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

Abstract

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.

Figure 1. Chemical structures of propiconazole (Pcz), uniconazole (Ucz). 24-epibrassinolide (eBL), and gibberellic acid₃ (GA₃).

Structure elements critical for inhibitor activity in Pcz and Ucz 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. In GA₃, the 4,10-lactone bridge is depicted in green and the carboxyl group is depicted in orange. Hydroxyl groups in 24-epibrassinolide and GA₃ are depicted in red. Structures were drawn using the ChemBioDraw 12.0.2 software and structures were compared to the ChemACX 12.12.1 database.

Figure 2. Photographic and SEM images of various media substrates.

(A–B) peat germinating mix, (C–D) perlite. (E–F) vermiculite. (G–H) Turface. (A,C,E,G) Photographic images exhibiting textural differences between media substrates. (B,D,F,H) Scanning election microscopy photos of substrates. Scale Bar (A,C,E,H) 1 cm and (B,D,F,H) 10 µm.

Figure 3. Plant growth response of maize seedlings treated with Pcz in different media substrates.

(A–E) B73 maize seedlings grown for 11d in the light in either (B) Turface, (C) peat germinating mix, (D) vermiculite, or (E) perlite. (F–I) 1∶1 (v/v) mixtures of (G) Turface/peat mix, (H) Turface/vermiculite, and (I) Turface/perlite. Plants were watered without (left), 20 µM (center) or 200 µM (right) of Pcz as needed. (A) Height of plants grown in pure media as well as (F) Turface 1∶1 mixtures was measured from the root-shoot transition zone to the highest leaf collar using ImageJ 1.43. (A, F) Error bars represent standard deviation. Size bars indicate 5 cm.

Figure 4. LC-MS, and standard curve of Pcz within solution.

(A) LC-MS spectrum of Pcz. (B) photometrically determined absorption spectrum of Pcz measured from 200 nm to 350 nm. (C) Standard curve of Pcz concentrations from 0 to 500 mM plotted against absorption at 225 nm. (D) Absorbance kinetics of Pcz on Turface (black bars) and vermiculite (white bars) measured at 225 nm. Absorbance of Pcz to substrates was tested from 0 to 23 h.

Figure 5. FTIR.

Diffuse Reflectance (DR- FTIR) spectra of (T) Turface and (TP) Turface saturated with Pcz are shown in the 4000 to 700 cm⁻¹ region. An expanded plot showing the spectra features of interest are plotted in the inset from 1750 to 1350 cm-1 showing the DR-FTIR spectra of (T), (TP), and (TP-T) the spectral subtraction of (TP) – (T). For comparison, the Attenuated Total Reflectance (ATR-FTIR) spectrum of (P) Pcz and (pP) pure Pcz are shown. The bands marked by a (*) are spectral features influenced by the presence of the surfactant in (P) Pcz.

Figure 6. Adsorption isotherms of Pcz to Turface and vermiculite.

Adsoprtion isotherms of Pcz absorbance to Turface and vermiculite determined over 250 h. Inset shows absorbance kinetics during the first 3 h of interaction. Error bars represent standard deviation.

Figure 7. Methanol inhibits Pcz adsorption to Turface.

Adsorption isotherms of Pcz absorbance to Turface over 72 h with Pcz in water, Pcz in 10% MeOH, and Pcz in 20% MeOH. Error bars represent standard deviation.

Figure 8. Growth responses of B73 seedlings to Pcz, Ucz, eBL and GA₃ on Turface and vermiculite media.

Light grown B73 seedlings grown on Turface (black bars) and vermiculite (grey bars) treated with 1 and 10 µM Pcz, Ucz, eBL and GA₃ for 9 d. Seedlings grown on vermiculite were treated with 1 and 10 µM concentration of each chemical. Seedlings grown on Turface were treated with 10 µM concentrations of each chemical. Error bars represent standard deviation. Statistically significant differences to controls are indicated with asterisks determined by student's t-test (p<0.01).

Figure 9. Plant growth responses of maize seedlings treated with Pcz or Ucz grown in an aeroponic culture system.

B73 maize seedlings were germinated and grown as described in Material and Methods and treated with (A–B) Mock conditions, 0.01 µM Pcz, 0.1 µM Pcz, 1 µM Pcz, or 10 µM Pcz. A second set of plants was treated with (C–D) Mock, 1 µM Pcz, 10 µM Pcz, 1 µM Ucz, or 10 µM Ucz. (A,C) plant height measured from the top of the mesocotyl to the second leaf collar. (B,D) length of the primary root measured from the root tip to the root-shoot transition zone. All plants were grown in aeroponic culture (see also Fig. S3). Error bars represent standard deviation. Statistical analysis was performed by “Post Hoc” and indicated by lowercase letters (p<0.05). (E–I) Photographic images of seedlings grown in aeroponic culture. Seedlings were grown in the presence of (F) 1 µM Pcz, (G) 10 µM Pcz, (H) 1 µM Ucz, and (I) 10 µM Ucz. Scale bars indicate 5 cm.