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).

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.

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.

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.

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.

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).

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.

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.

Unmethyl-esterified homogalacturonan and extensins seal Arabidopsis graft union.

BACKGROUND: Grafting is a technique widely used in horticulture. The processes involved in grafting are diverse, and the technique is commonly employed in studies focusing on the mechanisms that regulate cell differentiation or response of plants to abiotic stress. Information on the changes in the composition of the cell wall that occur during the grafting process is scarce. Therefore, this study was carried out for analyzing the composition of the cell wall using Arabidopsis hypocotyls as an example. During the study, the formation of a layer that covers the surface of the graft union was observed. So, this study also aimed to describe the histological and cellular changes that accompany autografting of Arabidopsis hypocotyls and to perform preliminary chemical and structural analyses of extracellular material that seals the graft union. RESULTS: During grafting, polyphenolic and lipid compounds were detected, along with extracellular deposition of carbohydrate/protein material. The spatiotemporal changes observed in the structure of the extracellular material included the formation of a fibrillar network, polymerization of the fibrillar network into a membranous layer, and the presence of bead-like structures on the surface of cells in established graft union. These bead-like structures appeared either "closed" or "open". Only three cell wall epitopes, namely: LM19 (un/low-methyl-esterified homogalacturonan), JIM11, and JIM20 (extensins), were detected abundantly on the cut surfaces that made the adhesion plane, as well as in the structure that covered the graft union and in the bead-like structures, during the subsequent stages of regeneration. CONCLUSIONS: To the best of our knowledge, this is the first report on the composition and structure of the extracellular material that gets deposited on the surface of graft union during Arabidopsis grafting. The results showed that unmethyl-esterified homogalacturonan and extensins are together involved in the adhesion of scion and stock, as well as taking part in sealing the graft union. The extracellular material is of importance not only due to the potential pectin-extensin interaction but also due to its origin. The findings presented here implicate a need for studies with biochemical approach for a detailed analysis of the composition and structure of the extracellular material.

Effect of Nanoparticles Surface Charge on the Arabidopsis thaliana (L.) Roots Development and Their Movement into the Root Cells and Protoplasts.

Increasing usage of gold nanoparticles (AuNPs) in different industrial areas inevitably leads to their release into the environment. Thus, living organisms, including plants, may be exposed to a direct contact with nanoparticles (NPs). Despite the growing amount of research on this topic, our knowledge about NPs uptake by plants and their influence on different developmental processes is still insufficient. The first physical barrier for NPs penetration to the plant body is a cell wall which protects cytoplasm from external factors and environmental stresses. The absence of a cell wall may facilitate the internalization of various particles including NPs. Our studies have shown that AuNPs, independently of their surface charge, did not cross the cell wall of Arabidopsis thaliana (L.) roots. However, the research carried out with using light and transmission electron microscope revealed that AuNPs with different surface charge caused diverse changes in the root's histology and ultrastructure. Therefore, we verified whether this is only the wall which protects cells against particles penetration and for this purpose we used protoplasts culture. It has been shown that plasma membrane (PM) is not a barrier for positively charged (+) AuNPs and negatively charged (-) AuNPs, which passage to the cell.

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.

Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus.

MAIN CONCLUSION: The new model orange callus line, similar to carrot root, was rich in carotenoids due to altered expression of some carotenogenesis-associated genes and possessed unique diversity of chromoplast ultrastructure. Callus induced from carrot root segments cultured in vitro is usually pale yellow (p-y) and poor in carotenoids. A unique, non-engineered callus line of dark orange (d-o) colour was developed in this work. The content of carotenoid pigments in d-o callus was at the same level as in an orange carrot storage root and nine-fold higher than in p-y callus. Carotenoids accumulated mainly in abundant crystalline chromoplasts that are also common in carrot root but not in p-y callus. Using transmission electron microscopy, other types of chromoplasts were also found in d-o callus, including membranous chromoplasts rarely identified in plants and not observed in carrot root until now. At the transcriptional level, most carotenogenesis-associated genes were upregulated in d-o callus in comparison to p-y callus, but their expression was downregulated or unchanged when compared to root tissue. Two pathway steps were critical and could explain the massive carotenoid accumulation in this tissue. The geranylgeranyl diphosphate synthase gene involved in the biosynthesis of carotenoid precursors was highly expressed, while the ß-carotene hydroxylase gene involved in ß-carotene conversion to downstream xanthophylls was highly repressed. Additionally, paralogues of these genes and phytoene synthase were differentially expressed, indicating their tissue-specific roles in carotenoid biosynthesis and metabolism. The established system may serve as a novel model for elucidating plastid biogenesis that coincides with carotenogenesis.

Organ and Tissue-Specific Localisation of Selected Cell Wall Epitopes in the Zygotic Embryo of Brachypodium distachyon.

The plant cell wall shows a great diversity regarding its chemical composition, which may vary significantly even during different developmental stages. In this study, we analysed the distribution of several cell wall epitopes in embryos of Brachypodium distachyon (Brachypodium). We also described the variations in the nucleus shape and the number of nucleoli that occurred in some embryo cells. The use of transmission electron microscopy, and histological and immunolocalisation techniques permitted the distribution of selected arabinogalactan proteins, extensins, pectins, and hemicelluloses on the embryo surface, internal cell compartments, and in the context of the cell wall ultrastructure to be demonstrated. We revealed that the majority of arabinogalactan proteins and extensins were distributed on the cell surface and that pectins were the main component of the seed coat and other parts, such as the mesocotyl cell walls and the radicula. Hemicelluloses were localised in the cell wall and outside of the radicula protodermis, respectively. The specific arrangement of those components may indicate their significance during embryo development and seed germination, thus suggesting the importance of their protective functions. Despite the differences in the cell wall composition, we found that some of the antibodies can be used as markers to identify specific cells and the parts of the developing Brachypodium embryo.

5-Azacitidine Induces Cell Death in a Tissue Culture of Brachypodium distachyon.

Morphological and histological observations revealed that, at a concentration of 50 µM, 5-azacitidine (5-azaC) totally inhibited the induction of embryogenic masses (EM), while the cultivation of explants (zygotic embryos; ZEs) in the presence of 5 µM of 5-azaC led to the formation of a callus with EM in 10% of the cases. Transmission electron microscopy (TEM) analyzes revealed the presence of the morphological and ultrastructural features that are typical for the vacuolar type of cell death in the callus cells that were treated. A TUNEL assay confirmed the presence of DNA double-strand breaks for the callus cells that had been treated with both 5 and 50 µM 5-azaC concentrations. Analysis of the gene expression of selected cell death markers demonstrated a reduced expression of metacaspase, protein executer 1 (EX1), and thioredoxin (TRX) in the callus cells that had been treated compared to the control culture. The strongest increase in the gene activity was characteristic for glutathione S-transferase (GST). Our studies also included an analysis of the distribution of some arabinogalactan proteins (AGPs) and extensin epitopes, which can be used as markers of cells that are undergoing death in a Brachypodium distachyon tissue culture.

Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture.

Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli.

Distribution of some pectic and arabinogalactan protein epitopes during Solanum lycopersicum (L.) adventitious root development.

BACKGROUND: The adventitious roots (AR) of plants share the same function as primary and lateral roots (LR), although their development is mainly an adaptive reaction to stress conditions. Regeneration of grafted plants is often accompanied by AR formation thus making the grafting technique a good model for studying AR initiation and development and their means of emergence. Pectins and arabinogalactan proteins (AGP) are helpful markers of particular cellular events, such as programmed cell death (PCD), elongation, proliferation or other differentiation events that accompany AR development. However, little is known about the distribution of pectins and AGPs during AR ontogeny, either in the primordium or stem tissues from which AR arise or their correspondence with these events during LR formation. RESULTS: AR were developed from different stem tissues such as parenchyma, xylem rays and the cambium, depending on the stem age and treatment (grafting versus cutting) of the parental tissue. Immunochemical analysis of the presence of pectic (LM8, LM19, LM20) and AGP (JIM8, JIM13, JIM16) epitopes in AR and AR-associated tissues showed differential, tissue-specific distributions of these epitopes. Two pectic epitopes (LM19, LM20) were developmentally regulated and the occurrence of the LM8 xylogalacturonan epitope in the root cap of the AR differed from other species described so far. AGP epitopes were abundantly present in the cytoplasmic compartments (mainly the tonoplast) and were correlated with the degree of cell vacuolisation. JIM8 and JIM13 epitopes were detected in the more advanced stages of primordium development, whereas the JIM16 epitope was present from the earliest division events of the initial AR cells. The comparison between AR and LR showed quantitative (AGP,) and qualitative (pectins) differences. CONCLUSION: The chemical compositions of adventitious and lateral root cells show differences that correlate with the different origins of these cells. In AR, developmental changes in the distribution of pectins and AGP suggest the turnover of wall compounds. Our data extend the knowledge about the distribution of pectin and AGP during non-embryogenic root development in a species that is important from an agronomic point of view.

Identification of symplasmic domains in the embryo and seed of Sedum acre L. (Crassulaceae).

MAIN CONCLUSION: Our study demonstrated that symplasmic communication between Sedum acre seed compartments and the embryo proper is not uniform. The presence of plasmodesmata (PD) constitutes the structural basis for information exchange between cells, and symplasmic communication is involved in the regulation of cell differentiation and plant development. Most recent studies concerning an analysis of symplasmic communication between seed compartments and the embryo have been predominantly performed on Arabidopsis thaliana. The results presented in this paper describe the analysis of symplasmic communication on the example of Sedum acre seeds, because the ultrastructure of the seed compartments and the embryo proper, including the PD, have already been described, and this species represents an embryonic type of development different to Arabidopsis. Moreover, in this species, an unusual electron-dense dome associated with plasmodesmata on the border between the basal cell/chalazal suspensor cells and the basal cell/the endosperm has been described. This prompted the question as to whether these plasmodesmata are functional. Thus, the aim of this study was to describe the movement of symplasmic transport fluorochromes between different Sedum seed compartments, with particular emphasis on the movement between the basal cell and the embryo proper and endosperm, to answer the following questions: (1) are seeds divided into symplasmic domains; (2) if so, are they stable or do they change with the development? The results have shown that symplasmic tracers movement: (a) from the external integument to internal integument is restricted; (b) from the basal cell to the other part of the embryo proper and from the basal cell to the endosperm is also restricted;

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.