Persulfidation and phosphorylation of transcription factor SlWRKY6 differentially regulate tomato fruit ripening.

Cysteine desulfhydrase (LCD) catalyzes the generation of the signaling molecule hydrogen sulfide (H2S) in plants. In this study, we found that H2S can inhibit tomato (Solanum lycopersicum) fruit ripening and SlWRKY6 undergoes differential protein persulfidation in SlLCD1-overexpressing leaves. Then, further study indicated that SlWRKY6 could be persulfidated by H2S at Cys396. By construction of slwrky6 mutants and SlWRKY6-OE lines, we found that SlWRKY6 positively regulates leaf senescence and fruit ripening by activating the transcription of ripening-related genes STAYGREEN 1 (SlSGR1) and Senescence-Associated Gene 12 (SlSAG12). In addition, SlWRKY6 interacted with kinase SlMAPK4 and was phosphorylated at Ser33. Dual luciferase transient expression assays and electrophoretic mobility shift assays indicated that SlWRKY6 persulfidation attenuated its transcriptional regulation of target genes SlSGR1 and SlSAG12, whereas SlWRKY6 phosphorylation by SlMAPK4 activated the transcription of target genes to promote fruit ripening. Moreover, we provided evidence that SlWRKY6 persulfidation attenuated its SlMAPK4-mediated phosphorylation to inhibit tomato fruit ripening. By transient expression of SlWRKY6, SlWRKY6C396A, SlWRKY6S33A and SlWRKY6S33D in slwrky6 fruits, we found that SlWRKY6 persulfidation attenuated the expression of SlSGR1 and SlSAG12 thereby delaying tomato fruit ripening, while SlWRKY6 phosphorylation increased the expression of target genes. As tomato fruits ripened, endogenous H2S production decreased, while SlMAPK4 expression increased. Therefore, our findings reveal a model in which SlWRKY6 persulfidation due to higher endogenous H2S levels in un-ripened fruit inhibits its ability to activate SlSGR1 and SlSAG12 expression, while SlWRKY6 phosphorylation by SlMAPK4 activates its transcriptional activity, thereby promoting tomato fruit ripening.

Hydrogen Peroxide Promotes Tomato Leaf Senescence by Regulating Antioxidant System and Hydrogen Sulfide Metabolism.

Hydrogen peroxide (H2O2) is relatively stable among ROS (reactive oxygen species) and could act as a signal in plant cells. In the present work, detached tomato leaves were treated with exogenous H2O2 at 10 mmol/L for 8 h to study the mechanism of how H2O2 regulates leaf senescence. The data indicated that H2O2 treatment significantly accelerated the degradation of chlorophyll and led to the upregulation of the expression of leaf senescence-related genes (NYC1, PAO, PPH, SGR1, SAG12 and SAG15) during leaf senescence. H2O2 treatment also induced the accumulation of H2O2 and malondialdehyde (MDA), decreased POD and SOD enzyme activities and inhibited H2S production by reducing the expression of LCD1/2 and DCD1/2. A correlation analysis indicated that H2O2 was significantly and negatively correlated with chlorophyll, the expression of leaf senescence-related genes, and LCD1/2 and DCD1/2. The principal component analysis (PCA) results show that H2S showed the highest load value followed by O2•-, H2O2, DCD1, SAG15, etc. Therefore, these findings provide a basis for studying the role of H2O2 in regulating detached tomato leaf senescence and demonstrated that H2O2 plays a positive role in the senescence of detached leaves by repressing antioxidant enzymes and H2S production.