Functional Characterization Of CitF3’H Involved In The Hydroxylation Biosynthesis Of Citrus Flavonoids

Flavonoids in citrus are a class of bioactive substances with significant pharmacological functions,and their diverse structures are important basis for exerting multiple biological activities.Hydroxylation is a major modification that enriches the structure of flavonoids,however,the flavonoid hydroxylase(FH)catalyzing this reaction in citrus has not been identified.In this study,we used Ougan(Citrus reticulata cv.Suavissima)as materials to identify the FH genes related to flavonoid hydroxyl biosynthesis in Citrus and characterize the function of the key members.The main research results are as follows:1.The phylogeny,gene structure,protein structure and gene expression pattern of CYP450 s members were analyzed in combination with the Citrus clementine genome and transcriptome.A total of 334 CYP450 s members were identified and divided into two families: Group A(239)and non-A(95),which differed significantly in the gene structure,protein conserved structural domain and conserved motif.The expression patterns of different CYP450 s members were also diverse in different development stages of Ougan fruit.2.Based on transcriptome sequencing and in combination with cluster analysis with identified FHs in other plants,30 members of CYP450 s were selected as candidate FH genes from the peel of Ougan.The heterologous expression in yeast cells showed that one of the candidates(accession number in the citrus genome: Ciclev10019637m)displayed 3’-hydroxylation toward naringin(flavanone)and apigenin(flavone),yielding eriodictyol and luteolin,respectively,and thus termed as Cit F3’H(Citrus flavonoid 3’-hydroxylase).3.Further functional studies of Cit F3’H were carried out using yeast eukaryotic expression and virus-induced gene silencing(VIGS)in citrus.Heterologous expression in yeast cells showed that Cit F3’H specifically catalyzed flavonoid substrates with hydroxyl groups at the 4’ position of the Bring,including flavanones,flavones,flavonols and dihydroflavonols,and showed the highest catalytic activity toward flavanones.During Ougan fruit maturation,the expression level of Cit F3’H in peels decreased at first and then increased,indicating that Cit F3’H might be involved in the 3’-hydroxylation of both flavanones and polymethoxylated flavones in citrus.The content of flavonoids with substituted groups at 3’ position decreased by 41% on average in Cit F3’H VIGS citrus.Taken together,these results demonstrated that Cit F3’H(CYP75B1),a member of the CYP450 s,was involved in the biosynthesis pathway of 3’-hydroxylation of flavonoids in citrus.