Global warming is increasing the frequency of heat stress, a major abiotic constraint on crop growth and productivity. Hydrogen sulfide (H₂S), a novel gasotransmitter, has been reported to enhance crops’ heat tolerance, yet its underlying mechanism remains poorly understood. Here, we provide genetic evidence confirming that L-cysteine desulfhydrase (SlLCD1, Solyc01g068160) was the enzymatic source of endogenous H₂S in tomato heat adaptation. Dual activation of H₂S signaling through both SlLCD1 overexpression and exogenous application enhanced tomato heat tolerance. Conversely, CRISPR/Cas9-generated SlLCD1 mutants (cr-sllcd1), deficient in heat-induced H₂S production, displayed heightened heat sensitivity with accelerated wilting and increased oxidative damage, which was rescued by exogenous H₂S application. Compared to wild-type plants, the mutants showed a compromised heat-induced increase in antioxidant enzyme activities and levels. This defect, along with the concomitant ROS accumulation and oxidative damage, was reversed by H₂S pretreatment, underscoring the critical role of the SlLCD1-H₂S module in maintaining ROS homeostasis during heat adaptation. Additionally, cr-sllcd1 mutants exhibited attenuated heat-induced stomatal closure and increased stomatal density. H₂S pretreatment rescued both of these defects, thereby optimizing the trade-off among transpirational cooling, water conservation, and photosynthetic efficiency. Overall, the SlLCD1-H₂S module confers heat tolerance by a dual mechanism, coordinately enhancing antioxidant capacity and fine-tuning stomatal dynamics. Our study elucidates an important component of the H₂S signaling pathway in plant heat tolerance and offers a promising tractable target for developing heat-tolerant tomato cultivars.

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