Hydrogen sulfide interferes with ethylene biosynthesis and signaling pathway in tomato by the mediation of <scp>SlERF</scp>.<scp>D2</scp> persulfidation

Hydrogen sulfide (H₂S), as a signaling molecule, is found to delay fruit ripening and senescence by antagonizing the biosynthesis and signaling of ethylene, whereas the mechanism remains unclear. In the current work, exogenous H₂S fumigation could alleviate tomato fruit ripening and an ethylene response factor SlERF.D2 was found to be persulfidated at Cys35 by mass spectrometry analysis. Meanwhile, ethylene biosynthesis related genes SlACS1 and SlACO3 were significantly downregulated at gene expression level in H₂S-treated fruit. By CRISPR/Cas9 and gene overexpression, we showed that overexpression of SlERF.D2 promoted fruit ripening by accelerating chlorophyll degradation and carotenoid accumulation and upregulating the expression of ripening related genes SlPAO, SlPPH, SlSGR1, SlACS1, SlACS2, SlACS4, SlEIN2, SlACO1, and SlACO3, while the mutation of slerf.d2 delayed fruit ripening. Additionally, slerf.d2 mutant showed delayed ethylene production during tomato fruit ripening. Moreover, SlERF.D2 was found to interact with the kinase SlMAPK4 and was phosphorylated at Ser42 by yeast two-hybrid screening, pull down and LC-MS/MS. By cis-element analysis, electrophoretic mobility shift assay and dual-luciferase assay, SlERF.D2 could activate the transcription of the ethylene pathway-associated gene SlACO3 and SlEIN2. Besides, we provided evidence that SlERF.D2 persulfidation weakened the transcriptional activity of SlERF.D2 on the target gene SlACO3 and SlEIN2. In contrast, SlMAPK4-mediated phosphorylation enhanced SlERF.D2's transcriptional activation activity on SlACO3 and SlEIN2. Therefore, the present research provides insights into the mechanism of H₂S in antagonizing the biosynthesis and signaling transduction of ethylene and reveals the importance of SlERF.D2 persulfidation and phosphorylation in dynamically regulating tomato fruit ripening.