Mechanism of plasmodesmata formation in characean algae in relation to evolution of intercellular communication in higher plants

It is generally accepted that higher plants evolved from ancestral forms of the modern charophytes. For this reason, we chose the characean alga, Chara corallina Klein ex Willd., em. R.D.W. (C. australis R. Br.), to determine whether this transition species produces plasmodesmata in a manner analogous to higher plants. As with higher plants and unlike most green algae, Chara utilizes a phragmoplast for cell division; however, in contrast with the situation in both lower and higher vascular plants, the developing cell plate and newly formed cell wall were found to be completely free of plasmodesmata. Only when the daughter cells had separated completely were plasmodesmata formed across the division wall. Presumably, highly localized activity of wall-degrading (or loosening) enzymes inserted into the plasma membrane play a central role in this process. In general appearance characean plasmodesmata are similar to those of higher plants with the notable exception that they lack an appressed endoplasmic reticulum. Further secondary modifications in plasmodesmal structure were found to occur as a function of cell development, giving rise to highly branched plasmodesmata in mature cell walls. These findings are discussed in terms of the evolution of the mechanism for plasmodesmata formation in algae and higher plants.This work was supported in part by National Foundation grant No. DCB-9016756 (W.J.L.). We thank the Electron Microscopy Center of Washington State University and the Zoology Department, University of California, Davis, for the use of their microscopy facilities.     

Behaviour of plasma membrane, cortical ER and plasmodesmata during plasmolysis of onion epidermal cells

During plasmolysis of onion epidermal cells, the contracting protoplast remains connected to the cell wall by an intricate, branched system of plasma membrane (PM) ‘Hechtian strands’ which stain strongly with the fluorescent probe DiOC₆. In addition, extensive regions of the cortical endoplasmic reticulum (ER) network remain anchored to the cell wall during plasmolysis and do not become incorporated into the contracting protoplast with the other cell organelles. These ER profiles become tightly encased by the PM as the latter contracts towards the centre of the cell. Thus, although the cortical ER is left outside the main protoplast body, it is nonetheless still bound by the PM of the cell. As well as being anchored to the wall, the cortical ER remains intimately linked with plasmodesmata and retains continuity between cells via the central desmotubules which become distended during plasmolysis. The PM also remains in close contact with the plasmodesmatal pore following plasmolysis. It is suggested that plasmodesmata, although sealed, may not be broken during plasmolysis, their substructure being preserved by continuity of both ER and PM through the plasmodesmatal pore. A structural model is presented which links the behaviour of PM, ER and plasmodesmata during plasmolysis.     

Substructure of freeze-substituted plasmodesmata

Summary The substructure of plasmodesmata in freeze-substituted tissues of developing leaves of the tobacco plant (Nicotiana tabacum L. var. Maryland Mammoth) was studied by high resolution electron microscopy and computer image enhancement techniques. Both the desmotubule wall and the inner leaflet of the plasmodesmatal plasma membrane are composed of regularly spaced electron-dense particles approximately 3 nm in diameter, presumably proteinaceous and embedded in lipid. The central rod of the desmotubule is also particulate. In plasmodesmata with central cavities, spoke-like extensions are present between the desmotubule and the plasma membrane in the central cavity region. The space between the desmotubule and the plasma membrane appears to be the major pathway for intercellular transport through plasmodesmata. This pathway may be tortuous and its dimensions could be regulated by interactions between desmotubule and plasma membrane particles.Abbreviations ER endoplasmic reticulum - PJF propane jet freezing - HPF high pressure freezing - CRT cathode ray tube - IP₃ inositoltrisphosphate     

Endoplasmic reticulum in the formation of the cell plate and plasmodesmata

Summary The association of endoplasmic reticulum (ER) with the developing cell plate has been analyzed in lettuce roots fixed in glutaraldehyde and post-fixed in a mixture of osmium tetroxide-potassium ferricyanide (OsFeCN). Electron microscopic observations show that elements of ER, which are selectively stained by the OsFeCN reagent, become loosely associated with aggregating dictyosome vesicles at the onset of plate formation. Subsequently the ER, in a tubular reticulate network, surrounds the vesicular aggregates creating a three dimensional membrane matrix. It is suggested that the ER (1) provides a structural framework that holds the vesicles in position and directs their fusion within the plane of the plate and/or (2) regulates the local release of calcium ions required for vesicle fusion.OsFeCN post-fixation also provides new information about the cell plate vesicles themselves. The results demonstrate that vesicles derived from dictyosomes undergo an abrupt increase in staining as they fuse at the plate.Finally the ER associated with developing and mature plasmodesmata has been examined. Electron micrographs reveal that the OsFeCN staining, seen traversing the cell plate in early stages, later becomes restricted from that portion of the ER extending through the plasmodesmatal canal. These structural observations support the idea that during formation of the plasmodesma a tubular element of ER is tightly furled upon itself and that its inner leaflet is compressed into a rod. The ER cisternal space appears occluded and thus it is argued that intercellular transport occurs through the cytoplasmic annulus of the plasmodesmata.     

Chara tomentosa Antheridial Plasmodesmata at Various Stages of Spermatogenesis

Chara tomentosa antheridial plasmodesmata are described during proliferation and spermiogenesis. In antheridial filament cells which are cycling completely synchronously, unplugged plasmodesmata are filled with light cytoplasm. The same plasmodesmata are observed after cessation of mitotic division followed by the onset of synchronous spermiogenesis. Walls separating cells at different cell cycle stages and dividing antheridial filaments into asynchronous domains are plugged with a dense osmophilic substance. Similarly plugged plasmodesmata are present between antheridial cells of different types, e.g., capitular cells and antheridial filaments. In mid spermiogenesis when abundant endoplasmic reticulum (ER) appears temporarily it penetrates into plasmodesmata enabling cell-to-cell transport via ER cisternae. In late spermiogenesis there are no cisternae in plasmodesmata. This revised version was published online in July 2006 with corrections to the Cover Date.     

Maize calreticulin localizes preferentially to plasmodesmata in root apex

Using a polyclonal antibody raised against calreticulin purified and sequenced from maize, we performed an immunocytological study to characterize putative domain-specific subcellular distributions of endoplasmic reticulum (ER)-resident calreticulin in meristematic cells of maize root tip. At the light microscopy level, calreticulin was immunolocalized preferentially at cellular peripheries, in addition to nuclear envelopes and cytoplasmic structures. Punctate labelling at the longitudinal walls and continuous labelling at the transverse walls was characteristic. Immunogold electron microscopy revealed plasmodesmata as the most prominently labelled cell periphery structure. In order to further probe the ER-domain-specific distribution of maize calreticulin at plasmodesmata, root apices were exposed to mannitol-induced osmotic stress. Plasmolysis was associated with prominent accumulations of calreticulin at callose-enriched plasmodesmata and pit fields while the contracting protoplasts were depleted of calreticulin. In contrast, other ER-resident proteins recognized by HDEL peptide and BiP antibodies localized exclusively to contracted protoplasts. This finding reveals that, in plasmolysed cells, calreticulin enriched ER domains at plasmodesmata and pit fields are depleted of other ER-resident proteins containing the HDEL retention peptide.     

Plasmodesmal Dynamics in Both Woody Poplar and Herbaceous Winter Wheat Under Controlled Short Day and in Field Winter Period

Electron microscopic observation revealed that poplar (Populus deltoides Marsh.) and winter wheat (Triticum aestivum L. cv. Seward 80004) plasmodesmatal structures significantly changed under short day (SD, 8 h light) and in winter period, and such changes differed also noticeably between these two woody and herbaceous plants. Under long day (LD, 16 h light), many plasmodesmata with strong stain appeared in the cell wall of both poplar apical buds and winter wheat young leaf tissues, and connections of cytoplasmic endoplasmic reticulum (ER) with the ER in some plasmodesmata were observed. In addition, the typical “neck type” plasmodesmata were observed in winter wheat young leaf tissues, and their central desmotubules (appressed-ER) could be clearly identified. Under SD, many poplar plasmodesmata showed only a partial structure in the cell wall and appeared to be discontinued; some plasmodesmata swelled in the mid-wall, forming the cavity, and no appressed-ER appeared. In winter wheat, however, no noticeable alterations of plasmodesmata occurred, and the plasmodesmatal structure essentially remained same as it was under LD. In winter period, poplar plasmodesmata had a similar morphology as those observed under SD, however, winter wheat manifested at least two types of significant plasmodesmatal alterations: one plugged by electron-dense materials and the other of reduced neck region compared to those under LD. The above dynamic difference of the two species plasmodesmata under SD and winter period revealed the difference of their dormancy development under those environmental conditions.     

Changes in ultrastructure of plasmodesmata during spermatogenesis in Chara vulgaris L.

Two types of plasmodesmata are found within an antheridium of Chara vulgaris: open plasmodesmata filled with electron-transparent cytoplasm, and plugged plasmodesmata, filled with an osmiophilic dense substance. Open plasmodesmata occur only between cells synchronized completely in respect of their advancement in cell-cycle progression or differentiation. Plugged plasmodesmata connect different types of cells or cells of the same type at various stages of the cell cycle. Open plasmodesmata may become plugged, and vice versa. These changes are connected with the limitation or extension of synchronization of cellular divisions and differentiation within the groups of cells in the antheridium.     

Localization of a myosin‐like protein to plasmodesmata

Myosin has been localized to plasmodesmata in root tissues of Allium cepa, Zea mays and Hordeum vulgare using a polyclonal antibody to animal myosin in both fluorescence and electron microscopy. Labelling was also observed throughout the cytoplasm, mainly associated with the endoplasmic reticulum and plasma membrane. On Western blots, bands of 180 and 110 kDa were consistently labelled in all three species. These bands were also labelled when the blot was incubated in actin prior to staining with antibodies to actin, raising the possibility that either of these proteins (180 kDa or 110 kDa) may be present in plasmodesmata. Pre-treatment of the tissue with 2,3-butanedione monoxime (BDM), an inhibitor of actin–myosin motility, resulted in a strong constriction of the neck region of plasmodesmata. These results indicate that a myosin-like protein may be present in plasmodesmata and may also play a role in the regulation of transport at the neck region.     

Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus

Summary Cell-to-cell communication in plants occurs through plasmodesmata, cytoplasmic channels that traverse the cell wall between neighboring cells. Plasmodesmata are also exploited by many viruses as an avenue for spread of viral progeny. In the case of tobacco mosaic virus (TMV), a virally-encoded movement protein (MP) enables the virus to move through plasmodesmata during infection. We have used thin section electron microscopy and immunocytochemistry to examine the structure of plasmodesmata in transgenic tobacco plants expressing the TMV MP. We observed a change in structure of the plasmodesmata as the leaves age, both in control and MP expressing [MP(+)] plants. In addition, the plasmodesmata of older cells of MP(+) plants accumulate a fibrous material in the central cavity. The presence of the fibers is correlated with the ability to label plasmodesmata with anti-MP antibodies. The developmental stage of leaf tissue at which this material is observed is the stage at which an increase in the size exclusion limit of the plasmodesmata can be measured in MP(+) plants. Using cell fractionation and aqueous phase partitioning studies, we identified the plasma membrane and cell wall as the compartments with which the MP stably associates. The nature of the interaction between the MP and the plasma membrane was studied using sodium carbonate and Triton X-100 washes. The MP behaves as an integral membrane protein. Identifying the mechanism by which the MP associates with plasma membrane and plasmodesmata will lead to a better understanding of how the MP alters the function of the plasmodesmata.Abbreviations MP movement protein - TMV tobacco mosaic virus     

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae)

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.     

Cytochemical localization of cellulase activity in pollen mother cells of david lily during meiotic prophase I and its relation to secondary formation of plasmodesmata

Summary Cellulase activity was localized at the ultrastructural level in pollen mother cells (PMCs) of David lily [Lilium davidii var.willmottiae (Wilson) Roffill] at different stages of meiotic prophase I. The enzyme was observed to appear at the early leptotene stage and reached its highest level at the subsequent zygotene stage, and its subcellular distribution revealed by the presence of electron-dense deposits of reaction product was found to be restricted exclusively to the endoplasmic reticulum (ER), the vesicles derived from that, and the cell wall, especially at the sites of secondary plasmodesmata and cytoplasmic channels where the wall was being digested. Other cytoplasmic organelles, such as dictyosomes and Golgi vesicles, lacked such deposits of reaction product. After zygotene the enzyme activity decreased abruptly, and at the pachytene stage only very few deposits could be observed in the cell wall. Our results indicate that cellulase is synthesized on rough ER and secreted directly via the smooth ER and ER-derived vesicles into the cell wall by exocytosis, where it brings about local wall breakdown, leading to the secondary formation of plasmodesmata and cytoplasmic channels.     

Fine structure of plasmodesmata in mature leaves of sugarcane

The fine structure of plasmodesmata in vascular bundles and contiguous tissues of mature leaf blades of sugarcane (Saccharum interspecific hybrid L62–96) was studied with the transmission electron microscope. Tissues were fixed in glutaraldehyde, with and without the addition of tannic acid, and postfixed in OsO₄. The results indicate that the fine structure of plasmodesmata in sugarcane differs among various cell combinations in a cell-specific manner, but that three basic structural variations can be recognized among plasmodesmata in the mature leaf: 1) Plasmodesmata between mesophyll cells. These plasmodesmata possess amorphous, electron-opaque structures, termed sphincters, that extend from plasma membrane to desmotubule near the orifices of the plasmodesmata. The cytoplasmic sleeve is filled by the sphincters where they occur; elsewhere it is open and entirely free of particulate or spokelike components. The desmotubule is tightly constricted and has no lumen within the sphincters, but between the sphincters it is a convoluted tubule with an open lumen. 2) Plasmodesmata that traverse the walls of chlorenchymatous bundle-sheath cells and mestome-sheath cells. In addition to the presence of sphincters, these plasmodesmata are modified by the presence of suberin lamellae in the walls. Although the plasmodesmata are quite narrow and the lumens of the desmotubules are constricted where they traverse the suberin lamellae, the cytoplasmic sleeves are still discernible and appear to contain substructural components there. 3) Plasmodesmata between parenchymatous cells of the vascular bundles. These plasmodesmata strongly resemble those found in the roots of Azolla, in that their desmotubules are closed for their entire length and their cytoplasmic sleeves appear to contain substructural components for their entire length. The structural variations exhibited by the plasmodesmata of the sugarcane leaf are compared with those proposed for a widely-adopted model of plasmodesmatal structure.Abbreviation ER endoplasmic reticulum This study was supported by National Science Foundation grants DCB 87-01116 and DCB 90-01759 to R.F.E. and a University of Wisconsin-Madison Dean's Fellowship to K. R.-B. We also thank Claudia Lipke and Kandis Elliot for photographic and artistic assistance, respectively.     

Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata-associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.     

Observations on structure and differentiation in plasmodesmata

Summary The fine structure of plasmodesmata in a number of plant tissues has been examined following fixation in glutaraldehyde. The structure of plasmodesmata is not constant. Variations occur between species, tissues and between different cellular situations in a single tissue. The nature of these variations is described and related to current theories of the formation and function of plasmodesmata. Evidence is presented that the young cell wall after division contains a large number of plasmodesmata, probably functionally and structurally identical, and that the development process involves characteristic modifications both to the distribution of plasmodesmata within the wall, and to the structure of individual plasmodesmata. The probable importance of the endoplasmic reticulum is stressed in relation to the formation and functioning of plasmodesmata.     

A 45 kDa protein isolated from the nodal walls of Chara corallina is localised to plasmodesmata

The cellular anatomy of the green alga, Chara corallina, was exploited to isolate putative plasmodesmataassociated proteins. In C. corallina , large internodal cells are symplastically connected via intervening nodal complexes of smaller cells which have plasmodesmata in their cell walls. Comparison of proteins extracted from walls with plasmodesmata (nodal complexes) with those from walls without plasmodesmata (external internodal walls) identified four putative plasmodesmata-associated proteins. These putative plasmodesmata-associated proteins were approximately 95, 45, 44 and 33 kDa. A monoclonal antibody (MAB45/22) was raised against the 45 kDa putative plasmodesmata-associated protein (CPAP45). Using immunofluorescence, this antibody co-localised with aniline blue induced fluorescence of callose in the source cell walls. MAB45/22 was localised to the plasmodesmata of C. corallina and, in particular, to the central cavity using immunogold cytochemistry. In contrast, a monoclonal antibody to callose specifically labelled the mouth of C. corallina plasmodesmata. MAB45/22 also labelled higher plant plasmodesmata.     

Ultrastructural analysis of leaf trichome plasmodesmata reveals major differences from mesophyll plasmodesmata

Functional studies on molecular transport through plasmodesmata in leaf mesophyll and trichome cells revealed significant differences in their basal size-exclusion limits and their response to microinjected tobacco mosaic virus movement protein (E. Waigmann et al., 1994, Proc. Natl. Acad. Sci. USA 91: 1433–1437; E. Waigmann and P. Zambryski, 1995, Plant Cell 7: 2069–2079). To address the basis for these functional differences, Nicotiana clevelandii trichome and mesophyll plasmodesmata were compared ultrastructurally. Trichome plasmodesmata increase in ultrastructural complexity from the tip to the base cell. Their neck regions, thought to control molecular traffic through plasmodesmata, are clearly distinct from necks of mesophyll plasmodesmata. In contrast to the electron-dense desmotubular area in mesophyll plasmodesmata, trichome plasmodesmata contain an electron-translucent circle in their center, surrounded by an electron-dense ring. This latter ring is connected to the inner leaflet of the plasma membrane by multiple spokes or filaments. Two monoclonal antibodies raised against a maize plasmodesmal protein preparation (A. Turner et al., 1994, J Cell Sci. 107: 3351–3361) interact with both trichome and mesophyll N. clevelandii plasmodesmata. Based on the localization pattern and the high degree of cross-reactivity, both antibodies likely recognize a conserved structural component of plasmodesmata, and may be useful to mark plasmodesma in a variety of plants and tissues. Received: 24 January 1997 / Accepted: 3 March 1997     

Cytokinesis and nodal anatomy in the charophycean green algaChara zeylanica

Summary Cell plate formation inChara zeylanica was compared with recent models of cytokinesis in higher plants in order to gain insight into the evolutionary origin of plant cytokinetic processes. Transmission electron microscopy (TEM) reveals that while cytokinesis inC. zeylanica bears many features in common with that in higher plants, there are significant differences. Unlike that in higher plants, cytokinesis inC. zeylanica begins with a congregation of smooth membrane tubules that are closely associated with endoplasmic reticulum (ER) and Golgi membranes. Mitochondria and other organelles excluded by the phragmoplast in higher plants are present as well. Unlike in higher plants, phragmoplast microtubules persist throughout cytokinesis inC. zeylanica, and the cell plate generally forms across the whole cell at once, though development is patchy, due to small regions developing at different rates; the ends of the plate form last. By identifying aspects of cytokinesis that are different inC. zeylanica and plants, our study indicates which cytokinetic features are more likely to be derived, and which are more likely to be ancestral. In addition, we demonstrated that all nodal cells ofC. zeylanica are interconnected via plasmodesmata, lending support to the idea that, whileChara spp. are generally considered to be filamentous organisms, nodal regions may be thought of as meristemlike tissues.Abbreviations HPF high-pressure freezing - KFe potassium ferricyanide - SCF stepwise chemical fixation - TEM transmission electron microscopy     

Actin associated with plasmodesmata

Summary We 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 m 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.Abbreviations BSA bovine serum albumin - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulphonic acid) - Mes 2(N-morpholino)ethanesulfonic acid - SDS sodium dodecyl sulphate - Tris tris-(hydroxymethyl)aminomethane