Molecular Characterization Of TaDFR Genes Involved In Anthocyanin Biosynthesis And Expressed In Purple Grain Of Triticum Aestivum L.

In this study, patterns of anthocyanin accumulation was investigated in the purple grain of wheat. Coloration of grain was not observed at day 15 after pollination (AP). the anthocyanin is accumulated in the cells of seed coat at day 20 AP, Further, the pigmentation occurs in the cells of pericarp at day 25 AP. Then, darkned colour was observed in these tissues at day 30 and day 35 AP. It is demonstrated that anthocyanin accumulation occurs in a tissue-specific manner during development of the purple grain.To understand the molecular mechanism of pigmentation in the grain, three TaDFR genes encoding the key enzyme dihydroflavonol 4-reductase in the biosynthetic pathway of anthocyanin were isolated from purple grain tissues. And they were designated as TaDFR1(AY555573), TaDFR2(AY707 917) and TaDFR3 (AY707918). Sequence analysis shows that these three genes share higher homology with DFR genes in other plant species. Southern hybridization showed that five hybridization bands were detectable in the genomes of four cultivars. These data suggest that there are at least three TaDFR genes in the genomes of wheat.The expression pattern of TaDFR genes was determined by Northern hybridization. Northern analysis showed that TaDFR genes are highly expressed in purple coleoptiles. But they are not expressed in roots, stems and leaves. By using poly(A)~+ RNA, TaDFRl transcripts were detected in the purple grains of Heimai 76, and lower level of its transcripts in the light purple grains of Luozhen 1. But TaDFR transcripts are not accumulated in the grains of Bainong 66 and Yangmai 158. In situ hybridization was carried out to localize TaDFR transcripts in grains. TaDFR transcripts were detected in seed coats, but not in the cells of pericarp, although the pigments are accumulated in both tissues of purple grains, implying that there is a mechanism controlling pigments biosynthesis in the pericarp of purple grains.Roles of TaDFRl was analyzied by transformed 35S::TaDFRl into Arabidopsis. It is indicated that the diversity of purple color in different transformants could be caused by the different levels of TaDFRl expression. Separation of anthocyanins by high-performance liquid chromatography (HPLC) showed that anthocyanin levels are much higher in transgenic leaves than in wild type leaves. The results demonstrate that the overexpression of TaDFRl is responsible for the formation of purple color in Arabidopsis plants.