Planta (1982)156:218-225
P l a n t a 9 Springer-Verlag 1982
Disoriented growth of pollen tubes of Lilium longitlorum Thunb. induced by prolonged treatment with the calcium-chelating antibiotic, chlorotetracycline Hans-Dieter Reiss * and Werner Herth Zellenlehre der Universitfit, Im Neuenheimer Feld 230, D-6900 Heidelberg, Federal Republic of Germany
Abstract. Pollen of L i l i u m 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. Vesicleand 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.
Key words: Calcium, dynamics - Chlorotetracycline - Disorientation of growth - L i l i u m - Organelle distribution - Pollen tube growth - Tip growth.
Introduction
Pollen tubes have become one of the standard systems used to investigate plant tip growth in vitro (review: Sievers and Schnepf 1981). In typical tip * To whom correspondence should be addressed
Abbreviations: CTC = chlorotetracycline; ER = endoplasmic reticulum
0032-0935/82/0156/0218/$01.60
growth, a polar distribution of cell organelles, with Golgi vesicles enriched at the very tip, characterizes the growing zone. This organelle zonation is disturbed by the Ca 2 +-ionophore A-23187, which stops pollen tube growth and disorients exocytosis (Herth 1978; Reiss and Herth 1979a). Other ionophores, e.g., X-537A for a broader range of cations and valinomycin for K +, do not disorganize the polar zonation of organelles in the tip region (Reiss and Herth 1980; Reiss 1980). Therefore, the polar distribution of cell organelles and oriented exocytosis seems to be somehow regulated by specific distributions of calcium ions, rather than by other ions. This concept is supported by electrophysiological data and autoradiography (review: Weisenseel and Kicherer 1981). We visualized and measured a tip-to-base gradient of calcium by chlorotetracycline (CTC)-fluorescence (Reiss and Herth 1978) and proton-induced X-ray microanalysis (Reiss 1980; Bosch et al. 1980; Reiss et al. 1982). Chlorotetracycline is an indicator for membrane-bound calcium in living cells (Reiss and Herth 1979b; Saunders and Hepler 1981; for review see Caswell 1979). CTC, however, is also an antibiotic drug, inhibiting protein biosynthesis (review: Neu 1978); CTC also enhances the level of membrane-bound Ca 2 + (Reiss 1980; Reiss et al. 1982). Exposing pollen tubes to CTC for a longer period should disturb the steps in tip growth dependent on Ca a +-dynamics. Material and methods Pollen of Lilium longiflorum Thunb. var. Ace was stored and cultivated as described previously (Franke et al. 1972; Herth 1978). After germination the tubes were treated with an aqueous solution of 1"10-4M chlorotetracycline ( C T C = " A u r e o mycin", Serva, Heidelberg, FRG), or the pollen germinated 12 h overnight in a 1-10-4M CTC-containing medium. The tubes were then investigated with an inverse light microscope
H.-D. Reiss and W. Herth: Disorientation of pollen tube growth
219
Fig. la, b, Pollen grain and pollen tube, treated with 1q0-4 M CTC during germination, a Light micrograph. The tube growth is oriented toward the own grain. Extensive wall thickenings are visible (arrows). The dear tip zone, normally seen by phase contrast, is destroyed, b Electron micrograph (longitudinal section). The tube flank shows a wall thickening (arrow). The lipid accumulation in the pollen grain is not atypical, ex, exine; lp, lipid bodies; n, vegetative nucleus; p, plastids; v, vacuoles. a x 500; b x 2,100
Fig. 2a-d. CTC effects in the tip region, a Pollen tube tip (cortical section) showing high vesicle accumulation and extensive irregular wall thickenings, b Median section near the tip with wall thickenings consisting of fibrous globules, e, d High magnifications of wall thickenings, e Asters show globular aggregates of fibrils, d Sometimes the wall globules still have the form of secretory vesicle contents (asters) and tend to form bigger aggregates (aster-row). In both figures (e and d) membranes are also found between the extruded wall material (arrows). cw, cell wall; lp, lipid body; m, mitochondrium; p, plastid; sv, secretory vesicle(s). a x 8,000; b x 5,000; e x 20,000; d x 4 0 , 0 0 0
Fig. 3a-c. CTC effects on the flanks, a Tube flank with extensive walI thickenings, showing aggregates of globular wall material (asters). In the adjacent cytoplasm accumulations of secretory vesicles and endoplasmic reticulum are frequent, b Wall protuberances into the cytoplasm. Often the wall globules form bigger aggregates (asters). The plasma membrane is very undulated. Many myeline-like structures are found between the plasma membrane and cell wall (arrows). c Bigger wall protuberance showing a dense core of fibrils (aster), which still have visible projections to the plasma membrane (arrows). cw, cell wall; d, dictyosome; er, endoplasmic reticulum; rn, mitochondrium; prn, plasma membrane, sv, secretory vesicle(s), a x 12,000; b x 45,000; c x 65,000
Fig. 4a-k. Ultrastructural effects of CTC on organelles, a The plasma membrane often shows multilayers of membranes (arrows). b, e Deep invaginations (arrows) of the plasma membrane, d Short piece of a solitary cortical microtubule (arrows). (No further microtubules could be detected in all other sections), e Extended parallel membrane configurations (partly myeline-like, partly ER-like) in the cytoplasm (arrows) surrounding an unusually formed plastid with big starch grains. (Most plastids look normal). f, g Dictyosomes, showing normal polarity from the regeneration to the secretion side, and vesicle production. Between small arrows: intercisternal elements. Big arrow: dictyosome-associated polysome, h-k Mitochondria with groups of unusual dilated cristae (arrows) besides normal cristae, cw, cell wall; er, endoplasmic reticulum; pro, plasma membrane; rs, regeneration side; ss, secretion side; st, starch grain; sv, secretory vesicle, a-e x 75,000; d, e x 60,000; f, g x 50,000; h, i x 70,000; k x 130,000
H.-D. Reiss and W. Herth: Disorientation of pollen tube growth (Zeiss IM 35) with phase contrast and epifluorescence (for further details see Reiss and Herth 1979b). For thin-section analysis, the tubes were fixed after 1-12 h of CTC application (for details of fixation and embedding see Reiss and Herth 1979a) and investigated with the Philips EM 400.
Results
Light microscopy. After about 30 min treatment by 1-10 -4 M chlorotetracycline (CTC), strong effects on tube growth and calcium/CTC-fluorescence become visible. The pollen tubes, normally growing linear, begin to bend, and many tubes finally grow back to the grain. Tubes which just start growing out of the grain at this time form only short tubes, coming in close contact with the grain surface. The growth rate is slowed down, then after about 2 h CTC-treatment, growth stops. Pollen germinates only slowly in a 1-10-4M CTC-containing medium, the first short tubes are detected after about 4 h. They also grow curved back to the grain (Fig. 1 a). No tube longer than 150 gm was found. Growth stops after the next 2 h; cytoplasmic streaming also slows down and stops some minutes later. After the first bending effects the tubes show wall thickenings in different parts of the tube (Fig. 1 a). The tube diameter does not increase in its basal parts, but many tubes show apical swelling. The clear cap often almost disappears, due to strong wall thickenings. The tip-to-base calcium gradient, normally visualized by CTC-fluorescence (Reiss and Herth 1978), is destroyed after 30 min of CTC-treatment of growing tubes. In CTC-germinated tubes a gradient was never observed.
Electron microscopy. There is almost no difference in the ultrastructural effects visible with short-time CTC-treatment or long-time CTC-treatment. All figures here are taken from 1.10-4 M CTC-germinated tubes, as this fixation series was better. Figure 1 b shows the survey of a pollen grain and its outgrown tube. The tube bent when it grew out of the grain. The tube tip is not visible in the same section, as the tube grew helically. The very tip is characterized by an abnormal high accumulation of secretory vesicles, together with extreme wall thickenings (Fig. 2a). Such vesicle accumulations are also observed in other tube areas, where wall thickenings occurred (Fig. 3a). There are endoplasmic reticulum (ER) accumulations in places where they are not observed in the controls (Fig. 3 a). The general zonation of the tip zone is irregular (Fig. 2 b).
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The wall thickenings consist of globular aggregates of fibrous wall material, extruded irregularly between a normal wall layer and the plasma membrane (Figs. 2b-d). The aggregates have the form and size of Golgi vesicle contents, or form bigger aggregates. Their fibrils are dense in the core, and loose at their flanks, with visible contacts to the plasma membrane (Figs. 3 b, c). Some wall thickenings form great protuberances into the cytoplasm (Figs. 3 b, c). In these areas the plasma membrane is very undulated and often multilayers of membranes, myeline like structures, or membrane invagination are seen (Figs. 4a-c). Between the wall thickenings, condensed membrane-like material is often found (Figs. 2c, d). Myeline-like membrane structures possibly derived from ER also occur deep in the cytoplasm (Fig. 4e). Next to the vesicle accumulations a high concentration of ER is seen (Fig. 3 a). The mitochondrial ultrastructure differs from the average: groups of unusual tubular cristae occur besides normal ones (Figs. 4h-k). Other organelles, like the dictyosomes, plastids, and lipid bodies, show almost no visible alterations (Figs. 1, 2 b, 4 f, g). Microtubules could not be detected after CTC-treatment, with the exception of a short cortical piece in one section (Fig. 4d), whereas control tubes showed cortical microtubules with the same fixation conditions (see Reiss and Herth 1979a). Discussion
It is well known from earlier experiments that calcium acts as a modulator for pollen germination and tube growth (review: Rosen 1968). The "population effect", which means that germination and growth will start only at a specific pollen density in the medium, was demonstrated as an effect of external Ca2+-ions brought in from the pollen grains (Brewbaker and Kwack 1963). The external Ca 2 +-ions also control and influence the pollen tube growth rate. An optimal growth rate was measured after adding 5.10 .5 M CaC12 to the medium (Reiss 1980). Higher amounts of Ca2+-ions often influence the growth pattern, i.e., inducing helical tube growth (Morr6 and Van der Woude 1974). Disoriented tube growth with thickened walls, as demonstrated by CTC-treatment, may also be induced by high external Ca 2 + (Morr6 and Van der Woude 1974; and own unpublished experiments with 1 0 - 3 M Ca2+-ions). At even higher Ca 2 +-concentrations than 10 - 3 M, tube growth is stopped (Reiss, unpublished). After short CTCtreatment the Ca z +-concentration also is enhanced at the tip: a twenty-fold higher calcium content
224 (up to 8 mg g-1) than in the control is detected in pollen tubes by proton-induced X-ray microanalysis (PIXE) after 10 min CTC-pretreatment (Reiss et al. 1982). This may be due to the ability of CTC to bind and fix calcium-ions on cellular membranes (Caswell 1979). High internal Ca/§ concentrations could have effects on the contractile machinery and could cause a depolymerization of microtubules (e.g., Schliwa 1976). Indeed, microtubules are not detected in CTC-treated tubes, and cytoplasmic streaming stops after about 2 h. However, microtubules do not seem to be essential guide elements for tip growth in pollen tubes (Franke et al. 1972). Therefore, the disoriented wall thickenings must be due to a general disorientation, as is also demonstrated by the accumulations of E R and vesicles in the wrong places. The bending growth of the tubes toward or along the grain after CTC-treatment might be explained if the grain contained more Ca / + and the pollen tubes grew in the direction of the higher Ca2§ This supports the abovementioned results, that calcium ions may influence the growth direction of pollen tubes. In vitro pollen tubes grow chemotropically toward a higher concentration of calcium (Mascarenhas and Machlis 1962; Rosen 1964). This was also demonstrated for some species, when pollen tubes in vivo grow through the stigma toward the embryo sac (Miki 1954; Mascarenhas 1966). We suppose that the binding of calcium on membranes by CTC disturbs the intracellular dynamic calcium-gradient, which may be built up from calcium ions which are part of the electrical inward current of the growing pollen tubes (Weisenseel and Kicherer 1981). The theory of a dynamic gradient nature is supported by the fact that the calcium distribution is not simply coupled to the membrane distribution, which can be demonstrated by fluorescamine fluorescence (visualizing the cellular membranes, see Poccia et al. 1979) and by the phosphorus distribution with the Heidelberg proton microprobe (Reiss 1980). Nevertheless, this interpretation is still under discussion (Sievers and Schnepf 1981). Wrong areas with artificially induced high membrane bound calcium may then induce the exocytosis of Golgi vesicles, because Ca 2 +-ions are thought to play a role in membrane fusion and possibly vesicle transport (Morr~ et al. 1974; Wilschut and Papahadjopoulos 1979). Similar effects are observed after treatment by the calcium-ionophore A-23187 (Reiss and Herth 1979a), which equilibrates the internal calcium gradient (Reiss and Herth 1978; Reiss et al. 1982). The aggregates of membrane material after
H.-D. Reiss and W. Herth: Disorientation of pollen tube growth CTC-treatment are possibly due to a normal lipidand vesicle-production rate, but here not having a 1 : 1 ratio to the increase in the plasma membrane surface, as in normal growth (Van der Woude and Morr6 1968). " C o a t e d regions" on the plasma membrane, as possible recycling structures (Farquhar 1978), do not occur more frequently than in the control tubes. The organelle ultrastructure shows less effects than in calcium-ionophoretreated tubes. The mitochondria show effects in both cases. Perhaps an extreme Ca 2 +-efflux from the mitochondria, caused by the calcium-complexing of CTC, induces such formations, but secondary effects are also possible, like, e.g., the inhibition of protein biosynthesis by CTC (Neu 1978). The final end of tube growth and exocytosis may be caused by an accumulation of the various effects. These results are further evidence that the oriented exocytosis in pollen tubes with its polar zonation of cell organelles depends mainly on the dynamics of the CaZ+-distribution, which is disturbed by fixing the Ca 2§ with CTC to membranes. The authors thank Professor E. Schnepf for valuable discussions. This work was supported by the Deutsche Forschungsgemeinschaft. References
Bosch, F., E1 Goresy, A., Herth, W., Martin, B., Nobiling, R., Povh, B., Reiss, H.-D., Traxel, K. (1980) The Heidelberg proton microprobe. Nucl. Sci. Appl. 1, 33-55 Brewbaker, J.L., Kwack, B.H. (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am. J. Bot. 50, 859-865 Caswell, A.H. (1979) Methods of measuring intracellular calcium. Int. Rev. Cytol. 56, 145-181 Farquhar, M.G. (1978) Recoveryof surface membrane in anterior pituitary cells. Variations in traffic detected with anionic and cationic ferritin. J. Cell Biol. 78, R35-R42 Franke, W.W., Herth, W., Van der Woude, W.J., MorrO, D.J. (1972) Tubular and filamentous structures in pollen tubes: possible involvement as guide elements in protoplasmic streaming and vectorial migration of secretory vesicles. Planta 105, 317-341 Herth, W. (1978) Ionophore A 23187 stops tip growth, but not cytoplasmic streaming, in pollen tubes of Liliurn long# florum. Protoplasma 96, 275-282 Mascarenhas, J.P. (1966) The distribution of ionic calcium in the tissues of the gynoecium of Antirrhinum majus. Protoplasma 62, 53-58 Mascarenhas, J.P., Machlis, L. (1962) Chemotropic response of Antirrhinurn majus pollen to calcium. Nature (London) 196, 292-293 Mike, H. (1954) A study of tropism of pollen tubes to the pistil. I. Tropism in Lilium. Bot. Mag. 67, 143-147 Morr6, D.J., Bracket, C.E., Van der Woude, W.J. (1974) Influence of calcium ions on the plant cell surface: membrane fusions and conformational changes. 32nd Annu. Proc. Electron Microsc. Soc. Am., St. Louis, Mo.
H.-D. Reiss and W. Herth: Disorientation of pollen tube growth Morr6, D.J., Van der Woude, W.J. (1974) Origin and growth of cell surface components. In: Macromolecules regulating growth and development; 30th Symp. Soc. Dev. Biol., pp. 81-111. Academic Press, New York London Neu, M.D. (1978) A symposium on the tetracyclines: a major appraisal. Introduction. Bull. N.Y. Acad. Med. 54, 141-249 Poccia, D.L., Palevitz, B.A., Campisi, J., Lyman, H. (1979) Fluorescence staining of living cells with fluorescamine. Protoplasma 98, 91-113 Reiss, H.-D. (1980) Calcium-Gradienten und Spitzenwachstum bei Pollenschlfiuchen von Lilium longiJ7orum. Dissertation, Universitfit Heidelberg Reiss, H.-D., Herth, W. (1978) Visualization of the Ca 2 +-gradient in growing pollen tubes of Lilium longiflorum with chlorotetracycline fluorescence. Protoplasma 97, 373-377 Reiss, H.-D., Herth, W. (1979a) Calcium ionophore A-23187 affects localized wall secretion in the tip region of pollen tubes of Lilium longiflorum. Planta 145, 225-232 Reiss, H.-D., Herth, W. (1979b) Calcium gradients in tip growing plant cells visualized by chlorotetracycline fluorescence. Planta 146, 615-621 Reiss, H.-D., Herth, W. (1980) Effects of the broad-range ionophore X-537A on pollen tubes of Lilium longijqorum. P1anta 147, 295-301 Reiss, H.-D., Herth, W., Schnepf, E., Nobiling, R. (1982) The tip-to-base calcium gradient in pollen tubes of Lilium longiflorum measured by proton-induced X-ray emission (PIXE). Protoplasma (in press) Rosen, W.G. (1964) Chemotropism and fine structure of pollen
225 tubes. In: Pollen physiology and fertilization, pp. 159 166, Linskens, H.F., ed. North Holland, Amsterdam Rosen, W.G. (1968) Ultrastructure and physiology of pollen. Annu. Rev. Plant Physiol. 19, 435462 Saunders, M.J., Hepler, P.K. (1981) Localization of membraneassociated calcium following cytokinin treatment in Funaria using chlorotetracycline. Planta 152, 272-281 Schliwa, M. (1976) The role of divalent cations in the regulation of microtubule assembly. In vitro studies on microtubules of the heliozoan axopodium using the ionophore A23187. J. Cell Biol. 70, 527-540 Sievers, A., Schnepf, E. (1981) Morphogenesis and polarity of tubular cells with tip growth. In: Cell Biology Monographs, vol. 8: Cytomorphogenesis in plants, pp. 265-299, Kiermayer, O., ed. Springer, Berlin Heidelberg New York Van der Woude, W.J., Mort6, D.J. (1968) Endoplasmic reticulure - dictyosome - secretory vesicle associations in pollen tubes of Lilium longiflorum Thunb. Proc. Ind. Acad. Sci. 77, 164-170 Weisenseel, M.H., Kicherer, R.M. (1981) Ionic currents as control mechanism in cytomorphogenesis. In: Cell Biology Monographs, vol. 8 : Cytomorphogenesis in plants, pp. 379399, Kiermayer, O., ed. Springer, Berlin Heidelberg New York Wilschut, J., Papahadjopoulos, D. (1979) Ca 2 +-induced fusion of phospholipid vesicles monitored by mixing of aqueous contents. Nature (London) 281,690-692 Received 22 March; accepted 4 August 1982
P l a n t a 9 Springer-Verlag 1982
Disoriented growth of pollen tubes of Lilium longitlorum Thunb. induced by prolonged treatment with the calcium-chelating antibiotic, chlorotetracycline Hans-Dieter Reiss * and Werner Herth Zellenlehre der Universitfit, Im Neuenheimer Feld 230, D-6900 Heidelberg, Federal Republic of Germany
Abstract. Pollen of L i l i u m 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. Vesicleand 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.
Key words: Calcium, dynamics - Chlorotetracycline - Disorientation of growth - L i l i u m - Organelle distribution - Pollen tube growth - Tip growth.
Introduction
Pollen tubes have become one of the standard systems used to investigate plant tip growth in vitro (review: Sievers and Schnepf 1981). In typical tip * To whom correspondence should be addressed
Abbreviations: CTC = chlorotetracycline; ER = endoplasmic reticulum
0032-0935/82/0156/0218/$01.60
growth, a polar distribution of cell organelles, with Golgi vesicles enriched at the very tip, characterizes the growing zone. This organelle zonation is disturbed by the Ca 2 +-ionophore A-23187, which stops pollen tube growth and disorients exocytosis (Herth 1978; Reiss and Herth 1979a). Other ionophores, e.g., X-537A for a broader range of cations and valinomycin for K +, do not disorganize the polar zonation of organelles in the tip region (Reiss and Herth 1980; Reiss 1980). Therefore, the polar distribution of cell organelles and oriented exocytosis seems to be somehow regulated by specific distributions of calcium ions, rather than by other ions. This concept is supported by electrophysiological data and autoradiography (review: Weisenseel and Kicherer 1981). We visualized and measured a tip-to-base gradient of calcium by chlorotetracycline (CTC)-fluorescence (Reiss and Herth 1978) and proton-induced X-ray microanalysis (Reiss 1980; Bosch et al. 1980; Reiss et al. 1982). Chlorotetracycline is an indicator for membrane-bound calcium in living cells (Reiss and Herth 1979b; Saunders and Hepler 1981; for review see Caswell 1979). CTC, however, is also an antibiotic drug, inhibiting protein biosynthesis (review: Neu 1978); CTC also enhances the level of membrane-bound Ca 2 + (Reiss 1980; Reiss et al. 1982). Exposing pollen tubes to CTC for a longer period should disturb the steps in tip growth dependent on Ca a +-dynamics. Material and methods Pollen of Lilium longiflorum Thunb. var. Ace was stored and cultivated as described previously (Franke et al. 1972; Herth 1978). After germination the tubes were treated with an aqueous solution of 1"10-4M chlorotetracycline ( C T C = " A u r e o mycin", Serva, Heidelberg, FRG), or the pollen germinated 12 h overnight in a 1-10-4M CTC-containing medium. The tubes were then investigated with an inverse light microscope
H.-D. Reiss and W. Herth: Disorientation of pollen tube growth
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Fig. la, b, Pollen grain and pollen tube, treated with 1q0-4 M CTC during germination, a Light micrograph. The tube growth is oriented toward the own grain. Extensive wall thickenings are visible (arrows). The dear tip zone, normally seen by phase contrast, is destroyed, b Electron micrograph (longitudinal section). The tube flank shows a wall thickening (arrow). The lipid accumulation in the pollen grain is not atypical, ex, exine; lp, lipid bodies; n, vegetative nucleus; p, plastids; v, vacuoles. a x 500; b x 2,100
Fig. 2a-d. CTC effects in the tip region, a Pollen tube tip (cortical section) showing high vesicle accumulation and extensive irregular wall thickenings, b Median section near the tip with wall thickenings consisting of fibrous globules, e, d High magnifications of wall thickenings, e Asters show globular aggregates of fibrils, d Sometimes the wall globules still have the form of secretory vesicle contents (asters) and tend to form bigger aggregates (aster-row). In both figures (e and d) membranes are also found between the extruded wall material (arrows). cw, cell wall; lp, lipid body; m, mitochondrium; p, plastid; sv, secretory vesicle(s). a x 8,000; b x 5,000; e x 20,000; d x 4 0 , 0 0 0
Fig. 3a-c. CTC effects on the flanks, a Tube flank with extensive walI thickenings, showing aggregates of globular wall material (asters). In the adjacent cytoplasm accumulations of secretory vesicles and endoplasmic reticulum are frequent, b Wall protuberances into the cytoplasm. Often the wall globules form bigger aggregates (asters). The plasma membrane is very undulated. Many myeline-like structures are found between the plasma membrane and cell wall (arrows). c Bigger wall protuberance showing a dense core of fibrils (aster), which still have visible projections to the plasma membrane (arrows). cw, cell wall; d, dictyosome; er, endoplasmic reticulum; rn, mitochondrium; prn, plasma membrane, sv, secretory vesicle(s), a x 12,000; b x 45,000; c x 65,000
Fig. 4a-k. Ultrastructural effects of CTC on organelles, a The plasma membrane often shows multilayers of membranes (arrows). b, e Deep invaginations (arrows) of the plasma membrane, d Short piece of a solitary cortical microtubule (arrows). (No further microtubules could be detected in all other sections), e Extended parallel membrane configurations (partly myeline-like, partly ER-like) in the cytoplasm (arrows) surrounding an unusually formed plastid with big starch grains. (Most plastids look normal). f, g Dictyosomes, showing normal polarity from the regeneration to the secretion side, and vesicle production. Between small arrows: intercisternal elements. Big arrow: dictyosome-associated polysome, h-k Mitochondria with groups of unusual dilated cristae (arrows) besides normal cristae, cw, cell wall; er, endoplasmic reticulum; pro, plasma membrane; rs, regeneration side; ss, secretion side; st, starch grain; sv, secretory vesicle, a-e x 75,000; d, e x 60,000; f, g x 50,000; h, i x 70,000; k x 130,000
H.-D. Reiss and W. Herth: Disorientation of pollen tube growth (Zeiss IM 35) with phase contrast and epifluorescence (for further details see Reiss and Herth 1979b). For thin-section analysis, the tubes were fixed after 1-12 h of CTC application (for details of fixation and embedding see Reiss and Herth 1979a) and investigated with the Philips EM 400.
Results
Light microscopy. After about 30 min treatment by 1-10 -4 M chlorotetracycline (CTC), strong effects on tube growth and calcium/CTC-fluorescence become visible. The pollen tubes, normally growing linear, begin to bend, and many tubes finally grow back to the grain. Tubes which just start growing out of the grain at this time form only short tubes, coming in close contact with the grain surface. The growth rate is slowed down, then after about 2 h CTC-treatment, growth stops. Pollen germinates only slowly in a 1-10-4M CTC-containing medium, the first short tubes are detected after about 4 h. They also grow curved back to the grain (Fig. 1 a). No tube longer than 150 gm was found. Growth stops after the next 2 h; cytoplasmic streaming also slows down and stops some minutes later. After the first bending effects the tubes show wall thickenings in different parts of the tube (Fig. 1 a). The tube diameter does not increase in its basal parts, but many tubes show apical swelling. The clear cap often almost disappears, due to strong wall thickenings. The tip-to-base calcium gradient, normally visualized by CTC-fluorescence (Reiss and Herth 1978), is destroyed after 30 min of CTC-treatment of growing tubes. In CTC-germinated tubes a gradient was never observed.
Electron microscopy. There is almost no difference in the ultrastructural effects visible with short-time CTC-treatment or long-time CTC-treatment. All figures here are taken from 1.10-4 M CTC-germinated tubes, as this fixation series was better. Figure 1 b shows the survey of a pollen grain and its outgrown tube. The tube bent when it grew out of the grain. The tube tip is not visible in the same section, as the tube grew helically. The very tip is characterized by an abnormal high accumulation of secretory vesicles, together with extreme wall thickenings (Fig. 2a). Such vesicle accumulations are also observed in other tube areas, where wall thickenings occurred (Fig. 3a). There are endoplasmic reticulum (ER) accumulations in places where they are not observed in the controls (Fig. 3 a). The general zonation of the tip zone is irregular (Fig. 2 b).
223
The wall thickenings consist of globular aggregates of fibrous wall material, extruded irregularly between a normal wall layer and the plasma membrane (Figs. 2b-d). The aggregates have the form and size of Golgi vesicle contents, or form bigger aggregates. Their fibrils are dense in the core, and loose at their flanks, with visible contacts to the plasma membrane (Figs. 3 b, c). Some wall thickenings form great protuberances into the cytoplasm (Figs. 3 b, c). In these areas the plasma membrane is very undulated and often multilayers of membranes, myeline like structures, or membrane invagination are seen (Figs. 4a-c). Between the wall thickenings, condensed membrane-like material is often found (Figs. 2c, d). Myeline-like membrane structures possibly derived from ER also occur deep in the cytoplasm (Fig. 4e). Next to the vesicle accumulations a high concentration of ER is seen (Fig. 3 a). The mitochondrial ultrastructure differs from the average: groups of unusual tubular cristae occur besides normal ones (Figs. 4h-k). Other organelles, like the dictyosomes, plastids, and lipid bodies, show almost no visible alterations (Figs. 1, 2 b, 4 f, g). Microtubules could not be detected after CTC-treatment, with the exception of a short cortical piece in one section (Fig. 4d), whereas control tubes showed cortical microtubules with the same fixation conditions (see Reiss and Herth 1979a). Discussion
It is well known from earlier experiments that calcium acts as a modulator for pollen germination and tube growth (review: Rosen 1968). The "population effect", which means that germination and growth will start only at a specific pollen density in the medium, was demonstrated as an effect of external Ca2+-ions brought in from the pollen grains (Brewbaker and Kwack 1963). The external Ca 2 +-ions also control and influence the pollen tube growth rate. An optimal growth rate was measured after adding 5.10 .5 M CaC12 to the medium (Reiss 1980). Higher amounts of Ca2+-ions often influence the growth pattern, i.e., inducing helical tube growth (Morr6 and Van der Woude 1974). Disoriented tube growth with thickened walls, as demonstrated by CTC-treatment, may also be induced by high external Ca 2 + (Morr6 and Van der Woude 1974; and own unpublished experiments with 1 0 - 3 M Ca2+-ions). At even higher Ca 2 +-concentrations than 10 - 3 M, tube growth is stopped (Reiss, unpublished). After short CTCtreatment the Ca z +-concentration also is enhanced at the tip: a twenty-fold higher calcium content
224 (up to 8 mg g-1) than in the control is detected in pollen tubes by proton-induced X-ray microanalysis (PIXE) after 10 min CTC-pretreatment (Reiss et al. 1982). This may be due to the ability of CTC to bind and fix calcium-ions on cellular membranes (Caswell 1979). High internal Ca/§ concentrations could have effects on the contractile machinery and could cause a depolymerization of microtubules (e.g., Schliwa 1976). Indeed, microtubules are not detected in CTC-treated tubes, and cytoplasmic streaming stops after about 2 h. However, microtubules do not seem to be essential guide elements for tip growth in pollen tubes (Franke et al. 1972). Therefore, the disoriented wall thickenings must be due to a general disorientation, as is also demonstrated by the accumulations of E R and vesicles in the wrong places. The bending growth of the tubes toward or along the grain after CTC-treatment might be explained if the grain contained more Ca / + and the pollen tubes grew in the direction of the higher Ca2§ This supports the abovementioned results, that calcium ions may influence the growth direction of pollen tubes. In vitro pollen tubes grow chemotropically toward a higher concentration of calcium (Mascarenhas and Machlis 1962; Rosen 1964). This was also demonstrated for some species, when pollen tubes in vivo grow through the stigma toward the embryo sac (Miki 1954; Mascarenhas 1966). We suppose that the binding of calcium on membranes by CTC disturbs the intracellular dynamic calcium-gradient, which may be built up from calcium ions which are part of the electrical inward current of the growing pollen tubes (Weisenseel and Kicherer 1981). The theory of a dynamic gradient nature is supported by the fact that the calcium distribution is not simply coupled to the membrane distribution, which can be demonstrated by fluorescamine fluorescence (visualizing the cellular membranes, see Poccia et al. 1979) and by the phosphorus distribution with the Heidelberg proton microprobe (Reiss 1980). Nevertheless, this interpretation is still under discussion (Sievers and Schnepf 1981). Wrong areas with artificially induced high membrane bound calcium may then induce the exocytosis of Golgi vesicles, because Ca 2 +-ions are thought to play a role in membrane fusion and possibly vesicle transport (Morr~ et al. 1974; Wilschut and Papahadjopoulos 1979). Similar effects are observed after treatment by the calcium-ionophore A-23187 (Reiss and Herth 1979a), which equilibrates the internal calcium gradient (Reiss and Herth 1978; Reiss et al. 1982). The aggregates of membrane material after
H.-D. Reiss and W. Herth: Disorientation of pollen tube growth CTC-treatment are possibly due to a normal lipidand vesicle-production rate, but here not having a 1 : 1 ratio to the increase in the plasma membrane surface, as in normal growth (Van der Woude and Morr6 1968). " C o a t e d regions" on the plasma membrane, as possible recycling structures (Farquhar 1978), do not occur more frequently than in the control tubes. The organelle ultrastructure shows less effects than in calcium-ionophoretreated tubes. The mitochondria show effects in both cases. Perhaps an extreme Ca 2 +-efflux from the mitochondria, caused by the calcium-complexing of CTC, induces such formations, but secondary effects are also possible, like, e.g., the inhibition of protein biosynthesis by CTC (Neu 1978). The final end of tube growth and exocytosis may be caused by an accumulation of the various effects. These results are further evidence that the oriented exocytosis in pollen tubes with its polar zonation of cell organelles depends mainly on the dynamics of the CaZ+-distribution, which is disturbed by fixing the Ca 2§ with CTC to membranes. The authors thank Professor E. Schnepf for valuable discussions. This work was supported by the Deutsche Forschungsgemeinschaft. References
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