The Mechanism Of OsPBA1-mediated The Degradation Of Storage Protein And The Occurrence Of PCD In The Aleurone Layers Of Germinated Rice Seeds

Seed germination is the starting point for plants to enter a new growth cycle.During the germination of cereal seeds,the embryo releases gibberellic acid(GA)to induce the synthesis of a series of acidic hydrolases(such as α-amylase,nuclease,proteases,etc.,)in the aleurone cells,while the storage proteins of aleurone cells are degraded into amino acids by proteases,which is the source of newly synthesized proteins and hydrolases.Subsequently,programmed cell death(PCD)occurred in the aleurone layers.It has been shown that 20 s proteasome β subunit 1(PBA1)is involved in the regulation of plant PCD.In this study,the seeds of rice(Oryza sativa L.)hybrid ’Teyou 128’ and its aleurone layers were used as experimental materials,by means of enzyme-linked immunosorbent assay,real-time fluorescence quantitative PCR,SDS-PAGE analysis and flow cytometry,the activity levels of OsPBA1,20S proteasome and 26S proteasome and the transcription level of OsPBA1 in the aleurone layers of germinated rice seeds were detected,and the changes of storage protein content in the aleurone cells of rice seeds under different treatments were measured,and then the mortality rate of rice aleurone cells was detected.The aim is to explore the mechanism of OsPBA1 regulating the degradation of storage proteins and the occurrence of PCD in the aleurone layers of germinated rice seeds.The results showed that:(1)During the germination of rice seeds,the storage proteins including albumin and globulin were degraded in the aleurone layers,and the activity levels of OsPBA1,20S proteasome and 26S proteasome and the gene transcription level of OsPBA1 were time-dependent.And the peak of OsPBA1 activity and OsPBA1 expression level appeared one day earlier than the peak of 26S proteasome activity,while the peak of 20S proteasome activity appeared one day later than the peak of 26S proteasome activity.It is speculated that there may be a correlation between OsPBA1,20S proteasome and 26S proteasome in regulating the degradation of storage proteins in the germinated rice seeds.(2)The inhibitory effects of caspase-1 specific inhibitor and caspase-3 specific inhibitor on OsPBA1 activity were consistent with their inhibitory effects on caspase-3activity,that is,the activities of OsPBA1 and caspase-3 were not only greatly inhibited by caspase-3 specific inhibitor,but also slightly inhibited by caspase-1 specific inhibitor.It is inferred that the OsPBA1 of rice aleurone layers has caspase-3-like activity,but not caspase-1-like activity.(3)Since the specific inhibition of CathB didn’t inhibit the activity of OsPBA1,there was no upstream or downstream relationship between OsPBA1 and OsCathB,which also had caspase-3-like activity.(4)Exogenous GA could stimulate the activities of OsPBA1,20S proteasome and26S proteasome in turn,while increased the degradation rate of total proteins,albumin and globulin,and accelerated the occurrence of PCD in rice aleurone layers,however,the GA synthesis inhibitor Uniconazole(Uni)reversed the activation of OsPBA1,20S proteasome,and 26S proteasome by GA.(5)The PBA1 activity inhibitor Ac-DEVD-CHO and 20S proteasome activity inhibitor Lactacystin not only inhibited the activities of OsPBA1,20S proteasome and 26S proteasome in the rice aleurone layers,but also hindered the degradation of multiple subunits of albumin and globulin and the occurrence of PCD of aleurone layers,and ultimately delayed the growth of germinated rice seeds.(6)The 26S proteasome inhibitor MG132 could induce the activity of OsPBA1 and20S proteasome.In addition,it inhibited the degradation of high-molecular-weight subunits of albumin and globulin to low-molecular-weight subunits on the one hand,and inhibited the degradation of low-molecular-weight subunits on the other hand,which accelerated the occurrence of PCD in the aleurone layers of rice seeds,resulting in the growth retardation of germinated rice seeds.(7)The inhibitory extent of PBA1 activity inhibitor and 20S proteasome activity inhibitor on the activities of OsPBA1,20S proteasome,and 26S proteasome decreased sequentially.Similarly,the increase extent of exogenous GA on the activities of OsPBA1,20S proteasome,and 26S proteasome,as well as 26S proteasome inhibitor on the activities of OsPBA1 and 20S proteasome also decreased in order,confirming the core position of OsPBA1 in the 20S proteasome and 26S proteasome in the rice aleurone layers.In summary,the activities of OsPBA1,20S proteasome,and 26S proteasome in rice aleurone layers are induced by GA,and the 26S proteasome degradation pathway with OsPBA1 as the core is responsible for the degradation of the subunits of two storage protein,albumin and globulin.OsPBA1 promotes the germination of rice seeds by regulating the process of PCD in the rice aleurone layers.