The Mechanism Of Cathepsin B Regulating Programmed Cell Death Of Aleurone Layer In The Germination Of Rice Seeds

Programmed cell death(PCD)is an autonomous way of cell death regulated by genes.It is well known that the protein storage vacuoles(PSVs)of aleurone cells fuse to lytic vacuoles(LVs),then the rupture of vacuoles occurs,and finally ends in the form of PCD during the germination of cereal seeds,therefore the PCD of aleurone layers is a key link of cereal germination.It is confirmed that the cathepsin B(Cath B)is associated with the occurrence of PCD in plant.In this study,the seeds of rice(Oryza sativa L.)were used as experimental materials.According to the methods of biochemistry,pharmacology,cell biology and molecular biology,the transcription level of Os Cath B1 in the aleurone layers of germinating rice seeds were detected by real-time fluorescent quantitative PCR technology.The effects of different inhibitors on the vacuolization process of rice aleurone cells were observed and compared.At the same time,the survival rate of rice aleurone cells and their protoplasts was measured and analyzed.Furthermore,the activity changes of Cath B,20 s proteasome βsubunit1(PBA1)and acid phosphatase(ACP)and the transcription level changes of the coding genes were tested.Based on the above tests,the effects of Cath B,PBA1 and ACP on the PCD of rice aleurone and its mechanism were explored.The results inculde:(1)Cath B exists in aleurone cells at the early stage of germinating rice seeds,and its activity level and the transcription level of Os Cath B1 showed a time-dependent change at the early stage of germination.(2)The change trend of Cath B activity in rice aleurone cells was consistent with that of caspase-3.Obviously,both the caspase-3 specific inhibitor Ac-DEVD-CHO and the Cath B specific inhibitor CA074 significantly inhibited the activity of Cath B and the activity of caspase-3,but have no effect on the activity of caspase-1.Therefore,it is speculated that Cath B has an activity similar to caspase-3.(3)The caspase-3 specific inhibitor Ac-DEVD-CHO dramatically inhibited the activity of PBA1 and reduced the protein content of PBA1,while the Cath B specific inhibitor CA07 could not inhibit the activity of PBA1.Thus PBA1 has an activity similar to caspase-3.However,there is no upstream and downstream relationship between PBA1 and Cath B.(4)The Cath B specific inhibitor and the caspase-3 specific inhibitor markedly inhibited the activity of ACP and reduce the protein content of ACP.This result indicated that both the decline of the activities of Cath B and PBA1 and the decrease of their protein contents down-regulate the activity of ACP and its protein content.(5)The changes of the transcription levels of Os Cath B1,Os PBA1 and Os ACP1 in the aleurone cells of water-cultured germinating seeds showed the same trend as the changes of their enzyme activity levels and protein contents.(6)Both CA074 and Ac-DEVD-CHO could significantly inhibited the vacuolation process of the aleurone layer.At the same time,both CA074 and Ac-DEVD-CHO significantly reduced the mortality of rice aleurone layer cells and delayed the germination of rice seeds,indicating that Cath B cooperating with PBA1 to regulated the occurrence of PCD in the the germination of rice aleurone layer.In summary,the above results demonstrated that both Cath B and PBA1 in the germinated rice aleurone layer cells possess caspase-3 activities.In particular,Cath B and PBA1 interact with each other,and coordinate with ACP to regulate the vacuolization process of the rice aleurone layer and the occurrence of PCD,which in turn influence the germination of rice seeds.