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

Abstract

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.

Figure 1

The majority of bri1 mutations cluster in the island and kinase domains. A schematic representation of BRI1 including all the known bri1 point mutations with their predicted effects. Symbols represent the following:  , Signal peptide;  , putative Leu-zipper motif;  , Cys pair;   , LRRs;    , 70-amino acid island;  , transmembrane domain;   , kinase domain. Asterisk, These alleles were published by Noguchi et al. (1999).

Figure 2

BRI1-GFP is expressed ubiquitously during early seedling development and is localized to the plasma membrane. A BRI1::GFP fusion protein was expressed from the BRI1 promoter in stably transformed wild-type Arabidopsis. A, Background chlorophyll fluorescence in a wild-type hypocotyl; B, BRI1::GFP fluorescence along the cell surface of hypocotyl cells; C, background fluorescence of a wild-type Arabidopsis root; D, BRI1::GFP at the cell surface in young root; E, BRI1::GFP at the cell surface in cotyledon epidermal cells (wild type has only stomata and guard cell chloroplast autofluorescence, which is not shown); F, BRI1::GFP localizes with the cytoplasm if cells are collapsed in 0.8 m mannitol, indicating that BRI1::GFP is in the plasma membrane. Bar = 20 μm.

Figure 3

BRI1 is a Ser/Thr kinase. Immunoprecipitated HA-BRI1, wild type, bri1-101, and bri1-113 were used for an in vitro kinase assay. A, The top presents an autoradiogram of the kinase assay. The bottom presents an immunoblot using anti-HA antibody of the same gel. Lane 1, Untransfected 293T cells; lane 2, wild-type BRI1; lane 3, bri1-101 kinase mutant; lane 4, bri1-113 island domain mutant. B, Autophosphorylation of BRI1 occurs on Ser/Thr residues. Anti-HA-BRI1 autophosphorylation was subjected to phosphoamino acid analysis. The positions of the internal phospho-Ser, phospho-Thr, and phospho-Tyr standards (visualized by ninhydrin staining) are indicated. The origin (+) and the directions of electrophoresis with the pH are as indicated.