Secondary Plasmodesmata Research Articles

Plasmodesmata dynamics in bryophyte model organisms: secondary formation and developmental modifications of structure and function

Main conclusionDeveloping bryophytes differentially modify their plasmodesmata structure and function. Secondary plasmodesmata formation via twinning appears to be an ancestral trait. Plasmodesmata networks in hornwort sporophyte meristems resemble those of angiosperms.All land-plant taxa use plasmodesmata (PD) cell connections for symplasmic communication. In angiosperm development, PD networks undergo an extensive remodeling by structural and functional PD modifications, and by postcytokinetic formation of additional secondary PD (secPD). Since comparable information on PD dynamics is scarce for the embryophyte sister groups, we investigated maturating tissues of Anthoceros agrestis (hornwort), Physcomitrium patens (moss), and Marchantia polymorpha (liverwort). As in angiosperms, quantitative electron microscopy revealed secPD formation via twinning in gametophytes of all model bryophytes, which gives rise to laterally adjacent PD pairs or to complex branched PD. This finding suggests that PD twinning is an ancient evolutionary mechanism to adjust PD numbers during wall expansion. Moreover, all bryophyte gametophytes modify their existing PD via taxon-specific strategies resembling those of angiosperms. Development of type II-like PD morphotypes with enlarged diameters or formation of pit pairs might be required to maintain PD transport rates during wall thickening. Similar to angiosperm leaves, fluorescence redistribution after photobleaching revealed a considerable reduction of the PD permeability in maturating P. patens phyllids. In contrast to previous reports on monoplex meristems of bryophyte gametophytes with single initials, we observed targeted secPD formation in the multi-initial basal meristems of A. agrestis sporophytes. Their PD networks share typical features of multi-initial angiosperm meristems, which may hint at a putative homologous origin. We also discuss that monoplex and multi-initial meristems may require distinct types of PD networks, with or without secPD formation, to control maintenance of initial identity and positional signaling.

Ultrastructural Observation of Cytokinesis and Plasmodesmata Formation in Brown Algae.

Similar to land plant cells, brown algal cells possess plasmodesmata with minute cytoplasmic tunnels, which enable the direct connection between adjacent cells. Plasmodesmata are distributed depending on the association of their formation with cytokinesis. Primary plasmodesmata are formed during cytokinesis, while secondary plasmodesmata appear on the cell wall septum following cytokinesis. Typically, the plasmodesmata of brown algae are cylindrical without the penetration of desmotubules from the endoplasmic reticulum, and there are no morphological differences between primary and secondary plasmodesmata. This present chapter describes the observation of cytokinesis and primary plasmodesmata formation in brown algae using electron microscopy as well as the examination of polysaccharide distribution using antibodies and enzyme-gold probes.

A correlative light electron microscopy approach reveals plasmodesmata ultrastructure at the graft interface.

Despite recent progress in our understanding of graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy (CLEM) approach to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing yellow FP or monomeric red FP in the endoplasmic reticulum (ER) allowed efficient targeting of the grafting interface for examination under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that four classes of PD were formed at the interface and that PD introgression into the cell wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these incomplete PD gives us insights into the process of secondary PD biogenesis. We found that the establishment of successful symplastic connections between the scion and rootstock occurred predominantly in the presence of thin cell walls and ER-plasma membrane tethering. The resolution reached in this work shows that our CLEM method advances the study of biological processes requiring the combination of light and electron microscopy.

Leaf Epidermis: The Ambiguous Symplastic Domain.

The ability to develop secondary (post-cytokinetic) plasmodesmata (PD) is an important evolutionary advantage that helps in creating symplastic domains within the plant body. Developmental regulation of secondary PD formation is not completely understood. In flowering plants, secondary PD occur exclusively between cells from different lineages, e.g., at the L1/L2 interface within shoot apices, or between leaf epidermis (L1-derivative), and mesophyll (L2-derivative). However, the highest numbers of secondary PD occur in the minor veins of leaf between bundle sheath cells and phloem companion cells in a group of plant species designated “symplastic” phloem loaders, as opposed to “apoplastic” loaders. This poses a question of whether secondary PD formation is upregulated in general in symplastic loaders. Distribution of PD in leaves and in shoot apices of two symplastic phloem loaders, Alonsoa meridionalis and Asarina barclaiana, was compared with that in two apoplastic loaders, Solanum tuberosum (potato) and Hordeum vulgare (barley), using immunolabeling of the PD-specific proteins and transmission electron microscopy (TEM), respectively. Single-cell sampling was performed to correlate sugar allocation between leaf epidermis and mesophyll to PD abundance. Although the distribution of PD in the leaf lamina (except within the vascular tissues) and in the meristem layers was similar in all species examined, far fewer PD were found at the epidermis/epidermis and mesophyll/epidermis boundaries in apoplastic loaders compared to symplastic loaders. In the latter, the leaf epidermis accumulated sugar, suggesting sugar import from the mesophyll via PD. Thus, leaf epidermis and mesophyll might represent a single symplastic domain in Alonsoa meridionalis and Asarina barclaiana.

The Enigma of Interspecific Plasmodesmata: Insight From Parasitic Plants.

Parasitic plants live in intimate physical connection with other plants serving as their hosts. These host plants provide the inorganic and organic compounds that the parasites need for their propagation. The uptake of the macromolecular compounds happens through symplasmic connections in the form of plasmodesmata. In contrast to regular plasmodesmata, which connect genetically identical cells of an individual plant, the plasmodesmata that connect the cells of host and parasite join separate individuals belonging to different species and are therefore termed “interspecific”. The existence of such interspecific plasmodesmata was deduced either indirectly using molecular approaches or observed directly by ultrastructural analyses. Most of this evidence concerns shoot parasitic Cuscuta species and root parasitic Orobanchaceae, which can both infect a large range of phylogenetically distant hosts. The existence of an interspecific chimeric symplast is both striking and unique and, with exceptions being observed in closely related grafted plants, exist only in these parasitic relationships. Considering the recent technical advances and upcoming tools for analyzing parasitic plants, interspecific plasmodesmata in parasite/host connections are a promising system for studying secondary plasmodesmata. For open questions like how their formation is induced, how their positioning is controlled and if they are initiated by one or both bordering cells simultaneously, the parasite/host interface with two adjacent distinguishable genetic systems provides valuable advantages. We summarize here what is known about interspecific plasmodesmata between parasitic plants and their hosts and discuss the potential of the intriguing parasite/host system for deepening our insight into plasmodesmatal structure, function, and development.

Morphological convergence in the inter-holdfast coalescence process among kelp and kelp-like seaweeds (Lessonia, Macrocystis, Durvillaea)

:In brown macroalgae, intraspecific holdfast coalescence has only been studied in two species of Lessonia (Lessonia spicata and Lessonia berteroana). In both species coalescence followed the same general pattern: once the connection between holdfasts was established, the contact areas showed significant cellular morphological modifications. Typical epidermal cells became polygonal and similar to cortical cells. In addition, coalescence involved the de novo formation of secondary plasmodesmata, establishing a direct cytoplasm connection within neighbouring cells, where dense materials, vacuoles and organelles can be observed. In the present study, we demonstrate intraspecific holdfast coalescence in two additional kelp species, Lessonia trabeculata and Macrocystis pyrifera, as well as in the kelp-like seaweed, Durvillaea antarctica. The process of holdfast fusions in these species is similar to that described previously and suggests that this is a generalized phenomenon among kelp and kelp-like brown algae. In addition, the formation of cytoplasmic connections between genetically different brown algal individuals is shown for the first time.

PIT MEMBRANES OF EPHEDRA RESEMBLE GYMNOSPERMS MORE THAN ANGIOSPERMS

Bordered pit pairs of Ephedra species were characterized using different types of microscopy. Pit membranes contained tori that did not stain for lignin. SEM and AFM views of the torus surface showed no plasmodesmatal openings, but branched, secondary plasmodesmata were occasionally noted using TEM in conjunction with ultrathin sections. The margo consisted of radial microfibrils as well as finer diameter tangential fibrils. The former formed fascicles of fibrils that merged into even thicker buttresses during the act of pit membrane aspiration. AFM showed a discontinuous layer of non-microfibrillar material on the surface of both torus and margo. It is hypothesized that this material is responsible for adhesion of the pit membrane to the surface of the pit border during the process of aspiration. Taken as a whole, intervascular pit membranes of Ephedra more closely resemble those of conifers than those of torus-bearing pit membranes of angiosperms.

Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.

Plasmodesmata distribution and sugar partitioning in nitrogen-fixing root nodules of Datisca glomerata

To understand carbon partitioning in roots and nodules of Datisca glomerata, activities of sucrose-degrading enzymes and sugar transporter expression patterns were analyzed in both organs, and plasmodesmal connections between nodule cortical cells were examined by transmission electron microscopy. The results indicate that in nodules, the contribution of symplastic transport processes is increased in comparison to roots, specifically in infected cells which develop many secondary plasmodesmata. Invertase activities are dramatically reduced in nodules as compared to roots, indicating that here the main enzyme responsible for the cleavage of sucrose is sucrose synthase. A high-affinity, low-specificity monosaccharide transporter whose expression is induced in infected cells prior to the onset of bacterial nitrogen fixation, and which has an unusually low pH optimum and may be involved in turgor control or hexose retrieval during infection thread growth.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-010-1285-8) contains supplementary material, which is available to authorized users.

Plasmodesmata Formation: Poking Holes in Walls with ise

Secondary plasmodesmata are cytoplasmic channels connecting adjacent plant cells that arise after cell division. How membrane-delimited channels penetrate cell walls is unknown, but now two genes, ISE1 and ISE2, are shown to be required for pathways that limit their formation.

Loss of INCREASED SIZE EXCLUSION LIMIT (ISE)1 or ISE2 Increases the Formation of Secondary Plasmodesmata

Plasmodesmata (PD) transport developmentally important nucleic acids and proteins between plant cells. Primary PD form during cell division and are simple, linear channels. Secondary PD form in existing cell walls after cell division and are simple, twinned, or branched. PD function undergoes a marked reduction at the mid-torpedo stage of Arabidopsis embryogenesis. Two mutants, increased size exclusion limit (ise)1 and ise2, fail to undergo this transition, and their null mutations are embryonically lethal. We investigated the ultrastructure of PD in early-, mid-, and late-torpedo-stage embryos and in young leaves. Wild-type (WT) embryos contain twinned and branched (T/B) PD at all stages, but ise1 and ise2 embryos contain significantly higher proportions of T/B PD than WT embryos. WT T/B PD formation occurs in a stage- and tissue-specific pattern that is reversed in ise1 embryos. Silencing ISE1 in Nicotiana benthamiana leaves increases the frequency of secondary PD in existing cell walls. Silencing ISE2 increases the proportion of T/B secondary PD formed. Silenced tissues exhibit increased PD-mediated movement of green fluorescent protein tracers. Thus, silencing of ISE1 and ISE2 phenocopies ise1 and ise2 mutant embryos: when wild-type ISE1 and ISE2 functions are lost, de novo production of PD occurs, leading to increased intercellular transport.

Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition

During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins.

Dynamics of plasmodesmal connectivity in successive interfaces of the cambial zone

Frequency, density and branching of plasmodesmata were counted in successive tangential and transverse walls in the cambial zone of tomato stems in order to examine development of the plasmodesmal network in a chronological order. Coincident with progress of cell development, plasmodesmal connectivity increased, both at the xylem- and phloem-side. In transverse walls, the number of secondary plasmodesmata enhanced considerably. The same held for tangential walls, with a superimposed plasmodesmal doubling during the first phase of phloem development. This plasmodesmal doubling was interpreted to result from the deposition of wall material between branched plasmodesmal strands. Structural plasmodesmal development was correlated with production of hydroxyl radicals which control local cell wall alterations. Successive phases of plasmodesmal deployment and modification were distinguished which may coincide with differential functional capacities as documented by intracellular injection of fluorochromes. Diffusion-driven symplasmic transport appeared to be transiently interrupted during cell maturation.

Peeking into pit fields — A new model of secondary plasmodesmata formation

Peeking into pit fields — A new model of secondary plasmodesmata formation

Peeking into Pit Fields: A Multiple Twinning Model of Secondary Plasmodesmata Formation in Tobacco

In higher plants, plasmodesmata (PD) are major conduits for cell-cell communication. Primary PD are laid down at cytokinesis, while secondary PD arise during wall extension. During leaf development, the basal cell walls of trichomes extend radially without division, providing a convenient system for studying the origin of secondary PD. We devised a simple freeze-fracture protocol for examining large numbers of PD in surface view. In the postcytokinetic wall, simple PD were distributed randomly. As the wall extended, PD became twinned at the cell periphery. Additional secondary pores were inserted at right angles to these, giving rise to pit fields composed of several paired PD. During wall extension, the number of PD increased fivefold due to the insertion of secondary PD. Our data are consistent with a model in which a subset of the original primary PD pores function as templates for the insertion of new secondary PD, spatially fixing the position of future pit fields. Many of the new PD shared the same wall collar as the original PD pore, suggesting that new PD pores may arise by fissions of existing PD progenitors. Different models of secondary PD formation are discussed. Our data are supported by a computational model, Plasmodesmap, which accurately simulates the formation of radial pit fields during cell wall extension based on the occurrence of multiple PD twinning events in the cell wall. The model predicts PD distributions with striking resemblance to those seen on fractured wall faces.

Cytokinin application to the shoot apical meristem of Sinapis alba enhances secondary plasmodesmata formation

A single application of cytokinin benzyladenine causes a threefold increase in the frequency of plasmodesmata in the vegetative shoot apical meristem (SAM) of Sinapis alba plants. This increase is observed 20 h after application within all cell layers (L1, L2, L3) as well as at the interfaces between these layers. Evidence is presented indicating that cytokinin promotes mainly the formation of new secondary plasmodesmata. A similar increase in the frequency of secondary plasmodesmata was observed in the Sinapis SAM during the floral transition induced by a single long day, suggesting that this effect of the long day is mediated by cytokinin.

Class 1 Reversibly Glycosylated Polypeptides Are Plasmodesmal-Associated Proteins Delivered to Plasmodesmata via the Golgi Apparatus

SE-WAP41, a salt-extractable 41-kD wall-associated protein that is associated with walls of etiolated maize (Zea mays) seedlings and is recognized by an antiserum previously reported to label plasmodesmata and the Golgi, was cloned, sequenced, and found to be a class 1 reversibly glycosylated polypeptide ((C1)RGP). Protein gel blot analysis of cell fractions with an antiserum against recombinant SE-WAP41 showed it to be enriched in the wall fraction. RNA gel blot analysis along the mesocotyl developmental axis and during deetiolation demonstrates that high SE-WAP41 transcript levels correlate spatially and temporally with primary and secondary plasmodesmata (Pd) formation. All four of the Arabidopsis thaliana (C1)RGP proteins, when fused to green fluorescent protein (GFP) and transiently expressed in tobacco (Nicotiana tabacum) epidermal cells, display fluorescence patterns indicating they are Golgi- and plasmodesmal-associated proteins. Localization to the Golgi apparatus was verified by colocalization of transiently expressed AtRGP2 fused to cyan fluorescence protein together with a known Golgi marker, Golgi Nucleotide Sugar Transporter 1 fused to yellow fluorescent protein (GONST1:YFP). In transgenic tobacco, AtRGP2:GFP fluorescence is punctate, is present only in contact walls between cells, and colocalizes with aniline blue-stained callose present around Pd. In plasmolyzed cells, AtRGP2:GFP remains wall embedded, whereas GONST1:YFP cannot be found embedded in cell walls. This result implies that the targeting to Pd is not due to a default pathway for Golgi-localized fusion proteins but is specific to (C1)RGPs. Treatment with the Golgi disrupting drug Brefeldin A inhibits Pd labeling by AtRGP2:GFP. Integrating these data, we conclude that (C1)RGPs are plasmodesmal-associated proteins delivered to plasmodesmata via the Golgi apparatus.

Plasmodesmata and the control of symplastic transport

In 1879 Eduard Tangle discovered cytoplasmic connections between cells in the cotyledons of Strychnos nuxvomica , which he interpreted to be protoplasmic contacts. This led him to hypothesize that ‘the protoplasmic bodies . . . are united by thin strands passing through connecting ducts in the walls, which put the cells into connection with each other and so unite them to an entity of higher order’ (Carr 1976). This challenged the then current view that cells functioned as autonomous units. It was after much research in many other species and cell types that Strasburger, in 1901, named these structures plasmodesmata (Carr 1976). During the division and differentiation of meristematic cells, plasmodesmata are formed across each developing cell plate, allowing cytoplasmic and endomembrane continuity to occur between all daughter cells, and ultimately, between all cells in a developing tissue (Mezitt & Lucas 1996). Those plasmodesmata that form during cell division are termed primary plasmodesmata (Jones 1976). Those that form de novo across existing cell walls are called secondary plasmodesmata (Ehlers & Kollmann 2001). The formation of secondary plasmodesmata allows cells to increase their potential for molecular trafficking and allows connections to be created between cells that are not related cytokinetically. As cells expand and differentiate, their fate determines the extent to which their cytoplasmic connectivity to other cells is maintained (Mezitt & Lucas 1996). Some cell types, such as those of the leaf mesophyll, remain closely connected to their neighbours, and may even lay down additional plasmodesmata to increase the continuity (Ding et al . 1992a). In other areas of the plant, for instance in vascular tissue, certain cells greatly reduce the number of plasmodesmata in their adjoining walls (Gamalei 1989). In this way, the cytoplasmic continuity can be altered depending on the tissue type (Botha & Evert 1988; Brown et al . 1995). However, although reductions in the number of plasmodesmata are common, only guard cells surrounding stomata (Erwee, Goodwin & van Bel 1985; Palevitz & Hepler 1985) and differentiating xylem elements (Lachaud & Maurousset 1996) lose all symplastic connections at maturity. In all other cells, some degree of intercellular connection is maintained. This plasmodesmal continuum that potentially exists throughout the whole plant is termed the symplast (Munch 1930). However, the symplast is not the open continuum that Munch originally hypothesized, but is divided into functional domains, each tightly regulated by different forms of plasmodesmata (Erwee & Goodwin 1985; Ehlers & Kollmann 2001). Plasmodesmata are now thought of as fluid, dynamic structures that can be modified both structurally and functionally to cope with the requirements of specific cells and tissues.

Primary and secondary plasmodesmata: structure, origin, and functioning.

In the multicellular organisms of higher plants, plasmodesmata provide pathways for intimate symplasmic communication between neighboring cells. The arguments summarized in the present review demonstrate that plasmodesmata are diverse and highly dynamic structures. Differences in the plasmodesmal origin and modifications of the plasmodesmal structure and functioning at the various cell interfaces are the basic means which give rise to a complicated and flexibile symplasmic network. This complex communication system is discussed to serve a significant role in the coordinated development and in the concerted physiological functioning of the cells within the plant tissues, organs, and organisms.

Developmental regulation of intercellular protein trafficking through plasmodesmata in tobacco leaf epidermis

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.