Digitonin-aided loading of Fluo-3 into embryogenic plant cells.

This paper describes a method to load embryogenic plant cells with Fluo-3 in its cell impermeant form with the aid of digitonin. Attempts to load cells with Fluo-3/AM were all unsuccessful. Presumably the indicator is cleaved outside the cells and cannot penetrate in its acidic form. At a low pH, Fluo-3 enters the plant cells but normal Ca2+ homeostasis seems to be disturbed. Successful loading of Fluo-3 was achieved by adding 0.1% digitonin during incubation with the Ca(2+)-indicator. A bright fluorescence was observed in the epidermal layer of heart and torpedo shaped somatic embryos of carrot with confocal scanning laser microscopy. Vacuoles were always without fluorescence which indicates that the dye, after loading, remains in the cytosol and does not leak out. The fluorescence intensity was sensitive to treatments with A23187 and EGTA. We conclude that Fluo-3 can be effectively loaded, with the aid of digitonin, into plant embryogenic cells in liquid culture. Therefore, we expect this technique to be very useful for the study of changes in cytosolic free Ca2+ levels during plant growth and development.

Hint of polar distribution in calcium channels under PIXE analysis.

Pollen tubes ofLilium longiflorum were treated for 10-30 min with 10(-5) M CoCl2, which binds to calcium channels in the plasma membrane and blocks them. Cobalt analyses were performed with the Heidelberg proton microprobe, using 3 MeV protons, a beam current of about 200 pA, and a spot size of 3×5 µm(2). X-ray spectra revealed that coblat has much higher concentrations in the cell than in the surrounding dried medium. The line scans, taken along the longitudinal cell axis in 1-µm steps, showed a cobalt gradient similar to the calcium gradient of the same cell. Based on our findings, we can conclude that neither do the cobalt signals come from the cell wall nor is the cobalt exclusively bound to the intracellular calcium-binding sites. There-fore, the present results suggest a polar distribution in calcium channels in the plasma membrane of pollen tubes.

Electron spectroscopic imaging (ESI) applied to the detection of potential Ca(2+)-binding sites in plant cells.

The applicability of the electron spectroscopic imaging technique for detection of the intracellular distribution of calcium in plant cells was tested with calyptra cells ofZea mays and with pollen tubes ofLilium longiflorum. After fixation in enhanced Ca(2+) levels and embedding in resin, ultrathin sections were analyzed for the elemental distribution. Calcium and phosphorus were enriched in cell wall, plasma membrane, endoplasmic reticulum, mitochondria, and Golgi vesicles, mainly in granular or globular deposits appearing electron dense in transmission electron microscopy. The results demonstrated that the ESI-technique allows exact localization of calcium enrichment relative to specific cell organelles.

Nifedipine-sensitive calcium channels are involved in polar growth of lily pollen tubes.

Pollen germination and tube growth of Lilium longiflorum in vitro are affected by 10(-5) M-nifedipine. Germinating 'tubes' form broad protuberances along the whole colpus. Short tubes show a high tendency to grow 'amoeboid-like' and to branch; or a second tube emerges in another region of the colpus. Longer tubes (greater than or equal to 100 micron) broaden irregularly or swell at their tips. The diameter of the tube can vary drastically within the same tube. With increasing time of treatment many tubes burst. Normal tip growth stops within 10 min, but protoplasmic streaming continues even after 15 h. More or less regularly, wall thickenings are formed along the whole tube or on the flanks of the germinating region after some hours. The internal calcium gradient, visualized by chlorotetracycline (CTC) fluorescence, is also disturbed. Nifedipine treatment results in uniform or irregular CTC fluorescence. Branching tubes temporarily show a new subapical CTC gradient. After 6-8 h of nifedipine treatment many cells are no longer stainable with CTC. The results indicate the presence of nifedipine-sensitive calcium channels in pollen tubes.

Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum.

Chlorotetracyclin (10(-4)M) has been used to observe the distribution of membrane-associated calcium during pollen germination of Lilium longiflorum. For comparison, the general membrane distribution has been determined with 4·10(-5) M fluorescamine. The pollen grains show a calcium gradient with either weak or strong chlorotetracycline-fluorescence intensity, but always increasing toward the germination colpus. This gradient intensifies during germination, reaching a maximum before the pollen tube emerges. The typical tip-to-base calcium gradient of the tube does not change during growth. Independent of the developmental stage, the pollen grains show a flat fluorescamine-fluorescence gradient with the highest intensity in one half of the grain. Pollen tubes reveal a tip-to-base membrane gradient, independent of their length. As an additional marker for membrane distribution, the distribution of phosphorus, measured by proton-induced X-ray emission in chemically fixed tubes, has been used. A tip-to-base phosphorus gradient, distinct from the calcium gradient measured with the same method, was detected.

Cllmodulin in tip-growing plant cells, visualized by fluorescing calmodulin-binding phenothiazines.

Calmodulin (CaM) was visualized light-microscopically by the fluorescent CaM inhibitors fluphenazine and chlorpromazine, both phenothiazines, during polar tip growth of pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya spec. and in the alga Acetabularia mediterranea, as well as during multipolar tip growth in Micrasterias denticulata. Young pollen tubes and root hairs showed tip fluorescence; at later stages and in the growing parts of the other subjects the fluorescence was almost uniform. After treatment with cytochalasin B, punctuate fluorescence occurred in the clear zone adjacent to the tip of pollen tubes. The observations indicate that there is CaM in all our tested systems detectable with this method. It may play a key role in starting polar growth. As in pollen tubes, CaM might be in part associated with the microfilament network at the tip, and thus regulate vesicle transport and cytoplasmic streaming.

The plasma membrane of the Funaria caulonema tip cell: morphology and distribution of particle rosettes, and the kinetics of cellulose synthesis.

Freeze-fracturing of Funaria hygrometrica caulonema cells leads to a cleavage within the plasma membrane. The extraplasmatic and the plasmatic fracture faces differ in their particle density. The plasmatic fracture face in caulonema tip cells or in tip cells of side branches, but never in other caulonema cells, is further characterized by the occurrence of particle rosettes. The highest density of rosettes is found at the cell apex but decreases steeply toward the cell base. The shape of the rosettes varies remarkably; 20% of them are found in an incomplete, presumably disintegrating or aggregating state. The complete rosette has a diameter of about 25 nm and consists of five to six particles. The size of the single particles varies between 4 nm to 10 nm. The rosettes are thought to posses cellulose-synthase activity. It is assumed that one rosette produces one elementary fibril; rough calculations, considering the number of rosettes and the estimated amount of cellulose produced in the tip region, indicate that an elementary fibrillar length of 900 nm is formed in 1 min by one rosette. The consequence of the kinetics on the life-time of the rosettes and the cellulose-synthase activity are discussed.

Disoriented growth of pollen tubes of Lilium longiflorum Thunb. induced by prolonged treatment with the calcium-chelating antibiotic, chlorotetracycline.

Pollen of Lilium longiflorum Thunb. was germinated for 12 h in growth medium containing 1·10(-4) M chlorotetracycline (CTC), or growing tubes were treated with 1·10(-4) M CTC for up to 2 h. These treatments have drastic effects: In the CTC-containing medium, out-growing tubes form only short tubes. Irregular wall thickenings are visible. Thirty minutes CTC-treatment cause growing tubes to bend and grow back toward the grain. Electron micrographs of CTC-treated tubes show that CTC affects the organelle distribution: The polar zonation of organelles is disturbed. Vesicle-and endoplasmic reticulum-accumulations are found in the wrong places, together with extensive wall thickenings and a very irregular plasma membrane. The structural details of most cell organelles look normal after CTC treatment, but the mitochondria possess unusual cristae, and microtubules are absent. The disoriented growth is interpreted as an effect of the ability of CTC to chelate intracellular calcium ions, to bind them to membranes, and thus to disturb the dynamics of the delicate Ca(2+)-equilibria thought to regulate oriented exocytosis.

Broad-range effects of ionophore X-537A on pollen tubes of Lilium longiflorum.

The effects of the broad-range cationophore X-537A on pollen tubes of Lilium longiflorum were investigated, using both light and electron microscopy. Pollen tube growth is completely inhibited within 30 min after the application of 5·10(-5) M ionophore X-537A; cytoplasmic streaming is stopped only after 60 min of ionophore treatment. Ultrastructurally, X-537A effects are a vacuolation of Golgi cisternae and a general vacuolation. The wall is thickened at the very tip. Coated vesicles and coated regions are enriched close to and at the plasma membrane. The results indicate that pollen tube tip growth needs a specific ion distribution.

Calcium ionophore A 23187 affects localized wall secretion in the tip region of pollen tubes of Lilium longiflorum.

The effects of the calcium inonophore A 23187 on growing pollen tubes of Lilium longiflorum Thunb. cv. Ace were investigated with the light and electron microscope. Tip growth is slowed down and stopped within 20 min after application of 5x10(-5) M ionophore A 23187. The main effects are the disappearance of the clear zone at the pollen tube tip and a thickening of the cell wall at the tip and at the pollen tube flanks. This effect on cell wall formation is confirmed under the electron microscope: The vesicular zone in treated pollen tubes is reduced, numerous vesicular contents are irregularly integrated in the pollen tube wall not only in the tip, but over a long distance of the pollen tube wall. In addition, effects on mitochondria and dictyosomes are observed. These results are interpreted as a disorientation of the Ca(2+)-based orientation mechanism of exocytosis after equilibration of the Ca(2+)-gradient.

Calcium gradients in tip growing plant cells visualized by chlorotetracycline fluorescence.

With chlorotetracycline (CTC)-fluorescence a tip-to-base Ca(2+) gradient is visualized in all tested, tip-growing plant cells: pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya and in the alga Acetabularia mediterranea. The fluorescence gradients in the different species vary in intensity and extension. Sometimes a punctate mobile CTC-fluorescence, in the size range of mitochondria, is observed. Bursting cells lose their fluorescence rapidly, indicating a cytoplasmic localization of the gradient. Only in Acetabularia is the wall also fluorescent with CTC. The results are interpreted as evidence for a general role of a calcium gradient in tip growth.