RESUMO
Brassinosteroid (BR)-regulated growth and development in Arabidopsis depends on BRASSINOSTEROID INSENSITIVE 1 (BRI1), the BR receptor that is responsible for initiating the events of BR signalling. We analysed the temporal and spatial regulation of BRI1 expression using stable transgenic lines that carried BRI1 promoter:reporter fusions. In both seedlings and mature plants the tissues undergoing elongation or differentiation showed elevated BRI1 gene activity, and it could be demonstrated that in the hypocotyl this was accompanied by accumulation of the BRI1 transcript and its receptor protein product. In seedlings the BRI1 promoter was also found to be under diurnal regulation, determined primarily by light repression and a superimposed circadian control. To determine the functional importance of transcriptional regulation we complemented the severely BR insensitive bri1-101 mutant with a BRI1-luciferase fusion construct that was driven by promoters with contrasting specificities. Whereas the BRI1 promoter-driven transgene fully restored the wild phenotype, expression from the photosynthesis-associated CAB3 and the vasculature-specific SUC2 and ATHB8 promoters resulted in plants with varying morphogenic defects. Our results reveal complex differential regulation of BRI1 expression, and suggest that by influencing the distribution and abundance of the receptor this regulation can enhance or attenuate BR signalling.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Brassinosteroides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas Quinases/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano/genética , Ritmo Circadiano/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Germinação/genética , Germinação/efeitos da radiação , Glucuronidase/metabolismo , Luminescência , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiação , Fatores de TempoRESUMO
Brassinosteroids (BRs) are steroidal phytohormones that regulate plant growth and development. Whereas in Arabidopsis the network-like routes of BR biosynthesis have been elucidated in considerable detail, the roles of some of the biosynthetic enzymes and their participation in the different subpathways remained to be clarified. We investigated the function of the cytochrome P450 monooxygenase CYP90A1/CPD, which earlier had been proposed to act as a BR C-23 hydroxylase. Our GC-MS and genetic analyses demonstrated that the cpd mutation arrests BR synthesis upstream of the DET2-mediated 5α reduction step and that overexpression of the C-23 hydroxylase CYP90C1 does not alleviate BR deficiency in the cpd mutant. In line with these results, we found that CYP90A1/CPD heterologously expressed in a baculovirus-insect cell system catalyzes C-3 oxidation of the early BR intermediates (22S)-22-hydroxycampesterol and (22R,23R)-22,23-dihydroxycampesterol, as well as of 6-deoxocathasterone and 6-deoxoteasterone. Enzyme kinetic data of CYP90A1/CPD and DET2, together with those of the earlier studied CYP90B1, CYP90C1, and CYP90D1, suggest that BR biosynthesis proceeds mainly via the campestanol-independent pathway.