SlCathB2 as a negative regulator mediates a novel regulatory pathway upon high-temperature stress response in tomato.

High-temperature stress (HS) is a major abiotic stress that affects the yield and quality of plants. Cathepsin B-like protease 2 (CathB2) has been reported to play a role in developmental processes and stress response, but its involvement in HS response has not been identified. Here, overexpression, virus-induced gene silencing (VIGS)and RNA-sequencing analysis were performed to uncover the functional characteristics of SlCathB2-1 and SlCathB2-2 genes for HS response in tomato. The results showed that overexpression of SlCathB2-1 and SlCathB2-2 resulted in reduced heat tolerance of tomato to HS while silencing the genes resulted in enhanced heat tolerance. RNA-sequencing analysis revealed that the heat shock proteins (HSPs) exhibited higher expression in WT than in SlCathB2-1 and SlCathB2-2 overexpression lines. Furthermore, the possible molecular regulation mechanism underlying SlCathB2-1 and SlCathB2-2-mediated response to HS was investigated. We found that SlCathB2-1 and SlCathB2-2 negatively regulated antioxidant capacity by regulating a set of genes involved in antioxidant defence and reactive oxygen species (ROS) signal transduction. We also demonstrated that SlCathB2-1 and SlCathB2-2 positively regulated ER-stress-induced PCD (ERSID) by regulating unfolded protein response (UPR) gene expression. Furthermore, SlCathB2-1 and SlCathB2-2 interacting with proteasome subunit beta type-4 (PBA4) was identified in the ERSID pathway using yeast two-hybrid (Y2H) analysis and bimolecular fluorescence complementation (BiFC) screening. Overall, the study identified both SlCathB2-1 and SlCathB2-2 as new negative regulators to HS and presented a new HS response pathway. This provided the foundation for the construction of heat-tolerant molecular mechanisms and breeding strategies aiming to improve the thermotolerance of tomato plants.