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.     

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. II. Capitular cells

In young antheridia, the structure of capitular cells is typical of meristematic cells. The cytoplasm is characterized by poorly developed ER system, numerous free ribosomes, active Golgi apparatus and plastids at the stage of proplastids. In the period of mitotic divisions, i.e. during formation of the initial cells of antheridial filaments, the nuclei of capitular cells have a changing structure. When capitular cells stop budding leading to the formation of successive antheridial filaments. DNA content in the nucleus is at 2C-4C level. The nucleolus with nucleolonema-like structure becomes gradually smaller in the course of the development of the anteheridium. During spermiogenesis capitular cells are vacuolated, cytoplasm contains numerous polysomes, mitochondria assume condensed structure, the incorporation of 3N-uridine and of labelled aminoacids increases. It has been suggested that capitular cells collaborate with other antheridial cells in the regulation of the course of spermiogenesis.     

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     

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.     

PIN2-like proteins may contribute to the regulation of morphogenetic processes during spermatogenesis in <Emphasis Type="Italic">Chara vulgaris</Emphasis>

Key message

PIN2-like auxin transporters are expressed, preferentially in a polarized manner, in antheridial cells of freshwater green alga Chara vulgaris , considered to be the closest relative of the present-day land plants.

Abstract

Chara vulgaris represents a group of advanced multicellular green algae that are considered as the closest relatives of the present-day land plants. A highly specialized structure of its male sex organs (antheridia) includes filaments consisting of generative cells, which after a series of synchronous divisions transform into mature sperm, and non-generative cells comprising outer shield cells, cylindrical manubria, and central complex of capitular cells from which antheridial filaments arise. Immunofluorescence observations indicate that PIN2-like proteins (PIN2-LPs), recognized by antibodies against PIN-FORMED2 (PIN2) auxin transporter in Arabidopsis thaliana, are expressed in both types of antheridial cells and, in most of them, preferentially accumulate in a polarized manner. The appearance of PIN2-LPs in germ-line cells is strictly confined to the proliferative period of spermatogenesis and their quantities increase steadily till antheridial filaments reach the 16-celled stage. An enhanced level of PIN2-LPs observed in the central cell walls separating two asynchronously developing parts of antheridial filaments (characterized by the plugged plasmodesmata) is correlated with an enhanced deposition of callose. Intense PIN2-LPs immunofluorescence maintained in the capitular cells and its altering polarity in manubria suggest a pivotal role of these cells in the regulation of auxin transport directionality during the whole time of antheridial ontogenesis. Immunohistochemical staining of IAA revealed a clear-cut correspondence between localization sites of auxins and PIN2-LPs. It seems probable then that a supplementary developmental mechanism has evolved in Chara, by which all antheridial elements may be integrated at the supra-cellular level via plasma membrane-targeted PIN2-LPs and auxin-mediated processes.

     

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.     

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.     

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     

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.     

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.     

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.     

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。     

Cytochemical Localization of Pectinase Activity in Pollen Mother Cells of Tobacco During Meiotic ProphaseⅠand Its Relation to the Formation of Secondary Plasmodesmata and Cytoplasmic Channels

Pectinase activity was localized at the ultrastructural level in pollen mother cells of tobacco (Nicotiana tabacum L.) during meiotic prophaseⅠto elucidate its role in the biogenesis of secondary plasmodesma (sPD) and cytoplasmic channel (CC). At the leptotene stage the enzyme was mainly present in the cisternae of smooth endoplasmic reticulum (SER) and their derived vesicles, but absent in the Golgi body and Golgi vesicles. Later at the zygotene stage, when sPDs and CCs were actively formed, strong pectinase activity was observed not only in the SER cisternae and their derived vesicles but also in the cell wall, especially in the vicinity of or within both simple and branched plasmodesmata, notably along the middle lamellae, which also characterized the sites of CCs being formed. The presence of exocytotic vesicles containing reaction products suggests that pectinase shares the same excretive pathway as that used by cellulase for its delivery into the wall, i.e. in active form via smooth endoplasmic reticulum (ER) and its derived vesicles by exocytosis. In combination with cellulase, pectinase also promotes the secondary formation of plasmodesmata and CCs by specifically digesting the pectin in middle lamella.     

Reorganization of the endoplasmic reticulum in epidermal cells of onion bulb scales after cold stress: Involvement of cytoskeletal elements

In the epidermal cells of onion (Allium cepa L.) bulb scales the endoplasmic reticulum (ER) can be subdivided into three domains: a peripheral tubular network, cisternae, and long tubular strands. The latter are the form in which the ER is moved in onion cells. During cold treatment the arrangement of the three domains changes drastically. The cisternae and long tubular strands disintegrate into short ER tubules which show rapid agitational motion. Long-distance movement is inhibited. The peripheral tubular ER network is presumably retained during cold treatment. Rewarming of previously chilled bulb scales initiates the reorganization of the ER into the three domains. The ER is partly relocated during recovery from cold treatment. Redistribution and reorganization of the ER is not affected by the microtubule-destabilizing herbicides oryzalin and trifluralin (5 M). Cytochalasin D (2M), however, inhibits not only the relocation of ER material, as is evident by the absence of long tubular ER strands, but also the movement of other cell organelles. The latter cluster on top of the cisternae in a manner which is characteristic of treatment with the actin-filament inhibitor. The array of actin filaments is similar in unstressed, cold-treated cells, and cells which recover from low temperatures in the presence of oryzalin or tap water alone. In the presence of cytochalasin D the actin filaments are severely fragmented. The results indicate that low temperatures most likely influence either the interaction of the force-generating system, probably myosin, with actin filaments, or the force-generating mechanism of the actomyosin-driven intracellular movement, but do not affect actin-filament integrity.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum     

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.     

Structural interactions of actin filaments and endoplasmic reticulum in honeybee photoreceptor cells

Summary Fluorescent phallotoxins and heavy meromyosin were used to reveal the organization of the actin cytoskeleton in honeybee photoreceptor cells, and the relationship of actin filaments to the submicrovillar, palisade-like cisternae of the endoplasmic reticulum (ER). Bundles of unipolar actin filaments (pointed end towards the cell center) protrude from the microvillar bases and extend through cytoplasmic bridges that traverse the submicrovillar ER. Within the cytoplasmic bridges, the filaments are regularly spaced and tightly apposed to the ER membrane. In addition, actin filaments are deployed close to the microvillar bases to form a loose web. Actin filaments are scarce in cell areas remote from the rhabdom; these areas contain microtubule-associated ER domains. The results suggest that the actin system of the submicrovillar cytoplasm shapes the submicrovillar ER cisternae, and that the distinct ER domains interact with different cytoskeletal elements.     

Evolution of the endoplasmic reticulum during spermiogenesis of the rooster: an electron microscopic study

The endoplasmic reticulum (ER) of rooster's spermatids was analyzed during spermiogenesis, which was subdivided into eight distinct steps on the basis of changes observed with the electron microscope in the nucleus, acrosome-perforatorium system, manchette, and flagellum. In steps 1 and 2, spermatids' ER cisternae presented the following specializations: A loose network of tubular cisternae was distributed throughout the cytoplasm. Six to eight tight networks of anastomosed tubular cisternae parallel to each other were closely stacked to form a discoid body (1.5-2.5 microns in diameter and 0.5-0.8-micron thick) in which spheroidal vesicles (0.4 micron in diameter) were inserted. Close to and connected with this body, called the alveolar body, there was a stack of annulate lamellae. Large, flattened ER cisternae were seen singly or in piles of two or three running parallel to the nuclear surface. A collection of tubular ER cisternae faced plaques of thickened plasma membranes. These elements of the ER system appear continuous with each other. During steps 3-5 of spermiogenesis, no modification of the alveolar body-annulate lamellae complex was noted; the large flattened ER cisternae disappeared, however, and the broad network of tubular cisternae developed markedly. During steps 6 and 7, the latter network of tubular cisternae fragmented into vesicles that swelled to give a vacuolated appearance to the cytoplasm. The alveolar body-annulate lamellae complex remained visible until late step 7, when it disintegrated just before spermiation. Thus the system of ER cisternae underwent marked structural modifications during spermiogenesis.     

Cell sizes and DNA level in antheridial filaments in relation to the initiation of spermatozoid differentiation in Chara vulgaris L

The observations carried out indicate that the exclusion of the S phase initiation from the course of telophase of the last mitotic division in the antheridial filaments of Chara vulgaris, leading to the formation of spermatids is not a simple result of the cell size reduction, gradually accomplished in the course of the successive cell cycles (of S+G2+M type). This critical moment of spermatogenesis is probably induced by the regulators operating at the level of an antheridium. In the conditions of the long (3-5 days) darkness resulting in the cell cycle arrest in antheridial filaments at the early stage of G2 phase there is detected the operation of some additional mechanisms synchronizing spermatogenesis, which enable some retarded antheridial filaments to pass the critical control points and to enter into the process of spermiogenesis insensitive to the lack of the light. The initiation of the differentiation is accomplished either after the cell division induced by the hypothetic inductors of spermiogenesis or -- more rarely -- with omitting mitosis, i.e. in the cells containing 2C DNA.     

The Ins and Outs of Nondestructive Cell-to-Cell and Systemic Movement of Plant Viruses

Propagation of viral infection in host plants comprises two distinct and sequential stages: viral transport from the initially infected cell into adjacent neighboring cells, a process termed local or cell-to-cell movement, and a chain of events collectively referred to as systemic movement that consists of entry into the vascular tissue, systemic distribution with the phloem stream, and unloading of the virus into noninfected tissues. To achieve intercellular transport, viruses exploit plasmodesmata, complex cytoplasmic bridges interconnecting plant cells. Viral transport through plasmodesmata is aided by virus-encoded proteins, the movement proteins (MPs), which function by two distinct mechanisms: MPs either bind viral nucleic acids and mediate passage of the resulting movement complexes (M-complexes) between cells, or MPs become a part of pathogenic tubules that penetrate through host cell walls and serve as conduits for transport of viral particles. In the first mechanism, M-complexes pass into neighboring cells without destroying or irreversibly altering plasmodesmata, whereas in the second mechanism plasmodesmata are replaced or significantly modified by the tubules. Here we summarize the current knowledge on both local and systemic movement of viruses that progress from cell to cell as M-complexes in a nondestructive fashion. For local movement, we focus mainly on movement functions of the 30 K superfamily viruses, which encode MPs with structural homology to the 30 kDa MP of Tobacco mosaic virus, one of the most extensively studied plant viruses, whereas systemic movement is primarily described for two well-characterized model systems, Tobacco mosaic virus and Tobacco etch potyvirus. Because local and systemic movement are intimately linked to the molecular infrastructure of the host cell, special emphasis is placed on host factors and cellular structures involved in viral transport.