Hydrogen sulfide (H₂S) is involved in multiple processes during plant growth and development. D-cysteine desulfhydrase (DCD) can produce H₂S with D-cysteine as the substrate; however, the potential developmental roles of DCD have not been explored during the tomato lifecycle. In the present study, SlDCD2 showed increasing expression during fruit ripening. Compared with the control fruits, the silencing of SlDCD2 by pTRV2-SlDCD2 accelerated fruit ripening. A SlDCD2 gene-edited mutant was constructed by CRISPR/Cas9 transformation, and the mutant exhibited accelerated fruit ripening, decreased H₂S release, higher total cysteine and ethylene contents, enhanced chlorophyll degradation and increased carotenoid accumulation. Additionally, the expression of multiple ripening-related genes, including NYC1, PAO, SGR1, PDS, PSY1, ACO1, ACS2, E4, CEL2, and EXP was enhanced during the dcd2 mutant tomato fruit ripening. Compared with the wild-type fruits, SlDCD2 mutation induced H₂O₂ and malondialdehyde (MDA) accumulation in fruits, which led to an imbalance in reactive oxygen species (ROS) metabolism. A correlation analysis indicated that H₂O₂ content was strongly positively correlated with carotenoids content, ethylene content and ripening-related gene expression and negatively correlated with the chlorophyll content. Additionally, the dcd2 mutant showed earlier leaf senescence, which may be due to disturbed ROS homeostasis. In short, our findings show that SlDCD2 is involved in H₂S generation and that the reduction in endogenous H₂S production in the dcd2 mutant causes accelerated fruit ripening and premature leaf senescence. Additionally, decreased H₂S in the dcd2 mutant causes excessive H₂O₂ accumulation and increased ethylene release, suggesting a role of H₂S and SlDCD2 in modulating ROS homeostasis and ethylene biosynthesis.

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