Figure 1.
Phylogenetic relationship of the group L glycosyltransferases from Arabidopsis thaliana.
The phylogenetic tree of Arabidopsis UGTs is adopted from the previous report [40]. Bootstrap values are indicated above the nodes. The glycosyltransferase sequences were retrieved from Carbohydrate-active enzymes database (http://www.cazy.org/GT1_eukaryota.html) and NCBI database. The asterisks indicate those glycosyltransferases with confirmed enzymatic activities toward phytohormone related compounds.
Figure 2.
SDS-PAGE analysis of the recombinant GST-UGT74D1 fusion protein.
Proteins were purified from E.coli, analyzed on a 12% (w/v) polyacrylamide gel and visualized with Coomassie Brilliant Blue staining. M: protein molecular weight marker; GST: glutathione-s-transferase; 74D1: fusion protein.
Figure 3.
HPLC and LC-MS analysis of reaction product from IBA.
(A) HPLC analysis. 1: the reaction was added with GST protein as control. 2: the reaction was added with UGT74D1 fusion protein and a new peak (peak b) was produced. Peak “a” represents the substrate IBA. (B) LC-MS analysis of peak b. (C) LC-MS analysis of IBA glucose conjugates produced by the catalysis of UGT74E2 which was used as positive control in this research. (D) Proposed enzymes and biosynthetic pathway for the synthesis of IBA-glucose ester from the aglycone IBA.
Figure 4.
HPLC analysis of reaction products from other auxins.
1: the reaction was added with GST protein as control. 2: the reaction was added with UGT74D1 fusion protein. Peak “a” represents the auxin substrates. Peak “b” represents the reaction products.
Table 1.
Specific activity of UGT74D1 toward auxins and related substrates.
Figure 5.
Analyses on factors affecting the activity of recombinant UGT74D1.
(A) The effects of temperature. (B) The effects of buffer and pH value. All the reaction mix (100 µl) contained 0.2 ug of recombinant UGT74D1, 5 mM UDP-glucose, 1 mM IBA, 2.5 mM MgSO4, 10 mM KCl, 14.4 mM 2-mercaptoethanol, 50 mM buffer and was incubated for 30 min as described in “Materials and Methods ”. The results represent means±SD from three replicates. The specific enzyme activity was defined as nmol of substrates converted into glucose conjugates per second (nanokatal, nkat) by 1 mg of protein.
Figure 6.
Analysis of the transgenic Arabidopsis plants overexpressing UGT74D1 using the CaMV35S promoter.
(A) RT-PCR analyses of the steady-state level of UGT74D1 mRNA in transgenic plants (OEs) and wild type (WT). (B) The glycosyltransferase activities in the crude protein extracts of transgenic plants and wild type were measured following the procedure described under “Materials and Methods”. The specific enzyme activity was expressed as pmol of IBA glucosylated to form IBA-Glc by 1 mg of protein per second of reaction time at 37°C.
Figure 7.
HPLC trace of IBA glucose conjugates of the extracts from the wild type (WT) and transgenic plants (OEs).
(+) and (−) represent the plant tissues incubated with or without IBA before extraction process. Peak “a” indicates the picloram added as an internal control at the beginning of the extraction process; Peak “b” indicates the IBA-glucose conjugates. The extracts were analyzed with a linear gradient of methanol in H2O from 10–70% (all solutions contained 0.01% H3PO4) over 30 min at 1 ml/min and monitored at 280 nm.
Figure 8.
Phenotypes of Transgenic Arabidopsis Plants.
(A) 5-week-old plant phenotypes of WT, mutants and overexpression lines. (B) 5-week-old rosette leaf phenotypes of wild type, mutants and overexpressor lines. (C) Seventh leaf and leaf transverse section of 5-week-old plants. (D) The flattening index of seventh leaf was calculated by dividing the projection area of intact curled leaves with that of manually uncurled leaves.
Table 2.
Phenotype comparison of overexpression lines of UGT74D1, UGT84B1 and UGT74E2 genes.