Cellular localization and levels of pectins and arabinogalactan proteins in olive (Olea europaea L.) pistil tissues during development: implications for pollen-pistil interaction.

Cell wall components in the pistil are involved in cell-cell recognition, nutrition and regulation of pollen tube growth. The aim of this work was to study the level, whole-organ distribution, and subcellular localization of pectins and arabinogalactan proteins (AGPs) in the olive developing pistil. Western blot analyses and immunolocalization with fluorescence and electron microscopy were carried out using a battery of antibodies recognizing different types of pectin epitopes (JIM7, JIM5, LM5, and LM6) and one anti-AGPs antibody (JIM13). In the olive pistil, highest levels of acid esterified and de-esterified pectins were observed at pollination. Moreover, pollination was accompanied by a slight decrease of the galactose-rich pectins pool, whereas arabinose-rich pectins were more abundant at that time. An increased expression of AGPs was also observed during pollination, in comparison to the pistil at the pre-anthesis stage. After pollination, the levels of pectins and AGPs declined significantly. Inmunofluorescence localization of pectins showed their different localization in the olive pistil. Pectins with galactose residues were located mainly in the cortical zones of the pistil, similar to the neutral pectins, which were found in the parenchyma and epidermis. In turn, the neutral pectins, which contain arabinose residues and AGPs, were localized predominantly in the stigmatic exudate, in the cell wall of secretory cells of the stigma, as well as in the transmitting tissue of the pistil during the pollination period. The differences in localization of pectins and AGPs are discussed in relation to their roles during olive pistil developmental course.

Arabinogalactan-proteins stimulate the organogenesis of guard cell protoplasts-derived callus in sugar beet.

Arabinogalactan proteins (AGPs) represent a class of proteoglycans implicated in the development and differentiation of cells and tissues both in planta and in vitro. Here we report that AGP-rich extracts isolated from media of embryogenic and non-embryogenic suspension cultures of sugar beet (Beta vulgaris L.) are able to enhance the organogenesis of guard protoplast-derived callus and to increase the number of shoots formed, in comparison to control cultures. Immunocytochemical detection of carbohydrate antigens in the extracts revealed the presence of epitopes that typify both AGP and pectin, the latter being frequently bound to AGPs or, in some cases, even contributing to the polysaccharide structure of proteoglycan molecules. The most abundant epitopes proved to be those recognized by the JIM13, LM2, and MAC207 antibodies, whereas some others could be found only in relatively small or trace amounts--these included epitopes recognized by JIM16, JIM5, and LM6. Surprisingly, the JIM4- and JIM8-binding epitopes that are expressed in the course of in vitro morphogenetic processes of many species could not be detected at all in sugar beet AGPs. This is the first report of the improvement of sugar beet protoplast-derived callus organogenesis by exogenous AGP-rich extracts, an achievement that will have great impact on the biotechnological applications of protoplast technology in this species.

Immunodetection of pectin and arabinogalactan protein epitopes during pollen exine formation of Beta vulgaris L.

We present the results of ultrastructural and immunocytochemical studies of sugar beet microsporocytes during the developmental phase that begins with the first meiotic metaphase and ends with the formation of young tetrads. The most prominent feature noted during this period of microsporogenesis was the presence of numerous cisternae of endoplasmic reticulum which frequently lie perpendicular to the surface of the plasma membrane and eventually fuse to it. Microscopic observations have been combined with the detection of several carbohydrate epitopes representing pectins and arabinogalactan proteins in the primexine and incipient exine. Pectin domains that possess both low and highly methylesterified epitopes, as well as pectin side chains enriched in (1-->4)-beta-D-galactose residues, are deposited in this young microspore wall. The epitopes of arabinogalactan protein that bind to JIM13, JIM8, and LM2 antibodies are localised within the callose wall surrounding posttelophase tetrads. The possibility of endoplasmic-reticulum involvement in the synthesis, transport, or metabolism of several microspore wall compounds is discussed.

Cell wall polysaccharides in differentiating anthers and pistils of Lolium perenne.

We are presenting the pattern of distribution of several carbohydrate epitopes, which constitute an important component of cell walls, within the anthers and pistils of a monocot grass species, perennial ryegrass (Lolium perenne L.). The results of immunocytochemical studies revealed that the flower organs are rich in (1-->3, 1-->4)-beta-D-glucans and possess surprisingly high amounts of methylesterified pectic domains that bind JIM7 antibody and pectin side chains rich in (1-->4)-beta-D-galactose residues which react with LM5 antibody. The presence of arabinogalactan protein epitopes binding JIM13 is restricted to microspores and ovule integuments. The results are discussed in terms of possible functions of cell wall polysaccharides and arabinogalactan proteins in the differentiation of flower organs.

Temporal and spatial distribution of pectin epitopes in differentiating anthers and microspores of fertile and sterile sugar beet.

We studied the possible involvement of several pectin epitopes in anther differentiation and microsporogenesis in fertile and cytoplasmically male sterile sugar beets. The spatial and temporal distribution of five structural motifs were traced with a panel of monoclonal antibodies in six stages: premeiosis, meiotic prophase, young and mature tetrads, young and expanding microspores. The composition of the walls of sporogenous cells and meiocytes differed than that in the tapetum, as evidenced by the presence of alpha-Fuc(1-->2)-beta-Gal and alpha-(1-->5)-L-Ara epitopes binding CCRC-M1 and LM6 antibodies. At meiotic prophase, the meiocyte walls were additionally marked by the appearance of poorly methyl-esterified domains of homogalacturonan and of (1-->4)-beta-Gal residues, detected by JIM5 and LM5. Some constituents of the meiocyte wall which reacted with JIM5 and JIM7 persisted on the surface of the special callose sheath during tetrad development. In newly formed primexine and exine layers of tetrads and microspores, epitopes that were bound by JIM5, JIM7 and LM5 were abundant. No differences in the deposition or relative abundance of pectins were found between fertile and sterile anthers until microspore release from the callose. Later, at the time of abortion, sterile microspores had much larger amounts of epitopes detected by JIM5 than their fertile counterparts.

Endophyte transmission via seeds of Lolium perenne L.: immunodetection of fungal antigens.

We traced the presence and distribution of Neotyphodium lolii within developing inflorescences and embryos of perennial ryegrass (Lolium perenne L.), cultivar "Grassland Nui," by in situ immunolocalization of fungal proteins. Evidence is presented that the fungus penetrates through the rachilla at the base of the ovary, and localizes in a very precise and specific manner in the ovular nucellus, but never enters the embryo sac or the integuments. Young embryos do not contain mycelium, but as they mature the hyphae penetrate through the scutellum from a neighboring "infection layer"-a remnant of nucellus heavily colonized by the fungus-that is readily visible as a discrete area directly adjoining the base of embryo cavity. Our observations document that N. lolii is transmitted to the embryo exclusively via sporophytic maternal tissue.

Guard cell wall: immunocytochemical detection of polysaccharide components.

The composition of guard cell walls in sugar beet leaves (Beta vulgaris L.) was studied by using histochemical staining and immunocytochemical detection of cell wall antigens. The findings were compared with those in the walls of epidermal and mesophyll cells. Probing of leaf sections with monoclonal antibodies against pectins, terminal fucosyl residues linked alpha-(1-->2) to galactose, beta-(1-->3)-glucans and arabinogalactan-proteins revealed several specific features of guard cells. Pectic epitopes recognized by JIM7 were homogeneously distributed in the wall, whereas pectins recognized by JIM5 were not found in the walls themselves, but were abundant in the cuticular layer. Large amounts of molecules bearing terminal fucose were located predominantly in ventral and lateral guard cell walls. Much smaller amounts were detected in dorsal walls of these cells, as well as in the walls of pavement and mesophyll cells. Conspicuous accumulation of these compounds was observed in the vicinity of the guard cell plasmalemma, whereas labelling was scarce in the areas of the wall adjacent to the cell surface. The presence of callose clearly marked the ventral wall between the recently formed, very young guard cells. Callose also appeared in some mature walls, where it was seen as punctate deposits that probably reflected a specific physiological state of the guard cells. Large amounts of arabinogalactan-proteins were deposited within the cuticle, and smaller amounts of these proteoglycans were also detected in other tissues of the leaf. The histochemical and immunocytochemical structure of the guard cell wall is discussed in the light of its multiple functions, most of which involve changes in cell size and shape.