Hydrogen Peroxide Regulates The Programmed Cell Death Of Rice Aleurone Layers Via Cooperating With OsVPE3

Programmed cell death(PCD)is an order and spontaneous phenomenon of cell death regulated by genes in organisms.The PCD of aleurone layers plays an important role in the germination of cereal seeds.It was found that both hydrogen peroxide(H2O2)and vacuole processing enzymes(VPEs)affected the process of vacuolation and the occurrence of PCD in the aleurone layers.In this study,the mechanism of endogenous H2O2 and OsVPE3 regulating the occurrence of PCD in the aleurone layers of rice(Oryza saliva L.)'Bo ? you767' seeds was explored by means of pharmacology,biochemistry,cell morphology and molecular biology,as well as the methods of microscopic technique.The results showed that:(1)Aleurone layers were the main part where rice seeds produced H2O2 and O2-.Both NADPH oxidase inhibitor DPI and H2O2 scavenger DMTU inhibited or eliminated the production of H2O2 and O2-,and the inhibitory effect of DPI was more significant than that of DMTU.(2)DPI and DMTU significantly slowed down the vacuolation of aleurone cells.With the the increase of treatment concentration,the process of vacuolation became slower,which showed a certain concentration effect,and the delayed effect of DPI was more obvious than that of DMTU.(3)The growth of radicle and plumule in the rice seeds cultured with both DMTU and DPI was inhibited,especially the inhibition of DPI on the growth of radicle was more significant than that on the growth of plumule.(4)The relative expression levers of and QsNOX9 were higher than that of other OsNOXs which are related to the germination of rice seeds.In addition,OsNOX8 was the only OsNOX that its gene expression was increased in the aleurone layers,thus it is speculated that OsNOX8 may be related to the PCD of aleurone layers.(5)DPI and DMTU delayed the occurrence of PCD by down-regulating the relative expression lever of OsNOXS and the content of H2O2 in the aleurone layers,and the inhibition effect of DPI was more significant than that of DMTU.(6)Exogenous H2O2 promoted the occurrence of PCD in the aleurone layers by increasing the transcript level of OsNOX8 and the content of endogenous H2O2 significantly.When DPI and DMTU were added into exogenous H2O2,respectively,the effects of exogenous H2O2 were effectively reversed.Compared with the co-treament of DMTU and H2O2,DPI combining with H2O2 treatment had a more significant effect of reversing exogenous H2O2.(7)GA promoted the PCD process of aleurone layers by inducing OsNOX8 gene expression and H2O2 production,which was effectively reversed by DPI.Therefore,OsNOX8 induced by GA regulates the production of H2O2 in the aleurone cells,and then affects the occurrence of PCD.(8)Both DPI and VPE inhibitor Ac-YVAD-CMK significantly inhibited the production of H2O2 in rice aleurone cells,and the addition of DPI into VPE inhibitor had a more significant inhibitory effect,which delayed the occurrence of PCD in the aleurone layers.It is suggested that OsVPE3 regulates PCD by up-regulating the production of H2O2.(9)DPI and VPE inhibitor Ac-YVAD-CMK significantly inhibited the expression level of OsVPE3 and the activity level of VPE.Moreover,the addition of VPE inhibitor into DPI had a more significant inhibitory effect,and delayed the occurrence of PCD in the aleurone layers,which is suggested that HZOZ produced by NADPH oxidase pathway may regulate PCD by up-regulating os VPE3.(10)GA up-regulated the expression level of OsVPE3 and the activity level of VPE,which was reversed by DPI and Ac-YVAD-CMK,respectively.In summary,the normal germination of rice seeds requires the participation of endogenous H2O2,and H2O2 produced by NADPH oxidase pathway plays a key role in regulating the germination of rice seeds.GA induces the up-regulation of OsNOX8 and OsVPE3 in rice aleurone layers,and the PCD of aleurone layers is co-regulated by H2O2 produced by NADPH oxidase pathway and OsVPE3.