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.     

Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.     

Blockage of Brassinosteroid Biosynthesis and Sensitivity Causes Dwarfism in Garden Pea

Endogenous brassinosteroids (BRs) in the dwarf mutants lka and lkb of garden pea (Pisum sativum L.) and comparable wild-type plants were quantified by gas chromatography-selected ion monitoring using deuterated internal standards. In young shoots of the lkb mutant, the levels of brassinolide, castasterone, and 6-deoxocastasterone were 23-, 22-, and 9-fold lower, respectively, than those of wild-type plants. Applications of brassinolide, castasterone, typhasterol, 3-dehydroteasterone, and teasterone normalized internode growth of lkb seedlings. These findings indicate that the lkb plants are BR-deficient mutants, probably as a consequence of a block in the BR biosynthetic pathway prior to the production of teasterone. Young shoots of lka plants contained only 50% less brassinolide and 5 times more castasterone than the equivalent wild-type tissues. The lka seedlings were approximately 100 times less responsive to brassinolide than the lkb mutant, and application of castasterone had only a marginal effect on lka internode growth, suggesting that the lka lesion results in impaired sensitivity to BR.     

BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis

Plant steroid hormones, brassinosteroids (BRs), are perceived by the plasma membrane-localized leucine-rich-repeat-receptor kinase BRI1. Based on sequence similarity, we have identified three members of the BRI1 family, named BRL1, BRL2 and BRL3. BRL1 and BRL3, but not BRL2, encode functional BR receptors that bind brassinolide, the most active BR, with high affinity. In agreement, only BRL1 and BRL3 can rescue bri1 mutants when expressed under the control of the BRI1 promoter. While BRI1 is ubiquitously expressed in growing cells, the expression of BRL1 and BRL3 is restricted to non-overlapping subsets of vascular cells. Loss-of-function of brl1 causes abnormal phloem:xylem differentiation ratios and enhances the vascular defects of a weak bri1 mutant. bri1 brl1 brl3 triple mutants enhance bri1 dwarfism and also exhibit abnormal vascular differentiation. Thus, Arabidopsis contains a small number of BR receptors that have specific functions in cell growth and vascular differentiation.     

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.     

Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase

Brassinosteroid (BR) mutants of Arabidopsis have pleiotropic phenotypes and provide evidence that BRs function throughout the life of the plant from seedling development to senescence. Screens for BR signaling mutants identified one locus, BRI1, which encodes a protein with homology to leucine-rich repeat receptor serine (Ser)/threonine (Thr) kinases. Twenty-seven alleles of this putative BR receptor have been isolated to date, and we present here the identification of the molecular lesions of 14 recessive alleles that represent five new mutations. BR-insensitive-1 (BRI1) is expressed at high levels in the meristem, root, shoot, and hypocotyl of seedlings and at lower levels later in development. Confocal microscopy analysis of full-length BRI1 fused to green fluorescent protein indicates that BRI1 is localized in the plasma membrane, and an in vitro kinase assay indicates that BRI1 is a functional Ser/Thr kinase. Among the bri1 mutants identified are mutants in the kinase domain, and we demonstrate that one of these mutations severely impairs BRI1 kinase activity. Therefore, we conclude that BRI1 is a ubiquitously expressed leucine-rich repeat receptor that plays a role in BR signaling through Ser/Thr phosphorylation.     

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.     

A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seed length

We have characterized a rice (Oryza sativa) dwarf mutant, dwarf11 (d11), that bears seeds of reduced length. To understand the mechanism by which seed length is regulated, the D11 gene was isolated by a map-based cloning method. The gene was found to encode a novel cytochrome P450 (CYP724B1), which showed homology to enzymes involved in brassinosteroid (BR) biosynthesis. The dwarf phenotype of d11 mutants was restored by the application of the brassinolide (BL). Compared with wild-type plants, the aberrant D11 mRNA accumulated at higher levels in d11 mutants and was dramatically reduced by treatment with BL, implying that the gene is feedback-regulated by BL. Precise determination of the defective step(s) in BR synthesis in d11 mutants proved intractable because of tissue specificity and the complex control of BR accumulation in plants. However, 6-deoxotyphasterol (6-DeoxoTY) and typhasterol (TY), but not any upstream intermediates before these compounds, effectively restored BR response in d11 mutants in a lamina joint bending assay. Multiple lines of evidence together suggest that the D11/CYP724B1 gene plays a role in BR synthesis and may be involved in the supply of 6-DeoxoTY and TY in the BR biosynthesis network in rice.     

BEN1, a gene encoding a dihydroflavonol 4-reductase (DFR)-like protein, regulates the levels of brassinosteroids in Arabidopsis thaliana

The ben1-1D (bri1-5 enhanced 1-1dominant) mutant was identified via an activation-tagging screen for bri1-5 extragenic modifiers. bri1-5 is a weak mutant allele of the brassinosteroid receptor gene, BRI1. Overexpression of BEN1 greatly enhances the defective phenotypes of bri1-5 plants. Removal of BEN1 by gene disruption in a Col-0 wild-type background, on the other hand, promotes the elongation of organs. Because BEN1 encodes a novel protein homologous to dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (BAN), BEN1 is probably involved in a brassinosteroid metabolic pathway. Analyses of brassinosteroid profiles demonstrated that BEN1 is indeed responsible for regulating the levels of several brassinosteroids, including typhasterol, castasterone and brassinolide. In vivo feeding and in vitro biochemical assays suggest that BEN1 is probably involved in a new mechanism to regulate brassinosteroid levels. These results provide additional insight into the regulatory mechanisms of bioactive brassinosteroids.     

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.     

Brassinosteroid homeostasis in Arabidopsis is ensured by feedback expressions of multiple genes involved in its metabolism

Homeostasis of brassinosteroids (BRs) is essential for normal growth and development in higher plants. We examined responsiveness of 11 BR metabolic gene expressions to the decrease or increase of endogenous BR contents in Arabidopsis (Arabidopsis thaliana) to expand our knowledge of molecular mechanisms underlying BR homeostasis. Five BR-specific biosynthesis genes (DET2, DWF4, CPD, BR6ox1, and ROT3) and two sterol biosynthesis genes (FK and DWF5) were up-regulated in BR-depleted wild-type plants grown under brassinazole, a BR biosynthesis inhibitor. On the other hand, in BR-excessive wild-type plants that were fed with brassinolide, four BR-specific synthesis genes (DWF4, CPD, BR6ox1, and ROT3) and a sterol synthesis gene (DWF7) were down-regulated and a BR inactivation gene (BAS1) was up-regulated. However, their response to fluctuation of BR levels was highly reduced (DWF4) or nullified (the other eight genes) in a bri1 mutant. Taken together, our results imply that BR homeostasis is maintained through feedback expressions of multiple genes, each of which is involved not only in BR-specific biosynthesis and inactivation, but also in sterol biosynthesis. Our results also indicate that their feedback expressions are under the control of a BRI1-mediated signaling pathway. Moreover, a weak response in the mutant suggests that DWF4 alone is likely to be regulated in other way(s) in addition to BRI1 mediation.     

Tyrosine phosphorylation in brassinosteroid signaling

Brassinosteroids (BRs) regulate plant growth and development through a complex signal transduction pathway involving BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, and its co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1). Both proteins are classified as Ser/Thr protein kinases. Recently, we reported that recombinant cytoplasmic domains (CD) of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases.¹ Two sites of Tyr autophosphorylation were identified that appear to have different effects on BRI1 function. Tyr-831 in the juxtamembrane domain is not essential for kinase activity but has a regulatory role, with phosphorylation of Tyr-831 causing inhibition of growth and delay of flowering. In contrast, Tyr-956 is located in subdomain IV of the kinase domain and is essential for kinase activity, and we are speculating that the free hydroxyl group at this position is essential and thus phosphorylation of Tyr-956 would inhibit BRI1 kinase activity. Expression of BRI1(Y831F)-Flag in the weak allele bri1-5 rescued the dwarf phenotype but plants had rounder leaves, increased shoot biomass, and flowered earlier than plants expressing the BRI1(wild type)-Flag in the bri1-5 background. To further elaborate on earlier results, we present additional phenotypic analysis of transgenic Arabidopsis plants expressing BRI1(Y831F)-Flag or site-directed mutants of other Tyr residues within the kinase domain. The results highlight the unique role of Tyr-831 in regulation of BR signaling in vivo. Elucidating the molecular basis for increased biomass accumulation in plants expressing BRI1(Y831F)-Flag may have applications for agriculture.Key words: brassinosteroids, LRR-RLK, autophosphorylation, tyrosine phosphorylation, signal transduction     

BAK1 and BKK1 regulate brassinosteroid-dependent growth and brassinosteroid-independent cell-death pathways

Brassinosteroids (BRs) are phytosteroid hormones controlling various physiological processes critical for normal growth and development. BRs are perceived by a protein complex containing two transmembrane receptor kinases, BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) [1-3]. BRI1 null mutants exhibit a dwarfed stature with epinastic leaves, delayed senescence, reduced male fertility, and altered light responses. BAK1 null mutants, however, only show a subtle phenotype, suggesting that functionally redundant proteins might be present in the Arabidopsis genome. Here we report that BAK1-LIKE 1 (BKK1) functions redundantly with BAK1 in regulating BR signaling. Surprisingly, rather than the expected bri1-like phenotype, bak1 bkk1 double mutants exhibit a seedling-lethality phenotype due to constitutive defense-gene expression, callose deposition, reactive oxygen species (ROS) accumulation, and spontaneous cell death even under sterile growing conditions. Our detailed analyses demonstrate that BAK1 and BKK1 have dual physiological roles: positively regulating a BR-dependent plant growth pathway, and negatively regulating a BR-independent cell-death pathway. Both BR signaling and developmentally controlled cell death are critical to optimal plant growth and development, but the mechanisms regulating early events in these pathways are poorly understood. This study provides novel insights into the initiation and crosstalk of the two signaling cascades.     

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.     

Transgenic rice plants ectopically expressing AtBAK1 are semi-dwarfed and hypersensitive to 24-epibrassinolide

Brassinosteroids (BRs) are endogenous plant hormones essential for plant growth and development. Brassinosteroid insensitive1 (BRI1)-assocaiated receptor kinase (BAK1) is one of the key components in the BR signal transduction pathway due to its direct association with the BR receptor, BRI1. Although BRI1 and its orthologs have been identified from both dicotyledonous and monocotyledonous plants, less is known about BAK1 and its orthologs in higher plants other than Arabidopsis. This article provides the first piece of evidence that AtBAK1 can greatly affect growth and development of rice plants when ectopically expressed, suggesting that rice may share similar BR perception mechanism via BRI1/BAK1 complex. Interestingly, transgenic rice plants displayed semi-dwarfism and shortened primary roots. Physiological analysis and cell morphology assay demonstrated that the observed phenotypes in transgenic plants were presumably caused by hypersensitivity to endogenous levels of BRs, different from BR insensitive and deficient rice mutants. Consistently, several known BR inducible genes were also upregulated in transgenic rice plants, further suggesting that BAK1 was able to affect BR signaling in rice. On the other hand, the transgenic plants generated by overproducing AtBAK1 may potentially have agricultural applications because the dwarfed phenotype is generally resistant to lodging, while the fertility remains unaffected.     

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.     

Somatic Embryogenesis Receptor Kinases Control Root Development Mainly via Brassinosteroid-Independent Actions in Arabidopsis thaliana

Brassinosteroids(BRs),a group of plant steroidal hormones,play critical roles in many aspects of plant growth and development.Previous studies showed that BRI1-mediated BR signaling regulates cell division and differentiation during Arabidopsis root development via interplaying with auxin and other phytohormones.Arabidopsis somatic embryogenesis receptor-like kinases(SERKs),as co-receptors of BRI1,were found to play a fundamental role in an early activation step of BR signaling pathway.Here we report a novel function of SERKs in regulating Arabidopsis root development.Genetic analyses indicated that SERKs control root growth mainly via a BR-independent pathway.Although BR signaling pathway is completely disrupted in the serk1 bak1 bkk1 triple mutant,the root growth of the triple mutant is much severely damaged than the BR deficiency or signaling null mutants.More detailed analyses indicated that the triple mutant exhibited drastically reduced expression of a number of genes critical to polar auxin transport,cell cycle,endodermis development and root meristem differentiation,which were not observed in null BR biosynthesis mutant cpd and null BR signaling mutant bri1-701.