Overexpression of the UGT73C6 alters brassinosteroid glucoside formation in Arabidopsis thaliana

In 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.The 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.Brassinosteroids (BRs) are a family of steroid hormones that regulate cell division and cell elongation in plants and participate in the control of growth and development [1]. BRs are synthesized from the sterol campesterol, which is modified by a cascade of hydroxylation and oxidation reactions to yield the biologically active BRs castasterone (CS) and brassinolide (BL) [2]. CS and BL bioactivity is conferred by their ability to bind to the BR-receptor BRI1 [3], which initiates a phosphorylation-dependent signal transduction cascade leading to nuclear acquisition of transcription facto