Evolutionarily conserved BIL4 suppresses the degradation of brassinosteroid receptor BRI1 and regulates cell elongation.
ABSTRACT: Brassinosteroids (BRs), plant steroid hormones, play important roles in plant cell elongation and differentiation. To investigate the mechanisms of BR signaling, we previously used the BR biosynthesis inhibitor Brz as a chemical biology tool and identified the Brz-insensitive-long hypocotyl4 mutant (bil4). Although the BIL4 gene encodes a seven-transmembrane-domain protein that is evolutionarily conserved in plants and animals, the molecular function of BIL4 in BR signaling has not been elucidated. Here, we demonstrate that BIL4 is expressed in early elongating cells and regulates cell elongation in Arabidopsis. BIL4 also activates BR signaling and interacts with the BR receptor brassinosteroid insensitive 1 (BRI1) in endosomes. BIL4 deficiency increases the localization of BRI1 in the vacuoles. Our results demonstrate that BIL4 regulates cell elongation and BR signaling via the regulation of BRI1 localization.
Project description:Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz-insensitive-long hypocotyls 2-1D (bil2-1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants (BIL2-OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2-OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria.
Project description:Brassinosteroids are essential phytohormones that have crucial roles in plant growth and development. Perception of brassinosteroids requires an active complex of BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE 1 (BAK1). Recognized by the extracellular leucine-rich repeat (LRR) domain of BRI1, brassinosteroids induce a phosphorylation-mediated cascade to regulate gene expression. Here we present the crystal structures of BRI1(LRR) in free and brassinolide-bound forms. BRI1(LRR) exists as a monomer in crystals and solution independent of brassinolide. It comprises a helical solenoid structure that accommodates a separate insertion domain at its concave surface. Sandwiched between them, brassinolide binds to a hydrophobicity-dominating surface groove on BRI1(LRR). Brassinolide recognition by BRI1(LRR) is through an induced-fit mechanism involving stabilization of two interdomain loops that creates a pronounced non-polar surface groove for the hormone binding. Together, our results define the molecular mechanisms by which BRI1 recognizes brassinosteroids and provide insight into brassinosteroid-induced BRI1 activation.
Project description:Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: brassinosteroid insensitive 1 (BRI1), which is the BR receptor, and its coreceptor BRI1-associated kinase 1 (BAK1). Both proteins are classified as serine/threonine protein kinases, but here we report that recombinant cytoplasmic domains of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases. With BRI1, Tyr-831 and Tyr-956 are identified as autophosphorylation sites in vitro and in vivo. Interestingly, Tyr-956 in kinase subdomain V is essential for activity, because the Y956F mutant is catalytically inactive and thus this site cannot be simply manipulated by mutagenesis. In contrast, Tyr-831 in the juxtamembrane domain is not essential for kinase activity but plays an important role in BR signaling in vivo, because expression of BRI1(Y831F)-Flag in transgenic bri1-5 plants results in plants with larger leaves (but altered leaf shape) and early flowering relative to plants expressing wild-type BRI1-Flag. Acidic substitutions of Tyr-831 restored normal leaf size (but not shape) and normal flowering time. This is an example where a specific tyrosine residue has been shown to play an important role in vivo in plant receptor kinase function. Interestingly, 6 additional LRR-RLKs (of the 23 tested) were also found to autophosphorylate on tyrosine in addition to serine and threonine, suggesting that tyrosine signaling should be considered with other plant receptor kinases as well.
Project description:Brassinosteroids are phytohormones involved in plant development and physiological processes. Brassinosteroids Insensitive 1 (BRI1) is required for BR perception and initiation of subsequent signal transduction in Arabidopsis. In this study, the orthologue of BRI1 in the model legume species Medicago truncatula, MtBRI1, was identified and characterised. Three allelic Tnt1 insertion mutants, mtbri1-1, mtbri1-2, and mtbri1-3, were obtained from the M. truncatula Tnt1 insertion population. mtbri1 mutants displayed characteristic bri1 mutant phenotypes: extreme dwarfness, dark green curled leaves, short primary roots, less lateral roots, and insensitive to exogenous brassinolide (BL). Moreover, mtbri1 mutants show decreased total nodule number and defects in nitrogen fixation. MtBRI1 is able to complement an Arabidopsis BRI1 mutant, bri1-5. Similar to the interaction of BRI1 and BAK1 in Arabidopsis, MtBRI1 interacts with MtSERK1 in vivo. Global gene expression profiling revealed that the expression of BR biosynthesis genes and SAUR genes are significantly altered in mtbri1 mutants. MapMan analysis indicated that genes involved in signaling, hormone, cell wall, and biotic stress responses are over-represented in differentially expressed genes. Taken together, the results indicate that MtBRI1 is the BR receptor in M. truncatula and that BR signaling may play a conserved role in balancing plant growth and defenses.
Project description:Brassinosteroids (BRs) are essential steroid hormones that have crucial roles in plant growth and development. BRs are perceived by the cell-surface receptor-like kinase brassinosteroid insensitive 1 (BRI1). In the absence of BRs, the cytosolic kinase domain (KD) of BRI1 is inhibited by its auto-inhibitory carboxyl terminus, as well as by interacting with an inhibitor protein, BRI1 kinase inhibitor 1 (BKI1). How BR binding to the extracellular domain of BRI1 leads to activation of the KD and dissociation of BKI1 into the cytosol remains unclear. Here we report the crystal structure of BRI1 KD in complex with the interacting peptide derived from BKI1. We also provide biochemical evidence that BRI1-associated kinase 1 (BAK1) plays an essential role in initiating BR signaling. Steroid-dependent heterodimerization of BRI1 and BAK1 ectodomains brings their cytoplasmic KDs in the right orientation for competing with BKI1 and transphosphorylation.
Project description:Plant steroid hormones, brassinosteroids (BRs), play important roles in plants. BRs regulate the expression of several thousand genes, half of which are induced and the other half repressed by the hormone. BRs signal through plasma membrane-localized receptor kinase brassinosteroid-insensitive 1 (BRI1), BRI1-associated receptor kinase (BAK1), and several intermediates to regulate the protein levels, cellular localizations, and/or DNA binding of BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) family transcription factors. Although BES1 is known to interact with other transcription factors, histone-modifying enzymes, and transcription elongation factors to activate BR-induced genes, how BES1 mediates the BR-repressed gene expression is not known. Here, we show that BES1 interacts with myeloblastosis family transcription factor-like 2 (MYBL2), a transcription repressor, to down-regulate BR-repressed gene expression. The loss-of-function mybl2 mutant enhances the phenotype of a weak allele of bri1 and suppresses the constitutive BR-response phenotype of bes1-D. The results suggest that suppression of BR-repressed gene expression is required for optimal BR response. Moreover, MYBL2 is a substrate of glycogen synthase kinase 3 (GSK3)-like kinase brassinosteroid-insensitive 2 (BIN2), which has been well established as a negative regulator in the BR pathway by phosphorylating and inhibiting the functions of BES1/BZR1. Unlike BIN2 phosphorylation of BES1/BZR1 leading to protein degradation, BIN2 phosphorylation stabilizes MYBL2. Such dual role of phosphorylation has also been reported in WNT signaling pathway in which GSK3 phosphorylation destabilizes ?-catenin and stabilizes Axin, a scaffolding protein facilitating the phosphorylation of ?-catenin by GSK3. Our results thus establish the mechanisms for BR-repressed gene expression and the integration of BR signaling and BR transcriptional network.
Project description:Brassinosteroids (BRs) are plant hormones that regulate many processes including cell elongation, leaf development, pollen tube growth and xylem differentiation. GSK3/shaggy-like kinases (GSK) are critical regulators of intracellular signalling initiated by the binding of BR to the BRI1 receptor complex. Three GSKs have already been shown to relay BR responses, including phosphorylation of the transcriptional regulator BES1. However, recent studies indicate that one or more yet unidentified protein kinases are involved in BR signalling. Here, we show that the in vivo protein kinase activity of the group-III GSK, ASKtheta, was negatively regulated by BRI1. Arabidopsis thaliana plants with enhanced ASKtheta activity displayed a bri1-like phenotype. ASKtheta overexpressors accumulated high levels of brassinolide, castasterone and typhasterol, and were insensitive to BR. ASKtheta localized to the nucleus and directly phosphorylated BES1 and BZR1. Moreover, the BES1/BZR1-like transcription factor BEH2 was isolated as an ASKtheta interaction partner in a yeast two-hybrid screen. ASKtheta phosphorylated BEH2 both in vitro and in vivo. Overall, these data provide strong evidence that ASKtheta is a novel component of the BR signalling cascade, targeting the transcription factors BES1, BZR1 and BEH2.
Project description:Brassinosteroids (BRs) 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 BR 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.
Project description:Brassinosteroids, which control plant growth and development, are sensed by the membrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Brassinosteroid binding to the BRI1 leucine-rich repeat (LRR) domain induces heteromerisation with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)-family co-receptor. This process allows the cytoplasmic kinase domains of BRI1 and SERK to interact, trans-phosphorylate and activate each other. Here we report crystal structures of the BRI1 kinase domain in its activated form and in complex with nucleotides. BRI1 has structural features reminiscent of both serine/threonine and tyrosine kinases, providing insight into the evolution of dual-specificity kinases in plants. Phosphorylation of Thr1039, Ser1042 and Ser1044 causes formation of a catalytically competent activation loop. Mapping previously identified serine/threonine and tyrosine phosphorylation sites onto the structure, we analyse their contribution to brassinosteroid signaling. The location of known genetic missense alleles provide detailed insight into the BRI1 kinase mechanism, while our analyses are inconsistent with a previously reported guanylate cyclase activity. We identify a protein interaction surface on the C-terminal lobe of the kinase and demonstrate that the isolated BRI1, SERK2 and SERK3 cytoplasmic segments form homodimers in solution and have a weak tendency to heteromerise. We propose a model in which heterodimerisation of the BRI1 and SERK ectodomains brings their cytoplasmic kinase domains in a catalytically competent arrangement, an interaction that can be modulated by the BRI1 inhibitor protein BKI1.
Project description:Brassinosteroids (BRs) are plant hormones, fundamental for the growth and development of plants. A trans-membrane protein receptor kinase, Brassinosteroid-Insensitive 1 (BRI1), is known to interact with BRs and be directly involved in plant development. This study investigates the structural organization of BRI1 orthologs in several taxa, with a specific interest in Triticum aestivum. True orthologs of Arabidopsis thaliana BRI1 (AtBRI1) from seven-plant species showed sequence identity ranging from 54% to 95% at the protein level. All gene sequences lacked introns, leading to speculation that post-transcriptional processing in TaBRI1 is similar to AtBRI1. Based on in silico analysis, a single copy of BRI1 was present in each of the three wheat genomes on the long arm of chromosome 3. Domain structure of BRI1 orthologs among different taxa showed multiple leucine rich repeats (LRRs), an island domain (ID), a juxtamembrane/transmembrane domain (JTMD), a catalytic kinase domain (KD), C and N-Terminal domains. The KD showed the highest level of conservation while the LRRs and JTMD were most variable. Phosphorylation of residues in the juxtamembrane domain, known to be involved in the activation of the KD, is conserved in TaBRI1. While TaBRI1 has well-defined differences in the ID and LRR domains, many residues involved in ligand binding are conserved. The activation loop present in the KD showed 100% conservation in all taxa. Despite residue differences, hydrophobicity was conserved in the BR binding pocket across taxa, suggesting that function may not differ as drastically as residue identity may suggest. Predicted 3D structure of AtBRI1 and TaBRI1 showed a conserved super helical assembly, a feature essential in protein-protein interactions. An unrooted phylogram showed TaBRI1 in the monocot clade to be distinct from that of dicots. New insight in the structure and functions of BRI1 may help in targeting BR pathway for crop improvement.