Gregory B. Clark

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.

Rapid and Efficient Suppression of Gene Expression in a Single-Cell Model System, Ceratopteris richardii

Germinating Ceratopteris richardii spores have proven to be a useful single-cell system for analyzing light and gravity regulation of development ([Cooke et al., 1995][1]; [Banks, 1999][2]; [Chatterjee and Roux, 2000][3]; [Murata and Sugai, 2000][4]), but the lack of a gene sequence database and of

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.

RNA-seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): evidence for modulation by calcium pumps and apyrase activity.

PREMISE OF THE STUDY Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spores downward polarity alignment, a polarization that is fixed by gravity ∼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. METHODS De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents. KEY RESULTS Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. CONCLUSIONS In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.