Characterization of a Putative Flavonoid 3’, 5’-Hydroxylase (PtF3’5’H1) in Populus
|Institution:||University of Victoria|
|Keywords:||F3'5'H; Populus; Anthocyanin; Proanthocyanin; Condensed tannin|
|Full text PDF:||http://hdl.handle.net/1828/6027|
Proanthocyanidins (PAs), also known as condensed tannins (CTs), are oligomers or polymers of flavan-3-ols. They have a very important role in plant-environment interactions, such as defense against herbivory and pathogens. They may also be important for light stress tolerance. In poplar, PAs can make up as much as 30% of the leaf dry weight. The synthesis of PAs in poplar was demonstrated to be inducible by both abiotic and biotic stresses. The B-ring hydroxylation pattern of flavan-3-ols directly affects the structure of PAs, and many studies have shown that B-ring hydroxylation of PAs is associated with their biological functions, including effects on leaf litter decomposition rate and anti-herbivore activity. Anthocyanins are very important colour pigments in plants, and share the intermediate leucoanthocyanidin with PAs. The role of anthocyanins in plant pollination, light stress tolerance, and seed dispersal has been well studied. A change in B-ring hydroxylation pattern can modify the colour of anthocyanins dramatically and also change their biological function. Flavonoid 3’-hydroxylase and flavonoid 3’, 5’-hydroxylase (F3’H and F3’5’H) are the two enzymes involved in determining the B-ring hydroxylation pattern of both PAs and anthocyanins. The objective of this study is to characterize the possible role of flavonoid 3’, 5’-hydroxylase in PA and anthocyanin biosynthesis in poplar. A candidate F3’5’H was identified in the Populus trichocarpa genome database based on previous expression profile experiments, and called PtF3’5’H1. The predicted protein shares high sequence similarity with previously characterized F3’5’H proteins from other plants. To test the function of PtF3’5’H1 directly, transgenic hybrid poplar plants overexpressing PtF3’5’H1 were generated. Preliminary LC-MS analysis showed that the hydroxylation pattern of the PA in the transgenic poplars was not significantly modified. Likewise, overexpression of PtF3’5’H1 in poplar did not change the overall amount of PAs. These results suggest that overexpression of PtF3’5’H1 in poplar is not sufficient to modify the B-ring hydroxylation pattern of PA, and that additional factors may be required. By contrast, the transgenic PtF3’5’H1 overexpressing poplar did show an alteration in anthocyanin profile. In leaves of transgenic poplars, several putative delphinidin derivatives were observed at greater levels than in the wild type, indicating that PtF3’5’H1 participates in the anthocyanin production in poplar. However, transiently introducing PtF3’5’H1 into Nicotiana benthamiana had no effect on the anthocyanin profile in this plant. I conclude that PtF3’5’H1 is very likely to be involved in the anthocyanin synthesis in poplar, while the function of PtF3’5’H1 in poplar PA synthesis has yet to be demonstrated.