Side chains of pectic polysaccharides are regulated in relation to cell proliferation and cell differentiation

The occurrence and function of the side chains occurring in the rhamnogalacturonan I domain of pectic poly- saccharides have been investigated during carrot cell development using monoclonal antibodies to defined epitopes of (1-->4)-beta-D-galactan and (1-->5)-alpha-L-arabinan. Immunolocalization studies of carrot root apices indicated that cell walls in the central region of the meristem contained higher levels of (1-->5)-alpha-arabinan than the cell walls of surrounding cells. In contrast (1-->4)-beta-galactan was absent from the cell walls of the central meristematic cells but appeared abundantly at a certain point during root cap cell differentiation and also appeared in cell walls of differentiating stele and cortical cells. This developmental pattern of epitope occurrence was also reflected in a suspension-cultured carrot cell line that can be induced to switch from proliferation to elongation by altered culture conditions. (1-->4)-beta-galactan occurred at a low level in cell walls of proliferating cells but accumulated rapidly in cell walls following induction, before any visible cell elongation, while (1-->5)-alpha-arabinan was present in cell walls of proliferating cells but was absent from cell walls of elongated cells. Immunochemical assays of the cultured cells confirmed the early appearance of (1-->4)-beta-galactan during the switch from cell proliferation to cell elongation. Anion-exchange chromatography confirmed that (1-->4)-beta-galactan was attached to acidic pectic domains and also indicated that it was separate from a distinct homogalacturonan-rich component. These results indicate that the neutral components of pectic polysaccharides may have important roles in plant cell development.     

Spatial regulation of pectic polysaccharides in relation to pit fields in cell walls of tomato fruit pericarp

Scanning electron microscopic examination of intact tomato (Lycopersicon esculentum) pericarp and isolated pericarp cell walls revealed pit fields and associated radiating ridges on the inner face of cell walls. In regions of the cell wall away from pit fields, equivalent ridges occurred in parallel arrays. Treatment of isolated cell walls with a calcium chelator resulted in the loss of these ridges, indicating that they contain homogalacturonan-rich pectic polysaccharides. Immunolabeling procedures confirmed that pit fields and associated radiating ridges contained homogalacturonan. Epitopes of the side chains of pectic polysaccharides were not located in the same regions as homogalacturonan and were spatially regulated in relation to pit fields. A (1-->4)-beta-galactan epitope was absent from cell walls in regions of pit fields. A (1-->5)-alpha-arabinan epitope occurred most abundantly at the inner face of cell walls in regions surrounding the pit fields.     

Localization of cell wall polysaccharides in nonarticulated laticifers ofAsclepias speciosa Torr.

Summary Asclepias speciosa Torr, has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1→4) β-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (l→5) α-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1→4) β-D-galac-tan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to β-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled byRicinus communis agglutinin, a lectin that binds to terminal β-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.     

Temporal and spatial regulation of pectic (1-->4)-beta-D-galactan in cell walls of developing pea cotyledons: implications for mechanical properties

Modifications in cell wall pectic polysaccharides are thought to influence cell-cell adhesion and the mechanical properties of plant tissues. Monoclonal antibodies to epitopes occurring in homo- galacturonan and side chains of rhamnogalacturonan I have been used in an immunolocalization study of cell wall architecture of developing pea cotyledons. Pectic (1-->4)-beta-D-galactan appears in cotyledon cell walls at a defined stage late in development (approximately 26-30 days after anthesis), whereas homogalacturonan and pectic (1-->5)-alpha-L-arabinan are present in cotyledon cell walls throughout development. (1-->4)-beta-galactan was restricted to a distinct thin layer at the plasma membrane face of the cell wall. Anion exchange and immunoaffinity chromatography indicated that the (1-->4)-beta-galactan was associated with acidic pectic components. Mechanical compressive testing of pea cotyledons, before and after (1-->4)-beta-galactan appearance, indicated that the cotyledons with the galactan-rich cell wall layer were twice as firm as those with no detectable (1-->4)-beta-galactan.     

Developmental regulation of pectic epitopes during potato tuberisation

We show, by immunogold labelling, that potato (Solanum tuberosum L. cv Karnico) pectic epitopes are developmentally regulated within regions of the stolon, in addition to showing tissue-specific differences in abundance and localisation. The (1-->4)-beta-D-galactan and (1-->5)-alpha-arabinan epitopes demarcate two distinct zones within stolons; galactans are enriched in primary walls of elongating cells proximal to the stolon hook, whilst arabinans predominate in younger cells distal to the hook. Low-methoxyl homogalacturonan epitopes are concentrated in the middle lamella and show a proximo-distal gradient in stolons similar to that of galactans, whilst high-methoxyl homogalacturonan is uniformly abundant. Calcium pectate is restricted to the middle lamella at cell corners and pit fields. Calcium-binding sites are uniformly present in stolon cell walls, but their total density is reduced and they become localised to a few cell corners in mature tubers, as determined by image-electron energy loss spectroscopy. During the transition from elongation growth to isodiametric expansion during tuberisation of the stolon hook, there were no detectable changes in pectic epitope abundance or localisation. As tubers matured, all epitopes increased in abundance in parenchymal cell walls, except for calcium pectate. We conclude that potentially significant changes in pectic composition occur as young cells distal to the stolon hook move into the zone of cell elongation proximal to the hook.     

Arabinogalactan-protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in Trifolium nigrescens Viv.

The formation of an extracellular matrix surface network (ECMSN), and associated changes in the distribution of arabinogalactan-protein and pectin epitopes, have been studied during somatic embryogenesis (SE) and callogenesis of Trifolium nigrescens Viv. Scanning electron microscopy observations revealed the occurrence of an ECMSN on the surface of cotyledonary-staged somatic embryos as well as on the peripheral, non-regenerating callus cells. The occurrence of six AGP (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) and four pectin (JIM5, JIM7, LM5, LM6) epitopes was analysed during early stages of SE, in cotyledonary-staged somatic embryos and in non-embryogenic callus using monoclonal antibodies. The JIM5 low methyl-esterified homogalacturonan (HG) epitope localized to ECMSN on the callus surface but none of the epitopes studied were found to localize to ECMSN over mature somatic embryos. The LM2 AGP epitope was detected during the development of somatic embryos and was also observed in the cell walls of meristematic cells from which SE was initiated. The pectic epitopes JIM5, JIM7, LM5 and LM6 were temporally regulated during SE. The LM6 arabinan epitope, carried by side chains of rhamnogalacturonan-I (RG-I), was detected predominantly in cells of embryogenic swellings, whilst the LM5 galactan epitope of RG-I was uniformly distributed throughout the ground tissue of cotyledonary-staged embryoids but not detected at the early stages of SE. Differences in the distribution patterns of low and high methyl-esterified HG were detected: low ester HG (JIM5 epitope) was most abundant during the early steps of embryo formation and highly methyl-esterified form of HG (JIM7 epitope) became prevalent during embryoid maturation.     

Ginseng root water-extracted pectic polysaccharides originate from secretory cavities

A range of molecular probes for cell wall polysaccharides has been used to explore the structure and location of water-extracted pectic polysaccharides occurring in fractions isolated from ginseng roots. The LM19 homogalacturonan (HG) epitope was abundant in an HG fraction and analysis of LM19 binding to a rhamnogalacturonan-I (RG-I) rich-fraction indicated that the LM19 epitope is sensitive to acetylation. A specific RG-I epitope (LM16), four arabinogalactan-protein (AGP) epitopes (LM2, LM14, JIM16, MAC207) and an extensin epitope (JIM20) were found to be abundant and co-located in several isolated polysaccharide fractions including an arabinogalactan fraction and two RG-I fractions. Detection of the RG-I, AGP and extensin epitopes identified in isolated polysaccharide fractions in sections of ginseng roots indicated that they were most abundant in secretory cavities found in the cortical regions of ginseng roots. In addition, the immunocytochemical study indicated that polysaccharide epitope masking is a widespread phenomenon in the primary cell walls of ginseng roots.     

Modification of cambial cell wall architecture during cambium periodicity in Populus tomentosa Carr.

Cambium periodicity is correlated with changes in the cambial cell wall, but the heterogeneity of cell wall structure and composition makes it difficult to give an accurate interpretation, especially for complex secondary vascular tissues. A combination of different methods is necessary to reveal the structure of this complex cell wall. In this study, the cell wall architecture and composition of active and dormant cambial cells in Populus tomentosa were investigated by a combination of light microscopy, rapid-freezing and deep-etching electron microscopy, Fourier-transform infrared microspectroscopy and immuno-histochemistry. The results showed that the architecture of dormant cambial cell walls displayed a multi-layered structure, denser fibril network, smaller pore size, and fewer crosslinks between microfibrils than active cambial cell walls. The FTIR spectra of cell walls from active and dormant cambium showed differences in the intensity of bands near 1,740, 1,629, 1,537, 1,240, and 830 cm⁻¹, which reflected differences in cell wall composition. Immuno-labeling indicated that high methyl-esterified homogalacturonan and (1 → 4)-β-d-galactan epitopes were abundant and distributed in active cambial cell walls, and relatively de-esterified homogalacturonan and (1 → 5)-α-l-arabinan epitopes had weak labeling in the active cambium, while almost no labeling or very weak labeling for high methyl-esterified homogalacturonan, (1 → 4)-β-d-galactan and (1 → 5)-α-l-arabinan epitopes occurred in dormant cambial cells, except for the de-esterified homogalacturonan epitope, which was abundant in dormant cambial cells. These results demonstrate that there are great differences, both in structure and composition, between active and dormant cambial cell walls, which reflect their dynamic changes during cambium activity.     

STRUCTURE AND ONTOGENY OF LATICIFERS IN CICHORIUM INTYBUS (COMPOSITAE)

The branched anastomosed laticifer system in the primary body of Cichorium intybus L. originates in embryos from files of laticiferous members at the boundary between phloic procambium and ground meristem. Upon seed germination, laticiferous members develop perforations in the end walls which become entirely resorbed. Perforations also develop in the longitudinal walls of contiguous laticiferous members and from lateral connections between developing laticifer branches. Additional laticiferous members originate as procambium differentiation proceeds, and their differentiation follows a continuous acropetal sequence in leaf primordia of the plumule. In roots, laticifers closely associated with sieve tubes in the secondary phloem originate from derivatives of fusiform initials in the vascular cambium. These laticifers develop wall perforations and in a mature condition resemble laticifers in the primary body. As the girth of the root increases, laticifers toward the periphery, unlike associated sieve tubes, resist crushing and obliteration. Laticifers vary in width from about 4 to 22 μm; the widest ones occur in involucral bracts and the narrowest ones in florets. There was no evidence that intrusive growth occurs during development of the laticifer system, although such growth may occur during development of occasional branches which extend through ground tissue independent of phloem and terminate in contact with the epidermis. Presence of amorphous callose deposits is related to aging of laticifers and mechanical injury.     

Gibberellin signaling

A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.     

THE STRUCTURE AND DEVELOPMENT OF AN UNUSUAL TYPE OF ARTICULATED LATICIFER IN MAMMILLARIA (CACTACEAE)

Although the laticifers of several species of Mammillaria can technically be classified as being of the articulated type, they differ significantly from all other reported articulated laticifers. They are derived from cells which differentiate only in older tissues, never in meristematic or young regions. The development involves the complete lysis of masses of cells, not just the perforation or resorption of the end walls in a single file of cells. At maturity, the laticifer lumen is lined with a one-to-several layered epithelium which may be quite thick. The laticifers increase in diameter with age, apparently by the lysis of the inner epithelial cells. Laticifers occur in the pith, cortex and tubercles of the vegetative body but were not observed in the roots, flower parts or in seedlings up to eight months old. Seven species were studied, all of which have “milky sap.” and the laticifers of each were virtually identical to the laticifers of the others.     

Distribution of pectin and arabinogalactan protein epitopes during organogenesis from androgenic callus of wheat

The distribution of several arabinogalactan protein and pectic epitopes were studied during organogenesis in androgenic callus of wheat. In cell wall of mature and degenerating parenchyma cells, the arabinogalactan epitopes JIM4, JIM14, JIM16 or LM2 were expressed differently according to the cells location. LM2 was observed also in meristematic cells of regenerated shoot buds and leaves. Anti-pectin JIM7 labelled the wall of meristematic cells but fluorescence was strongest in outer walls of surface cells of callus and shoot buds coated by extracellular matrix surface network (ECMSN). During leaves growth the ECMSN disappeared, and JIM7 fluorescence decreased. JIM5 epitope was abundant in the cell walls lining the intercellular spaces of callus parenchyma and in tricellular junctions within regenerated buds and leaves.     

An impregnated suberized wall layer in laticifers of theConvolvulaceae,and its resemblance to that in walls of oil cells

Summary The cell wall in laticifers of theConvolvulaceae, Calystegia silvatica, C. soldanella, C. tuguriorum, Convolvulus cneorum, C. verecundus, C. sabaticus subsp.mauritanicus, andIpomoea indica, contains an impregnated layer that surrounds the cells. The impregnated layer lies inside the primary wall of the laticifer, separated from the protoplast by a third (tertiary) layer of variable thickness. Histochemical and cytochemical staining give a positive reaction for suberin. The layer is often differentiated into dark and translucent regions, the latter frequently being composed of lamellae. The ultrastructure of this layer and its position within the cell wall of the laticifer is comparable to the condition found in oil cells where the walls contain a suberized layer. A suberized layer within the wall is unique for a laticifer system.     

MITOSIS IN THE NON-ARTICULATED LATICIFER OF EUPHORBIA MARGINATA

The multinucleate condition in the non-articulated laticifers of embryos of Euphorbia marginata arises as a result of mitosis. Successive stages of mitosis in the nuclei of the laticifer appear in the form of a wave. No sequence of mitotic stages has been noticed in the neighboring longitudinal tiers of cells. This difference in the mitotic pattern in the laticifer and other parenchymatous cells of the embryo suggests that the synthesis of factor(s) responsible for triggering mitosis occurs within the laticifer and does not diffuse to the surrounding cells. The mitotic waves originate distally from the meristems, either in the cotyledonary or hypocotyl portion of the laticifer, and move uni- or bidirectionally along its longitudinal axis. The mitotic stimulus does not start simultaneously in all the laticifers. The variable velocity of the mitotic substance results in aphasic mitotic waves in laticifers of the same embryo. Mitotic aberrations have not been observed in the dividing nuclei of the laticifer. A chromosome estimation made from a polar view of metaphase does not suggest polyploidization in the observed laticifers.     

Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil

Plant sexual reproduction involves the growth of tip-polarized pollen tubes through the female tissues in order to deliver the sperm nuclei to the egg cells. Despite the importance of this crucial step, little is known about the molecular mechanisms involved in this spatial and temporal control of the tube growth. In order to study this process and to characterize the structural composition of the extracellular matrix of the male gametophyte, immunocytochemical and biochemical analyses of Arabidopsis pollen tube wall have been carried out. Results showed a well-defined localization of cell wall epitopes with highly esterified homogalacturonan and arabinogalactan-protein mainly in the tip region, weakly methylesterified homogalacturonan back from the tip and xyloglucan and (1→5)-α-L-arabinan all along the tube. Here, we present complementary data regarding (1) the ultrastructure of the pollen tube cell wall and (2) the immunolocalization of homogalacturonan and arabinan epitopes in 16-h-old pollen tubes and in the stigma and the transmitting tract of the female organ. Discussion regarding the pattern of the distribution of the cell wall epitopes and the possible mechanisms of cell adhesion between the pollen tubes and the female tissues is provided.Key words: arabinan, cell adhesion, cell wall, homogalacturonan, pistil, pollen tube growth, transmitting tractFertilization of flowering plants requires the delivery of the two sperm cells, carried by the fast growing tip-polarized pollen tube, to the egg cell. At every stage of the pollen tube development within the stigma, style and ovary, pollen tubes are guided to the ovules via multiple signals that need to pass through the cell wall of the pollen tube to reach their targets.^1–6The analysis of Arabidopsis pollen tube cell wall has recently been reported.⁷ Results showed a well-defined localization of cell wall epitopes with highly methylesterified homogalacturonan (HG) and arabinogalactan-protein (AGP) mainly in the tip region, weakly methylesterified HG back from the tip and xyloglucan and arabinan all along the tube. In addition, according to the one letter nomenclature of xyloglucan,⁸ the main motif of Arabidopsis pollen tube xyloglucan was XXFG harboring one O-acetyl group. In order to bring new information regarding the possible interaction between the pollen tubes and the female tissues, the ultrastructural organization of the pollen tube cell wall, the cytological staining and immunolocalization of the cell wall epitopes of the pistil and especially the transmitting tract (TT), a specialized tissue where pollen tubes grow, were carried out.     

F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments

Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1-->4)-beta-D-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner.     

Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvatochromism in the rubber-producing plant, Eucommia ulmoides Oliver

A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry.     

FOSSIL LATICIFERS FROM EOCENE BROWN COAL DEPOSITS OF THE GEISELTAL

Thick mats of cellular remains from Eocene brown coal deposits of the Geiseltal near Halle, DDR, were determined to be fossil nonarticulated laticifers. Nuclear magnetic resonance analyses of intact strands showed they consisted of eis-1,4-configuration rubber representing the polymerized isoprenoid contents of individual laticifers. Only remains of laticifers are present; other cells are absent as a result of biodegradation. The long laticifers, often with a surrounding cell wall, retained a tubular shape during their preservation. The isoprenoid content, which filled the entire lumen, possessed a cribriform structural character. The interstices within the rubber represent areas of former protoplasm of the cell. Various configurations in the protoplasm molded by the rubber during the initial phase of fossilization appear as negative images of former nuclei, organelles, and possibly membrane surfaces. The laticifer axes possess branches of several configurations comparable in morphology to those in branched, nonarticulated laticifers in extant plants. Acetone extracts of the rubber contents analyzed by gas-liquid chromatography identified the presence of several hydrocarbons which form a characteristic profile for the laticifer. It is suggested that the distinctive cellular micromorphology, rubber configuration, and hydrocarbon profile of these laticifers can be employed as markers in comparative studies with extant plants to identify the generic or species origin of these laticifers.     

Intercellular pectic protuberances in Asplenium: new data on their composition and origin

BACKGROUND AND AIMS: Projections of cell wall material into the intercellular spaces between parenchymatic cells have been observed since the mid-19th century. Histochemical staining suggested that these intercellular protuberances are probably pectic in nature, but uncertainties about their origin, composition and biological function(s) have remained. METHODS: Using electron and light microscopy, including immunohistochemical methods, the structure and the presence of some major cell wall macromolecules in the intercellular pectic protuberances (IPPs) of the cortical parenchyma have been studied in a specimen of the Asplenium aethiopicum complex. KEY RESULTS: IPPs contained pectic homogalacturonan, but no evidence for pectic rhamnogalacturonan-I or xylogalacturonan epitopes was obtained. Arabinogalactan-proteins and xylan were not detected in cell walls, middle lamellae or IPPs of the cortical parenchyma, whereas xyloglucan was only found in its cell walls. Extensin (hydroxyproline-rich glycoproteins) LM1 and JIM11 and JIM20 epitopes were detected specifically in IPPs but not in their adjacent cell walls or middle lamellae. CONCLUSIONS: It is postulated that IPPs do not originate exclusively from the middle lamellae because extensins were only found in IPPs and not in surrounding cell walls, intercellular space linings or middle lamellae, and because IPPs and their adjacent cell walls are discontinuous.