EXO70A1-mediated vesicle trafficking is critical for tracheary element development in Arabidopsis.

Exocysts are highly conserved octameric complexes that play an essential role in the tethering of Golgi-derived vesicles to target membranes in eukaryotic organisms. Genes encoding the EXO70 subunit are highly duplicated in plants. Based on expression analyses, we proposed previously that individual EXO70 members may provide the exocyst with functional specificity to regulate cell type- or cargo-specific exocytosis, although direct evidence is not available. Here, we show that, as a gene expressed primarily during tracheary element (TE) development, EXO70A1 regulates vesicle trafficking in TE differentiation in Arabidopsis thaliana. Mutations of EXO70A1 led to aberrant xylem development, producing dwarfed and nearly sterile plants with very low fertility, reduced cell expansion, and decreased water potential and hydraulic transport. Grafting of a mutant shoot onto wild-type rootstock rescued most of these aboveground phenotypes, while grafting of a wild-type shoot to the mutant rootstock did not rescue the short root hair phenotype, consistent with the role of TEs in hydraulic transport from roots to shoots. Histological analyses revealed an altered pattern of secondary cell wall thickening and accumulation of large membrane-bound compartments specifically in developing TEs of the mutant. We thus propose that EXO70A1 functions in vesicle trafficking in TEs to regulate patterned secondary cell wall thickening.

Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation.

The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.

Arabidopsis VILLIN2 and VILLIN3 are required for the generation of thick actin filament bundles and for directional organ growth.

In plant cells, actin filament bundles serve as tracks for myosin-dependent organelle movement and play a role in the organization of the cytoplasm. Although virtually all plant cells contain actin filament bundles, the role of the different actin-bundling proteins remains largely unknown. In this study, we investigated the role of the actin-bundling protein villin in Arabidopsis (Arabidopsis thaliana). We used Arabidopsis T-DNA insertion lines to generate a double mutant in which VILLIN2 (VLN2) and VLN3 transcripts are truncated. Leaves, stems, siliques, and roots of vln2 vln3 double mutant plants are twisted, which is caused by local differences in cell length. Microscopy analysis of the actin cytoskeleton showed that in these double mutant plants, thin actin filament bundles are more abundant while thick actin filament bundles are virtually absent. In contrast to full-length VLN3, truncated VLN3 lacking the headpiece region does not rescue the phenotype of the vln2 vln3 double mutant. Our results show that villin is involved in the generation of thick actin filament bundles in several cell types and suggest that these bundles are involved in the regulation of coordinated cell expansion.

Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules.

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.

Golgi body motility in the plant cell cortex correlates with actin cytoskeleton organization.

The actin cytoskeleton is involved in the transport and positioning of Golgi bodies, but the actin-based processes that determine the positioning and motility behavior of Golgi bodies are not well understood. In this work, we have studied the relationship between Golgi body motility behavior and actin organization in intercalary growing root epidermal cells during different developmental stages. We show that in these cells two distinct actin configurations are present, depending on the developmental stage. In small cells of the early root elongation zone, fine filamentous actin (F-actin) occupies the whole cell, including the cortex. In larger cells in the late elongation zone that have almost completed cell elongation, actin filament bundles are interspersed with areas containing this fine F-actin and areas without F-actin. Golgi bodies in areas with the fine F-actin exhibit a non-directional, wiggling type of motility. Golgi bodies in areas containing actin filament bundles move up to 7 µm s⁻¹. Since the motility of Golgi bodies changes when they enter an area with a different actin configuration, we conclude that the type of movement depends on the actin organization and not on the individual organelle. Our results show that the positioning of Golgi bodies depends on the local actin organization.

Flexibility contra stiffness: the phragmoplast as a physical barrier for beads but not for vesicles.

In plant cells, Golgi vesicles are transported to the division plane to fuse with each other, forming the cell plate, the initial membrane-bordered cell wall separating daughter cells. Vesicles, but not organelles, move through the phragmoplast, which consists of two opposing cylinders of microtubules and actin filaments, interlaced with endoplasmic reticulum membrane. To study physical aspects of this transport/inhibition process, we microinjected fluorescent synthetic 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol (DOPG) vesicles and polystyrene beads into Tradescantia virginiana stamen hair cells. The phragmoplast was nonselective for DOPG vesicles of a size up to 150 nm in diameter but was a physical barrier for polystyrene beads having a diameter of 20 and 40 nm and also when beads were coated with the same DOPG membrane. We conclude that stiffness is a parameter for vesicle transit through the phragmoplast and discuss that cytoskeleton configurations can physically block such transit.

Expression and functional analyses of EXO70 genes in Arabidopsis implicate their roles in regulating cell type-specific exocytosis.

During exocytosis, Golgi-derived vesicles are tethered to the target plasma membrane by a conserved octameric complex called the exocyst. In contrast to a single gene in yeast and most animals, plants have greatly increased number of EXO70 genes in their genomes, with functions very much unknown. Reverse transcription-polymerase chain reactions were performed on all 23 EXO70 genes in Arabidopsis (Arabidopsis thaliana) to examine their expression at the organ level. Cell-level expression analyses were performed using transgenic plants carrying ß-glucuronidase reporter constructs, showing that EXO70 genes are primarily expressed in potential exocytosis-active cells such as tip-growing and elongating cells, developing xylem elements, and guard cells, whereas no expression was observed in cells of mature organs such as well-developed leaves, stems, sepals, and petals. Six EXO70 genes are expressed in distinct but partially overlapping stages during microspore development and pollen germination. A mutation in one of these genes, EXO70C1 (At5g13150), led to retarded pollen tube growth and compromised male transmission. This study implies that multiplications of EXO70 genes may allow plants to acquire cell type- and/or cargo-specific regulatory machinery for exocytosis.

Caspase inhibitors affect the kinetics and dimensions of tracheary elements in xylogenic Zinnia (Zinnia elegans) cell cultures.

BACKGROUND: The xylem vascular system is composed of fused dead, hollow cells called tracheary elements (TEs) that originate through trans-differentiation of root and shoot cambium cells. TEs undergo autolysis as they differentiate and mature. The final stage of the formation of TEs in plants is the death of the involved cells, a process showing some similarities to programmed cell death (PCD) in animal systems. Plant proteases with functional similarity to proteases involved in mammalian apoptotic cell death (caspases) are suggested as an integral part of the core mechanism of most PCD responses in plants, but participation of plant caspase-like proteases in TE PCD has not yet been documented. RESULTS: Confocal microscopic images revealed the consecutive stages of TE formation in Zinnia cells during trans-differentiation. Application of the caspase inhibitors Z-Asp-CH2-DCB, Ac-YVAD-CMK and Ac-DEVD-CHO affected the kinetics of formation and the dimensions of the TEs resulting in a significant delay of TE formation, production of larger TEs and in elimination of the 'two-wave' pattern of TE production. DNA breakdown and appearance of TUNEL-positive nuclei was observed in xylogenic cultures and this was suppressed in the presence of caspase inhibitors. CONCLUSIONS: To the best of our knowledge this is the first report showing that caspase inhibitors can modulate the process of trans-differentiation in Zinnia xylogenic cell cultures. As caspase inhibitors are closely associated with cell death inhibition in a variety of plant systems, this suggests that the altered TE formation results from suppression of PCD. The findings presented here are a first step towards the use of appropriate PCD signalling modulators or related molecular genetic strategies to improve the hydraulic properties of xylem vessels in favour of the quality and shelf life of plants or plant parts.

Probing cytoplasmic organization and the actin cytoskeleton of plant cells with optical tweezers.

In interphase plant cells, the actin cytoskeleton is essential for intracellular transport and organization. To fully understand how the actin cytoskeleton functions as the structural basis for cytoplasmic organization, both molecular and physical aspects of the actin organization have to be considered. In the present review, we discuss literature that gives an insight into how cytoplasmic organization is achieved and in which actin-binding proteins have been identified that play a role in this process. We discuss how physical properties of the actin cytoskeleton in the cytoplasm of live plant cells, such as deformability and elasticity, can be probed by using optical tweezers. This technique allows non-invasive manipulation of cytoplasmic organization. Optical tweezers, integrated in a confocal microscope, can be used to manipulate cytoplasmic organization while studying actin dynamics. By combining this with mutant studies and drug applications, insight can be obtained about how the physical properties of the actin cytoskeleton, and thus the cytoplasmic organization, are influenced by different cellular processes.

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers.

Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

Biosynthesis of callose and cellulose by detergent extracts of tobacco cell membranes and quantification of the polymers synthesized in vitro.

The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca(2+) and 8 mM Mg(2+). Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 microm in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

The plant exocyst.

The exocyst is an octameric vesicle tethering complex that functions upstream of SNARE mediated exocytotic vesicle fusion with the plasma membrane. All proteins in the complex have been conserved during evolution, and genes that encode the exocyst subunits are present in the genomes of all plants investigated to date. Although the plant exocyst has not been studied in great detail, it is likely that the basic function of the exocyst in vesicle tethering is conserved. Nevertheless, genomic and genetic studies suggest that the exocyst complex in plants may have more diversified roles than that in budding yeast. In this review, we compare the knowledge about the exocyst in plant cells to the well-studied exocyst in budding yeast, in order to explore similarities and differences in expression and function between these organisms, both of which have walled cells.

Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space.

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant's final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March-April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.

Establishing in vitro Zinnia elegans cell suspension culture with high tracheary element differentiation.

The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and for studying cellulose synthesis. There is, however, the problem of non-uniformity and significant variations in the yields of TEs (%TE). One possible cause for this variability in the %TE could be the lack of a standardized experimental protocol in various research laboratories for establishing the Zinnia culture. Mesophyll cells isolated from the first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were cultured in vitro and differentiated into TEs. The xylogenic culture medium was supplied with 1mg/l each of benzylaminopurine (BA) and alpha-naphthalene acetic acid (NAA). Application of this improved culture method resulted in stable and reproducible amounts of TE as high as 76% in the Zinnia culture. The increase was mainly due to conditioning of the mesophyll cell culture and adjustments of the phytohormonal balance in the cultures. Also, certain biochemical and cytological methods have been shown to reliably monitor progress of TE differentiation. We conclude that, with the adoption of current improvement in the xylogenic Z. elegans culture, higher amounts of tracheary elements can be produced. This successful outcome raises the potential of the Zinnia system as a suitable model for cellulose and xylogenesis research.

Microtubules and cellulose microfibrils: how intimate is their relationship?

The recent visualization of the motion of fluorescently labeled cellulose synthase complexes by Alexander Paredez and colleagues heralds the start of a new era in the science of the plant cell wall. Upon drug-induced complete depolymerization, the movement of the complexes does not become disordered but instead establishes an apparently self-organized novel pattern. The ability to label complexes in vivo has provided us with the ideal tool for tackling the intriguing question of the underlying default mechanisms at play.

CLASP stabilization of plus ends created by severing promotes microtubule creation and reorientation.

Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP's function in promoting microtubule amplification by severing and array reorientation.

Actin based processes that could determine the cytoplasmic architecture of plant cells.

Actin polymerisation can generate forces that are necessary for cell movement, such as the propulsion of a class of bacteria, including Listeria, and the protrusion of migrating animal cells. Force generation by the actin cytoskeleton in plant cells has not been studied. One process in plant cells that is likely to depend on actin-based force generation is the organisation of the cytoplasm. We compare the function of actin binding proteins of three well-studied mammalian models that depend on actin-based force generation with the function of their homologues in plants. We predict the possible role of these proteins, and thus the role of actin-based force generation, in the production of cytoplasmic organisation in plant cells.

Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation.

The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment.

The cytoskeleton in plant cell growth: lessons from root hairs.

In this review, we compare expansion of intercalary growing cells, in which growth takes place over a large surface, and root hairs, where expansion occurs at the tip only. Research that pinpoints the role of the cytoskeleton and the cytoplasmic free calcium in both root hairs and intercalary growing cells is reviewed. From the results of that research, we suggest experiments to be carried out on intercalary growing cells to test our hypotheses on plant cell expansion. Our main hypothesis is that instability of the cortical actin cytoskeleton determines the location where expansion takes place and the amount of expansion. Contents Summary 409 I. How do plant cells expand their surface? 409 II. Immunolocalization of epitopes in fixed root hairs for light-microscopy 410 III. The cytoskeleton in growing root hairs 412 1. Microtubules 412 2. Actin filaments 413 3. Free cytoplasmic calcium concentration 413 IV. The role of cytoskeletal elements and cytoplasmic free alcium in intercalary expanding root cells 414 1. Microtubules 414 2. Actin filaments 415 3. Free cytoplasmic calcium concentration 416 Acknowledgements 416 References 416.