Abstract

High-temperature stress severely limits the growth, development, and productivity of tomatoes. Understanding the molecular mechanisms underlying its thermotolerance is crucial for breeding heat-resistant varieties. This study employed a stepwise experimental strategy to systematically elucidate the role of the chlorophyll a/b-binding protein SlCAB3 in tomato thermotolerance. First, a high-temperature responsive transcription factor,SlDREBA4, previously identified in our lab, was used in a yeast two-hybrid screen to identify potential interacting proteins, including SlCAB3. The interaction betweenSlDREBA4andSlCAB3was further validated using tobacco in vivo luciferase complementation imaging (LCI) and in vitro pull-down assays. Subsequently, the expression patterns ofSlCAB3under heat stress were analyzed, and its biological function was further evaluated through overexpression, gene silencing, and knockout experiments. Additionally, reactive oxygen species (ROS) accumulation, antioxidant enzyme activities, chlorophyll content, and the expression of stress-responsive genes were measured to comprehensively assess their physiological and molecular regulatory roles. The results indicate thatSlCAB3encodes a typical chlorophyll a/b-binding protein and is rapidly induced by heat stress. Overexpression ofSlCAB3significantly enhances plant thermotolerance, evidenced by reduced heat damage, increased chlorophyll content, decreased ROS accumulation, elevated antioxidant enzyme activities, and upregulation of antioxidant-related genes. Conversely, silencingSlCAB3produces opposite effects. Moreover, co-expression ofSlCAB3withSlDREBA4further improves thermotolerance, accompanied by enhanced expression of heat shock protein-related and antioxidant-related genes. In conclusion,SlCAB3is a positive regulator of tomato thermotolerance, and the interaction module formed withSlDREBA4may collectively enhance heat resistance by strengthening antioxidant defense and heat stress response mec