Dynamic Changes Of Sugar And Organic Acid And Expression Analysis Of Metabolic Related Genes In ’Lane Late’ Navel Orange Fruit During Development

‘Lane Late’navel orange(Citrus sinensis(L.)Osb.)is a late maturing variety of Washington navel orange.However,due to the short cultivation time in China,there are few studies on fruit quality of‘Lane Late’Navel Orange,especially on organic acid metabolism.In this study,‘Lane Late’Navel Orange and‘Newhall’Navel Orange were used as the main materials to analyze the changes of sugar and acid during fruit development,and the genes related to organic acid accumulation in fruits were analysed by q PCR;At the same time,the effects of low temperature on the accumulation of organic acids in fruits were studied.And the results of this study are summarized below:1.Changes of total soluble solids and organic acid during Fruit DevelopmentWe measured the content of total soluble solids and organic acid during fruit development.It was found that the content of soluble sugar increased to 11.3% with fruit development and then stabilized;The organic acid content decreased to 0.85% with fruit development and then stabilized.but the organic acid content of‘Lane Late’navel orange was significantly higher than that of‘Newhall’Navel Orange before ripening.At the same time,citric acid is the main organic acid accumulated by‘Lane Late’Navel Orange during fruit development,and the difference of organic acid content between‘Lane Late’navel orange and‘Newhall’navel orange is mainly caused by the difference of citric acid content.2.Screening and expression analysis of citric acid metabolism related genesThrough homologous sequence alignment and gene annotation screening,a total of15 genes encoding citric acid metabolism related enzymes were finded(CsACO1,CsACO2,CsACO3,CsPEPC1,CsPEPC2,CsCS1,CsCS2,CsNADP-IDH1,CsNADP-IDH2,CsNADP-IDH3,CsGAD1,CsGAD2,CsGAD3,CsGS1,CsGS2).q RT-PCR analysis of these genes and some previously reported genes(CsPH8,CsCIT1)showed that the expression of CsNADP-IDH2 and CsGS1 in‘Lane Late’navel orange was significantly lower than that in‘Newhall’navel orange,while the expression of CsPH8 in‘Lane Late’navel orange was significantly higher than that in‘Newhall’navel orange in most periods;The combined action of CsNADP-IDH2,CsGS1 and CsPH8 may lead to the high accumulation of organic acids in ‘ Lane Late ’ Navel Orange during fruit development.3.Effect of low temperature on citric acid accumulation of‘Lane Late’navel orange and its mechanismThe citric acid content of ‘ Lane Late ’ navel orange after 15 days of low temperature treatment was significantly lower than that after 15 days of normal temperature treatment,indicating that the citric acid degradation rate of‘Lane Late’navel orange was accelerated at after low temperature treatment;The expressions of CsNADP-IDH1 and CsGAD2 were significantly up-regulated after low temperature treatment,indicating that these two genes may be involved in the accelerated process of citric acid degradation at low temperature.The promoters of CsNADP-IDH1 and CsGAD2 in‘Lane Late’navel orange were cloned and analyzed.It was found that the promoter sequences of CsNADP-IDH1 and CsGAD2 were consistent with those in the sweet orange genome,and contained LTR,MYB,MYC and other elements related to low temperature response,which may respond to low temperature induction through the action of corresponding transcription factors.The CDS of CsNADP-IDH1 and CsGAD2 in ‘ Lane Late ’ navel orange were cloned.We found that CsNADP-IDH1 had an Ic D domain and was a typical isocitrate dehydrogenase,while CsGAD2 had a Gad domain and was a typical glutamate decarboxylase;CsNADP-IDH1 could be clustered with At NADP-IDH1 in Arabidopsis,and CsGAD2 could be clustered with At GADs.The transient transformation experiment of sweet orange callus showed that the citric acid content of sweet orange callus overexpressing CsNADP-IDH1 and CsGAD2 decreased significantly;While the expression level of CsNADP-IDH1 and CsGAD2 were significantly negatively correlated with citric acid content during the degradation period of citric acid in‘Lane Late’navel orange and‘Newhall’navel orange.These evidences together suggest that‘Lane Late’navel orange accelerates the degradation of citric acid in fruit by up regulating the expression of CsNADP-IDH1 and CsGAD2 under low temperature.