Immunohistochemical Detection of the Wall Components on the Example of Shoot Apical Meristem of Fagopyrum esculentum and Fagopyrum tataricum.

Immunohistochemistry is a method that allows the detection of individual components of cell walls in an extremely precise way at the level of a single cell and wall domains. The cell wall antibodies detect specific epitopes of pectins, arabinogalactan proteins (AGP), hemicelluloses, and extensins. The presented method visualization of the selected pectic and AGP epitopes using antibodies directed to wall components is described. The method of the analysis of the chemical composition of the wall is present on the example of the shoot apical meristems of Fagopurum esculentum and Fagopyrum tataricum. Recommended protocols for immunostaining and examination on fluorescence microscopy level are presented.

Apoplastic and Symplasmic Markers of Somatic Embryogenesis.

Somatic embryogenesis (SE) is a process that scientists have been trying to understand for many years because, on the one hand, it is a manifestation of the totipotency of plant cells, so it enables the study of the mechanisms regulating this process, and, on the other hand, it is an important method of plant propagation. Using SE in basic research and in practice is invaluable. This article describes the latest, but also historical, information on changes in the chemical composition of the cell wall during the transition of cells from the somatic to embryogenic state, and the importance of symplasmic communication during SE. Among wall chemical components, different pectic, AGP, extensin epitopes, and lipid transfer proteins have been discussed as potential apoplastic markers of explant cells during the acquisition of embryogenic competence. The role of symplasmic communication/isolation during SE has also been discussed, paying particular attention to the formation of symplasmic domains within and between cells that carry out different developmental processes. Information about the number and functionality of plasmodesmata (PD) and callose deposition as the main player in symplasmic isolation has also been presented.

The Impact of Gold Nanoparticles on Somatic Embryogenesis Using the Example of Arabidopsis thaliana.

Although the influence of nanoparticles (NPs) on developmental processes is better understood, little is known about their impact on somatic embryogenesis (SE). This process involves changes in the direction of cell differentiation. Thus, studying the effect of NPs on SE is essential to reveal their impact on cell fate. This study aimed to examine the influence of gold nanoparticles (Au NPs) with different surface charges on the SE of 35S:BBM Arabidopsis thaliana, with particular emphasis on the spatiotemporal localization of pectic arabinogalactan proteins (AGPs) and extensin epitopes in cells changing the direction of their differentiation. The results show that under the influence of nanoparticles, the explant cells of 35S:BBM Arabidopsis thaliana seedling origin did not enter the path of SE. Bulges and the formation of organ-like structures were observed in these explants, in contrast to the control, where somatic embryos developed. Additionally, spatiotemporal changes in the chemical composition of the cell walls during the culture were observed. Under the influence of Au NPs, the following effects were observed: (1) explant cells did not enter the SE pathway, (2) the impacts of Au NPs with different surface charges on the explants were variable, and (3) the compositions of the analyzed pectic AGPs and extensin epitopes were diverse in the cells with different developmental programs: SE (control) and non-SE (treated with Au NPs).

Rhodamine-Tagged Polymethacrylate Dyes as Alternative Tools for Analysis of Plant Cells.

A rhodamine B (RhB)-based initiator for atom transfer radical polymerization (ATRP) was synthesized and applied for preparation of poly(2-trimethylammoniumethyl methacrylate) (PChMA), poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(2-trimethylsilyloxyethyl methacrylate) (PHEMATMS). Polymer fluorescence was confirmed by determination of quantum yield by comparative method with piroxicam as the standard exhibiting dependency of emission intensity on the polymer chain hydrophilicity and the kind of solvent. The RhB functionalized polymers were used for biological tests in plant materials except for RhB-PHEMATMS because of weak fluorescence. These two polymers slightly differed in cellular localization. RhB-PChMA was mostly observed in cell walls of root tissues and cotyledon epidermis. It was also observed in cytoplasm and cell organelles of root cap cells and rhizodermis, in contrast with cytoplasm of cotyledon epidermis. RhB-PHEMA was also present in apoplast. A strong signal in protoxylem cell walls and a weak signal in cell walls of rhizodermis and cortex were visible. Moreover, it was also present in cell walls of cotyledon epidermis. However, RhB-PHEMA was mostly observed in cytoplasm and cell organelles of all root tissues and epidermis of cotyledons. Both RhB-polymers did not cause cell death which means that they can be used in living plant material.

Symplasmic communication - supracellular system of regulation of cell differentiation / Komunikacja symplastowa ­ nadkomórkowy system regulacji róznicowania komórek.

Plasmodesmata (PD), discovered more than 120 years ago, are still a mystery about their role in regulating plant cell differentiation. Research in recent years has verified our idea about the structure of PD and their function in the exchange of information between cells of the plant body. The involvement of PD in the movement of proteins, including transcription factors, hormones, and various types of RNA, indicates that they play an important role in regulating cell differentiation. The movement of molecules through PD is called symplasmic communication, and its limitations or absence are an essential element in controlling the direction of cell differentiation.

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.

Analysis of the Distribution of Symplasmic Tracers During Zygotic and Somatic Embryogenesis.

Plasmodesmata (PD) are membraneous channels that span cell walls of adjacent cells to establish the symplasm. These connections are unique to plants and enable the cell-to-cell exchange of information via the symplasm. However, not every plant cell is connected to its neighbor. Absence of PD and lack of communication (symplasmic isolation) are important regulators of cell differentiation. To determine cell-to-cell symplasmic connectivity, the distribution of fluorescent tracers can be analyzed. Here, we describe in detail the entire procedure for conducting such analysis using fluorescence and confocal microscopy to study molecular fluxes in fluorescence recovery after photobleaching (FRAP) experiments. Studies using fluorochromes and fluorescent-labeled dextrans successfully inform the degree of symplasmic connectivity between cells in zygotic and somatic embryos. Small molecules, such as water and ions, travel through PD but also transcription factors and different types of RNA. Studies of symplasmic communication are important to determine the spatio-temporal correlation between cell differentiation and the exchange of information between cells. This information is necessary to determine the role of symplasmic communication during embryogenesis, which is a very important stage in plant development and morphogenesis.

Gold Nanoparticles-Induced Modifications in Cell Wall Composition in Barley Roots.

The increased use of nanoparticles (NP) in different industries inevitably results in their release into the environment. In such conditions, plants come into direct contact with NP. Knowledge about the uptake of NP by plants and their effect on different developmental processes is still insufficient. Our studies concerned analyses of the changes in the chemical components of the cell walls of Hordeum vulgare L. roots that were grown in the presence of gold nanoparticles (AuNP). The analyses were performed using the immunohistological method and fluorescence microscopy. The obtained results indicate that AuNP with different surface charges affects the presence and distribution of selected pectic and arabinogalactan protein (AGP) epitopes in the walls of root cells.

Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot.

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.

Similarities and Differences in the GFP Movement in the Zygotic and Somatic Embryos of Arabidopsis.

Intercellular signaling during embryo patterning is not well understood and the role of symplasmic communication has been poorly considered. The correlation between the symplasmic domains and the development of the embryo organs/tissues during zygotic embryogenesis has only been described for a few examples, including Arabidopsis. How this process occurs during the development of somatic embryos (SEs) is still unknown. The aim of these studies was to answer the question: do SEs have a restriction in symplasmic transport depending on the developmental stage that is similar to their zygotic counterparts? The studies included an analysis of the GFP distribution pattern as expressed under diverse promoters in zygotic embryos (ZEs) and SEs. The results of the GFP distribution in the ZEs and SEs showed that 1/the symplasmic domains between the embryo organs and tissues in the SEs was similar to those in the ZEs and 2/the restriction in symplasmic transport in the SEs was correlated with the developmental stage and was similar to the one in their zygotic counterparts, however, with the spatio-temporal differences and different PDs SEL value between these two types of embryos.

Cell Wall Composition as a Marker of the Reprogramming of the Cell Fate on the Example of a Daucus carota (L.) Hypocotyl in Which Somatic Embryogenesis Was Induced.

Changes in the composition of the cell walls are postulated to accompany changes in the cell's fate. We check whether there is a relationship between the presence of selected pectic, arabinogalactan proteins (AGPs), and extensins epitopes and changes in cell reprogramming in order to answer the question of whether they can be markers accompanying changes of cell fate. Selected antibodies were used for spatio-temporal immunolocalization of wall components during the induction of somatic embryogenesis. Based on the obtained results, it can be concluded that (1) the LM6 (pectic), LM2 (AGPs) epitopes are positive markers, but the LM5, LM19 (pectic), JIM8, JIM13 (AGPs) epitopes are negative markers of cells reprogramming to the meristematic/pluripotent state; (2) the LM8 (pectic), JIM8, JIM13, LM2 (AGPs) and JIM11 (extensin) epitopes are positive markers, but LM6 (pectic) epitope is negative marker of cells undergoing detachment; (3) JIM4 (AGPs) is a positive marker, but LM5 (pectic), JIM8, JIM13, LM2 (AGPs) are negative markers for pericycle cells on the xylem pole; (4) LM19, LM20 (pectic), JIM13, LM2 (AGPs) are constitutive wall components, but LM6, LM8 (pectic), JIM4, JIM8, JIM16 (AGPs), JIM11, JIM12 and JIM20 (extensins) are not constitutive wall components; (5) the extensins do not contribute to the cell reprogramming.

Tocopherols mutual balance is a key player for maintaining Arabidopsis thaliana growth under salt stress.

Enhanced channeling carbon through pathways: shikimate/chorismate, benzenoid-phenylopropanoid or 2-C-methyl-D-erythritol 4-phosphate (MEP) provides a multitude of secondary metabolites and cell wall components and allows plants response to environmental stresses. Through the biosynthetic pathways, different secondary metabolites, like tocopherols (TCs), are bind to mutual dependencies and metabolic loops, that are not yet fully understood. We compared, in parallel, the influence of α- and γ-TCs on metabolites involved in osmoprotective/antioxidative response, and physico-chemical modification of plasma membrane and cell wall. We studied Arabidopsis thaliana Columbia ecotype (WT), mutant vte1 deficient in α- and γ-TCs, mutant vte4 over-accumulating γ-TC instead of α-TC, and transgenic line tmt over-accumulating α-TC; exposed to NaCl. The results indicate that salt stress activates ß-carboxylation processes in WT plants and in plants with altered TCs accumulation. In α-TC-deficient plants, NaCl causes ACC decrease, but does not change SA, whose concentration remains higher than in α-TC accumulating plants. α/γ-TCs contents influence carbohydrates, poliamines, phenolic (caffeic, ferrulic, cinnamic) acids accumulation patterns. Salinity results in increased detection of the LM5 galactan and LM19 homogalacturonan epitopes in α-TC accumulating plants, and the LM6 arabinan and MAC207 AGP epitopes in α-TC deficient mutants. Parallel, plants with altered TCs composition show decreased both the cell turgor and elastic modulus determined at the individual cell level. α-TC deficient plants reveal lower values of cell turgor and elastic modulus, but higher cell hydraulic conductivity than α-TC accumulating plants. Under salt stress, α-TC shows stronger regulatory effect than γ-TC through the impact on chloroplastic biosynthetic pathways and ROS/osmotic-modulating compounds.

Symplasmic Isolation Contributes to Somatic Embryo Induction and Development in the Tree Fern Cyathea delgadii Sternb.

In this report, we describe studies on symplasmic communication and cellular rearrangement during direct somatic embryogenesis (SE) in the tree fern Cyathea delgadii. We analyzed changes in the symplasmic transport of low-molecular-weight fluorochromes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) and fluorescein (delivered to cells as fluorescein diacetate, FDA), within stipe explants and somatic embryos originating from single epidermal cells and developing during 16-d long culture. Induction of SE is preceded by a restriction in fluorochrome distribution between certain explant cells. Microscopic analysis showed a series of cellular changes like a decrease in vacuole size, increase in vacuole numbers, and increased density of cytoplasm and deposition of electron-dense material in cell walls that may be related with embryogenic transition. In somatic embryos, the limited symplasmic communication between cells was observed first in linear tri-cellular embryos. Further development of the fern embryo was associated with the formation of symplasmic domains corresponding to the four segments of the plant body. Using symplasmic tracers, we provided evidence that the changes in plasmodesmata permeability are corelated with somatic-to-embryogenic transition and somatic embryo development.

Extracellular matrix and wall composition are diverse in the organogenic and non-organogenic calli of Actinidia arguta.

KEY MESSAGE: Differences in the composition and the structural organisation of the extracellular matrix correlate with the morphogenic competence of the callus tissue that originated from the isolated endosperm of kiwifruit. The chemical composition and structural organisation of the extracellular matrix, including the cell wall and the layer on its surface, may correspond with the morphogenic competence of a tissue. In the presented study, this relationship was found in the callus tissue that had been differentiated from the isolated endosperm of the kiwiberry, Actinidia arguta. The experimental system was based on callus samples of exactly the same age that had originated from an isolated endosperm but were cultured under controlled conditions promoting either an organogenic or a non-organogenic pathway. The analyses which were performed using bright field, fluorescence and scanning electron microscopy techniques showed significant differences between the two types of calli. The organogenic tissue was compact and the outer walls of the peripheral cells were covered with granular structures. The non-organogenic tissue was composed of loosely attached cells, which were connected via a net-like structure. The extracellular matrices from both the non- and organogenic tissues were abundant in pectic homogalacturonan and extensins (LM19, LM20, JIM11, JIM12 and JIM20 epitopes), but the epitopes that are characteristic for rhamnogalacturonan I (LM5 and LM6), hemicellulose (LM25) and the arabinogalactan protein (LM2) were detected only in the non-organogenic callus. Moreover, we report the epitopes, which presence is characteristic for the Actinidia endosperm (LM21 and LM25, heteromannan and xyloglucan) and for the endosperm-derived cells that undergo dedifferentiation (loss of LM21 and LM25; appearance or increase in the content of LM5, LM6, LM19, JIM11, JIM12, JIM20, JIM8 and JIM16 epitopes).

Development of Embryo Suspensors for Five Genera of Crassulaceae with Special Emphasis on Plasmodesmata Distribution and Ultrastructure.

The suspensor in the majority of angiosperms is an evolutionally conserved embryonic structure functioning as a conduit that connects ovule tissues with the embryo proper for nutrients and growth factors flux. This is the first study serving the purpose of investigating the correlation between suspensor types and plasmodesmata (PD), by the ultrastructure of this organ in respect of its full development. The special attention is paid to PD in representatives of Crassulaceae genera: Sedum, Aeonium, Monanthes, Aichryson and Echeveria. The contribution of the suspensor in transporting nutrients to the embryo was confirmed by the basal cell structure of the suspensor which produced, on the micropylar side of all genera investigated, a branched haustorium protruding into the surrounding ovular tissue and with wall ingrowths typically associated with cell transfer. The cytoplasm of the basal cell was rich in endoplasmic reticulum, mitochondria, dictyosomes, specialized plastids, microtubules, microbodies and lipid droplets. The basal cell sustained a symplasmic connection with endosperm and neighboring suspensor cells. Our results indicated the dependence of PD ultrastructure on the type of suspensor development: (i) simple PD are assigned to an uniseriate filamentous suspensor and (ii) PD with an electron-dense material are formed in a multiseriate suspensor. The occurrence of only one or both types of PD seems to be specific for the species but not for the genus. Indeed, in the two tested species of Sedum (with the distinct uniseriate/multiseriate suspensors), a diversity in the structure of PD depends on the developmental pattern of the suspensor. In all other genera (with the multiseriate type of development of the suspensor), the one type of electron-dense PD was observed.

In Vitro Tissue Culture in Brachypodium: Applications and Challenges.

Brachypodium distachyon has become an excellent model for plant breeding and bioenergy grasses that permits many fundamental questions in grass biology to be addressed. One of the constraints to performing research in many grasses has been the difficulty with which they can be genetically transformed and the generally low frequency of such transformations. In this review, we discuss the contribution that transformation techniques have made in Brachypodium biology as well as how Brachypodium could be used to determine the factors that might contribute to transformation efficiency. In particular, we highlight the latest research on the mechanisms that govern the gradual loss of embryogenic potential in a tissue culture and propose using B. distachyon as a model for other recalcitrant monocots.

Symplasmic isolation marks cell fate changes during somatic embryogenesis.

Cell-to-cell signalling is a major mechanism controlling plant morphogenesis. Transport of signalling molecules through plasmodesmata is one way in which plants promote or restrict intercellular signalling over short distances. Plasmodesmata are membrane-lined pores between cells that regulate the intercellular flow of signalling molecules through changes in their size, creating symplasmic fields of connected cells. Here we examine the role of plasmodesmata and symplasmic communication in the establishment of plant cell totipotency, using somatic embryo induction from Arabidopsis explants as a model system. Cell-to-cell communication was evaluated using fluorescent tracers, supplemented with histological and ultrastructural analysis, and correlated with expression of a WOX2 embryo reporter. We showed that embryogenic cells are isolated symplasmically from non-embryogenic cells regardless of the explant type (immature zygotic embryos or seedlings) and inducer system (2,4-dichlorophenoxyacetic acid or the BABY BOOM (BBM) transcription factor), but that the symplasmic domains in different explants differ with respect to the maximum size of molecule capable of moving through the plasmodesmata. Callose deposition in plasmodesmata preceded WOX2 expression in future sites of somatic embryo development, but later was greatly reduced in WOX2-expressing domains. Callose deposition was also associated with a decrease DR5 auxin response in embryogenic tissue. Treatment of explants with the callose biosynthesis inhibitor 2-deoxy-D-glucose supressed somatic embryo formation in all three systems studied, and also blocked the observed decrease in DR5 expression. Together these data suggest that callose deposition at plasmodesmata is required for symplasmic isolation and establishment of cell totipotency in Arabidopsis.

Composition of the Reconstituted Cell Wall in Protoplast-Derived Cells of Daucus is Affected by Phytosulfokine (PSK).

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.

Aluminum Alters the Histology and Pectin Cell Wall Composition of Barley Roots.

Aluminum (Al) is one of the most important crust elements causing reduced plant production in acidic soils. Barley (Hordeum vulgare L.) is considered to be one of the crops that is most sensitive to Al, and the root cell wall is the primary target of Al toxicity. In this study, we evaluate the possible involvement of specific pectic epitopes in the cells of barley roots in response to aluminum exposure. We targeted four different pectic epitopes recognized by LM5, LM6, LM19, and LM20 antibodies using an immunocytochemical approach. Since Al becomes available and toxic to plants in acidic soils, we performed our analyses on barley roots that had been grown in acidic conditions (pH 4.0) with and without Al and in control conditions (pH 6.0). Differences connected with the presence and distribution of the pectic epitopes between the control and Al-treated roots were observed. In the Al-treated roots, pectins with galactan sidechains were detected with a visually lower fluorescence intensity than in the control roots while pectins with arabinan sidechains were abundantly present. Furthermore, esterified homogalacturonans (HGs) were present with a visually higher fluorescence intensity compared to the control, while methyl-esterified HGs were present in a similar amount. Based on the presented results, it was concluded that methyl-esterified HG can be a marker for newly arising cell walls. Additionally, histological changes were detected in the roots grown under Al exposure. Among them, an increase in root diameter, shortening of root cap, and increase in the size of rhizodermal cells and divisions of exodermal and cortex cells were observed. The presented data extend upon the knowledge on the chemical composition of the cell wall of barley root cells under stress conditions. The response of cells to Al can be expressed by the specific distribution of pectins in the cell wall and, thus, enables the knowledge on Al toxicity to be extended by explaining the mechanism by which Al inhibits root elongation.

Hydroxyproline-Rich Glycoproteins as Markers of Temperature Stress in the Leaves of Brachypodium distachyon.

Plants frequently encounter diverse abiotic stresses, one of which is environmental thermal stress. To cope with these stresses, plants have developed a range of mechanisms, including altering the cell wall architecture, which is facilitated by the arabinogalactan proteins (AGP) and extensins (EXT). In order to characterise the localisation of the epitopes of the AGP and EXT, which are induced by the stress connected with a low (4 °C) or a high (40 °C) temperature, in the leaves of Brachypodium distachyon, we performed immunohistochemical analyses using the antibodies that bind to selected AGP (JIM8, JIM13, JIM16, LM2 and MAC207), pectin/AGP (LM6) as well as EXT (JIM11, JIM12 and JIM20). The analyses of the epitopes of the AGP indicated their presence in the phloem and in the inner bundle sheath (JIM8, JIM13, JIM16 and LM2). The JIM16 epitope was less abundant in the leaves from the low or high temperature compared to the control leaves. The LM2 epitope was more abundant in the leaves that had been subjected to the high temperatures. In the case of JIM13 and MAC207, no changes were observed at the different temperatures. The epitopes of the EXT were primarily observed in the mesophyll and xylem cells of the major vascular bundle (JIM11, JIM12 and JIM20) and no correlation was observed between the presence of the epitopes and the temperature stress. We also analysed changes in the level of transcript accumulation of some of the genes encoding EXT, EXT-like receptor kinases and AGP in the response to the temperature stress. In both cases, although we observed the upregulation of the genes encoding AGP in stressed plants, the changes were more pronounced at the high temperature. Similar changes were observed in the expression profiles of the EXT and EXT-like receptor kinase genes. Our findings may be relevant for genetic engineering of plants with increased resistance to the temperature stress.