Symplasmic isolation ofChara vulgaris antheridium and mechanisms regulating the process of spermatogenesis

Summary The antheridium ofChara vulgaris L. is connected by plasmodesmata with the thallusvia a basal cell. Prior to the initiation of spermatozoid differentiation these plasmodesmata are spontaneously broken, resulting in symplasmic isolation of the antheridium.Premature plasmolytically evoked symplasmic isolation of the antheridium leads to a 2–4 fold reduction in the length of antheridial filaments and the elimination of 1–2 cell cycles from the first stage of spermatogenesis.Autoradiographic and cytophotometric studies have shown that, as a result of induced symplasmic isolation of the antheridium, endomitotic DNA synthesis was blocked both in the young manubria (after 24 hours) and in the capitular cells (after 48 hours). In the antheridial filaments DNA synthesis was inhibited together with either elimination of divisions and induction of spermatid differentiation or developmental block. We propose that breakage of plasmodesmata connecting the antheridium with the thallus is a signal which releases, in all antheridia, mechanisms that (i) block endomitotic DNA synthesis in the manubria, (ii) restrict the growth rate and the divisions of antheridial filament cells, and (iii) induce spermiogenesis in these antheridia in which the manubria attained the sufficient level of polyploidy.This work is supported by the Polish Academy of Sciences within the project CPBP 04.01.5.05.     

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.     

Plasmodesmal changes are related to different developmental stages of antheridia of Chara species

Summary During the development of the antheridia of Chara species, dynamic changes in the occurrence and ultrastructure of plasmodesmata are observed which are closely correlated to particular developmental phases and presumably regulate the morphogenetic events in the antheridia. The disappearance of plasmodesmata between shield cells and between shied cells and the basal cell leads to a cessation in symplasmic transport around the antheridum and determines its concentric or centrifugal character via centrally situated capitular cells. Unplugged plasmodesmata are present between fully synchronously developing antheridial filament cells and obviously coordinate the development of the cells. In the middle phase of spermiogenesis, rough endoplasmic reticulum in antheridial filaments passes uncompressed through wide plasmodesmata and provides an additional transport pathway for developmental control factors. Plugged plasmodesmata link cells of different types or cells of the same type which are at different phases of cell cycle and guarantee their individual development. The plugging of plasmodesmata is a reversible process that depends on the morphogenetic situation. Plasmodesmata connecting the basal cell and the subbasal cell as well as the basal cell and capitular cells are transformed successively from the simple into the complex type and might be the pathways for an import of gibberellins and nutrients into the strong sink tissues of the developing antheridium. There is a symplasmic connection between the antheridum and the thallus via a basal cell. Prior to the initiation of spermatozoid differentiation (spermiogenesis), plasmodesmata connecting the basal cell with a subbasal cell and the basal cell with capitular cells are spontaneously broken, resulting in symplasmic isolation of the antheridium that is probably a signal which triggers the induction of spermatozoid differentiation. Premature plasmolytically evoked symplasmic isolation of the antheridium leads to the elimination of 1 to 2 cell cycles from the proliferative stage of spermatogenesis. Autoradiographic studies demonstrate that both natural and induced symplasmic isolation drastically decreases the entry of isotopically labeled gibberellic acid into antheridia of Chara species that may be the consequence of the elimination of the hormone's transport through plasmodesmata.Correspondence and reprints: Department of Cytophysiology, University of ód, ulica Pilarskiego 14, 90-231 ód, Poland.Received March 11, 2002; accepted September 19, 2002; published online August 26, 2003     

The formation of symplasmic domains by plugging of plasmodesmata: a general event in plant morphogenesis?

Summary The plasmodesmal network was examined in multicellular protoplast-derived calluses of the dicotyledonSolanum nigrum which had not yet formed any visible adventitious organs and in globular proembryogenic structures developed from scutellar calluses of the monocotyledonMolinia caerulea. Electron microscopical analyses revealed that both calluses and proembryos consisted of small, undifferentiated cells. The interconnecting plasmodesmata at many cell interfaces were structurally inconspicuous in both systems; in particular cell walls, however, all plasmodesmata were occluded with an osmiophilic, dense material. As the blocking material was obviously located in the microchannels of the plasmodesmal cytoplasmic sleeves, the plugged plasmodesmata can be assumed to be nonfunctional. Thus, selective occlusion of all the plasmodesmata in specific cell walls resulted in the symplasmic disconnection of particular adjacent cells. Complex patterns of symplasmic continuity and discontinuity were established within the developing tissues. Some cells or groups of cells were entirely symplasmically disconnected from the surrounding cells by plugged plasmodesmata and might function as independent domains. However, blockage of plasmodesmata was achieved by the surrounding cells rather than by those cells belonging to the isolated domains. The demarcation of symplasmic domains might be a general prerequisite for differential morphogenesis, since they were found to be established very early in the course of morphogenetic processes.     

葫芦藓精子发生过程中胞间连丝与胞质桥的超微结构

从超微结构水平上对葫芦藓(Funaria hygrometrica Hedw.)精子发生过程中胞间连接系统的结构及其变化动态进行了研究.结果表明,同一区中的相邻生精细胞由大量胞质桥相连,而不同区的细胞之间则不存在胞质桥.胞间连丝存在于套细胞之间以及套细胞与生精细胞之间,但它在生精细胞间不存在.在精子器发生的后期,当精子细胞壁开始降解时,同一个精子器中所有的精子细胞似乎都由扩大的胞质桥相互连接.胞质桥一直保持到精子分化的后期,最终精子细胞同步分化成精子.胞间连丝与胞质桥具有不同的内部结、分布以及生物发生机制,这表明它们在精子器的发育过程中可能扮演着不同的角色.     

Plasmodesmata between synchronously and asynchronously developing cells of the antheridial filaments ofChara vulgaris L.

Summary Plasmodesmata connecting synchronously developing cells are filled with electron-transparent, homogenous ground cytoplasm. At the middle lamella, their average diameter is about 67 nm; the relative area occupied by plasmodesmata is calculated to be about 8 to 9% of the wall.Plasmodesmata occurring between cells which develop asynchronously are plugged by an electron-dense homogenous material. The plug fits tightly to the plasmalemma inside the plasmodesmatal canal. Occasionally (in 8% of the walls), the closing plugs are also found between synchronously dividing cells. Generally, the plugging takes place in the walls formed at the first stages of development of the antheridial filaments and is probably an irreversible process.It is supposed that the plugging of plasmodesmata is the cause of the appearance of two or more synchronous cell groups within a single filament.     

Autoradiographic studies on the role of plasmodesmata in the transport of gibberellin

Translocation of [¹⁴C]gibberellic acid into antheridial cells of Chara vulgaris L. was investigated in relation to the presence of symplasmic connections between the antheridium and the thallus. It was found that manubria, capitular cells, and antheridial filaments were about three-fold more strongly labelled in young antheridia connected to the thallus by plasmodesmata than in older antheridia in which spontaneous symplasmic isolation had occurred. Plasmolytically induced symplasmic isolation of young antheridia severely diminished the radioactivity of all the cells, down to the level characteristic for spontaneously isolated antheridia. It is concluded that plasmodesmata are the main channel of gibberellin transport into antheridia. The change in the character of symplasmic connections during the course of morphogenesis might, among other events, constitute a signal determining a shift of cell metabolism in a new direction, in response to a rapid change in gibberellin level.Abbreviations GA_(n) gibberellin (A_n) - GA₃ gibberellic acid - IAA indole-3-acetic acid This study was supported by the Polish Academy of Sciences research project CPBP 04.01.5.05.     

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.     

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.     

Translocation in the staminal hairs ofSetcreasea purpurea

Summary Investigations into plant intercellular communication were initiated through an examination of plasmodesmata and cell-to-cell passage of molecular probes in the staminal hairs ofSetcreasea purpurea. Plasmodesmata connecting staminal hair cells of small buds are filled with an electron-opaque homogenous material. To examine the permeation selectivity of plasmodesmata, molecular probes made up of fluorescein isothiocyanate (FITC) complexed with amino acids and peptides were injected into the staminal hair cells and the spread of these fluorescent molecules through the symplast, was monitored. Molecules composed of FITC complexed to single amino acids with polar and aliphatic R groups travel rapidly, while those which include peptides travel slowly. Dye molecules composed of an amino acid with an aromatic side group do not pass from cell to cell at all. It is hypothesized that the material occluding the plasmodesmata constitutes the diffusion barrier, by presenting a hydrophilic environment which allows passage of molecules with maximum molecular weights of 700–800 daltons, but which retains those with aromatic side groups.     

Biological role of endoreplication in the process of spermatogenesis inChara vulgaris L.

Summary During cell division in antheridial filaments ofChara vulgaris an increase in DNA content occurs in both shield cells and manubria within an antheridium, reaching 16C–64C and 8C–32C levels, respectively. Endoreplication ceases prior to the formation of spermatids and initiation of spermiogenesis, probably as a result of symplasmic isolation of the antheridium from the thallus. As the DNA content of the nuclei increases, the shield cells³H-leucine incorporation increases, and they grow intensively in the tangential plane. Translation decreases considerably after termination of shield cell growth. DNA content of mature manubria is half of that in shield cells, although their size is 10 times that of manubria. Translational activity of manubria also increases as DNA content rises and cells grow. However, during spermiogenesis, this activity remains at its maximum, which is associated with the secretory function of the manubria. Spermiogenesis is also accompanied by far-reaching ultrastructural changes within the manubrial cytoplasm.The level of endopolyploidy in both shield cells and manubria of antheridia formed in the spring is higher by one replication cycle, than in autumnal antheridia. AMO-1618, at a concentration of 10^–5M reduces the DNA content in the autumnal manubria. The higher the manubrial level of endopolyploidy in spermiogenesis, the greater their size, and the higher the translational activity and number of joined spermatids. The number of spermatozoids in the antheridium is also positively correlated with the internal volume of an antheridium, which is itself dependent on the endopolyploidy level of shield cells.The results obtained confirm the assumption that endoreplication favours the higher growth dynamics and potential translational activity, which occurs in the dynamic growth phase only in shield cells, while in manubria, i.e. cells producing substances necessary to spermatozoids development, it remains high until the end of spermiogenesis.     

Absence of plasma membrane H+-ATPase in plasmodesmata located in pit-fields of the young reactive pulvinus ofMimosa pudica L.

Summary Immunocytochemical techniques were employed to study the spatial distribution of the plasma membrane H⁺-ATPase within various cell types of the young reactive primary pulvinus ofMimosa pudica L. These cells were interconnected by large numbers of plasmodesmata, being concentrated within pit-fields. Although we could routinely detect evidence of the H⁺-ATPase along the plasma membrane, immunolabelling was rarely, if ever, observed along the plasma membranes of the plasmodesmata. This finding is discussed with respect to the likely specialized supramolecular structure of the plasmodesma.Abbreviations SEL size exclusion limit of plasmodesmata     

Occurrence of plasmodesmata between differentiating vessels and other xylem cells inSorbus torminalis L. Crantz and their fate during xylem maturation

Summary The development of pit-pairs between differentiating xylem cells has been examined by transmission electron microscopy in young shoots ofSorbus torminalis. In some vessel-to-tracheid pits, as well as in previously studied intertracheid pits, a thickening of the pit membrane containing branched plasmodesmata was observed. A secondary wall-like cap was deposited over the thickening prior to cytoplasmic autolysis; some plasmodesmata, parallel to the plane of section, appeared to perforate the cap. At the end of the cell maturation stage, the central part of the primary wall thickening was hydrolysed, while the cap, including plasmodesmata remnants, appeared unaltered. In half-bordered pit-pairs between a parenchyma cell and a vessel or a tracheid, similar structures could be observed beside the conducting elements. When the vessel or tracheid matured, sealing of the pit membrane plasmodesmata resulted from the formation of a protective layer on the parenchyma-side rather than from the deposition of a cap on the conducting cell-side. These observations provide the first information on the presence of symplasmic connections in pits between differentiating vessels and neighbouring xylem cells. InS. torminalis, xylem differentiation is probably highly coordinated within a symplasmic domain; the persistence of such connections may account for the lack of specialization ofSorbus wood.     

葫芦藓精子发生过程中胞间连丝与胞质桥的超微结构

从超微结构水平上对葫芦藓(Funaria hygrometrica Hedw.)精子发生过程中胞间连接系统的结构及其变化动态进行了研究。结果表明,同一区中的相邻生精细胞由大量胞质桥相连,而不同区的细胞之间则不存在胞质桥。胞间连丝存在于套细胞之间以及套细胞与生精细胞之间, 但它在生精细胞间不存在。在精子器发生的后期,当精子细胞壁开始降解时,同一个精子器中所有的精子细胞似乎都由扩大的胞质桥相互连接。胞质桥一直保持到精子分化的后期,最终精子细胞同步分化成精子。胞间连丝与胞质桥具有不同的内部结、分布以及生物发生机制,这表明它们在精子器的发育过程中可能扮演着不同的角色。     

Stigma development in Nicotiana tabacum. Cell death in transgenic plants as a marker to follow cell fate at high resolution

Pistil development was studied in transgenic tobacco plants in which the stigma is ablated by expression of a stigma-specific cytotoxic gene. These plants offer a tool to investigate the process of differentiation of the secretory zone, in that cell death caused by barnase activity provides a marker to follow cell fate at high resolution. After fusion of the carpel walls in the region most distal from the ovary, the epidermal cells begin to divide in both wild-type and stigmaless plants. Divisions of the L1 layer of the pistil are immediately followed by the morphogenetic events that lead to three different cell types: rounded-angular cells showing an equal number of anti- and periclinal divisions, cells that are more oblong forming the transition zone, and the square cells of the transmitting tissue dividing mostly anticlinally with respect to the original carpel wall. In the stigmaless plants, cell death caused by the expression ofSTIG 1-barnase begins at stage –1 and proceeds gradually, but is always associated with round epidermal cells and with angular-rounded cells underneath them. Studies at the ultrastructural level show that cell death caused by barnase activity occurs first in solitary cells and gradually extends to groups of cells.In situ hybridizations using the STIG 1 RNA probe in wild-type pistils confirm these results. Most likely, the cells in whichSTIG 1 is expressed are those that have just differentiated into the secretory cell type. Our results indicate that the transition zone or neck is autonomously differentiated from the secretory zone and the transmitting tissue. Furthermore, our results indicate that in both wild-type and stigmaless pistils secretion of lipids most likely occurs through the plasmodesmata. This observation suggests that bulk transport can occur via plasmodesmata.     

Subcellular localization of ubiquitin in plant protoplasts and the function of ubiquitin in selective degradation of outer-wall plasmodesmata in regenerating protoplasts

Immunocytochemical localizations in Vicia faba L. protoplasts and cultures of regenerating Solanum nigrum L. protoplasts support former observations that in plant cells ubiquitin occurs within the cytoplasm, the nucleus, the chloroplasts and at the plasmalemma, but not within the vacuole or the cell wall. Immunoresponses were also observed within mitochondria and associated with the endoplasmic reticulum, which is in accordance with previous findings on animal cells. Moreover, the tonoplast membrane system was found to be labelled. For regenerating S. nigrum protoplasts, evidence is given that ubiquitin plays a role in selective degradation even of whole subcellular structures. Most of the discontinuous plasmodesmata formed in the newly deposited outer cell walls during the early stages of culture disappear later on, except for those near the periphery of division walls or of non-division walls, which are probably used for the formation of continuous cell connections during further culture. Outer-wall plasmodesmata which are destined to disappear show high immunoreactivity to ubiquitin antibody, but no conspicuous immunolabelling was observed with the remaining plasmodesmata. Thus, the selective disintegration of whole plasmodesmatal structures is obviously regulated by ubiquitination of plasmodesmatal proteins. A model for the mechanism of degradation of outer-wall plasmodesmata during extension growth of the cell wall is presented.Dedicated to Professor Dr. Andreas Sievers on the occasion of his retirementThis work was supported by grants to R. K. (Deutsche Forschungsgemeinschaft) and to M. S. (Bennigsen-Foerder Preis des Landes Nordrhein-Westfalen). We thank Dipl.— Biol. Kirsten Leineweber for help with the V. faba protoplast isolation and Dr. Olaf Parge, Institut für Psychologie und Sozialforschung, Kiel, Germany, for giving assistance with the statistical analysis.     

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.     

Studies on the leaf of Amaranthus retroflexus (Amaranthaceae): ultrastructure,plasmodesmatal frequency,and solute concentration in relation to phloem loading

Both the mesophyll and bundle-sheath cells associated with the minor veins in the leaf of Amaranthus retroflexus L. contain abundant tubular endoplasmic reticulum, which is continuous between the two cell types via numerous plasmodesmata in their common walls. In bundle-sheath cells, the tubular endoplasmic reticulum forms an extensive network that permeates the cytoplasm, and is closely associated, if not continuous, with the delimiting membranes of the chloroplasts, mitochondria, and microbodies. Both the number and frequency of plasmodesmata between various cell types decrease markedly from the bundle-sheath — vascular-parenchyma cell interface to the sicve-tube member — companion-cell interface. For plants taken directly from lighted growth chambers, a stronger mannitol solution (1.4 M) was required to plasmolyze the companion cells and sieve-tube members than that (0.6 M) necessary to plasmolyze the mesophyll, bundle-sheath, and vascular-parenchyma cells. Placing plants in the dark for 48 h reduced the solute concentration in all cell types. Judging from the frequency of plasmodesmata between the various cell types of the vascular bundles, and from the solute concentrations of the various cell types, it appears that assimilates are actively accumulated by the sieve-tube — companion-cell complex from the apoplast.     

Studies on graft unions. I. Plasmodesmata between cells of plants belonging to different unrelated taxa

Summary In order to answer the question whether plasmodesmata exist between the cells of stock and scion in a graft union of higher plants, a heteroplastic graft has been selected with species-specific cell markers.Vicia faba was used as scion andHelianthus annuus as stock. In the mixed callus of the graft union, individual cells of the two partners differ in the structure of the nuclei, plastids and microbodies. In the fused cell walls betweenVicia andHelianthus cells, plasmodesmata interconnect the protoplasts of the unrelated cells. Two types of plasmodesma occur: single strands and branched connections. The fine structure of the interspecific cell bridges, which have been secondarily formed in non-division walls, is similar to that of normal plasmodesmata in division walls. Some questions concerning compatibility/incompatibility in the heterograft are discussed.     

Integument cell differentiation in dandelions (<Emphasis Type="Italic">Taraxacum</Emphasis>, Asteraceae,Lactuceae) with special attention paid to plasmodesmata

The aim of the paper is to determine what happens with plasmodesmata when mucilage is secreted into the periplasmic space in plant cells. Ultrastructural analysis of the periendothelial zone mucilage cells was performed on examples of the ovule tissues of several sexual and apomictic Taraxacum species. The cytoplasm of the periendothelial zone cells was dense, filled by numerous organelles and profiles of rough endoplasmic reticulum and active Golgi dictyosomes with vesicles that contained fibrillar material. At the beginning of the differentiation process of the periendothelial zone, the cells were connected by primary plasmodesmata. However, during the differentiation and the thickening of the cell walls (mucilage deposition), the plasmodesmata become elongated and associated with cytoplasmic bridges. The cytoplasmic bridges may connect the protoplast to the plasmodesmata through the mucilage layers in order to maintain cell-to-cell communication during the differentiation of the periendothelial zone cells.