Identification And Functional Analysis Of Sugar Transporter Genes,PpTST1 And MdSUT4.1 Involved In Fruit Sugar Accumulation

Sugar content is a key determinant factor of fruit taste and therefore the genetic modification of fruit sugar content is a primary target of fruit crop breeding programs.For fruits,most of the soluble sugars are stored in the vacuole,which suggests that tonoplast sugar transporters play an important role in fruit sugar accumulation.In this study,two vacuolar sugar transporter genes were identified and analyzed to participate in fruit sugar accumulation in peach and apple using both forward and reverse genetics approaches.The main results are as follows:Firstly,we reported the identification and functional analysis of a tonoplast sugar transporter gene controlling fruit sugar content in peach.Investigation of fruit sugar component and content of 61 peach cultivars revealed that sucrose is the predominant sugar in ripe peach fruits.Based on the annotation of peach reference genome,a sugar transporter gene?Prupe₅G005300?,designated PpTST1,was identified to be located in a previously reported QTL interval on Chr5 controlling fruit sucrose content.RT-qPCR analysis showed that the expression profile of PpTST1 exhibited a consistency with sugar accumulation pattern.In addition,a nonsynonymous G/T variant in the third exon of PpTST1 was identified,and it was used to develop a dCAPS marker.Genotyping of peach cultivars with this dCAPS marker revealed that cultivars with either G/T or T/T genotypes had significantly higher sucrose and total sugar content than cultivars with the G/G genotype.Subcellular localization analysis showed that PpTST1 was located in vacuolar membrane.Furthermore,the repression of PpTST1 gene using VIGS significantly inhibited sugar accumulation in peach fruits.All these results demonstrated PpTST1 participated in peach fruit sugar accumulation.Secondly,a sucrose transporter gene MdSUT4.1 was identified to play an important role in fruit sugar accumulation in apple.There were six MdSUT members in the apple genome,and they were classified into three subgroups,SUT1,SUT2 and SUT4,according to phylogenic tree analysis.Subsequently,we developed SSR marker for all MdSUT genes and conducted candidate gene-based association study among353 accessions.As a result,only a SUT4 member,MdSUT4.1 was significantly associated with fructose content in apple fruit.All testing accessions were divided into three genotypes:?AG?_9/9,?AG?_6/9,and?AG?_{6/6 or 11},based on the?AG?_n microsatellite located 15 bp upstream of the start codon of MdSUT4.1.The mean value of both fructose and glucose content were significantly lower in ripe fruits of the first two genotypes compared to the last genotype.Subcellular localization analysis showed that MdSUT4.1 was located to vacuolar membrane.Overexpression of MdSUT4.1 in strawberry and apple callus had an overall negative impact on sugar accumulation.Hence,all these results indicated that MdSUT4.1 is involved in the regulation of fruit sugar accumulation in apple.Lastly,we developed a fructose sensor.Based on the principle of FRET and the backbone of sugar sensor,we designed Cra fructose sensor.Then we removed its DNA binding domain and kept pocket like sugar binding domain to develop FLIP_Cra-N66AA.The monitoring results of fluorescent signal change induced by fructose addition in vivo and in vitro indicated that FLIP_Cra-N66AA could be used for fructose signal testing.In addition,FLIP_Cra-N66AA could act as the template for optimizing fructose sensor,which can be used to test sensor efficiency in E.coli.Altogether,our study identified two sugar transporter genes involved in fruit sugar accumulation and their tagged molecular markers were developed as well.These results are not only helpful for understanding the mechanisms underlying fruit sugar accumulation,but provide molecular tools for genetic improvement of fruit sweetness in future breeding programs of peach and apple,as well as other fruit tree crops.