Analysis of phosphorylation of the BRI1/BAK1 complex in arabidopsis reveals amino acid residues critical for receptor formation and activation of BR signaling

The plasma membrane-localized BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1) are a well-known receptor pair involved in brassinosteroids (BR) signaling in Arabidposis. The formation of a receptor complex in response to BRs and the subsequent activation of cytoplasmic domain kinase activity share mechanistic characteristics with animal receptor kinases. Here, we demonstrate that BRI1 and BAK1 are BR-dependently phosphorylated, and that phosphorylated forms of the two proteins persist for different lengths of time. Mutations of either protein abolished phosphorylation of the counterpart protein, implying transphosphorylation of the receptor kinases. To investigate the specific amino acids critical for formation of the receptor complex and activation of BAK1 kinase activity, we expressed several versions of BAK1 in yeast and plants. L32E and L46E substitutions resulted in a loss of binding of BAK1 to BRI1, and threonine T455 was essential for the kinase activity of BAK1 in yeast. Transgenic bri1 mutant plants overexpressing BAK1(L46E) displayed reduced apical dominance and seed development. In addition, transgenic wild type plants overexpressing BAK1(T455A) lost the phosphorylation activity normally exhibited in response to BL, leading to semi-dwarfism. These results suggest that BAK1 is a critical component regulating the duration of BR efficacy, even though it cannot directly bind BRs in plants.     

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.     

Is kinase activity essential for biological functions of BRI1?

Brassinosteroids (BRs) are a major group of plant hormones that regulate plant growth and development. BRI1, a protein localized to the plasma membrane, functions as a BR receptor and it has been proposed that its kinase activity has an essential role in BR-regulated plant growth and development. Here we report the isolation and molecular characterization of a new allele of bri1, bri1-301, which shows moderate morphological phenotypes and a reduced response to BRs under normal growth conditions. Sequence analysis identified a two-base alteration from GG to AT, resulting in a conversion of 989G to 989I in the BRI1 kinase domain. An in vitro assay of kinase activity showed that bri1-301 has no detectable autophosphorylation activity or phosphorylation activity towards the BRI1 substrates TTL and BAK1. Furthermore, our results suggest that bri1-301, even with extremely impaired kinase activity, still retains partial function in regulating plant growth and development, which raises the question of whether BRI1 kinase activity is essential for BR-mediated growth and development in higher plants.     

BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling

The Arabidopsis BAK1 (BRI1 Associated receptor Kinase 1) was identified by a yeast two-hybrid screen as a specific interactor for BRI1, a critical component of a membrane brassinosteroid (BR) receptor. In yeast, BAK1/BRI1 interaction activates their kinase activities through transphosphorylation. BAK1 and BRI1 share similar gene expression and subcellular localization patterns and physically associate with each other in plants. Overexpression of the BAK1 gene leads to a phenotype reminiscent of BRI1-overexpression transgenic plants and rescues a weak bri1 mutant. In contrast, a bak1 knockout mutation gives rise to a weak bri1-like phenotype and enhances a weak bri1 mutation. We propose that BAK1 and BRI1 function together to mediate plant steroid signaling.     

BAK7 displays unequal genetic redundancy with BAK1 in brassinosteroid signaling and early senescence in arabidopsis

BRI1-Associated kinase1 (BAK1), a five leucine-rich-repeat containing receptor-like serine/threonine kinase, has been shown to have dual functions: mediating brassinosteroid (BR) signaling and acting in the BR-independent plant defense response. Sequence analysis has revealed that BAK1 has two homologs, BAK7 and BAK8. Because BAK8 deviates from the canonical RD kinase motif, we focused on the functional analysis of BAK7. The expression pattern and tissues in which BAK7 appeared partially overlapped with those observed for BAK1. Expression levels of BAK7 increased in the bak1 mutant. Overexpression of BAK7 rescued the bri1 mutant phenotype, indicating that BAK7 can compensate for BAK1 in BR-mediated processes, especially in the absence of BAK1. However, root and hypocotyl elongation patterns of transgenic plants overexpressing BAK1 or BAK7 appeared to be different from the patterns observed in a BRI1 overexpressor. Furthermore, the sensitivity of transgenic plants overexpressing BAK7 to brassinazole, a biosynthetic inhibitor of brassinolide (BL), did not change compared to that of wild-type plants. In addition, we generated transgenic plants expressing BAK7 RNA interference constructs and found severe growth retardation and early senescence in these lines. Taken together, these results suggest that BAK7 is a component of the BR signaling pathway, with varying degrees of genetic redundancy with BAK1, and that it affects plant growth via BL-independent pathways in vivo.     

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.     

BRL1, a leucine-rich repeat receptor-like protein kinase, is functionally redundant with BRI1 in regulating Arabidopsis brassinosteroid signaling

BRI1-like receptor kinase (BRL1) was identified as an extragenic suppressor of a weak bri1 allele, bri1-5, in an activation-tagging genetic screen for novel brassinosteroid (BR) signal transduction regulators. BRL1 encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Sequence alignment revealed that BRL1 is closely related to BRI1, which is involved in BR perception. Overexpression of a BRL1 cDNA, driven by a constitutive CaMV 35S promoter, recapitulates the bri1-5 suppression phenotypes, and partially complements the phenotypes of a null bri1 allele, bri1-4. Analysis of a BR-specific feedback response gene, CPD, indicates that BRL1 functions in BR signaling. BRL1 expression pattern overlaps with, but is distinct from, that of BRI1. In addition, both the expression level and in vitro kinase autophosphorylation activity of BRL1 are significantly lower than those of BRI1. bri1-5 brl1-1 double mutant plants have enhanced developmental defects relative to bri1-5 mutant plants, revealing that BRL1 plays a partially redundant role with BRI1 in controlling Arabidopsis growth and development. These findings enhance our understanding of functional redundancy and add an additional layer of complexity to RLK-mediated BR signaling transduction in Arabidopsis.     

BAK1, an Arabidopsis LRR receptor-like protein kinase,interacts with BRI1 and modulates brassinosteroid signaling

Brassinosteroids regulate plant growth and development through a protein complex that includes the leucine-rich repeat receptor-like protein kinase (LRR-RLK) brassinosteroid-insensitive 1 (BRI1). Activation tagging was used to identify a dominant genetic suppressor of bri1, bak1-1D (bri1-associated receptor kinase 1-1Dominant), which encodes an LRR-RLK, distinct from BRI1. Overexpression of BAK1 results in elongated organ phenotypes, while a null allele of BAK1 displays a semidwarfed phenotype and has reduced sensitivity to brassinosteroids (BRs). BAK1 is a serine/threonine protein kinase, and BRI1 and BAK1 interact in vitro and in vivo. Expression of a dominant-negative mutant allele of BAK1 causes a severe dwarf phenotype, resembling the phenotype of null bri1 alleles. These results indicate BAK1 is a component of BR signaling.     

The Arabidopsis transthyretin-like protein is a potential substrate of BRASSINOSTEROID-INSENSITIVE 1

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) is a Leu-rich-repeat (LRR) receptor kinase that functions as a critical component of a transmembrane brassinosteroid (BR) receptor. It is believed that BRI1 becomes activated through heterodimerization with BAK1, a similar LRR receptor kinase, in response to BR signal. A yeast two-hybrid screen using the kinase domain of BRI1 identified an Arabidopsis thaliana Transthyretin-Like protein (TTL) as a potential BRI1 substrate. TTL interacts with BRI1 in a kinase-dependent manner in yeast and is phosphorylated by BRI1 in vitro. TTL displays a similar expression pattern with BRI1 and is associated with the plasma membrane. Overexpression of the TTL gene results in a phenotype that was observed in weak bri1 mutants and null bak1 mutants. By contrast, two T-DNA insertional mutations in the TTL gene promote plant growth and enhance BR sensitivity. We hypothesized that TTL might directly regulate certain biochemical activities near the plasma membrane to control plant growth.     

Role of Specific Phosphorylation Sites of <Emphasis Type="Italic">Arabidopsis</Emphasis> Brassinosteroid-Insensitive 1 Receptor Kinase in Plant Growth and Development

Brassinosteroid-insensitive 1 (BRI1), the receptor of brassinosteroids (BRs), is a dual-function serine/threonine/tyrosine protein kinase which initiates BR signaling and regulates plant growth via its protein kinase activity. Previous research has identified phosphorylation sites of Arabidopsis BRI1 in vivo and in vitro, but the significance of which to BR signaling and plant development has not been discussed comprehensively. To investigate this, we systematically characterized Arabidopsis BRI1 site-directed mutants in the weak bri1-5 background. For vegetative organ development regulation, we demonstrated that Thr-1039, Ser-1042, and Ser-1044 were critical for vegetative development because mutants with eliminated phosphorylation at these residues exhibited aberrant leaf growth, whereas Ser-1172 and Ser-1187 slightly inhibited leaf growth. For reproductive organ development regulation, first, the notion that Thr-1039, Ser-1042, and Ser-1044 were essential for normal plant height is supported by the evidence that mutations preventing phosphorylation at Thr-1039, Ser-1042, and Ser-1044 decreased plant height. Second, comparison of seed yield-related traits showed that unphosphorylated Ser-1168-Ala, Ser-1172-Ala, and Ser-1179-Ala+Thr-1180-Ala mutants reduced seed yield dramatically, whereas eliminating phosphorylation at Ser-1042 caused increased seed production. In addition, we found that Ser-1042 and Ser-1044 were essential for BR signaling. The unphosphorylated Ser-1042-Ala and Ser-1044-Ala mutants displayed hyposensitive phenotypes accompanied with decreased accumulation of dephosphorylated BRI1-EMS suppressor 1 (BES1) protein and increased Constitutive Photomorphogenesis Dwarf expression levels as well as limited inhibition of hypocotyl and root elongation under exogenous brassinolide. Taken together, our data suggest that BRI1 phosphorylation at specific sites differentially affects growth and development which may provide novel approaches to precisely regulate economic yield through modifying specific BRI1 phosphorylation sites in crop species.     

Suppressor of <Emphasis Type="Italic">bri1-120</Emphasis> mutant allele revealed interrelated and independent actions of brassinosteroid and light signaling

Brassinosteroids (BRs) are plant hormones that affect diverse aspects of plant development. Various BR-biosynthetic or BR-signaling mutants contribute to BR functions and signaling events in many plant species. The BR receptor brassinosteroid-Insensitive 1 (BRI1) plays critical roles in BR signaling. We previously identified a weak bri1 mutant allele, bri1-120, that has a mutation site in the extracellular domain of BRI1. Here, genetic suppressor screening revealed that a PHYB gene mutation led to suppression of ethyl methanesulfonate (EMS)-mutagenized bri1-120. The morphology of bri1-120phyB-1 indicated that compact and rounded phenotypes of bri1-120 were suppressed. However, BR sensitivity of the bri1-120phyB-1 was only recovered in hypocotyl elongation, and overexpression of PHYB in bri1-120 did not enhance bri1-120 phenotypes. To further investigate the relationship between BR and light signalings, we examined the seed germination pattern and hypocotyl growth of bri1-120phyB-1 as compared to that of each single mutant under various light conditions. Seed germination in bri1-120phyB-1 was higher than in both the single mutants. Hypocotyl length in bri1-120phyB-1 was intermediate between that of bri1-120 and phyB-1, whereas sensitivity to red light in bri1-120phyB-1 remained the same as in phyB-1. These results suggest that BR and light signalings affect diverse cellular responses both together and independently, depending on the specific cellular processes.     

The multifaceted function of BAK1/SERK3: Plant immunity to pathogens and responses to insect herbivores

Almost a decade ago BRI1-associated kinase 1 (BAK1) was identified as a co-receptor of brassinosteroid (BR) insensitive 1 (BRI1), the receptor for BRs, which plays an essential role in transducing BR signaling to regulate plant development. BAK1 is also critical in resistance to various pathogens. BAK1 rapidly binds to certain receptors for pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) after the perception of pathogen elicitors and is required for the full elicitation of pathogen-induced defense responses, such as the activation of the mitogen-activated protein kinase 6 (MPK6) and production of reactive oxygen species. Thus, BAK1 functions in both BR signaling and PAMP-triggered immunity (PTI). Recently BAK1 was also found to play an important role in mediating defense responses against an insect herbivore (Manduca sexta) of Nicotiana attenuata. In this interaction, BAK1 positively modulates wound- or herbivore feeding-induced accumulation of jasmonic acid (JA) and JA-isoleucine (JA-Ile). This mini-review summarizes recent advances in our understanding of the functions of BAK1 in resistance to pathogens and herbivores.Key words: BAK1, defense, herbivore, immunity, insect, jasmonate, pathogen, wounding     

The activation of the Arabidopsis P-ATPase 1 by the brassinosteroid receptor BRI1 is independent of threonine 948 phosphorylation

The plasma membrane-spanning receptor brassinosteroid insenstive 1 (BRI1) rapidly induces plant cell wall expansion in response to brassinosteroids such as brassinolide (BL). Wall expansion is accompanied by a rapid hyperpolarization of the plasma membrane, which is recordable by measuring the fluorescence lifetime (FLT) of the green fluorescent protein (GFP) fused to BRI1. For the BL induction of hyperpolarization and wall expansion, the activation of the plasma membrane P-type H⁺-ATPase is necessary. Furthermore, the activation of the P-ATPase requires BRI1 kinase activity and appears to be mediated by a BL-modulated association of BRI1 with the proton pump. Here, we show that BRI1 also associates with a mutant version of the Arabidopsis P-ATPase 1 (AHA1) characterized by an exchange of a well-known regulatory threonine for a non-phosphorylatable residue in the auto-inhibitory C-terminal domain. Even more important, BRI1 is still able to activate this AHA1 mutant in response to BL. This suggests a novel mechanism for the enzymatic activation of the P-ATPase by BRI1 in the plasma membrane. Furthermore, we demonstrate that the FLT of BRI1-GFP can be used as a non-invasive probe to analyze long-distance BL signaling in Arabidopsis seedlings.Key words: BRI1, fluorescence lifetime, membrane potential, P-ATPase, cell wall expansionUsing spectro-microscopic technologies, we recently started the quantitative analysis of the properties and subcellular function of GFP fusion of the plasma membrane-localized brassinosteroid (BR) receptor, BRI1, in living plant cells of Arabidopsis thaliana and tobacco (Nicotiana benthamiana) leaf cells.^1,2 Brassinosteroids, such as brassinolide (BL), are involved in responses to biotic and abiotic stresses and developmental processes, including cell elongation.³ The present model of the BR response pathway includes the binding of BRs to BRI1, resulting in the autophosphorylation of the receptor and the subsequent recruitment of the co-receptor BRI1-associated receptor kinase 1 (BAK1). This association is followed by trans-phosphorylation between BRI1 and BAK1 and results in the activation of downstream BR signaling processes leading to differential gene expression and, finally, to the execution of the specific responses.⁴ However, the molecular events that take place in the plasma membrane immediately after the perception of BL and initiate cell elongation still have to be included in this model.⁵ We recently reported a rapid BRI1-GFP-dependent cell wall expansion in Arabidopsis seedlings, which is attributed to wall loosening and water incorporation into the wall, and precedes cell elongation.^1,2 This expansion response was accompanied by a change in the FLT of BRI1-GFP, which reflects an alteration in the plasma membrane potential (E_m).^2,6 For both the FLT change in BRI1-GFP and the wall expansion, the activity of the plasma membrane P-ATPase is crucial. Notably, H⁺-pump activation was shown to depend on the kinase activity of BRI1.² This suggests a fast BRI1-dependent response pathway in the plasma membrane which links BL perception via P-ATPase activation and E_m hyperpolarization to wall expansion. In this report, we demonstrate that the phosphorylation of a conserved threonine in the auto-inhibitory domain of AHA1 is not required for the enzymatic activation by BRI1 suggesting a novel mechanism by which BRI1 may initiate the activation of the P-ATPase. Furthermore, we show that the FLT of BRI1-GFP is a useful and senstitive probe for the non-invasive analysis of systemic signaling processes in living plants.