Overexpression of the UGT73C6 alters brassinosteroid glucoside formation in Arabidopsis thaliana

Sigrid Husar,Yi Li,Shozo Fujioka,Rainer Schuhmacher,Luisa Elias,Rudolf Krska,Florian Kalaivanan,Franz Berthiller,Wilfried Rozhon,Gillian S Higgins,Fabian E Vaistij,Hideharu Seto,Brigitte Poppenberger,Dianna Bowles,Mamoona Khan

doi:10.1186/1471-2229-11-51

Abstract

BackgroundBrassinosteroids (BRs) are signaling molecules that play essential roles in the spatial regulation of plant growth and development. In contrast to other plant hormones BRs act locally, close to the sites of their synthesis, and thus homeostatic mechanisms must operate at the cellular level to equilibrate BR concentrations. Whilst it is recognized that levels of bioactive BRs are likely adjusted by controlling the relative rates of biosynthesis and by catabolism, few factors, which participate in these regulatory events, have as yet been identified. Previously we have shown that the UDP-glycosyltransferase UGT73C5 of Arabidopsis thaliana catalyzes 23-O-glucosylation of BRs and that glucosylation renders BRs inactive. This study identifies the closest homologue of UGT73C5, UGT73C6, as an enzyme that is also able to glucosylate BRs in planta.ResultsIn a candidate gene approach, in which homologues of UGT73C5 were screened for their potential to induce BR deficiency when over-expressed in plants, UGT73C6 was identified as an enzyme that can glucosylate the BRs CS and BL at their 23-O-positions in planta. GUS reporter analysis indicates that UGT73C6 shows over-lapping, but also distinct expression patterns with UGT73C5 and YFP reporter data suggests that at the cellular level, both UGTs localize to the cytoplasm and to the nucleus. A liquid chromatography high-resolution mass spectrometry method for BR metabolite analysis was developed and applied to determine the kinetics of formation and the catabolic fate of BR-23-O-glucosides in wild type and UGT73C5 and UGT73C6 over-expression lines. This approach identified novel BR catabolites, which are considered to be BR-malonylglucosides, and provided first evidence indicating that glucosylation protects BRs from cellular removal. The physiological significance of BR glucosylation, and the possible role of UGT73C6 as a regulatory factor in this process are discussed in light of the results presented.ConclusionThe present study generates essential knowledge and molecular and biochemical tools, that will allow for the verification of a potential physiological role of UGT73C6 in BR glucosylation and will facilitate the investigation of the functional significance of BR glucoside formation in plants.

Highlights

Brassinosteroids (BRs) are signaling molecules that play essential roles in the spatial regulation of plant growth and development
Over-expression of UGT73C6 results in BR deficiency in A. thaliana UGT73C5 is a member of UGT subfamily 73C, which is comprised of seven genes, six of which are clustered in a tandem repeat on Chromosome 2 (Figure 1a)
It was of interest to investigate the phenotypic consequences of over-expressing other members of the UGT73C gene cluster. 15 to 25 independent transgenic lines expressing each of the UGTs 73C1, 73C2, 73C3, 73C4 and 73C6 under control of the constitutive Cauliflower Mosaic Virus 35 S (CaMV35S) promoter were generated and analyzed for steady-state levels of transcripts using semi-quantitative RT-PCRs. 3 to 5 lines with high expression levels were chosen for each UGT to assess effects on plant growth and development

Summary

Introduction

Brassinosteroids (BRs) are signaling molecules that play essential roles in the spatial regulation of plant growth and development. Whilst it is recognized that levels of bioactive BRs are likely adjusted by controlling the relative rates of biosynthesis and by catabolism, few factors, which participate in these regulatory events, have as yet been identified. Hydroxylation is one means of catabolic inactivation and is catalyzed by the Arabidopsis thaliana cytochrome P450 monooxygenase BAS1 [6]. Another class of BR conjugates, which are inactive, are glucosides. Whilst enzymes mediating C-2 glucosylation of BRs are still unknown, we could previously show that 23-Oglucosylation of CS and BL in A. thaliana is catalyzed by UGT73C5, a UDP-glycosyltransferase (UGT) [8]. An increase in BR-23-O-glucosylation activity in UGT73C5 over-expressing plants correlated with reduced levels of typhasterol (TY), 6-deoxocastasterone (6-deoxoCS) and CS and with BR-deficient phenotypes, showing that 23-O-glucosylation reduces BR bioactivity [8]

Methods

Results

Conclusion