Evolutionary and functional analyses of the 2-oxoglutarate-dependent dioxygenase genes involved in the flavonoid biosynthesis pathway in tobacco

Main conclusion This study illustrates the differences in the gene structure of 2-oxoglutarate-dependent oxygenaseinvolved in flavonoid biosynthesis(2ODD-IFB), and their potential roles in regulating tobacco flavonoid biosynthesisand plant growth. Flavonol synthase(FLS), anthocyanidin synthase(ANS), and flavanone3β-hydroxylase belong to the 2-oxoglutarate-dependent (2ODD) oxygenasefamily, and each performs crucial functions in the biosynthesisof flavonoids. We identified two NtFLS genes, two NtANS genes, and four NtF3H genes from Nicotiana tabacumgenome, as well as their homologous genes in the N. sylvestris and N. tomentosiformis genomes. Our phylogenetic analysis indicated that these three types of genes split from each other before the divergence of gymnospermsand angiosperms. FLS evolved faster in the eudicotplants, whereas ANS evolved faster in the monocot plants. Gene structure analysis revealed two fragment insertions occurred at different times in the intron one position of tobacco FLS genes. Homologous protein modeling revealed distinct structures in the N terminus of the tobacco 2ODD oxygenases. We found that the expression patterns of genes encoding tobacco 2ODD oxygenasesin flavonoids biosynthesis(2ODD-IFB) did not determine the accumulation patterns of flavonoidsamong various tobacco tissues, but strongly affected the concentration of flavonoidsin the tissues, where they were biosynthesized. More carbon resource flowed to the flavonol biosynthesiswhen NtANS gene was silenced, otherwise more anthocyanidinaccumulated when NtFLS gene was repressed. This study illustrates the 2ODD-IFB gene structure evolution, differences among their protein structures, and provides a foundation for regulating plant development and altering flavonoidcontent and/or composition through the manipulation of plant 2ODD-IFB genes.