Brassinosteroids, the steroid hormones of plants, are perceived at the plasma membrane by a leucine-rich repeat receptor serine/threonine kinase called BRI1. We report a BRI1-interacting protein, BKI1, which is a negative regulator of brassinosteroid signaling. Brassinosteroids cause the rapid dissociation of BKI1-yellow fluorescent protein from the plasma membrane in a process that is dependent on BRI1-kinase. BKI1 is a substrate of BRI1 kinase and limits the interaction of BRI1 with its proposed coreceptor, BAK1, suggesting that BKI1 prevents the activation of BRI1.
The LRR receptor serine/threonine kinases are a major eukaryotic receptor family, for which the central regulatory mechanism of endosomal trafficking remains largely unadressed. We show that the steroid receptor BRI1 localizes to both plasma membrane and early endosomal compartments, even when observed at low, endogenous expression levels, and that its localization and turnover are independent of ligand. However, increasing endosomal localization of BRI1 enhances activation of the pathway and genomic responses. Our data indicate distinct signaling and trafficking mechanisms within this receptor class and show that the use of endosomes as signaling compartments is an unexpectedly broad phenomenon in eukaryotes.Supplemental material is available at http://www.genesdev.org.
SUMMARY Plants encounter a variety of stresses and must fine-tune their growth and stress-response programs to best suit their environment. BES1 functions as a master regulator in the brassinosteroid (BR) pathway that promotes plant growth. Here, we show that BES1 interacts with the ubiquitin receptor protein DSK2 and is targeted to the autophagy pathway during stress via the interaction of DSK2 with ATG8, a ubiquitin-like protein directing autophagosome formation and cargo recruitment. Additionally, DSK2 is phosphorylated by the GSK3-like kinase BIN2, a negative regulator in the BR pathway. BIN2 phosphorylation of DSK2 flanking its ATG8 interacting motifs (AIMs) promotes DSK2-ATG8 interaction, thereby targeting BES1 for degradation. Accordingly, loss-of-function dsk2 mutants accumulate BES1, have altered global gene expression profiles, and have compromised stress responses. Our results thus reveal that plants coordinate growth and stress responses by integrating BR and autophagy pathways and identify the molecular basis of this crosstalk.
The leucine-rich-repeat receptor serine/threonine kinase, BRI1, is a cell-surface receptor for brassinosteroids (BRs), the steroid hormones of plants, yet its activation mechanism is unknown. Here, we report a unique autoregulatory mechanism of BRI1 activation. Removal of BRI1's C terminus leads to a hypersensitive receptor, indicated by suppression of dwarfism of BR-deficient and BR-perception mutants and by enhanced BR signaling as a result of elevated phosphorylation of BRI1. Several sites in the C-terminal region can be phosphorylated in vitro, and transgenic Arabidopsis expressing BRI1 mutated at these sites demonstrates an essential role of phosphorylation in BRI1 activation. BRI1 is a ligand-independent homo-oligomer, as evidenced by the transphosphorylation of BRI1 kinase in vitro, the dominant-negative effect of a kinase-inactive BRI1 in transgenic Arabidopsis, and coimmunoprecipitation experiments. Our results support a BRI1-activation model that involves inhibition of kinase activity by its C-terminal domain, which is relieved upon ligand binding to the extracellular domain.
Phytohormones have essential roles in coordinately regulating a large array of developmental processes. Studies have revealed that brassinosteroids (BRs) and abscisic acid (ABA) interact to regulate hundreds of expression in genes, governing many biological processes. However, whether their interaction is through modification or intersection of their primary signaling cascades, or by independent or parallel pathways remains a big mystery. Using biochemical and molecular markers of BR signaling and ABA biosynthetic mutants, we demonstrated that exogenous ABA rapidly inhibits BR signaling outputs as indicated by the phosphorylation status of BES1 and BR-responsive gene expression. Experiments using a bri1 null-allele, bri1-116, and analysis of subcellular localization of BKI1-YFP further revealed that the BR receptor complex is not required for ABA to act on BR signaling outputs. However, when the BR downstream signaling component BIN2 is inhibited by LiCl, ABA failed to inhibit BR signaling outputs. Also, using a set of ABA insensitive mutants, we found that regulation of ABA on the BR primary signaling pathway depends on the ABA early signaling components, ABI1 and ABI2. We propose that the signaling cascades of ABA and BR primarily cross-talk after BR perception, but before their transcriptional activation. This model provides a reasonable explanation for why a large proportion of BR-responsive genes are also regulated by ABA, and provides an insight into the molecular mechanisms by which BRs could interact with ABA.cross-talk ͉ gene expression ͉ phosphorylation ͉ seed germination ͉ signal transduction U nlike animals, plants are sessile and need to constantly regulate their developmental and physiological processes to respond to various internal and external stimuli. Studies have revealed that many biological processes result from integrating multiple hormonal signals, and extensive cross-talk among different hormonal signaling pathways is present in plants (1, 2). Recently, a large set of microarray data verified that many genes are coregulated by multiple hormones, suggesting the importance of hormones to coordinately regulate biological processes in plants (3,4). A few studies have also elucidated specific molecular mechanisms of hormonal cross-talk. They include the role of auxin and ethylene in regulating root meristem development (1), the antagonistic relationship between abscisic acid (ABA) and gibberellins (GAs) on seed dormancy and germination (5, 6), and integration of the primary signaling pathways of auxin and brassinosteroids (BRs) by auxin response factor 2 (ARF2) (7).Studies have indicated that BRs and ABA can coregulate the expression of hundreds of genes (4), and they interact physiologically in controlling many developmental processes (5,(8)(9)(10). It is also well known that ABA is required to establish seed dormancy during embryo maturation and to inhibit seed germination (5), whereas BRs promote seed germination, likely through enhancing the embryo growth potential to antagonize the effect...
The development of anther and pollen is important for male reproduction, and this process is coordinately regulated by many external and internal cues. In this study, we systematically examined the male reproductive phenotypes of a series of brassinosteroid biosynthetic and signaling mutants and found that, besides the expected cell-expansion defects, these mutants also showed reduced pollen number, viability, and release efficiency. These defects were related with abnormal tapetum and microspore development. Using both real-time quantitative RT-PCR and microarray experiments, we found that the expression of many key genes required for anther and pollen development was suppressed in these mutants. ChIP analysis demonstrated that BES1, an important transcription factor for brassinosteroid signaling, could directly bind to the promoter regions of genes encoding transcription factors essential for anther and pollen development, SPL/NZZ, TDF1, AMS, MS1, and MS2. Taken together, these data lead us to propose that brassinosteroids control male fertility at least in part via directly regulating key genes for anther and pollen development in Arabidopsis. Our work provides a unique mechanism to explain how a phytohormone regulates an essential genetic program for plant development.male fertility | regulatory network | signaling | chromatin immunoprecipitation
Strigolactones (SLs), a class of the most recently identified terpenoid phytohormones, play essential roles in plant development, specifically in suppressing shoot branching. MAX2, a subunit of an SCF E3 ligase and a positive regulator that inhibits shoot branching, is likely a key SL signaling component. Here, we provide genetic and biochemical evidence to demonstrate that BES1 interacts with MAX2 and acts as its substrate to regulate SL-responsive gene expression. Additional AtD14, a putative receptor of SLs, can promote BES1 degradation. Knockdown of BES1 and its homologs dramatically suppressed the branching phenotype of max2-1 mutant. These results portray an SL signaling cascade from the putative receptor to downstream transcription factors. In addition, we demonstrate that the SL and brassinosteroid (BR) signaling pathways distinctly regulate the same transcription factor, BES1, to control specific developmental processes.
Arabidopsis glycogen synthase kinase 3 (GSK3)-like kinases have versatile functions in plant development and in responding to abiotic stresses. Although physiological evidence suggested a potential role of GSK3-like kinases in abscisic acid (ABA) signaling, the underlying molecular mechanism was largely unknown. Here we identified members of Snf1-related kinase 2s (SnRK2s), SnRK2.2 and SnRK2.3, that can interact with and be phosphorylated by a GSK3-like kinase, brassinosteroid insensitive 2 (BIN2). bin2-3 bil1 bil2, a loss-offunction mutant of BIN2 and its two closest homologs, BIN2 like 1 (BIL1) and BIN2 like 2 (BIL2), was hyposensitive to ABA in primary root inhibition, ABA-responsive gene expression, and phosphorylating ABA Response Element Binding Factor (ABF) 2 fragment by in-gel kinase assays, whereas bin2-1, a gain-of-function mutation of BIN2, was hypersensitive to ABA, suggesting that these GSK3-like kinases function as positive regulators in ABA signaling. Furthermore, BIN2 phosphorylated SnRK2.3 on T180, and SnRK2.3 T180A had decreased kinase activity in both autophosphorylation and phosphorylating ABFs. Bikinin, a GSK3 kinase inhibitor, inhibited the SnRK2.3 kinase activity and its T180 phosphorylation in vivo. Our genetic analysis further demonstrated that BIN2 regulates ABA signaling downstream of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS receptors and clade A protein phosphatase 2C but relies on SnRK2.2 and SnRK2.3. These findings provide significant insight into the modulation of ABA signaling by Arabidopsis GSK3-like kinases.signal transduction | phosphorylation cascades | kinase activation
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