Marianne Dauwalder

The Golgi apparatus and an early stage in cell plate formation.

Glutaraldehyde-fixed, osmium postfixed root apex cells in successive stages of cytokinesis show the involvement of small vesicular bodies in cell plate formation. These bodies apparently are produced by the Golgi apparatus; beginning by metaphase, they invade the spindle, cluster in the equatorial regions of the cell, and seem to set up some condition essential for the fusion of large Golgi vesicles in the same region. The plate is formed by this fusion, during which the small vesicles are incorporated into it. Plate formation proceeds centrifugally, with spindle fibers and clusters of the small vesicular bodies marking the edges, around which vesicle-producing Golgi apparatus are grouped. Puromycin affects either the production of the small vesicular bodies or their movement away from the Golgi cisternae. Colchicine precludes fusion of the larger Golgi vesicles, apparently by preventing clustering of the small ones, perhaps indirectly through effects on the spindle.

The Golgi apparatus, the plasma membrane, and functional integration.

Publisher Summary This chapter discusses the Golgi apparatus, the plasma membrane, and functional integration. Through the years many different suggestions have been made concerning the activities of the Golgi apparatus. The information available was largely morphological and often led to conflicting interpretations. Although the role of the Golgi apparatus in secretion has been well supported, there is as yet no generally accepted explanation for its presence in some form in all eukaryotic cells. Although principal attention has been directed to membrane interactions, some aspects of the role of the Golgi apparatus in secretion as defined in the more limited sense is described in the chapter. A broad spectrum of functionally important materials is involved, and these may act at varying distances from the site of their production. Cellular function and sociological behavior are to a great extent affected by elements outside the cell associations with other cells, characteristics of surrounding matrix materials, and numerous circulating or environmental factors.

Distribution of calmodulin in pea seedlings: immunocytochemical localization in plumules and root apices

Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in young etiolated pea (Pisum sativum L.) seedlings. A fairly uniform staining was seen in the nucleoplasm and background cytoplasm of most cell types. Cell walls and nucleoli were not stained. In addition, patterned staining reactions were seen in many cells. In cells of the plumule, punctate staining of the cytoplasm was common, and in part this stain appeared to be associated with the plastids. A very distinctive staining of amyloplasts was seen in the columella of the root cap. Staining associated with cytoskeletal elements could be shown in division stages. By metaphase, staining of the spindle region was quite evident. In epidermal cells of the stem and along the underside of the leaf there was an intense staining of the vacuolar contents. Guard cells lacked this vacuolar stain. Vacuolar staining was sometimes seen in cells of the stele, but the most distinctive pattern in the stele was associated with young conducting cells of the xylem. These staining patterns are consistent with the idea that the interactions of plastids and the cytoskeletal system may be one of the Ca2+-mediated steps in the response of plants to environmental stimuli. Nuclear functions may also be controlled, at least in part, by Ca2+.

Purification and immunolocalization of an annexin-like protein in pea seedlings.

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds 45Ca2+ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.

Immunolocalization and histochemical evidence for the association of two different Arabidopsis annexins with secretion during early seedling growth and development.

Annexins are a multigene, multifunctional family of calcium-dependent, membrane-binding proteins found in animal and plant cells. In plants, annexins have been localized in the cytoplasm and at the cell periphery of highly secretory cell types, and in the tip region of polarly growing cells. Consequently, one proposed function for annexins in plant cells is participation in the Golgi-mediated secretion of new wall materials. In Arabidopsis, there are eight different annexin cDNAs, which share between 30% and 81% deduced amino acid sequence identity. We have used two monospecific Arabidopsis anti-annexin antibodies, raised against divergent 31-mer peptides from AnnAt1 and AnnAt2 and a previously characterized pea anti-annexin p35 antibody, for Western blot and immunolocalization studies in Arabidopsis. Western blot analyses of various Arabidopsis protein fractions showed that the two Arabidopsis antibodies are able to specifically recognize annexins in both soluble and membrane fractions. Immunofluorescence results with the three annexin antibodies show staining of secretory cells, especially at the cell periphery in developing sieve tubes, outer root cap cells, and in root hairs, consistent with previous results. In developmentally different stages some staining was also seen near the apical meristem, in some leaf cells, and in phloem-associated cells. Autoradiography following 3H-galactose incorporation was used to more clearly correlate active secretion of wall materials with the localization patterns of a specific individual annexin protein in the same cells at the same developmental stage. The results obtained in this study provide further support for the hypothesis that these two Arabidopsis annexins function in Golgi-mediated secretion during early seedling growth and development.

Staining of cells of Zea mays root apices with the osmium-zinc iodide and osmium impregnation techniques.

In the three cell types designated as secreting, the OZI procedure stained only 1 to 3 cisternae at the proximal face of the Golgi apparatus, Neither secretory vesicles nor secreted materials showed staining. In other cells the Golgi apparatus was most commonly stained at both faces with unstained cisternae “sandwiched” between the stained ones. This latter pattern was also seen in dividing cells even though in these cells the apparatus produces materials to be “secreted” into the cell plate. Certain specialized internal membranes of the amyloplasts were also stained, as were, variably, the nuclear envelope, the endoplasmic reticulum, and some vacuoles. With osmium no staining of the Golgi apparatus was observed, the same internal plastid membranes were stained, and the nuclear envelope and endoplasmic reticulum showed variable staining. Neither procedure stained lipid droplets. The significance of these staining reactions for Golgi apparatus is discussed.

Assembly, Continuity, and Exchanges in Certain Cytoplasmic Membrane Systems

Basic to the definition of a cell is a membrane separating the activities within it from the surrounding environment. In even the simplest organisms, the plasma membrane, or plasmalemma, bounding the cellular mass is characterized by definable structure and distinctive physiological properties. The structure, composition, and properties of membranes have all been subjects of extensive, interrelated studies. This work will de al with questions about their assembly, continuity, and exchange. Only brief consideration will be given to structure, composition, and properties to provide a basic understanding that will lend coherence to the other questions.

Immunological and biochemical evidence for nuclear localization of annexin in peas.

Immunofluorescent localization of annexins using an anti-pea annexin polyclonal antibody (anti-p35) in pea (Pisum sativum) leaf and stem epidermal peels showed staining of the nuclei and the cell periphery. Nuclear staining was also seen in cell teases prepared from pea plumules. The amount of nuclear stain was reduced both by fixation time and by dehydration and organic solvent treatment. Observation with confocal microscopy demonstrated that the anti-p35 stain was diffusely distributed throughout the nuclear structure. Immunoblots of purified nuclei, nuclear envelope matrix, nucleolar, and chromatin fractions showed a cross-reactive protein band of 35 kDa. These data are the first to show annexins localized in plant cell nuclei where they may play a role in nuclear function.

Redistribution of annexin in gravistimulated pea plumules

We used immunocytochemistry to investigate the effects of gravistimulation on annexin localization in etiolated pea plumule shoots. In longitudinal sections, an asymmetric annexin immunostaining pattern was observed in a defined group of cells located just basipetal to apical meristems at the main shoot apex and at all of the axillary buds, an area classically referred to as the leaf gap. The pattern was observed using both protein-A-purified anti-annexin and affinity-purified anti-annexin antibodies for the immunostaining. A subset of the cells with the annexin staining also showed an unusually high level of periodic acid Schiff (PAS) staining in their cell walls. Prior to gravistimulation, the highest concentration of annexin was oriented toward the direction of gravity along the apical end of these immunostained cells. In contrast, both at 15 and 30 min after gravistimulation, the annexin immunostain became more evenly distributed all around the cell and more distinctly cell peripheral. The asymmetry along the lower wall of these cells was no longer evident. In accord with current models of annexin action, we interpret the results to indicate that annexin-mediated secretion in the leaf gap area is preferentially toward the apical meristem prior to gravistimulation, and that gravistimulation results in a redirection of this secretion. These data are to our knowledge the first to show a correlation between the vector of gravity and the distribution of annexins in the cells of flowering plants.

Immunolocalization of an annexin-like protein in corn

Although calcium has been proposed to be an important regulatory element in plant gravitropic growth, as yet no specific function of Ca2+ in growth regulation has been discovered. Our recent studies on a Ca(2+)-binding protein in pea seedlings called p35 indicate that it is a member of the annexin family of proteins and may play a key role in growth regulation through its function in delivering polysaccharides needed for wall construction. We previously reported the isolation of p35 from pea plumules and the production of polyclonal antibodies to it. Immunolocalizaton analyses of p35 in pea tissues revealed high levels of staining in secretory cell types such as developing vascular cells and outer root cap cells. To test how general was the occurrence and distribution of this annexin-like protein in plant cells we initiated an analysis of annexins in the monocot corn using immunological techniques. Our results indicate the immunochemical properties and localization of corn annexins are very similar to those reported for pea. They are consistent with the postulate that annexins may play a general role in the regulation of the secretion of wall polysaccharides needed for growth, and thus could be an important target of calcium action during gravitropic growth.