ABSTRACT
A new study provides insight into microtubule turnover during plant cell division. Using clever molecular-genetic and imaging strategies, the authors demonstrate that the recently discovered CORD4 and 5 proteins associate with phragmoplast microtubules and control recruitment and activity of the microtubule-severing protein katanin.
Subject(s)
Cytokinesis , Plant Cells , Katanin , Microtubules , PlantsABSTRACT
How organisms attain their specific shapes and modify their growth patterns in response to environmental and chemical signals has been the subject of many investigations. Plant cells are at high turgor pressure and are surrounded by a rigid yet flexible cell wall, which is the primary determinant of plant growth and morphogenesis. Cellulose microfibrils, synthesized by plasma membrane-localized cellulose synthase complexes, are major tension-bearing components of the cell wall that mediate directional growth. Despite advances in understanding the genetic and biophysical regulation of morphogenesis, direct studies of cellulose biosynthesis and its impact on morphogenesis of different cell and tissue types are largely lacking. In this study, we took advantage of mutants of three primary cellulose synthase (CESA) genes that are involved in primary wall cellulose synthesis. Using field emission scanning electron microscopy, live cell imaging and biophysical measurements, we aimed to understand how the primary wall CESA complex acts during shoot apical meristem development. Our results indicate that cellulose biosynthesis impacts the mechanics and growth of the shoot apical meristem.
Subject(s)
Arabidopsis/metabolism , Cell Wall/enzymology , Cell Wall/metabolism , Glucosyltransferases/metabolism , Meristem/metabolism , Arabidopsis/enzymology , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Meristem/enzymology , Meristem/growth & developmentABSTRACT
The capacity for sustained cell division within the plant meristem is a critical determinant of organ structure and performance. This capacity is diminished in mutants lacking the microtubule-associated protein CLASP and when brassinosteroid signaling is increased. Here, we discovered that CLASP is both targeted by and promotes activity of the brassinosteroid pathway in Arabidopsis root apical meristems. We show that enhanced brassinosteroid signaling reduces CLASP transcript and protein levels, dramatically shifts microtubule organization, and reduces the number of cells in the meristem. In turn, CLASP, which tethers sorting nexin 1 vesicles to microtubules, sustains brassinosteroid signaling by fostering retrieval of endocytosed BRI1 receptors to the plasma membrane. clasp-1 null mutants have dampened brassinosteroid (BR)-mediated transcriptional activity and responses. Global transcript profiling confirmed the collapse of cell-cycle activity in clasp-1 and identified CLASP-mediated hormone crosstalk. Together, these findings reveal an unprecedented form of negative feedback supporting meristem homeostasis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Brassinosteroids/metabolism , Cell Proliferation/physiology , Meristem/physiology , Microtubule-Associated Proteins/metabolism , Plant Roots/physiology , Arabidopsis Proteins/genetics , Brefeldin A/pharmacology , Cloning, Molecular , Dinitrobenzenes/pharmacology , Down-Regulation , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/physiology , Microtubule-Associated Proteins/genetics , Microtubules , Signal Transduction , Sulfanilamides/pharmacologyABSTRACT
Cellulose synthesis at the plasma membrane is a critical process in plant growth and development. The displacement of cellulose synthase complexes (CSCs) by the rigid cellulose polymers they produce is a measure of enzyme activity. Connections between cortical microtubules and CSCs have been identified but it remains unclear how these affect CSC displacement speed. In this study, we applied a high throughput automated particle tracking method using near-total internal reflection fluorescence microscopy to measure the speed of CSCs. We found CSC speeds did not vary according to their proximity to microtubules, and that inhibiting microtubule polymerization could have opposite effects on CSC speed, depending on the nature of inhibition. While CSC speed increased in the temperature-sensitive mor1-1 mutant, it decreased after treatment with the drug oryzalin. Moreover, introducing the mor1-1 mutation into the CesA1 mutant any1 increased CSC speed, suggesting that microtubule dynamics affect CSC speed by a mechanism other than Cellulose Synthase A (CesA) catalytic activity. CSC speed varied widely in a range of mutants with reduced growth anisotropy, indicating that the relationship between CSC speed and anisotropy is complex. We conclude that microtubules affect CSC speed by finely tuned mechanisms that are independent of their physical association with CSCs.
ABSTRACT
Microtubules are dynamic filaments, the assembly and disassembly of which are under precise control of various associated proteins, including motor proteins and regulatory enzymes. In Arabidopsis thaliana, two such proteins are the ARMADILLO-REPEAT KINESIN 1 (ARK1), which promotes microtubule disassembly, and the NIMA-RELATED KINASE 6 (NEK6), which has a role in organizing microtubule arrays. Previous yeast two-hybrid and in vitro pull-down assays determined that NEK6 can interact with ARK1 through the latter protein's Armadillo-repeat (ARM) cargo domain. To explore the function of the ARM domain, we generated fluorescent reporter fusion proteins to ARK1 lacking the ARM domain (ARK1ΔARM-GFP) and to the ARM domain alone (ARM-GFP). Both of these constructs strongly associated with the growing plus ends of microtubules, but only ARK1ΔARM-GFP was capable of inducing microtubule catastrophe and rescuing the ark1-1 root hair phenotype. These results indicate that neither the ARM domain nor NEK6's putative interaction with it is required for ARK1 to induce microtubule catastrophe. In further exploration of the ARK1-NEK6 relationship, we demonstrated that, despite evidence that NEK6 can phosphorylate ARK1 in vitro, the in vivo distribution and function of ARK1 were not affected by the loss of NEK6, and vice versa. Moreover, NEK6 and ARK1 were found to have overlapping but non-identical distribution on microtubules, and hormone treatments known to affect NEK6 activity did not stimulate interaction. These findings suggest that ARK1 and NEK6 function independently in microtubule dynamics and cell morphogenesis. Despite the results of this functional analysis, we found that overexpression of the ARM domain led to complete loss of NEK6 transcription, suggesting that the ARM domain might have a regulatory role in NEK6 expression.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Kinesins/metabolism , Microtubules/metabolism , NIMA-Related Kinases/metabolism , Amino Acids, Cyclic/pharmacology , Arabidopsis/cytology , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Gibberellins/pharmacology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kinesins/genetics , Microtubules/genetics , Mutation , Phosphorylation , Plants, Genetically Modified , Protein Interaction Domains and MotifsABSTRACT
Peroxules are thin protrusions from spherical peroxisomes produced under low levels of reactive oxygen species (ROS) stress. Whereas, stress mitigation favors peroxule retraction, prolongation of the ROS stress leads to the elongation of the peroxisome into a tubular form. Subsequently, the elongated form becomes constricted through the binding of proteins such as dynamin related proteins 3A and 3B and eventually undergoes fission to increase the peroxisomal population within a cell. The events that occur in the short time window between peroxule initiation and the tubulation of the entire peroxisome have not been observed in living plant cells. Here, using fluorescent protein aided live-imaging, we show that peroxules are formed after only 4 min of high light (HL) irradiation during which there is a perceptible increase in the cytosolic levels of hydrogen peroxide. Using a stable, double transgenic line of Arabidopsis thaliana expressing a peroxisome targeted YFP and a mitochondrial targeted GFP probe, we observed sustained interactions between peroxules and small, spherical mitochondria. Further, it was observed that the frequency of HL-induced interactions between peroxules and mitochondria increased in the Arabidopsis anisotropy1 mutant that has reduced cell wall crystallinity and where we show accumulation of higher H2O2 levels than wild type plants. Our observations suggest a testable model whereby peroxules act as interaction platforms for ROS-distressed mitochondria that may release membrane proteins and fission factors. These proteins might thus become easily available to peroxisomes and facilitate their proliferation for enhancing the ROS-combating capability of a plant cell.
ABSTRACT
Plants employ five DNA-dependent RNA polymerases (Pols) in transcription. One of these polymerases, Pol III, has previously been reported to transcribe 5S rRNA, tRNAs, and a number of small RNAs. However, in-depth functional analysis is complicated by the fact that knockout mutations in Pol subunits are typically lethal. Here, we report the characterization of the first known viable Pol III subunit mutant,nrpc7-1 This mutant was originally isolated from a forward genetic screen designed to identify enhancers of the autoimmune mutantsnc1, which contains a gain-of-function mutation in a nucleotide-binding leucine-rich repeat (NLR) immune receptor-encoding gene. Thenrpc7-1mutation occurs in an intron-exon splice site and results in intron retention in someNRPC7transcripts. There is a global disruption in RNA equilibrium innrpc7-1, exemplified by the altered expression of a number of RNA molecules, some of which are not reported to be transcribed by Pol III. There are developmental defects associated with the mutation, as homozygous mutant plants are dwarf, have stunted roots and siliques, and possess serrated leaves. These defects are possibly due to altered small RNA stability or activity. Additionally, thenrpc7-1mutation confers anNLR-specific alternative splicing defect that correlates with enhanced disease resistance, highlighting the importance of alternative splicing in regulating NLR activity. Altogether, these results reveal novel roles for Pol III in maintaining RNA homeostasis, adjusting the expression of a diverse suite of genes, and indirectly modulating gene splicing. Future analyses using thenrpc7-1mutant will be instrumental in examining other unknown Pol III functions.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/enzymology , Arabidopsis/genetics , Genetic Pleiotropy , Mutation/genetics , Protein Subunits/genetics , RNA Polymerase III/genetics , Arabidopsis/immunology , Arabidopsis Proteins/metabolism , Base Sequence , Cell Nucleus/metabolism , Chromosomes, Plant/genetics , Cloning, Molecular , Plant Immunity , Protein Subunits/metabolism , RNA Polymerase III/metabolism , RNA Splicing/genetics , RNA, Plant/metabolism , Subcellular Fractions/metabolismABSTRACT
Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.
Subject(s)
Arabidopsis/cytology , Fluorescent Antibody Technique/methods , Microscopy, Electron, Transmission/methods , Microtubules/ultrastructure , Cell Survival , Freezing , Glutaral , Luminescent Proteins/metabolism , Microtubules/metabolism , Osmium , Pressure , Staining and Labeling , Tissue FixationABSTRACT
Adventitious roots (AR) are post embryonic lateral organs that differentiate from non-root tissues. The understanding of the molecular mechanism which underlies their differentiation is important because of their central role in vegetative plant propagation. Here it was studied how the expression of different microtubule (MT)-associated proteins (MAPs) is affected during AR induction, and whether expression differences are dependent on MT organization itself. To examine AR formation when MTs are disturbed we used two mutants in the MT severing protein KATANIN. It was found that rate and number of AR primordium formed following IBA induction for three days was reduced in bot1-1 and bot1-7 plants. The reduced capacity to form ARs in bot1-1 was associated with altered expression of MAP-encoding genes along AR induction. While the expression of MAP65-4, MAP65-3, AURORA1, AURORA2 and TANGLED, increased in wild-type but not in bot1-1 plants, the expression of MAP65-8 and MDP25 decreased in wild type plants but not in the bot1-1 plant after two days of IBA-treatment. The expression of MOR1 was increased two days after AR induction in wild type and bot1-1 plants. To examine its expression specifically in AR primordium, MOR1 upstream regulatory sequence was isolated and cloned to regulate GFP. Expression of GFP was induced in the primary root tips and lateral roots, in the pericycle of the hypocotyls and in all stages of AR primordium formation. It is concluded that the expression of MAPs is regulated along AR induction and that reduction in KATANIN expression inhibits AR formation and indirectly influences the specific expression of some MAPs.
Subject(s)
Adenosine Triphosphatases/metabolism , Adventitia/growth & development , Arabidopsis/growth & development , Gene Expression Regulation, Plant/drug effects , Indoles/pharmacology , Microtubule-Associated Proteins/metabolism , Plant Roots/growth & development , Adenosine Triphosphatases/genetics , Adventitia/drug effects , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Katanin , Microtubule-Associated Proteins/genetics , Mutation , Plant Roots/drug effects , Promoter Regions, GeneticABSTRACT
Induction of adventitious roots (ARs) in recalcitrant plants often culminates in cell division and callus formation rather than root differentiation. Evidence is provided here to suggest that microtubules (MTs) play a role in the shift from cell division to cell differentiation during AR induction. First, it was found that fewer ARs form in the temperature-sensitive mutant mor1-1, in which the MT-associated protein MOR1 is mutated, and in bot1-1, in which the MT-severing protein katanin is mutated. In the two latter mutants, MT dynamics and form are perturbed. By contrast, the number of ARs increased in RIC1-OX3 plants, in which MT bundling is enhanced and katanin is activated. In addition, any1 plants in which cell walls are perturbed made more ARs than wild-type plants. MT perturbations during AR induction in mor1-1 or in wild-type hypocotyls treated with oryzalin led to the formation of amorphous clusters of cells reminiscent of callus. In these cells a specific pattern of polarized light retardation by the cell walls was lost. PIN1 polarization and auxin maxima were hampered and differentiation of the epidermis was inhibited. It is concluded that a fine-tuned crosstalk between MTs, cell walls, and auxin transport is required for proper AR induction.
Subject(s)
Arabidopsis/growth & development , Microtubules/physiology , Plant Roots/growth & development , Arabidopsis/drug effects , Arabidopsis/metabolism , Arabidopsis/ultrastructure , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , Cell Differentiation , Cell Division , Cell Wall/metabolism , Dinitrobenzenes/pharmacology , Indoleacetic Acids/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/physiology , Microtubules/genetics , Microtubules/metabolism , Mutation , Plant Roots/cytology , Plant Roots/metabolism , Plant Roots/ultrastructure , Sulfanilamides/pharmacology , TemperatureABSTRACT
Plants use robust mechanisms to optimize organ size to prevailing conditions. Modulating the transition from cell division to elongation dramatically affects morphology and size. Although it is well established that auxin, cytokinin and brassinosteroid mediate these transitions, recent works show that the cytoskeleton, which is normally thought to act downstream of these hormones, plays a key role in this regulatory process. In particular, the microtubule-associated protein CLASP has a dual role in meristem maintenance. CLASP modulates levels of the auxin efflux carrier PIN2 by tethering SNX1 endosomes to cortical microtubules, which in turn fine tunes auxin maxima in the root apical meristem. CLASP is also required for transfacial microtubule bundle formation at the sharp cell edges, a feature strongly associated with maintaining the capacity for further cell division.
Subject(s)
Microtubules/metabolism , Cell Division/physiology , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Meristem/metabolism , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1's function is redundant in cells other than those forming root hairs.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/metabolism , Kinesins/physiology , Microtubules/metabolism , Arabidopsis/growth & development , Arabidopsis/ultrastructure , Arabidopsis Proteins/analysis , Arabidopsis Proteins/metabolism , Binding Sites , Dinitrobenzenes/pharmacology , Kinesins/analysis , Kinesins/metabolism , Microtubules/ultrastructure , Plant Roots/growth & development , Plant Roots/metabolism , Plant Roots/ultrastructure , Protein Structure, Tertiary , Sulfanilamides/pharmacologyABSTRACT
The nuclear envelope in plant cells has long been known to be a microtubule organizing center (MTOC), but its influence on microtubule organization in the cell cortex has been unclear. Here we show that nuclear MTOC activity favors the formation of longitudinal cortical microtubule (CMT) arrays. We used green fluorescent protein (GFP)-tagged gamma tubulin-complex protein 2 (GCP2) to identify nuclear MTOC activity and GFP-tagged End-Binding Protein 1b (EB1b) to track microtubule growth directions. We found that microtubules initiate from nuclei and enter the cortex in two directions along the long axis of the cell, creating bipolar longitudinal CMT arrays. Such arrays were observed in all cell types showing nuclear MTOC activity, including root hairs, recently divided cells in root tips, and the leaf epidermis. In order to confirm the causal nature of nuclei in bipolar array formation, we displaced nuclei by centrifugation, which generated a corresponding shift in the bipolarity split point. We also found that bipolar CMT arrays were associated with bidirectional trafficking of vesicular components to cell ends. Together, these findings reveal a conserved function of plant nuclear MTOCs and centrosomes/spindle pole bodies in animals and fungi, wherein all structures serve to establish polarities in microtubule growth.
Subject(s)
Arabidopsis/metabolism , Microtubule-Organizing Center/metabolism , Arabidopsis/ultrastructure , Arabidopsis Proteins/metabolism , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cell Polarity , Centrosome/metabolism , Centrosome/ultrastructure , Cytoplasm/metabolism , Cytoplasm/ultrastructure , Genes, Reporter , Microtubule-Associated Proteins/metabolism , Microtubule-Organizing Center/ultrastructure , Microtubules/metabolism , Microtubules/ultrastructure , Plant Epidermis/metabolism , Plant Epidermis/ultrastructure , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Plant Roots/metabolism , Plant Roots/ultrastructure , Recombinant Fusion Proteins , Spindle Pole Bodies/metabolism , Spindle Pole Bodies/ultrastructure , Tubulin/metabolismABSTRACT
Giant internodal cells of characean green algae have been widely used for studying cellular physiology. This review emphasizes their significance for understanding cytoarchitecture and cytoplasmic reorganization. The cytoarchitecture of internodal cells undergoes pronounced, cytoskeleton-dependent changes during development and in response to environmental cues. Under bright light, internodes develop alternating bands of acid and alkaline pH at their surface that correlate with the differential size and abundance of cortical organelles and, in the genus Chara, with the size and distribution of convoluted plasma membrane domains known as charasomes. Wounding induces responses ranging from chloroplast detachment to deposition of wound walls. These properties and the possibility for mechanical manipulation make the internodal cell ideal for exploring plasma membrane domains, organelle interactions, vesicle trafficking, and local cell wall deposition. The significance of this model system will further increase with the application of molecular biological methods in combination with metabolomics and proteomics.
Subject(s)
Chara/cytology , Models, Biological , Chara/ultrastructure , Cytoplasm/metabolism , EnvironmentABSTRACT
Proteins detrimental to endoplasmic reticulum (ER) morphology need to be efficiently exported. Here, we identify two mechanisms that control trafficking of Arabidopsis thalianaGLL23, a 43 kDa GDSL-like lipase implicated in glucosinolate metabolism through its association with the ß-glucosidase myrosinase. Using immunofluorescence, we identified two mutants that showed aberrant accumulation of GLL23: large perinuclear ER aggregates in the nuclear cage (nuc) mutant; and small compartments contiguous with the peripheral ER in the cytoplasmic bodies (cyb) mutant. Live imaging of fluorescently tagged GLL23 confirmed its presence in the nuc and cyb compartments, but lack of fluorescent signals in the wild-type plants suggested that GLL23 is normally post-translationally modified for ER export. NUC encodes the MVP1/GOLD36/ERMO3 myrosinase-associated protein, previously shown to have vacuolar distribution. CYB is an ER and Golgi-localized p24 type I membrane protein component of coat protein complex (COP) vesicles, animal and yeast homologues of which are known to be involved in selective cargo sorting for ER-Golgi export. Without NUC, GLL23 accumulates in the ER this situation suggests that NUC is in fact active in the ER. Without CYB, both GLL23 and NUC were found to accumulate in cyb compartments, consistent with a role for NUC in GLL23 processing and indicated that GLL23 is the likely sorting target of the CYB p24 protein.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Alleles , Arabidopsis/genetics , Arabidopsis/ultrastructure , Arabidopsis Proteins/genetics , COP-Coated Vesicles/metabolism , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Cytoplasm/metabolism , Endoplasmic Reticulum/metabolism , Gene Expression , Genes, Reporter , Golgi Apparatus/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Electron , Mutation , Protein Transport , Proteomics , Seedlings/genetics , Seedlings/metabolism , Seedlings/ultrastructureABSTRACT
Cellulose microfibrils are critical for plant cell specialization and function. Recent advances in live cell imaging of fluorescently tagged cellulose synthases to track cellulose synthesis have greatly advanced our understanding of cellulose biosynthesis. Nevertheless, cellulose deposition patterns remain poorly described in many cell types, including those in the process of division or differentiation. In this study, we used field emission scanning electron microscopy analysis of cryo-planed tissues to determine the arrangement of cellulose microfibrils in various faces of cells undergoing cytokinesis or specialized development, including cell types in which cellulose cannot be imaged by conventional approaches. In dividing cells, we detected microfibrillar meshworks in the cell plates, consistent with the concentration at the cell plate of cellulose synthase complexes, as detected by fluorescently tagged CesA6. We also observed a loss of parallel cellulose microfibril orientation in walls of the mother cell during cytokinesis, which corresponded with the loss of fluorescently tagged cellulose synthase complexes from these surfaces. In recently formed guard cells, microfibrils were randomly organized and only formed a highly ordered circumferential pattern after pore formation. In pit fields, cellulose microfibrils were arranged in circular patterns around plasmodesmata. Microfibrils were random in most cotyledon cells except the epidermis and were parallel to the growth axis in trichomes. Deposition of cellulose microfibrils was spatially delineated in metaxylem and protoxylem cells of the inflorescence stem, supporting recent studies on microtubule exclusion mechanisms.
Subject(s)
Arabidopsis/cytology , Arabidopsis/metabolism , Cellulose/metabolism , Cell Differentiation/physiology , Cytokinesis , Glucosyltransferases/metabolism , Microscopy, Electron, Scanning , Plant Cells/metabolism , Plant Roots/cytology , Plant Roots/metabolism , Plant Stems/cytology , Plant Stems/metabolismABSTRACT
Movement of secretory organelles is a fascinating yet largely mysterious feature of eukaryotic cells. Microtubule-based endomembrane and organelle motility utilizing the motor proteins dynein and kinesin is commonplace in animal cells. In contrast, it has been long accepted that intracellular motility in plant cells is predominantly driven by myosin motors dragging organelles and endomembrane-bounded cargo along actin filament bundles. Consistent with this, defects in the acto-myosin cytoskeleton compromise plant growth and development. Recent findings, however, challenge the actin-centric view of the motility of critical secretory organelles and distribution of associated protein machinery. In this review, we provide an overview of the current knowledge on actin-mediated organelle movement within the secretory pathway of plant cells, and report on recent and exciting findings that support a critical role of microtubules in plant cell development, in fine-tuning the positioning of Golgi stacks, as well as their involvement in cellulose synthesis and auxin polar transport. These emerging aspects of the biology of microtubules highlight adaptations of an ancestral machinery that plants have specifically evolved to support the functioning of the acto-myosin cytoskeleton, and mark new trends in our global appreciation of the complexity of organelle movement within the plant secretory pathway.
Subject(s)
Cell Membrane/ultrastructure , Cytoskeleton/ultrastructure , Microtubules/physiology , Plant Cells/ultrastructure , Actin Cytoskeleton/physiology , Arabidopsis/cytology , Cell Membrane/metabolism , Cytoskeleton/metabolism , Endosomes/metabolism , Glucosyltransferases/metabolism , Indoleacetic Acids/metabolism , Kinesins/metabolism , Microtubule-Organizing Center/metabolism , Microtubules/ultrastructure , Myosins/metabolism , Organelles/metabolism , Plant Cells/physiology , Nicotiana/cytologyABSTRACT
Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1's permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature.
Subject(s)
Arabidopsis/genetics , Glucosyltransferases/genetics , Alleles , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Catalytic Domain/genetics , Cell Wall/genetics , Cell Wall/metabolism , Cellulose/genetics , Cellulose/metabolism , Mutation , Phenotype , Plant Roots/genetics , Plant Roots/metabolism , Plant Stems/genetics , Plant Stems/metabolism , Seeds/genetics , Seeds/metabolism , TemperatureABSTRACT
Polarized movement of auxin generates concentration gradients within plant tissues to control cell division patterns and growth direction by modulating microtubule organization. In this study, we identify a reverse mechanism, wherein microtubules influence polar auxin transport. We show that the microtubule-associated protein CLASP interacts with the retromer component sorting nexin 1 (SNX1) to mediate an association between endosomes and microtubules. clasp-1 null mutants display aberrant SNX1 endosomes, as do wild-type plants treated with microtubule-depolymerizing drugs. Consistent with SNX1's role in trafficking of the auxin efflux carrier PIN-FORMED2 (PIN2), clasp-1 mutant plants have enhanced PIN2 degradation, and PIN2 movement to lytic vacuoles is rapidly induced by depolymerization of microtubules. clasp-1 mutants display aberrant auxin distribution and exhibit numerous auxin-related phenotypes. In addition to mechanistically linking auxin transport and microtubules, our data identify a ubiquitous endosome-microtubule association in plants.