Integrated Proteomic and Functional Analyses Reveal the Roles of Organelle-Specific Small Heat Shock Proteins (sHSPs) in Tomato Thermotolerance

Global warming-induced extreme heatwaves present a severe threat to global tomato yield and production stability. To elucidate the molecular regulatory mechanisms underlying heat stress tolerance in tomato (Solanum lycopersicum), this study utilized label-free quantitative proteomics to profile alterations in protein abundance in tomato leaves in response to heat stress. A total of 294 differentially expressed proteins (DEPs) were identified, with function enrichment in the systematic activation of core stress-responsive biological processes, including the mitogen-activated protein kinase (MAPK) signaling cascade, the endoplasmic reticulum protein processing, and glutathione metabolism. Among them, heat shock protein (HSP) family members exhibited the most significant changes, particularly two small heat shock proteins (sHSPs), designated as SlsHSP1 and SlHSP17.4. Functional validation showed that silencing either SlsHSP1 or SlHSP17.4 drastically impaired heat tolerance in tomato plants. Specifically, silenced lines displayed excessive reactive oxygen species (ROS) accumulation and reduced antioxidant enzyme activities, with SlsHSP1-silenced plants showing more severe heat-induced phenotypic damage. Subcellular localization assays further demonstrated SlsHSP1 was located in the ER and SlHSP17.4 in the nucleus. Collectively, this study unravels multiple heat defense regulatory networks in tomato, in which organelle-specific sHSPs like SlsHSP1 and SlHSP17.4 synergistically maintain protein homeostasis and cellular redox balance, conferring broad-spectrum stress resistance in plants under high-temperature stress.