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.     

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.     

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.     

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.     

CRISPR/Cas9介导靶向敲除拟南芥BRI1突变体的鉴定

以拟南芥(Arabidopsis thaliana)油菜素内酯受体BRI1为目的基因,利用CRISPR/Cas9基因编辑技术定向编辑拟南芥BRI1,以期获得更多BRI1的突变体,为后续BRI1功能的进一步深入研究奠定基础。通过筛选转基因植株,对编辑后的BRI1进行测序分析,结果显示该突变体中BRI1基因序列由于新碱基的插入导致提前终止。同BRI1强突变体bri1-710一样,相比于野生型对照均对BL处理不敏感,但相比于bri1-710,该突变体植株较大,暗示BRI1 N端可能在BR信号途径中有重要作用。因此该研究可为后续进一步研究拟南芥及其他同源物种的BRI1功能提供可靠的参考依据。     

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.     

Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis

Plant steroid hormones, brassinosteroids (BRs), are perceived by a cell surface receptor kinase, BRI1, but how BR binding leads to regulation of gene expression in the nucleus is unknown. Here we describe the identification of BZR1 as a nuclear component of the BR signal transduction pathway. A dominant mutation bzr1-1D suppresses BR-deficient and BR-insensitive (bri1) phenotypes and enhances feedback inhibition of BR biosynthesis. BZR1 protein accumulates in the nucleus of elongating cells of dark-grown hypocotyls and is stabilized by BR signaling and the bzr1-1D mutation. Our results demonstrate that BZR1 is a positive regulator of the BR signaling pathway that mediates both downstream BR responses and feedback regulation of BR biosynthesis.     

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.     

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     

Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles of BRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However, bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore, BRI1 is required for the homeostasis of endogenous BR levels.     

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.     

Arabidopsis ubiquitin conjugase UBC32 is an ERAD component that functions in brassinosteroid-mediated salt stress tolerance

Plants modify their growth and development to protect themselves from detrimental conditions by triggering a variety of signaling pathways, including the activation of the ubiquitin-mediated protein degradation pathway. Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is an important aspect of the ubiquitin-proteasome system, but only a few of the active ERAD components have been reported in plants. Here, we report that the Arabidopsis thaliana ubiquitin-conjugating enzyme, UBC32, a stress-induced functional ubiquitin conjugation enzyme (E2) localized to the ER membrane, connects the ERAD process and brassinosteroid (BR)-mediated growth promotion and salt stress tolerance. In vivo data showed that UBC32 was a functional ERAD component that affected the stability of a known ERAD substrate, the barley (Hordeum vulgare) powdery mildew O (MLO) mutant MLO-12. UBC32 mutation caused the accumulation of bri1-5 and bri1-9, the mutant forms of the BR receptor, BRI1, and these mutant forms subsequently activated BR signal transduction. Further genetic and physiological data supported the contention that UBC32 plays a role in the BR-mediated salt stress response and that BR signaling is necessary for the plant to tolerate salt. Our data indicates a possible mechanism by which an ERAD component regulates the growth and stress response of plants.     

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.     

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.     

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.     

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.