Interaction between endogenous H₂O₂ and OsVPE3 in the GA-induced PCD of rice aleurone layers

Endogenous hydrogen peroxide (H₂O₂) is involved in regulating the gibberellic acid-induced programmed cell death (PCD) of the aleurone layers by cooperating with OsVPE3 during rice seed germination. Preliminary experiments revealed that H₂O₂ produced by the NOX pathway is the key factor affecting rice germination. Histochemical analysis indicated that H₂O₂ is located in the aleurone layer. Both the H₂O₂ scavenger DMTU and the NOX inhibitor DPI decreased H₂O₂ content and significantly slowed down vacuolation in a dose-dependent manner. Interestingly, DMTU down-regulated the OsNOX8 transcript or DMTU and DPI decreased the intracellular H₂O₂ level, resulting in a delay of PCD. In contrast, GA and H₂O₂ up-regulated the OsNOX8 transcript and intracellular H₂O₂ level, leading to premature PCD, and the effects of GA and H₂O₂ were reversed by DMTU and DPI, respectively. These results showed that the imbalance of intracellular H₂O₂ levels leads to the delayed or premature PCD. Further experiments indicated that GA up-regulated the OsVPE3 transcript and VPE activity, and the effect was reversed by DPI. Furthermore, Ac-YVAD-CMK significantly blocked H₂O₂ accumulation, and DPI + Ac-YVAD-CMK had a more significant inhibitory effect compared with DPI alone, resulting in the delayed PCD, suggesting that OsVPE3 regulates PCD by promoting H₂O₂ generation. Meanwhile, DPI significantly inhibited the OsVPE3 transcript and VPE activity, and in turn delayed PCD occurrence, suggesting that the H₂O₂ produced by the NOX pathway may regulate PCD by up-regulating the OsVPE3 transcript. Thus, the endogenous H₂O₂ produced by the NOX pathway mediates the GA-induced PCD of rice aleurone layers by interacting with OsVPE3.