| Plant mitochondria play a central role in metabolism and various biosynthetic reactions,such as oxidative phosphorylation(OXPHOS),a pathway in which nutrients are oxidized to form adenosine triphosphate(ATP),to meet the structural demands of growth and development.Under normal physiological conditions,an estimated 0.2%-2%of the total oxygen consumed during electron transport is not reduced to water,but due to electron leakage in complexes I-III of the respiratory chain.This results in the production of reactive oxygen species(ROS),such as superoxide and hydrogen peroxideStudies in Arabidopsis thaliana have shown that ROS levels in chloroplasts and mitochondria are co-regulated by the chloroplastic stromal APX(sAPX),in addition,cytosolic APX(cAPX)is assisted by cross-compartment protection.However,the regulatory mechanism of mitochondrial ROS level in woody plants is not clear enough.This study discusses the specificity of subcellular localization of PtomitAPX by analyzing the secondary structure of protein;the expression profile of PtomitAPX is analyzed by qRT-PCR;observation of morphological defects in transgenic plants and cells to explore the main role of PtomitAPX in plant growth and development;furthermore,the effect of the gene on mitochondrial function and mitochondrial ROS level is observed by CLSM;moreover,the oxidation degree,the content of H2O2,the content of non-enzymatic reductant,the content of ATP and the activities of respiratory complexes of isolated high purity mitochondria are measured in vitro;detection of apoptosis in transgenic suspension cell lines by flow cytometry;in addition,through comparative analysis of transcriptome data of wild type and transgenic cell lines,the role of the gene in plant antioxidant system and growth and development is further explored,which makes an important foundation for further revealing the antioxidant mechanism of plant mitochondria.The main results of this study are as follows:1.The results of secondary structure analysis of PtomitAPX and PtosAPX show that there are significant differences in the 19 N-te(H2O2).Ascorbate peroxidase(APX)is one of the key enzymes that scavenge H2O2 in plant cells.rminal portion of the signal peptide between two proteins;the signal peptide of PtomitAPX has the characteristics of mitochondrial localization:which is rich in positively charged amino acid residues(lysine,arginine,histidine),and these positively charged amino acid residues are concentrated in the alpha helix;PtosAPX signal peptide has the characteristics of chloroplastic localization;PtosAPX has only one positively charged amino acid,and the 19 N-terminal portion of the signal peptide has no alpha helix.2.In this study,the expression profiles of PtomitAPX,PtosAPX and PtotAPX are analyzed by qRT-PCR under hydrogen peroxide stress,salt stress,high temperature stress,drought stress,low temperature stress and high light stress.PtomitAPX can respond to oxidative stress,salt stress and high temperature stress,while PtosAPX mainly respond to high light stress;the activity of PtomitAPX is not significantly different from that of PtosAPX in the study of enzymatic kinetics;the content of APX in mitochondria detected by ELISA shows that the content of PtomitAPX is significantly higher than that of PtosAPX.Therefore,PtomitAPX plays a major role in mitochondria,and the expression profile of PtosAPX is similar to that of chlAPX,while the expression profile of PtomitAPX has mitochondrial characteristics.3.Severe growth retardation is observed in antisense transgenic plants,and slight growth retardation is observed in overexpressed transgenic plants,requiring additional 6-BA to grow normally;results of confocal laser scanning microscopy(CLSM)show that the mitochondrial morphology of antisense and overexpressed transgenic plants is abnormal and the membrane potential difference is decreased.4.Antisense and overexpressed transgenic suspension cells show different degrees of growth retardation;the results of TEM and CLSM show,in mitochondria of antisense transgenic cells,the mitochondrial morphology is abnormal,the membrane potential difference is decreased,the motor ability is lost,the ATP content is decreased,and the respiratory complex activities are decreased;the results of CLSM and mitochondrial physiological detection show that ROS burst and oxidation increased in mitochondria,suggesting that the structural and functional abnormalities of mitochondria in antisense transgenic cells are caused by oxidative damage to mitochondria;mitochondrial structure and function of overexpressed transgenic cells are abnormal,mitochondrial ATP content is decreased,and mitochondrial respiratory complex activities are not significantly different from that of wild type,suggesting that the abnormal mitochondrial structure and function of overexpressed transgenic cells is caused by the lack of H2O2 content;the addition of exogenous AsA and H2O2 alleviate the abnormality of mitochondrial structure and function of antisense and overexpressed suspension cells,respectively;the results of flow cytometry show that the number of living cells in antisense transgenic cell lines decreased significantly under untreated conditions compared with wild type cells;exogenous H2O2 can aggravate this phenomenon,while exogenous AsA can partly alleviate this phenomenon;the number of living cells in overexpressed transgenic cell lines remained at a high level(?98%).5.The results of RNA-seq analysis show that a large number of genes related to phosphorylation are down-regulated in antisense and overexpressed transgenic cell lines under untreated conditions,indicating that the change of PtomitAPX expression has an impact on mitochondrial function;the phosphorylation of antisense transgenic cell lines is significantly restored after AsA treatment,while that of overexpressed transgenic cell lines is significantly restored after H2O2 treatment.Our results indicate that PtomitAPX acts as an irreplaceable controller for preventing ROS accumulation,which affects the structure and function of mitochondria as well as plant growth. |
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