The Role Of Mitochondrial Membrane Channel Protein VDAC And Its Regulation Mechanism During Aging Of Elm(Ulmus Pumila L.)Seeds

Seed is an important carrier for germplasm conservation and plays an important role in the continuation of species,but the seed viability gradually declines after maturation,which is called seed ageing.Previous studies in our laboratory have found reactive oxygen species(ROS)mediated programmed cell death(PCD)in the process of seed aging,but the specific mechanism is still unclear.It has been reported that mitochondrial membrane voltage-dependent anion channel(VDAC)is an important element in mitochondrial pathway PCD,and plays a key role in initiating cell death.In this study,elm(Ulmus pumila L.)seeds were used as experimental materials to study the role of VDAC in seeds aging process and its regulatory mechanism.The main results are as follows:During the aging process of elm seeds,mitochondrial morphology and function changed with ROS eruption.The results of ROS donor and inhibitor pre-treatments showed that the accumulation of ROS was tightly related to the decrease of seed vigor.Confocal microscopy with mitochondrial specific fluorescence probe showed that the aggregation of mitochondria in early aging stages was related to the production of ROS.The mitochondrial proteome was then analyzed by two-dimensional electrophoresis(2-DE),and 37 proteins were identified by mass spectrometry,whose abundance changed with the ROS eruption and the aggregation of mitochondria.Among these proteins,a large number of proteins related to TCA cycle,protein metabolism and antioxidation were significantly down-regulated during aging.It was further found that changes of 16 proteins were in response to ROS,including VDAC.Thirteen proteins including VDAC were identified increased in carbonylation modification level.In vitro experiments showed that carbonylation modification caused the functional changes of VDAC channels.Biochemical tests showed that the up-regulation of VDAC expression and carbonylation modification were accompanied by the release of mitochondrial cytochrorne c(Cyt c).In conclusion,we speculate that ROS-induced changes in VDAC function may be an intermediate link in ROS-mediated mitochondrial pathway apoptosis.Pharmacological studies of elm seeds and CDT experiments on transgenic Arabidopsis seeds confirmed the key role of UpVDAC3 channel function in seed aging.Using GST pull down assay,glyceraldehyde-3-phosphate dehydrogenase(GAPDH)was screened to interact with UpVDAC3 during seed aging.The interaction between VDAC and GAPDH in elm seeds at different aging stages was further analyzed by Co-IP and Duolink PLA,and it was found that the interaction between VDAC and GAPDH increased after seed aging and peaked at 2 days.Dot blotting analysis showed that glutathionylation modification of UpGAPDH1 promoted its interaction with UpVDAC3.Western blotting and PLA detection showed that the glutathionylation level of GAPDH increased after seed aging,and also peaked at 2 days.In order to further explore the interaction between these two proteins,the glutathionylation sites of UpGAPDH1 were analyzed.Biochemical analysis and transgenic experiments combined with site-directed mutagenesis assay showed that the interaction between glutathionylated UpGAPDH1 and UpVDAC3 could induce the release of mitochondrial Cyt c and cell death.The reason for glutathionylation of UpGAPDH1 may be the elevation of GSH and ROS levels caused by the elevation of free metals.These results suggest that the interaction between GAPDH and VDAC mediated by glutathionylation may have participated in the gated regulation of VDAC.Studies have shown that metal-binding proteins are more prone to carbonylation.Metal-binding proteins in mitochondria of elm seeds were captured by IMAC metal affinity chromatography and compared with the proteins with increased carbonylation level during the aging process of elm seeds.The results showed that metal-binding proteins were the main target for carbonylation modification of mitochondrial proteins.Inaddition,VDAC was found to have metal-binding abilities to Fe,Cu and Zn.Biochemical analysis showed that H204A-and HZ19A-UpVDAC3 lost metal binding ability and showed insensitivity to metal induced carbonylation modification.In order to verify the effect of carbonylation modification of VDAC on cell death during seed aging,normal and metal-binding site mutant UpVDAC3 were overexpressed in yeast and Arabidopsis respectively,and oxidative stress or seed aging experiments were carried out.The results showed that the mutation of metal-binding site could alleviate the effect of UpVDAC3 overexpression on cell death and seed aging.In addition,the interaction between UpGAPDHl and UpVDAC3 makes the latter more sensitive to metal-induced carbonylation modification,suggesting that the carbonylation modification of VDAC may also participate in the gated regulation of VDAC.These studies suggest that ROS may regulate the activity of VDAC channel and induce the release of apoptotic factors in mitochondria through the oxidative modification induced interaction between VDAC and other proteins or the oxidative modification of VDAC itself during the aging process of elm seeds.