Overexpression of the UGT73C6 alters brassinosteroid glucoside formation in Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>Brassinosteroids (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 <it>Arabidopsis thaliana </it>catalyzes 23-<it>O</it>-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 <it>in planta</it>.</p> <p>Results</p> <p>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-<it>O</it>-positions <it>in planta</it>. GUS reporter analysis indicates that <it>UGT73C6 </it>shows over-lapping, but also distinct expression patterns with <it>UGT73C5 </it>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-<it>O</it>-glucosides in wild type and <it>UGT73C5 </it>and <it>UGT73C6 </it>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.</p> <p>Conclusion</p> <p>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.</p

Genetic Variation in Plant CYP51s Confers Resistance against Voriconazole, a Novel Inhibitor of Brassinosteroid-Dependent Sterol Biosynthesis

<div><p>Brassinosteroids (BRs) are plant steroid hormones with structural similarity to mammalian sex steroids and ecdysteroids from insects. The BRs are synthesized from sterols and are essential regulators of cell division, cell elongation and cell differentiation. In this work we show that voriconazole, an antifungal therapeutic drug used in human and veterinary medicine, severely impairs plant growth by inhibiting sterol-14α-demethylation and thereby interfering with BR production. The plant growth regulatory properties of voriconazole and related triazoles were identified in a screen for compounds with the ability to alter BR homeostasis. Voriconazole suppressed growth of the model plant <em>Arabidopsis thaliana</em> and of a wide range of both monocotyledonous and dicotyledonous plants. We uncover that voriconazole toxicity in plants is a result of a deficiency in BRs that stems from an inhibition of the cytochrome P450 CYP51, which catalyzes a step of BR-dependent sterol biosynthesis. Interestingly, we found that the woodland strawberry <em>Fragaria vesca,</em> a member of the <em>Rosaceae</em>, is naturally voriconazole resistant and that this resistance is conferred by the specific CYP51 variant of <em>F. vesca</em>. The potential of voriconazole as a novel tool for plant research is discussed.</p> </div

FvCYP51 confers voriconazole resistance.

<p>(<b>A</b>) Western blot analysis of lines expressing YFP-tagged versions of <i>AtCYP51A2</i> or <i>FvCYP51</i> under control of the 35S promoter (left panel). Staining of the membrane with coomassie brillant blue R250 shows loading of the samples. (<b>B</b>) Phenotype of <i>AtCYP51</i> and <i>FvCYP51</i> over-expressing lines and wild-type plants grown on ½ MS media or media complemeted with 1 µM voriconazole for 3 weeks. Two representative plants are shown for each line. Hypocotyl length (<b>C</b>) and biomass (<b>D</b>) of the same lines grown on ½ MS supplemented with the indicated concentration of voriconazole. Plants were grown under long day conditions for 10 day (<b>C</b>) or 2 weeks (<b>D</b>). Data are the mean ± SD of at least 20 plants measured.</p

Voriconazole alters the sterol and BR profile of arabidopsis plants.

<p>Sterol and BR contents were quantified by GC-MS in two independent experiments in which arabidopsis seedlings grown either on media containing 3 µM voriconazole or on unsupplemented media for 10 d were compared. Sterol (µg/g fw) and BR levels (ng/g fw) are shown in the table. nd, not detected (below detection limit). For illustration changes are marked in the pathway, which was adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053650#pone.0053650-Kim2" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053650#pone.0053650-Bishop1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053650#pone.0053650-Ohnishi1" target="_blank">[50]</a>. ↑, up; ↓, down; ∼, changes less than 2-fold. Biosynthetic enzymes of the cytochrome P450 family are in bold.</p

Voriconazole does not affect GA action.

<p>(<b>A</b>) In contrary to GA biosynthesis inhibitors voriconazole does not interfere with germination. 50 to 60 seeds were incubated on ATS media containing 25 µM of the indicated compounds, in long-day conditions at 24±2°C. Germination was assessed after 4 days. Data are the mean ± SD from 3 independent experiments. Paclobutrazole and unicozole treated samples are statistically highly significantly different from the other samples (ANOVA P-value <0.01%). (<b>B</b>) Application of GA does not rescue voriconazole induced growth inhibition. Seedling development in the presence of voriconazole, 24-epiBL or GA₃. Plants were grown on ATS media supplemented with the indicated compounds for 12 days.</p

Voriconazole inhibits growth of monocotyledonous and dicotyledonous crop plants.

<p>Phenotypes of plants grown on control media (left) or media containing 1 µM voriconazole (right). The days after germination are indicated in brackets. The white scale bar represents 1 cm.</p

Voriconazole, itraconazole and fluconazole induce phenotypes indicative of BR deficiency in arabidopsis and cress.

<p>(<b>A</b>) Arabidopsis plants grown on plates containing 5 µM of the indicated compounds under long-day conditions for 7 days. (<b>B</b>) Hypocotyl length of cress seedlings grown for 3 days on plates containing different concentrations of the indicated compounds. Data are the mean ± SD of 30 seedlings measured.</p

Structures of the triazoles voriconazole, fluconazole and itraconazole and the BR biosynthesis inhibitor Brz2001.

<p>Structures of the triazoles voriconazole, fluconazole and itraconazole and the BR biosynthesis inhibitor Brz2001.</p

Voriconazole is rapidly incorporated.

<p>Arabidopsis seedlings were incubated for the indicated periods of time in ATS media supplemented with 25 µM voriconazole and the <i>in situ</i> contents of voriconazole were quantified by HPLC-DAD analysis. The means and standard deviations were calculated from four measurements.</p