Associate Professor David Collings

To understand plant growth and development, it is often necessary to investigate the organization of plant cells and plant cell walls. Plant cell walls are often fluorescently labeled for confocal imaging with the dye propidium iodide using a pseudo-Schiff reaction. This reaction binds free amine groups on dye molecules to aldehyde groups on cellulose that result from oxidation with periodic acid. We tested a range of fluorescent dyes carrying free amine groups for their ability to act as pseudo-Schiff reagents. Using the low-pH solution historically used for the Schiff reaction, these alternative dyes failed to label cell walls of Arabidopsis cotyledon vascular tissue as strongly as propidium iodide but replacing the acidic solution with water greatly improved fluorescence labeling. Under these conditions, rhodamine-123 provided improved staining of plant cell walls compared to propidium iodide. We also developed protocols for pseudo-Schiff labeling with ATTO 647N-amine, a dye compatible for super-resolution Stimulated Emission Depletion (STED) imaging. ATTO 647N-amine was used for super-resolution imaging of cell wall ingrowths that occur in phloem parenchyma transfer cells of Arabidopsis, structures whose small size is only slightly larger than the resolution limit of conventional confocal microscopy. Application of surface-rendering software demonstrated the increase in plasma membrane surface area as a consequence of wall ingrowth deposition and suggests that STED-based approaches will be useful for more detailed morphological analysis of wall ingrowth formation. These improvements in pseudo-Schiff labeling for conventional confocal microscopy and STED imaging will be broadly applicable for high-resolution imaging of plant cell walls.

Interlocked grain occurs when the orientation of xylem fibres oscillates, alternating between left- and right-handed spirals in successive wood layers. The cellular mechanisms giving rise to interlocked grain, thought to involve the slow rotation of fusiform initials within the vascular cambium, remain unclear. We suggest that observations of wood structure at the cellular level, but over large areas, might reveal these mechanisms. We assayed timber from several commercially important tropical angiosperms from the genus Khaya (African mahogany) that exhibit interlocked grain using X-ray computed microtomography followed by orthogonal slicing and image processing in ImageJ. Reconstructed tangential longitudinal sections were processed with the ImageJ DIRECTIONALITY plug-in to directly measure fibre orientation and showed grain deviations of more than 10¿ from vertical in both left- and right-handed directions. Grain changed at locally constant rates, separated by locations where the direction of grain change sharply reversed. Image thresholding and segmentation conducted on reconstructed cross sections allowed the identification of vessels and measurement of their location, with vessel orientations then calculated in Matlab and, independently, in recalculated tangential longitudinal sections with the DIRECTIONALITY plug-in. Vessel orientations varied more than fibre orientations, and on average deviated further from vertical than fibres at the locations where the direction of grain change reversed. Moreover, the reversal location for vessels was shifted ~400 µm towards the pith compared with the fibres, despite both cell types arising from the same fusiform initials within the vascular cambium. We propose a simple model to explain these distinct grain patterns. Were an auxin signal to control both the reorientation of cambial initials, as well as coordinating the end-on-end differentiation and linkage of xylem vessel elements, then it would be possible for fibres and vessels to run at subtly different angles, and to show different grain reversal locations.

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na+]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na+]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na+]i, whereas no significant differences in branchial [Na+]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na+]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.

The velamen is a multicelled epidermal layer found in orchid roots. This layer consists of dead cells that have helical secondary wall thickenings and is usually thicker in epiphytic orchids with aerial roots than terrestrial orchids, consistent with the velamen¿s role in water capture. While velamen struture has been widely studied as a taxonomic character, few studies have looked at its development. We used confocal microscopy to investigate cell wall formation in the epiphytic orchid Miltoniopsis. Development of the helical thickenings begins with microtubule-dependant cellulose deposition but as the secondary wall ridges develop, parallel microtubules bind either side of the ridge. Cellulose organization was also investigated using pontamine staining which showed bifluorescence of the cellulose strands. These data may provide insight into the mechanical efficiency of the velamen layer in orchid roots.

Injury to the anterior thalamic nuclei (ATN) may affect both hippocampus and retrosplenial cortex thus explaining some parallels between diencephalic and medial temporal lobe amnesias. We found that standard-housed rats with ATN lesions, compared with standard-housed controls, showed reduced spine density in hippocampal CA1 neurons (basal dendrites, -11.2%; apical dendrites, -9.6%) and in retrospenial granular b cortex (Rgb) neurons (apical dendrites, -20.1%) together with spatial memory deficits on cross maze and radial-arm maze tasks. Additional rats with ATN lesions were also shown to display a severe deficit on spatial working memory in the cross-maze, but subsequent enriched housing ameliorated their performance on both this task and the radial-arm maze. These enriched rats with ATN lesions also showed recovery of both basal and apical CA1 spine density to levels comparable to that of the standard-housed controls, but no recovery of Rgb spine density. Inspection of spine types in the CA1 neurons showed that ATN lesions reduced the density of thin spines and mushroom spines, but not stubby spines; while enrichment promoted recovery of thin spines. Comparison with enriched rats that received pseudo-training, which provided comparable task-related experience, but no explicit spatial memory training, suggested that basal CA1 spine density in particular was associated with spatial learning and memory performance. Distal pathology in terms of reduced integrity of hippocampal and retrosplenial microstructure provides clear support for the influence of the ATN lesions on the extended hippocampal system. The reversal by postoperative enrichment of this deficit in the hippocampus but not the retrosplenial cortex may indicate region-specific mechanisms of recovery after ATN injury.

Turgor regulation is the process by which walled organisms alter their internal osmotic potential to adapt to osmotic changes in the environment. Apart from a few studies on freshwater oomycetes, the ability of stramenopiles to turgor regulate has not been investigated. In this study, turgor regulation and growth were compared in two species of the stramenopile alga Vaucheria, Vaucheria erythrospora isolated from an estuarine habitat, and Vaucheria repens isolated from a freshwater habitat. Species were identified using their rbcL sequences and respective morphologies. Using a single cell pressure probe to directly measure turgor in Vaucheria after hyperosmotic shock, V. erythrospora was found to recover turgor after a larger shock than V. repens. Threshold shock values for this ability were >0.5 MPa for V. erythrospora and <0.5 MPa for V. repens. Recovery was more rapid in V. erythrospora than V. repens after comparable shocks. Turgor recovery in V. erythrospora was inhibited by Gd3+ and TEA, suggesting a role for mechanosensitive channels, nonselective cation channels, and K+ channels in the process. Growth studies showed that V. erythrospora was able to grow over a wider range of NaCl concentrations. These responses may underlie the ability of V. erythrospora to survive in an estuarine habitat and restrict V. repens to freshwater. The fact that both species can turgor regulate may indicate a fundamental difference between members of the Stramenopila, as research to date on oomycetes suggests they are unable to turgor regulate. © 2013 Phycological Society of America.

Viruses of the genus Mastrevirus (family Geminiviridae) are transmitted by leafhoppers and infect either mono- or dicotyledonous plants. Here we have determined the full length sequences of 49 dicot-infecting mastrevirus isolates sampled in Australia, Eritrea, India, Iran, Pakistan, Syria, Turkey and Yemen. Comprehensive analysis of all available dicot-infecting mastrevirus sequences showed the diversity of these viruses in Australia to be greater than in the rest of their known range, consistent with earlier studies, and that, in contrast with the situation in monocot-infecting mastreviruses, detected inter-species recombination events outnumbered intra-species recombination events. Consistent with Australia having the greatest diversity of known dicot-infecting mastreviruses phylogeographic analyses indicating the most plausible scheme for the spread of these viruses to their present locations, suggest that most recent common ancestor of these viruses is likely nearer Australia than it is to the other regions investigated. © 2013 Elsevier Inc.

Using confocal microscopy, tracheid wall organisation was investigated with pontamine fast scarlet 4B (P4B), a cellulose-specific stain that fluoresced red following green excitation. P4B fluorescence was present throughout unlignified walls (cambium, ray cells, resin canals and parenchyma cells) and in two concentric bands around opposite and compression wood tracheids. Scanning electron micrographs demonstrated these bands as the S1 and S3 layers of normal wood, and the S1 and inner S2 layers of compression wood. Fluorescence was also strongly dependent on the polarisation of the excitation light, a phenomenon referred to as bifluorescence. Compared to two other cell wall stains, Congo red and calcofluor white, P4B showed a higher specificity for the S1 and S3 layers and increased bifluorescence. These results suggest that P4B is an ideal tool with which to investigate the orientation of cellulose microfibrils in the S1 and S3 layers of the tracheid wall. © 2012 Springer-Verlag.

The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system. © 2013 Springer Science+Business Media, LLC.

¿ Introduction: There has been an increasing interest in very early selection of radiata pine to reduce the breeding cycle for solid wood products. For such selection, new approaches are required to assess wood quality in wood from very young stems. ¿ Methods: Nursery seedlings of clones of radiata pine were grown in leant condition using two leaning strategies for 18-20 months. Opposite wood and compression wood were isolated from the leaning stems and tested for dynamic modulus of elasticity, density, longitudinal shrinkage, volumetric shrinkage and compression wood area using new methods evolved for testing small size samples quickly and reliably. The methods were tested for their efficiency in differentiating clones by their wood properties. ¿ Results: Leaning of stems provided distinct opposite and compression wood for testing. Automated image analysis method used for compression wood area assessment was found to be a quick and effective method for processing large number of samples from young stems. Compression wood was characterised by high basic density, high longitudinal shrinkage and low volumetric shrinkage than that of opposite wood. Acoustic velocity in opposite wood had a strong negative association with longitudinal shrinkage. The study signifies the importance of preventing mixing of opposite wood with compression wood while assessing wood quality in young stems thus making leaning a critical strategy. The comparison of wood properties of opposite wood revealed significant differences between clones. Opposite wood of the clone with the lowest dynamic modulus of elasticity exhibited the highest longitudinal shrinkage. ¿ Conclusion: Significant differences in measurable wood properties between clones suggest the prospects of early selection for solid wood products. © 2013 INRA and Springer-Verlag France.

Mycobacterium avium subsp. paratuberculosis is a bacterium sometimes found in human blood and tissue samples that may have a role in the etiology of Crohn's disease in humans. To date, however, there have been few studies examining the interactions of these bacteria with human cells. Using the THP-1 human monocytic cell line, this study shows that the uptake and trafficking of M. avium subsp. paratuberculosis in human cells are cholesterol dependent and that these bacteria localize to cholesterolrich compartments that are slow to acidify. M. avium subsp. paratuberculosis bacteria containing phagosomes stain for the late endosomal marker Rab7, but recruitment of the Rab7-interacting lysosomal protein that regulates the fusion of bacteriumcontaining phagosomes with lysosomal compartments and facilitates subsequent bacterial clearance is significantly reduced. Disruption of phagosome acidification via this mechanism may contribute to M. avium subsp. paratuberculosis persistence in human cells, but there was no evidence that internalized M. avium subsp. paratuberculosis also affects the survival of bacteria taken up during a secondary phagocytic event. © 2012, American Society for Microbiology.

Microtubule-associated proteins of the highly conserved XMAP215/Dis1 family promote both microtubule growth and shrinkage, and move with the dynamic microtubule ends. The plant homologue, MOR1, is predicted to form a long linear molecule with five N-terminal TOG domains. Within the first (TOG1) domain, the mor1-1 leucine to phenylalanine (L174F) substitution causes temperature-dependent disorganization of microtubule arrays and reduces microtubule growth and shrinkage rates. By expressing the two N-terminal TOG domains (TOG12) of MOR1, both in planta for analysis in living cells and in bacteria for in vitro microtubule-binding and polymerization assays, we determined that the N-terminal domain of MOR1 is crucial for microtubule polymer binding. Tagging TOG12 at the N-terminus interfered with its ability to bind microtubules when stably expressed in Arabidopsis or when transiently overexpressed in leek epidermal cells, and impeded polymerase activity in vitro. In contrast, TOG12 tagged at the C-terminus interacted with microtubules in vivo, rescued the temperature-sensitive mor1-1 phenotype, and promoted microtubule polymerization in vitro. TOG12 constructs containing the L174F mor1-1 point mutation caused microtubule disruption when transiently overexpressed in leek epidermis and increased the affinity of TOG12 for microtubules in vitro. This suggests that the mor1-1 mutant protein makes microtubules less dynamic by binding the microtubule lattice too strongly to support rapid plus-end tracking. We conclude from our results that a balanced microtubule affinity in the N-terminal TOG domain is crucial for the polymerase activity of MOR1. ©2012.

Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) is a multi-component, ssDNA virus, which causes widespread banana crop losses throughout tropical Africa and Australasia. We determined the full genome sequences of 12 BBTV isolates from the Kingdom of Tonga and analysed these together with previously determined BBTV sequences to show that reassortment and both inter- and intra-component recombination have all been relatively requent occurrences during BBTV evolution. We found that whereas DNA-U3 components display evidence of complex inter- and intra-component recombination, all of the South Pacific DNA-R components have a common intra-component recombinant origin spanning the replicationassociated protein gene. Altogether, the DNA-U3 and DNA-M components display a greater degree of inter-component recombination than the DNA-R, -S, -C and -M components. The breakpoint distribution of the inter-component recombination events reveals a primary recombination hotspot around the 59 side of the common region major and, in accordance with recombination hotspots detectable in related ssDNA viruses, a secondary recombination hotspot near the origin of virion-strand replication. © 2012 SGM.

Beak and feather disease virus (BFDV) is a circular ssDNA virus that causes psittacine beak and feather disease and has almost global presence. Here, we report for the first time the presence of in Nouvelle-Calédonie (New Caledonia). One hundred and sixty-eight exotic and 79 endemic birds were sampled in Nouvelle-Calédonie, 26 were found to be positive for BFDV. We characterized the full genomes of 26 isolates and phylogenetic analysis placed nine of the isolates into the BFDV-J strain, with the remaining 17 isolates from Deplanche's Rainbow Lorikeet (Trichoglossus haematodus deplanchii) forming a novel strain, BFDV-P. Of more concern was the discovery of an infected bird from the vulnerable and endemic New Caledonian Parakeet (Cyanoramphus saisseti). Our results reveal that there have been at least two introductions of BFDV into Nouvelle-Calédonie. © 2012 SGM.

Monocotyledonous plant infecting mastreviruses (family Geminiviridae) have been found in the Old World. The greatest diversity of these viruses has been found in Africa but this may simply reflect the more extensive sampling that has been done there. To provide a better understanding of mastrevirus diversity in Australia, we have sequenced the genomes of 41 virus isolates found in naturalised and native grasses and identified four new species in addition to the four previously characterised species. Two of these species, which were recovered from a single Sporobolus plant, are highly divergent and are most closely related to the African streak viruses. This, coupled with the discovery of divergent dicotyledonous plant infecting mastreviruses in Australia brings into question the hypothesis that mastreviruses may have originated in Africa. We found that the patterns of inter- and intra-species recombination and the recombination hotspots mirror those found in both their African monocot-infecting counterparts and dicot-infecting mastrevirus. © 2012 Elsevier B.V.

Dragonfly cyclovirus (DfCyV), a new species of ssDNA virus discovered using viral metagenomics in dragonflies (family Libellulidae) from the Kingdom of Tonga. Metagenomic sequences of DfCyV were similar to viruses of the recently proposed genus Cyclovirus within the family Circoviridae. Specific PCRs resulted in the recovery of 21 DfCyV genomes from three dragonfly species (Pantala flavescens, Tholymis tillarga and Diplacodes bipunctata). The 1741 nt DfCyV genomes share > 95% nucleotide identity and are classified into 11 subtypes representing a single strain. The DfCyV genomes share 48-63% genome-wide nucleotide identity with cycloviruses identified in human faecal samples. Recombination analysis revealed three recombinant DfCyV genomes, suggesting that recombination plays an important role in cyclovirus evolution. To our knowledge, this is the first report of a circular ssDNA virus identified in insects, and the data may help elucidate evolutionary links among novel Circoviridae recently identified in animals and environmental samples. © 2011 SGM.

Summary Plasmodesmata are plasma membrane-lined channels through which cytoplasmic molecules move from cell-to-cell in plants. Most plasmodesmata contain a desmotubule, a central tube of endoplasmic reticulum (ER), that connects the ER of adjacent cells. Here we demonstrate that molecules of up to 10.4 kDa in size can move between the ER lumen of neighbouring leaf trichome or epidermal cells via the desmotubule lumen. Fluorescent molecules of up to 10 kDa, microinjected into the ER of Nicotiana trichome cells, consistently moved into the ER and nuclei of neighbouring trichome cells. This movement occurred more rapidly than movement via the cytoplasmic pathway. A fluorescent 3-kDa dextran microinjected into the ER of a basal trichome cell moved into the ER and nuclei of epidermal cells across a barrier to cytoplasmic movement. We constructed a 10.4-kDa recombinant ER-lumenal reporter protein (LRP) from a fragment of the endogenous ER-lumenal binding protein AtBIP1. Following transient expression of the LRP in the ER of Tradescantia leaf epidermal cells, it often moved into the nuclear envelopes of neighbouring cells. However, green fluorescent protein targeted to the ER lumen (ER-GFP) did not move from cell to cell. We propose that the ER lumen of plant cells is continuous with that of their neighbours, and allows movement of small ER-lumenal molecules between cells. © 2011 Blackwell Publishing Ltd.

The textbook image of the plant vacuole sitting passively in the centre of the cell is not always correct. We observed vacuole dynamics in the epidermal cells of red onion (Allium cepa) bulbs, using confocal microscopy to detect autofluorescence from the pigment anthocyanin. The central vacuole was penetrated by highly mobile transvacuolar strands of cytoplasm, which were also visible in concurrent transmitted light images. Tubular vacuoles also extended from the large central vacuole and radiated through the cortical cytoplasm. These tubules were thin, having a diameter of about 1.5 µm, and were connected to the central vacuole as shown by fluorescence recovery after photobleaching (FRAP) experiments. The tubules were bounded by the tonoplast, as revealed by transient expression of green fluorescent protein (GFP) targeted to the vacuolar membrane and through labeling with the dye MDY-64. Expression of endoplasmic reticulum-targeted GFP demonstrated that the vacuolar tubules were distinct from the cortical endoplasmic reticulum. Movement of the tubular vacuoles depended on actin microfilaments, as microfilament disruption blocked tubule movement and caused their collapse into minivacuoles. The close association of the tubules with GFP-tagged actin microfilaments suggests that the tubules are associated with myosin, and that tubules likely move along microfilaments. Tubular vacuoles do not require anthocyanin for their formation, as tubules were also present in white onion cells that lack anthocyanin. The function of these tubular vacuoles remains unknown, but as they greatly increase the surface area of the tonoplast, they might increase transport rates between the cytoplasm and vacuole.

The actin cytoskeleton and associated actin-binding proteins form a complex network involved in a number of fundamental cellular processes including intracellular trafficking. In plants, both actin and myosin have been localised to plasmodesmata, and thus it is likely that other actin-binding proteins are also associated with plasmodesmata structure or function. A 75-kDa protein, enriched in plasmodesmata-rich cell wall extracts from the green alga Chara corallina, was sequenced and found to contain three peptides with similarity to the animal actin-binding protein tropomyosin. Western blot analysis with anti-tropomyosin antibodies confirmed the identity of this 75-kDa protein as a tropomyosin-like protein and further identified an additional 55-kDa protein, while immunofluorescence microscopy localised the antibodies to plasmodesmata and to the subcortical actin bundles and associated structures. The anti-tropomyosin antibodies detected a single protein at 42.5 kDa in Arabidopsis thaliana extracts and two proteins at 58.5 and 54 kDa in leek extracts, and these localised to plasmodesmata and the cell plate in A. thaliana and to plasmodesmata in leek tissue. Tropomyosin is an actin-binding protein thought to be involved in a range of functions associated with the actin cytoskeleton, including the regulation of myosin binding to actin filaments, but to date no tropomyosin-like proteins have been conclusively identified in plant genomes. Our data suggests that a tropomyosin-like protein is associated with plasmodesmata. © 2009 Elsevier GmbH. All rights reserved.

During cytokinesis in onion (Allium cepa L.) and leek (Allium porrum L.), all the peroxisomes present within the dividing cell aggregate in the phragmoplast adjacent to the developing cell plate. In order to understand the functional implications of this novel arrangement, especially in onion, which has hitherto been regarded as a model system in which to study plant cell division, we investigated how widespread the phenomenon was in selected monocots and dicots. During monocot cytokinesis, peroxisomes lacked aggregation in some taxa, notably grasses, and underwent partial aggregation in other taxa. However, complete aggregation of a cell's entire complement of peroxisomes was restricted to the genus Allium. Although peroxisomal aggregation has been suggested to function in the formation of the cell plate, the distribution of partial and complete aggregation in monocots did not match known differences in primary cell wall biochemistry. No aggregation was seen during cytokinesis in dicots. Through quantification of peroxisome distribution in cells whose actin microfilaments were disrupted with either latrunculin or cytochalasin, we demonstrated that peroxisomal aggregation is a microfilament-dependent process in Allium and the closely related plant Tristagma, which shows only partial aggregation. We speculate whether analysis of the peroxisomal proteome might reveal novel function(s) for aggregated peroxisomes during cytokinesis. © 2008 by The University of Chicago. All rights reserved.

In plant cells, the cytoskeleton comprises distinct and highly dynamic arrays of microtubules and actin microfilaments. The basic structures and proteins of both the microtubules (25¿¿nm-diameter polymers of a- and ß-tubulin heterodimers), and the microfilaments (7¿¿nm-diameter polymers of 42¿kDa actin monomers) are conserved in all eukaryotic organisms, and occur in all cell types. The third cytoskeletal array present in animal cells, intermediate filaments, are of a more varied composition and their presence has not (yet) been demonstrated in plant cells. The basic organization of microtubules and microfilaments in various plant cells was determined over several decades from static images of fixed material. These images often demonstrated that microfilaments co-align with microtubules. As functional and molecular studies have become more prevalent, it has become apparent that co-ordination of dynamic microtubules and microfilaments is necessary for many facets of growth and development, and that cross-talk exists between them. Numerous studies have shown such interactions in animal cells (Gavin 1997; Goode et al. 2000; Dehmelt and Halpain 2003), and it is the diversity of these processes in plants that forms the subject of this review. As such, this review takes a broad approach to the topic. Defining microtubule-microfilament cross-talk (or microfilament-microtubule cross-talk for those of an actin persuasion) as any type of relationship between microtubules and microfilaments, the review commences with a reassessment of early work into colocalization between microtubules and microfilaments (Sect.), which leads to information about microtubule-microfilament interactions (Sect.). In this review, the term "interactions" implies a direct, physical relationship between the two components of the cytoskeleton, whereas "cross-talk" is used in a more encompassing way that includes indirect interactions. Section considers proteins that might mediate direct microtubule-microfilament interactions. However, taking the broad view of microtubule-microfilament cross-talk leads to discussion of systems where both microtubules and microfilaments play a role, but without any direct involvement with one another. Such microtubule-microfilament co-ordination seemingly occurs in organelle movement and shaping (Sect.). A further component of microtubule-microfilament cross-talk involves indirect, but specific interplay between the networks via the Rop-signalling pathway (Sect.). The cytoskeleton performs numerous fundamental roles within plant cells, and plant biologists have demonstrated that the microtubules and microfilaments function independently in many of these. However, as this review documents, on the occasions when these two networks come together, and there is interplay between them, dissecting the tangled cross-wires of the microtubules and microfilaments can become difficult. © 2008 Springer-Verlag Berlin Heidelberg.

Dynamin-related proteins are large GTPases that deform and cause fission of membranes. The DRP1 family of Arabidopsis thaliana has five members of which DRP1A, DRP1C, and DRP1E are widely expressed. Likely functions of DRP1A were identified by studying rsw9, a null mutant of the Columbia ecotype that grows continuously but with altered morphology. Mutant roots and hypocotyls are short and swollen, features plausibly originating in their cellulose-deficient walls. The reduction in cellulose is specific since non-cellulosic polysaccharides in rsw9 have more arabinose, xylose, and galactose than those in wild type. Cell plates in rsw9 roots lack DRP1A but still retain DRP1E. Abnormally placed and often incomplete cell walls are preceded by abnormally curved cell plates. Notwithstanding these division abnormalities, roots and stems add new cells at wild-type rates and organ elongation slows because rsw9 cells do not grow as long as wild-type cells. Absence of DRP1A reduces endocytotic uptake of FM4-64 into the cytoplasm of root cells and the hypersensitivity of elongation and radial swelling in rsw9 to the trafficking inhibitor monensin suggests that impaired endocytosis may contribute to the development of shorter fatter roots, probably by reducing cellulose synthesis. © 2008 The Author(s).

Transversely oriented cortical microtubules in elongating cells typically reorient themselves towards longitudinal directions at the end of cell elongation. We have investigated the reorientation mechanism along the outer epidermal wall in maturing leek (Allium porrum L.) leaves using a GFP-MBD microtubule reporter gene and fluorescence microscopy. Incubating leaf segments for 14-18 h with the anti-actin or anti-actomyosin agents, 20 µm cytochalasin D or 20 mm 2,3-butanedione monoxime, inhibited the normal developmental reorientation of microtubules to the longitudinal direction. Observation of living cells revealed a small subpopulation of microtubules with their free ends swinging into oblique or longitudinal directions, before continuing to assemble in the new direction. Electron microscopy confirmed that longitudinal microtubules are partly detached from the plasma membrane. Incubating leaf segments with 0.2% 1°-butanol, an activator of phospholipase D, which has been implicated in plasma membrane-microtubule anchoring, promoted the reorientation, presumably by promoting microtubule detachment from the membrane. Stabilizing microtubules with 10 µm taxol also promoted longitudinal orientation, even in the absence of cytoplasmic streaming. These results were consistent with confocal microscopy of live cells before and after drug treatments, which also revealed that the slow (days) global microtubule reorientation is superimposed over short-term (hours) regional cycling in a clockwise and an anti-clockwise direction. We propose that partial detachment of transverse microtubules from the plasma membrane in maturing cells exposes them to hydrodynamic forces of actomyosin-driven cytoplasmic streaming, which bends or shifts pivoting microtubules into longitudinal directions, and thus provides an impetus to push microtubule dynamics in the new direction. © 2008 The Authors.

The cytoskeleton governs many critical processes in expanding plant cells, including the delivery of wall components and the establishment and maintenance of growth direction. This work describes how cytoskeletal arrays assemble, and how their spatial organization and dynamics regulate the anisotropic properties of plant cell walls. We describe the mechanisms that construct and organize transverse microtubule arrays, and explore how these arrays, and the direction of elongation, are influenced by hormones. We then consider how cortical microtubules regulate the mechanical properties of the load-bearing cellulose microfibrils, through interacting with cellulose synthase complexes, and by coordinating the secretion of wall proteins. Actin microfilaments form part of the machinery that controls polar auxin transport, and have critical functions in vesicle transport. In recent years, it has become increasingly clear that microtubules and actin microfilaments work in concert to coordinate cell expansion. This microfilament-microtubule coordination is mediated through the activity of Rop GTPase signalling switches. We highlight this process in the growth of pavement cells found in the epidermal layers of leaves. © 2007 Springer-Verlag Berlin Heidelberg.

¿ Elongation of diffusely expanding plant cells is thought to be mainly under the control of cortical microtubules. Drug treatments that disrupt actin microfilaments, however, can reduce elongation and induce radial swelling. To understand how microfilaments assist growth anisotropy, we explored their functional interactions with microtubules by measuring how microtubule disruption affects the sensitivity of cells to microfilament-targeted drugs. ¿ We assessed the sensitivity to actin-targeted drugs by measuring the lengths and diameters of expanding roots and by analysing microtubule and microfilament patterns in the temperature-sensitive Arabidopsis thaliana mutant microtubule organization 1 (mor1-1), along with other mutants that constitutively alter microtubule arrays. ¿ At the restrictive temperature of mor1-1, root expansion was hypersensitive to the microfilament-disrupting drugs latrunculin B and cytochalasin D, while immunofluorescence microscopy showed that low doses of latrunculin B exacerbated microtubule disruption. Root expansion studies also showed that the botero and spiral1 mutants were hypersensitive to latrunculin B. ¿ Hypersensitivity to actin-targeted drugs is a direct consequence of altered microtubule polymer status, demonstrating that cross-talk between microfilaments and microtubules is critical for regulating anisotropic cell expansion. © New Phytologist (2006).

MICROTUBULE ORGANIZATION 1 (MOR1) is a plant member of the highly conserved MAP215/Dis1 family of microtubule-associated proteins. Prior studies with the temperature-sensitive mor1 mutants of Arabidopsis (Ambidopsis thaliana), which harbor single amino acid substitutions in an N-terminal HEAT repeat, proved that MOR1 regulates cortical microtubule organization and function. Here we demonstrate by use of live cell imaging and immunolabeling that the mor1-1 mutation generates specific defects in the microtubule arrays of dividing vegetative cells. Unlike the universal cortical microtubule disorganization in elongating mor1-1 cells, disruption of mitotic and cytokinetic microtubule arrays was not detected in all dividing cells. Nevertheless, quantitative analysis identified distinct defects in preprophase bands (PPBs), spindles, and phragmoplasts. In nearly one-half of dividing cells at the restrictive temperature of 30°C, PPBs were not detected prior to spindle formation, and those that did form were often disrupted, mor1-1 spindles and phragmoplasts were short and abnormally organized and persisted for longer times than in wild-type cells. The reduced length of these arrays predicts that the component microtubule lengths are also reduced, suggesting that microtubule length is a critical determinant of spindle and phragmoplast structure, orientation, and function. Microtubule organizational defects led to aberrant chromosomal arrangements, misaligned or incomplete cell plates, and multinucleate cells. Antiserum raised against an N-terminal MOR1 sequence labeled the full length of microtubules in interphase arrays, PPBs, spindles, and phragmoplasts. Continued immunolabeling of the disorganized and short microtubules of mor1-1 at the restrictive temperature demonstrated that the mutant mor1-1L174F protein loses function without dissociating from microtubules, providing important insight into the mechanism by which MOR1 may regulate microtubule length. © 2005 American Society of Plant Biologists.

The Arabidopsis radial swelling mutant rsw10 showed ballooning of root trichoblasts, a lower than wild-type level of cellulose and altered levels of some monosaccharides in non-cellulosic polysaccharides. Map-based cloning showed that the mutated gene (At1g71100) encodes a ribose 5-phosphate isomerase (RPI) and that the rsw10 mutation replaces a conserved glutamic acid residue with lysine. Although RPI is intimately involved with many biochemical pathways, media supplementation experiments suggest that the visible phenotype results from a defect in the production of pyrimidine-based sugar-nucleotide compounds, most likely uridine 5'-diphosphate-glucose, the presumed substrate of cellulose synthase. Two of three RPI sequences in the nuclear genome are cytoplasmic, while the third has a putative chloroplast transit sequence. The sequence encoding both cytoplasmic enzymes could complement the mutation when expressed behind the CaMV 35S promoter, while fusion of the RSW10 promoter region to the GUS reporter gene established that the gene is expressed in many aerial tissues as well as the roots. The prominence of the rsw10 phenotype in roots probably reflects RSW10 being the only cytosolic RPI in this tissue and the gene encoding the plastid RPI being relatively weakly expressed. We could not, however, detect a decrease in total RPI activity in root extracts. The rsw10 phenotype is prominent near the root tip where cells undergo division, endoreduplication and cell expansion and so are susceptible to a restriction in de novo pyrimidine production. The two cytosolic RPIs probably arose in an ancient duplication event, their present expression patterns representing subfunctionalization of the expression of the original ancestral gene. © 2006 The Authors.

The endoplasmic reticulum (ER) of plant cells undergoes a drastic reorganization during cell division. In tobacco NT-1 cells that stably express a GFP construct targeted to the ER, we have mapped the reorganization of ER that occurs during mitosis and cytokinesis with confocal laser scanning microscopy. During division, the ER and nuclear envelope do not vesiculate. Instead, tubules of ER accumulate around the chromosomes after the nuclear envelope breaks down, with these tubules aligning parallel to the microtubules of the mitotic spindle. In cytokinesis, the phragmoplast is particularly rich in ER, and the transnuclear channels and invaginations present in many interphase cells appear to develop from ER tubules trapped in the developing phragmoplast. Drug studies, using oryzalin and latrunculin to disrupt the microtubules and actin microfilaments, respectively, demonstrate that during division, the arrangement of ER is controlled by microtubules and not by actin, which is the reverse of the situation in interphase cells. © 2006 Elsevier SAS. All rights reserved.

Determination of the precise role(s) of actin microfilaments in the control of cell shape and elongation in the root tips of the model genetic system Arabidopsis thaliana (L.) Heynh is frustrated by inadequate microscopy imaging techniques. In this paper, we documented both microfilaments and microtubules in the root tips of Arabidopsis by double immunofluorescence labelling and computer-generated reconstruction of confocal image series. Our procedure, which complements the use of recently developed fluorescent reporter proteins, revealed hitherto undescribed aspects of the Arabidopsis microfilament cytoskeleton that may provide important clues about mechanisms behind cell elongation. We found that preservation of extensive arrays of transverse cortical microfilaments depends on unperturbed microtubule organization. Compared with ordinary epidermal cells, cells situated in the trichoblast or hair-forming cell files were comparatively devoid of endoplasmic microfilaments when in the distal elongation zone, well before hair formation begins. Computer-aided reconstructions also revealed that the nonexpanding end walls of cells in the distal elongation zone have radially oriented microtubules and randomly arranged microfilaments. In dividing cells, microfilaments became more prominent in the cell cortex, and subtle differences between microtubule and microfilament organization were seen within the phragmoplast. These observations will form the basis of understanding the roles of the cytoskeleton in controlling elongation in root tissues. In light of the many Arabidopsis mutants with altered root morphology, our methods offer a reliable approach to assess the function of cytoskeletal proteins and signalling systems in root morphogenesis. © 2005 NRC.

The normal tip-growing pattern exhibited by root hairs of legumes is disrupted when the hair is exposed to Nod factors generated by compatible bacteria capable of inducing nodule formation. Since microtubules (MTs) play an important role in regulating directionality and stability of apical growth in root hairs [T.N. Bibikova et al. (1999) Plant J 17:657-665], we examined the possibility that Nod factors might affect the MT distribution patterns in root hairs of Medicago sativa L. We observed that Nod factor application caused rapid changes in the pattern of MTs starting as early as 3 min after perfusion. Within 3 to 10 min after Nod factor application, first endoplasmic and then cortical MTs depolymerised, initially at the proximal ends of cells. Twenty minutes after exposure to Nod factors, a transverse band of microtubules was seen behind the tip, while almost all other MTs had depolymerised. By 30 min, very few MTs remained in the root hair and yet by 1 h the MT cytoskeleton re-formed. When Nod factors were applied in the presence of 10 µM oryzalin or 5 µM taxol, the MTs appeared disintegrated while the morphological effects, such as bulging and branching, became enhanced. Compared to the treatments with oryzalin or taxol alone, the combinatory treatments exhibited higher growth rates. Since microtubule reorganization is one of the earliest measurable events following Nod factor application we conclude that microtubules have an important role in the early phases of the signalling cascade. Microtubule involvement could be direct or a consequence of Nod factor-induced changes in ion levels.

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion (Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek (Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the ß-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

The organisation of plant microtubules into distinct arrays during the cell cycle requires interactions with partner proteins. Having recently identified a 90-kDa phospholipase D (PLD) that associates with microtubules and the plasma membrane [Gardiner et al. (2001) Plant Cell 13: 2143], we exposed seeds and young seedlings of Arabidopsis to 1-butanol, a specific inhibitor of PLD-dependent production of the signalling molecule phosphatidic acid (PA). When added to agar growth media, 0.2% 1-butanol strongly inhibited the emergence of the radicle and cotyledons, while 0.4% 1-butanol effectively blocked germination. When normal seedlings were transferred onto media containing 0.2% and 0.4% 1-butanol, the inhibitor retarded root growth by about 40% and 90%, respectively, by reducing cell elongation. Inhibited plants showed significant swelling in the root elongation zone, bulbous or branched root hairs, and modified cotyledon morphology. Confocal immunofluorescence microscopy of root tips revealed that 1-butanol disrupted the organisation of interphase cortical microtubules. Butanol isomers that do not inhibit PLD-dependent PA production, 2- and 3-butanol, had no effect on seed germination, seedling growth, or microtubule organisation. We propose that production of PA by PLD may be required for normal microtubule organisation and hence normal growth in Arabidopsis.

In Zea mays L., changes in orientation of stems are perceived by the pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. Gravity is perceived in the bundle sheath cells, which contain amyloplasts that sediment to the new cell base when a change in the gravity vector occurs. The mechanism by which the mechanical signal is transduced into a physiological response is so far unknown for any gravity perceiving tissue. It is hypothesized that this involves interactions of amyloplasts with the plasma membrane and/or ER via cytoskeletal elements. To gain further insights into this process we monitored amyloplast movements in response to gravistimulation. In a pharmacological approach we investigated how the dynamics of plastid sedimentation are affected by actin and microtubule (MT) disrupting drugs. Dark grown caulonemal filaments of the moss Physcomitrella patens respond to gravity vector changes with a reorientation of tip growth away from the gravity vector. MT distributions in tip cells were monitored over time and MTs were seen to accumulate preferentially on the lower flank of the tip 30 min after a 90° turn. Using a self-referencing Ca 2+ selective ion probe, we found that growing caulonemal filaments exhibit a Ca 2+ influx at the apical dome, similar to that reported previously for other tip growing cells. However, in gravistimulated Physcomitrella filaments the region of Ca 2+ influx is not confined to the apex, but extends about 60µm along the upper side of the filament. Our results indicate an asymmetry in the Ca 2+ flux pattern between the upper and side of the filament suggesting differential activation of Ca 2+ permeable channels at the plasma membrane. © 2003 COSPAR. Published by Elsevier Ltd. All rights reserved.

Peroxisomal shape, distribution, motility, and interactions with cytoskeletal elements were examined during interphase in living leek (Allium porrum L.) epidermal cells transiently transformed with a construct encoding the green fluorescent protein bearing a carboxy-terminal type 1 peroxisomal targeting signal. Confocal laser scanning microscopy and time-course analysis revealed that labeled peroxisomes were either spherical or rod-shaped and possessed several types of motility including random oscillations, slow and fast directional and bidirectional movements, and stop-and-go movements. Co-localization studies indicated that most peroxisomes were in close association with actin filaments, while treatment of cells with the actin-disrupting drug cytochalasin D blocked all types of peroxisomal movements. In contrast, the overall spatial organization of peroxisomes and the microtubule cytoskeleton were different, and the microtubule-destabilizing agent oryzalin had no obvious effect on peroxisomal motility. These data indicate that the peroxisome in plant cells is a highly dynamic compartment that is dependent upon the actin cytoskeleton, not microtubules, for its subcellular distribution and movements.

In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pHc) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pHc has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pHc in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pHc changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pHc changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pHc has an important role in the early signaling pathways of maize stem gravitropism.

The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissimum L. and Nicotiana benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimido-benzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the basis for advancing studies on the role of actin in gravity perception and signaling.

Profilins are low-molecular-mass (12-15 kDa) cytosolic proteins that are major regulators of actin assembly in all eukaryotic cells. In general, profilins from evolutionarily diverse organisms share the ability to bind to G-actin, poly-(L-proline) (PLP) and prolinerich proteins, and polyphosphoinositides. However, the functional importance of each of these interactions remains unclear and might differ between organisms. We investigated the importance of profilin's interaction with its various ligands in plant cells by characterizing four maize (Zea mays) profilin 5 (ZmPRO5) mutants that had single amino acid substitutions in the presumed sites of ligand interaction. Comparisons in vitro with wild-type ZmPRO5 showed that these mutations altered ligand association specifically. ZmPRO5-Y6F had a 3-fold increased affinity for PLP, ZmPRO5-Y6Q had a 5-fold decreased affinity for PLP, ZmPRO5-D8A had a 2-fold increased affinity for PtdIns(4,5)P2 and ZmPRO5-K86A had a 35-fold decreased affinity for G-actin. When the profilins were microinjected into Tradescantia stamen hair cells, ZmPRO5-Y6F increased the rate of nuclear displacement in stamen hairs, whereas ZmPRO5-K86A decreased the rate. Mutants with a decreased affinity for PLP (ZmPRO5-Y6Q) or an enhanced affinity for Ptdlns(4,5)P2 (ZmPRO5-D8A) were not significantly different from wild-type ZmPRO5 in affecting nuclear position. These results indicate that plant profilin's association with G-actin is extremely important and further substantiate the simple model that profilin acts primarily as a G-actin-sequestering protein in plant cells. Furthermore, interaction with proline-rich binding partners might also contribute to regulating profilin's effect on actin assembly in plant cells.

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipid-metabolizing enzyme and a Ca2+-dependent lipid binding domain and is identical to Arabidopsis PLDd. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.

Through reverse transcription-polymerase chain reaction and Northern blot analysis, we identified TBK5, a novel plant-specific kinesin-like protein (KLP) that is highly expressed in interphase tobacco BY-2 cells. TBK5 mRNA was present at a high level throughout the growth cycle, even in cells that had entered the stationary phase, where cell proliferation had ceased. However, transcripts for five other tobacco KLPs that we have identified were preferentially expressed in mitotic cells, and either not or only slightly accumulated in cells that had entered the stationary phase. Thus, TBK5 appears to be a KLP whose cellular function most closely relates to the cortical array of microtubules that plays a key role in plant cell morphogenesis. The predicted structure of TBK5 is characterized by a central motor domain that is phylogenetically distant from those of other reported KLPs, coiled-coil domains located on both sides of the motor domain, and a basic C-terminal domain. In addition, TBK5 has a putative neck domain which is closely related to the neck domain of KLPs with C-terminal motor domains, previously shown to control the direction of KLP movement towards the minus ends. Antibodies against truncated TBK5 recognized a polypeptide with a molecular mass of 74 kDa in cytoplasmic extracts of interphase cells, and this polypeptide cosedimented with microtubules assembled in the cytoplasmic extracts. The 74 kDa polypeptide corresponding to TBK5 dissociated from microtubules with high concentrations of NaCl but was not dissociated by MgATP. We hypothesize that TBK5 functions in the regulation of the arrangement of cortical microtubules.

Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

When lysed in an actin stabilizing buffer, protoplasts made from tobacco BY-2 suspension culture cells formed plasma membrane ghosts that retained both cortical actin and microtubules. Distinct cytoskeletal arrays occurred: the most common ghost array (type I) derived from protoplasts in interphase and had random actin and microtubules, although the alignment of the actin was dependent, at least partially, on microtubule organization. Type II ghosts were larger and more irregular in shape than type I ghosts, and were characterized by a lack of microtubules and the presence of distinctive arrays of actin bundles in concentric arcs. These ghosts derived from protoplasts lacking cortical microtubules produced when wall digestion occurred while the cells were in cell division, or from protoplasts isolated in the presence of 100 µM propyzamide. Because type II ghosts derived from protoplasts of similar size to those that give rise to type I ghosts, and because type II ghosts retained ordered actin arrays while the parent protoplasts had random cortical actin, type II ghosts apparently form differently to type I ghosts. We speculate that instead of the protoplast being sheared off to produce a round ghost, the plasma membrane tears and collapses onto the slide, ordering the actin bundles in the process. One implication of this model would be that cortical microtubules provide structural support to the plasma membrane of the protoplast so that only in their absence do the type II ghosts form.

Microtubule and filamentous(F)-actin organization in the potato suspension culture line HH260 was studied by fluorescence microscopy in double-labelled cells. During interphase, microtubules and F-actin were randomly arrayed in isodiametric cells but were aligned transversely to the direction of growth in elongated cells. Microtubules and F-actin coaligned in preprophase bands which were, however, comparatively rare and diffuse. Interestingly, more than half of the cells in telophase contained phragmoplasts that were either horseshoe-shaped or straight, instead of being round. We traced the cause of this difference to preprophase, where misplaced nuclear localization away from the central axis of cells may give rise to acentrally placed spindles and, subsequently, to acentrally placed phragmoplasts and cell plates. Further, we hypothesize that it is the uneven fusion of the expanding cell plates with the parent plasma membrane, and the accompanying depolymerization of those parts of the phragmoplasts, that gives the incomplete phragmoplasts observed.

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K+ and Ca2+ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.

Characterization of gravitropic bending in the maize stem pulvinus, a tissue that functions specifically in gravity responses, demonstrates that the pulvinus is an ideal system for studying gravitropism. Gravistimulation during the second of three developmental phases of the pulvinus induces a gradient of cell elongation across the non-growing cells of the pulvinus, with the most elongation occurring on the lower side. This cell elongation is spatially and temporally separated from normal internodal cell elongation. The three characterized growth phases in the pulvinus correspond closely to a specialized developmental sequence in which structural features typical of cells not fully matured are retained while cell maturation occurs in surrounding internodal and nodal tissue. For example, the lignification of supporting tissue and rearrangement of transverse microtubules to oblique that occur in the internode when cell elongation ceases are delayed for up to 10 d in the adjacent cells of the pulvinus, and only occurs as a pulvinus loses its capacity to respond to gravistimulation. Gravistimulation does not modify this developmental sequence. Neither wall lignification nor rearrangement of transverse microtubules occurs in the rapidly elongating lower side or non-responsive upper side of the pulvinus until the pulvinus loses the capacity to bend further. Gravistimulation does, however, lead to the formation of putative pit fields within the expanding cells of the pulvinus.