Genome-wide identification of the chickpea chitinase family reveals CaCHI4, a Class IV chitinase, as a positive regulator of salt tolerance

Abstract

Chickpea (Cicer arietinum L.) is a vital legume crop, yet its productivity is severely constrained by soil salinity. While chitinases (CHIs) are well-recognized for their roles in biotic defense, their involvement in abiotic stress adaptation remains largely unexplored in chickpea. In this study, we systematically identified 25 CaCHI genes across the chickpea genome. Among them, CaCHI4, a tandemly duplicated Class IV chitinase localized to the apoplast, was identified as a key salt-responsive candidate. Overexpression of CaCHI4 in Arabidopsis thaliana enhanced salt tolerance, as evidenced by improved germination rates, increased biomass, and enhanced primary root growth. Physiological analyses revealed that CaCHI4 maintains cellular homeostasis by augmenting antioxidant enzyme activities (SOD, POD, and CAT), thereby reducing reactive oxygen species (ROS) accumulation and lipid peroxidation. Furthermore, CaCHI4 promoted active stomatal closure and preserved photosystem II (PSII) photochemistry under stress. Consistently, its overexpression in chickpea hairy roots also conferred superior salt tolerance, alleviating leaf wilting and resulting in a more robust systemic phenotype. Integrated transcriptomic profiling (RNA-Seq) further demonstrated that CaCHI4 orchestrates a hierarchical regulatory network, up-regulating signaling components such as receptor-like kinases (MALE-RLK) and key transcription factors (DREB2C, NAC048) to execute downstream stress responses. In conclusion, this study identifies the tandemly duplicated CaCHI4 as a pivotal player with a non-canonical role in salinity resilience, elucidating its physiological and molecular regulatory mechanisms and providing a promising genetic candidate for the improvement of legume crop resilience in saline environments.