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 transmemhrane receptor BRI1 can direct...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 transmemhrane 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 MSBPI specifically interacts with the extraeellular 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 intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI 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 BAKI 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.展开更多
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 pa...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 hyS, hyh, or hy5hyh 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 ex- pression 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 photomorpho- genesis during the dark-light transition.展开更多
Overexpression of membrane steroid binding protein 1 （MSBP1） stimulates the root gravitropism and antigravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hyp...Overexpression of membrane steroid binding protein 1 （MSBP1） stimulates the root gravitropism and antigravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hypocotyls and root tips. The inhibitory effects by 1-N-naphthylphthalamic acid （NPA）, an inhibitor of polar auxin transport, are suppressed under the MSBP1 overexpression, suggesting the positive effects of MSBP1 on polar auxin transport. Interestingly, sub-cellular localization studies showed that MSBP1 is also localized in endosomes and observations of the membraneselective dye FM4-64 revealed the enhanced vesicle trafficking under MSBP1 overexpression. MSBPl-overexpressing seedlings are less sensitive to brefeldin A （BFA） treatment, whereas the vesicle trafficking was evidently reduced by suppressed MSBP1 expression. Enhanced MSBP1 does not affect the polar localization of PIN2, but stimulates the PIN2 cycling and enhances the asymmetric PIN2 redistribution under gravi-stimulation. These results suggest that MSBP1 could enhance the cycling of PIN2-containing vesicles to stimulate the auxin redistribution under gravi-stimulation, providing informative hints on interactions between auxin and steroid binding protein.展开更多
基金Acknowledgments This study was supported by the Chinese Academy of Sciences and National Natural Science Foundation of China （Grants 30425029, 30421001, 90717001）. We greatly thank Prof Hong Ma （Penn. State University, USA） for critical reading and writing improvement and Prof Nam-Hai Chua （The Rockefeller University, USA） for helpful comments. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, and Prof Sheng Luan （University of California, Berkeley, USA） for providing the construct pATC940. We thank Prof Hong-Quan Yang （SIPPE, CAS） for providing LexA yeast two-hybrid system and Prof Zhi-Yong Wang （The Stanford University, USA） for providing the BRI1 antibody. We thank Mr Xiao-Shu Gao for the help on Confocal Laser Scanning Microscopy.
文摘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 transmemhrane 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 MSBPI specifically interacts with the extraeellular 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 intracellnlar kinase domain, sug- gesting that MSBP1 may suppress BR signaling through interacting with BAK1. Subcellular localization studies re- vealed that both MSBPI 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 BAKI 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.
文摘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 hyS, hyh, or hy5hyh 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 ex- pression 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 photomorpho- genesis during the dark-light transition.
基金This work was supported by the National Natural Science Foundation of China （No. 90717001, 30721061, 30425029） and Science and Technology Commission of Shanghai Municipality （08XD14049）.We thank Jian Xu （Utrecht University, Netherlands） for providing Arabidopsis seeds containing DR5-GUS and PIN2-EGFP expression cassettes. No conflict of interest declared,
文摘Overexpression of membrane steroid binding protein 1 （MSBP1） stimulates the root gravitropism and antigravitropism of hypocotyl, which is mainly due to the enhanced auxin redistribution in the bending regions of hypocotyls and root tips. The inhibitory effects by 1-N-naphthylphthalamic acid （NPA）, an inhibitor of polar auxin transport, are suppressed under the MSBP1 overexpression, suggesting the positive effects of MSBP1 on polar auxin transport. Interestingly, sub-cellular localization studies showed that MSBP1 is also localized in endosomes and observations of the membraneselective dye FM4-64 revealed the enhanced vesicle trafficking under MSBP1 overexpression. MSBPl-overexpressing seedlings are less sensitive to brefeldin A （BFA） treatment, whereas the vesicle trafficking was evidently reduced by suppressed MSBP1 expression. Enhanced MSBP1 does not affect the polar localization of PIN2, but stimulates the PIN2 cycling and enhances the asymmetric PIN2 redistribution under gravi-stimulation. These results suggest that MSBP1 could enhance the cycling of PIN2-containing vesicles to stimulate the auxin redistribution under gravi-stimulation, providing informative hints on interactions between auxin and steroid binding protein.
