Robyn L. Overall

Actin associated with plasmodesmata

SummaryWe have used several methods to localise actin associated with plasmodesmata. In meristematic plant material fixed in 0.1% glutaraldehyde/1% paraformaldehyde and embedded in LR White resin, actin was localised (in TEM using 5 nm gold-labelled secondary antibody to C4 anti-actin primary antibody) in the neck region by the plasma membrane and endoplasmic reticulum, and also down the length of the plasmodesma, deep in the cell wall. When the chemical fixation was replaced by rapid freezing in liquid propane (without cryoprotectants) and substitution in acetone, the plasmodesmata were labelled in similar positions, but with less background label on sections. While only 8–20% of plasmodesmata were labelled, the label was 10 to 100 fold denser over plasmodesmata than over the surrounding wall indicating specific association with plasmodesmata. We presume the apparent extracellular location of some label was due to the size of the antibodies between the site of attachment and the observed position of the gold particle. Gold label was found in similar locations in material fixed in 3% paraformaldehyde, infiltrated with sucrose, frozen, sectioned (10–12 μm thick), then labelled with antibodies before resin embedding. Furthermore, cell walls in epidermal peels stained with rhodamine-phalloidin showed localised patches of fluorescence, presumably at the site of plasmodesmata (or primary pit-fields), which were connected on either side to fluorescent strands of actin in the cytoplasm. Suspension cultured cells ofNicotiana plumbaginifolia similarly stained showed very faint, narrow fluorescent strands crossing the walls of sister cells, which may indicate actin associated with individual plasmodesmata, shown in TEM to be sparsely distributed in these walls. In addition, the neck regions of cytochalasin-treated plasmodesmata were greatly enlarged and lacked the normal extracellular ring of particles. We propose that actin associated with plasmodesmata stabilizes the neck region and possibly also the cytoplasmic sleeve, and may be actively involved in regulating cell-to-cell transport.

A model of the macromolecular structure of plasmodesmata

Plasmodesmata are minute channels that traverse the plant cell wall to provide a cytoplasmic pathway for communication between neighbouring cells. Recently, these connections have been shown to transport molecules much larger than previously thought possible. Indeed, plasmodesmata now appear to be highly dynamic structures that can actively and selectively transport very large molecules between cells. Emerging physiological and molecular data must now be incorporated with information from electron microscopy to generate a dynamic model for their structure.

CALLOSE DEPOSITION AT PLASMODESMATA

SummaryThe transport of ions and metabolites through plasmodesmata has been thought to be controlled at the neck region where the cytoplasmic annulus is constricted and where callose has also been localised. In order to determine the possible structural and functional effects of callose, its deposition was inhibited through incubation of the plant tissue with 2-deoxy-D-glucose (DDG) for 1 h prior to fixation in 2.5% glutaraldehyde. The inhibition of callose formation was monitored through aniline blue-induced fluorescence of callose. The neck region of the plasmodesmata fromAllium cepa L. roots treated with DDG exhibited a funnel-shaped configuration. This is in contrast to the plasmodesmata from tissue not incubated with DDG, which exhibited constricted necks similar to those previously reported. Both initial dissection and glutaraldehyde fixation induced neck constriction in plasmodesmata, however, dissection of tissue increased the frequency of constrictions. The inhibition of callose formation by chemical means showed that the neck constrictions and raised collars in this area are artefacts due to physical wounding and glutaraldehyde fixation. The external electron-dense material observed when tannic acid is included in the primary fixative appears to be unrelated to the deposition of callose at the neck region.

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata

Abstract. The concentration of cytoplasmic free calcium ([Ca2+]cyt) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca2+]cyt were applied by cold treatment, and ion injection was also used to increase [Ca2+]cyt, by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca2+]cyt was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca2+]cyt (from 107 to 210u2009nM). In response to iontophoresis of Ca2+, plasmodesmata were observed to go from “open” (low resistance) to “shut” (high resistance) and then back “open” within 10u2009s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca2+]cyt.

Localization of a centrin-like protein to higher plant plasmodesmata

Antibodies against centrin, the ubiquitous calcium-binding contractile protein, recognized a 17 kDa protein in extracts of onion root tips and cauliflower florets. Using immunofluorescence microscopy, anti-centrin antibodies were localized to the developing cell plate of onion and cauliflower root tip cells. In cauliflower florets, these antibodies localized to the walls in a punctate manner, consistent with the distribution of plasmodesmata as shown by colocalization with callose. Anti-centrin antibodies were localized to plasmodesmata of onion root tips and cauliflower florets with immunogold electron microscopy. Furthermore, this label was concentrated around the necks of plasmodesmata. In contrast, an antibody against calmodulin, which is a closely related calcium-binding protein, did not label plasmodesmata. We propose that centrin is a component of calcium-sensitive contractile nanofilaments in the neck region of plasmodesmata and facilitates the calcium-induced regulation of intercellular transport.

CELL-TO-CELL COMMUNICATION VIA PLANT ENDOMEMBRANES

Cell‐to‐cell communication was investigated in epidermal cells cut from stem internodal tissue of Nicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans were microinjected using iontophoresis into the cytoplasm andcortical endomembrane network of these cells. The microinjected endomembrane network was similar in location and structure to the endoplasmic reticulum (ER) as revealed by staining with 3,3′‐dihexyloxacarbocyanine iodide (DiOC6). No cell‐to‐cell movement of dextrans was observed following cytoplasmic injections but injection of dextrans into the endomembrane network resulted in rapid diffusion of the probes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellular communication via the desmotubule through plasmodesmata.

Electrical and mechanical fields orient cortical microtubules in higher plant tissues

Abstract Microtubules are a major component of the plant cytoskeleton. The factors which control the orientation of microtubules in plants are unknown. In this paper we describe preliminary findings that non-injurious electrical fields or mechanical fields, applied to pea roots, can cause a re-orientation of cortical microtubules which then lie in a plane approximately perpendicular to the direction of the applied field, within 6 hours. These results provide the first experimental evidence that physical fields can control the orientation of microtubules in plant tissues.

Re-orientation of cortical F-actin is not necessary for wound-induced microtubule re-orientation and cell polarity establishment

SummaryTo assess the relative roles of cortical actin and microtubule re-orientation in the establishment of new cell polarity, we have examined the kinetics of cortical actin re-orientation around a wedge-shaped wound in pea roots. Cortical actin re-orients from a transverse alignment to an approximately longitudinal orientation between 5 and 24h after wounding, that is, after the re-alignment of microtubules, which is known to occur before 5h post-wounding. F-actin in root cortical cells does not appear to be necessary for the establishment of new cell polarity around wounds, since normal MT re-alignment, and new planes of cell division are still established around a wound in cytochalasin treated roots. The cytochalasin treatment appeared to totally disrupt cortical and cytoplasmic F-actin in cells of the root cortex. However, in the apparent absence of F-actin in these cells, the rate of wound-induced cell division, but not cell expansion, is slower, and we suggest that an effect on the phragmosomal actin is involved. Finally, we demonstrate that new cell polarity around a wound is not established if microtubules are disrupted by the herbicide oryzalin, but after re-establishment of these arrays following a wash-out of the drug, the typical new planes of cell expansion are observed. We conclude that microtubules play a critical role in establishing and maintaining cell polarity in this system, and that cortical F-actin has a minor and presently unclear function in these processes.

Centrin homologues in higher plants are prominently associated with the developing cell plate

SummaryCentrin and calmodulin are members of the EF-hand calcium-binding superfamily of proteins. In this study we compared localisation and immunoblotting of centrin with calmodulin in several monocot (onion and wheat) and dicot (mung bean andArabidopsis) plants. We confirmed that an anti-calmodulin antibody recognised a 17 kDa protein in all species tested and localises to the cytoplasm, mitotic matrix and with microtubules of the preprophase band and phragmoplast. In contrast, immunoblotting using anti-centrin antibodies shows that plant centrins vary from 17 to 20 kDa. Immunofluorescence microscopy with anti-centrin antibodies revealed only weak centrin immunoreactivity in the cytoplasm, nucleus, nuclear envelope, phragmoplast and mitotic matrix in meristematic cells. There was a slightly more intense perinuclear labelling in large differentiating onion cells and between separating anaphase chromosomes. While centrin is known to localise to the mitotic spindle poles in animal and algal cells, there was no appreciable immunoreactivity at the spindle poles in higher plants. In contrast, there was an intense immunofluorescence signal with anti-centrin antibodies in the developing cell plate. Further characterisation of the cell plate labelling by immunogold electron microscopy shows centrin immunoreactivity was closely associated with vesicles in the cell plate. Our observations suggest that centrin may play a role in cell plate formation.

Substructure of Plasmodesmata

Plasmodesmata now appear to be highly dynamic structures which are able to select and transport even large molecules from cell to cell (for review, see Zambryski1995). It has become crucial to understand the substructural architecture of plasmodesmata and the molecular interactions that control the intercellular passage of molecules and viruses. Our images of plasmodesmal structure have been necessarily static, leading to the generation of similarly static models of their architecture. The present challenge is to incorporate the emerging data on the molecular composition and operation of plasmodesmata into a dynamic model of their substructure.