Propiconazole Is a Specific and Accessible Brassinosteroid (BR) Biosynthesis Inhibitor for Arabidopsis and Maize

Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA3 co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species

Pcz specificity towards BR biosynthesis inhibition.

<p>(<b>A–C</b>) 3-day old Ws-2 were transferred to ½ MS media containing either 1 µM Pcz, 0.1 µM BL, 10 µM GA₃, or co-applications of 1 µM Pcz with 0.1 µM BL or 10 µM GA₃ and grown for 3 more days. (<b>A</b>) Seedlings at the end of treatment. (<b>B</b>) Close-up of the cotyledons and true leaves in the same order as (<b>A</b>). (<b>C</b>) Average root lengths at day 3 of treatment (<i>n</i>>15). (<b>D–F</b>) Col-0 and <i>bzr1-1D</i> (Col-0) grown on ½ MS media in the dark for 7 days. (<b>D</b>) Col-0 or (<b>E</b>) <i>bzr1-1D</i> grown on medium with 2 µM Pcz or 2 µM Brz. (<b>F</b>) Hypocotyl lengths of Col-0 and <i>bzr1-1D</i> grown on medium with 2, 4, 8 µM Pcz or 2 µM Brz (n≥10). (<b>C</b>, <b>F</b>) Error bars represent standard deviation. (<b>C</b>) Lowercase letters indicate significant differences among treatments determined by “Post-hoc” test (<i>p</i><0.05). (<b>F</b>) Asterisks indicate significant difference to the respective mock determined by Student's <i>t</i>-test (<i>p</i><0.01). Scale bar (<b>A</b>, <b>D</b>, <b>E</b>) 1 cm and (<b>B</b>) 0.5 cm.</p

Tissue-specific response of dark and light grown W22 seedlings to Pcz and Ucz.

<p>(<b>A</b>) Maize seedlings grown for 8 d at 29°C in the dark treated with (left to right): 0 µM Pcz, 0 µM Ucz, 1 µM Pcz, 1 µM Ucz, 10 µM Pcz, and 10 µM Ucz, respectively. (<b>B</b>) W22 seedlings grown in the light for 3 weeks at concentrations of (left to right): 0, 0.2, 1, and 5 µM Pcz, respectively. (<b>C–F</b>) W22 seedlings grown for 8 d at 29°C in the dark with Pcz or Ucz at concentrations of 0, 0.5, 1, 5, 10, 20, or 30 µM. Lengths of the (<b>C</b>) mesocotyl, (<b>D</b>) true leaves, (<b>E</b>) coleoptile, and (<b>F</b>) primary root of W22 seedlings grown in the dark with indicated concentrations of Pcz or Ucz (<i>n</i>>15). (<b>G–H</b>) Analysis of W22 maize seedlings grown in the light for 3 weeks at concentrations of 0, 0.2, 1, or 5 µM Pcz (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036625#pone.0036625.s005" target="_blank">Table S5</a>). (<b>G</b>) Plant height and (<b>H</b>) primary root length was measured (<i>n</i>>15). Error bars represent standard deviation. (<b>C–H</b>) Statistical analysis determined by “Post-hoc” test is shown (<b>C–F</b>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036625#pone.0036625.s001" target="_blank">Table S1</a> and (<b>G–H</b>) indicated by lowercase letters (<i>p</i><0.05). Scale bar (<b>A–B</b>) 10 cm.</p

Impact of the genetic diversity on the tissue specific sensitivity towards Pcz and Ucz.

<p>(<b>A–C</b>) Maize inbred seedlings (<b>A</b>) Mo20W, (<b>B</b>) A619, and (<b>C</b>) B73 grown in vermiculite for 8 d at 29°C in the dark treated with (left to right): 0 µM Pcz, 1 µM Pcz, 10 µM Pcz, 0 µM Ucz, 1 µM Ucz, and 10 µM Ucz, respectively. (<b>D–G</b>) Length of the (<b>D</b>) mesocotyl, (<b>E</b>) true leaves, (<b>F</b>) coleoptile, and (<b>G</b>) primary root of Mo20W, A619, and B73 maize seedlings treated with 0 µM Pcz, 1 µM Pcz, 10 µM Pcz, 0 µM Ucz, 1 µM Ucz, or 10 µM Ucz, respectively (<i>n</i>>15). (<b>D–G</b>) Error bars represent standard deviation. Statistical analysis determined by “Post-hoc” test is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036625#pone.0036625.s002" target="_blank">Table S2</a> (<i>p</i><0.05). Scale bar (<b>A–C</b>) 6 cm.</p

Sensitivity of <i>Arabidopsis</i> wild type, BR biosynthetic mutant <i>dwf7-1</i>, and signaling mutant <i>bri1-5</i> to Pcz and Brz.

<p>(<b>A–C</b>) Seedlings of (<b>A</b>) Ws-2, (<b>B</b>) the BR biosynthetic mutant <i>dwf7-1</i> (Ws-2), and (<b>C</b>) the BR signaling mutant <i>bri1-5</i> (Ws-2) grown on ½ MS media with either 1 µM Pcz or Brz or 10 µM Brz. (<b>D–E</b>) Close-up of the shoot apices harboring cotyledons and the first pair of true leaves of (<b>D</b>) <i>dwf7-1</i> and (<b>E</b>) <i>bri1-5</i>. (<b>F–H</b>) Average root lengths of (<b>F</b>) Ws-2, (<b>G</b>) <i>dwf7-1</i> and (<b>H</b>) <i>bri1-5</i> measured at the end of treatments (<i>n</i>>10). (<b>F–H</b>) Error bars represent standard deviation and lowercase letters indicate significant differences among treatments determined by “Post-hoc” test (<i>p</i><0.05). Scale bar (<b>A–E</b>) 1 cm.</p

Chemical structures of brassinazole, propiconazole, paclobutrazole, and uniconazole.

<p>Structure elements critical for inhibitor activity have been color-coded: (blue) nitrogen atoms in the azole ring; (purple) chlorine atom(s) of the phenyl ring; and (red) either primary/secondary hydroxyl group or 1,3-dioxlane. Structures were drawn using the ChemBioDraw 12.0.2 software and structures were compared to the ChemACX 12.12.1 database.</p

Resistance of <i>dwf4-1</i> to Pcz and Brz.

<p>(<b>A–B</b>) Seedlings of (<b>A</b>) Ws-2 and (<b>B</b>) <i>dwf4-1</i> grown on ½ MS media for 7 days, and then transferred to media containing 1 µM Pcz or Brz for 3 more days of growth. (<b>C</b>) Average root lengths of Ws-2 and <i>dwf4-1</i> measured at the end of treatments (<i>n</i>>10). Error bars represent standard deviation and lowercase letters indicate significant differences among treatments determined by “Post-hoc” test (<i>p</i><0.05). Scale bar (<b>A</b>–<b>B</b>) 1 cm.</p

Influence of the genetic variation on the BR sensitivity in maize root.

<p>Maize inbred seedlings W22, Mo20W, A619, and B73 grown in vermiculite for 8 d at 29°C in the dark in the presence or absences of 1.25, 5, or 20 µM BL, respectively. Average length of the primary root measured at the end of treatment (<i>n</i>>15). Error bars represent standard deviation. Statistical analysis determined by “Post-hoc” test (<i>p</i><0.05) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036625#pone.0036625.s003" target="_blank">Table S3</a>.</p

Responses of <i>Arabidopsis</i> seedlings to inhibitor treatments and BL Complementation.

<p>(<b>A–C</b>) 3-day old Ws-2 seedlings were transferred to ½ MS media containing 0, 0.1, 0.5, 1, or 5 µM of Pcz and incubated for 5 more days. (<b>A</b>) Seedlings at the end of treatment (5 d). (<b>B</b>) Close-up of the cotyledons and true leaves in the same order as (<b>A</b>). (<b>C</b>) Average root lengths at day 3 of treatment are illustrated (<i>n</i>>15). (<b>D</b>) 3-day old Ws-2 seedlings were transferred to ½ MS media containing 1 µM of Pcz, Brz, Ucz, or co-applications of inhibitors (1 µM) with 0.1 µM BL or 0.1 µM BL alone and incubated for 4 more days. Average root lengths at the end of treatment (n>15). (<b>C–D</b>) Error bars represent standard deviation and lowercase letters indicate significant differences among treatments determined by “Post-hoc” test. Scale bar (<b>A</b>) 1 cm and (<b>B</b>) 0.5 cm.</p

Soilless plant growth media influence the efficacy of phytohormones and phytohormone inhibitors.

Plant growth regulators, such as hormones and their respective biosynthesis inhibitors, are effective tools to elucidate the physiological function of phytohormones in plants. A problem of chemical treatments, however, is the potential for interaction of the active compound with the growth media substrate. We studied the interaction and efficacy of propiconazole, a potent and specific inhibitor of brassinosteroid biosynthesis, with common soilless greenhouse growth media for rice, sorghum, and maize. Many of the tested growth media interacted with propiconazole reducing its efficacy up to a hundred fold. To determine the molecular interaction of inhibitors with media substrates, Fourier Transform Infrared Spectroscopy and sorption isotherm analysis was applied. While mica clay substrates absorbed up to 1.3 mg of propiconazole per g substrate, calcined clays bound up to 12 mg of propiconazole per g substrate. The efficacy of the gibberellic acid biosynthesis inhibitor, uniconazole, and the most active brassinosteroid, brassinolide, was impacted similarly by the respective substrates. Conversely, gibberellic acid showed no distinct growth response in different media. Our results suggest that the reduction in efficacy of propiconazole, uniconazole, and brassinolide in bioassays when grown in calcined clay is caused by hydrophobic interactions between the plant growth regulators and the growth media. This was further confirmed by experiments using methanol-water solvent mixes with higher hydrophobicity values, which reduce the interaction of propiconazole and calcined clay