Genome-wide identification and characterization of CsHSP60 gene family associated with heat and drought responses in tea plants (Camellia sinensis)

Heat and drought are the stressors with significant adverse impacts on the yield stability of tea plants. The heat shock proteins 60 (HSP60s) play important roles in protecting plants under heat stress. However, the mechanism of HSP60s under heat and drought stresses remains unclear. Here, we identified 19 CsHSP60s (namely CsHSP60-1 to CsHSP60-19) in tea plants and classified them into three groups based on phylogenetic analysis. In addition, studies on gene duplication events during the evolutionary process demonstrated that CsHSP60 members were subjected to purify selection. Analysis of cis-acting elements revealed the presence of numerous stress and hormone-responsive elements within the promoter regions of CsHSP60s. Real-time quantitative fluorescent PCR (qRT-PCR) analyses demonstrated that CsHSP60s rapidly responded to heat and combined heat and drought stress while exhibiting a delayed response to drought stress. The inhibition of eight CsHSP60 genes via antisense oligodeoxynucleotide (AsODN) resulted in more severe damage and ROS accumulation. Specifically, CsHSP60-9, CsHSP60-16, and CsHSP60-19 exhibited notable reductions in Fv/Fm values and displayed increased accumulation of H₂O₂ and O₂^·-. These observations indicated a potential role for CsHSP60 in mitigating ROS accumulation under stress conditions, thereby enhancing tea plants' resilience to heat and drought stresses. Using a yeast two-hybrid (Y2H) assay, we identified that CsHSP60-2 and CsHSP60-16 physically interact with CsCPN10-4 and CsCPN10-5, respectively. These interactions suggest a cooperative chaperone activity between CsHSP60 and CsCPN10 in response to combined heat and drought stress. These findings lay a foundation for further understanding the involvement of HSP60s in the tolerance mechanisms to compound heat and drought stresses.