Application of high-throughput sequencing technologies to determine the molecular basis of drought tolerance in chickpea (Cicer arietinum L.)

Drought stress is one of the most important abiotic stresses, which adversely effects chickpea (Cicer arietinum L.) production across the globe. It is responsible for substantial yield loses up to 50%, which led to stagnated productivity of chickpea for the past six decades. In chickpea, terminal drought stress leads to significant reduction in the seed yield (58-95%). The deleterious effects of terminal drought stress are mainly manifested as increased flower and pod abortion, reduced pod production, and reduced seed size. For the past 20 years, root traits such as deep and profuse rooting system have been proposed as a main breeding target for improving drought tolerance in chickpea. Due to the complexity of drought stress, traditional breeding approaches have been largely unsuccessful in exploiting root traits for developing tolerant chickpea cultivars. Drought tolerance is a complex quantitative trait, which is influenced by number of genetic and environmental interactions. It is mainly controlled by the several drought responsive genes or gene networks and shows genotypic divergence depending on the plant phenology. In addition, plants show genotype-, tissue- and stage-specific variations during their response to drought stress. Hence, understanding the physiological and genetic basis of drought stress in a genotype-, tissue- and stage-specific manner is essential to decipher the complexity of drought stress. Till date, no study was focused on deciphering the molecular mechanisms that underlie root and reproductive growth during drought stress in chickpea. Therefore, this study was designed to employ RNA-Sequencing (RNA-Seq) for investigating genome-wide transcriptome changes in the roots and reproductive tissues during different developmental stages in response to drought stress. Further, the study also investigated the role of drought responsive microRNAs during reproductive development. This will improve an overall understanding of molecular mechanisms that control root and reproductive development during drought stress.<br><br>To enable the employment of RNA-Seq, two chickpea genotypes with contrasting drought tolerance (ICC 8261 – drought tolerant; ICC 283 – drought sensitive) were challenged with drought stress. Analysis of physiological data revealed a phenotypic divergence in the root traits of tolerant and sensitive genotypes. Further, RNA-Seq revealed significant transcriptome changes in the roots of both genotypes during three stages of plant development (VS – Vegetative stage; RTS – Reproductive Transition stage (ICC 283/Drought susceptible genotype); RS – Reproductive stage). Gene enrichment and pathway analysis was performed to identify key molecular mechanisms that underlie phenotypical changes observed in the roots of both genotypes. Resulting analysis indicated a genotype- and stage-specific activation of gene or gene networks involved in transcriptional regulation (AP2/ERF, bHLH, C2C2-Dof, DREB, MYB), signal transduction (MEKK1, CPK3, PERK4, HSL2, CIPK1), ROS (Reactive Oxygen Species) production (RBOHD, RBOHH) and scavenging (GST, GST-L1-like, DHAR3, PER47, PER72-like), transport facilitation (ABCB19, ABCB12-like, ABCB15-like, ABCG22-like, PIP2-1, TIP2-2), nodulation (LYK3, CLAVATA1) and jasmonate biosynthesis (LOX, AOC3). Further, stage-specific activation/repression of components involved in phytohormone signalling such as Abscisic acid (NCED3, CYP707A3, PYL4), Auxin (PIN), Cytokinin (IPT5, AHP4) led us to propose a model of hormonal cross-talk that might lead to strong root growth during early stages and conservative root growth during reproductive stages in tolerant genotype during drought stress.<br><br>To explore the molecular basis of reproductive development in tolerant and sensitive genotypes, a physiological assay was performed to challenge the plants with drought stress. Reproductive tissues collected from five different stages (Shoot Apical Meristem – SAM; Flower Bud – FB; Partially Opened Flower – POF; Fully Opened Flower – FOF; Young Pod - YP) were used to perform RNA-Seq and analyse genome-wide transcriptome changes in both genotypes. Further, GO, KEGG enrichment and network analysis was performed to decipher the genes and gene pathways involved in reproductive growth under drought stress. The results indicated genotype- and stage-specific activation and repression of multiple genes families involved in dynamic signal transduction (NPK1, YODA, CPK17, SUB3, SUB5, PRK1, PRK3, PRK4), transcriptional regulatory networks (WRKY/ERF/STZ module, ARF19, NAC029, PERANTHIA, CRABSCLAW), pollen development (b-fructofuranosidases, ACOS5, CYP703A2, CYP704B1, FAR2, ABCG26, CalS5), pod development (SWEET3, EXO) and circadian clock function (LHY, ELF4) that may control reproductive success during drought stress. Additionally, genotype-specific regulation of Auxin (AUX1, ARF2, XTH22, PID) and Jasmonate (LOX2.1-2, TIFY-10A, MYB21) signalling during FB and POF may positively regulate flower development, while ABA-dependent (NCED1, AB15, RD29B) and –independent (DREB1A) signalling during YP might provide molecular basis to help reduce pod abortion in chickpea.<br><br>During reproductive growth, microRNAs (miRNAs) control various aspects such as vegetative to reproductive phase transition, meristem initiation, flower development, floral organ growth and seed development. However, drought induced regulation of miRNAs during reproductive development has never been evaluated in chickpea. To explore the miRNA regulation, small RNA-component was sequenced during three reproductive stages (SAM, POF, YP) from the same tissue that was used for transcriptome sequencing. This allowed us to perform integrated miRNA mRNA expression analysis to identify putative regulatory modules that control reproductive development under drought stress. The results indicated differential regulation of 287 miRNAs in treatment-, genotype- and stage-specific manner. The miRNAs were involved in controlling the targets related to abiotic stimulus, signal transduction, transport facilitation, floral organ growth (pollen and pistil development), secondary metabolism and phytohormone signalling pathways. Genotype- and stage-specific activation/repression of regulatory modules controlling transport activity (Ca-miR7716-5p:TIP4-1, Ca-miR5227:KPNB1, Ca-miR1130:ABCG3, Ca-miR166g-5p:CHX20, Ca-miR8657a:ABCB21-like), reproductive transition and flower development (Ca-miR166i:ATHB14/ATHB15/REVOLUTA, miR167d-5p:ARF6/ARF8, Ca-miR319q:TCP4-like, Ca-miR159c:GAMYB-like and Ca-miR172c:APETALA2/AP2-like), floral organ growth (Ca-miR390b:EMS1, Ca-miR39h:GRF1-like/GRF4/GRF4-like/GRF9, Ca-miR2916b-5p:POLLENLESS3/GSL12-like) might play a critical role in reproductive success during drought stress. The study also identified 16 novel miRNAs that were involved in response to ethylene stimulus, response to abiotic stimulus, reproductive development, kinase and transporter activities. Finally, genotype-specific expression of two regulatory modules involved in ABA-dependent (Ca-miR166h-3p:ABI5) and –independent signalling (Ca-miR2912a:CYP707A1, Ca-miR6267a:ATAF1) during YP may form the molecular basis for normal pod development during drought stress.<br><br>In summary, the potential gene or gene networks identified in this study were based on their role in other plant species. Hence, functional analyses of candidate genes and miRNAs using transgenic over-expression or CRISPR-Cas9 mediated gene editing will provide a better understanding of their role in enhancing drought tolerance in chickpea. Furthermore, the candidate genes can be used in conjunction with genome-wide association study (GWAS), QTL mapping for rapid dissection of drought stress tolerance. Nevertheless, this study is the first documentation of genotype- and stage-specific transcriptome profiling of chickpea roots and reproductive tissues in response to drought stress using RNA-Seq. Further, expression profiling of miRNAs using small RNA-Seq allowed us to identify putative regulatory modules involved in maintaining normal reproductive development under drought stress. This will further aid in better understanding of complex molecular mechanisms that control drought stress tolerance in chickpea.