Sugar Accumulation During Fruit Development In ’Liuyuezao’ Pummelo And Genes Expression Related To Sugar Component Transformation

’Liuyuezao’ pummelo(Citrus maxima[Burm.]Merr.cv.’Liuyuezao’)is a new early-ripening cultivar bred from a mutant of ’Guanximiyou’ and has gained recognition because of its tender flesh and delicate taste.Composition,content and proportion of sugar in fruit are the key factors that determine fruit quality and commodity value.Research group found the mature ’Liuyuezao’ has a higher sugar content than its parent’Guanximiyou’ which suggested that there are differences in sugar metabolism between two cultivars.At present,the accumulation pattern of soluble sugar during fruit growth and development of ’Liuyuezao’pummelo has not been systematically analyzed yet.In this study,’Liuyuezao’ was selected as the experiment materials,’Guanximiyou’ was as control.The content changes of soluble sugar(sucrose,fructose,glucose)and organic acid(citric acid,malic acid,ascorbic acid,succinic acid,quinic acid,tartaric acid)during fruit growth and development in two cultivars were measured by UPLC to explore the sugar accumulation rule in ’Liuyuezao’.qRT-PCR was used to compare and analyze the expression levels of sugar component transformation related genes in the fruit of two pummelo cultivars.The key differential expressed genes were cloned and sequenced.Subcellular localization of key genes was performed through transient transformation system of tobacco.The induction effect of different concentrations and different soluble sugar components on genes was explored through feeding juice sacs by exogenous sugars,in order to understand the sugar accumulation mechanism during the fruit development of ’Liuyuezao’ pummelo and to provide theoretical basis and practical basis for explaining the fruit flavor and quality differences among different pummelo cultivars.Main research results were as follows:1.Changes of sugar and acid components in ’Liuyuezao’ during fruit growth and development.The accumulation patterns of sucrose were similar between the two pummelo cultivars.Except for 50 DAF,the sucrose content in’Guanximiyou’ was higher than that in ’Liuyuezao’ at the same period.Fructose and glucose contents decreased gradually in ’Guanximiyou’ but a unimodal trend in ’Liuyuezao’ during fruit development and higher than’Guanximiyou’ in ’Liuyuezao’.The total sugar content in ’Liuyuezao’ was higher than that in ’Guanximiyou’ during the whole growth stage.The ratio of sucrose and hexose(fructose and glucose)in ’Liuyuezao’ in maturity stage(125 DAF)was about 1.5:1,higher than that in ’Guanximiyou’ in maturity stage(155 DAF),which was 1:1.The content of citric acid in’Guanximiyou’ was higher than that in ’Liuyuezao’.The ascorbic acid content in ’Liuyuezao’ was always higher than that in ’Guanximiyou’.Citric acid is the main organic acids component in pummelo fruit among six tested organic acids.2.Analysis of gene expression involved in sugar component transformation during fruit development in ’Liuyuezao’ pummelo.Genes involved in sugar component transformation including twelve INVs,six SUSs,four FRKs and three HXKs were screened from the published pummelo genome.The relative expression level of INV6 was the highest among twelve INVs.The expression level of INV6 in ’Liuyuezao’juice sacs on 110,125 and 140 DAF was significantly higher than that in’Guanximiyou’.Among six SUSs,the expression levels of SUS2 and SUS3 were high.On 80,and from 125 to 155 DAF,the expression level of SUS3 in ’Liuyuezao’ was significantly higher than that in ’Guanximiyou’.Except for FRK4 with low expression level,the expression of the other three FRK genes in ’Liuyuezao’ fruits reached the lowest level at maturity stage(125 DAF).In ’Guanximiyou’,the expression levels of FRK1,FRK2 and FRK3 showed an declining trend during fruit development with fluctuating.Compared with ’Guanximiyou’,the expression of HXK2 from 95 to 125 DAF in ’Liuyuezao’ is significantly higher than that in ’Guanximiyou’.In ’Liuyuezao’,the expression levels of almost all INVs,SUSs,FRKs and HXKs were negatively correlated with glucose content,while in’Guanximiyou’ were positively correlated with fructose and glucose.Compared with ’Guanximiyou’,the high expression of INV6 and SUS3 may promote the accumulation of hexose in ’Liuyuezao’ fruits,while the increase of hexose may up-regulate the expression levels of FRK3 and HXK2.Overall,INV6,SUS3,FRK3 and HXK2 are proposed to be the candidate genes for sugar metabolism in pummelo fruit.3.Cloning and analysis of key genes involved in sugar component transformation in ’Liuyuezao’ pummelo fruit.The ORF of INV6,SUS3,FRK3,HXK2 were cloned and sequenced.The ORF lengths of INV6,SUS3,FRK3 and HXK2 were 1527,2130,1146,1497 bp,respectively.INV6 and HXK2 belong to stable acid hydrophilic proteins,SUS3 belongs to unstable acid hydrophilic proteins,FRK3 belongs to stable acid hydrophobic proteins.INV6 mainly consists of extended chain and random curling,of which 50.79%is random curling.SUS3,FRK3 and HXK2 are dominated by α-helix and random curling.All these four genes have a β-angle of around 6-7%.4.Preliminary study on the function of key genes involved in sugar component transformation in ’Liuyuezao’ fruit.Transient transformation of tobacco confirmed that FRK3 and HXK2 were localized in chloroplasts.Exogenous sugar feeding induced the expression of INV6.Induced strength was sucrose>glucose>fructose from large to small.The relative expression level of INV6 reached the maximum after feeding 10mM sucrose for 12 hours,which was 3.85 times that of control.Exogenous sugar feeding induced the expression of SUS3.Induced strength was sucrose>glucose>fructose.The relative expression level of SUS3 reached the maximum after feeding 10mM sucrose for 12 hours,which was 2.39 times that of control.Exogenous sugar feeding induced the expression of FRK3.Induced strength was fructose>glucose>sucrose.The relative expression of FRK3 reached the maximum after feeding 20mM of fructose for 12 hours,which was 3.57 times that of control.Exogenous sugar feeding induced HXK2 expression.The induced strength of fructose and glucose was similar.The relative expression of HXK2 reached the maximum after feeding 20mM glucose for 12 hours,which was 1.63 times that of control.