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First published online October 14, 2004
doi: 10.1242/10.1242/dev.01403

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

Ana Caño-Delgado^1,*, Yanhai Yin^1,*, Cong Yu^2,*, Dionne Vafeados¹, Santiago Mora-García¹, Jin-Chen Cheng², Kyoung Hee Nam^2,3, Jianming Li^2, and Joanne Chory^1,

¹ Howard Hughes Medical Institute and Plant Biology Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, CA 92037, USA
² Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 North University, Ann Arbor, MI 48109-1048, USA
³ Major in Biological Science, Sookmyung Women's University, 53-12 Chungpa-dong 2 Ka, Yongsan-gu, Seoul, 140-742, Korea

View larger version (86K): [in a new window]   Fig. 1. BRL genes encode homologs of BRI1. (A) Phylogenetic tree of members of the BRI1-family. Full-length protein sequences were aligned with ClustalX and manually adjusted. The N-J tree was calculated with MEGA2.1 (Kumar et al., 2001) for 1000 bootstrap repetitions. Branch lengths are proportional to the number of substitutions per 100 residues (indicated by the bar below the tree) and the branchpoint values indicate the percentage bootstrap support. The comparison included the Arabidopsis family members BRI1 [AtBRI1 (Li and Chory, 1997), AAC49810], AtBRL1 (NP_175957), AtBRL2 [(Clay and Nelson 2001), NP_178304], AtBRL3 (NP_187946), the corresponding sequences from rice, OsBRI1 [(Yamamuro et al., 2000), BAB68053], OsBRL1 (BAD01717) and OsBRL2 (AAK52544), and those reported for tomato [LeBRI1, cu3 (Montoya et al., 2002), Q8GUQ5], pea [PsBRI1, lka (Nomura et al., 2003), Q8GUQ5] and barley [HvBRI1, uzu (Chono et al., 2003), BAD06331]. Arabidopsis CLV1 (Q9SYQ8) was included as an outgroup RLK. The Arabidopsis proteins are highlighted in blue. (B) Diagram of the BRI1 and BRL proteins showing the location of the T-DNA insertions. Like BRI1, no introns are present in any of the BRL genes. (C) Alignment of the Arabidopsis proteins. In the extracellular region, the LRR domain is underlined in red, the arrowheads indicate the beginning of each LRR, the 70-amino acid island domain region is underlined in purple and the paired cysteines flanking the LRR-domain are indicated by black dots. The Ser/Thr kinase domain is underlined in black. Identical and similar amino acids are highlighted by black and gray boxes, respectively.

View larger version (47K): [in a new window]   Fig. 2. BRL1 and BRL3, but not BRL2, are able to rescue the phenotype of a weak bri1 mutant. (A) Arabidopsis wild-type rosette phenotype. (B) Complementation of bri1-301 mutant by BRI1 genomic DNA. (C) bri1-301 shows a weak dwarf phenotype. (D) pBRI-BRL1 rescues the dwarf bri1-301 phenotype. (E) pBRI-BRL2 fails to rescue the bri1-301 phenotype. (F) pBRI-BRL3 rescues the dwarf bri1-301 phenotype. (G,H) Point mutations in the island domain (G597E, G) and the kinase domain (E1056K, H) of BRL1 destroy the bri1-rescuing activity of pBRI1-BRL1.

View larger version (40K): [in a new window]   Fig. 3. BRL1 and BRL3 are membrane-localized receptors that bind BL. (A) Specific [3H] –BL binding for immunoprecipitated membrane fractions of BRL1 and (B) BRL3. Shown are the Kd values from one representative experiment of three replicates. BRI1-GFP was used as a positive control in our experiments (Kd=55.5 nM±0.08). (C) Western blot developed with anti-GFP antibodies showing the amount of BRI1::GFP, BRL1::GFP, BRL2::GFP and BRL3::GFP fusion proteins in the immunoprecipitated membrane fractions used for the [3H] –BL binding assays. (D) Subcellular localization of the BRL receptors. Confocal images of hypocotyl epidermal cells of light-grown 7-day-old seedlings carrying 35S::BRL1-GFP and 35S::BRL3-GFP fusion constructs show that both proteins are localized to the plasma membrane. Scale bar: 25 µm. (E) The specific [3H] –BL binding activity for each BRL protein and BRI1. BRL2 does not bind BL specifically. In these graphs, BRL1 binding appears lower than BRL3 or BRI1 due to the level of protein expressed in the transgenic lines used for this experiment.

View larger version (81K): [in a new window]   Fig. 4. BRL1 and BRL3 are expressed in the vascular tissues. BRL expression was assessed by GUS staining in 7-day-old seedlings, in embryos and in expanded leaves. (A) pBRL1::GUS is expressed in the columella root cap cells and in mature vascular tissues. (B) pBRL3::GUS is expressed in the roots in the two protophloem cell files in the elongation zone. (C) pBRI1:GUS is ubiquitously expressed in roots, especially in the elongation zone. (D) pBRL1::GUS is expressed in the mid-vein and lower part of the cotyledons. (E) pBRL3::GUS is preferentially expressed in the secondary veins at the tip of cotyledons. (F) pBRI1::GUS expression in the petioles and the lower part of cotyledons and leaves. (G) pBRL1::GUS expression in leaves is predominantly in the mid-vein. (H) pBRL3::GUS expression in leaves is predominantly in the secondary and tertiary veins and reduced in the mid-vein. (I) Strong expression of pBRI1::GUS in the petioles. (J) GUS staining in the embryos of pBRL1::GUS transgenic seeds. (K) Transverse section of the root tip of seedlings shows that pBRL1::GUS expression is confined to the vascular cylinder. en, endodermis; pe, pericycle; st, stele. (L) The root tip of a five-day-old pBRL1::GUS transgenic seedling showing that, after germination, BRL1 expression appears associated with the stele. (M) Transverse section at the base of the inflorescence stem in mature plants shows pBRL1::GUS expression in the procambial cells in the vascular bundles. (N) Detail of a leaf expressing pBRL3::GUS shows higher expression in the secondary and tertiary veins compared to the mid-vein. (O) Close-up showing the leaf vasculature stained with pBRL3::GUS reveals specific expression in the phloem sieve elements. (P) The root tip of a 5-day-old pBRL3::GUS transgenic seedling showing that, after germination, BRL3 expression appears associated with the protophloem. (Scale bars: 100 µm in A,B,C; 10 µm in J; 25 µm in K,L,M,N,P.

View larger version (75K): [in a new window]   Fig. 5. Phenotypic analysis of brl mutants and genetic combinations of double and triple mutants with bri1. Mature plants of (A) Col-0 wild type, (B) brl1-2 (C), and brl3 mutants were taken for histological analysis of the vascular tissues. Since no apparent phenotype was observed for any of the brl1 alleles, only brl1-2 is shown. (D) bri1-5 mature plants (left) compared with the double mutant bri1-5 brl1-1 (right), where bri1-5 dwarfism is enhanced. (E) Cross section of an inflorescence stem showing the organization of a Toluidine Blue-stained vascular bundle of Ws-2 ecotype. (F) brl1-1 shows an increased amount of phloem compared to the xylem in the vascular bundles. (G) bri1-5 shows an increased differentiation of the phloem at the expense of the xylem and enlarged phloem cap cells compared with wild-type plants. (H) The aberrant vasculature in the bri1-5 brl1-1 double mutant consists of small vascular bundles where the small phloem cells are placed between the xylem and less differentiated than the wild type, and has dramatically enlarged phloem cap cells. (I) Schematic representation of the Arabidopsis vascular system at the basal part of the inflorescence stem of a mature plant. In wild-type plants, there are six to eight vascular bundles arranged in a radial pattern. The activity of the procambial cells (yellow) gives rise to phloem (red) tissue in the outer part and the xylem (blue) in the inner part of the vascular bundle. Pictures shown in this figure show the anatomy of a single vascular bundle (black box). Cross section of an inflorescence stem showing the organization of a Toluidine Blue-stained vascular bundle of Col-0 ecotype. (J,K) brl1-2 and brl3 in Col-0 background show normal vascular bundles. (L) A biosynthetic mutant, det2-1, shows similar defects to those of the bri1 mutants. (M) Plants overexpressing the BRI1 receptor (Wang et al., 2001) show increased xylem differentiation in the vascular bundles. (N,O) bri1-101 mutant phenotype and triple mutants bri1-101 brl1-2 brl3. (P) Vascular bundle in bri1-101 mutants. (Q) Vascular bundle bri1-101 brl1-2 brl3 mutants showing reduced vascular differentiation and unusual formation of phloem fibers. Phc, phloem cap cell; ph, phloem; x, xylem; if, interfascicular xylem). Scale bar: 10 mm in A-D,N,O; 20 µm in E-M,P,Q.

View larger version (33K): [in a new window]   Fig. 6. A model for BR action in vascular development. Schematic representation of the vascular bundles in which BRs act as a vascular patterning signal that promotes xylem while repressing phloem differentiation. (B) In the case of defective BR-signaling (as shown in bri1 and brl1-1 mutants), increased phloem differentiation occurs at the expense of xylem. This model supports the idea that the specification of phloem and xylem in the vascular bundles is tightly linked. Whether BRs are perceived by receptors of the BRI1-family on the plasma membrane of cells that secrete BRs (autocrine) or cells that do not secrete BRs (paracrine) remains unknown.

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