Steven D. Clouse

A Brassinosteroid-Insensitive Mutant in Arabidopsis thaliana Exhibits Multiple Defects in Growth and Development

Brassinosteroids are widely distributed plant compounds that modulate cell elongation and division, but little is known about the mechanism of action of these plant growth regulators. To investigate brassinosteroids as signals influencing plant growth and development, we identified a brassinosteroid-insensitive mutant in Arabidopsis thaliana (L.) Henyh. ecotype Columbia. The mutant, termed bri1, did not respond to brassinosteroids in hypocotyl elongation and primary root inhibition assays, but it did retain sensitivity to auxins, cytokinins, ethylene, abscisic acid, and gibberellins. The bri1 mutant showed multiple deficiencies in developmental pathways that could not be rescued by brassinosteroid treatment, including a severely dwarfed stature; dark green, thickened leaves; male sterility; reduced apical dominance; and de-etiolation of darkgrown seedlings. Genetic analysis suggests that the Bri1 phenotype is caused by a recessive mutation in a single gene with pleiotropic effects that maps 1.6 centimorgans from the cleaved, amplified, polymorphic sequence marker DHS1 on the bottom of chromosome IV. The multiple and dramatic effects of mutation of the BRI1 locus on development suggests that the BRI1 gene may play a critical role in brassinosteroid perception or signal transduction.

Sequential Transphosphorylation of the BRI1/BAK1 Receptor Kinase Complex Impacts Early Events in Brassinosteroid Signaling

Brassinosteroids (BRs) regulate plant development through a signal transduction pathway involving the BRI1 and BAK1 transmembrane receptor kinases. The detailed molecular mechanisms of phosphorylation, kinase activation, and oligomerization of the BRI1/BAK1 complex in response to BRs are uncertain. We demonstrate that BR-dependent activation of BRI1 precedes association with BAK1 in planta, and that BRI1 positively regulates BAK1 phosphorylation levels in vivo. BRI1 transphosphorylates BAK1 in vitro on specific kinase-domain residues critical for BAK1 function. BAK1 also transphosphorylates BRI1, thereby quantitatively increasing BRI1 kinase activity toward a specific substrate. We propose a sequential transphosphorylation model in which BRI1 controls signaling specificity by direct BR binding followed by substrate phosphorylation. The coreceptor BAK1 is then activated by BRI1-dependent transphosphorylation and subsequently enhances signaling output through reciprocal BRI1 transphosphorylation. This model suggests both conservation and distinct differences between the molecular mechanisms regulating phosphorylation-dependent kinase activation in plant and animal receptor kinases.

Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase.

Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography–tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.

Brassinosteroid Signal Transduction: From Receptor Kinase Activation to Transcriptional Networks Regulating Plant Development

Brassinosteroid (BR) signal transduction research has progressed rapidly from the initial discovery of the BR receptor to a complete definition of the basic molecular components required to relay the BR signal from perception by receptor kinases at the cell surface to activation of a small family of transcription factors that regulate the expression of more than a thousand genes in a BR-dependent manner. These mechanistic advances have helped answer the intriguing question of how a single molecule, such as a hormone, can have dramatic pleiotropic effects on a broad range of diverse developmental pathways and have shed light on how BRs interact with other plant hormones and environmental cues to shape the growth of the whole plant. This review summarizes the current state of BR signal transduction research and then examines recent articles uncovering gene regulatory networks through which BR influences both vegetative and reproductive development.

Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis

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.

Genetic Evidence for an Indispensable Role of Somatic Embryogenesis Receptor Kinases in Brassinosteroid Signaling

The Arabidopsis thaliana Somatic Embryogenesis Receptor Kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant role with BAK1. Here we provide genetic and biochemical evidence to demonstrate that SERKs are absolutely required for early steps in BR signaling. Overexpression of four of the five SERKs—SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1—suppressed the phenotypes of an intermediate BRI1 mutant, bri1-5. Overexpression of the kinase-dead versions of these four genes in the bri1-5 background, on the other hand, resulted in typical dominant negative phenotypes, resembling those of null BRI1 mutants. We isolated and generated single, double, triple, and quadruple mutants and analyzed their phenotypes in detail. While the quadruple mutant is embryo-lethal, the serk1 bak1 bkk1 triple null mutant exhibits an extreme de-etiolated phenotype similar to a null bri1 mutant. While overexpression of BRI1 can drastically increase hypocotyl growth of wild-type plants, overexpression of BRI1 does not alter hypocotyl growth of the serk1 bak1 bkk1 triple mutant. Biochemical analysis indicated that the phosphorylation level of BRI1 in serk1 bak1 bkk1 is incapable of sensing exogenously applied BR. As a result, the unphosphorylated level of BES1 has lost its sensitivity to the BR treatment in the triple mutant, indicating that the BR signaling pathway has been completely abolished in the triple mutant. These data clearly demonstrate that SERKs are essential to the early events of BR signaling.

Brassinosteroid signal transduction: clarifying the pathway from ligand perception to gene expression.

Recent genetic screens for novel components of brassinosteroid signaling have revealed proteins with cell surface, cytoplasmic, and nuclear localization that function as either positive activators or negative regulators of the brassinosteroid response. Initial microarray experiments have expanded the number of known brassinosteroid-regulated genes, providing a useful resource for better understanding terminal events in signal transduction.

Arabidopsis mutants reveal multiple roles for sterols in plant development

The molecular genetic, and biochemical analysis of sterol-deficient mutants in Arabidopsis strongly suggests an essential role for sterols in regulating multiple events in plant development, independent of their conversion to brassinosteroids (BRs). Embryogenesis, cell elongation, vascular

Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

BackgroundTransmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs), representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated.ResultsAs a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs) of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC) at Ohio State University for full accessibility by the Arabidopsis research community.ConclusionsMost of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

Autophosphorylation of Tyr-610 in the receptor kinase BAK1 plays a role in brassinosteroid signaling and basal defense gene expression

BAK1 is a leucine-rich repeat receptor-like kinase that functions as a coreceptor with the brassinosteroid (BR) receptor BRI1 and the flagellin receptor FLS2, and as a negative regulator of programmed cell death. BAK1 has been shown to autophosphorylate on numerous serine/threonine sites in vitro as well as to transphosphorylate associated receptor kinases both in vitro and in planta. In the present study we identify Tyr-610 in the carboxyl-terminal domain of BAK1 as a major site of autophosphorylation that is brassinolide-induced in vivo and requires a kinase-active BAK1. Expression of BAK1(Y610F)-Flag in transgenic plants lacking the endogenous bak1 and its functional paralogue, bkk1, produced plants that were viable but extremely small and generally resembled BR signaling mutants, whereas an acidic substitution for Tyr-610 to mimic phosphorylation restored normal growth. Several lines of evidence support the notion that BR signaling is impaired in the BAK1(Y610F)-Flag plants, and are consistent with the recently proposed sequential transphosphorylation model for BRI1/BAK1 interaction and activation. In contrast, the FLS2-mediated inhibition of seedling growth by the flg22 elicitor occurred normally in the Y610F-directed mutant. However, expression of many defense genes was dramatically reduced in BAK1(Y610F) plants and the nonpathogenic hrpA mutant of Pseudomonas syringae was able to grow rapidly in the mutant. These results indicate that phosphorylation of Tyr-610 is required for some but not all functions of BAK1, and adds significantly to the emerging notion that tyrosine phosphorylation could play an important role in plant receptor kinase signaling.