Over‐expression of an Arabidopsis gene encoding a glucosyltransferase of indole‐3‐acetic acid: phenotypic characterisation of transgenic lines

Abstract

SummaryAn analysis of the multigene family of Group 1 glucosyltransferases (UGTs) of Arabidopsis thaliana revealed a gene, UGT84B1, whose recombinant product glucosylated indole-3-acetic acid (IAA) in vitro. Transgenic Arabidopsis plants constitutively over-expressing UGT84B1 under the control of the CaMV 35S promoter have been constructed and their phenotype analysed. The transgenic lines displayed a number of changes that resembled those described previously in lines in which auxin levels were depleted. A root elongation assay was used as a measure of auxin sensitivity. A reduced sensitivity of the transgenic lines compared to wild-type was observed when IAA was applied. In contrast, application of 2,4-dichlorophenoxyacetic acid (2,4-D), previously demonstrated not to be a substrate for UGT84B1, led to a wild-type response. These data suggested that the catalytic specificity of the recombinant enzyme in vitro was maintained in planta. This was further confirmed when levels of IAA metabolites and conjugates were measured in extracts of the transgenic plants and 1-O-IAGlc was found to be elevated to approximately 50 pg mg À1 FW, compared to the trace levels characteristic of wild-type plants. Surprisingly, in the same extracts, levels of free IAA were also found to have accumulated to some 70 pg mg À1 FW compared to approximately 15 pg mg À1 FW in extracts of wild-type plants. Analysis of leaves at different developmental stages revealed the auxin gradient, typical of wild-type plants, was not observed in the transgenic lines, with free IAA levels in the apex and youngest leaves at a lower level compared to wild-type. In total, the data reveal that significant changes in auxin homeostasis can be caused by overproduction of an IAA-conjugating enzyme.

N-Glucosylation of Cytokinins by Glycosyltransferases of Arabidopsis thaliana

“…The constitutive overexpression of these UGTs has been found to lead to a diversity of effects, dependent on the aglycone that is glucosylated (54,56). For example, when (24,25,27,28) was analyzed by the neighbor-joining method using the distance matrix derived from the multiple alignment of CLUSTAL W (EMBL, Heidelberg, Germany).…”

“…UGT84B1 recognizing indole-3-acetic acid (IAA) was constitutively overexpressed, the transgenic lines showed many features typical of an auxin-deficient phenotype (56). The lack of gravitropism displayed by the root system of these plants was recovered by application of an auxin analogue 2,4-D, which was not glucosylated by the UGT (55,56).…”

“…The lack of gravitropism displayed by the root system of these plants was recovered by application of an auxin analogue 2,4-D, which was not glucosylated by the UGT (55,56). Metabolite profiling of the transgenic lines confirmed very high levels of the IAAglucose ester, but levels of IAA were also elevated, suggesting that changing the IAA conjugate levels in the plant caused a complex set of events to occur (56). As yet, we have only a preliminary analysis of the transgenic lines described in this study overexpressing the UGT76C1.…”

An Expression and Bioinformatics Analysis of the Arabidopsis Serine Carboxypeptidase-Like Gene Family

“…Similarly, indole-3-acetic acid (IAA)-Glc synthesized by indole-3-acetic acid glucosyltransferase serves as an activated form of IAA, which is used in a transesterification reaction to form IAA-inositol (Michalczuk and Bandurski, 1982;Jackson et al, 2001Jackson et al, , 2002Ljung et al, 2002). This latter reaction is analogous to sinapoylmalate biosynthesis and preliminary indications suggest that it may be catalyzed by an SCPL enzyme (Kowalczyk et al, 2003).…”

“…In the course of evolutionary history, the genes that encode these proteins have diversified to coordinate these metabolic pathways unique to plants. Examples that have been particularly well studied include the Cyt P450-dependent monooxygenases, MYB transcription factors, terpene synthases, and UDPglucosyltransferases (Meyer et al, 1996;Mizutani et al, 1998;Jackson et al, 2001;Li et al, 2001b;Lim et al, 2001;Stracke et al, 2001;Aubourg et al, 2002;Jackson et al, 2002;Naur et al, 2003). SCPL proteins appear to represent a similar case of recruitment and diversification in plant lineages that has received limited attention to date.…”