Regulation By Nitric Oxide On Softening And Mitochondrial Single-stranded Binding Protein In Peach Fruit

Fruit softening is closely related to the degradation of cell wall polysaccharides,and sufficient energy supply can delay fruit softening and inhibit cell wall degradation.Mitochondria are important organelles for energy metabolism in cells and an important place for the generation and accumulation of reactive oxygen species(ROS).ROS induces oxidative stress in mitochondria and leads to damage and mutation of mitochondrial DNA(mtDNA),resulting in mitochondrial senescence and apoptosis.Mitochondrial single-stranded binding protein(mtSSB)is an important protein involved in mitochondrial DNA replication and damage repair,but its mechanism of action in plants is unclear.Nitric oxide(NO)is an important reactive nitrogen in living organisms and mediates various physiological functions in plants.Studies have shown that NO is involved in regulating the physiological functions of mitochondria.Exogenous NO at an appropriate concentration can reduce the oxidative damage of mitochondria,maintain the basic function of mitochondria,and thus maintain the storage firmness of fruits.In this paper,"Xintaihong"peach fruits were used as the experimental material,and soaked with double distilled water,15?mol?L^-1 NO and 20?mol?L^-1 c-PTIO(NO scavenger),respectively.The effects of different treatments on cell wall metabolism,mitochondrial energy metabolism,mitochondrial ROS content,and mtSSB(a key factor in mtDNA replication)were studied in peaches storage at 0°C.The conclusions were as follows:(1)NO treatment inhibited the decrease of CDTA-pectin,Na₂CO₃-pectin,cellulose and hemicellulose and the increase of H₂O-pectin by inhibiting the activities of polygalacturonase,pectin methylesterase,?-galactosidase,cellulase,xyloglucan endotransglycosylase and glucosidase,thereby inhibiting the hydrolysis of the peach fruit cell wall,maintaining the firmness of the fruit during storage,and prolonging the fruit storage time.(2)NO treatment maintained the mitochondrial respiratory control rate,reduced the oxygen consumption and content of cytochrome c,reduced the content of ROS(superoxide anion,hydrogen peroxide and hydroxyl radical),and increased activities of H⁺-ATP synthase and Ca²⁺-ATP synthase and the activities of pyruvate dehydrogenase,citrate synthase,aconitase,isocitrate dehydrogenase,?-ketoglutarate dehydrogenase,succinate thiokinase,succinate dehydrogenase,fumarate and malate dehydrogenase in tricarboxylic acid cycle.NO treatment also maintained mitochondrial DNA copy number as well as mitochondrial DNA integrity.It was shown that NO can reduce mitochondrial oxidative damage and maintain mitochondrial function by regulating the content of ROS in mitochondria,and at the same time reduce mitochondrial DNA damage,alleviate mitochondrial damage and apoptosis to improve mitochondrial energy metabolism.(3)The mtSSB gene in peach fruit was cloned by reverse transcription-polymerase chain reaction(RT-PCR)technology,the full-length CDS region of 648 bp was obtained,and it encoded 215 amino acids.Biological analysis of the mtSSB sequence showed that the molecule of this protein is C₁₀₄₆H₁₆₇₈N₃₀₂O₃₂₁S₆.It was hydrophilic,and mainly composed of?-helix and irregular coils,and was a tetrameric structure with no transmembrane region and signal peptide region.And it had the high homology with almond(Prunus Dulcis)and sweet cherry(Prunus avium),and the targeting organelle was mitochondria.The recombinant protein constructed in vitro was successfully expressed in BL21(DE3).At the same time,the results showed that NO treatment upregulate mtSSB gene expression and increase mtSSB copy number.