Calcium Gradients in Tip Growing Plant Cells Visualized by Chlorotetracycline Fluorescence Hans-Dieter Reiss and W e r n e r H e r t h Zellenlehre, Universitfit Heidelberg, Im Neuenheimer Feld 230, D 6900 Heidelberg, Federal Republic of Germany
Abstract. With chlorotetracycline (CTC)-fluorescence a tip-to-base Ca2+gradient is visualized in all tested, tip-growing plant cells: pollen tubes o f Lilium longiflorum, r o o t hairs o f Lepidium sativum, moss caulonem a o f Funaria kygrometrica, fungal h y p h a e o f AcMya 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 o f a calcium gradient in tip growth. Key words: Ca 2 + gradient - Cells (tip growth) - Chlorotetracycline-fluorescence G r o w t h (tip) - Tip growth.
Introduction We previously reported the visualization o f a tip-tob a s e C a 2 + gradient in the tip region o f growing pollen tubes of Lilium longiflorum with chlorotetracycline (CTC)-fluorescence (Reiss and Herth, 1978). Such a Ca z + gradient, t h o u g h t to be involved in oriented exocytosis, was also postulated f r o m electrophysiological measurements (Weisenseel etat., t975; Weisenseel and Jaffe, 1976), f r o m inhibition o f pollen tube tip growth with the calcium-ionophore A-23187 (Herth, 1978), and f r o m the ultrastructural effects o f this iono p h o r e (Reiss and Herth, 1979). W i t h ~5Ca autoradiography, a Ca z+ accumulation in pollen tube tips was d e m o n s t r a t e d (Jaffe et al., 1975). First results with the Heidelberg p r o t o n m i c r o p r o b e confirm the presence o f a Ca 2 + gradient in pollen tubes (Bosch et al., 1979). We tested with CTC-calcium fluorescence to determine whether a Ca z + gradient is also present in
Abbreviation : CTC- chlorotetracycline
other tip growing plant cells, namely r o o t hairs, fungal hyphae, moss caulonema, and Acetabularia stalks (Sievers, 1963 ; Girbardt, 1969; Bopp, 1959 ; Schweiger, 1969; Herth etal., 1972).
Materials and Methods Cress (Lepidium sativum) seeds were germinated in Petri dishes between water soaked filter paper. The moss Funaria hygrometrica was grown on a specific agar medium (Schmiedel, 1978). Hyphae of the fungus Achlya spec. were cultivated in water. The alga Acetabularia mediterraneawas cultivated as usual in enriched sea water. Parallel to untreated controls, probes were treated with chlorotetracycline (CTC ; = "Aureomycin" ; Serva, Heidelberg), dissolved in the specific media or water to give a final concentration of 10 4 M CTC. This concentration was tested to combine best fluorescence with no visible a~ltJbiotic effects during the exposure time (see also Reiss and Herth, 1978). From t to 10 min after CTC application the different probes were observed and photographed with the Zeiss microscope IM 35 with phase contrast or epifluorescence equipment (HBO 50 W lamp, interference filter combination, BP 450-490/FT 510/LP 520 or 2 FI: G 365/FT 395/ LP 420; automatic exposure time 10-50 s).
Results All objects still grow and show no obvious effects, e~g., cytoplasmic streaming effects, within 10 min after C T C application and during the exposure time. Later antibiotic effects are observed (Reiss and Herth, in prep.)
Cress Root Hairs I R o o t hairs o f Lepidium sativum are rather sensitive to experimental handling. M a n y root hairs burst 1 For Hordeum root hairs, Weisenseel recently also demonstrated a similar CTC-fluorescence gradient (lecture at the congress of Deutsche Entwicklungsphysiologiscbe Gesellschaft, Heidelberg, April [979)
Fig. l a - c . Lepidium root hairs. Bars indicate 100 gm. a Root (R) and root hairs treated 2 min with 1 0 - 4 M CTC in phase contrast. Note the many bursting hairs (arrows). b Fluorescence micrograph of the same region. Some root hairs show an intense fluorescent cap at the very tip (arrow-heads). The bursting hairs are not fluorescent (arrows). c Fluorescence gradient of a root hair viewed in oil immersion. R, root; v, vacuole, a, b x 300; c x 4 8 0
Fig. 2a-d. Funaria caulonema. Bars indicate 50 gm. a Tip region of untreated caulonema cell. The very tip (t) is free from chloroplasts (p); (phase contrast), b Autofluorescence of the same region. Only the chloroplasts are visible (p). e Caulonema ceils treated 5 min with 10 -4 M CTC in phase contrast. The zonation of the cells still is normal. Some cells burst (large arrow). Note the oblique cross wall between the small arrows, d Fluorescence micrograph of same region as Fig. 2c. The region from the tip toward the vacuole is visible with a clear fluorescence gradient (between the lines). Bursting cells lose their fluorescence (large arrow). The cross wall region between the small arrows is also fluorescent, p, chloroplasts; t, tip; v, vacuole, a, b x 500; e, d x 400
H.-D. Reiss and W. Herth: Calcium Gradients in Plant Tip Growth (Fig. la) and show no autofluorescence. With 10 . 4 M CTC, the root hairs become fluorescent and the tip region shows the most intense fluorescence, decreasing slowly to the hair base (Figs. 1 b, c). Sometimes a limited region at the tip, probably corresponding to the vesicle zone, is observed (Fig. 1 b). In this region punctate fluorescence may be observed. The root hairs differ in fluorescence intensity. Bursting hairs lose their fluorescence, indicating that the cytoplasm and not the wall is fluorescent (Fig. l b). After 5 min the fluorescence is lost due to bleaching out of the CTC fluorescence.
Moss Caulonema The end cells of Funaria hygrometrica caulonema show a clear zonation into a chloroplast-free tip zone, a chloroplast-rich cytoplasmic region, and the vacuole region (Figs. 2a, c). With autofluorescence only the chloroplasts are visible (Fig. 2b). With 10-4 M CTC, there appears a fluorescence gradient with high C T C fluorescence intensity at the very tip, surpassing the added fluorescence of CTC and chloroplasts in the remaining region. The oblique cross-wall region is also fluorescent. Bursting caulonema cells lose their CTC fluorescence (Fig. 2d, arrow), and again the fluorescence bleaches out rapidly.
Fungal Hyphae The hyphae of Achlya spec. (Fig. 3a) are not fluorescent without CTC application. With 10 -4 M CTC a weak but long stretched gradient extends from the tip to the base (Fig. 3b). Sometimes a strong and corpuscular fluorescence is discovered at the very tip and its flanks (Fig. 3c). Normally the young branch hyphae show a more intense CTC fluorescence gradient (Fig. 3d) which, in few cases, the fluorescence optimum is not located in the furthest tip. The fluorescence is also bleached out rapidly.
Fig. 3a-d. Achlya hyphae. Bars indicate 100 gtm. a Cell treated about 3 min with 10-4M CTC; (phase contrast), b Fluorescence micrograph of the same region. A weak long stretched fluorescence gradient is visible, c CTC treated hyphae with an intense and punctate fluorescence at the very tip and tip flanks (arrow). The vacuole (v) shows no fluorescence, d Short branch hyphae show more intense fluorescence gradients than the main hyphae, mh, main hypha; sb, side hyphae; v, vacuole, a, b x 260; ex330; dx380
Fig. 4a-d. Acetabularia stalks and whorls. Bars indicate 100 gm. a Light grown tip whorl treated 2 rain with 10 _4 M CTC. Note the intense wall fluorescence (arrow-heads). The main cytoplasmic fluorescence is due to the chloroplasts, b Clear CTC-fluorescence gradient in the second whorl of light grown Acetabularia. e Stalk tip region of dark grown Acetabularia, treated 3 min with 10 + M CTC. Beside wall fluorescence (arrow-heads), this tip reveals a thin fluorescent cytoplasmic layer decreasing to the tip flanks (arrow). d Tip whorl of dark grown Acetabularia with CTC-fluorescence. Beside the wall fluorescence (arrows), a weak CTC-fluorescence in the whorl tips (arrow-heads) and a stronger fluorescence at the whorl base is observed a x 500; b x 200 ; e,d x 300
Acetabularia Stalks and Whorls
About 4-20 mm long stalks of Acetabularia mediterranea show an intense autofluorescence of chloroplasts up to the stalk tip and up the tips of the youngest whorl. The tips of the second as well as the third whorl show no autofluorescence. With 10 -4 M CTC the cell wall of the whorls and the total stalk wall (which are not autofluorescent) react with a very stable fluorescence (Figs. 4 a, c, d). The tips of the second whorl are stained by CTC with a visible gradient (Fig. 4b) and are bleached out very rapidly. In the stalk tip there is a thin cytoplasmic layer under the tip and at the tip flanks with intense CTC fluorescence (Fig. 4c). This is more easily visible in dark grown Acetabularia, where the autofluorescent chloroplasts in the very tip are rare (Koop et al., 1978), but is also present in light-grown Acetabularia as visualized with filter combination 2 F1. This is also the case for the youngest whorl at the tip (Fig. 4d).
Discussion The results show that in all tested objects, as long as autofluorescence does not disturb the picture, CTC-fluorescence gradients exist in the region close to the tip. In contrast to the very intense and less bleaching fluorescence gradient in growing lily pollen tubes (Reiss and Herth, 1978), the fluorescence gradients in the now tested systems are weaker and vary in their extension. This can not be explained at the moment, but could be due to the more rapid growth of pollen tubes than of the other tip growing cells [up to 300 p~m h- t for lily pollen tubes (own measurements), 80 gm h- t for cress root hairs (Moerz, 1977), 48 gm h- 1 for Funaria caulonema (Schmiedel, 1978), 35 gm h- 1 for Acetabularia (own measurements), for Achlya hyphae not determined]. The relative long zone of longitudinal fluorescence gradient may reflect the localization of the Ca 2+ sources responsible for the stronger and narrower zone of fluorescence at the actual growth region. The real intensity at the tip is difficult to document in photographs, due to the long exposure time and the rapid bleaching out of the fluorescence. In the microprobe analysis the Ca z + gradient in the tip region is rather steep (Bosch et al., 1979). The fluorescence gradient did not show the same intensity in each cell of a sample. This might be due to the different growth states of the cells. Furthermore, CTC may act as an antibiotic inhibitor with different cells displaying different sensitivities (Baloun and Hudak, 1979). In Acetabularia the
H.-D. Reiss and W. Herth: Calcium Gradients in Plant Tip Growth
greatest part of CTC seems to react with the cell wall, perhaps causing access problems. The often observed punctate and moving CTC fluorescence agrees with the result in the pollen tubes (Reiss and Herth, 1978) and with the postulated mechanism of CTC which complexes with Ca z + and cytoplasmic membranes (Caswell and Hutchinson, 1971; Tfiljedal, 1978; further discussion in Reiss and Herth, 1978). These observations lead us to the conclusion that, generally, tip growth needs a specific distribution of Ca 2+. This gradient, visualized with CTC, is obviously one of the causal elements in oriented tip growth as its destruction leads to growth inhibition (Herth, 1978; Schmiedel, 1978; Reiss and Herth, 1979). This effect seems Ca 2+specific as the effects of the broad range ionophore X-537A are different (Reiss and Herth, in prep.). The preliminary results of the proton microprobe indicate that other ions may also be involved (Reiss, unpublished), It is not yet clear whether the Ca z + gradient is passively coupled with a specific zonation of cytoplasmic organelles and elements, or if it organizes this zonation itself. The authors thank Profs. E. Schnepf, H.G. Schweiger and A. Gibor for valuable discussions, Dr. G. Schmiedel, Frl. G. Deichgr/iber, Dr. S. Berger and Dr. H. Spring for the probes of Funaria, Achlya and Acetabularia. This work was supported by the Deutsche Forschungsgemeinschaft.
References Baloun, J., Hudak, J.: Nuclear degeneration induced by chlorotetracycline. Experientia 35, 201-202 (1979) Bopp, M.: Versuche zur Analyse yon Wachstum und Differenzierung des Labmoosprotonemas. Planta 53, 178-197 (1959) Bosch, F., D6bbeling, H., McKenzie, C.D., Martin, B., Nobiling, R., Pelte, D., Povh, B., Traxel, K., Petzelt, Ch., Herth, W., Reiss, H.D.: Microanalysis of elemental distributions in cells performed with a proton microprobe. MPI H - 1979 - V3 (Max-Planck-Institut fiir Kernphysik, Heidelberg), 1-10 (1979) CasweI1, A.H., Hutchinson, J.D.: Selectivity of cation chelation to tetracyclines: evidence for special conformation of calcium chelate. Biochem. Biophys. Res. Commun. 43, 625-630 (1971) Girbardt, M.: Die Ultrastruktur der Apikalregion yon Pilzhyphen. Protoplasma 67, 413441 (1969) Herth, W. : Ionophore A 23187 stops tip growth, but not cytoplasmic streaming in pollen tubes of Lilium longiflorum. Protoplasma 96, 275-282 (1978) Herth, W., Franke, W.W., VanDerWoude, W.J. : Cytochalasin stops tip growth in plants. Naturwissenschaften 59, 38-39 (1972) Jaffe, L.A., Weisenseel, M.H., Jaffe, L.F.: Calcium accumulation within the growing tips of pollen tubes. J. Cell. Biol. 67, 488-492 (1975) Koop, H.-U., Schmidt, R., Heunert, H.-H., Milthaler, B. : Chloroplast migration : a new circadian rhythm in Acetabularia. Protoplasma 97, 301-310 (1978) Moerz, G.: Wachstum der Wurzelhaare. Staatsexamensarbeit, Universitfit Heidelberg 1977
H.-D. Reiss and W. Herth: Calcium Gradients in Plant Tip Growth Reiss, H.D., Herth, W. : Visualization of the Ca 2 + gradient in growing pollen tubes of Lilium longiflorum with chlorotetracycline fluorescence. Protoplasma 97, 373 377 (1978) Reiss, H.D., Herth, W.: Ionophore A 23187 affects localized wall secretion in the tip region of pollen tubes of Lilium longiflorum. Planta 145, 225 232 (1979) Schmiedel, G. : Zellpolaritfit und Verzweigung im Caulonema von Funaria hygrometrica Sibth. Dissertation, Universitfit Heidelberg 1978 Schweiger, H.G. : Cell biology of Acetabularia. Curr. Top. Microbiol. ImmunoI. 50, 1 36 (1969) Sievers, A.: Beteiligung des Golgi-Apparates bei der Bildung der Zellwand von Wurzelhaaren. Protoplasma 56, 188-192 (1963) T/iljedal, I.-B.: Chlorotetracycline as a fluorescent Ca z+ probe
621 in pancreatic islet cells. Methodological aspects and effects of alloxan, sugars, methylxanthines, and Mg 2+. J. Cell Biol. 76, 652 674 (1978) Weisenseel, M.H., Jaffe, L.F.: The major growth current through lily pollen tubes enters as K + and leaves as H +. Planta 133, 1-7 (1976) Weisenseel, M.H., Nucitelli, R., Jaffe, L.F.: Large electrical currents traverse growing pollen tubes. J. Cell Biol. 66, 556-567 (1975)
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