Filament Disruption in Funaria Protonemata: Occlusion of Plasmodesmata

Plasmodesmata are occluded when Funaria chloronemata are fragmented by the development of tmema cells (TCs). The TC deposits a new wall layer along the cross wall toward the neighbouring non-sister cell (NC). This wall layer cuts off the plasmodesmata and its connection with the cross wall is soon lost. The plasmodesmata become isolated when the NC forms a new wall layer along the former cross wall. At the end of TC development, before its disintegration, the sister cell (SC) also deposits a new wall layer along the cross wall toward the TC, cutting off the plasmodesmata. For some time the plasmalemma of the plasmodesmata remains connected to the NC or the TC, whereas the desmotubule soon disappears. Relicts of the plasmalemma remain even after the isolation of the plasmodesmata and the disintegration of the TC. During the decay of the plasmodesmata, a cylinder of electron-dense material is frequently formed along the border of the plasmodesmatal channel. This may extend over the surface of the cell wall. Eventually, the plasmodesmatal channel is filled with wall material. Callose is only observed around functional plasmodesmata and does not seem to play a role in their occlusion.     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

Ultrastructural specializations of the cell wall sleeve around plasmodesmata

Specialized structures were observed in the sleeve of wall surrounding plasmodesmata in the rhizome tips of Nephrolepis exaltata and root tips of Spirodela oligorrhiza, Azolla pinnata, and Hordeum vulgare. Material was prepared either by fixation in glutaraldehyde/paraformaldehyde/tannic acid followed by cell wall digestion, or by rapid freezing and freeze substitution, prior to resin embedding. Two structures were identified: rings of electron-opaque material encircling the neck region of plasmodesmata and electron-opaque helices spiraling around the length of plasmodesmata. In some instances, particle-like subunits were observed in the electron-opaque rings. The possible role of these structures as external sphincters involved in the control of permeability of plasmodesmata is discussed.     

Distribution and structure of the plasmodesmata in mesophyll and bundle-sheath cells of Zea mays L.

In leaf blades of Zea mays L. plasmodesmata between mesophyll cells are aggregated in numerous thickened portions of the walls. The plasmodesmata are unbranched and all are characterized by the presence of electron-dense structures, called sphincters by us, near both ends of the plasmodesmatal canal. The sphincters surround the desmotubule and occlude the cytoplasmic annulus where they occur. Plasmodesmata between mesophyll and bundle-sheath cells are aggregated in primary pit-fields and are constricted by a wide suberin lamella on the sheath-cell side of the wall. Each plasmodesma contains a sphincter on the mesophyll-cell side of the wall. The outer tangential and radial walls of the sheath cells exhibit a continuous suberin lamella. However, on the inner tangential wall only the sites of plasmodesmatal aggregates are consistently suberized. Apparently the movement of photosynthetic intermediates between mesophyll and sheath cells is restricted largely or entirely to the plasmodesmata (symplastic pathway) and transpirational water movement to the cell walls (apoplastic pathway).Abbreviation ER endoplasmic reticulum     

Rapid detection of plasmodesmata in purified cell walls

Summary Plasmodesmata are complex channels within the plant cell wall, which create plasma membrane and symplastic continuity between neighbouring cells. To detect plasmodesmata in cell wall preparations fromNicotiana cle elandii, we have used 3,3-dihexyl-oxacarbocyanine iodide (DiOC₆), a cationic amphiphilic fluorescent probe, widely employed for general studies of membrane structure and dynamics. Punctate fluorescent staining was readily seen in pit fields, small depressions within the cell wall known to be rich in plasmodesmata. Scanning electron microscopy was used to demonstrate that the punctate staining corresponded to plasmodesmata. Treatment of cell wall fragments with chloroform-methanol to remove lipids did not alter the staining of plasmodesmata. In contrast, pronase E-sodium dodecyl sulfate treatment completely abolished staining, indicating that the DiOC₆ labelling of plasmodesmata may be protein rather than lipid specific. Although not membrane mediated, DiOC₆ staining of plasmodesmata is a simple, rapid, and specific tool for the detection of plasmodesmata in isolated cell walls and will prove useful for studies of plasmodesmal location, structure, and composition.     

Structure and development of cell connections in the phloem ofMetasequoia glyptostroboides needles I. Ultrastructural aspects of modified primary plasmodesmata in Strasburger cells

Summary Symplasmic contacts of Strasburger cells in the mature needle ofMetasequoia glyptostroboides were analysed with special regard to changes of plasmodesmata in fine structure and distribution. In meristematic cells simple primary plasmodesmata are evenly distributed throughout the entire wall, whereas in mature Strasburger cells plasmodesmata are aggregated in defined, dome-shaped wall thickenings. The elongated, often multiple-branched cytoplasmic strands show a distinct neck region besides a considerably dilated sleeve region confluent with cavities, which have formed at branching sites of plasmodesmata in various planes of the wall thickening. Most branches radiating from these cavities connect the protoplasts of the adjacent cells; occasionally some strands are discontinuous. The desmotubules of both, continuous and discontinuous plasmodesmal branches exhibit great variability in structure and number: they may be partially dilated, multiple-stranded and branched within single plasmodesmal branches. Fine structurally, plasmodesmata of Strasburger cells show great resemblance with developing sieve pores of conifers. This characteristic fine structure implicates a special role of the endomembrane system for phloem loading in theMetasequoia leaf.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

PLASMODESMATA AND AN ASSOCIATED CELL WALL COMPONENT IN BARLEY ALEURONE TISSUE

A unique cell wall component has been observed in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). This wall component has been shown to be localized adjacent to the plasmalemma. Unlike the surrounding cell wall matrix it is resistant to “Onozuka” cellulase and remains intact during gibberellic acid-stimulated hydrolase release. After treatment of the tissue with gibberellic acid followed by digestion with “Onozuka” cellulase this resistant wall component can be isolated free of protoplast. Study of its surface features revealed the presence of numerous tubular extensions, 120 nm wide, connecting adjacent resistant walls. These tubes resembled light microscope images of plasmodesmata in size and appearance. E.M. sections of resistant walls showed the presence of unit membrane lining the inner surface of the wall tubes. It was concluded that the resistant wall constitutes a modified wall layer that is secreted uniformly across all plasmalemma surfaces, including those in the wall (plasmodesmata). The presence of wall tubes surrounding plasmodesmata enhances the apparent size of the plasmodesmata in the light microscope. This may account for previous inconsistencies in the literature between light and electron microscope determinations of plasmodesmata diameters.     

Studies on graft unions

Summary The occurrence of plasmodesmata in the graft interfaces of two heteroplastic grafts (Impatiens walleriana onImpatiens olivieri andHelianthus annum onVicia faba) has been studied. For both systems two types of intercellular strand are described: 1. Continuous plasmodesmata interconnecting the cells of stock and scion and 2. half plasmodesmata traversing the wall part of one partner cell without connection to the abutting cell. Single strands or branched forms occur in both types of plasmodesma. In the case of half plasmodesmata, branchings with extended median nodules predominate. The distribution of half and continuous plasmodesmata varies with the different areas of a graft interface: in the region of bridging vascular tissues most cell connections are continuous. In areas where cortex or pith-derived callus cells and those of misaligned tissues (cortex/vascular tissue; cortex/pith; pith/vascular tissue) match, discontinuous strands predominate.Branched half plasmodesmata also occur in presumably fused walls between related callus cells; they are typical structures secondarily formed in non-division walls.The results are discussed with regard to compatibility/incompatibility phenomena in heterografts and the development and function of interspecific cell bridges.     

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.     

CELL WALL CHEMISTRY AND FINE STRUCTURE IN LEPTOIDS OF DENDROLIGOTRICHUM (BRYOPHYTA): THE END WALL

Leptoids (sieve elements) of Dendroligotrichum are characterized by a highly oblique end wall which is composed of cellulose (birefringent; IKI-H₂SO₄-positive), polyuronides (toluidine blue-positive), pectins (hydroxylamine-positive) and natural aldehydes (silver hexamine and silver proteinate-positive). Cytochemically the end wall appears identical to the unevenly thickened lateral wall. Electron cytochemical localization of aldehydes with silver proteinate reveals two distinct wall layers in comparison to the 3-layered lateral wall. Plasmodesmata are present in the end wall with a frequency of 15-20 per μm². A characteristic feature of end wall plasmodesmata is an expanded median cavity which is 0.12-0.15 μm in diameter. Frequently an electron-dense substance, whose chemical nature and origin are unknown, occludes the plasmodesmata.     

Elektronenoptische Untersuchungen anEndomycopsis fibuliger auf festen Nährböden

Summary The fine structure of the filamentous and mycelial cell ofEndomycopsis fibuliger grown on the agar medium and fixed in osmium tetroxide is described. The cell wall is relatively thick. Of particular interest is the occurrence of numerous vesicles underside of the cell wall proper. They have become dilated frequently at the cell surface, probably as a result of the amylase secretion. The wall presumably is penetrated by plasmodesmata. The plasmodesmata and the intercellular connections are associated with the endoplasmic reticulum. The cytoplasmic matrix is generally granular and contains nonmembrane-limited patches of lipoid.Mitochondria are numerous and variable in shape, and have been observed in different stages of development. The mitochondria profiles are delimited from the surrounding cytoplasm by single or sometimes double electron-dense lines.The double dark lines of a unit membrane at the nuclear surface are not always clear.     

An ultrastructural study of abscission zone cells with special reference to the mechanism of enzyme secretion

Summary The ultrastructural changes which occur during the foliar abscission ofImpatiens sultani Hook. andColeus blumei Benth. have been described. In both cases fracture of the separation zone results from a modification of the wall and cleavage along the line of the middle lamella. This process starts at the corners of the cells and in regions rich in plasmodesmata.During the period of wall breakdown, cellular integrity is maintained and the membrane degradation described by others was not observed. Plasmolysis studies confirmed that the separation zone cells retained their selective permeability characteristics until well after wall fracture. Quantitative data are presented to show that there is an increase in the frequency of rough endoplasmic reticulum, dictyosomes and dictyosome vesicles during the period when wall-hydrolyzing enzyme secretion is occurring. These changes are interpreted as reflecting an increase in demand for the secretory machinery of the endomembrane system. The possible involvement of plasmodesmata in this process is also discussed.     

Modified plasmodesmata inSorghum (Sorghum bicolor L Moench) leaf tissues infected by maize dwarf mosaic virus

The early acute response (EAR), a type of hypersensitive response, is defined by small chlorotic spots at the base of the youngest leaf of sorghum (Sorghum bicolor L. Moench) cultivar HOK, and usually appears within five days after inoculation with maize dwarf mosaic virus strain A (MDMV-A). These chlorotic spots become necrotic one to two days later and the leaf tissues are rapidly killed. In leaf tissues showing EAR, plasmodesmal fields contained many modified plasmodesmata of various sizes and structures within thickened cell walls. The membranous vesicles and tubules, derived from the extended terminal structures of modified plasmodesmata, were blocked by callose-like deposits in the area between the cell wall and plasmalemma. Also observed were two opposite-directed channels united via a central cavity at the middle lamella of the cell wall, one end of which was connected to the plasmalemma, but the other end sealed off to form a bulbous extension. The localized structure, an extraprotoplasmic sac containing aggregates of elongated virus-like particles associated with the modified plasmodesmata, was located between the plasmalemma and the cell wall. The sac was bound by membranes, and appeared to be sealed and completely excluded from the protoplasm. Extraprotoplasmic sacs appeared to be derived from the terminal extension of modified plasmodesmata, and these modification seem to be related to restriction of the viral spread.     

Stomata and plasmodesmata

Summary In developing epidermal tissue ofPhaseolus vulgare L. complete plasmodesmatal connections occurred between guard cells and epidermal cells and between sister guard cells of a stoma but they were not seen in fully differentiated tissue. However, incomplete, aborted plasmodesmata were occasionally seen in the common guard/epidermal cell wall, usually connected to the epidermal cell protoplast, in mature tissue. Plasmodesmatal connections between neighbouring epidermal cells were commonly observed in tissue at all stages of development. In all locations, the plasmodesmata were usually unbranched occurring singly or in small pit fields; very rarely branched, incomplete plasmodesmata were also seen in the wall between mature guard and epidermal cells. The significance of these findings were related to stomatal functioning and to the development of plasmodesmata in general.     

PLASMODESMATAL FREQUENCY AND OTHER STRUCTURAL ASPECTS OF ASSIMILATE COLLECTION AND PHLOEM LOADING IN LEAVES OF SONCHUS OLERACEUS (ASTERACEAE), A SPECIES WITH MINOR VEIN TRANSFER CELLS

Leaves of Sonchus oleraceus (Asteraceae) were examined with the electron microscope to determine plasmodesmatal frequencies and other structural features relating to the collection of photoassimilate and its subsequent loading into minor veins. Few plasmodesmata occur between mesophyll cells, which contain chloroplasts that are sometimes connected to both the plasmalemma and the tonoplast by membranous tubules. The minor veins consist of tracheary elements, sieve-tube members, vascular parenchyma cells, and companion cells. The latter two cell types are transfer cells, with some of the fingerlike wall ingrowths in companion cells being traversed lengthwise by plasmodesmata. The frequencies of plasmodesmata at the mesophyllbundle sheath boundary and within are higher at some interfaces than at corresponding interfaces in nine other species, including some that previously had been characterized as loading assimilate via the symplast. It is thus premature to designate all species containing transfer cells in their minor veins as loading assimilate only via the apoplast.     

A novel block of plant virus movement genes

Hibiscus green spot virus (HGSV) is a recently discovered and so far poorly characterized bacilliform plant virus with a positive‐stranded RNA genome consisting of three RNA species. Here, we demonstrate that the proteins encoded by the ORF2 and ORF3 in HGSV RNA2 are necessary and sufficient to mediate cell‐to‐cell movement of transport‐deficient Potato virus X in Nicotiana benthamiana. These two genes represent a specialized transport module called a ‘binary movement block’ (BMB), and ORF2 and ORF3 are termed BMB1 and BMB2 genes. In agroinfiltrated epidermal cells of N. benthamiana, green fluorescent protein (GFP)‐BMB1 fusion protein was distributed diffusely in the cytoplasm and the nucleus. However, in the presence of BMB2, GFP‐BMB1 was directed to cell wall‐adjacent elongated bodies at the cell periphery, to cell wall‐embedded punctate structures co‐localizing with callose deposits at plasmodesmata, and to cells adjacent to the initially transformed cell. Thus, BMB2 can mediate the transport of BMB1 to and through plasmodesmata. In general, our observations support the idea that cell‐to‐cell trafficking of movement proteins involves an initial delivery to membrane compartments adjacent to plasmodesmata, subsequent entry of the plasmodesmata cavity and, finally, transport to adjacent cells. This process, as an alternative to tubule‐based transport, has most likely evolved independently in triple gene block (TGB), double gene block (DGB), BMB and the single gene‐coded transport system.     

Unusual electron-dense dome associates with compound plasmodesmata in the embryo-suspensor of genus Sedum (Crassulaceae)

Plasmodesmata ensure the continuity of cytoplasm between plant cells and play an important part in the intercellular communication and signal transduction. During the development of the suspensor of both Sedum acre L. and Sedum hispanicum L., changes in the ultrastructure of plasmodesmata and adjoining cytoplasm are observed. Numerous simple plasmodesmata are present in the inner wall of the two-celled embryo separating the basal cell from the apical cell. From the early-globular to the torpedo stage of embryo development, the part of the wall separating the basal cell from the first layer of the chalazal suspensor cells is perforated by unusual, compound plasmodesmata. The role and the sort of transport through these plasmodesmata are discussed.     

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     

Ultrastructural characterization of apospory in Panicum maximum

The nucellar ultrastructure of apomictic Panicum maximum was analyzed during the meiocytic stage and during aposporous embryo sac formation. At pachytene the megameiocyte shows a random cell organelle distribution and sometimes only an incomplete micropylar callose wall. The chalazal nucellar cells are meristematic until the tetrad stage. They can turn into initial cells of aposporous embryo sacs. The aposporous initials can be recognized by their increased cell size, large nucleus, and the presence of many vesicles. The cell wall is thin with few plasmodesmata. If only a sexual embryo sac is formed, the nucellar cells retain their meristematic character. The aposporous initial cell is somewhat comparable to a vacuolated functional megaspore. It shows large vacuoles around the central nucleus and is surrounded by a thick cell wall without plasmodesmata. In the mature aposporous embryo sac the structure of the cells of the egg apparatus is similar to each other. In the chalazal part of the egg apparatus the cell walls are thin and do not hamper the transfer of sperm cells. Structural and functional aspects of nucellar cell differentiation and aposporous and sexual embryo sac development are discussed.     

The formation of cytoplasmic channels between tapetal cells inZea mays

Summary In this report we show that large cytoplasmic channels form between the tapetal cells ofZea mays (maize) during the period of tapetal cell differentiation. Tapetal cells are connected by plasmodesmata through their cellulosic cell walls prior to the first meiotic division of the meiocytes. As the tapetal cellulose wall is degraded at the onset of meiosis, both plasmodesmata and cytoplasmic channels measuring 50–200 nm are detectable between tapetal cells. By the time the meiotic tetrad is formed, the cytoplasmic channels are well-established and vary in size from 100–400 nm. The channels, with an average diameter of 200–300 nm, persist after the microspores are released from the callose wall and throughout the period of exine development in microsporogenesis. The channels could potentially allow for free exchange of cytoplasm and organelles. As the tapetal cells begin to pull apart and become vacuolate prior to microspore mitosis, the connecting channels are no longer detectable.