SUMMARY Brassinosteroids (BRs) regulate a wide range of developmental and physiological processes in plants through a receptor-kinase signaling pathway that controls the BZR transcription factors. Here we use transcript profiling and chromatin-immunoprecipitation microarray (ChIP-chip) experiments to identify 953 BR-regulated BZR1 target (BRBT) genes. Functional studies of selected BRBTs further demonstrate roles in BR-promotion of cell elongation. The BRBT genes reveal numerous molecular links between the BR signaling pathway and downstream components involved in developmental and physiological processes. Furthermore, the results reveal extensive crosstalk between BR and other hormonal and light signaling pathways at multiple levels. For example, BZR1 not only controls the expression of many signaling components of other hormonal and light pathways, but also co-regulates common target genes with light-signaling transcription factors. Our results provide a genomic map of steroid hormone actions in plants, which reveals a regulatory network that integrates hormonal and light signaling pathways for plant growth regulation.
Brassinosteroids bind to the extracellular domain of the receptor kinase BRI1 to activate a signal transduction cascade that regulates nuclear gene expression and plant development. Many components of the brassinosteroid signaling pathway have been identified and studied in detail. However, the substrate of BRI1 kinase that transduces the signal to downstream components remains unknown. Proteomic studies of plasma membrane proteins lead to the identification of three homologous BR-signaling kinases (BSK1, BSK2 and BSK3). The BSKs are phosphorylated by BRI1 in vitro and interact with BRI1 in vivo. Genetic and transgenic studies demonstrate that the BSKs represent a small family of kinases that activate BR signaling downstream of BRI1. These results demonstrate that BSKs are the substrates of BRI1 kinase that activate downstream BR signal transduction. One-sentence summaryBrassinosteroid signaling kinases identified by proteomics Cell-surface receptor kinases activate cellular signal transduction pathways upon perception of extracellular signals, thereby mediating cellular responses to the environment and to other cells. The Arabidopsis genome encodes over 400 receptor-like kinases (RLKs) (1). Some of these RLKs function in growth regulation and plant responses to hormonal and environmental signals. However, the molecular mechanism of RLK signaling to immediate downstream components remains poorly understood, as no RLK substrate that mediates signal transduction has been established in Arabidopsis (2). BRI1 is an RLK that functions as the major receptor for the steroid hormones brassinosteroids (2). Brassinosteroids bind the extracellular domain of BRI1 to activate its kinase activity, initiating a signal transduction cascade that regulates nuclear gene expression and a wide range of developmental and physiological processes ( fig. S1) (3). Many components of the BR signaling pathway have been identified and much detail has been revealed about how BR activates BRI1(4-8) and how phosphorylation by downstream GSK3-like kinase BIN2 regulates the activity of the nuclear transcription factors that mediate §To whom correspondence should be addressed:
Brassinosteroid (BR) regulates gene expression and plant development through a receptor kinase-mediated signal transduction pathway1. Despite many components of the pathway identified, how the BR signal is transduced from the cell surface to the nucleus remains unclear2. Here we describe a complete BR signaling pathway by elucidating the key missing steps of the pathway. We show that phosphorylation of BSK1 by the BR receptor kinase BRI1 promotes BSK1 binding to the BSU1 phosphatase, and BSU1 inactivates the GSK3-like kinase BIN2 by dephosphorylating a conserved phospho-tyrosine residue (pTyr200). Mutations that affect phosphorylation/dephosphorylation of BIN2 pTyr200 (bin2-1, bin2-Y200F, and quadruple loss-of-function of BSU1-related phosphatases) support an essential role for BSU1-mediated BIN2 dephosphorylation in BR-dependent plant growth. These results demonstrate direct sequential BR activation of BRI1, BSK1, and BSU1, and inactivation of BIN2, leading to accumulation of unphosphorylated BZR transcription factors in the nucleus. This study establishes a fully connected BR signaling pathway and provides new insight into the mechanism of GSK3 regulation.
Brassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BRI1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.
When brassinosteroid (BR) levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. BR promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here we identified protein phosphatase 2A (PP2A) as BZR1-interacting proteins using tandem affinity purification. Genetic analyses demonstrated a positive role of PP2A in BR signalling and BZR1 dephosphorylation. Members of the B'regulatory subunits of PP2A directly interact with BZR1's putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function of PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the BR-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.
Extracellular ATP is a known receptor agonist in animals and was previously shown to alter plant growth, and so we investigated whether ATP derivatives could function outside plant cells as signaling agents. Signaling responses induced by exogenous nucleotides in animal cells typically include increases in free cytoplasmic calcium concentration ([Ca 2þ ] cyt ). We have evaluated the ability of exogenously applied adenosine 59-[g-thio]triphosphate (ATPgS), adenosine 59-[b-thio]diphosphate (ADPbS), and adenosine 59-O-thiomonophosphate to alter [Ca 2þ ] cyt in intact apoaequorin transgenic Arabidopsis thaliana seedlings. ATPgS and ADPbS increase [Ca 2þ ] cyt , and this increase is enhanced further when the nucleotides are added with the elicitor oligogalacturonic acid. Exogenous treatment with ATP also increases the level of transcripts encoding mitogen-activated protein kinases and proteins involved in ethylene biosynthesis and signal transduction. The increase in [Ca 2þ ] cyt induced by nucleotide derivatives can be ablated by Ca 2þ -channel blocking agents and by the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid (BAPTA), and the changes in gene expression can be partially blocked by these agents. These observations suggest that extracellular ATP can activate calcium-mediated cell-signaling pathways in plants, potentially playing a physiological role in transducing stress and wound responses.
The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we performed a proteomics study of BR-regulated proteins in Arabidopsis using two-dimensional DIGE coupled with LC-MS/MS. We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR-insensitive mutant bri1-116 and BR-hypersensitive mutant bzr1-1D identified five proteins (PATL1, PATL2, THI1, AtMDAR3, and NADP-ME2) affected both by BR treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knock-out mutants or immunoblotting. Interestingly about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed genespecific kinetic relationships between RNA and protein responses. Furthermore BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in two-dimensional DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses.
Signal transduction involves posttranslational modifications and protein-protein interactions, which can be studied by proteomics. In Arabidopsis, the steroid hormone (brassinosteroid (BR)) binds to the extracellular domain of a receptor kinase (BRI1) to initiate a phosphorylation/ dephosphorylation cascade that controls gene expression and plant growth. Here we detected early BR signaling events and identified early response proteins using prefractionation and two-dimensional (2-D) DIGE. Proteomic changes induced rapidly by BR treatments were detected in phosphoprotein and plasma membrane (PM) fractions by 2-D DIGE but not in total protein extracts. LC-MS/MS analysis of gel spots identified 19 BR-regulated PM proteins and six proteins from phosphoprotein fractions. These include the BAK1 receptor kinase and BZR1 transcription factor of the BR signaling pathway. Both proteins showed spot shifts consistent with BR-regulated phosphorylation. In addition, in vivo phosphorylation sites were identified for BZR1, two tetratricopeptide repeat proteins, and a phosphoenolpyruvate carboxykinase (PCK1). Overexpression of a novel BR-induced PM protein (DREPP) partially suppressed the phenotypes of a BR-deficient mutant, demonstrating its important function in BR responses. Our study demonstrates that prefractionation coupled with 2-D DIGE is a powerful approach for studying signal transduction. Molecular & Cellular Proteomics 7:728 -738, 2008.Perception and response to extracellular signals are crucial for growth and survival of all organisms. Signals are transduced intracellularly through mechanisms that include posttranslational protein modification and protein-protein interaction. Detection of such signaling events using proteomics methods can identify proteins involved in signal transduction but is technically challenging because of the low abundance of signaling proteins. Brassinosteroids (BRs) 1 are plant hormones that play important roles in multiple plant developmental processes. Mutant plants with a defect in BR synthesis or signal transduction show a wide range of phenotypes including dwarfism, reduced fertility, light-grown morphology in the dark, and delayed senescence (1). BRs structurally resemble animal steroid hormones but act through a distinct signaling mechanism (2). Although animal steroid hormones are perceived by nuclear receptors, BRs are perceived by a cell surface receptor-like kinase named BRI1 (2, 3). BRs bind to the extracellular domain of BRI1 to activate its kinase and downstream BR signal transduction (4), which involves another receptor-like kinase named BAK1 (5, 6); a GSK3-like kinase, BIN2 (7); a phosphatase, BSU1 (8); and two transcription factors, BZR1 and BZR2 (also known as BES1) (9, 10). In the absence of BR, the inhibitory BIN2 kinase phosphorylates BZR1 and BZR2, and phosphorylation inhibits the function of BZR1 and BZR2 through multiple mechanisms, which include inhibition of DNA binding activity, degradation by the proteasome, and cytoplasmic retention by the 14-3-3 proteins that b...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.