes involved in cell wall modification and root morphogenesis such as XHTs and casparian strip membrane proteins that could potentially confer salt tolerance were identified. Further, the directional efflux of auxin which is controlled by <i>PIN-FORMED</i> genes modulates the agravitropic root growth in response to salt stress (Ca18322; FC: 11.95â†‘).
Apart from roots that are directly exposed to saline soil water, the flowering stage is very crucial for crop productivity. Salinity has the maximum adverse effect on the reproductive processes. Chickpea exhibits great variation amongst the accessions in terms of flowering time, pod filling and number of flowers. These traits have been described as a measure of salt tolerance. The gene expression profiling of the flower tissues is therefore important to understand the comparative molecular events between the tolerant and the sensitive genotype that lead to salt tolerance/sensitivity.
Deep RNA-Sequencing of flower tissues (fully developed but unopened flower) from the tolerant and sensitive genotype resulted in a total of ~700 million reads. Bioinformatic analysis led to identification of 2,022 potential differentially expressed genes (DEGs) between the tolerant and sensitive genotypes. It was observed that genes involved in flowering time and pollen development such as ABORTED MICROSPORES (AMS) (Ca14533; FC: 427.5 â†‘), rho-GTPase (Ca05726; FC: 3.36 â†‘) and pollen receptor kinase were significantly differentially regulated in the tolerant genotype, suggesting their role in dealing with salt stress. In addition to this, a suite of important genes such as cytochrome P450, MYB proteins (Ca05149; FC: 5.24 â†‘), bHLH, thaumatin (Ca30893; FC: 1418.3 â†‘) and serine/threonine kinase, which are important regulators of transcriptional responses toegulators of salinity tolerance were identified to be differentially expressed. Further, the induced action of auxin genes enhance the expression of H<sup>+</sup>/ATPase (Ca14828; FC: 3.18 â†‘) which polarise the membrane and activate genes like expansin involved in cell wall loosening. This important mechanism of cell wall loosening regulates the floral organ development during the salt stress.
Apart from understanding the coding landscapes of the transcriptome, emerging definitive evidence now suggest that long non-coding RNAs (lncRNAs) may have a potential role in regulating the functional RNA in response to abiotic stress. To understand this and extensively characterize low and rarely expressed long non-coding RNAs in chickpea, 12 poly(A+) and 12 poly(A-) RNA-Sequencing libraries from flower tissues of salt tolerant (JG11) and salt sensitive (ICCV2) genotypes were sequenced to nearly 1,167 million reads with and average of 50 million reads per sample. An <i>ab initio</i> transcriptome assembly comprising of 32982 transcripts from poly(A+) RNA and 34082 transcripts from poly(A-) RNA was generated. In total, 7364 lncRNA were identified in response to salt stress. Out of these, 3616 lncRNA (2686 lincRNA + 930 NATs) were from poly(A+) samples and 3738 lncRNA (3153 lincRNA + 615 NATS) were characterized from poly(A-) samples. The comparative homology analysis across six plant model species including <i>Arabidosis</i>, <i>Medicago</i>, <i>Glycine</i>, <i>Phaseolus</i>, <i>Oryza </i>and <i>Vitis </i>revealed 2,345 lncRNAs specific to chickpea. Based on the orthologous inference, 26 lncRNAs with homology were identified. Among them, with COOLAIR (XLOC_016687; FC: 5.5 â†‘), IPS1 (XLOC_016933; FC: 2.1 â†‘) and AT4 (XLOC_007084, FC: 8.0 â†‘) lncRNAs and were significantly induced in the tolerant genotype as shown in response to the salt stress. A total of 178 lncRNAs were differentially expressed amongst the genotypes with more number being differentially expressed (110) and 14-fold up-regulated in the tolerant genotype during the salt stress. The gene set enrichment analysis showed that these lncRNAs were significantly involved in epigenetic gene regulation and therefore may play an important role in post-transcriptional RNA modification and mRNA cleavage. The tissue-specificity scores of DE-lncRNAs significantly correlated with highly induced coding genes like DNA (cytosine-5)-methyltransferase (Ca07939; FC: 13.9 â†‘), cytochrome P450 (Ca09486; FC: 98.3 â†‘) and pollen receptor kinase suggesting that lncRNAs may lead to epigenetic modification of gene expression in response to the salt stress. The gene set enrichment analysis and functional annotation of both, coding and non-coding transcripts, genes suggests that they may be the potential options futilised for improving chickpea crop improvement in terms of salt tolerance.
RNA-Sequencing has escalated the high-resolution trait mapping that has led to the rapid crop improvement. The deep-sequencing of two parental lines in this study will provide enough genomic resources to assist the approaches like "Bulked segregant RNA-Seq (BSR-Seq)" and targeted gene expression for salt tolerance. The genes involved in regulation of various salt tolerance pathways can be potential candidate(s) in future molecular breeding programmes. The comprehensive set of long noncoding RNAs in this study can be used to develop epigenetic biomarkers that will aid in future revolutionary approaches like "epigenomics-assisted breeding (EAB)". The robust suite of genes in the present study unveils the complete landscape of chickpea transciptome which will provide in-depth knowledge of lncRNA guided gene regulation to promote plant growth and productivity.