The Dual Function Brassinosteroid Signaling Components Sterol Bki1 Mediated By A New Signal Transduction Pathway Studies

Brassinosteroids (BRs) are a class of plant steroid hormones and play important roles in a wide range of developmental and physiological processes such as stem elongation, vascular differentiation, male fertility, seed size and germination, flowering time, senescence, and resistance to biotic and abiotic stresses. To regulate these biological processes, BR signaling is initiated by BR binding to a cell surface localized receptor kinase, BRASSINOSTEROID INSENSITIVE 1 (BRI1). In the absence of BRs, BRI1 is inactive, largely due to the negative regulator BRI1 KINASE INHIBITOR 1 (BKI1). BKI1 is localized at the plasma membrane (PM), and interacts with BRI1 to inhibit BRI1 activity. When BRs bind to BRI1,BKI1 is phosphorylated and dissociates from the PM to the cytosol, releasing its inhibition on BRI1. This subsequently activates the BR signal transduction pathway. However, the underlying mechanism behind BKI1's dissociation and its function in the cytosol is unknown. Here, we studied the dissociation mechanism of BKI1 and its function in the cytosol, and elucidated the molecular and biochemical mechanism of the BR signal transduction pathway. Our three main results are shown below.Firstly, we elucidated the dissociation mechanism of BKI1 from the PM. In order to study the dissociation mechanism of BKI1 from PM, the truncated BKI1 C and N terminal regions were overexpressed, and their transgenic plants were analyzed with specific reporter genes and proteins. We found that the N-terminus of BKI1 is responsible for PM localization, and its C-terminus regulates BKI1's dissociation from the PM and is involved in its function in the cytosol. In addition, over-experssion of the C-terminus can also enhance the output of BR signaling. Moreover, the phosphorylation sites of BKI1 by BRI1 kinase were identified by mass spectophotometer (MS) analysis, leading to the discovery that the S270 and S274 sites are key phosphorylation sites for BKI1's dissociation. Mutations of S270 and S274 prevented the phosphorylation of BKI1 by BRI1 kinase, leading to a constant BKI1 localization at the PM, the dwarf phenotype of their transgenic plants, and their insensitivity to BRs.Secondly, we studied the interaction mechanism of BKI1 with 14-3-3 proteins. While further examining the function of cytosolic BKI1, a 14-3-3 protein was identified to interact with the cytosolic BKI1 via LC-MS/MS anlysis. The 14-3-3s interacts with the phosphorylated BKI1 through the motif containing S270 and S274. Interestingly, the over-expression of 14-3-3s can rescue the BKI1 inhibition on plant growth. Moreover, by observing the effect of 14-3-3s on the plasma membrane localization of BKI1-YFP, we proposed that the 14-3-3s can enhance the dissociation of the phosphorylated BKI1 from the PM.Thirdly, we found the positive function of cytosolic-localized BKI1 in BR signaling. To investigate the physiological function of BKI1, a transgenic line with low levels of BKI1 expression was identified and analyzed. Through physiological experimentation, it was found that, under low levels of BRs, BKI1-YFP is mainly localized to the PM as a negative regulator of the BR signaling while, in response of high levels of BRs, BKI1 enhances BR signlaing output as a positive regulator. Furthermore, we found that the mechanism of BR signaling output enhancement is through the phosphorylated BKI1 releasing the inhibition of 14-3-3 on BES1.Based on the above findings, we propose that the steroid-induced, cytosolic and phosphorylated BKI1 positively regulates BR signaling by interacting with a subset of 14-3-3 proteins. BKI1, along with this subset of 14-3-3 proteins, play dual roles in BR signaling by directly linking the plasma membrane components and the downstream transcription factors. In the absence of BRs, BKI1 negatively regulates BR signaling by inhibiting BRI1 activity; in the presence of BR, BKI1 is phosphorylated and positively regulates BR signaling by competing with BES1 for interacting with 14-3-3s. Furthermore,14-3-3 protein also play dual role in BR signaling. In the absence of BRs,14-3-3 retains the phosphorylated BES1 in the cytosol and inhibits BR signaling; in the presence of BR, it interacts with the phosphorylated BKI1 to release its inhibition on BES1 and BR signaling. Thus, a novel, concise, and efficient BR signaling pathway, in which the proteins BKI1 and 14-3-3 play important roles, has been established.