The Mechanism Of OsVPE3-and Microfilaments-Mediated Programmed Cell Death In Rice Aleurone Layers

Programmed cell death(PCD)throughout the plants' growth and development process,is a genetically regulated and autonomously well-ordered way of death.PCD in cereal crops' aleurone layers is a crucial part of seed germination.The structure changes of microfilaments(MFs)influenced the vacuolar morphology during the process of PCD in cereal aleurone layers.Importantly,the activated activity and regulated expression levels of vacuolar processing enzyme(VPE)play significant roles in the morphological changes of vacuoles and the process of vacuolization.In this study,to explore the effects of OsVPE3 and MFs on the PCD of aleurone layers,rice(Oryza sativa.L)'you?128' seeds were chosen as experimental materials.The morphological changes of MFs and vacuoles were detected,meanwhile,investigating and analyzing the expression levels of OsVPEs,the activity level of VPE and the survival rate of aleurone layers in rice by means of pharmacology,biochemistry,cell morphology and molecular biology,as well as the methods of fluorescent tags and fluorescence staining,combining with qRT-PCR and laser scanning confocal microscope(LSCM)technology.The results indicated that:(1)the relative expression level of OsVPE3 is the highest in four OsVPEs which are related to the germination of rice seeds.OsVPE3 is the OsVPE that play a relatively significant role in the germinating process of rice seeds.The relative expression levels and activity of OsVPE3 in rice aleurone layers were significantly higher than that of embryo and endosperm,and the relative expression levels of OsVPE3 also changed subsequently in the germination.(2)The activity of OsVPE3 showed almost same variation trend with that of caspase-1.The caspase-1 specific inhibitor Ac-YVAD-CMK inhibited effectively the activities of VPE,caspase-1 and caspase-3,and caspase-3 specific inhibitor Ac-DEVD-CHO only inhibited the activity of caspase-3,namely OsVPE3 with the caspase-1-like activity instead of caspase-3.Therefore,it is speculated that OsVPE3 may regulate the process of PCD via caspase-3-like activity in the aleurone layers of rice.(3)GA promoted the occurrence of PCD through up-regulating the relative transcript levels and activity levels of OsVPE3,conversely,ABA down-regulated the relative transcript and activity levels of OsVPE3.Therefore,there is an antagonism between GA and ABA,and ABA reverses the effect of GA on promoting the death of aleurone cells,then delays the process of PCD.(4)Morphological and structural changes of MFs,namely the depolymerization and polymerization of MFs,occur in the whole process of vacuoles fusion.Promoting or inhibiting the depolymerization of MFs accelerated or delayed the process of vacuoles fusion.Meanwhile,inhibiting the VPE activity delayed the depolymerization of MFs,subsequently inhibited the vacuoles fusion.Thus,VPE regulated the fusion of the vacuoles in the rice aleurone layers by mediating microfilaments.(5)Promoting the depolymerization of MFs contributes to induce the up-regulation of the relative transcript level of OsVPE3 and activity level of VPE,inhibiting the VPE activity or caspase-l-like activity reverses the up-regulated effect of MFs depolymerization on the VPE activity,and further delaying the process of PCD in rice aleurone layers.It is speculated that MFs-mediated OsVPE3 regulate the PCD of aleurone layers in rice.All above results confirmed that both of OsVPE3 and MFs accelerate the programmed cell death in rice aleurone layers.GA induces OsVPE3 in the aleurone layers of rice,and the OsVPE3 further activates caspase-3-like.Furthermore,OsVPE3 regulates the PCD process of rice aleurone layers by interacting with MFs.