Functional characterization of flavonoid glycosyltransferases and an acid phosphatase from poplar (Populus spp.)




Veljanovski, Vasko

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Plants have evolved a wide variety of physical and biochemical defense mechanisms to protect against herbivores and pathogens. When wounded, hybrid poplar (Populus trichocarpa X P. deltoides) upregulates a suite of defense-related genes, some of which encode anti-herbivore proteins. Among the most strongly insect- and wound- induced genes in poplar is an acid phosphatase gene (AP). APs are enzymes that function in hydrolyzing phosphate from P-monoesters and anhydrides and are involved in the remobilization of phosphate from these pools. However, APs may also play a role in the defense against insects by acting as anti-insect proteins. In poplar, AP mRNA induction occurs within 1.5 hours, which is similar to other known poplar defense genes. In the work described in this thesis, a 2 to 3-fold increase in the extractable AP activity was demonstrated in the leaves of saplings 4 days post wounding. These results suggest the poplar AP is part of the defense response against leaf-eating herbivores. In another type of defense reaction, when hybrid poplar is infected by the pathogen Melampsora medusae, which causes poplar leaf rust, flavonoid pathway genes are induced. This induction leads to the accumulation of proanthocyanidins (PAs), compounds with antimicrobial activity. The expression of several flavonoid-specific glycosyltransferase (UGTs) genes were correlated with these PA genes, suggesting a role for them in PA biosynthesis. Therefore, the second objective of this thesis was to functionally analyze these UGT genes. UGTs are enzymes which catalyze glycosylation reactions, which is typically one of the last steps in the biosynthesis of plant phenolic compounds. Active recombinant proteins for two highly induced poplar UGTs (PtUGT1 and PtUGT2) were generated, and sequence analysis grouped these proteins with others involved in the glycosylation of flavonols and anthocyanidins (UGT78 family), and not PA precursors as expected from microarray data. Enzymatic analysis of one of these proteins (PtUGT1) supports this phylogenetic grouping. By contrast, PtUGT2 does not use any known flavonoid substrates. To investigate the role of PtUGT1 in planta, transgenic poplars were produced that suppressed the expression of this gene using RNA interference. Phytochemical analysis of these knockdown plants were found to display decreased levels of PAs. Tissue survey analysis also implicates the PtUGT1 gene in PA biosynthesis since phytochemical analysis correlates with gene expression of PtUGT1 in the various tissues tested. Thus the data suggests that this UGT gene may be involved in PA biosynthesis.



Biology, Forest Biology, Plant Biology