Plant steroid hormones, brassinosteroids (BRs), are perceived by a cell surface receptor kinase, BRI1, but how BR binding leads to regulation of gene expression in the nucleus is unknown. Here we describe the identification of BZR1 as a nuclear component of the BR signal transduction pathway. A dominant mutation bzr1-1D suppresses BR-deficient and BR-insensitive (bri1) phenotypes and enhances feedback inhibition of BR biosynthesis. BZR1 protein accumulates in the nucleus of elongating cells of dark-grown hypocotyls and is stabilized by BR signaling and the bzr1-1D mutation. Our results demonstrate that BZR1 is a positive regulator of the BR signaling pathway that mediates both downstream BR responses and feedback regulation of BR biosynthesis.
Brassinosteroid (BR) homeostasis and signaling are crucial for normal growth and development of plants. BR signaling through cell-surface receptor kinases and intracellular components leads to dephosphorylation and accumulation of the nuclear protein BZR1. How BR signaling regulates gene expression, however, remains unknown. Here we show that BZR1 is a transcriptional repressor that has a previously unknown DNA binding domain and binds directly to the promoters of feedback-regulated BR biosynthetic genes. Microarray analyses identified additional potential targets of BZR1 and illustrated, together with physiological studies, that BZR1 coordinates BR homeostasis and signaling by playing dual roles in regulating BR biosynthesis and downstream growth responses.
Brassinosteroids (BRs) are a class of steroid hormones essential for normal growth and development in plants. BR signaling involves the cell-surface receptor BRI1, the glycogen synthase kinase-3-like kinase BIN2 as a negative regulator, and nuclear proteins BZR1 and BZR2͞BES1 as positive regulators. The interactions among these components remain unclear. Here we report that BRs induce dephosphorylation and accumulation of BZR1 protein. Experiments using a proteasome inhibitor, MG132, suggest that phosphorylation of BZR1 increases its degradation by the proteasome machinery. BIN2 directly interacts with BZR1 in yeast two-hybrid assays, phosphorylates BZR1 in vitro, and negatively regulates BZR1 protein accumulation in vivo. These results strongly suggest that BIN2 phosphorylates BZR1 and targets it for degradation and that BR signaling causes BZR1 dephosphorylation and accumulation by inhibiting BIN2 activity. B rassinosteroids (BRs) are a class of steroid hormones that play important roles in plant growth and development (1-3). Deficiency in BR biosynthesis or signaling causes dramatic growth defects that include dwarfism, reduced apical dominance and fertility, delayed flowering and senescence, and photomorphogenesis in the dark (4-6). BRs are perceived by a cell-surface receptor kinase and transduced via an undefined signal transduction pathway, leading to changes in gene expression and growth (7,8). BR signaling thus resembles the nongenomic steroid actions observed in some animal systems (9), but differs from the well-studied genomic steroid actions mediated by the nuclear receptors in animals (10).Extensive genetic screens for recessive BR-insensitive Arabidopsis mutants have identified only one gene, brassinosteroid insensitive 1 (BRI1), that is essential for BR response. BRI1 encodes a leucine-rich-repeat receptor kinase localized to the plasma membrane (11-13). Molecular biochemical studies have shown that BRI1 functions as the BR receptor. BRI1 perceives BRs through its extracellular domain and transduces the signal by phosphorylating downstream signaling proteins that have yet to be identified (7).Studies of the semidominant dwarf mutant brassinosteroid insensitive 2 (bin2) have led to the identification of a potential downstream component that negatively regulates BR response. BIN2 encodes a member of the glycogen synthase kinase-3 (GSK3)-like kinases (14, 15). The increased kinase activity by the semidominant bin2-1 mutation and the phenotypes of transgenic plants with altered BIN2 expression levels indicate that BIN2 is a negative regulator for BR response and cell elongation (16). In animals, GSK3-like kinases play key roles as negative regulators in a variety of signaling pathways (17). Extracellular signals, such as insulin or growth factors, inhibit GSK3 kinases, allowing dephosphorylation of the substrates and activation of downstream responses (18,19). As a negative regulator of BR response, BIN2 might function in a manner similar to the animal GSK3 kinases. BIN2 may phosphorylate and inactiva...
The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TIMING OF CAB EXPRESSION 1 (TOC1) and CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY). CCA1 and LHY are Myb transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data support that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA-binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression, demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNAbinding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the pseudoreceiver domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify previously unexplored potential TOC1 targets and output pathways. Taken together, these results define a biochemical action for the core clock protein TOC1 and refine our perspective on how plant clocks function.ost organisms that experience day/night cycles have a circadian clock that phases cellular processes and behavior to specific times of day while also anticipating daily diurnal changes to confer a fitness advantage (1). The basic molecular architecture of most clocks consists of negative-feedback loops where positive and negative components control each other's expression to generate oscillations with an approximate 24-h period (1).At the core of the Arabidopsis clock, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) (2) and LATE ELONGATED HY-POCOTYL (LHY) (3) are morning-expressed Myb transcription factors that directly bind the evening element (4, 5) in the promoters of evening-expressed genes and act as transcriptional repressors. CCA1 and LHY have redundant functions (6, 7), are often coexpressed, and make up the negative arm of the first described feedback loop in Arabidopsis by binding the promoter of TIMING OF CAB EXPRESSION 1 (TOC1) (4). Genetically, TOC1 is the positive component of this feedback loop because CCA1/LHY morning reactivation is dependent on TOC1 (4), yet TOC1 overexpression also results in lower CCA1/LHY expression (8, 9), confusing our understanding of TOC1's role in the core clock feedback loop. TOC1 is an evening-expressed protein (10) that is part of a five-member family called the PSEUDO-RESPONSE REGULATORS (PRRs) that are expressed in succession from the morning to the night in the order PRR9, PRR7, PRR5, PRR3, and then TOC1 (11). Sequence similari...
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.
Summary Insults to endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as a previously uncharacterized and important facet of eukaryotic translational control.
Spatiotemporal control of the formation of organ primordia and organ boundaries from the stem cell niche in the shoot apical meristem (SAM) determines the patterning and architecture of plants, but the underlying signaling mechanisms remain poorly understood. Here we show that brassinosteroids (BRs) play a key role in organ boundary formation by repressing organ boundary identity genes. BR-hypersensitive mutants display organ-fusion phenotypes, whereas BR-insensitive mutants show enhanced organ boundaries. The BR-activated transcription factor BZR1 directly represses the CUP-SHAPED COTYLEDON (CUC) family of organ boundary identity genes. In WT plants, BZR1 accumulates at high levels in the nuclei of central meristem and organ primordia but at a low level in organ boundary cells to allow CUC gene expression. Activation of BR signaling represses CUC gene expression and causes organ fusion phenotypes. This study uncovers a role for BR in the spatiotemporal control of organ boundary formation and morphogenesis in the SAM.hormone | steroid
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