Callose deposition analysis with special emphasis on plasmodesmata ultrastructure during megasporogenesis in Sedum (Crassulaceae).

In this study, the results of the first detection of callose within the ovules of the representatives of the family Crassulaceae are presented. This study was carried out on three species of the genus Sedum. Data analysis showed differences in the callose deposition pattern between Sedum hispanicum and Sedum ser. Rupestria species during megasporogenesis. Callose was present mostly in the transversal walls of dyads and tetrads in S. hispanicum. Furthermore, a complete loss of callose from the cell walls of the linear tetrad and a gradual and simultaneous deposition of callose within the nucellus of S. hispanicum were observed. The findings of this study showed the presence of hypostase with callose in the ovules of S. hispanicum, which is not common in other angiosperms. The remaining species tested in this study-Sedum sediforme and Sedum rupestre-showed a typical, well-known callose deposition pattern for plants with the monospore type of megasporogenesis and the Polygonum type of embryo sac. The functional megaspore (FM) in all studied species was located most chalazally. FM is a mononuclear cell, which wall is callose-free in the chalazal pole. The study presents the causes of different patterns of callose deposition within Sedum and their relationship with the systematic position of the study species. Moreover, embryological studies present an argument for excluding callose as a substance that forms an electron-dense material near the plasmodesmata in megaspores of S. hispanicum. This research expands the knowledge about the embryological processes of succulent plants from the family Crassulaceae.

Embryogenesis in Crassulaceae: structural aspect of suspensor development / Embriogeneza u Crassulaceae: strukturalny aspekt rozwoju wieszadelka.

The suspensor in the majority of angiosperms is an evolutionally conserved embryonic organ functioning as a conduit that connects ovule tissues with the embryo proper for nutrients and growth regulators flux. In this article the present knowledge on the embryo-suspensor ultrastructure and function in representatives of Crassulaceae genera: Sedum, Jovibarba, Sempervivum, Aeonium, Monanthes, Aichryson and Echeveria. The role of the suspensor in the transport of nutrients from the tissues of the ovule to the proper embryo is confirmed by the structure of the basal cell, especially the nature of the micropylar part of its wall, the "transfer wall". The basal suspensor cell is a site of intense metabolic activity. The special attention is paid to the plasmodesmata. The correlation between types of suspensors and structure of plasmodesmata was investigated. Final conclusions are given and the presented data summarized.

Ultrastructural Analysis and Three-Dimensional Reconstruction of Plasmodesmata.

Array tomography (AT) is a new high-throughput imaging method for high-resolution imaging of ultrastructure and for 3-D reconstruction of cells and organelles. Here, we describe the entire procedure for obtaining a spatial image of the distribution of plasmodesmata (PD). As example, the protocol is applied here to reconstruct the number and arrangement of PD between cells undergoing differentiation during Arabidopsis somatic embryogenesis.

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.

Computational Tools for Serial Block Electron Microscopy Reveal Plasmodesmata Distributions and Wall Environments.

Plasmodesmata are small channels that connect plant cells. While recent technological advances have facilitated analysis of the ultrastructure of these channels, there are limitations to efficiently addressing their presence over an entire cellular interface. Here, we highlight the value of serial block electron microscopy for this purpose. We developed a computational pipeline to study plasmodesmata distributions and detect the presence/absence of plasmodesmata clusters, or pit fields, at the phloem unloading interfaces of Arabidopsis (Arabidopsis thaliana) roots. Pit fields were visualized and quantified. As the wall environment of plasmodesmata is highly specialized, we also designed a tool to extract the thickness of the extracellular matrix at and outside of plasmodesmata positions. We detected and quantified clear wall thinning around plasmodesmata with differences between genotypes, including the recently published plm-2 sphingolipid mutant. Our tools open avenues for quantitative approaches in the analysis of symplastic trafficking.

Multiple C2 domains and transmembrane region proteins (MCTPs) tether membranes at plasmodesmata.

In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)-plasma membrane (PM) contacts coincide with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER-PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-to-cell signalling.

Sphingolipid biosynthesis modulates plasmodesmal ultrastructure and phloem unloading.

During phloem unloading, multiple cell-to-cell transport events move organic substances to the root meristem. Although the primary unloading event from the sieve elements to the phloem pole pericycle has been characterized to some extent, little is known about post-sieve element unloading. Here, we report a novel gene, PHLOEM UNLOADING MODULATOR (PLM), in the absence of which plasmodesmata-mediated symplastic transport through the phloem pole pericycle-endodermis interface is specifically enhanced. Increased unloading is attributable to a defect in the formation of the endoplasmic reticulum-plasma membrane tethers during plasmodesmal morphogenesis, resulting in the majority of pores lacking a visible cytoplasmic sleeve. PLM encodes a putative enzyme required for the biosynthesis of sphingolipids with very-long-chain fatty acid. Taken together, our results indicate that post-sieve element unloading involves sphingolipid metabolism, which affects plasmodesmal ultrastructure. They also raise the question of how and why plasmodesmata with no cytoplasmic sleeve facilitate molecular trafficking.

Response of plasmodesmata formation in leaves of C4 grasses to growth irradiance.

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C4 leaves is a key requirement for C4 photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C4 species using our PD quantification method, which combines three-dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP-ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 µmol m-2  s-1 ), and high light (1,000 µmol m-2  s-1 ), was observed both at seedling and established growth stages. We found that the effect of growth irradiance on C4 leaf PD density depended on plant age and species. The high light treatment resulted in two to four-fold greater PD density per unit leaf area than at low light, due to greater area of PD clusters and greater PD size in high light plants. These results along with our finding that the effect of light on M-BS PD density was not tightly linked to photosynthetic capacity suggest a complex mechanism underlying the dynamic response of C4 leaf PD formation to growth irradiance.

Direct Comparison of Leaf Plasmodesma Structure and Function in Relation to Phloem-Loading Type.

The export of photosynthetically produced sugars from leaves depends on plasmodesmatal transport of sugar molecules from mesophyll to phloem. Traditionally, the density of plasmodesmata (PD) along this phloem-loading pathway has been used as a defining feature of different phloem-loading types, with species proposed to have either many or few PD between the phloem and surrounding cells of the leaf. However, quantitative determination of PD density has rarely been performed. Moreover, the structure of PD has not been considered, even though it could impact permeability, and functional data are only available for very few species. Here, a comparison of PD density, structure, and function using data from transmission electron microscopy and live-cell microscopy was conducted for all relevant cell-cell interfaces in leaves of nine species. These species represent the three principal phloem-loading types currently discussed in literature. Results show that relative PD density among the different cell-cell interfaces in one species, but not absolute PD density, is indicative of phloem-loading type. PD density data of single interfaces, even combined with PD diameter and length data, did not correlate with the intercellular diffusion capacity measured by the fluorescence loss in photobleaching method. This means that PD substructure not visible on standard transmission electron micrographs may have a strong influence on permeability. Furthermore, the results support a proposed passive symplasmic loading mechanism in the tree species horse chestnut (Aesculus hippocastanum), white birch (Betula pubescens), orchard apple (Malus domestica), and gray poplar (Populus x canescens) as functional cell coupling and PD structure differed from active symplasmic and apoplasmic phloem-loading species.

Sieve Elements: The Favourite Habitat of Phytoplasmas.

The sieve elements are the only plant compartments, where phytoplasmas can survive and propagate. Therefore, this chapter is focussed on the specific molecular and cell-biological properties of the sieve element. Sieve element-companion cell complexes arise from (pro)cambial mother cells induced by key genes known to be decisive for sieve-element differentiation. The special anatomy, cell biology, and plasma-membrane outfit of sieve elements allows them to act collectively as a tube system that is able to drive a mass flow against the flow induced by transpiration. Plasmodesmal corridors are vital for the translocation of photoassimilates and systemic signals and for survival of the enucleate sieve elements. Of paramount importance is the Ca2+-dependent gating of plasmodesmata by callose and proteins. Hence, some of the complex, regulatory mechanisms to maintain Ca2+ homoeostasis in sieve elements are presented. Finally, the peculiarities of the chemical and physical sieve-element environment offered to phytoplasmas are discussed.

Evolution of root apical meristem structures in vascular plants: plasmodesmatal networks.

PREMISE OF THE STUDY: The apical meristem generates indeterminate apical growth of the stem and root of vascular plants. Our previous examination showed that shoot apical meristems (SAMs) can be classified into two types based on plasmodesmatal networks (PNs), which are important elements in symplasmic signaling pathways within the apical meristem. Here, we examined the PNs of root apical meristems (RAMs) in comparison with those of SAMs. METHODS: Root apical meristems of 18 families and 22 species of lycophytes and euphyllophytes were analyzed. Plasmodesmata (PD) in cell walls in median longitudinal sections of RAMs were enumerated using transmission electron micrographs, and the PD density per 1 µm2 of each cell wall was calculated. KEY RESULTS: Root apical meristems with prominent apical cells of monilophytes (euphyllophytes) and Selaginellaceae (lycophytes) had high PD densities, while RAMs with plural initial cells of gymnosperms and angiosperms (euphyllophytes), and of Lycopodiaceae and Isoetaceae (lycophytes) had low PD densities. This correlation between structures of apical meristems and PD densities is identical to that in SAMs already described. CONCLUSIONS: Irrespective of their diversified structures, the RAMs of vascular plants can be classified into two types with respect to PNs: the fern (monilophyte) type, which has a lineage-specific PN with only primary PD, and the seed-plant type, which has an interspecific PN with secondary PD in addition to primary PD. PNs may have played a key role in the evolution of apical meristems in vascular plants.

Sugar transport played a more important role than sugar biosynthesis in fruit sugar accumulation during Chinese jujube domestication.

MAIN CONCLUSION: Sugar transport, including the symplasmic pathway in plasmodesmata and apoplasmic pathway mediated by sugar transporters, accelerated sugar accumulation in cultivated jujube, while sugar metabolism-related genes played weak roles in jujube domestication. The fruit of Chinese jujube (Ziziphus jujuba Mill.) is high in sugar concentration. By contrast, wild type-sour jujube (Z. jujuba Mill. var. spinosa Hu) contains markedly less sugar. It is unknown whether sugar transport or sugar metabolism drove sugar accumulation during jujube domestication. Using a combination of ultrastructural observations, phylogenetic analysis, testing for soluble sugars, and transcriptional analysis, the sugar accumulation mechanism was studied in the developmental stages of cultivated jujube and sour jujube. Our results indicate that the symplasmic transport pathway in plasmodesmata is present in cultivated jujube, but not in sour jujube. Sugar transporter genes have higher frequencies of duplication than sugar metabolism-related genes. Gene expression patterns indicate that sugar transporter genes, especially ZjSUT2, ZjSWEET1, ZjSWEET7, ZjSWEET11, ZjSTP3, and ZjSTP13a, rather than sugar metabolism-related genes showed higher expression levels in cultivated jujube versus sour jujube during fruit sugar accumulation. These findings suggest that sugar transport, including apoplasmic and symplasmic transport, rather than sugar biosynthesis, is associated with the difference in sugar accumulation between jujube and sour jujube, and that it may drive jujube domestication. This study provides valuable genetic information for jujube improvement, and offers new insights into fruit tree domestication related to sugar accumulation.

Mechanisms of phloem loading.

The complex form of higher plants requires continuous, balanced transport of nutrients in the phloem. The initial step of transferring sugars, amino acids, and other materials from photosynthetic cells to the conducting sieve tubes is known as phloem loading. Three phloem loading mechanisms have been described. The first involves release of sucrose into the apoplast and subsequent retrieval by the phloem. The initial release step in this process is now known to be mediated by a new class of transporters, the SWEET proteins. In the other two loading mechanisms, polymer trapping and diffusion, sucrose passes into the phloem through cytoplasmic channels, the plasmodesmata. Recent models have shed additional light on these mechanisms and their ability to sustain the growth of even the tallest trees.

Ultrastructural and cytochemical aspects of female gametophyte development in Sedum hispanicum L. (Crassulaceae).

Until now, development of the female gametophyte has been investigated only in some species of Crassulaceae using a light microscope. To the best of our knowledge, this is the first report that describes the process of megasporogenesis and megagametogenesis in Crassulaceae in detail. To achieve this, we performed embryological studies on Sedum hispanicum L. (Crassulaceae). Cytochemical analysis detected the presence of proteins, lipids, and insoluble polysaccharides in individual cells of the gametophyte. The development of the embryo sac conforms to the monosporic or Polygonum-type in anatropous, crassinucellate, and bitegmic ovules. One megaspore mother cell initiates the process of megasporogenesis. Prior to the first meiotic division, the nucleus is centrally located within the meiocyte. Other organelles seem to be distributed evenly over the micropylar and chalazal parts during the development. Most storage reserves detected during megasporogenesis were observed in the megaspore mother cell. Three mitotic divisions within the chalazal functional megaspore resulted in the enlargement of the eight-nucleated embryo sac. In the seven-celled gametophyte, three chalazally located antipodes degenerated. A mature embryo sac was formed by the egg apparatus and central cell. When the antipodes degenerated, both synergids became organelle-rich and more active. The concentration of lipid droplets, starch grains, and proteins increased during megagametogenesis in the growing gametophyte. In the cellular embryo sac, the central cell can be distinguished by its largest accumulation. Our data confirm the hypothesis that plasmodesmata with electron-dense dome are formed during development of the female gametophyte in S. hispanicum and not just during the stages of embryogenesis. We observed these structures in megaspores and coenocytic embryo sac walls. Functions of observed plasmodesmata are discussed.

Comparative proteomic profiling of the choline transporter-like1 (CHER1) mutant provides insights into plasmodesmata composition of fully developed Arabidopsis thaliana leaves.

In plants, intercellular communication and exchange are highly dependent on cell wall bridging structures between adhering cells, so-called plasmodesmata (PD). In our previous genetic screen for PD-deficient Arabidopsis mutants, we described choline transporter-like 1 (CHER1) being important for PD genesis and maturation. Leaves of cher1 mutant plants have up to 10 times less PD, which do not develop to complex structures. Here we utilize the T-DNA insertion mutant cher1-4 and report a deep comparative proteomic workflow for the identification of cell-wall-embedded PD-associated proteins. Analyzing triplicates of cell-wall-enriched fractions in depth by fractionation and quantitative high-resolution mass spectrometry, we compared > 5000 proteins obtained from fully developed leaves. Comparative data analysis and subsequent filtering generated a list of 61 proteins being significantly more abundant in Col-0. This list was enriched for previously described PD-associated proteins. To validate PD association of so far uncharacterized proteins, subcellular localization analyses were carried out by confocal laser-scanning microscopy. This study confirmed the association of PD for three out of four selected candidates, indicating that the comparative approach indeed allowed identification of so far undescribed PD-associated proteins. Performing comparative cell wall proteomics of Nicotiana benthamiana tissue, we observed an increase in abundance of these three selected candidates during sink to source transition. Taken together, our comparative proteomic approach revealed a valuable data set of potential PD-associated proteins, which can be used as a resource to unravel the molecular composition of complex PD and to investigate their function in cell-to-cell communication.

Phloem Loading through Plasmodesmata: A Biophysical Analysis.

In many species, Suc en route out of the leaf migrates from photosynthetically active mesophyll cells into the phloem down its concentration gradient via plasmodesmata, i.e. symplastically. In some of these plants, the process is entirely passive, but in others phloem Suc is actively converted into larger sugars, raffinose and stachyose, and segregated (trapped), thus raising total phloem sugar concentration to a level higher than in the mesophyll. Questions remain regarding the mechanisms and selective advantages conferred by both of these symplastic-loading processes. Here, we present an integrated model-including local and global transport and kinetics of polymerization-for passive and active symplastic loading. We also propose a physical model of transport through the plasmodesmata. With these models, we predict that (1) relative to passive loading, polymerization of Suc in the phloem, even in the absence of segregation, lowers the sugar content in the leaf required to achieve a given export rate and accelerates export for a given concentration of Suc in the mesophyll and (2) segregation of oligomers and the inverted gradient of total sugar content can be achieved for physiologically reasonable parameter values, but even higher export rates can be accessed in scenarios in which polymers are allowed to diffuse back into the mesophyll. We discuss these predictions in relation to further studies aimed at the clarification of loading mechanisms, fitness of active and passive symplastic loading, and potential targets for engineering improved rates of export.

Suppressing a Putative Sterol Carrier Gene Reduces Plasmodesmal Permeability and Activates Sucrose Transporter Genes during Cotton Fiber Elongation.

Plasmodesmata (PDs) play vital roles in cell-to-cell communication and plant development. Emerging evidence suggests that sterols are involved in PD activity during cytokinesis. However, whether sterols contribute to PD gating between established cells remains unknown. Here, we isolated GhSCP2D, a putative sterol carrier protein gene from elongating cotton (Gossypium hirsutum) fibers. In contrast to wild-type fiber PDs, which opened at 5 to 10 d postanthesis (DPA) and closed only at 15 to 25 DPA, plants with suppressed GhSCP2D expression had reduced sterol contents and closed PDs at 5 through 25 DPA The GhSCP2D-suppressed fibers exhibited callose deposition at the PDs, likely due to reduced expression of GhPdBG3-2A/D, which encodes a PD-targeting ß-1,3-glucanase. Both GhPdBG3-2A/D expression and callose deposition were sensitive to a sterol biosynthesis inhibitor. Moreover, suppressing GhSCP2D upregulated a cohort of SUT and SWEET sucrose transporter genes in fiber cells. Collectively, our results indicate that (1) GhSCP2D is required for GhPdBG3-2A/D expression to degrade callose at the PD, thereby contributing to the establishment of the symplasmic pathway; and (2) blocking the symplasmic pathway by downregulating GhSCP2D activates or increases the expression of SUTs and SWEETs, leading to the switch from symplasmic to apoplasmic pathways.

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves.

Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.

Isolation of Plasmodesmata.

Plasmodesmata (PD) are plasma membrane lined pores that cross the plant cell wall and connect adjacent cells. Plasmodesmata are composed of elements of the endoplasmic reticulum, plasma membrane, cytosol, and cell wall and thus, as multicomposite structures that are embedded in the cell wall, they are notoriously difficult to isolate from whole plant tissue. However, understanding PD structure, function, and regulation necessitates identification of their molecular components and therefore proteomic and lipidomic analyses of PD fractions are an essential strategy for plasmodesmal biology. Here we outline a simple two-step purification procedure that allows isolation of PD-derived membranes from Arabidopsis suspension cells. The method involves isolation of purified cell wall fragments containing intact PD which is followed by enzymatic degradation of the cell wall to release the PD. This membrane-rich fraction can be subjected to protein and lipid extraction for molecular characterization of PD components. The first step of this procedure involves the isolation of cell wall fragments containing intact PD, free from contamination from other cellular compartments. Purified PD membranes are then released from the cell wall matrix by enzymatic degradation. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.