Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps.

The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.

The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle transport, vesicle fusion and tip extension in pollen tubes.

The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine, all antagonists of Ca(2+) function in cells, have been studied in growing pollen tubes of Tradescantia virginiana. All four drugs inhibit pollen-tube growth but bring about different ultrastructural changes at the growing tips and within the cytoplasm. The results strongly support the hypothesis that Ca(2+) plays a vital role in the mechanism of pollen-tube tip growth. The effect of ruthenium red provides evidence that sequestration of Ca(2+) by mitochondria critically adjusts the concentration of these ions at tube tips. Fluorescein isothiocyanate appears to be a potent inhibitor of vesicle fusion at the plasma membrane, with vesicles accumulating in the tip at rates equivalent to those determined previously for their production. Both vesicle fusion and tip extension are regulated by Ca(2+) but appear to be independently controlled processes.

The inhibition of dictyosome vesicle formation in higher plant cells by cytochalasin D.

We have examined the effect of cytochalasin D on secretory processes in plant root tips and pollen tubes. While confirming that inhibition of vesicle transport is the immediate effect of the drug, we now present quantitative evidence to show that vesicle formation by elements of the Golgi apparatus in plants, the dictyosomes, is progressively inhibited. Total inhibition of vesicle formation occurs within exposure times ranging from one-half to two hours. It is concluded that vesicle formation is a cytochalasin-sensitive process.

Membrane recycling and the control of secretory activity in pollen tubes.

Pollen tubes of Tradescantia growing on media containing 10(-3) M-Ca2+ and 10(-2)M-Ca2+ exhibit growth rates of 28 micrometers min-1 and 7 micrometers min-1, respectively. The rates of vesicle production by the dictyosomes in these tubes were determined from the rates at which vesicles accumulated in the cytoplasm after treatment with cytochalasin D. Although the vesicle requirements for these two growth rates are considerably different, it was found that vesicle production rates were the same. The results indicate that, in pollen tubes, membrane recycling is occurring and that dictyosome activity is not regulated according to the vesicle requirements for tube growth.

The effect of cycloheximide on dictyosome activity in Tradescantia pollen tubes determined using cytochalasin D.

Dictyosome activity in Tradescantia pollen tubes has been determined using a recently developed method based on the assumption that the rate of vesicle accumulation around the dictyosomes, after treatment with cytochalasin D, is equivalent to the actual rate of vesicle production. In tubes germinated in the presence of 1.0 micrograms/ml cycloheximide, reduced dictyosome activity could be detected as early as 10 min after sowing, although tube extension was not halted until later. After 30 min vesicle production had completely ceased. These observations are discussed in relation to previous reports on the effect of cycloheximide on pollen tube growth, and in relation to the synthesis and transfer of membrane proteins to secretory vesicles and the plasma membrane. It is concluded that the ability of pollen to germinate and produce short tubes in the presence of cycloheximide, does not necessarily indicate that protein synthesis is not a requirement for early pollen tube growth, as protein shortages would not be expected to become apparent over time periods less than the dictyosome turnover time and the secretory vesicle residence time.

Determination of secretory vesicle production rates by dictyosomes in pollen tubes of Tradescantia using cytochalasin D.

Pollen tubes of Tradescantia were grown in vitro and exposed to 0.3 microgram/ml cytochalasin D for 5 or 10 min. Fine-structural observations revealed no visible effect of the drug on the organelles. Stereological analysis, using a method recently developed by Rose (1980) to obtain sphere size-distributions corrected for section thickness, revealed substantial increase in the number of secretory vesicles present in the cytoplasm around the dictyosomes. Equating the rate of vesicle accumulation with the rate of vesicle production, a total of 5388 vesicles per minute are formed by a growing tube. This corresponds to 2.4 vesicles per minute per dictyosome, and a turnover rate of 3.7 min for a single dictyosome cisterna, or about 15-18.5 min for a complete dictyosome. The calculated vesicle production rate agrees well with that required to sustain the observed growth rate of such tubes, based on the addition of membrane or wall material to the tube tip.

Differentiation of the tapetum in Avena. I. The cell surface.

The development of the tapetal cell surface and associated structures in Avena has been followed from cell formation to senescence. Plasmodesmata initially connect the tapetal cells to each other, the pollen mother cells, and the inner loculus wall cells. These connexions are subsequently severed, those to the sporogenous cells being broken first at the pollen mother cell surface during callose wall formation. Loss of cellulose from the tapetal walls was followed using the decline in the ability of the wall to bind the fluorescent brightener, Calcofluor White M2R New. Subplasma-membrane microtubules persist after loss of the cellulose wall. The tapetal plasma membrane facing the meiocytes then develops a series of depressions, or cups, over its surface, which are later the site of pro-orbicule formation. Sporopollenin is laid down over the pro-orbicules, to form orbicules, and over other tapetal cell surfaces. No morphological evidence was found for the intracytoplasmic formation of pro-orbicules or polymerized sporopollenin precursors. These observations on Avena are compared with those on other plants. The changes in the cell wall and associated structures, plasmodesmata and microtubules, are considered in detail, while the general significance of cell wall loss to the water relations of the tissue are assessed. Proposals that pro-orbicule formation results from non-specific accumulation of lipid at a free cell surface are rejected, instead this formation is considered to be related to the presence of a specially modified plasmamembrane surface.

Nuclear and cytoplasmic genome relationships in the genus Avena: analysis by isoelectric focusing of ribulose biphosphate carboxylase subunits.

Comparisons of the isoelectric points of small and large subunits of ribulose biphosphate carboxylase extracted from a number of diploid, tetraploid, and hexaploid Avena species have been used to obtain information on the nuclear and cytoplasmic genome relationships within the genus. All species tested had small subunits with similar isoelectric points, so their analysis provided no information of taxonomic value. Three types of large subunits could be distinguished by this method, and the distribution of each among the available species provides strong evidence against the involvement of a C genome diploid (such as A. ventricosa) as the maternal parent in the formation of either tetraploid or hexaploid species. One type of large subunit was confined to the perennial tetraploid, A. macrostachya, and its position in the genus and possible origin are discussed. The value of this approach in studying genome relationships within the genus Avena and related genera is assessed.

An atypical crista resembling a "tight junction" in bean root mitochondria.

The conformation and structure of an atypical crista found in a small percentage of the mitochondria in root tip cells of Phaseolus vulgaris L. have been studied electron microscopically in material fixed in glutaraldehyde followed by osmium tetroxide. In its transformation into an atypical crista, a normal crista elongates, broadens, and flattens, and the inner leaflets of its apposed unit membranes appear to fuse in a manner analogous to the formation of "tight junctions" between certain animal cells. The result is a large platelike, quintuple-layered structure, 240-260 A thick, whose long axis parallels that of the mitochondrion. The outer layers of the "plate," bordering on the mitochondrial matrix, are thickened and exhibit striking patterns in the micrographs. The structure of the plate is compared with that previously described for tight junctions between animal cells.

Isolation, properties, and structure of fraction I protein from Avena sativa L.

1. A method is described for the extraction and purification of Fraction I protein from Avena sativa L. leaves. 2. The protein possesses ribulose diphosphate carboxylase activity. Chromatography on gels of Sephadex G-200 separates phosphoribulokinase and ribose phosphate isomerase from the carboxylase. 3. The S°20w was calculated to be 18.2, the Stokes radius (determined by gel filtration on a cabibrated column) 74 Å, the molecular weight 5.7×10(5), and the frictional ratio 1.35. 4. An amino acid analysis is presented. 5. Electron microscope observations of negatively-stained Avena Fraction I protein molecules are compatible with the suggestion that they consist of 24 protomers disposed on the surface of an octahedral shell with 4:3:2 symmetry, and of diameter approximately 105 Å.