Studies On Salt Stress-Induced Programmed Cell Death In Rice Root Tip Cells And Related Mitochondrial Proteome Changes

Salt stressed rice root tip cells were used to investigate the changes of reactive oxygen species(ROS) and antioxidant enzymes at the early stages of programmed cell death(PCD). The results indicated that 500 mmol/L NaCl could lead to specific features of PCD in root tip cells,such as DNA ladder,nuclear condense and deformation,and transferase mediated dUTP nick end labeling positive reaction,which were initiated at 4 h of treatment and progressed thereafter.Cytochrome c release from mitochondria into cytoplasm was also observed,which occurred at 2 h and was earlier than the above nuclear events.In the very early phase of PCD,an immediate burst in hydrogen peroxide and superoxide anion production rate was accompanied by two-phase changes of superoxide dismutases and ascorbate peroxidase.A short period of increase in the activity was followed by prolonged impairment.The results indicated that salt can induce PCD in rice root tip cells,and in the early phase of rice root cell PCD,salt stress-induced oxidative burst increased the antioxidant enzyme activity,which,in turn,scavenged the ROS and abrogated PCD.Also, when the stress is prolonged,the antioxidant system is damaged and accumulated ROS induces the PCD process,which leads to cytochrome c release and nuclear change.In the above study,salt stress-induced PCD model was established in rice root tip cells. Next,by using rice seedling Wuyunjing 8^th,effects of lanthanum(inhibitor of calcium channel) on salt stress-induced PCD early events were studied.The results indicated that low concentration(10μmol/L),but not high concentration(100μmol/L) of LaCl₃ could effectively prevent salt stress-induced PCD.Further study demonstrated that in the early stage of salt-induced PCD process,10μmol/L of La³⁺ could prevent the increase of cytoplasmic calcium level,inhibit ROS production,and enhance the ROS-scavenging enzymes activities such as SOD and APX.20 mmol/L of imidazole,the inhibitor of NADPH oxidase,could alleviate PCD occurrence obviously,and such alleviation could be enhanced by addition of La³⁺,indicating the involvement of NADPH oxidase in salt stress-induced PCD process.Taken together,lanthanum could prevent salt stress-induced PCD occurrence in the rice root tip cells by blocking calcium influx under stress,which was followed by inhibiting calcium-dependent NADPH oxidase activity to prevent O₂^—production and,enhancing the cytosolic antioxidative enzyme activities to scavenge the reactive oxygen species.In order to found more mechanisms functioned under salt stress-induced PCD in rice root tip cells,the role of mitochondria during the early phase of PCD(2h-PCD) was analyzed in rice roots.After 2h-PCD inductions,the integrity of mitochondria slightly decreased,which was consistent with the little release of cytochrome c.2h-PCD partially inhibited electron transport,resulting in oxidative burst in mitochondria.However,ATP production is maintained constant.In order to reveal the molecular basis of this difference,soluble proteins and electron transport chain complexes of mitochondria were analyzed by IEF/SDS-PAGE and BN/SDS-PAGE two-dimensional gel electrophoresis,respectively.Both the two methods demonstrated that the complexâ… ,â…¢,andâ…£in electron transport chain were down-regulated in response to 2h-PCD.Interestingly,ATP synthase was up-regulated during the early phase of PCD,which was verified by immunoblotting.This suggested that the increases of ATP synthase subunits perhaps are the basis of higher ATP level at the early PCD induction stage.