RESUMEN
Membrane Steroid Binding Protein 1 (MSBP1) can bind steroids in vitro and negatively regulates brassinosteroid (BR) signaling, as well as cell elongation and expansion. Detailed analysis of the MSBP1 expression pattern based on quantitative real-time RT-PCR and promoter-GUS fusion studies revealed that MSBP1 expression in hypocotyls is stimulated by various light conditions. Interestingly, MSBP1 expression is greatly suppressed in hy5, hyh, or hy5 hyh mutants but enhanced in cop1 mutants. Further analysis employing a yeast one-hybrid assay, an electrophoretic mobility shift assay (EMSA), and a Chromatin IP (ChIP) assay confirmed the direct binding of Long Hypocotyl 5 (HY5) and HY5 Homolog (HYH) to the promoter region of MSBP1, indicating that MSBP1 is involved in light-regulated hypocotyl growth by serving as a direct target for HY5 and HYH. In addition, hy5 and hy5 hyh mutants show altered BR responses to light, which is consistent with the suppressed expression of MSBP1 in these mutants. These results suggest that light triggers MSBP1 expression through direct binding to and activation by HY5 and HYH, thereby inhibiting hypocotyl elongation. The findings also provide informative clues regarding the mechanisms for the negative regulation of BR sensitivity and photomorphogenesis during the dark-light transition.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Brasinoesteroides/biosíntesis , Proteínas Portadoras/metabolismo , Luz , Morfogénesis/efectos de la radiación , Proteínas Nucleares/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Proteínas Portadoras/genética , Proteínas de Unión al ADN , Factores de Transcripción GATA/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Mutación , Proteínas Nucleares/deficiencia , Regiones Promotoras Genéticas/genética , Transducción de Señal/efectos de la radiación , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
In order to study Brassica napus fatty acid (FA) metabolism and relevant regulatory networks, a systematic identification of fatty acid (FA) biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization (>8000 EST clones from seed). The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17-21 d after flowering (DAF), whereas FA biosynthesis-related genes were highly expressed primarily at 21 DAF. Hormone (auxin and jasmonate) signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.
Asunto(s)
Arabidopsis/genética , Brassica napus/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas/fisiología , Metabolismo de los Lípidos/fisiología , Semillas/genética , ADN Complementario , Etiquetas de Secuencia Expresada , Ácidos Grasos/genética , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas/genética , Metabolismo de los Lípidos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
Brassinosteroids (BRs) are perceived by transmembrane receptors and play vital roles in plant growth and development, as well as cell in responses to environmental stimuli. The transmembrane receptor BRI1 can directly bind to brassinolide (BL), and BAK1 interacts with BRI1 to enhance the BRI1-mediated BR signaling. Our previous studies indicated that a membrane steroid-binding protein 1 (MSBP1) could bind to BL in vitro and is negatively involved in BR signaling. To further elucidate the underlying mechanism, we here show that MSBP1 specifically interacts with the extracellular domain of BAK1 in vivo in a BL-independent manner. Suppressed cell expansion and BR responses by increased expression of MSBP1 can be recovered by overexpressing BAK1 or its intracellular kinase domain, suggesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies revealed that both MSBP1 and BAK1 are localized to plasma membrane and endocytic vesicles and MSBP1 accelerates BAK1 endocytosis, which results in suppressed BR signaling by shifting the equilibrium of BAK1 toward endosomes. Indeed, enhanced MSBP1 expression reduces the interaction between BRI1 and BAK1 in vivo, demonstrating that MSBP1 acts as a negative factor at an early step of the BR signaling pathway.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Colestanoles/farmacología , Endocitosis , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Esteroides Heterocíclicos/farmacología , Brasinoesteroides , Dimerización , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
The Shanghai RAPESEED Database (RAPESEED, http://rapeseed.plantsignal.cn/) was created to provide the solid platform for functional genomics studies of oilseed crops with the emphasis on seed development and fatty acid metabolism. The RAPESEED includes the resource of 8462 unique ESTs, of which 3526 clones are with full length cDNA; the expression profiles of 8095 genes and the Serial Analysis of Gene Expression (SAGE, 23,895 unique tags) and tag-to-gene data during seed development. In addition, a total of approximately 14,700 M3 mutant populations were generated by ethylmethanesulfonate (EMS) mutagenesis and related seed quality information was determined using the Foss NIR System. Further, the TILLING (Targeting Induced Local Lesions IN Genomes) platform was established based on the generated EMS mutant population. The relevant information was collected in RAPESEED database, which can be searched through keywords, nucleotide or protein sequences, or seed quality parameters, and downloaded.