RESUMO
P4B (2-phenyl-1-[4-(6-(piperidin-1-yl) pyridazin-3-yl) piperazin-1-yl] butan-1-one) is a novel cellulose biosynthesis inhibitor (CBI) discovered in a screen for molecules to identify inhibitors of Arabidopsis (Arabidopsis thaliana) seedling growth. Growth and cellulose synthesis inhibition by P4B were greatly reduced in a novel mutant for the cellulose synthase catalytic subunit gene CESA3 (cesa3pbr1). Cross-tolerance to P4B was also observed for isoxaben-resistant (ixr) cesa3 mutants ixr1-1 and ixr1-2. P4B has an original mode of action as compared with most other CBIs. Indeed, short-term treatments with P4B did not affect the velocity of cellulose synthase complexes (CSCs) but led to a decrease in CSC density in the plasma membrane without affecting their accumulation in microtubule-associated compartments. This was observed in the wild type but not in a cesa3pbr1 background. This reduced density correlated with a reduced delivery rate of CSCs to the plasma membrane but also with changes in cortical microtubule dynamics and orientation. At longer timescales, however, the responses to P4B treatments resembled those to other CBIs, including the inhibition of CSC motility, reduced growth anisotropy, interference with the assembly of an extensible wall, pectin demethylesterification, and ectopic lignin and callose accumulation. Together, the data suggest that P4B either directly targets CESA3 or affects another cellular function related to CSC plasma membrane delivery and/or microtubule dynamics that is bypassed specifically by mutations in CESA3.
RESUMO
Growth of etiolated Arabidopsis hypocotyls is biphasic. During the first phase, cells elongate slowly and synchronously. At 48 h after imbibition, cells at the hypocotyl base accelerate their growth. Subsequently, this rapid elongation propagates through the hypocotyl from base to top. It is largely unclear what regulates the switch from slow to fast elongation. Reverse genetics-based screening for hypocotyl phenotypes identified three independent mutant lines of At1g70990, a short extensin (EXT) family protein that we named EXT33, with shorter etiolated hypocotyls during the slow elongation phase. However, at 72 h after imbibition, these dark-grown mutant hypocotyls start to elongate faster than the wild type (WT). As a result, fully mature 8-day-old dark-grown hypocotyls were significantly longer than WTs. Mutant roots showed no growth phenotype. In line with these results, analysis of native promoter-driven transcriptional fusion lines revealed that, in dark-grown hypocotyls, expression occurred in the epidermis and cortex and that it was strongest in the growing part. Confocal and spinning disk microscopy on C-terminal protein-GFP fusion lines localized the EXT33-protein to the ER and cell wall. Fourier-transform infrared microspectroscopy identified subtle changes in cell wall composition between WT and the mutant, reflecting altered cell wall biomechanics measured by constant load extensometry. Our results indicate that the EXT33 short EXT family protein is required during the first phase of dark-grown hypocotyl elongation and that it regulates the moment and extent of the growth acceleration by modulating cell wall extensibility.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Hipocótilo/crescimento & desenvolvimento , Proteínas de Membrana/fisiologia , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Cotilédone/metabolismo , Estiolamento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Hipocótilo/metabolismo , Proteínas de Membrana/genética , Filogenia , Raízes de Plantas/metabolismo , Alinhamento de Sequência , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Perturbation of cellulose synthesis in plants triggers stress responses, including growth retardation, mediated by the cell wall integrity-sensing receptor-like kinase (RLK) THESEUS1 (THE1). The analysis of two alleles carrying T-DNA insertions at comparable positions has led to conflicting conclusions concerning the impact of THE1 signaling on growth. Here we confirm that, unlike the1-3 and other the1 alleles in which cellular responses to genetic or pharmacological inhibition of cellulose synthesis are attenuated, the1-4 showed enhanced responses, including growth inhibition, ectopic lignification, and stress gene expression. Both the1-3 and the1-4 express a transcript encoding a predicted membrane-associated truncated protein lacking the kinase domain. However, the1-3, in contrast to the1-4, strongly expresses antisense transcripts, which are expected to prevent the expression of the truncated protein as suggested by the genetic interactions between the two alleles. Seedlings overexpressing such a truncated protein react to isoxaben treatment similarly to the1-4 and the full-length THE overexpressor. We conclude that the1-4 is a hypermorphic allele; that THE1 signaling upon cell wall damage has a negative impact on cell expansion; and that caution is required when interpreting the phenotypic effects of T-DNA insertions in RLK genes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/citologia , Parede Celular/metabolismo , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Alelos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Benzamidas/farmacologia , Parede Celular/genética , Celulose/biossíntese , DNA Bacteriano , Regulação da Expressão Gênica de Plantas , Genes Dominantes , Lignina/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Transdução de SinaisRESUMO
In higher plants, cellulose is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs). Arabidopsis thaliana GH9A1/KORRIGAN1 is a membrane-bound, family 9 glycosyl hydrolase that is important for cellulose synthesis in both primary and secondary cell walls. Most previously identified korrigan1 mutants show severe phenotypes such as embryo lethality; therefore, the role of GH9A1 in cellulose synthesis remains unclear. Here, we report a novel A577V missense mutation, designated jiaoyao1 (jia1), in the second of the glycosyl hydrolase family 9 active site signature motifs in GH9A1. jia1 is defective in cell expansion in dark-grown hypocotyls, roots, and adult plants. Consistent with its defect in cell expansion, this mutation in GH9A1 resulted in reduced cellulose content and reduced CSC velocity at the plasma membrane. Green fluorescent protein-GH9A1 is associated with CSCs at multiple locations, including the plasma membrane, Golgi, trans-Golgi network, and small CESA-containing compartments or microtubule-associated cellulose synthase compartments, indicating a tight association between GH9A1 and CSCs. GH9A1A577V abolishes the endoglucanase activity of GH9A1 in vitro but does not affect its interaction with CESAs in vitro, suggesting that endoglucanase activity is important for cellulose synthesis. Interestingly, jia1 results in both cellulose microfibril and microtubule disorganization. Our study establishes the important role of endoglucanase in cellulose synthesis and cellulose microfibril organization in plants.
RESUMO
While the molecular basis for cytokinin action is quite well understood in flowering plants, little is known about the cytokinin signal transduction in early diverging land plants. The genome of the bryophyte Physcomitrella patens (Hedw.) B.S. encodes three classical cytokinin receptors, the CHASE domain-containing histidine kinases, CHK1, CHK2, and CHK3. In a complementation assay with protoplasts of receptor-deficient Arabidopsis thaliana as well as in cytokinin binding assays, we found evidence that CHK1 and CHK2 receptors can function in cytokinin perception. Using gene targeting, we generated a collection of CHK knockout mutants comprising single (Δchk1, Δchk2, Δchk3), double (Δchk1,2, Δchk1,3, Δchk2,3), and triple (Δchk1,2,3) mutants. Mutants were characterized for their cytokinin response and differentiation capacities. While the wild type did not grow on high doses of cytokinin (1 µM benzyladenine), the Δchk1,2,3 mutant exhibited normal protonema growth. Bud induction assays showed that all three cytokinin receptors contribute to the triggering of budding, albeit to different extents. Furthermore, while the triple mutant showed no response in this bioassay, the remaining mutants displayed budding responses in a diverse manner to different types and concentrations of cytokinins. Determination of cytokinin levels in mutants showed no drastic changes for any of the cytokinins; thus, in contrast to Arabidopsis, revealing only small impacts of cytokinin signaling on homeostasis. In summary, our study provides a first insight into the molecular action of cytokinin in an early diverging land plant and demonstrates that CHK receptors play an essential role in bud induction and gametophore development.
Assuntos
Bryopsida/metabolismo , Citocininas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Adaptação Fisiológica , Bioensaio , Bryopsida/efeitos dos fármacos , Bryopsida/genética , Butadienos/farmacologia , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Hemiterpenos/farmacologia , Mutação/genética , Pentanos/farmacologia , Fenótipo , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
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
Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Peptídeos/metabolismo , Plantas/metabolismo , Parede Celular/metabolismo , Raízes de Plantas/metabolismoRESUMO
Imbibed Arabidopsis (Arabidopsis thaliana) seeds are encapsulated by mucilage that is formed of hydrated polysaccharides released from seed coat epidermal cells. The mucilage is structured with water-soluble and adherent layers, with cellulose present uniquely in an inner domain of the latter. Using a reverse-genetic approach to identify the cellulose synthases (CESAs) that produce mucilage cellulose, cesa5 mutants were shown to be required for the correct formation of these layers. Expression of CESA5 in the seed coat was specific to epidermal cells and coincided with the accumulation of mucilage polysaccharides in their apoplast. Analysis of sugar composition showed that although total sugar composition or amounts were unchanged, their partition between layers was different in the mutant, with redistribution from adherent to water-soluble mucilage. The macromolecular characteristics of the water-soluble mucilage were also modified. In accordance with a role for CESA5 in mucilage cellulose synthesis, crystalline cellulose contents were reduced in mutant seeds and birefringent microfibrils were absent from adherent mucilage. Although the mucilage-modified5 mutant showed similar defects to cesa5 in the distribution of sugar components between water-soluble and adherent mucilage, labeling of residual adherent mucilage indicated that cesa5 contained less cellulose and less pectin methyl esterification. Together, the results demonstrate that CESA5 plays a major and essential role in cellulose production in seed mucilage, which is critical for the establishment of mucilage structured in layers and domains.
Assuntos
Adesivos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Celulose/biossíntese , Glucosiltransferases/metabolismo , Sementes/enzimologia , Adesividade , Alelos , Arabidopsis/citologia , Arabidopsis/ultraestrutura , Metabolismo dos Carboidratos , Diferenciação Celular , Cristalização , Substâncias Macromoleculares/metabolismo , Monossacarídeos/metabolismo , Mutação/genética , Fenótipo , Epiderme Vegetal/citologia , Epiderme Vegetal/enzimologia , Epiderme Vegetal/ultraestrutura , Sementes/citologia , Sementes/ultraestrutura , Solubilidade , Coloração e Rotulagem , ÁguaRESUMO
The de-methylesterification of the pectic polysaccharide homogalacturonan (HG) by pectin methylesterases (PMEs) is a critical step in the control of plant cell expansion and morphogenesis. Plants have large gene families encoding PMEs but also PME inhibitors (PMEIs) with differ in their biochemical properties. The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate pectin methylesterase activity in studies assessing its role in the control of morphogenesis. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity at acidic pH in a variety of cell wall extracts and in purified PME preparations, but does not affect the much stronger PME activity at neutral pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, growing Arabidopsis thaliana seedlings in the presence of purified PMEI3 caused a dose-dependent inhibition of root growth associated with the overall inhibition of HG de-methylesterification of the root surface. This suggests an essential in vivo role for PME activity at acidic pH in HG de-methylesterification and growth control. These results show that purified recombinant PMEI3 is a powerful tool to study the connection between pectin de-methylesterification and cell expansion.
RESUMO
N-linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N-linked glycosylation shares a common pathway with assembly of a dolichol-linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an alpha-1,2-mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol-linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N-glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low-level accumulation of Man(3)GlcNAc(2) and Man(4)GlcNAc(2) glycans, structures that are seldom detected in wild-type plants. In addition, the lew3 mutant has low levels of normal high-mannose-type glycans, but increased levels of complex-type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N-glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild-type. These results demonstrate that protein N-glycosylation plays crucial roles in plant development and the response to abiotic stresses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Parede Celular/metabolismo , Manosiltransferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Celulose/biossíntese , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes Essenciais , Teste de Complementação Genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Manosiltransferases/genética , Dados de Sequência Molecular , Transpiração Vegetal , Regiões Promotoras Genéticas , RNA de Plantas/genética , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Xilema/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
Plant cell walls are highly dynamic and heterogeneous structures, which vary between cell types, growth stages but also between microdomains within a single cell wall. In this review, we summarize the imaging techniques using fluorescent tags that are currently being used and which should in the coming years revolutionize our understanding of the dynamics of cell wall architecture and the cellular processes involved in the synthesis of cell wall components.
RESUMO
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
Plant growth and development depend on anisotropic cell expansion. Cell wall yielding provides the driving force for cell expansion, and is regulated in part by the oriented deposition of cellulose microfibrils around the cell. Our current understanding of anisotropic cell expansion combines hypotheses generated by more than 50 years of research. Here, we discuss the evolving views of researchers in the field of cellulose synthesis, and highlight several unresolved questions. Recent results using live-cell imaging have illustrated novel roles for cortical microtubules in cellulose synthesis, and further research using these approaches promises to reveal exciting links between the cytoskeleton, intracellular trafficking, and anisotropic growth.
Assuntos
Células/ultraestrutura , Fenômenos Fisiológicos Vegetais , Anisotropia , Parede Celular/fisiologia , Parede Celular/ultraestrutura , Celulose/química , Microfibrilas/fisiologia , Microfibrilas/ultraestrutura , Microtúbulos/fisiologia , Microtúbulos/ultraestruturaRESUMO
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.