Brassinosteroids (BRs) are plant steroid hormones, which play important roles in a wide range of physiological and developmental processes, including cell expansion, cell division, vascular differentiation, seed germination, photomorphogenesis, stress tolerance and disease resistance. Molecular genetic studies have identified several components of the BR signaling pathway, through which BRs bind the cell surface receptors to regulate nuclear gene expression. However, there are still gaps in our knowledge of the current BR signaling pathway. In particular, the substrates of the receptor kinases that transduce the BR signal from plasma membrane to cytoplasmic components remain unknown. Despite the success of genetic studies, the function of many genes cannot be discovered by forward genetic approaches, largely due to genetic redundancy. Therefore, alternative approaches are required to significantly advance our understanding of the BR signaling pathway. Thus, proteomic studies, which use two-dimensional difference gel electrophoresis followed by mass spectrometry, have been used to identify BR-regulated proteins in Arabidopsis. The goal of this research is to characterize plasma membrane-localized BR-responsive proteins that are likely to mediate early BR signaling events. Functional studies of these proteins will advance our understanding of BR signal transduction and provide molecular tools for improving plant productivity.
The broader impacts of this project include establishment of effective proteomic methods and training graduate students and postdocs to develop their research skills in molecular genetic and proteomic approaches for studying plant signal transduction. This project will also provide opportunities for undergraduate students and high school students to participate in modern life science research. Through teachers in local high schools and community colleges, undergraduate and high school students from under-represented minority groups will be identified. These students will be instilled with a fascination for scientific research through summer and academic year research experiences. This will have a positive influence on their choice of future career.
Brassinosteroid is a major growth-promoting hormone that is required for a wide range of important developmental processes in plants. Brassinosteroid is perceived at the cell surface by the receptor kinase BRI1, which is one of the over two hundred leucine-rich repeat receptor-like kinases (LRR-RLK) in Arabidopsis. BR activation of BRI1 initiates a chain of signal transduction events that lead to alteration of gene expression in the cell nucleus. However, the biochemical nature of this signal transduction chain was not fully understood. Although genetic studies identified a cytoplasmic kinase and a phosphatase involved in this process, direct interaction between these cytoplasmic components and the membrane-anchored receptors was not observed, and therefore how the BRI1 receptor kinase regulates these cytoplasmic components was an outstanding question. To answer this question, we searched for brassinosteroid-regulated membrane-associated proteins using a powerful proteomic tool called two-dimensional difference gel electrophoresis (2-D DIGE). Our research identified three homologous BR-signaling kinases (renamed BSK1, BSK2, and BSK3), which become phosphorylated upon brassinosteroid treatment of plants. BSKs are kinase proteins that are localized on the inner side of the plasma membrane but contain no transmembrane domain. The effects of loss of BSK3 and overexpression of BSKs indicated an important function of BSKs in brassinosteroid signaling. We further showed that BRI1 interacts with BSK1 and phosphorylates BSK1 at the Serine-230 residue. Upon BRI1 phosphorylation, BSK1 and a related kinase CDG1 interact with and activate the BSU1 phosphatase. We further showed that BSU1 interacts with and inactivates BIN2, a member of the glycogen synthase kinase 3 (GSK3) family kinases, which otherwise phosphorylates and inactivates BZR1, the DNA-binding protein that regulates brassinosteroid-responsive gene expression. As such, our identification of BSKs filled a major gap in the brassinosteroid signaling chain, and our detailed biochemical studies fully connected, with mechanistic details, the brassinosteroid signaling chain from the cell surface receptor to the nuclear gene regulator. This study made key contributions to the first complete and so far best-understood molecular chain connecting a cell surface RLK with nuclear DNA-binding protein in plants. Therefore, this study not only greatly advanced our understanding of the biochemical mechanisms of steroid regulation of plant development, but also provides a paradigm for understanding hundreds of RLK pathways in plants, many of which are known to control important processes such as development, immunity, and responses to the environment. Our study also demonstrates an effective proteomic strategy for studying signal transduction using a combination of sub-cellular fractionation and 2-D DIGE, which can potentially benefit broadly studies of cellular regulatory mechanisms. Through this project, three postdocs have obtained extensive training in proteomics and signal transduction research and have successfully moved on to independent academic positions; a high school student was named semifinalist of the Intel Science Competition, and several high school and undergraduate students have gained valuable research experience and developed interest in science.
