UBC Theses and Dissertations
Characterization of two udp glycosyltransferase genes from hybrid poplar Kenawy, Ahmed
Glycosyltransferases (GTs) play important roles in plant growth and development. The biological functions of many GTs are unknown. In the present study, two putative GT genes (PopGT1 and PopGT2) were cloned and their biological roles in growth and development of Arabidopsis and hybrid-poplar were investigated. In silico, in vitro, and in vivo methods were used to characterize the two encoded proteins. Phylogenetic analysis, enzyme activity assays, and transcript abundance were studied. In addition, plant growth and development, leaf morphology, stem anatomy, cell wall composition, biomechanical properties, soluble carbohydrate, and phenolic metabolite contents were determined. The results indicated that PopGT1 showed high similarity to tobacco salicylic acid glycosyltransferase, and both PopGT1 and PopGT2 (annotated as AtUGT74F2) were clustered within phylogenetic group L of family-1 GTs (UGTs). In vitro characterization of the two recombinant proteins indicated that PopGT1 glycosylated several flavonoids, showed only trace activities towards cinnamic and indole butyric acid, and accepted UDP-glucose as a sugar donor. The optimum temperature and pH for in vitro PopGT1 activity was 35 ºC and pH 7.5, respectively. PopGT2 showed no enzymatic activity towards any substrates. The two coding sequences (PopGT1 and PopGT2) were cloned in the pSM3 expression vector and over-expressed in Arabidopsis plants to investigate their in vivo functions. Phenotypically, plant height, stem diameter, rosette diameter, and stem number increased significantly in the transgenic plants. In addition, rosette morphology and root gravitropism were altered. Transgenic plants flowered earlier than the control plants. Chemically, cell wall compositions and phenolic metabolite contents changed significantly. In parallel, transgenic trees showed changes in leaf morphology, stem diameter, phloem fibre arrangement, and early bud break. Wood density was reduced revealing a brittle-stem phenotype. Marginal increases in lignin and reductions in cellulose content were apparent. Salireposide content was reduced in the bark of transgenic trees. The results indicated that altering the expression of both genes in Arabidopsis and poplar affected plant growth and development, cell wall composition, phenolic metabolite profiles, and wood biomechanical properties. PopGT1 showed in vivo substrate specificity towards kaempferol and promiscuous in vitro enzyme activity. However, the substrate of PopGT2 remains unclear.
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