Protein BRASSINOSTEROID INSENSITIVE 1Silencing of OsI-BAK1 in rice plants produced a high number of undeveloped green braassinosteroid unfilled grains compared to the untransformed plants. Histological analyses demonstrated that embryos were either absent or retarded in their development in these unfilled rice grains of OsI-BAK1 RNAi plants. Down regulation of OsI-BAK1 caused a reduction in cell brassinosteroid insensitive 1 and enlargement in leaf bulliform cells. Furthermore, transgenic rice plants overexpressing OsI-BAK1 were demonstrated to have corrugated and ciclo winstrol y oxandrolona oral leaves probably due to increased cell number that caused abnormal bulliform cell structure which inswnsitive enlarged and plugged deep into leaf epidermis. The current findings suggest that OsI-BAK1 may play an important role in the brassinosteroid insensitive 1 processes of rice grain filling and leaf cell including the bulliform brassinosteroid insensitive 1. National Center for Biotechnology InformationU. Didn't get brassinosteroud message?
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. 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.
Among the bri1 mutants identified are mutants in the kinase domain, and we demonstrate that one of these mutations severely impairs BRI1 kinase activity. Receptor protein kinases RPKs activate a complex array of intracellular signaling pathways in response to the extracellular environment van der Geer et al.
RPKs are single-pass transmembrane proteins that contain an amino-terminal signal sequence, extracellular domains unique to each receptor, and a cytoplasmic kinase domain. In general, ligand binding induces homo- or heterodimerization of RPKs, and the resultant close proximity of the cytoplasmic domains results in kinase activation by transphosphorylation.
Although plants have many proteins similar to RPKs, no ligand has been identified for these receptor-like kinases RLKs. The known interactors for mammalian LRR receptors are peptide hormones, such as nerve growth factor and gonadotropin Braun et al. CLV1 is involved in the control of cell division and differentiation in the shoot apical meristem with CLV3 being the putative peptide ligand Fletcher et al. BRs are a unique class of plant steroids found throughout the plant kingdom that exhibit multiple effects when applied exogenously, including cell expansion of young aerial tissues, especially the hypocotyl and leaf petioles Mandova, Classical animal steroid hormone receptors belong to a subfamily of nuclear receptors that are ligand-dependent transcription factors that regulate gene expression Beato et al.
There is also evidence for action of steroid hormones outside the nucleus involving membrane receptors and protein phosphorylation Wehling, Additionally, progesterone stimulates Tyr phosphorylation in human sperm via a putative cell surface receptor Tesarik et al. Three bri1 mutations are in glycines of the island domain, and one is a missense mutation in the first LRR following the amino acid island domain Li and Chory, ; Noguchi et al.
In this paper we present the identity of additional recessive alleles of BRI1 , which highlights the importance of certain domains for BRI1 function. Additionally, we used a green fluorescent protein fusion to show the localization of BRI1 to the plasma membrane and to determine its pattern of expression within the plant.
We previously conducted a screen for BR-insensitive mutants, and isolated 18 new bri1 alleles Li and Chory, To identify regions of functional significance, we sequenced BRI1 from 14 of these mutants.
The molecular lesions of bri1 alleles are reported in Table I and are schematically represented in Figure 1 , including already published bri1 alleles Li and Chory, ; Noguchi et al. We identified three new mutations in the kinase domain; bri and bri are missense alleles altering amino acids Ala to Thr and Arg to Gln, respectively.
The bri allele has a mutation at codon 1, of a non-conserved Asp to Asn in a region of the protein that generally contains negatively charged residues in protein kinases. These new mutants re-emphasize the necessity of the BRI1 kinase domain. In the BRI1 extracellular domain, we identified two new mutations: The latter mutation occurs after the 25th LRR and before the second Cys pair, indicating a new region in BRI1 whose integrity is necessary for function.
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: Asterisk, These alleles were published by Noguchi et al. To determine the spatial pattern of expression and subcellular localization of BRI1, a green fluorescent protein GFP fusion with the C terminus of full-length BRI1 was made in the context of a BRI1 genomic clone containing, in addition to coding sequence, 1.
This translational fusion BRI GFP was able to rescue bri data not shown , indicating that the fusion protein was functional. The expression of BRI GFP is ubiquitous in young tissue, especially in the meristem. GFP fluorescence is localized to the cell surface in the hypocotyl, root, and cotyledons of young light-grown seedlings. The hypocotyl cells of wild-type vector alone seedlings Fig.
The cotyledons show a similar pattern with the BRI GFP fluorescence illuminating the surface of epidermal cells Fig. Roots have very low background fluorescence due to the lack of chloroplasts, and in Figure 2 C, the fluorescence of the image was enhanced to show that a root-tip was in the field. In stark contrast to this wild-type root is the transgenic root Fig. In these young cells in the root tip, the cytoplasm is not pushed up against the plasma membrane because the vacuoles are small.
GFP in all tissues in both light- and dark-grown seedlings data not shown. Expression continues in younger tissue, but BRI GFP fluorescence decreases in non-growing older tissue data not shown.
BRI1-GFP is expressed ubiquitously during early seedling development and is localized to the plasma membrane. GFP at the cell surface in cotyledon epidermal cells wild type has only stomata and guard cell chloroplast autofluorescence, which is not shown ; F, BRI GFP localizes with the cytoplasm if cells are collapsed in 0. GFP is in the plasma membrane. To determine if BRI1 is localized to the cell wall or the plasma membrane, a plasmolysis experiment was performed.
Negative osmotic pressure results in the evacuation of fluid from inside the cell, and the plasma membrane is internalized with the cellular organelles, leaving the cell wall unaltered. When roots from a transgenic seedling were placed in 0. GFP fluorescence was internalized with the plasma membrane and the rest of the cell Fig. The mutants tested were bri , a kinase domain mutant altering Gly to Glu in subdomain IX, and bri , which has a mutation in the extracellular island domain Fig.
Transfected BRI1 is readily detectable as an approximately kD protein by immunoblot of whole cell lysates using the 12CA5 monoclonal antibody and enhanced chemiluminescence data not shown. The kinase activity of BRI1 proteins was determined by an in vitro kinase assay. Figure 3 A shows that although the wild-type and bri proteins are competent for autophosphorylation, the kinase activity of bri is significantly reduced. Quantitation revealed that the kinase activity of bri was 45 times lower than that of the wild type in this assay, whereas bri expression was only one-half of the wild-type levels Fig.
Phosphoamino acid analysis performed with the in vitro autophosphorylated receptor showed that phospho-Ser and phospho-Thr were present, thus confirming this prediction Fig.
Immunoprecipitated HA-BRI1, wild type, bri, and bri 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 T cells; lane 2, wild-type BRI1; lane 3, bri kinase mutant; lane 4, bri island domain mutant.
The positions of the internal phospho-Ser, phospho-Thr, and phospho-Tyr standards visualized by ninhydrin staining are indicated. The results from these assays showed that the fusion protein possessed kinetic parameters typical of protein kinases. We chose to express BRI1 in an animal cell system because similarities exist between animal and plant cells with regard to the expression and the peptide signal-mediated membrane targeting of large receptor proteins.
We reasoned that proper membrane expression of the full-length protein in a cellular context would allow the study of hormonal regulation of receptor activation. BRI1 expressed in T cells by transient transfection was used for in vivo labeling experiments data not shown and was also immunoprecipitated and used for an in vitro kinase assay. The in vitro kinase assay revealed that both the wild-type protein and the product of an allele that contains a mutation in the extracellular domain were active protein kinases, at least in vitro, under conditions of antibody-mediated dimerization.
On the other hand, the product of the bri allele, which contains a mutation in the kinase domain, exhibited greatly reduced kinase activity. This assay confirms the predicted activity of the kinase domain of BRI1 and can now be used as a biochemical tool to identify direct substrates of the activated receptor.
The demonstration that BRI1 encodes an active protein kinase provides more substantial ground to explain the effects of the intracellular domain mutations in BRI1. Although mutations in the intracellular domain of a transmembrane receptor might affect several processes, such as receptor homo- and heterodimerization, interaction with other proteins, receptor stability, and enzymatic activity, most mutations found in the intracellular domain of BRI1 are consistent with the latter possibility: Phosphoamino acid analyses of in vitro phosphorylated BRI1 revealed the presence of phosphorylated Ser and Thr only, as originally predicted from the inspection of primary amino acid sequence of this kinase.
In addition to activation loop phosphorylation, which generally serves to increase kinase activity, sites elsewhere may be phosphorylated to recruit phosphopeptide-binding proteins into an activated receptor complex, as happens for receptor protein-Tyr kinases. Three modular protein domains are now known that selectively bind to peptide sequences containing phosphorylated Ser or Thr: We have not detected either basal or steroid-induced phosphorylation of BRI1 in T cells labeled with [ 32 P]orthophosphate, despite the fact that many variables have been explored and that phosphorylation of several other protein kinases and kinase substrates was readily detected under similar conditions data not shown.
Based on what has been learned from RPKs in other systems and on the observation that at least part of the autophosphorylation of HAESA RLK5 is intermolecular Horn and Walker, , we speculate that BRI1 is found in the inactive monomeric state when expressed in T cells and that the formation of kinase-active BRI1 dimers is induced during the immunoprecipitation reaction that precedes the in vitro kinase assay.
This model explains why, under the conditions of the latter assay, a ligand is not required to promote stimulation of receptor kinase activity. Brassinolide and other steroids may have failed to promote BRI1 dimerization and activation in intact cells for several reasons, such as a missing accessory steroid carrier protein or presenting molecule in the heterologous T cell system.
There are several putative domains in the extracellular region of BRI1. Analysis of mutant alleles indicates domains of functional importance, including the amino-terminal Cys pair, the amino acid island, the LRR domain, and the region between the LRR and the second Cys pair Fig.
The island domain contains three missense mutations, bri , bri , and bri , which affect three separate Gly residues. Although the bri mutant fails to respond to the steroid, it was shown in this paper to be an active kinase in vitro. This mutant could be an active kinase in vitro due to antibody-mediated dimerization, but in vivo a mutation of Gly in the island domain Gly might inhibit positive regulation of the kinase domain. The requirement for this Gly could be due to the small size of this residue, which allows for a conformational change of the protein when the ligand is bound to activate the kinase domain.
This Gly is mutated in the bri allele, thereby supporting the hypothesis that the Glys represent a structural requirement for LRR transmembrane protein function. The LxxLxLxx x is any amino acid domain within the LRR corresponds with the solvent face of the protein with the Leu resides facing away from the solvent face Jones and Jones, The x or variable amino acids could lead to specificity of the protein binding to the LRR.
The strong clv1 alleles, clv and clv , are both missense mutations in this solvent face domain. In contrast to this, bri is a weak allele Noguchi et al. It is interesting that this mutation is in the first of the four LRR after the island domain. This could indicate that many of the important molecular interactions occur close to the transmembrane domain. This idea is supported by bri , a mutation also in this region. Conversely, the clustering of mutations closer to the transmembrane domain could indicate that the protein interactions with the first 21 LRRs are strong enough that altering one amino acid does not significantly destabilize binding.
BRI1 is expressed in all tissues in the seedling root, hypocotyl, cotyledons, and leaves; Fig. Although there is no tissue-specific expression of BRI1, there is temporal regulation. Fully expanded leaves and elongated root or inflorescence cells express BRI GFP at low levels data not shown. This expression of the transgene correlates with physiological data that showed that exogenously applied BRs promote growth only in younger tissues Mandova,