Phytohormone inhibitor treatments phenocopy brassinosteroid-gibberellin dwarf mutant interactions in maize

Abstract

Phytohormone biosynthesis produces metabolites with profound effects on plant growth and development. Modulation of hormone levels during developmental events, in response to the environment, by genetic polymorphism, or by chemical application, can reveal the plant processes most responsive to a phytohormone. Applications of chemical inhibitors and subsequent measurements of specific phytohormones can determine whether, and which, phytohormone is affected by a molecule. In many cases, the sensitivity of biochemical testing has determined multiple pathways affected by a single inhibitor. Genetic studies are not subject to this problem, and a wealth of data about the morphological impacts of hormone biosynthetic inhibition have accumulated through the study of enzyme mutants. In this work, we sought to assess the specificity of three triazole inhibitors of cytochrome P450s by determining their abilities to recapitulate the phenotypes of single and double mutants affected in the production of brassinosteroid (BR) and gibberellin (GA) biosynthesis. The GA biosynthetic inhibitors uniconazole (UCZ) and paclobutrazol (PAC) were applied to the BR biosynthetic mutant nana plant2 (na2), and all double-mutant phenotypes were recovered in the UCZ treatment. PAC was unable to suppress the retention of pistils in the tassels of na2 mutant plants. The BR biosynthetic inhibitor propiconazole (PCZ) suppressed tiller outgrowth in the GA biosynthetic mutant dwarf5 (d5). All treatments were additive with genetic mutants for effects on plant height. Due to additional measurements performed here but not in previous studies of the double mutants, we detected new interactions between GA and BR biosynthesis affecting the days to tassel emergence and tassel branching. These experiments, a refinement of our previous model, and a discussion of the extension of this type of work are presented.

Keywords: Zea mays; biochemical genetics; flowering; paclobutrazol; propiconazole; uniconazole.

Figure 1

Structures of chemicals used in this study. Chemical structure of (a) propiconazole, (b) paclobutrazol, and (c) uniconazole

Figure 2

All three plant growth regulators are known to affect more than one pathway. (a) Brassinosteroid, (b) gibberellin, (c) cytokinin, and (d) ABA metabolic pathways. Metabolites are indicated in black text and enzymes by black arrows with respective names in gray text. Functionally characterized enzymes in maize are indicated in parentheses. Previously identified enzymes inhibited by PCZ (yellow), PAC (purple), and UCZ (green) are indicated by colored ovals

Figure 3

Effects of plant growth regulators and genotype on internode length. Mean internode lengths in cm and SD of (a) mock‐treated, (b) PCZ‐treated, (c) PAC‐treated, and (d) UCZ‐treated na2‐1 and wild‐type siblings. Asterisks indicate statistical difference by Student's t test at p < .05 between na2‐1 and wild‐type siblings at the respective internode

Figure 4

Effects of na2‐1 or PCZ on maize plant architecture. Mock‐treated (a) wild‐type plant and (b) na2‐1 plant. (c) PCZ‐treated wild‐type plant. Scale bar corresponds to 20 cm

Figure 5

Effects of plant growth regulators and genotype on internode length. Mean internode lengths in cm and SD of (a) mock‐treated, (b) PCZ‐treated, (c) PAC‐treated, and (d) UCZ‐treated d5 and wild‐type siblings. Asterisks indicate statistical difference by Student's t test at p < .05 between d5 and wild‐type siblings at the respective internode

Figure 6

Effects of d5, PAC, and UCZ on maize plant architecture. Mock‐treated (a) wild‐type plant and (b) d5 plant. (c) PAC‐treated and (d) UCZ‐treated wild‐type plant. Scale bar corresponds to 20 cm

Figure 7

Summary of inhibitor tests of the genetic model of BR–GA interaction during maize growth. Check marks indicate concordance of inhibitor treatment and combined inhibitor and mutant with respective biosynthetic single‐ and double‐mutant analysis presented in Best, Hartwig et al. (2016). PCZ is indicated in yellow, UCZ in blue, and PAC in red. Dashed lines indicate trait measured; lines with arrows indicate positive regulation; lines with blocked arrows indicate negative regulation; and lines with diamond heads indicate synergistic positive regulation. GA mutants were epistatic to BR mutants for the presence of pistils in tassel (POPIT), and both PCZ and UCZ treatments were concordant with this interaction, while PAC was not. GA inhibits anther‐ear (AE), and both UCZ and PAC phenocopied the mutants. BR mutants were epistatic to GA mutants for tiller outgrowth (T), and both PCZ and UCZ were concordant for this interaction, while PAC was not. GA and BR were additive for plant height (PH), and all three inhibitors were concordant for this phenotype

Figure 8

Proposed interactions between BR and GA based on inhibitor treatments of the d5 and na2‐1 mutants. Check marks indicate concordance of inhibitor treatments with single‐mutant results and concordance between combined inhibition of both pathways achieved with mutant–inhibitor combinations. PCZ is indicated in yellow, UCZ in blue, and PAC in red. Dashed lines indicate trait measured; lines with arrows indicate positive regulation; lines with blocked arrows indicate negative regulation; and lines with diamond heads indicate synergistic positive regulation. (a) GA and BR were additive for days to tassel (DT), and all three inhibitors were concordant with this observation. Inhibition of GA in na2‐1 mutants decreased total node number (TN) and node of the top ear (NTE), but inhibition of BR in d5 mutants did not. (b) GA and BR were additive for length of the upper leaf (LUL) in all combinations. (c) GA and BR reduced the angle of the lower leaf (ALL) and length of the lower leaf (LLL), and all three inhibitors additively affected this phenotype. GA inhibited the width of the lower leaf (WLL), and both UCZ and PAC were concordant. (d) GA and BR were additive for promotion of tassel length (TL) and tassel branch number (TB), and all three inhibitors were concordant for these phenotypes