RESUMO
In many flowering plants, asymmetric division of the zygote generates apical and basal cells with different fates. In Arabidopsis thaliana, the apical cell generates the embryo while the basal cell divides anticlinally, leading to a suspensor of six to nine cells that remain extra-embryonic and eventually senesce. In some genetic backgrounds, or upon ablation of the embryo, suspensor cells can undergo periclinal cell divisions and eventually form a second twin embryo. Likewise, embryogenesis can be induced from somatic cells by various genes, but the relationship with suspensor-derived embryos is unclear. Here, we addressed the nature of the suspensor to embryo fate transformation and its genetic triggers. We expressed most known embryogenesis-inducing genes specifically in suspensor cells. We next analyzed morphology and fate-marker expression in embryos in which suspensor division was activated by different triggers to address the developmental paths towards reprogramming. Our results show that reprogramming of Arabidopsis suspensor cells towards embryonic identity is a specific cellular response that is triggered by defined regulators, follows a conserved developmental trajectory and shares similarity to the process of somatic embryogenesis from post-embryonic tissues.
Assuntos
Proteínas de Arabidopsis/metabolismo , Sementes/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Sementes/genéticaRESUMO
Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Divisão Celular Assimétrica , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Arabidopsis/enzimologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula , Citosol/enzimologia , Citosol/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Sistema de Sinalização das MAP Quinases , Complexos Multiproteicos/química , Fenótipo , Fosforilação , Estômatos de Plantas/citologia , Ligação Proteica , Proteínas Quinases/metabolismo , Especificidade por SubstratoRESUMO
Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.
Assuntos
Arabidopsis/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brefeldina A/farmacologia , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transporte Proteico , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Land plants are called 'embryophytes' and thus, their collective name is defined by their ability to form embryos. Indeed, embryogenesis is a widespread phenomenon in plants, and much of our diet is composed of embryos (just think of grains, beans or nuts; Figure 1). However, in addition to embryos as a source of nutrition, they are also a fascinating study object. Some of the most fundamental decisions on fate and identity, as well as patterning and morphogenesis, are taken during the first days of plant life. Yet, embryos are diverse in structure and function, and embryogenesis in plants is by no means restricted to the zygote - the product of fertilization. In this Primer, we discuss the adventures of the young plant. We will consider what it means to be a plant embryo and how to become one. We will next highlight how the study of early embryogenesis can reveal principles underlying oriented cell division and developmental pattern formation in plants.
Assuntos
Células Vegetais/metabolismo , Plantas/embriologia , Sementes/crescimento & desenvolvimento , Divisão Celular , MorfogêneseRESUMO
BAK1 is a coreceptor and positive regulator of multiple ligand binding leucine-rich repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity, and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here, we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand binding receptors to prevent BAK1 receptor complex formation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Arabidopsis/efeitos dos fármacos , Brassinosteroides/metabolismo , Morte Celular/efeitos dos fármacos , Flagelina/farmacologia , Proteínas de Repetições Ricas em Leucina , Ligantes , Mutação/genética , Moléculas com Motivos Associados a Patógenos/metabolismo , Fenótipo , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transdução de SinaisRESUMO
Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observation-fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of Förster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nanoestruturas , Raízes de Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transferência Ressonante de Energia de FluorescênciaRESUMO
A combination of coimmunoprecipitation (coIP) of tagged proteins followed by protein identification and quantitation using Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) has proven to be a reliable method to qualitatively characterize membrane-bound receptor complexes from plants. Success depends on a range of parameters, such as abundance and stability of the complex and functionality of the tagged receptors, efficiency of the protein complex isolation procedure, MS equipment, and analysis software in use. In this Chapter, we focus on the use of one of the green fluorescent protein-tagged receptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, of which SERK3, also known as BRASSINOSTEROID INSENSITIVE1 (BRI1) ASSOCIATED KINASE1 (BAK1), is a coreceptor of BRI1. Like BRI1 itself, SERK3 is a leucine-rich repeat receptor kinase (LRR RK) with a single-pass transmembrane domain. The latest updated laboratory protocol is presented as well as examples of data analysis and typical results obtained. Potential drawbacks of the procedure employed for plant membrane proteins will be pointed out.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Plântula/genética , Transdução de Sinais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Cromatografia Líquida , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação/métodos , Anotação de Sequência Molecular , Plantas Geneticamente Modificadas , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Plântula/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Somatic embryogenesis receptor kinases (SERKs) are ligand-binding coreceptors that are able to combine with different ligand-perceiving receptors such as BRASSINOSTEROID INSENSITIVE1 (BRI1) and FLAGELLIN-SENSITIVE2. Phenotypical analysis of serk single mutants is not straightforward because multiple pathways can be affected, while redundancy is observed for a single phenotype. For example, serk1serk3 double mutant roots are insensitive toward brassinosteroids but have a phenotype different from bri1 mutant roots. To decipher these effects, 4-d-old Arabidopsis (Arabidopsis thaliana) roots were studied using microarray analysis. A total of 698 genes, involved in multiple biological processes, were found to be differentially regulated in serk1-3serk3-2 double mutants. About half of these are related to brassinosteroid signaling. The remainder appear to be unlinked to brassinosteroids and related to primary and secondary metabolism. In addition, methionine-derived glucosinolate biosynthesis genes are up-regulated, which was verified by metabolite profiling. The results also show that the gene expression pattern in serk3-2 mutant roots is similar to that of the serk1-3serk3-2 double mutant roots. This confirms the existence of partial redundancy between SERK3 and SERK1 as well as the promoting or repressive activity of a single coreceptor in multiple simultaneously active pathways.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Mutação/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Transcrição Gênica , Alelos , Brassinosteroides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucosinolatos/farmacologia , Metaboloma/efeitos dos fármacos , Análise Multivariada , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
In plants, embryogenesis generally occurs through the sexual process of double fertilization, which involves a haploid sperm cell fusing with a haploid egg cell to ultimately give rise to a diploid embryo. Embryogenesis can also occur asexually in the absence of fertilization, both in vitro and in vivo. Somatic or gametic cells are able to differentiate into embryos in vitro following the application of plant growth regulators or stress treatments. Asexual embryogenesis also occurs naturally in some plant species in vivo, from either ovule cells as part of a process defined as apomixis, or from somatic leaf tissue in other species. In both in vitro and in vivo asexual embryogenesis, the embryo precursor cells must attain an embryogenic fate without the act of fertilization. This review compares the processes of in vitro and in vivo asexual embryogenesis including what is known regarding the genetic and epigenetic regulation of each process, and considers how the precursor cells are able to change fate and adopt an embryogenic pathway.
Assuntos
Epigênese Genética , Desenvolvimento Vegetal/genética , Plantas/genética , Reprodução Assexuada/genética , Apomixia/genética , Fertilização/genética , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/genética , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
In this issue of Science Signaling, Somssich and co-workers use fluorescence techniques to show the dynamics that occur during the activation of two different receptor complexes in living plant cells.
Assuntos
Nicotiana/metabolismo , Peptídeos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisAssuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ligação Competitiva , Proteínas de Ligação a DNA/metabolismo , Estômatos de Plantas/crescimento & desenvolvimento , Estômatos de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases involved in several, seemingly unrelated, plant-signaling pathways. In Arabidopsis thaliana, functional and genetic analysis of four SERK proteins has indicated that they are only partly redundant; their functions overlap but each performs a specific subset of signaling roles. The molecular basis for the functional specificity within this highly homologous protein family is currently not known. Sequence analysis of SERK proteins from different plant species indicates that the SERKs are a highly conserved protein family present in monocots, dicots, and non-vascular plants. Residues in the extracellular domain that are important for interaction with other receptor kinases are highly conserved, even among SERK members without a function in the corresponding pathways. SERK2, for instance, does not function in the brassinosteroid pathway, does not interact with BRI1, but is conserved in its BRI1-interacting domain. Further sequence analysis indicates that SERK3/BAK1 and SERK4/BKK1 have diverged from the original SERK protein in both their extracellular and cytoplasmic domains. Functional analysis of chimeric SERK proteins shows that different domains provide the SERK proteins with different functional specificity. For instance, the SERK1 or SERK2 extracellular domains are essential for SERK function in male sporogenesis, while the SERK3 extracellular and cytoplasmic domains are essential for SERK3 activity in brassinosteroid and flagellin signaling. The emerging picture is that SERKs are ancient genes, whose products have been recruited as co-receptors in the newly evolved signaling pathways. The SERK ligand-binding and protein-protein interaction domains are highly conserved, allowing all SERKs to form complexes, albeit with different affinity. However, specific functional residues must have been altered, in both the extracellular and intracellular domains, to allow for the observed differences in functionality.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Arabidopsis/enzimologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Biologia Computacional , Células Germinativas Vegetais/enzimologia , Células Germinativas Vegetais/crescimento & desenvolvimento , Dados de Sequência Molecular , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Alinhamento de SequênciaRESUMO
The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development.
Assuntos
Matriz Extracelular/metabolismo , Proteômica/métodos , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , AnimaisRESUMO
Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Ácidos Indolacéticos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/química , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
BACKGROUND: Transmembrane leucine-rich repeat (LRR) receptors are commonly used innate immune receptors in plants and animals but can also sense endogenous signals to regulate development. BAK1 is a plant LRR-receptor-like kinase (RLK) that interacts with several ligand-binding LRR-RLKs to positively regulate their functions. BAK1 is involved in brassinosteroid-dependent growth and development, innate immunity, and cell-death control by interacting with the brassinosteroid receptor BRI1, immune receptors, such as FLS2 and EFR, and the small receptor kinase BIR1, respectively. RESULTS: Identification of in vivo BAK1 complex partners by LC/ESI-MS/MS uncovered two novel BAK1-interacting RLKs, BIR2 and BIR3. Phosphorylation studies revealed that BIR2 is unidirectionally phosphorylated by BAK1 and that the interaction between BAK1 and BIR2 is kinase-activity dependent. Functional analyses of bir2 mutants show differential impact on BAK1-regulated processes, such as hyperresponsiveness to pathogen-associated molecular patterns (PAMP), enhanced cell death, and resistance to bacterial pathogens, but have no effect on brassinosteroid-regulated growth. BIR2 interacts constitutively with BAK1, thereby preventing interaction with the ligand-binding LRR-RLK FLS2. PAMP perception leads to BIR2 release from the BAK1 complex and enables the recruitment of BAK1 into the FLS2 complex. CONCLUSIONS: Our results provide evidence for a new regulatory mechanism for innate immune receptors with BIR2 acting as a negative regulator of PAMP-triggered immunity by limiting BAK1-receptor complex formation in the absence of ligands.
Assuntos
Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Imunidade Vegetal , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Morte Celular , Regulação da Expressão Gênica de Plantas , Ligantes , Mutação , Fosforilação , Proteínas Quinases/genéticaRESUMO
Brassinosteroids (BRs) are key regulators in plant growth and development. The main BR-perceiving receptor in Arabidopsis (Arabidopsis thaliana) is BRASSINOSTEROID INSENSITIVE1 (BRI1). Seedling root growth and hypocotyl elongation can be accurately predicted using a model for BRI1 receptor activity. Genetic evidence shows that non-ligand-binding coreceptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family are essential for BRI1 signal transduction. A relatively simple biochemical model based on the properties of SERK loss-of-function alleles explains complex physiological responses of the BRI1-mediated BR pathway. The model uses BRI1-BR occupancy as the central estimated parameter and includes BRI1-SERK interaction based on mass action kinetics and accurately describes wild-type root growth and hypocotyl elongation. Simulation studies suggest that the SERK coreceptors primarily act to increase the magnitude of the BRI1 signal. The model predicts that only a small number of active BRI1-SERK complexes are required to carry out BR signaling at physiological ligand concentration. Finally, when calibrated with single mutants, the model predicts that roots of the serk1serk3 double mutant are almost completely brassinolide (BL) insensitive, while the double mutant hypocotyls remain sensitive. This points to residual BRI1 signaling or to a different coreceptor requirement in shoots.
Assuntos
Proteínas de Arabidopsis/metabolismo , Modelos Teóricos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Algoritmos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Brassinosteroides/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocótilo/efeitos dos fármacos , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Microscopia Confocal , Mutação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Esteroides Heterocíclicos/farmacologiaRESUMO
Brassinosteroid (BR) hormones are primarily perceived at the cell surface by the leucine-rich repeat receptor-like kinase brassinosteroid insensitive1 (BRI1). In Arabidopsis thaliana, BRI1 has two close homologs, BRI1-LIKE1 (BRL1) and BRL3, respectively, which are expressed in the vascular tissues and regulate shoot vascular development. Here, we identify novel components of the BRL3 receptor complex in planta by immunoprecipitation and mass spectrometry analysis. Whereas BRI1 associated kinase1 (BAK1) and several other known BRI1 interactors coimmunoprecipitated with BRL3, no evidence was found of a direct interaction between BRI1 and BRL3. In addition, we confirmed that BAK1 interacts with the BRL1 receptor by coimmunoprecipitation and fluorescence microscopy analysis. Importantly, genetic analysis of brl1 brl3 bak1-3 triple mutants revealed that BAK1, BRL1, and BRL3 signaling modulate root growth and development by contributing to the cellular activities of provascular and quiescent center cells. This provides functional relevance to the observed protein-protein interactions of the BRL3 signalosome. Overall, our study demonstrates that cell-specific BR receptor complexes can be assembled to perform different cellular activities during plant root growth, while highlighting that immunoprecipitation of leucine-rich repeat receptor kinases in plants is a powerful approach for unveiling signaling mechanisms with cellular resolution in plant development.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Ciclo Celular , Cromatografia Líquida , Genes Reporter , Complexos Multiproteicos , Mutação , Fenótipo , Floema/citologia , Floema/genética , Floema/crescimento & desenvolvimento , Floema/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão , Espectrometria de Massas em TandemRESUMO
The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main ligand-perceiving receptor for brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana). Binding of BRs to the ectodomain of plasma membrane (PM)-located BRI1 receptors initiates an intracellular signal transduction cascade that influences various aspects of plant growth and development. Even though the major components of BR signaling have been revealed and the PM was identified as the main site of BRI1 signaling activity, the very first steps of signal transmission are still elusive. Recently, it was shown that the initiation of BR signal transduction requires the interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors. In addition, the resolved structure of the BRI1 ectodomain suggested that BRI1-ASSOCIATED KINASE1 [BAK1](SERK3) may constitute a component of the ligand-perceiving receptor complex. Therefore, we investigated the spatial correlation between BRI1 and BAK1(SERK3) in the natural habitat of both leucine-rich repeat receptor-like kinases using comparative colocalization analysis and fluorescence lifetime imaging microscopy. We show that activation of BR signaling by exogenous ligand application resulted in both elevated colocalization between BRI1 and BAK1(SERK3) and an about 50% increase of receptor heterooligomerization in the PM of live Arabidopsis root epidermal cells. However, large populations of BRI1 and BAK1(SERK3) colocalized independently of BRs. Moreover, we could visualize that approximately 7% of the BRI1 PM pool constitutively heterooligomerizes with BAK1(SERK3) in live root cells. We propose that only small populations of PM-located BRI1 and BAK1(SERK3) receptors participate in active BR signaling and that the initiation of downstream signal transduction involves preassembled BRI1-BAK1(SERK3) heterooligomers.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Brefeldina A/metabolismo , Brefeldina A/farmacologia , Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Multimerização Proteica , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Triazóis/farmacologiaRESUMO
Computational models are useful tools to help understand signalling pathways in plant cells. A systems biology approach where models and experimental data are combined can provide experimentally verifiable predictions and novel insights. The brassinosteroid insensitive 1 (BRI1) receptor is one of the best-understood receptor systems in Arabidopsis with clearly described ligands, mutants and associated phenotypes. Therefore, BRI1-mediated signalling is attractive for mathematical modelling approaches to understand and interpret the spatial and temporal dynamics of signal transduction cascades in planta. To establish such a model, quantitative data sets incorporating local protein concentration, binding affinity and phosphorylation state of the different pathway components are essential. Computational modelling is increasingly employed in studies of plant growth and development. In this section, we have focused on the use of quantitative imaging of fluorescently labelled proteins as an entry point in modelling studies.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Modelos Biológicos , Proteínas Quinases/metabolismo , Brassinosteroides/metabolismo , Receptor Cross-Talk , Transdução de SinaisRESUMO
Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK's) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein in vitro. Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state.