RBOH-dependent hydrogen peroxide signaling mediates melatonin-induced anthocyanin biosynthesis in red pear fruit

Although several studies have confirmed that exogenous melatonin promotes anthocyanin accumulation, the molecular mechanism of this remains elusive. Here, the signaling cross-talk between melatonin and NADPH oxidase (RBOH) -mediated ROS during anthocyanin biosynthesis were investigated. We found that application of exogenous melatonin not only induced anthocyanin biosynthesis, but also increased endogenous H₂O₂ and O₂^‾ content in pear fruits. The effect of melatonin on anthocyanin biosynthesis was abolished by inhibitors of RBOH. We also observed that genes encoding RBOH (PuRBOHF) were ubiquitously and highly expressed after melatonin treatment. Transient PuRBOHF overexpression significantly enhanced anthocyanin accumulation and activated transcription of anthocyanin biosynthesis genes, whereas PuRBOHF silencing repressed melatonin-promoted anthocyanin accumulation and H₂O₂ production. Moreover, RBOH-derived H₂O₂ induced PuMYB10 transcription, and PuRBOHF enhanced the PuMYB10-induced activation of the PuUFGT promoter. PuMYB10, in turn, activated PuRBOHF transcription, revealing a positive feedback loop. These results provide molecular evidence supporting the essential roles of PuRBOHF-dependent H₂O₂ in melatonin-induced anthocyanin accumulation in pears.