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

Abstract 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.