Understanding the drought mechanism and designing strategies to mitigate drought stress in chickpea (Cicer arietinum L.)
Creators: name: BADHAN, Sapna
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310504
subject: Epigenetics (incl. genome methylation and epigenomics)
classificationCode: 310505
subject: Gene expression (incl. microarray and other genome-wide approaches)
classificationCode: 310804
subject: Plant developmental and reproductive biology

description: Chickpea (Cicer arietinum L.) is a valued pulse crop that is consumed globally and is becoming increasingly important as agricultural productivity is predicted to drastically affected by climate change in coming years. Chickpea is also important as a nitrogen-fixing leguminous crop cultivated in rotation with cereal crops such as wheat and barley. This rotation strategy replenishes soil nitrogen whilst providing a break from pests and diseases. Abiotic stresses such as drought, salinity and heat are key factors limiting chickpea productivity worldwide. Drought is the major cause of the global decline in chickpea production. In plants, extreme drought conditions adversely affect growth, physiology and reproduction leading to reduced yield.  To date, research has provided only limited insight into different genetic pathways related to drought tolerance/response. The aims of this thesis were first, to conduct RNA sequencing of leaf tissues from drought-tolerant and sensitive chickpea genotypes and identify candidate genes and pathways associated with drought tolerance/sensitivity. Secondly, to identify DNA methylation patterns that potentially regulate drought tolerance/sensitivity of selected chickpea genotypes. Thirdly, to perform gene knockout of selected drought tolerance associated genes using modern technique CRISPR/Cas9 DNA free editing to understand their role in drought tolerance. The first chapter of the thesis comprises the literature review discussing the challenges in chickpea production and examines the different mechanisms currently known to confer drought tolerance. The experimental chapter 2 describes the identification of drought tolerance associated genes by RNA sequencing of the leaf tissue from two contrasting chickpea genotypes under drought stress. In Chapter 2, to identify the genes/pathways that are differentially expressed under drought stress in the leaves of drought-tolerant and sensitive genotypes, leaf tissue from the shoot apical meristem developmental stage of drought-tolerant (ICC8261) and drought-sensitive (ICC283) genotypes were analysed using RNA sequencing. It was found that in the tolerant genotype, genes associated with ethylene response, MYB-related protein, xyloglucan endotransglycosylase, alkane hydroxylase MAH-like, BON-1-related, peroxidase 3-related, a cysteine-rich and transmembrane domain, vignain and mitochondrial uncoupling were explicitly upregulated, while in the sensitive genotype, the same genes were observed to be downregulated. The results confirm that the sensitive genotype was more affected by the drought stress compared to the tolerant genotype.

The crosstalk between the different hormones and transcriptional factors involved in the resistance and vulnerability to drought in both genotypes makes them perfect candidates for future studies. As a genetic donor, the tolerant genotype ICC8261 can be used to further enhance the drought-tolerant characteristics of the chickpea germplasm. This study will help to understand the putative role of candidate genes in response to drought stress in chickpea. Future research can use more systematic experimental approaches to define the individual role of these identified genes, their roles, pathways involved and application in a field study for promising drought-resilient chickpea crops. 
The work conducted in Chapter 3 aimed to study the correlation between DNA methylation and gene expression under drought stress in the consensus genes identified in Chapter 2. The purpose of this study was to obtain a clear picture of the drought mechanism involved in two contrasting genotypes of chickpeas using both, RNA sequencing and RRBS (Reduced representation bisulphite sequencing). To conduct RRBS under drought stress, leaf tissues from the shoot apical meristem of the drought-sensitive and drought-tolerant genotypes plants employed in Chapter 2 were used again. Compared to the tolerant genotype, the findings showed a larger number of differentially methylated regions (DMRs) and bases in the sensitive genotype. It was reported that in the sensitive genotype, hypermethylation in the upstream/promoter region can be associated with the downregulation of gene expression. In comparison, in the tolerant genotype, hypomethylation was interrelated to the upregulation of gene expression in the upstream/promoter region. In both genotypes, gene ontology (GO) was enriched with GO terms related to cellular metabolic processes, biosynthetic processes, oxidation-reduction processes, gene expression control, transcription regulation, DNA prototype, oxidation-reduction processes, signal transduction, lipid metabolic processes and intracellular signal transduction. This is the first study to examine DEG-associated differentially methylated cytosines (DMCs) and differentially methylated regions (DMRs) in these two significant chickpea genotypes. An indication of the association of DNA methylation with gene expression under drought stress is the differential methylated regions and genes associated with the DMRs in contrasting genotypes. For both genotypes, the results of the study produced insightful datasets that will contribute to future studies aimed at unmasking the function of these strong epigenetic regulatory mechanisms involved in drought tolerance and sensitivity.
 
The final aim of the study was to use CRISPR/Cas9 DNA-free editing in chickpea protoplast for knockout of the 4CL and RVE7 genes. This is the first research using CRISPR/Cas9 DNA-free gene editing of candidate genes linked to drought in chickpea protoplast. In several plant-based applications, the genome editing method CRISPR/Cas9 has already proven its high proficiency, adaptability and simplicity. This, along with many of the available genome and transcriptome databases, represents a great opportunity for plant breeding and research in coming years. It is possible to facilitate the delivery of pre-assembled ribonucleoprotein complexes (RNPs) consisting of the Cas9 enzyme and synthetically engineered sgRNA using conventional transformation methods or using modern nanoparticles to attain targeted mutagenesis in plants. There is minimal research focused on CRISPR/Cas9 gene editing in legumes; to date, no studies have employed in chickpea. The two genes selected for CRISPR editing were related to drought tolerance. 4CL (4-coumarate ligase) is a central enzyme in the lignin biosynthesis pathway for phenylpropanoid metabolism. This is known to manage the aggregation of lignin in other plants under stress conditions. RVE7 is a transcription factor for MYB that is part of the circadian regulatory rhythm in plants. A novel approach to achieving selective mutagenesis in chickpeas is the knockout of these selected genes in the chickpea protoplast using DNA free CRISPR/Cas9 editing. The results showed high efficiency of editing in the RVE7 gene in vivo. For the 4CL gene, the in vitro digestion worked well compared to the in vivo knockout. The results obtained in this study show the potential for using CRISPR/Cas9 in chickpea, leading to improved stress tolerance and enhanced productivity. Further validation of the role of these genes in drought stress is proposed. The study provides a comprehensive dataset as well as CRISPR/Cas9 editing method which can be exploited to improve chickpea productivity through a targeted breeding program.