Sterol biosynthesis in Arabidopsis. A simplified pathway illustrates the synthesis of C5-sterols including campesterol, sitosterol, and stigmasterol in Arabidopsis. Three atypical sterols (5α-cholesta-8,14-dien-3β-ol [CH], (24R)-5α-ergosta-8,14-dien-3β-ol [ER], and (24R)-5α-stigmasta-8,14-dien-3β-ol [ST]) accumulated in fk mutants are shown.

Fenpropimorph inhibits sterol C-14 reductase in wild-type plants and causes sterol composition change including the accumulation of 8,14-diene sterols. Values shown are the content of each chemical (nano- or micrograms per gram fresh weight) for plants treated with 100 μg mL⁻¹ fenpropimorph (top), with 10 μg mL⁻¹ fenpropimorph (middle), or mock treated (bottom).

Both BL and sterols are activators of gene expression. Seedlings were grown in Murashige and Skoog liquid medium in continuous white light (90 μE m⁻² s⁻¹) at 25°C on a shaker. Two-DAG seedlings were treated with BL (10⁻⁶ m) or sterols (10⁻⁶ m) for 4 h. RNA blots were hybridized to probes specific to TCH4, Meri-5, β-TUBLIN (TUB), and KOR as described in “Materials and Methods.” Five micrograms of RNA was used for each sample. Ethidium bromide-stained rRNA bands were used as loading controls.

Marker gene expression in fk and other BR-deficient mutants. Gene expression was determined in mutants blocked in BR-specific pathway (det2 and dwf4), in sterol-specific pathway (dim1, fk-X224 and fk-J79), in BR perception (bri1), and in their respective wild-type plants (Col for det2 and bri1, C24 for dim1, Landsberg erecta for fk-X224, BE for fk-J79, and En-2 for dwf4). RNA gel-blot analyses were performed using 8-DAG seedlings grown on Murashige and Skoog plates under a photoperiod of 16 h of light (90 μE m⁻² s⁻¹) and 8 h of dark at 25°C. Five micrograms of RNA was used for each sample. Ethidium bromide-stained rRNA bands were used as loading controls. Expression data for En-2 and dwf4 were obtained by reverse transcription-PCR.

Phenocopy of fk mutants using fenpropimorph. A, Wild-type plants were grown on Murashige and Skoog plates supplemented with 0, 10, 50, or 100 μg mL⁻¹ of fenpropimorph for 3 weeks under a photoperiod of 16 h light (90 μE m⁻² s⁻¹) and 8 h dark at 25°C. B to D, The effects of fenpropimorph on Arabidopsis seedling development. The effects of fenpropimorph on the elongation of hypocotyl (B), petiole (C), and main root (D) were determined using wild-type (BE) plants grown on Murashige and Skoog plates under a photoperiod of 16-h-light (90 μE m⁻² s⁻¹) and 8-h-dark cycle at 25°C for 8 d. The error bars represent ses of the means derived from the measurements of 15 plants. E, Fenpropimorph (Fen) affects the expression of genes involved in cell division and expansion. RNA was extracted from wild-type (BE) plants grown on 1× Murashige and Skoog plates under a photoperiod of 16 h of light (90 μE m⁻² s⁻¹) and 8 h of dark at 25°C for 8 d. Five micrograms of RNA was loaded in each lane. Ethidium bromide-stained rRNA bands were used as loading controls. F, Transient activation of TCH4 and FK by BL. Wild-type (Col) seedlings of 3- or 8-DAG were grown in Murashige and Skoog liquid medium in continuous white light (90 μE m⁻² s⁻¹) at 25°C on a shaker. Samples were treated with BL (10⁻⁷ m) for 0, 1, 2, 4, 8, 16, or 32 h. Five micrograms of RNA was loaded in each lane. Ethidium bromide-stained rRNA bands were used as loading controls.

Regulation of FK expression. A, The expression of reporter gene FK::GUS in wild-type, fk-J79, and det2 plants. Shown are 4-DAG seedlings stained with 5-bromo-4-chloro-3-indolyl-β-glucuronic acid for 24 h. B, Quantitative GUS analysis. Fifteen seedlings were pooled for each sample, and cell extract was used in an enzymatic assay using 4-methylumbelliferyl β-d-glucuronide (see “Materials and Methods”). The error bars represent ses of the means derived from three replicates. C, fk mutant is hypersensitive to auxin and is insensitive to cytokinin as reflected by exaggerated callus production and diminished shoot regeneration, respectively. For callus induction, root explants were sliced into 1-cm long sections, and five sections of each genotype were placed onto a CIM. CIM consisted of 1× Murashige and Skoog salts, B5 vitamins, 0.5 mg L⁻¹ 2,4-dichlorophenoxyacetic acid, 0.05 mg L⁻¹ benzylaminopurine, 0.05 mg L⁻¹ kinetin, and 0.7% (w/v) phytagar (Invitrogen). Callus induction was conducted for 10 d under 50 μE m⁻² s⁻¹ white light at 25°C. For shoot induction, callus was transferred to SIM and grown for 2 to 4 weeks under the same condition. SIM consisted of 1× Murashige and Skoog salts, 5 mg L⁻¹ isopentenyl adenine, 0.15 mg L⁻¹ IAA, and 0.7% (w/v) phytagar (Invitrogen). D, FK expression was enhanced by IAA, BL, gibberellin (GA), and cytokinin (BA). RNA gel-blot analysis was performed using 3-DAG wild-type seedlings grown in Murashige and Skoog liquid medium in continuous white light (90 μE m⁻² s⁻¹) at 25°C on a shaker. RNA samples were extracted from seedlings treated with hormone (10⁻⁶ m) for 4, 8, 16, or 32 h. Five micrograms of RNA was loaded in each lane. Ethidium bromide-stained rRNA bands were used as loading controls. E, Sterols enhance the expression of FK. RNA gel-blot analyses were performed using 2-DAG seedlings grown in Murashige and Skoog liquid medium in continuous white light (90 μE m⁻² s⁻¹) at 25°C on a shaker. Seedlings were treated with BL (10⁻⁶ m) or sterols (10⁻⁶ m sitosterol, stigmasterol, CH, ER, or ST) for 4 h. Five micrograms of RNA was loaded in each lane. Ethidium bromide-stained rRNA bands were used as loading controls.