Identification And Characterization Of Flavonoid Modification Genes From Citrus

Citrus fruits are generally consumed freshly due to its pleasant taste and high level of vitamins,fibers,minerals,flavonoids and other nutrients.Citrus genera contain diverse classes with dissimilar concentration of secondary metabolites.Sweet orange(Citrus sinensis)is the most commonly consumed and cultivated citrus among citrus germplasm.Sweet orange also possess enormous types of secondary metabolites;however the molecular mechanism underlying the biosynthesis and regulation of these secondary metabolites is less understood.Previous transcriptome data from our group indicated that a CYTOCHROME P450 gene and a UDP-GLUCOSYL TRANSFERASE gene expression were positively correlated metabolites accumulation and respond to different abiotic stresses(such as drought stress and high light stress).In this study,we cloned one CYTOCHROME P450 75B1(Cs CYP75B1)gene and one UDP-GLUCOSYL TRANSFERASE(Cs UGT78D3)gene from sweet orange to overexpress in the Arabidopsis thaliana to evaluate their function and possible role under stress.The overexpression of Cs CYP75B1 gene leads to high accumulation of flavonoids contents in all transgenic Arabidopsis lines and many flavonoids biosynthesis genes were significantly up-regulated 2-12 folds than wild type.Moreover,all transgenic lines have higher antioxidant activity and capacity as compared to wild type and empty vector plants.In addition,when the transgenic lines exposed to drought stress,they showed significant increment in root length and confer drought tolerance.After 14 days of drought stress,all transgenic lines showed strong drought tolerance by accumulating higher contents of antioxidant flavonoids,anthocyanins and phenolics with higher total antioxidant activity and antioxidant enzymatic activities.Furthermore,significantly lower level of reactive oxygen species(ROS),electrolytic leakage and malondialdehyde were observed in all transgenic lines than wild type and empty vector Arabidopsis plants.These outcomes demonstrate that overexpression citrus Cs CYP75B1 gene is involved in biosynthesis of antioxidant flavonoids and confers drought tolerance with better ROS scavenging mechanism in all transgenic lines than wild type.Overexpression of Cs UGT78D3 showed dark brown color to transgenic Arabidopsis seeds with high accumulation of proanthocyanidins in the seed coats than wild type seeds.In addition,significantly higher contents of flavonoids and anthocyanins were observed in the leaves of all transgenic lines.The gene expression data revealed that many anthocyanin and flavonoid pathway genes were 4-15 folds induced in all transgenic lines.Moreover,when transgenic lines were exposed to 14 days of high light stress,they showed dark purple color phenotype in leaves and possesses high level of total anthocyanin and flavonoid contents whereas wild type and empty vector plants showed serve chlorosis with lower level of anthocyanins.Additionally,all transgenic lines showed tolerance to 14 days of high light stress than wild type and empty vector lines.Furthermore,the transgenic lines leaves have higher antioxidant activity with lower superoxide radicals than wild types plants.Our study concluded that the citrus Cs UGT78D3 gene is involved in proanthocyanidins accumulation in seed coats and confers high light stress tolerance by accumulating total anthocyanin contents with better antioxidant potential in the transgenic Arabidopsis.Our both studies concluded that overexpression of secondary metabolic gene not only enhances the total metabolic level but also improves the tolerance level of plants due to strong antioxidant and photoprotective activities of flavonoids and anthocyanins respectively.The enhancement of antioxidant capacity of these genes is promising in the application of nutritional improvements of fruits in future.