Epibrassinolide

For expression analysis of B2 protein coding gene in wheat, T. aestivum cv. PBW343 seeds were surface sterilized, placed in the culture room maintained at 22 ± 1°C with 16:8 h light and dark phase with a light intensity of 100–125 μmol m^-2s^-1. For gene expression analysis, 12-day-old seedlings were given heat stress at 37°C for 2 h and then at 42°C for another 2 h maintained in a growth chamber. In addition, seedlings were incubated in 150 mM NaCl solution for simulating salt stress, 2% mannitol for drought stress and at 4°C for cold stress for 24 h. For hormone treatments, seedlings were immersed in abscisic acid (ABA; 10 μM, Sigma, USA), brassinosteroid (BR; 1 μM epibrassinolide, Sigma), salicylic acid (SA; 100 μM) and CaCl₂ (10 mM) solutions for 4 h (Khurana et al., 2012 (link)). All samples were frozen in liquid nitrogen and stored at -80°C until RNA isolation. Arabidopsis thaliana ecotype Columbia was used as wild-type for the generation of transgenic plants and for gene expression analysis. Plants were grown in Petriplates containing half strength of MS medium and in pots containing vermiculite, with a 16:8 h light and dark phase at 22 ± 1°C under 100–125 μmol m^-2s^-1 photoperiod.

Karrikins (KAR₁, KAR₂), GR24^5DS, and GR24^ent-5DS were prepared as described (Flematti et al., 2007 (link); Goddard-Borger et al., 2007 (link); Scaffidi et al., 2014 (link)) and dissolved as 10 mM stock solutions in acetone. Epibrassinolide (Sigma E1641), gibberellic acid (GA₄ from L. N. Mander, Australian National University), 3-indoleacetic acid (Sigma I2886), (+)-cis, trans-abscisic acid (AG Scientific A-1103) and (±)-jasmonic acid (Sigma J2500) were dissolved in acetone as 5, 10, 10, 10, and 50 mM stock solutions, respectively.

For hypocotyl growth assays, sterilized seeds were grown on 1/2 MS medium with or without 30 mM sucrose in the dark for 5 days. To assess potential interactions between sucrose and BR, the medium also contained BR (Epibrassinolide) (Sigma-Aldrich, St. Louis, MO, USA), the TOR inhibitor AZD8055 (1 μM) (M1666, AbMole, Houston, TX, USA), the S6K inhibitor LY2584702 tosylate (AbMole, M4864), or a mock solution of dimethyl sulfoxide (DMSO). At the end of the designated incubation period, the etiolated seedlings were carefully removed from the agar plates and placed on a flat surface for photography with a digital camera. The hypocotyl lengths of individual seedlings were measured using ImageJ software (http://rsb.info.nih.gov/ij/, accessed on 10 May 2021).

Four-week-old wild-type (WT) Populus “Nanlin 895” were incubated with the hormone solutions. The concentrations for the treatments were 0 (control), 1, 10, and 50 μM BL (Epibrassinolide; Sigma–Aldrich, St. Louis, MO, United States). BL-treated stems were subjected to PdC3H17 expression analysis by quantitative real-time polymerase chain reaction (qRT-PCR). Three biological replicates were performed independently.
To analyze the responsiveness of BRs or auxin with BRs on xylem formation, at least three independent lines of root-removed control and transgenic seedlings were grown on the 1/2 MS medium with or without PCZ/(PCZ + IBA) at various determined concentrations for 21 days (8 h for gene expression analysis), and 0.3-cm segments were taken from their basal stems. These segments were fixed in 4% paraformaldehyde (Sigma-Aldrich) at 4°C for 4 days, dehydrated in graded ethanol series, and embedded into paraplast. The 5-μm sections were obtained using a Leica RM2235 rotary microtome and adhered to Superfrost microscope slides (Thermo). The sections were stained with 0.1% toluidine blue and observed using an Olympus X51 light microscope (Olympus). Radial widths of xylem were measured in three independent replicates using the SmileView software (JEOL).

The 7-day-old rice seedlings in 50 mL polypropylene conical tubes containing 30 mL water were first treated with varying levels of epibrassinolide (BR; Sigma-Aldrich, St. Louis, MO, USA), ethephon (Sunmoon Green Science, Seoul, Korea), 6-benzylaminopurine (BA; Sigma-Aldrich), and indole-3-acetic acid (IAA; Sigma-Aldrich). The BR biosynthesis inhibitor brassinazole (100 µM; Sigma-Aldrich) and GA biosynthesis inhibitor paclobutrazol (10 µM; Sigma-Aldrich) were used to inhibit BR and GA biosynthesis, respectively. GA₃ was purchased from Duchefa Biochemie (Harrlem, The Netherlands). BR, BA, and IAA were dissolved in 0.1% ethanol. The control (C) contained 0.1% ethanol in water, except for ethephon, where the control was water. Treatments were applied for 1 day under cool daylight fluorescent lamps (60 μmol m^–2 s^–1) (Philips) with a 14 h light/10 h dark photoperiod at 28 °C/24 °C (day/night), followed by treatment with 0.5 mM CdCl₂ for 3 days under continuous light conditions (60 μmol photons m^–2 s^–1) for melatonin detection by high-performance liquid chromatography (HPLC). The upper parts of leaves and stems were harvested and stored in liquid nitrogen for further analyses.

To examine the response of hypocotyl epidermis cells to 1-naphthylphthalamic acid (NPA; Sigma-Aldrich 399728), seedlings of wild-type were grown on 1/2 MS with or without 10 μM NPA for 7 days. The morphology of hypocotyl epidermis cells was observed by DIC.
To analyze the formation of ovule primordia upon 1-naphthylacetic acid (NAA; Sigma-Aldrich 317918), picloram (Sigma-Aldrich 1918021) and Epibrassinolide (eBL; Sigma-Aldrich 78821439) treatments, the floral buds larger than stage 10 were removed and the remaining inflorescences were immersed in 0.01% (v/v) Silwet L-77 solutions containing 2 μM eBL, 2 μM NAA, or 5 μM picloram as previously described [65 (link)]. The control inflorescences were treated with 0.01% Silwet L-77 solutions containing corresponding volume ethanol or DMSO. After 24 h, the inflorescences were washed with distilled water to remove residual chemicals. The pistils were collected 2 days later.
For qRT-PCR analysis, the whole wild-type inflorescences were immersed in the mock (containing 0.5% DMSO or 1% ethanol), 50 μM picloram and 100 μM NAA solutions, respectively, as described above. The pistils were collected after 1 h for later analysis.

The sensitivity to BR was investigated with the leaf unrolling test (Wada et al., 1985 (link); as described by Chono et al. (2003) (link)). Samples were planted in vermiculite grown in darkness at 24°C for 8–9 days for mutant and WT plants. Leaf segments of 1.5 cm were incubated for 72 h in 10⁻⁵ M Epibrassinolide (Sigma-Aldrich) with water as control. The level of unrolling was measured using digital calipers. At least seven samples were used for the mutant and WT, respectively, and the experiment was repeated three times.