Control of specific gene expression by gibberellin and brassinosteroid

Abstract

We identified a recessive, brassinolide-insensitive mutant caused by a deletion allele (bri1-201) of the brassinosteroid (BR) receptor BRI1. The bri1-201 mutant displayed altered expression levels of genes differentially regulated by gibberellin (GA). RNA-blot analysis revealed that BR and GA antagonistically regulate the accumulation of mRNAs of the GA-responsive GASA1 gene, as well as the GA-repressible GA5 gene. Expression studies with cycloheximide indicated that the antagonistic effects of GA and BR on GA5 require de novo protein synthesis. Reporter transgene analyses and RNA-blot analysis showed that BR and GA modulate GA5 expression, at least in part, at the transcriptional level, and that the signals are independent and subtractive.

Figure 1

Phenotype of the bri1-201 mutant under long-day conditions (16 h light/8 h dark). A, Comparative development of bri1-201 and WT. B, bri1-201 35 d after germination. C through F, WT (C and D) and bri1-201 flowers (E and F). In D and F, sepals and petals were removed to show reproductive organs. Bars represent 1 mm.

Figure 2

bri1-201 exhibits insensitivity to EBR and constitutive skotomorphogenesis. A, Two-week-old WT and bri1-201 were transferred to plates without (con) or with (+EBR) 0.5 μm EBR and grown for 5 d. B, Root length of WT, bri1-1 and bri1-201 seedlings grown 5 d without (control) or with (+EBR) 0.2 μm EBR. Each value represents the mean of 50 independent measurements. C, Skotomorphogenesis of WT and mutant seedlings. Seedlings were germinated and grown for 5 d in darkness on Murashige and Skoog medium.

Figure 3

RNA-blot analysis of GA5, GA4, GASA1, GAI, and RGA mRNA accumulation in bri1-201 and WT upon GA₃ treatment. Poly(A⁺) RNA (1 μg lane⁻¹) from 16-d-old plants grown on Murashige and Skoog medium without or with 50 μm GA₃ added for the last 48 h. Ribonucleic ³²P-CTP antisense probes were synthesized using T7 RNA polymerase from partial cDNA 3′ sequences cloned in the pGEM-Teasy vector. GA5, GA4, and GASA1 hybridizations were performed on the same filter, whereas GAI and RGA hybridizations were performed on independent filters. Radioactive signals were quantified on all membranes and standardized (WT con = 100) by comparison to signals obtained after subsequent blotting with the EF1-α probe.

Figure 4

GASA1 and GA5 mRNA accumulation is antagonistically controlled by GA and BR. Two-week-old seedlings grown on Murashige and Skoog were transferred to 50 mL liquid one-half-strength Murashige and Skoog in flasks for 1 d prior to treatment. RNA-blot analyses were performed and normalized with EF1-α as described in Figure 3. A, WT, bri1-201 and cpd seedlings were treated with 1 μm EBR for 48 h. B, cpd seedlings were treated with GA or BR in presence (CHX) or not of translational inhibitors. CHX (50 μm) and chloramphenicol (50 μm) were added to the medium 2 h before GA or BR treatments (50 μm GA₃ or 1 μm EBR for 16 h).

Figure 5

Effects of BR and GA on GA5-LUC expression. A, LUC imaging of the GA5-LUC reporter in 16-d-old WT-GA5-LUC and cpd-GA5-LUC seedlings treated with 1 μm EBR for various times. Insert in A, GA5 transcript accumulation monitored by RNA-blot analysis from 16-d-old cpd seedlings treated with 1 μm EBR for 2 h. Hybridization conditions and normalization with EF1-α were performed as described in Figure 3. B, One-week-old cpd-GA5-LUC seedlings grown on Murashige and Skoog plates were transferred to liquid one-half-strength Murashige and Skoog in presence or absence of ancymidol (1 mg L⁻¹) and grown for 7 more d. BR (0.1 μm EBR) and GA (50 μm GA₃) treatments were performed for 16 h by adding the hormones to the medium. C, GA5 transcript accumulation in 16-d-old cpd-ga1-1 double mutant seedlings treated with 1 μm EBR for 16 h. Hybridization conditions and normalization with EF1-α were performed as described in Figure 3. In A and B, bioluminescent LUC images (displayed in pseudocolors) acquired by CCD camera are shown below the quantification (average gray values) of LUC images.