RESUMO
During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo-vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP-labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co-localisation studies using GFP-CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast-associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP-CESA from doughnut-shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP-CESA density diminished, whereas GFP-CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP-CESA in clathrin-containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose-deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Glucosiltransferases/genética , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Divisão Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/ultraestrutura , Clatrina/metabolismo , Citocinese , Genes Reporter , Glucosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Isoenzimas , Microscopia Confocal , Microtúbulos/ultraestrutura , Modelos Biológicos , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão , Plântula/citologia , Plântula/genética , Plântula/metabolismoRESUMO
Cellulose synthesis is driven by large plasma membrane-inserted protein complexes, which in plants have 6-fold symmetry. In Arabidopsis (Arabidopsis thaliana), functional cellulose synthesis complexes (CSCs) are composed of at least three different cellulose synthase catalytic subunits (CESAs), but the actual ratio of the CESA isoforms within the CSCs remains unresolved. In this work, the stoichiometry of the CESAs in the primary cell wall CSC was determined, after elimination of CESA redundancy in a mutant background, by coimmunoprecipitation and mass spectrometry using label-free quantitative methods. Based on spectral counting, we show that CESA1, CESA3, and CESA6 are present in a 1:1:1 molecular ratio.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Glucosiltransferases/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Domínio Catalítico , Parede Celular/metabolismo , Glucosiltransferases/genética , Imunoprecipitação , Isoenzimas , Espectrometria de Massas , Proteínas de Membrana , Proteômica , Plântula/enzimologia , Plântula/genéticaRESUMO
Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis in plants or bacteria also requires the activity of an endo-1,4-ß-d-glucanase, the exact function of which in the synthesis process is not known. Here, we show, to our knowledge for the first time, that a leaky mutation in the Arabidopsis (Arabidopsis thaliana) membrane-bound endo-1,4-ß-d-glucanase KORRIGAN1 (KOR1) not only caused reduced CSC movement in the plasma membrane but also a reduced cellulose synthesis inhibitor-induced accumulation of CSCs in intracellular compartments. This suggests a role for KOR1 both in the synthesis of cellulose microfibrils and in the intracellular trafficking of CSCs. Next, we used a multidisciplinary approach, including live cell imaging, gel filtration chromatography analysis, split ubiquitin assays in yeast (Saccharomyces cerevisiae NMY51), and bimolecular fluorescence complementation, to show that, in contrast to previous observations, KOR1 is an integral part of the primary cell wall CSC in the plasma membrane.
RESUMO
It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.
Assuntos
Arabidopsis/anatomia & histologia , Arabidopsis/crescimento & desenvolvimento , Celulose/ultraestrutura , Hipocótilo/citologia , Hipocótilo/crescimento & desenvolvimento , Epiderme Vegetal/citologia , Epiderme Vegetal/crescimento & desenvolvimento , Anisotropia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Parede Celular/química , Celulose/metabolismo , Glucosiltransferases/metabolismo , Hipocótilo/metabolismo , Microfibrilas/química , Microfibrilas/metabolismo , Microfibrilas/ultraestrutura , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Epiderme Vegetal/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The growth of plants, like that of other walled organisms, depends on the ability of the cell wall to yield without losing its integrity. In this context, plant cells can sense the perturbation of their walls and trigger adaptive modifications in cell wall polymer interactions. Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) THESEUS1 (THE1) was previously shown in Arabidopsis to trigger growth inhibition and defense responses upon perturbation of the cell wall, but so far, neither the ligand nor the role of the receptor in normal development was known. Here, we report that THE1 is a receptor for the peptide rapid alkalinization factor (RALF) 34 and that this signaling module has a role in the fine-tuning of lateral root initiation. We also show that RALF34-THE1 signaling depends, at least for some responses, on FERONIA (FER), another RALF receptor involved in a variety of processes, including immune signaling, mechanosensing, and reproduction [1]. Together, the results show that RALF34 and THE1 are part of a signaling network that integrates information on the integrity of the cell wall with the coordination of normal morphogenesis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Hormônios Peptídicos/genética , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Hormônios Peptídicos/metabolismo , Raízes de Plantas/genética , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases. In addition to the CESA proteins, cellulose biosynthesis almost certainly requires the action of other proteins, although few have been identified and little is known about the biochemical role of those that have been identified. One of these proteins is KORRIGAN (KOR1). Mutant analysis of this protein in Arabidopsis thaliana showed altered cellulose content in both the primary and secondary cell wall. KOR1 is thought to be required for cellulose synthesis acting as a cellulase at the plasma membrane-cell wall interface. KOR1 has recently been shown to interact with the primary cellulose synthase rosette complex however direct interaction with that of the secondary cell wall has never been demonstrated. Using various methods, both in vitro and in planta, it was shown that KOR1 interacts specifically with only two of the secondary CESA proteins. The KOR1 protein domain(s) involved in the interaction with the CESA proteins were also identified by analyzing the interaction of truncated forms of KOR1 with CESA proteins. The KOR1 transmembrane domain has shown to be required for the interaction between KOR1 and the different CESAs, as well as for higher oligomer formation of KOR1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Celulase/metabolismo , Glucosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Membrana Celular/metabolismo , Parede Celular/metabolismo , Celulase/química , Proteínas de Membrana/química , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Plant development is highly plastic and dependent on light quantity and quality monitored by specific photoreceptors. Although we have a detailed knowledge of light signaling pathways, little is known about downstream targets involved in growth control. Cell size and shape are in part controlled by cellulose microfibrils extruded from large cellulose synthase complexes (CSCs) that migrate in the plasma membrane along cortical microtubules. Here we show a role for the red/far-red light photoreceptor PHYTOCHROME B (PHYB) in the regulation of cellulose synthesis in the growing Arabidopsis hypocotyl. In this organ, CSCs contains three distinct cellulose synthase (CESA) isoform classes: nonredundant CESA1 and CESA3 and a third class represented by partially redundant CESA2, CESA5, and CESA6. Interestingly, in the dark, depending on which CESA subunits occupy the third position, CSC velocity is more or less inhibited through an interaction with microtubules. Activation of PHYB overrules this inhibition. The analysis of cesa5 mutants shows a role for phosphorylation in the control of CSC velocity. These results, combined with the cesa5 mutant phenotype, suggest that cellulose synthesis is fine tuned through the regulated interaction of CSCs with microtubules and that PHYB signaling impinges on this process to maintain cell wall strength and growth in changing environments.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , DNA Complementar/genética , Genes de Plantas , Glucosiltransferases/metabolismo , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Luz , Microfibrilas/metabolismo , Microtúbulos/metabolismo , Fitocromo B/metabolismoRESUMO
It has not yet been reported how the secondary CESA (cellulose synthase) proteins are organized in the rosette structure. A membrane-based yeast two-hybrid (MbYTH) approach was used to analyze the interactions between the CESA proteins involved in secondary cell wall synthesis of Arabidopsis and the findings were confirmed in planta by bimolecular fluorescence complementation (BiFC) assay. Results indicated that although all CESA proteins can interact with each other, only CESA4 is able to form homodimers. A model is proposed for the secondary rosette structure. The RING-motif proved not to be essential for the interaction between the CESA proteins.
Assuntos
Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Glucosiltransferases/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos/fisiologia , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Plantas/metabolismo , Ligação Proteica , Multimerização Proteica/fisiologia , Domínios RING Finger/fisiologia , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by plasma membrane-bound complexes containing cellulose synthase proteins (CESAs). Here, we establish a role for the cytoskeleton in intracellular trafficking of cellulose synthase complexes (CSCs) through the in vivo study of the green fluorescent protein (GFP)-CESA3 fusion protein in Arabidopsis thaliana hypocotyls. GFP-CESA3 localizes to the plasma membrane, Golgi apparatus, a compartment identified by the VHA-a1 marker, and, surprisingly, a novel microtubule-associated cellulose synthase compartment (MASC) whose formation and movement depend on the dynamic cortical microtubule array. Osmotic stress or treatment with the cellulose synthesis inhibitor CGA 325'615 induces internalization of CSCs in MASCs, mimicking the intracellular distribution of CSCs in nongrowing cells. Our results indicate that cellulose synthesis is coordinated with growth status and regulated in part through CSC internalization. We find that CSC insertion in the plasma membrane is regulated by pauses of the Golgi apparatus along cortical microtubules. Our data support a model in which cortical microtubules not only guide the trajectories of CSCs in the plasma membrane, but also regulate the insertion and internalization of CSCs, thus allowing dynamic remodeling of CSC secretion during cell expansion and differentiation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Glucosiltransferases/metabolismo , Complexo de Golgi/fisiologia , Microtúbulos/fisiologia , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/análise , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Transporte Proteico , Proteínas Recombinantes de Fusão/análiseRESUMO
In all land plants, cellulose is synthesized from hexameric plasma membrane complexes. Indirect evidence suggests that in vascular plants the complexes involved in primary wall synthesis contain three distinct cellulose synthase catalytic subunits (CESAs). In this study, we show that CESA3 and CESA6 fused to GFP are expressed in the same cells and at the same time in the hypocotyl of etiolated seedlings and migrate with comparable velocities along linear trajectories at the cell surface. We also show that CESA3 and CESA6 can be coimmunoprecipitated from detergent-solubilized extracts, their protein levels decrease in mutants for either CESA3, CESA6, or CESA1 and CESA3, CESA6 and also CESA1 can physically interact in vivo as shown by bimolecular fluorescence complementation. We also demonstrate that CESA6-related CESA5 and CESA2 are partially, but not completely, redundant with CESA6 and most likely compete with CESA6 for the same position in the cellulose synthesis complex. Using promoter-beta-glucuronidase fusions we show that CESA5, CESA6, and CESA2 have distinct overlapping expression patterns in hypocotyl and root corresponding to different stages of cellular development. Together, these data provide evidence for the existence of binding sites for three distinct CESA subunits in primary wall cellulose synthase complexes, with two positions being invariably occupied by CESA1 and CESA3, whereas at least three isoforms compete for the third position. Participation of the latter three isoforms might fine-tune the CESA complexes for the deposition of microfibrils at distinct cellular growth stages.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Arabidopsis/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Celulose/química , Genes de Plantas , Glucosiltransferases/genética , Microfibrilas , Modelos Genéticos , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Isoformas de ProteínasRESUMO
Isoxaben is a pre-emergence herbicide that inhibits cellulose biosynthesis in higher plants. Two loci identified by isoxaben-resistant mutants (ixr1-1, ixr1-2, and ixr2-1) in Arabidopsis have been reported previously. IXR1 was recently shown to encode the cellulose synthase catalytic subunit CESA3 (W.-R. Scheible, R. Eshed, T. Richmond, D. Delmer, and C. Somerville [2001] Proc Natl Acad Sci USA 98: 10079-10084). Here, we report on the cloning of IXR2, and show that it encodes another cellulose synthase isoform, CESA6. ixr2-1 carries a mutation substituting an amino acid close to the C terminus of CESA6 that is highly conserved among CESA family members. Transformation of wild-type plants with the mutated gene and not with the wild-type gene conferred increased resistance against the herbicide. The simplest interpretation for the existence of these two isoxaben-resistant loci is that CESA3 and CESA6 have redundant functions. However, loss of function procuste1 alleles of CESA6 were previously shown to have a strong growth defect and reduced cellulose content in roots and dark-grown hypocotyls. This indicates that in these mutants, the presence of CESA3 does not compensate for the absence of CESA6 in roots and dark-grown hypocotyls, which argues against redundant functions for CESA3 and CESA6. Together, these observations are compatible with a model in which CESA6 and CESA3 are active as a protein complex.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/crescimento & desenvolvimento , Benzamidas/farmacologia , Celulose/metabolismo , Glucosiltransferases/genética , Herbicidas/farmacologia , Proteínas de Schizosaccharomyces pombe , Fatores Ativadores da Transcrição , Alelos , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Mapeamento Cromossômico , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Expressão Gênica , Genótipo , Glucanos/metabolismo , Glucosiltransferases/metabolismo , Hipocótilo/efeitos dos fármacos , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Isoenzimas/genética , Isoenzimas/metabolismo , Lignina/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fenótipo , Filogenia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transformação GenéticaRESUMO
The cell wall is the major limiting factor for plant growth. Wall extension is thought to result from the loosening of its structure. However, it is not known how this is coordinated with wall synthesis. We have identified two novel allelic cellulose-deficient dwarf mutants, kobito1-1 and kobito1-2 (kob1-1 and kob1-2). The cellulose deficiency was confirmed by the direct observation of microfibrils in most recent wall layers of elongating root cells. In contrast to the wild type, which showed transversely oriented parallel microfibrils, kob1 microfibrils were randomized and occluded by a layer of pectic material. No such changes were observed in another dwarf mutant, pom1, suggesting that the cellulose defect in kob1 is not an indirect result of the reduced cell elongation. Interestingly, in the meristematic zone of kob1 roots, microfibrils appeared unaltered compared with the wild type, suggesting a role for KOB1 preferentially in rapidly elongating cells. KOB1 was cloned and encodes a novel, highly conserved, plant-specific protein that is plasma membrane bound, as shown with a green fluorescent protein-KOB1 fusion protein. KOB1 mRNA was present in all organs investigated, and its overexpression did not cause visible phenotypic changes. KOB1 may be part of the cellulose synthesis machinery in elongating cells, or it may play a role in the coordination between cell elongation and cellulose synthesis.