Hans-Dieter Reiss

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

With chlorotetracycline (CTC)-fluorescence a tip-to-base Ca2+ 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.

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 Ca2+-based orientation mechanism of exocytosis after equilibration of the Ca2+-gradient

Quantitative analysis of calcium gradients and activity in growing pollen tubes ofLilium longiflorum

SummaryPollen tubes ofLilium longiflorum were loaded with quin-2 to determine the cytoplasmic free calcium. Quin-2-fluorescence was detected at 500 nm with alternating excitation at 340 nm and 360 nm. The calcium2+-concentration was obtained using the intensity ratio R=I340/I360. The analysis exhibits a [Ca2+] of nearly 10−7mol·l−1 in the tip region and about 2·10−8 mol·l−1at the tube base, near the pollen grain. The data give evidence for the existence of a continuous gradient of free calcium within growing pollen tubes of various length.

The tip-to-base calcium gradient in pollen tubes ofLilium longiflorum measured by proton-induced X-ray emission (PIXE)

SummaryThe intracellular calcium distribution in pollen tubes ofLilium longiflorum was measured by proton-induced X-ray emission (PIXE) with the Heidelberg proton microprobe, which is a new method for biological application. Studies by electron-induced X-ray emission (EIXE) were done in comparison. Independent of the preparation technique, the pollen tubes show a tip-to-base calcium gradient. The shape of the calcium gradient and the total calcium content depend on the preparation technique. The calcium ionophore A-23187 destroys the calcium gradient and leads to a loss of most cell calcium. Chlorotetracycline (CTC) treatment increases the amount of membrane-bound calcium.

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.

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

Chlorotetracyclin (10-4M) 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.

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 Ca2+-equilibria thought to regulate oriented exocytosis.

Quin-2 fluorescence in lily pollen tubes: Distribution of free cytoplasmic calcium

SummaryUsing the fluorescent calcium probe Quin-2, we could demonstrate a tip-to-base gradient of free calcium within growing pollen tubes ofLilium longiflorum. This result shows that it is possible to visualize calcium ions using Quin-2 in plant cells which are surrounded by a cell wall and that the calcium gradients demonstrated by CTC fluorescence (indicating membrane-bound calcium) and PIXE (showing total calcium) is paralleled by a gradient of free calcium in pollen tubes.

Localization of calcium during somatic embryogenesis of carrot (Daucus carota L.).

SummaryThe distribution of free cytosolic Ca2+ was studied during somatic embryogenesis of carrot using confocal scanning laser microscopy with fluo-3 as a fluorescent Ca2+ indicator. Chlorotetracycline fluorescence, antimonate precipitation and proton induced X-ray emission analysis were used as additional methods to confirm the results obtained with fluo-3. The process of embryogenesis was found to coincide with a rise in the level of free cytosolic Ca2+. The level of Ca2+ was low in proembryogenic masses and relatively high in later stages of embryogenesis. The highest signal was found in the protoderm of embryos from the late globular to the torpedo-shaped stage. A gradient in fluorescence intensity was often observed along the longitudinal axis of the embryos. The most conspicuous intracellular signal was found in the nucleus. Other organelles did not take up the dye and were always without fluorescence. The changes in [Ca2+]c are discussed in relation to physiological processes which are known to be important during somatic embryogenesis.