Identification And Functional Characterization Of Major Glucosyltransferase Genes Involved In Rice Flavonoid Etabolism

Majority of the biochemical diversity existing within the natural plant kingdom is the consequence of various modifications,the mechanisms that mostly alters the biological function of their precursor molecules.Glycosylation is one of the major modifications of secondary metabolites mediated by the UDP-dependent glycosyltransferases(UGTs).Flavonoids represent one of the diverse groups of special(secondary)metabolites,ubiquitously found in plants and have been implicated to play a vital role in a vast range of biological processes.Despite flavonoid classes,especially flavonols have extensively been studied in model plant species such as Arabidopsis and regulation of their metabolism has largely been uncovered.However,flavones,a flavonoids sub-class,most commonly glycosylated(conjugation with sugar moieties)by the UGTs are still overlooked,and the natural variation of their metabolism in model cereal crops such as rice remains to be elucidated.In the present study,we describe and report on the dissection of genetic and biochemical bases behind the natural variation of major flavonoids in rice.Application of LC-MS-based widely-targeted metabolomics method facilitated the comprehensive profiling of metabolites and subsequent detection of flavonoids,including flavones,flavonols,and flavanones in 7 plant species,encompassing pre-angiosperms,monocots and eudicots.Specie/plant-specific accumulation patterns based glycosylated flavonoids abundance was observed mainly in monocots such as rice,which further provided a basis to unravel the regulation of the natural variation of its flavones metabolism.Afterward,metabolite-based genome-wide association study(mGWAS)disclosed the genetic architecture corresponding to the UDP-dependent glycosyltransferases,underlying three prominent loci(on chromosome 1 and 5),which were associated and might contribute to the natural variation of various flavonoids conjugates contents in rice.Phylogenetic analysis displayed the characterization of most of the putative candidate rice UGT genes in a unique clade of flavonoid glucosyltransferases.Functional interpretation and verification of the candidate associations through the in vitro biochemical assays displayed five OsUGT genes,OsUGT706D2,OsUGT706B1,OsUGT706C3,OsUGT706C1,OsUGT705A1,which achieved specific activities on flavonols,and OsUGT706D1,OsUGT706E1,OsUGT705A2,OsUGT706C4,as four flavone active OsUGTs.Further validation via the in vivo transgenic and subsequent metabolic contentsanalysis showed that the naturally occurring variation of levels of rice flavone conjugates,in principal is determined by OsUGT706D1,characterize as flavone 7-Oglucosyltransferase.Assessment of the association between flavonoids accumulation and its biosynthetic gene nucleotide polymorphisms(SNPs)depicted the possible functional allelic variants in the coding sequence region of OsUGT706D1,which might be the cause of natural variation in rice flavone(apigenin)7-O-glucosides.Besides,protein 3Dhomology model performed for allelic OsUGT706D1,disclosed multiple distant amino acid residues than the one allelic candidate,Pro83 Leu,near the putative enzyme acceptor substrate binding pocket,which caused no changes in the protein structure and thus,was suggested not a vital specificity determinant for the catalytic activities of OsUGT706D1.Comparative genomic analysis of F7 GlcTs in the evolutionary context and biochemical evaluation revealed their conserved evolution in angiosperms.Taken together,our study states that the forward genomics-based strategy driven gene-metabolite analysis not only aids a potential tool to provide better understandings into the biochemical and genetic regulation of flavones metabolism,but also represent an important way for functional gene identification and combined omics-based crop genetic improvement.