Regulation Mechanism Of Nitric Oxide On Sorbitol Dehydrogenase Activiyt In Peach Fruit

Fruit quality mainly depends on sugar, acid, protein, fat, vitamin, aroma, mineralelements and so on. Sugar and acid contents and sugar-acid ratio have important effects oninternal quality formation, and are important parameters of determining fruit qualities. UsingFeicheng peach as tested materials, the effects of NO on changes in contents of soluble sugarsand related enzyme activities in peach fruits were studied in this paper. The method ofhigh-performance liquid chromatography （HPLC） method with evaporative light scatteringdetection （ELSD） for separating sorbitol, fructose, glucose and sucrose mixture wasestablished, and the gene encoding sorbitol dehydrogenase was cloned and expressed inEscherichia Coli, the inhibitory kinetics of sorbitol dehydrogenase （SDH） activity by NO wasstudied. Site-directed mutagenesis and circular dichroism spectrum were applied for studyingthe changes in spatial conformation of SDH caused by NO. A possible inhibition mechanismfor SDH activity by NO was also discussed in this paper.It was found that treatment with10μmol L^-1NO significantly decreased the content ofsoluble sugar and the the ratio of sugar and acid in fruit, effectively delayed mutualtransformation between sugars, especially kept high content of sucrose and low contents offructose and glucose, significantly improved the activities of sorbitol dehydrogenase andsorbitol oxidase during storage. Besides, treatment with10μmol L^-1NO also increasedsucrose phosphate synthetase activity, and had time dependent inhibitions for convertingenzyme （acid invertase and neutral invertase） and sucrose synthase （SS） synthesis.Fructose, sorbitol, glucose and sucrose were seperated with HPLC-ELSD. The samplewere separated on a Phenomenex Luna5u NH2100A column （250mm×4.60mm,5micron）eluted with acetonitrile-water （82.5:17.5） as mobile phase. Flow rate:1ml min^-1; columntemperatrue:30°C; injection:20μL. Evaporative light scattering detector was adopted, drifttube temperature was set at82°C, and the carrier gas flow （N2） was set at2L min^-1. Fructose,sorbitol, glucose and sucrose were completely separated. A good linearity was obtained withthe correlation coefficients, R=0.9965-0.9999. LODs （S/N=3） and LOQs （S/N=10） were0.0008⁰.0047μg and0.0020μg⁰.0118μg, recovery （%） was96.05%¹03.32%, RSD（%） was0.27%².06%.Results of qRT-PCR showed that the expression of SDH gene in fruit harvested in60days and90days after flower （DAF） was5.1times and16.5times, respectively, than that infruits in30days DAF. The SDH cloned in this paper was identical with the sequence reportedin GenBank. The recombinant SDH protein expessed in Escherichia Coli had normalcatalytical activity. The optimum conditions for the maximum SDH activity were that, in thecondition of0.4687mg mL^-1recombinant SDH, the temperature of reaction was35°C, thepH was9.0, the sorbitol concentration was0.20mol L^-1, and K_mwas0.6667mol L^-1forsorbitol. It was found that fluorescence intensity of SDH decreased with the increase of NO.Results from circular dichroism spectrum showed that absorption peaks in208nm,218nm,222nm dropped by14.64%²8%,19.78%³5.68%,12%⁴2.24%for SDH treated with lowconcentration NO.It was found that SDH activity was strongly inhibited by NO under the optimum reactionconditions. The type of inhibition by NO was validated on the basis of studying initialreaction rates of SDH-catalyzed oxidation of sorbitol in the presence of NO. The inhibitionreaction was found to follow an apparent non-competitive inhibition by Lineweaver-Burkmethod and the non-competitive inhibition constant Kiwas17.5μmol L^-1. And then, apossible inhibition mechanism for SDH activity by NO was discussed.