Membrane steroid-binding protein 1 （MSBP1） negatively regulates brassinosteroid signaling by enhancing the endocytosis of BAK1

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.

Heterotrimeric G protein α subunit is involved in rice brassinosteroid response

Heterotrimeric G proteins are known to function as messengers in numerous signal transduction pathways.The nullmutation of RGA(rice heterotrimeric G protein α subunit),which encodes the α subunit of heterotrimeric G proteinin rice,causes severe dwarfism and reduced responsiveness to gibberellic acid in rice.However,less is known aboutheterotrimeric G protein in brassinosteroid(BR)signaling,one of the well-understood phytohormone pathways.In thepresent study,we used root elongation inhibition assay,lamina inclination assay and coleoptile elongation analysis todemonstrated reduced sensitivity of dl mutant plants(caused by the null mutation of RGA)to 24-epibrassinolide(24-epiBL),which belongs to brassinosteroids and plays a wide variety of roles in plant growth and development.Moreover,RGA transcript level was decreased in 24-epiBL-treated seedlings in a dose-dependent manner.Our results show thatRGA is involved in rice brassinosteroid response,which may be beneficial to elucidate the molecular mechanisms of Gprotein signaling and provide a novel perspective to understand BR signaling in higher plants.

Brassinosteroid Signaling and Application in Rice

Combined approaches with genetics, biochemistry, and proteomics studies have greatly advanced our understanding of brassinosteroid （BR） signaling in Arabidopsis. However, in rice, a model plant of monocot and as well an important crop plant, BR signaling is not as well characterized as in Arabidopsis. Recent studies by forward and reverse genetics have identified a number of either conserved or specific components of rice BR signaling pathway, bringing new ideas into BR signaling regulation mechanisms. Genetic manipulation of BR level or BR sensitivity to improve rice yield has established the great significance of BR research achievements.

Brassinosteroid Signaling Recruits Histone 3 Lysine-27 Demethylation Activity to FLOWERING LOCUS C Chromatin to Inhibit the Floral Transition in Arabidopsis

The steroid hormone brassinosteroid （BR） plays a broad role in plant growth and development. As the retarded growth in BR-insensitive and BR-deficient mutants causes a strong delay in days to flowering, BR signaling has been thought to promote the floral transition inArabidopsis. In this study, using a developmental measure of flowering time, we show that BR signaling inhibits the floral transition and promotes vegetative growth in the Arabidopsis accessions Columbia and Enkheim-2. We found that BR signaling promotes the expression of the potent floral repressor FLOWERING LOCUS C （FLC） and three FLC homologs to inhibit flow- ering. In the presence of BR, the transcription factor BRASSINAZOLE-RESISTANT1 （BZR1）, together with BES1 -INTERACTING MYC-like proteins （BIMs）, specifically binds a BR- responsive element in the first intron of FLC and further recruits a histone 3 lysine 27 （H3K27） demethylase to downregulate levels of the repressive H3K27 trimethylation mark and thus antagonize Polycomb silencing at FLC, leading to its activation. Taken together, our findings demonstrate that BR signaling inhibits the floral transition inArabidopsis by a novel molecular mechanism in which BR signals are transduced into FLC activation and consequent floral repression.

Ribonuclease H-like gene SMALL GRAIN2 regulates grain size in rice through brassinosteroid signaling pathway

Grain size is a key agronomic trait that determines the yield in plants.Regulation of grain size by brassinosteroids(BRs)in rice has been widely reported.However,the relationship between the BR signaling pathway and grain size still requires further study.Here,we isolated a rice mutant,named small grain2(sg2),which displayed smaller grain and a semi-dwarf phenotype.The decreased grain size was caused by repressed cell expansion in spikelet hulls of the sg2 mutant.Using map-based cloning combined with a Mut Map approach,we cloned SG2,which encodes a plant-specific protein with a ribonuclease H-like domain.SG2 is a positive regulator downstream of GLYCOGEN SYNTHASE KINASE2(GSK2)in response to BR signaling,and its mutation causes insensitivity to exogenous BR treatment.Genetical and biochemical analysis showed that GSK2 interacts with and phosphorylates SG2.We further found that BRs enhance the accumulation of SG2 in the nucleus,and subcellular distribution of SG2 is regulated by GSK2 kinase activity.In addition,Oryza sativa OVATE family protein 19(OsOFP19),a negative regulator of grain shape,interacts with SG2 and plays an antagonistic role with SG2 in controlling gene expression and grain size.Our results indicated that SG2 is a new component of GSK2-related BR signaling response and regulates grain size by interacting with Os OFP19.

Silencing the SLB3 transcription factor gene decreases drought stress tolerance in tomato

BRI1-EMS-SUPPRESSOR 1(BES1)transcription factor is closely associated with the brassinosteroid(BR)signaling pathway and plays an important role in plant growth and development.SLB3 is a member of BES1 transcription factor family and its expression was previously shown to increase significantly in tomato seedlings under drought stress.In the present study,we used virus-induced gene silencing(VIGS)technology to downregulate SLB3 expression to reveal the function of the SLB3 gene under drought stress further.The downregulated expression of SLB3 weakened the drought tolerance of the plants appeared earlier wilting and higher accumulation of H2 O2 and O2^–·,decreased superoxide dismutase(SOD)activity,and increased proline(PRO)and malondialdehyde(MDA)contents and peroxidase(POD)activity.Quantitative real-time PCR(qRT-PCR)analysis of BR-related genes revealed that the expression of SlCPD,SlDWARF and BIN2-related genes was significantly upregulated in SLB3-silenced seedlings under drought stress,but that the expression of TCH4-related genes was downregulated.These results showed that silencing the SLB3 gene reduced the drought resistance of tomato plants and had an impact on the BR signaling transduction which may be probably responsible for the variation in drought resistance of the tomato plants.

Spatiotemporal Dynamics of the BRI1 Receptor and its Regulation by Membrane Microdomains in Living Arabidopsis Cells

The major brassinosteroid （BR） receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 （BRI1） plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRII. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFIotl-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFIotl amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bril-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.

Arabidopsis MSBP1 Is Activated by HY5 and HYH and Is Involved in Photomorphogenesis and Brassinosteroid Sensitivity Regulation

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.