Isolation And Functional Analysis Of Genes Governing Flavonoid Neohesperidoside Metabolism In Citrus

Citrus fruit flavor is determined by a complex combination of soluble compounds,and as one of the important traits,bitter flavor has attracted much research attention.As one subgroup of flavonoids,neohesperidoside(NEO)is mainly responsible for the ‘primary bitterness' of citrus fruit,and its metabolic differences in citrus fruit have been a hotspot of research in recent years.The complex systematic relationships,abundant mutant and germplasm resources and distinct differences in secondary metabolism make citrus a highly promising material for studying flavonoids.Therefore,it is of great significance to evaluate citrus germplasm resources at both physiological and molecular level,and to clarify the mechanisms underlying the bitterness of citrus fruit.The research was carried in three parts: firstly,the flavonoids(especially flavanone glycosides)in different tissues of sweet orange fruit were qualitatively and quantitatively determined during fruit development;secondly,1,2RhaT(1,2-rhamnosyltransferase gene)from different citrus germplasms was cloned and the relevance between flavonoid accumulation and gene polymorphisms of 1,2RhaT was analyzed,and F1 hybrid progenies were also employed to provide more genetic basis;finally,a metabolic branch with direct competition to bitter NEO was found to be catalyzed by d GlcT,and the functional characterization and expression analysis of d GlcT illuminated the competitive relationship between the accumulation of flavonoid-7-O-di-glucoside and NEO(yielded from 1,2RhaT function)during fruit development.The main results were as follows:1.Two red-flesh(‘Cara Cara' navel orange and ‘Honganliu')and two blonde-flesh(‘Seike' navel orange and ‘Anliu')sweet orange(C.sinensis)were selected as the research materials.LC-MS system together with a standard addition test were used to detected the composition of flavanone glycosides(FGs)and polymethoxyflavones(PMFs)in different fruit tissues of sweet orange,and HPLC system was employed to quantify the flavonoids during different fruit development stages of sweet orange.The results showed that despite of some similar characteristics to NEO,the detectable FGs in sweet orange fruit are non-bitter rutinosides,suggesting that the metabolism of NEO in sweet orange was not genetically inherited as an offspring of pummelo(C.maxima).In addition,sequence analysis revealed that the identical candidate gene 1,2RhaT from sweet orange had a high homology(92%)to Cm1,2RhaT(AY048882),while two frameshift mutations were found at the nucleotide positions of 278 and 1299,which might lead to the absence of bitter NEO in sweet orange.2.The PCR results of 1,2RhaT and its high homologous sequences in different citrus germplasms together with the analysis of citrus gneome databases showed that citrus harbors two types of genes annotated as 1,2RhaT-like.The first type had abundant sequence polymorphisms and was named as d GlcT-1,whose complete coding sequence shared an extremely high homology(amino acid sequence;88%)to the gene of Cm1,2RhaT and only existed in several pummeo cultivars;the other type included 2 genes named as d GlcT-2 and d GlcT-3,which shared 67% amino acid sequence homology to the Cm1,2RhaT and were arranged as tandem repeats in different citrus genome.3.The correlation analysis in the genotypes of 1,2RhaT and 1,2RhaT-like genes(d GlcT-1,d GlcT-2 and d GlcT-3)and the phenotype of naringin accumulation was performed with the genomic DNA of 50 selected germplasms of 3 citrus genus,including Citrus,Poncirus and Fortunella.The results showed only the gene 1,2RhaT was directly related to phenotype of naringin accumulation in Citrus and Poncirus genotypes.In addition,the F1 hybrid progenies(‘Red tangerine' × ‘Trifoliata orange' and ‘Hirado Butun' pummelo × ‘Fairchild' tangelo)of citrus were used for the genetic analysis of naringin biosynthesis.The results showed that the progenies harboring the complete 1,2RhaT sequence had normal naringin sythesis,and otherwise naringin was undetectable,suggesting that 1,2RhaT plays a critical role in the metabolism of naringin in germplasms of citrus genus,Citrus and Poncirus.However,a significant different regularity was found in Fortunella,which can not synthesize naringin and flavanone glycosides even though it has intact 1,2RhaT,suggesting that the lack of NEO is not due to 1,2RhaT.In addition,these results were further supported by the genetic transformation of Cm1,2RhaT from ‘Fenghuangyu'(C.maxima)into the Hong kong kumquat(F.hindisti).4.The above results showed that the candidate genes(1,2RhaT-like)might be incapable of regulating the accumulation of naringin.Therefore,we analyzed the function differences between 1,2RhaT-like proteins and Cm1,2RhaT in tobacco BY2 cells by over-expressing these genes.The biotransformation and feeding assays showed that BY2-Cmd GlcT-1 and BY2-Csd GlcT-2 could effectively catalyze flavonoid-7-O-glucosides into flavonoid-7-O-di-glucosides but not the formation of NEO,a group of flavonoid compounds with bitter taste.In addition,enzymatic reaction tests feeding with various flavonoid aglycone and glucoside substrates showed that Csd GlcT had a broad spectrum of substrates,with flavanones as its preferential substrates rather than flavones and flavonols.Despite of a catalytic ability distinctly different from that of 1,2RhaT,a direct competitive inhibition is apparent in sharing the same substrate of flavanone/flavone-7-O-glucosides.The RNA-seq and q RT-PCR data suggested that a strong competitive inhibition is present in the biosynthesis of NEO or rutinoside with flavonoid-7-O-di-glucosides at the young fruit stage of citrus,leading to the low accumulation of flavonoid-7-O-di-glucosides in this stage.However,the inadequate supply of flavonoid aglycone substrate may cause the interruption of flavonoid glycoside biosynthesis during fruit maturation of citrus.The above results about the metabolic mechanism of NEO especially naringin contribute to a solid theoretical foundation for understanding the ‘primary' bitterness forming mechanism in citrus fruit.Furthermore,the discovery of d GlcT will provide an indirect way to regulate the accumulation of bitter NEO and control the bitterness of pummelo fruit.