Directional cell-to-cell communication in theArabidopsis root apical meristem II. Dynamics of plasmodesmatal formation

Summary Cell development in the root apical meristem is thought to be regulated by position-dependent information, but as yet, the underlying mechanism for this remains unknown. In order to examine the potential involvement of the symplasmic transmission of positional signals, plasmodesmatal frequency and distribution was quantitatively analyzed in root apical meristem cell walls ofArabidopsis thaliana during root development. A consistent distribution pattern of plasmodesmata was observed in the root apex over four weeks. While cells within initial tiers were uniformly interconnected, more symplasmic connections between the initial tiers and their immature-cell (primary-meristem) derivatives were observed than within the initial tiers. Immature cells were connected across transverse walls by primary plasmodesmata according to a tissue-specific pattern. Cells of the immature vascular tissue and cortex had the highest plasmodesmatal frequencies, followed by the immature epidermis and root cap. Although the numbers of plasmodesmata in transverse walls (primary plasmodesmata) was reduced in all tissues as the root aged, the tissue-specific distribution remained constant. The extent of symplasmic coupling across the boundaries of each tissue appeared to be limited by fewer secondary plasmodesmata in longitudinal walls. The frequency of all plasmodesmata decreased as the root aged. The primary plasmodesmata within each tissue increased at one week and then dramatically decreased with root age; the frequency of secondary plasmodesmata in longitudinal walls also decreased, but more gradually. These findings are discussed with respect to the roles likely played by plasmodesmata in facilitating transport of position-dependent information during root development.     

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.     

Directional cell-to-cell communication in theArabidopsis root apical meristem I. An ultrastructural and functional analysis

Summary As a foundation for studies on directional intercellular communication and its regulation in apical development, the network of plasmodesmata inArabidopsis root apical meristems was characterized by quantitative electron microscopy and dye-coupling analysis, using symplasmic probes, and real-time imaging in confocal laser scanning microscopy. A tissue-specific plasmodesmatal network, which interconnected the cells in the root apical meristem, was characterized by the following features, (a) Plasmodesmatal distribution and density were found to be tissue-specific, (b) Primary and secondary plasmodesmata were differentially grouped and regulated. Primary plasmodesmata were formed in large numbers in the transverse walls of each tissue, and were subject to deletion during cell differentiation. Secondary plasmodesmata were mostly distributed in longitudinal walls between cell files and common walls between neighboring tissues; they also provided a symplasmic path between different initial tiers in the meristem. Small fluorescent tracers moved through the plasmodesmatal network of the root apical meristem in two distinct phases. At low concentrations molecules trafficked in a non-tissue-specific manner, whereas at higher concentrations, their distribution reflected the presence of tissue-specific movement consistent with plasmodesmatal distribution. These findings are discussed in terms of the role of tissue-specific plasmodesmatal domains in the control of root development.     

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.     

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.     

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.     

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.     

Distribution of tobamovirus movement protein in infected cells and implications for cell-to-cell spread of infection

The intercellular and intracellular distribution of the movement protein (MP) of the Ob tobamovirus was examined in infected leaf tissues using an infectious clone of Ob in which the MP gene was translationally fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria. In leaves of Nicotiana tabacum and N. benthamiana, the modified virus caused fluorescent infection sites that were visible as expanding rings. Microscopy of epidermal cells revealed subcellular patterns of accumulation of the MP:GFP fusion protein which differed depending upon the radial position of the cells within the fluorescent ring. Punctate, highly localized fluorescence was associated with cell walls of all of the epidermal cells within the infection site, and apparently represents association of the fusion protein with plasmodesmata; furthermore, fluorescence was retained in cell walls purified from infected leaves. Within the brightest region of the fluorescent ring, the MP:GFP was observed in irregularly shaped inclusions in the cortical regions of infected cells. Fluorescent filamentous structures presumed to represent association of MP:GFP with microtubules were observed, but were distributed differently within the infection sites on the two hosts. Within cells containing filaments, a number of fluorescent bodies, some apparently streaming in cytoplasmic strands, were also observed. The significance of these observations is discussed in relation to MP accumulation, targeting to plasmodesmata, and degradation.     

Characterization of the Cell Wall Microdomain Surrounding Plasmodesmata in Apple Fruit

In fleshy fruits ripening is generally associated with a loss in tissue firmness resulting from depolymerization of wall components and separation of adjacent cells. In the regions of the wall that contain plasmodesmata, the usual sequences of ripening events, i.e. depolymerization of the middle lamellae and splitting of the walls, are not observed. In the present study we attempted to characterize in apple (Malus domestica Borkh.) fruit the structural microdomain of the cell wall that surrounds the plasmodesmata by in muro visualization of the cell wall components. Anionic sites of galacturonic acids were labeled with cationic gold. Low-esterified homogalacturonans were labeled with the monoclonal antibody JIM 5. In addition, a polyclonal antibody directed toward [beta](1->3)-glucopyranose was used to target callose in situ. The results indicated that the plasmodesmata-wall complexes were surrounded by a pectic microdomain. This domain was composed of low-esterified homogalacturonans that were not involved in calcium cross-bridging but were probably surrounded by a cationic environment. These structural features may result in the prevention of normal cell wall separation in regions containing plasmodesmata. However, observations by low-temperature scanning electron microscopy suggested that splitting of these walls ruptured the plasmodesmata and ultimately resulted in the spatial separation of adjacent cells.     

Intercellular communication inAzolla roots: II. Electrical coupling

Summary There is a predictable and well defined variation in numbers of plasmodesmata in roots ofAzolla. As the apical cell of the root ages, it lays down walls with progressively fewer plasmodesmata, thereby gradually cutting itself off from the rest of the root (Gunning 1978). Electrical coupling was examined between the apical cell and an adjacent merophyte in roots of various lengths. The apical cell becomes increasingly electrically isolated from the rest of the root as it ages. Electrical coupling is strongly correlated with the number of the plasmodesmata between the coupled cells. The resistance of a plasmodesma, as estimated from equivalent electrical circuits, was 150–600 times more resistive than a value based on theoretical considerations. No evidence was found for a change in the physiology of plasmodesmata as the root ages. Coupling experiments, both on root hairs and at the apex, gave some suggestion that plasmodesmata may be less resistive towards the apical cell than away from it.     

Cell number,cell growth,antheridiogenesis, and callose amount is reduced and atrophy induced by deoxyglucose in <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

Fluorescence staining and morphometrical measurements revealed that callose was a component of newly formed cell plates of symmetrically dividing cells and asymmetrically dividing antheridial mother cells during gibberellic acid-induced antheridiogenesis as well as in walls of young growing cells of Anemia phyllitidis gametophytes. Callose in cell walls forms granulations characteristic of pit fields with plasmodesmata. 2-deoxy-d-glucose (DDG), eliminated callose granulations and reduced its amount estimated by measurements of fluorescence intensity. This effect was accompanied by reduction of antheridia and cell numbers as well as size and atrophy of particular cells and whole gametophytes. It is suggested that inhibition of glucose metabolism and/or signalling, might decrease callose synthesis in A. phyllitidis gametophytes leading to its elimination from cell plates of dividing cells and from walls of differentiating ones as well as from plasmodesmata resulting in inhibition of cytokinesis, cell growth and disruption of the intercellular communication system, thus disturbing developmental programs and leading to cell death.     

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.     

Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves

Summary The sink-source transition in tobacco leaves was studied noninvasively using transgenic plants expressing the green-fluorescent protein (GFP) under control of theArabidopsis thaliana SUC2 promoter, and also by imaging transgenic plants that constitutively expressed a tobacco mosaic virus movement protein (MP) fused to GFP (MP-GFP). The sink-source transition was measured on intact leaves and progressed basipetally at rates of up to 600 m/h. The transition was most rapid on the largest sink leaves. However, leaf size was a poor indicator of the current position of the sink-source transition. A quantitative study of plasmodesmatal frequencies revealed the loss of enormous numbers of simple plasmodemata during the sink-source transition. In contrast, branched plasmodesmata increased in frequency during the sink-source transition, particularly between periclinal cell walls of the spongy mesophyll. The progression of plasmodesmal branching, as mapped by the labelling of plasmodesmata with MP-GFP fusion, occurred asynchronously in different cell layers, commencing in trichomes and appearing lastly in periclinal cell walls of the palisade layer. It appears that dividing cells retain simple plasmodesmata for longer periods than nondividing cells. The rapid conversion of simple to branched plasmodesmata is discussed in relation to the capacity for macromolecular trafficking in developing leaf tissues.     

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.     

Scanning electron microscopy in nematode-induced giant transfer cells.

A study of giant cells induced by the root-knot nematode, Meloidogyne incognita, in roots of Impatiens balsamina was made by scanning electron microscopy. The cytoplasmic contents of giant cells were removed by a procedure based on KOH digestion, to reveal inner wall structure. Wall ingrowths typical of transfer cells are present in giant cells from six days onwards after induction. They develop on walls adjacent to vascular tissues, and their distribution and development was examined. Pit fields contianing plasmodesmata become elaborated in walls between giant cells, but pit fields are lost between giant cells and cells outside them. The distribution of plasmodesmata in pit fields suggests that de novo formation of plasmodesmata occurs in walls between giant cells. Various aspects of giant cell formation and function are discussed and wall ingrowth development is compared in giant cells and normal transfer cells.     

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.     

SECONDARY PULVINUS OF ROBINIA PSEUDOACACIA (LEGUMINOSAE): STRUCTURAL AND ULTRASTRUCTURAL FEATURES

The structure of the secondary pulvinus of Robinia pseudoacacia has been examined together with ultrastructural features of motor cells both in open and closed pulvini, to identify ultrastructural changes associated with leaflet movement. Pulvini have a central vascular core bordered by thick-walled collenchyma cells, which in turn are surrounded by several layers of cortical parenchyma cells. Cortical motor cells exhibit ultrastructural features similar to those reported in homologous cells of other pulvini. The vacuolar compartment contains two kinds of vacuoles: nontannin vacuoles, which change both in number and size during leaflet movement, and tannin vacuoles, which may act as an ion reservoir. No differences in wall thickness were found between flexor and extensor motor cells. Thick walls of collenchyma cells show numerous pits with plasmodesmata through which the phloem parenchyma cells and the inner cortical motor cells are connected. Tannin vacuoles and calcium oxalate crystals are common inclusions of phloem parenchyma cells. The tissue arrangement and the occurrence of pits with plasmodesmata in the central cylinder cells provide evidence of symplastic continuity through the central cylinder between the extensor and flexor regions of the motor organs. The greater amplitude of Robinia leaflet movements may be related to the extension of motor regions, the scarcity of lignification in the central vascular core, and the thin flexor walls.     

Directional cell-to-cell communication in theArabidopsis root apical meristem III. Plasmodesmata turnover and apoptosis in meristem and root cap cells during four weeks after germination

Summary Plasmodesmata frequency and distribution in root cap cells ofArabidopsis thaliana root tips were characterized during four weeks after germination to understand the symplasmic control of apoptosis. Apoptotic cells in some of the root apical-meristem cells and in root cap cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reaction and characterized by electron microscopy. Starting at the second week after germination, cells in the outermost layers of the root cap showed typical apoptotic features, including nuclear DNA fragmentation, chromatin condensation, cytoplasmic vacuolation, and organelle destruction. Intercellular connections, indicated by the frequency and number of plasmodesmata per cell length, were significantly reduced in the walls of outer root cap cells. This shows that cells become symplasmically isolated during the apoptosis process. In apoptotic root cap cells, the majority of nonfunctional plasmodesmata were observed to be associated with degenerated endoplasmic reticulum; this state was prior to the detection of any nuclear DNA fragmentation. Other nonfunctional plasmodesmata were sealed by heterogeneous cell wall materials. However, in immature epidermal and cortical cells in 4-week-old arrested roots the endoplasmic reticulum associated with plasmodesmata became disconnected as a result of protoplast condensation and shrinkage. No degenerated endoplasmic reticulum was observed in these cells. These observations suggest that the apoptotic processes in the root body and the root cap are different.     

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     

SOME ULTRASTRUCTURAL OBSERVATIONS ON ENDODERMAL CELL DEVELOPMENT IN ZEA MAYS ROOTS

The fine structure of primary, secondary, and tertiary stages of Zea endodermal cell development was investigated. The casparian strip formed in situ in the anticlinal walls and remained at a fixed point relative to the endodermis-pericycle boundary. The only protoplasmic structure that had a constant spatial association with the developing strip was the plasmalemma. Plasmodesmata appeared to be more numerous on the tangential walls than on radial walls; only rarely were they located in the casparian strip. The suberized lamella developed on inner and outer tangential walls before it appeared on the radial walls. No cytoplasmic organelles were found to have any particular spatial association with this layer. The suberized lamella was about 0.04 μm thick except near plasmodesmata and along the adaxial margin of the casparian strip, where it was thicker. Occasionally it failed to form along the abaxial margin of the strip. The adherent affinity between plasmalemma and casparian strip was lost after the strip was covered by suberized lamella. The secondary wall became asymmetrically thickened by differential deposition of successive lamellae. A thin layer of secondary wall material extended across the floor of each pit. Pit cavities often contained mitochondria, and plasmodesmata were restricted to the pits. The plasmodesmata were constricted where they entered the thin layer of secondary wall material and where they penetrated the suberized lamella. The various stages of cell development tended to be asynchronous. No passage cells were observed. Endodermal cell development in Zea closely resembles that described for barley.