Effects Of Mitochondrial Damage And Cytochrome C Release On Programmed Cell Death In Wheat Endosperm Under Waterlogging Stress

Preliminary studies showed that the development of endosperm in wheat（Triticum aestivum L.）undergo programmed cell death（PCD）process,and waterlogging can accelerate its progress.In order to further reveal the physiological and cytological mechanisms of the advancement of PCD process in wheat endosperm cells under waterlogging stress,the experiment used Huamai 8 as material,which was waterlogged on the day of flowering.The dynamic changes of mitochondria in endosperm cells were studied at microscopic and ultramicroscopic level.The release of cytochrome c in endosperm was studied using immunofluorescence and Western blot techniques.The gene expression of mitochondrial permeability transition pore（MPTP）in endosperm was studied by real-time quantitative PCR.In addition,the effects of ascorbic acid,cyclosporine A,and hydrogen peroxide treatment on the physiological and biochemical and PCD processes of highly waterlogging tolerant cultivar Huamai 8 and waterlogging susceptible cultivar Huamai 9 were studied.1.0 DWT（days waterlogging treatment）,the ultrastructure showed that the mitochondrial structure in the early developmental endosperm cells was intact,the cristae were clear,the mitochondria were slightly deformed at 12 DAF（12 days after flowering）,the cristae was fuzzy,and most of the mitochondria were degraded and only a small amount of mitochondria on the edge of the cell at 20 DAF.The results of mitochondrial membrane potential and membrane absorbance showed that the mitochondrial membrane potential decreased,membrane permeability increased,and mitochondrial damage increased with the development of endosperm cells.7 DWT,part of the mitochondria in the endosperm cells expanded significantly at 10 DAF,and most of the mitochondria showed electron-transparent regions within the mitochondria,and undegraded mitochondria surrounded by the envelope structure at DAF.Mitochondrial membrane potential and membrane absorbance was lower than the no-waterlogging treatment group at the same period.The mitochondrial of normal growing wheat endosperm cells is gradually destroyed along with the development of the endosperm PCD,and the damage is exacerbated by waterlogging stress and thetime is advanced.2.Under normal growth conditions,immunofluorescence results showed that cytochrome c localized mainly on mitochondria in endosperm cells at 10 DAF and cytochrome c released from mitochondria to cytoplasm at 12 DAF,15 DAF.Cytochrome c was further released at 20 DAF.Ultracytochemical localization of cytochrome c was found on the mitochondria at 8 DAF,and cytochrome c could be detected both mitochondria in vitro and in vivo at 15 DAF.It was shown that cytochrome c content gradually increased in the cytoplasm of endosperm cells after12 DAF by WB.After waterlogging treatment,immunofluorescence and Western blotting showed that cytochrome c released from mitochondria to cytoplasm was observed at 10 DAF,and the cytochrome c released content was higher than that in the omitted waterlogging treatment at the same time.Cytochrome c was released from mitochondria to cytoplasm at 12 DAF in normal wheat endosperm cells.Under waterlogging stress,cytochrome c released from mitochondria at 10 DAF and released more than the same period.Waterlogging stress resulted in the earlier release of cytochrome c and increased release.3.The results of real-time quantitative PCR showed that the expression of VDAC2 in wheat endosperm cells remained almost unchanged,the expression of VDAC3 increased first and then decreased,the expression of ANT1 decreased gradually,and the expression of protein folding related genes HSP70 and HSP90 decreased first and then increased.Under waterlogging treatment,the expression of VDAC2 and VDAC3 increased significantly at 8 DAF,and the expression of ANT1 peaked at 10 DAF,and the expression of HSP70 and HSP90 genes were lower than that of the no-waterlogging group.MPTP may participate in the regulation of PCD in endosperm cells under waterlogging stress,and that the protein folding disorder may not be the primary cause of mitochondrial impairment under waterlogging.4.After treatment with exogenous H₂O₂,compared with the control group,the content of H₂O₂ and O^2-in Huamai 8 increased,SOD and CAT activity increased,mitochondrial membrane permeability increased,cytochrome c release increased,and caspase-like protease activity increased.The change trend in Huamai 9 was similar,but compared with Huamai 8,the O^2-accumulation was more and the CAT activity in the cells was smaller.After treatment with exogenous ascorbic acid,H₂O₂ content in Huamai 8 and Huamai 9 decreased.After treatment with exogenous cyclosporin A,partial inhibition of increased ROS content,mitochondrial membrane permeability.Release of cytochrome c and caspase-like protease activity increased which caused by H₂O₂ treatment.The above results show that exogenous H₂O₂ treatment exacerbates the PCD of wheat endosperm cells,and more ROS accumulation in Huamai 9endosperm,the antioxidant enzyme system is weaker,probably it is one of the mechanism of no waterlogging resistance.In summary,waterlogging stress exacerbates mitochondrial damage and cytochrome c release in wheat endosperm cells,which may be one of the reasons that waterlogging exacerbates PCD in the endosperm cells.Mitochondria play an important role in the regulation of PCD of wheat endosperm cells under normal growth and waterlogging stress.The accumulation of ROS and the weak antioxidant system in Huamai 9 endosperm may be one of the reasons for its poor resistance to waterlogging.