Regulatory Mechanisms By Nitric Oxide On Hexose Metabolism And Hexokinase In Mitochondria Of Postharvest Peach Fruit

Hexokinase?HK?can interact with mitochondrial permeability transition pore?MPTP?to regulate the opening of MPTP,which is involved in mediating apoptosis,but its mechanism of effect in plants is still uncertain.As a small molecule of plant physiological signal,nitric oxide?NO?mediates many physiological activities in plants such as regulation of mitochondrial function,respiration and glycolytic metabolism.Exogenous NO can delay fruit senescence and prolong fruit storage.Studying the regulation by NO on HK protein and hexose metabolism was helpful to understanding the roles of HK and hexose metabolism in the postharvest preservation of Feicheng peach fruits.It was important for clarifying the mechanism of how NO extend fruit storage.In this paper,Feicheng peach was used as the experimental material.By designing primers and gene cloning,the part of consensus sequence of the HKII gene searched from the NCBI was completely consistent with the sequenced Feicheng peach sequence.Based on the RACE experiment and nested PCR,the full-length HKII gene had 1835 bp including complete reading frame with 1496 bp and encoding 497 amino acids.The bioinformatics analysis showed that the HKII protein was a hydrophobic transmembrane protein consisting of 15 segments?helices and 11 segments of?-sheets and random coils with molecular weight of 50 kDa.The homologic and phylogenetic analysis of amino acid sequence showed that HKII protein had highest homology with the loquat?96.98%?and was highly conservative.The TMHMM predicted that HKII was a transmembrane protein mostly outside the membrane.The result of fluorescence spectra showed that NO quenched the fluorescence of HKII protein through a static process.NO could bind HKII strongly with a 1:1 molar ratio.Analysis of the glutathionylation and nitration of the HKII gene revealed 4 cysteines and 12 tyrosines were exposed on the surface of the protein.According to the bond energy analysis,the glutathionylation reaction was easier to happen than the nitration.The physiological and biochemical studies about the hexose metabolism of Feicheng peach fruits showed that sucrose content in peaches treated with 15?mol L^-1 NO was declined significantly and the activities of AI,NI,SS in decomposition,HK,FRK of 15?mol L^-1 NO treated peaches were increased in the early stage of storage.Exogenous NO increased the rate of sucrose consumption and glycolysis to provide energy for respiratory climacteric.Hexose content,the activities of HK,PGI,gene expression of HK in peaches treated with 15?mol L^-1 NO declined significantly and the activities of FRK,SPS increased.However,treatment of c-PTIO increased the contents of fructose and glucose and the activities of SPS and PGI.The study indicated that NO limited the rate of fruit glycolysis at the late stage of storage and prolonged the shelf life of peaches.