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Herbicides and the microtubular apparatus of Nicotiana tabacum pollen tube: immunofluorescence and immunogold labelling studies
Toxicology in Vitro 15 (2001) 143±151
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Herbicides and the microtubular apparatus of Nicotiana tabacum pollen tube: immuno¯uorescence and immunogold labelling studies E. Ovidi a,*, G. Gambellini b, A.R. Taddei b, G. Cai c, C. Del Casino c, M. Ceci a, S. Rondini a, A. Tiezzi a,b a Dipartimento Scienze Ambientali, UniversitaÁ degli Studi della Tuscia, Via S. Camillo de Lellis blocco D, 01100 Viterbo, Italy Centro Interdipartimentale Microscopia Elettronica (CIME), UniversitaÁ degli Studi della Tuscia, Via S. Camillo de Lellis blocco D, 01100 Viterbo, Italy c Dipartimento Scienze Ambientali, UniversitaÁ degli Studi di Siena, Via Mattioli 4, 53100 Siena, Italy
b
Accepted 12 November 2000
Abstract Herbicides are chemical compounds widely used in agriculture. As their intensive application is becoming a cause of environmental pollution, detailed and more sophisticated investigations are needed to understand better their consequences at the biological level. After herbicides are dispersed in the ®elds, they establish chemical interactions with both target and non-target plants. In both cases, herbicides can interact with the plant reproductive apparatus; consequently they could play a role during the fertilisation process in higher plants. Using an antibody to the a-tubulin subunit in immuno¯uorescence and immunoelectron microscopy techniques, we investigated the distribution of microtubules in Nicotiana tabacum pollen tubes grown under in vitro conditions in the presence of ®ve dierent herbicides selected among those used frequently in central Italy. Herbicides have a speci®c eect on the microtubular apparatus of both pollen tube and generative cell. In addition to other tests and assays, these results suggest that the microtubule cytoskeleton of pollen tubes can be used as a bioindicator for studying the toxicity eects induced by herbicides. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Herbicides; Microtubules; Pollen tube; Generative cell; Immuno¯uorescence; Immunogold labelling
1. Introduction In recent years, the application of herbicides in many countries has increased. Herbicides have an important role in the development of agricultural production and in the enhancement of products, both in terms of quantity and quality, contributing therefore to a general wellbeing. On the other hand, the large and intensive use of herbicides is also becoming a factor contributing to environmental pollution (Chevreuil et al., 1996; Kim and Feagley, 1998; Abdel-Rahman et al., 1999). The mechanism of toxicological action on plants and animals is not yet completely understood for many herbicides. As Abbreviations: GC, generative cell; IEM, immunoelectron microscopy; IF, immuno¯uorescence; W, GC wall * Corresponding author. Tel.: +39-761-357130; fax: +39-761357105. E-mail address: [email protected] (E. Ovidi).
a consequence, the lack of detailed evaluations of the role(s) of herbicides at the biological level could also represent a problem for human health (Munger et al., 1997; Gorell et al., 1998). Although the ¯owering apparatus is not the most important target of many herbicides, the latter can have incidental eects during the fertilisation process in higher plants. When herbicides are dispersed in the ®elds, they also reach the stigmas, anthers and pollen surface of dierent plant species. The eects of these chemical interactions are not yet clearly elucidated. Fungicides, for instance, another class of chemical compounds widely used in agriculture that are dispersed in the ®eld like herbicides, have an active role on sexual plant reproduction, as has been known for a long time (Church and Williams, 1978; Ries, 1978; Marcucci and Filiti, 1984; Bonomo and Tiezzi, 1986). This evidence suggests that herbicides and fungicides could alter some physiological parameters within the growing pollen tube.
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Eucaryotic cells contain a complex network of ®lamentous proteins collectively called the cytoskeleton, which participates in many cellular functions, including cell division and organelle motility. Microtubules from dierent sources have been studied intensively in terms of ultrastructure, biochemistry and molecular biology, whereas many functions of microtubules were studied using antimicrotubular drugs, alkaloids and other chemicals. It has been established that speci®c binding sites for dierent chemical compounds are present along microtubules and that alkaloids and other chemicals may induce polymerisation or depolymerisation of microtubules (Wilson and Jordan, 1994). Some classes of herbicides, such as dinitroanilines, phosphoric amides and N-phenylcarbamates, are microtubule-depolymerizing chemical compounds (Morejohn and Fosket, 1991). Depending on the economic relevance of herbicide utilisation, the study of the chemical interactions between herbicides and microtubules is becoming the subject of intensi®ed investigation (Anthony et al., 1998). Pollen is the male gametophyte of higher plants, which is constituted at maturity by two or three cells, depending on the species. During the reproductive process, pollen grains produce a cell protrusion, known as the pollen tube, which acts as a cellular channel to convey the sperm cells from the pollen grain to the female gametophyte (Tiezzi, 1991). It has been well established that the pollen tube contains a complex cytoskeletal apparatus that plays a central role during pollen tube growth (for a recent review see Geitmann and Emons, 2000). Microtubules are one of the main components of the pollen tube cytoskeleton and they extend along the longitudinal axis of the tube, individually or associated into bundles, showing a cortical distribution (Del Casino et al., 1993). The pollen grain contains a vegetative nucleus and, depending on the plant species, a generative cell or two sperm cells that move from the grain to the tube apex. The generative/sperm cells are spindle-shaped and contain essentially a microtubular apparatus, which consists of bundles of microtubules organised into a characteristic basket-like structure (Del Casino et al., 1992; Palevitz and Tiezzi, 1992). In the present paper, we investigate the eects of ®ve herbicides on the microtubular apparatus of Nicotiana tabacum pollen tubes. These herbicides are selected among those mostly used in central Italy, and particularly in the countryside around the town of Viterbo. Using an anti a-tubulin antibody, we carried out both immuno¯uorescence observations by confocal laser scanning microscopy and immunogold labelling in electron microscopy investigations. Results show that herbicides used at ®eld concentrations, as suggested by the suppliers, have an eect on the microtubular apparatus of both the pollen tube and the generative cell, although in different ways. All herbicides induce depolymerisation of
pollen tube microtubules, whereas a dierent behaviour for the generative cell microtubules is observed. In particular, some herbicides interact with the generative cell microtubules inducing a complete depolymerisation of the microtubular cytoskeleton and considerable modi®cations of the cytoplasmic cytoarchitecture. Other herbicides interact with the generative cell microtubules without inducing complete depolymerisation, thus allowing the generative cell to preserve its cytoplasmic organisation. The aim of this study is to determine whether the pollen tube cell system, and particularly the microtubular cytoskeleton of the pollen tube, might represent a well-grounded indicator to assess the eects of herbicides. Present results indicate that microtubules can be used as valuable bioindicators for assaying the herbicide toxicity, hence contributing, in addition to other toxicological tests (Kristen, 1997), to the assessment of herbicide eects as they are introduced in the environment. 2. Materials and methods 2.1. Pollen cultures Pollen grains from N. tabacum were obtained from plants grown at the Botanical Garden of Tuscia University in Viterbo Italy, dehydrated and kept at 20 C until use. For germination, pollen grains were hydrated overnight in a moist chamber at room temperature and then suspended in BK medium (Brewbaker and Kwack, 1963) containing 15% sucrose in petri dishes (0.2±0.4 mg pollen/ml BK medium) at room temperature under gentle oscillation. Controls were represented by pollen grains cultured in BK medium containing 15% sucrose without the addition of herbicides. The distribution of microtubules was investigated by immuno¯uorescence techniques on samples collected, respectively, after 4 and 23 h of germination. Immunogold investigations were carried out on samples collected after 4 h of germination. Three hours after germination, herbicides were added to the culture medium and the eects of herbicide treatment were tested by immuno¯uorescence techniques after 1 and 20 h of subsequent culturing. Immunogold labelling and electron microscopy observations were carried out on samples collected after 1 h of herbicide treatment. 2.2. Herbicides Herbicides used in the test belong to dierent classes of chemical compounds able to aect a large number of plants. They were selected among those mainly used in central Italy and particularly in the countryside around the town of Viterbo. All herbicides, tested at ®eld concentration according to the indications of the suppliers (Muccinelli, 1998),
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were added to the petri dishes containing pollen tubes growing in BK medium: . Illoxan (methyl (RS)-2-[4-(2,4-dichlorophenoxy) phenoxy] propionate) from Roussel-Hoechst Agrovet. Final concentration: 2.73 mg/ml. . Banvel 21S (3,6-dichloro-o-anisic acid) from Siapa. Final concentration: 0.35 mg/ml. . Tre¯an (2,6-dinitro-N,N-dipropyl-4-tri¯uoromethylaniline) from DowElanco. Final concentration: 1.53 mg /ml. . Ronstar [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphennyl)-1,3,4-oxadiazol-2(3H)-one] from RhonePoulenc Agro. Final concentration: 0.85 mg/ml. . Roundup 400 [N-(phosphonomethyl) glicine] from Monsanto Italia. Final concentration: 3.44 mg/ml.
2.3. Fluorescence techniques For immuno¯uorescence (IF) investigations, we used the procedure described by Del Casino et al. (1993). Brie¯y, pollen tubes were ®xed in fresh 3% PFA prepared in PM (50 mm PIPES pH 6.9, 1 mm ethylene glycol-bis(baminoethyl ether) N,N,N0 , N0 -tetraacetic acid (EGTA), and 0.5 mm MgCl2) containing 15% sucrose for 30 min. After three washes in PM, samples were incubated for 10 min in the dark in 2% cellulysin (Calbiochem, CA, USA). Following three washes, the samples were post®xed in cold acetone ( 20 C) for 5 min and rinsed in Tris-buered saline (TBS), pH 7.2. After two washes in buer, a monoclonal antibody to the a-tubulin subunit (Amersham, UK) diluted 1:200 was applied to the samples for 1 h at 37 C. Pollen tubes were rinsed in three changes of the buer and samples incubated in second antibody ¯uorescein isothiocyanate (FITC)-conjugated goat antimouse (Cappel, ICN, Germany) diluted 1:400 for 1 h. Subsequently, samples were rinsed three times in buer and mounted on slides in 5% n-propyl gallate in glycerol to minimise fading of the ¯uorescent conjugate. Staining of nucleic acids was carried out with propidium iodide (Molecular Probes, Inc. Eugene, OR, USA) diluted 1:7000 and added to samples immediately before observation at the microscope. Depending on samples, optical sections (0.8±1 mm each) were collected by a Leica confocal laser scanning microscope model 40TD. Images were printed by an Epson Stylus Colour 800 printer. In control experiments, immuno¯uorescence observations were carried out after 4 and 23 h of pollen culture, respectively. In herbicide-treated samples, immuno¯uorescence investigations were carried out after 1 and 20 h of herbicide treatment, respectively. 2.4. Immunogold labelling For immunoelectron microscopy (IEM) observations, we followed the procedure described by Tirlapur et al.
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(1995). Pollen tubes were ®xed with a mixture of 2.5% glutaraldehyde+1.6% paraformaldehyde in 0.1 m phosphate buer, pH 6.9 for 20 min at room temperature. After rinsing in the same buer for 10 min, samples were dehydrated in a graded ethanol series and embedded in medium grade LR White resin (Multilab Supplies, Surrey, UK). The resin was polymerized in tightly capped gelatine capsules for 48 h at 50 C. Ultrathin sections were obtained using a Reichert Ultracut ultramicrotome with a diamond knife, and collected on nickel grids. For immunogold staining (IGS) non-speci®c antigens were blocked with 0.5% bovine serum albumin (BSA) in 0.05 m Tris±HCl buer pH 7.6 for 15 min. Sections were incubated for 3 h in a moist chamber with a primary antibody to the a tubulin subunit (Amersham) diluted 1:500 in 0.05 m Tris±HCl buer, pH 7.6 containing 0.2% BSA. The grids were washed in 0.05 m Tris±HCl, pH 7.6 for 20 min and then in 0.05 m Tris±HCl, pH 7.6 containing 0.1% BSA for 10 min. Sections were incubated with a secondary goat anti-mouse antibody conjugated to 10 nm gold particles (Aurion, Wageningen, The Netherlands), diluted 1:20 in 0.02 m Tris±HCl buer, pH 8.2. After rinsing in 0.05 m Tris±HCl buer containing 0.1% BSA for 10 min and in 0.05 m Tris±HCl buer for 20 min, the grids were washed three times with distilled water (for 5 min). Sections were subsequently stained with uranyl acetate and lead citrate and observed with a Jeol JEM EX II transmission electron microscope at 120 kV. Primary antibody was omitted in control sections. In control experiments, immunogold labelling observations were carried out after 4 h of pollen culture, whereas in herbicide-treated materials analyses were accomplished after 3 h of uninhibited growth and 1 h of herbicide treatment. 3. Results 3.1. Control: 4 h of germination in BK medium After 4 h of culture in control conditions, around 60% of pollen grains were germinated. The generative cell was generally present within the tube. IF techniques allowed the selective staining of microtubules within both the pollen tube and the generative cell (GC). Pollen tube microtubules were distributed along the tube and located for the most part in the peripheral region of the tube cytoplasm (Plate 1a). Within the GC (Plate 1b1), bundles of microtubules formed a basket-like structure and, as shown by a cross-section of the same GC (Plate 1b2), microtubules located in the peripheral region of the cytoplasm with a regular distribution. At IEM level GC, as shown by a cross-section (Plate 1c), is located within the pollen tube and surrounded by
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a wall. Into the GC, the nucleus comprises a large part of the cell volume. Gold particles were observed both in the pollen tube cytoplasm and within the GC. In the pollen tube, gold particles are frequently located in the peripheral region of the tube cytoplasm (data not shown). At higher magni®cation, as shown by crosssection (Plate 1d), groups of gold particles speci®cally located within the GC cytoplasm. 3.2. Control: 23 h of germination in BK medium After 23 h of culture, very long pollen tubes were present in the samples. In some cases, the GC stretched its spindle-like shape, suggesting that the division into two sperm cells was occurring. Some sperm cells were also observed. When investigated at IF level, a microtubular apparatus was still present within the tube (data not shown), the organisation and distribution of which resembled that observed in the 4-h cultured pollen tube. Within the elongated GC, a basket-like structure of microtubules matching that present within the GC after 4 h of culture was observed (Plate 1e).
3.3. Pollen tubes grown in the presence of herbicides All the herbicides tested had an eect on the vegetative cell microtubules. At IF level, ¯uorescent spots were distributed in the pollen tube cytoplasm (Plate 1f), whereas, at IEM level, gold particles were randomly distributed in the pollen tube cytoplasm (data not shown). Herbicides aected the GC microtubules in dierent ways. Results for individual herbicides are reported below. 3.3.1. Illoxan Pollen cultured for 3 h in BK medium and for one additional hour after Illoxan treatment was clearly aected by the herbicide. Compared with the equivalent control, pollen tubes were shorter and the GC round up and increased in volume. IF Ð Within the GC, microtubules were depolymerised and a diused ¯uorescence was present. In some cases short ®laments were observed (Plate 2a). IEM Ð In comparison with the control, only gold particles were present in the cytoplasm. In contrast to the control, label was present in the nucleus (Plate 2b).
Plate 1. (a) Microtubules in the vegetative cell of the pollen tube. Single microtubules are arranged along the longitudinal axis of the tube (380). (b1) Microtubules in the generative cell. Within the generative cell bundles of microtubules form a basket-like structure (470). (b2) Cross-section of the same GC. Microtubules bundles locate in the peripheral region of the GC cytoplasm and are distributed regularly (470). (c) TEM micrograph with immunogold label for tubulin. Cross-section of pollen tube. VCC, vegetative cell cytoplasm; W, GC wall; GCC, generative cell cytoplasm; N, nucleus (23,000). (d) Groups of gold particles are located in the generative cell cytoplasmic lobes (GCC). W, wall; N, nucleus (100,000). (e) GC after 23 h of culture showing cell division. Microtubules still maintain their basket-like structure within the spindle-like shaped GC (450). (f) Pollen tube microtubules aected by herbicide treatment: depolymerised tubulin is identi®ed as bright spots. All tested herbicides provided similar staining (420).
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Twenty hours of Illoxan treatment aected the growth of the tubes, since they were shorter with respect to the equivalent control. IF Ð the immunostaining pattern con®rmed what was already observed after 1 h of Illoxan treatment. The herbicide strongly aected the GC microtubules and a diuse ¯uorescence was present (Plate 2c). In a few cases short ®laments were observed. As shown by double staining with propidium iodide, the nuclear material was distributed in the entire cell volume (Plate 2d). 3.3.2. Banvel 21S Pollen tubes germinated for 1 h in the presence of Banvel 21S showed similar length to those in control conditions. The GC substantially preserved the spindlelike shape, although a small increase in volume was sometimes observed. IF Ð The GC microtubular apparatus was partially aected. Microtubules formed the characteristic basket-like structure; however, some ¯uorescent spots were clearly observed, thus suggesting
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the presence of both polymerised and depolymerised tubulin (Plate 3a). IEM Ð at EM level, the GC morphology was very similar to the control material. Many gold particles were present in the cytoplasmic lobes (Plate 3b). Pollen tubes germinated for 20 h in the presence of Banvel 21S were shorter than the pollen tubes of the equivalent control. IF Ð The elongated spindle-like shape of the GC resembled that observed in the control. Within the GC, only ¯uorescent spots were observed (Plate 3c). Double staining by propidium iodide showed nuclear material ®lling a large part but not all of the GC volume (Plate 3d). 3.3.3. Tre¯an In comparison with the equivalent control, pollen tubes germinated for 1 h in BK medium containing Tre¯an were shorter. The generative cell changed its shape and increased in volume. IF Ð within the GC (Plate 4a) only a diuse ¯uorescence and some brighter
Plate 2. (a) Pollen tubes cultured for one hour in presence of Illoxan labeled with anti-tubulin. A diuse ¯uorescence and few short microtubule ®laments are present within the generative cell (480). (b) TEM micrograph with immunogold label for tubulin. Few gold particles are present both in the cytoplasm and in the nucleus (arrow). C, cytoplasm; N, nucleus; (85,000). (c) Twenty h of Illoxan treatment. Within the generative cell microtubules are completely depolymerised (480). (d) Propidium iodide staining of the same GC as in (c) Nuclear material is distributed in the entire cell volume (480).
Plate 3. (a) Pollen tubes cultured for 1 h in the presence of Banvel 21S. Microtubules maintain the characteristic basket-like structure within the generative cell (480). (b) TEM micrograph with immunogold label for tubulin. Samples treated with Banvel 21S were very similar to the control. C, cytoplasm; N, nucleus; W, GC wall (50,000). (c) After 20 h of treatment with Banvel 21S, the GC still preserves its spindle-like shape; the GC microtubular apparatus appeared depolymerised and ¯uorescence spots were observed (300). (d) The nuclear material does not ®ll the entire cellular volume as seen after the staining with propidium iodide (300).
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spots were observed, thus con®rming the depolymerisation of microtubules. IEM Ð at IEM level, gold particles localised in the GC cytoplasm (Plate 4b). After 20 h of germination in BK medium containing Tre¯an, pollen tubes were shorter when compared with the control. IF Ð the GC maintained the shape already observed in the pollen germinated for 1 h in BK medium containing Tre¯an. No microtubules in the pollen tube were observed whereas a diuse ¯uorescence staining within the GC con®rmed the complete depolymerisation of microtubules (Plate 4c). Propidium iodide staining showed the nuclear material as widely diused in the entire volume of the cell (Plate 4d). 3.3.4. Ronstar In comparison with the equivalent control, pollen tubes grown for 1 h in BK medium containing Ronstar were shorter. The GC did not substantially increase in volume and maintained the spindle-like shape. IF Ð short microtubules showed a random distribution within the GC; some ¯uorescent spots were also detected (Plate 5a). IEM Ð clusters of gold particles were observed within the cytoplasmic lobes (Plate 5b).
After 20 h of incubation in BK medium containing Ronstar, pollen tubes were shorter in comparison with the control. The GC increased in volume while preserving the spindle-like shape. IF Ð within the GC, microtubules were not detected and ¯uorescence spots of varying size were observed in the peripheral region of the GC cytoplasm (Plate 5c). As shown by propidium iodide staining, the nuclear material was not distributed in the entire GC volume (Plate 5d). 3.3.5. Roundup 400 In comparison with the control, pollen tubes germinated for 1 h in BK medium containing Roundup 400 were shorter. The GC increased in volume and preserved the spindle-like shape. IF Ð within the GC, a diuse ¯uorescent staining mainly localised in the peripheral region of the cytoplasm. No microtubules were observed (Plate 6a). IEM Ð At IEM level gold particles were detected within the GC cytoplasm (Plate 6b). In comparison with the control, after 20 h of incubation in BK medium containing Roundup 400, pollen tubes were shorter. The GC increased in volume and changed their characteristic spindle-like shape to an
Plate 4. (a) 1 h of treatment with Tre¯an. The GC has rounded up and only some brighter spots were observed after labeling for tubulin (470). (b) At IEM level, gold particles are localised in the GC cytoplasm. C, cytoplasm; N, nucleus; W, GC wall (60,000). (c) A diuse ¯uorescence was present in the GC of pollen tube grown for 20 h in the presence of Tre¯an con®rming a complete depolymerisation of MTs (470). (d) Propidium iodide staining showed the nuclear material to be widely diused in the entire volume of the cell (470).
Plate 5. (a) After 1 h of Ronstar treatment short microtubules showed a random distribution (470). (b) Clusters of gold particles were observed within the cytoplasmic lobes. C, cytoplasm; N, nucleus; W, GC wall (80,000). (c) Twenty h after treatment only ¯uorescent spots were detected in the peripheral region of the GC cytoplasm (470). (d) As shown by propidium iodide staining, the nuclear material was not distributed in the entire GC volume (470).
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Plate 6. (a) After 1 h of Rondup 400 treatment in the peripheral region of the GC cell a diuse ¯uorescence was present (470). (b) At IEM level gold particles were detected within the GC cytoplasm. C, cytoplasm; N, nucleus; W, GC wall (75,000). (c) After 20 h of treatment within the GC, no microtubules were found and a diuse ¯uorescent staining was detected (470). (d) As shown by propidium iodide, the nuclear material was distributed in the entire cell volume (470).
elongated cylinder-like shape. IF Ð Within the GC, no microtubules were found and a diuse ¯uorescent staining was detected. In particular, an intense staining was observed in the central region of the GC (Plate 6c). As shown by propidium iodide staining, the nuclear material was distributed in the entire cell volume (Plate 6d). 4. Discussion In previous experiments (unpublished results), pollen grains, which were directly cultured in herbicide-containing medium, did not germinate or germinated at very low percentage. Here we cultured the pollen grains for 3 h before adding the herbicides; after 3 h of culturing, as shown in controls, pollen tubes elongated for hundreds of micrometers and the microtubular cytoskeleton of both the vegetative and generative cell was fully organised (for review see Geitmann and Emons, 2000). The examined herbicides exert their toxicological eects on dierent compartments and structures of the cell, depending on their chemical nature (as described by suppliers information). Our results clearly show that many of the tested herbicides aect the pollen tube growth. Therefore it is possible that the pollen tube microtubular apparatus does not represent the main target for the toxicological action of herbicides. However, the chemical action of herbicides on microtubules is consistent and reproducible and it is clearly revealed at both IF and IEM level. Within the tube, the vegetative cell microtubules are already depolymerised after 1 h of herbicide treatment, thus con®rming a lower resistance of this population of microtubules to chemical stress conditions (Pierson and Cresti, 1992). In the absence of microtubules, the pollen tubes continue to grow, thus con®rming that microtubules do not have a key role in the pollen tube growth process (AÊstrom et al., 1995; Cai et al., 1997). However, the pollen tube length is reduced when compared with the controls.
The generative cell and its microtubular cytoskeleton show dierent behaviour after treatment with each herbicide. Depending on their eects on the generative cell microtubules, we can divide the examined herbicides into three dierent groups. One group consisted of Illoxan, Tre¯an and Roundup 400, a second includes Ronstar, and the third consisted of Banvel 21S. Although there exist some minor dierences among the individual components, all herbicides of the ®rst group aect the GC and its microtubular cytoskeleton in a consistent manner. After 1 h of culturing in the presence of herbicides, the GC changes its shape and the depolymerisation of microtubules is prominent. We can conclude that the chemical activity of these herbicides occurs within a short time and is dramatic. The increase in volume and the presence of nuclear material dispersed in the entire cell volume after 20 h of culturing in the presence of herbicides suggests a physiological alteration of cytoplasmic components and damages of the nuclear envelope. In particular, this is con®rmed by Illoxan treatment: as shown by gold particles, tubulin molecules result in the nuclear zone probably mixed to nuclear materials. The chemical eect of Ronstar is not immediate and after 1 h of culturing in the presence of the herbicide, the GC preserves the spindle-like shape and the microtubular cytoskeleton is only partially aected. Microtubules are still present and, as shown by the gold particles speci®cally clustered into the cytoplasmic lobes, they maintain their characteristic organisation in bundles within the cytoplasm. After 20 h of culturing in the presence of Ronstar, the GC is signi®cantly aected. The cell increases in volume and microtubules are not evident within the GC. The ¯uorescence is not diuse; intense ¯uorescent spots speci®cally localised in the peripheral region of the GC cytoplasm suggest the presence of depolymerised tubulin. The spreading of nuclear material into the GC but not in the entire cell volume suggests that the structural integrity of cellular compartments is preserved.
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Banvel 21S hardly aects the pollen tubes. However, the pollen tube length is more similar to the control than that of pollen tubes grown in the presence of other herbicides. After 1 h of herbicide treatment, the GC essentially maintains the spindle-like shape although a slight increase of the cell volume is observed. The GC microtubular apparatus is partially aected and a basketlike structure of microtubules is still present. The occurrence of ¯uorescent spots suggests that a process of microtubule depolymerisation takes place. After 20 h of herbicide treatment, the GC does not seem particularly aected. The GC has an elongated shape resembling that of dividing GCs (see control, Plate 1e). Microtubules are not present, whereas ¯uorescent spots suggest the presence of depolymerised tubulin mainly in the peripheral region of the GC cytoplasm. The herbicide does not induce the diusion of nuclear material in the entire volume of the cell, thus con®rming the integrity of the nuclear envelope and a less disruptive action of Banvel 21S in comparison with the other herbicides. In terms of structural and biochemical properties of the pollen tube microtubules, these data con®rm the presence of dierent patterns of microtubules within the pollen tube and the GC. It has been already shown that dierent tubulin isoforms are present in both the microtubular apparatus of pollen tubes and of GCs and that they are localised dierently (AÊstrom, 1992; Del Casino et al., 1993). It is likely that the dierent resistance of microtubules to the stress conditions induced by herbicides and the characteristic staining patterns could be dependent on speci®c biochemical properties related to dierent tubulin isoforms. Herbicide resistance is also generally related to dierence in the amino acid composition of tubulins, since spontaneous mutation in tubulin genes can confer resistance to dinitroaniline herbicides (Anthony et al., 1998). From a general point of view, it is evident that many of the examined herbicides aected also the GC wall, although the particular embedding resin did not allow detailed investigations at ultrastructural level to be carried out. In some cases, the nuclear membrane seems to be aected, thus con®rming a general chemical action of the herbicides.
(Kristen and Kappler, 1995), have also been introduced (for a review see Kristen, 1997) in addition to speci®c assays on animal cells cultures. The pollen tube proved to be a highly sensitive indicator for the toxicity assessment of herbicides and synthetic compounds because the germination and elongation of pollen tubes is aected by treatment with these molecules. Data shown in this work con®rm that the pollen tube is a good model for toxicological tests and, in addition, introduce the study of its microtubular apparatus as an additional parameter for a deeper evaluation of the toxicological activity of herbicides. Further, results reported here also represent matters for general consideration. In terms of agriculture and environment, the evidence of consistent biological damage induced by herbicides on in vitro growing pollen tubes raises questions concerning their role in the higher plant fertilisation process. In fact, it could be possible that they reduce the level of pollen fertility and consequently the number of fertilised eggs in non-target plants. In addition, since some plants could be more naturally resistant to herbicides than others, they could contribute to plant selection in the area aected by the herbicide treatment. In terms of human health, in recent times herbicides, in addition to their use in agriculture, have been applied to plants growing within towns and cities and as a consequence the number of people interacting with such chemicals has greatly increased. It is evident that the use of herbicides must be carefully evaluated and planned by administrators, and scienti®c research should supply tools for a wider and complete evaluation and planning. With this in view, the results reported here can represent a further contribution. Acknowledgements The authors wish to thank Professor Li Yi-Qin of the Department of Biological Sciences and Biotechnology of Tsinghua University, Beijing, China, for helpful suggestions during the preparation of the manuscript.
References 5. Further considerations For a long time, animals have been used to evaluate the toxicity of many kinds of chemical compounds. The use of animals in these assays implies problems such as reproducibility, diculties and complexity of the tests as well as high costs. For ethical and economic reasons, during recent years in vitro procedures have been applied to examine the toxicological eects of chemical compounds (Clemedson et al., 1996). Toxicological tests using plant cells, such as the pollen tube growth test
Abdel-Rahman, A.R., Wauchope, R.D., Truman, C.C., Dowler, C.C., 1999. Runo and leaching of atrazine and alachlor on a sandy soil as aected by application in sprinkler irrigation. Journal of Environmantal Science and Health B Ð Pesticides and Food Contaminants 34, 381±396. Anthony, R.G., Waldin, T.R., Ray, J.A., Bright, S.W., Hussey, P.J., 1998. Herbicide resistance caused by spontaneous mutation of the cytoskeletal protein tubulin. Nature 393, 260±263. AÊstrom, 1992. Acetylated a-tubulin in the pollen tube MTs. Cell Biology International Reports 16, 871±881. AÊstrom, H, Sorri, O., Raudaskoski, M., 1995. Role of microtubules in the movement of the vegetative nucleus and generative cell in tobacco pollen tubes. Sexual Plant Reproduction 8, 61±69.
E. Ovidi et al. / Toxicology in Vitro 15 (2001) 143±151 Bonomo, R., Tiezzi, A., 1986. In¯uenza di regolatori di crescita e fungicidi in polline di melo in vivo ed in vitro. Rivista della Orto¯orofrutticoltura italiana 70, 43±52. Brewbaker, J.L., Kwack, B.H., 1963. The essential role of calcium ion in pollen germination and pollen tube growth. American Journal of Botany 50, 859±865. Cai, G., Moscatelli, A., Cresti, M., 1997. Cytoskeletal organization and pollen tube growth. Trends in Plant Science 2, 86±91. Chevreuil, M., Garmouma, M., Teil, M.J., Chesteriko, A., 1996. Occurrence of organochlorines (PCBs, pesticides) and herbicides (triazines, phenylureas) in the atmosphere and in the fallout from urban and rural stations of Paris area. Science of the Total Environment 182, 25±37. Church, B.M., Williams, R.R., 1978. The toxicity to apple pollen of several fungicides as demonstrated by in vivo and in vitro techniques. Journal of Horticultural Science 52, 429±436. Clemedson, C., McFarlane-Abdulla, E., Andersson, M., Barile, F.A., Calleeja, M.C., ChesneÂ, C., Clothier, R., Cottin, M., Curren, R., Dierickx, P., Ferro, M., FiskesjoÈ, G., Garza-OcanÄas, L., GoÂmezLechoÂn, M.J., GuÈlden, M., Isomaa, B., Janus, J., Judge, P., Kahru, A., Kemp, R.B., Kerszmann, G., Kristen, U., Kunimoto, M., KaÈrenlampi, S., Lavrijsen, K., Lewan, L., Lilius, H., Malmsten, A., Ohno, T., Persoone, G., Pettersson, R., Roguet, R., Romert, L., Sandberg, M., Sawyer, T.W., Seibert, H., Shrivastsva, R., SjoÈstroÈm, M., Stammati, A., Tanaka, N., Torres-Alinis, O., Voss, J.U., Wakuri, S., Walum, E., Wang, X., Zucco, F., Ekwall, B., 1996. MEIC evaluation of acute systemic toxicity. Part II. In vitro results from 68 toxicity assays used to test the ®rst 30 references chemicals and a comparative cytotoxicity analysis. ATLA 24, 273±311. Del Casino, C., Tiezzi, A., Wagner, W.T., Cresti, M., 1992. The organization of the cytoskeleton in the generative cell and sperms of Hyacinthus orientalis. Protoplasma 168, 41±50. Del Casino, C., Li, Y.Q., Moscatelli, A., Scali, M., Tiezzi, A., Cresti, M., 1993. Distribution of microtubules during growth of tobacco pollen tubes. Biology of the Cell 79, 125±132. Geitmann, A., Emons, A.M.C., 2000. The cytoskeleton in plant and fungal cell tip growth. Journal of Microscopy 198, 218±245. Gorell, J.M., Johnson, C.C., Rybicki, B.A., Peterson, E.L., Ricchardson, R.J., 1998. The risk of Pakinson's disease with exposure to pesticides, farming, well water, and rural living. Neurology 50, 1346±1350.
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Kim, J.H., Feagley, S.E., 1998. Adsorpion and leaching of tri¯uralin, metolachlor, and metribuzin in a commerce soil. Journal of Environmental Science and Health B Ð Pesticides and Food Contaminants 33, 529±546. Kristen, U., 1997. Use of higher plants as screens for toxicity assessment. Toxicology in vitro 11, 181±191. Kristen, U., Kappler, R., 1995. The pollen tube growth test. In: O'Hare, S., Atterwill, C.K. (Eds.), In vitro Testing Protocols. Methods in Molecular Biology. Humana Press, Totowa, NJ, pp. 189±198. Marcucci, M.C., Filiti, N., 1984. Germination of pear and apple pollen as in¯uenced by fungicides. Gartenbauwissenschaft 49, 28±32. Morejohn, L.C., Fosket, D.E., 1991. The biochemistry of compounds with anti-microtubule activity in plant cells. Pharmacology and Therapeutics 51, 217±230. Muccinelli, M., 1998. Prontuario dei ®tofarmaci, 8th Edition. Edagricole. Munger, R., Isacson, P., Hu, S., Burns, T., Hanson, J., Lynch, C.F., Cherryholmes, K., VanDorpe, P., Hausler Jr., W.J., 1997. Intrauterine growth retardation in Iowa communities with herbicide-contaminated drinking water supplies. Environmental Health Perspectives 105, 308±314. Palevitz, B.A., Tiezzi, A., 1992. The organization and function of the generative cell and sperm cytoskeleton. International of Review Cytology 140, 149±185. Pierson, E.S., Cresti, M., 1992. Cytoskeleton and cytoplasmatic organization of pollen and pollen tubes. Internatinal of Review Cytology 140, 73±125. Ries, S.M., 1978. Germination of apple pollen is in¯uenced by fungicides. Fruit Varietes Journal 32, 12±16. Tiezzi, A., 1991. The pollen tube cytoskeleton. Electron Microscopy Reviews. Subcellular and Biomolecular Structure 4, 205±219. Tirlapur, U.K., Faleri, C., Milanesi, C., Moscatelli, A., Scali, M., Del Casino, C., Tiezzi, A., Cresti, M., 1995. High-resolution confocal imaging and colloidal-gold immuno-electron microscopy of changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum L. European Journal of Cell Biology 67, 209±217. Wilson, L., Jordan, M.A., 1994. Pharmacological probes of microtubules function. In: Hyams, J.S., Lloyd, C.W. (Eds.), Microtubules, Modern Cell Biology Series. Wiley-Liss, New York, pp. 59±83.
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Herbicides and the microtubular apparatus of Nicotiana tabacum pollen tube: immuno¯uorescence and immunogold labelling studies E. Ovidi a,*, G. Gambellini b, A.R. Taddei b, G. Cai c, C. Del Casino c, M. Ceci a, S. Rondini a, A. Tiezzi a,b a Dipartimento Scienze Ambientali, UniversitaÁ degli Studi della Tuscia, Via S. Camillo de Lellis blocco D, 01100 Viterbo, Italy Centro Interdipartimentale Microscopia Elettronica (CIME), UniversitaÁ degli Studi della Tuscia, Via S. Camillo de Lellis blocco D, 01100 Viterbo, Italy c Dipartimento Scienze Ambientali, UniversitaÁ degli Studi di Siena, Via Mattioli 4, 53100 Siena, Italy
b
Accepted 12 November 2000
Abstract Herbicides are chemical compounds widely used in agriculture. As their intensive application is becoming a cause of environmental pollution, detailed and more sophisticated investigations are needed to understand better their consequences at the biological level. After herbicides are dispersed in the ®elds, they establish chemical interactions with both target and non-target plants. In both cases, herbicides can interact with the plant reproductive apparatus; consequently they could play a role during the fertilisation process in higher plants. Using an antibody to the a-tubulin subunit in immuno¯uorescence and immunoelectron microscopy techniques, we investigated the distribution of microtubules in Nicotiana tabacum pollen tubes grown under in vitro conditions in the presence of ®ve dierent herbicides selected among those used frequently in central Italy. Herbicides have a speci®c eect on the microtubular apparatus of both pollen tube and generative cell. In addition to other tests and assays, these results suggest that the microtubule cytoskeleton of pollen tubes can be used as a bioindicator for studying the toxicity eects induced by herbicides. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Herbicides; Microtubules; Pollen tube; Generative cell; Immuno¯uorescence; Immunogold labelling
1. Introduction In recent years, the application of herbicides in many countries has increased. Herbicides have an important role in the development of agricultural production and in the enhancement of products, both in terms of quantity and quality, contributing therefore to a general wellbeing. On the other hand, the large and intensive use of herbicides is also becoming a factor contributing to environmental pollution (Chevreuil et al., 1996; Kim and Feagley, 1998; Abdel-Rahman et al., 1999). The mechanism of toxicological action on plants and animals is not yet completely understood for many herbicides. As Abbreviations: GC, generative cell; IEM, immunoelectron microscopy; IF, immuno¯uorescence; W, GC wall * Corresponding author. Tel.: +39-761-357130; fax: +39-761357105. E-mail address: [email protected] (E. Ovidi).
a consequence, the lack of detailed evaluations of the role(s) of herbicides at the biological level could also represent a problem for human health (Munger et al., 1997; Gorell et al., 1998). Although the ¯owering apparatus is not the most important target of many herbicides, the latter can have incidental eects during the fertilisation process in higher plants. When herbicides are dispersed in the ®elds, they also reach the stigmas, anthers and pollen surface of dierent plant species. The eects of these chemical interactions are not yet clearly elucidated. Fungicides, for instance, another class of chemical compounds widely used in agriculture that are dispersed in the ®eld like herbicides, have an active role on sexual plant reproduction, as has been known for a long time (Church and Williams, 1978; Ries, 1978; Marcucci and Filiti, 1984; Bonomo and Tiezzi, 1986). This evidence suggests that herbicides and fungicides could alter some physiological parameters within the growing pollen tube.
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(00)00064-3
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Eucaryotic cells contain a complex network of ®lamentous proteins collectively called the cytoskeleton, which participates in many cellular functions, including cell division and organelle motility. Microtubules from dierent sources have been studied intensively in terms of ultrastructure, biochemistry and molecular biology, whereas many functions of microtubules were studied using antimicrotubular drugs, alkaloids and other chemicals. It has been established that speci®c binding sites for dierent chemical compounds are present along microtubules and that alkaloids and other chemicals may induce polymerisation or depolymerisation of microtubules (Wilson and Jordan, 1994). Some classes of herbicides, such as dinitroanilines, phosphoric amides and N-phenylcarbamates, are microtubule-depolymerizing chemical compounds (Morejohn and Fosket, 1991). Depending on the economic relevance of herbicide utilisation, the study of the chemical interactions between herbicides and microtubules is becoming the subject of intensi®ed investigation (Anthony et al., 1998). Pollen is the male gametophyte of higher plants, which is constituted at maturity by two or three cells, depending on the species. During the reproductive process, pollen grains produce a cell protrusion, known as the pollen tube, which acts as a cellular channel to convey the sperm cells from the pollen grain to the female gametophyte (Tiezzi, 1991). It has been well established that the pollen tube contains a complex cytoskeletal apparatus that plays a central role during pollen tube growth (for a recent review see Geitmann and Emons, 2000). Microtubules are one of the main components of the pollen tube cytoskeleton and they extend along the longitudinal axis of the tube, individually or associated into bundles, showing a cortical distribution (Del Casino et al., 1993). The pollen grain contains a vegetative nucleus and, depending on the plant species, a generative cell or two sperm cells that move from the grain to the tube apex. The generative/sperm cells are spindle-shaped and contain essentially a microtubular apparatus, which consists of bundles of microtubules organised into a characteristic basket-like structure (Del Casino et al., 1992; Palevitz and Tiezzi, 1992). In the present paper, we investigate the eects of ®ve herbicides on the microtubular apparatus of Nicotiana tabacum pollen tubes. These herbicides are selected among those mostly used in central Italy, and particularly in the countryside around the town of Viterbo. Using an anti a-tubulin antibody, we carried out both immuno¯uorescence observations by confocal laser scanning microscopy and immunogold labelling in electron microscopy investigations. Results show that herbicides used at ®eld concentrations, as suggested by the suppliers, have an eect on the microtubular apparatus of both the pollen tube and the generative cell, although in different ways. All herbicides induce depolymerisation of
pollen tube microtubules, whereas a dierent behaviour for the generative cell microtubules is observed. In particular, some herbicides interact with the generative cell microtubules inducing a complete depolymerisation of the microtubular cytoskeleton and considerable modi®cations of the cytoplasmic cytoarchitecture. Other herbicides interact with the generative cell microtubules without inducing complete depolymerisation, thus allowing the generative cell to preserve its cytoplasmic organisation. The aim of this study is to determine whether the pollen tube cell system, and particularly the microtubular cytoskeleton of the pollen tube, might represent a well-grounded indicator to assess the eects of herbicides. Present results indicate that microtubules can be used as valuable bioindicators for assaying the herbicide toxicity, hence contributing, in addition to other toxicological tests (Kristen, 1997), to the assessment of herbicide eects as they are introduced in the environment. 2. Materials and methods 2.1. Pollen cultures Pollen grains from N. tabacum were obtained from plants grown at the Botanical Garden of Tuscia University in Viterbo Italy, dehydrated and kept at 20 C until use. For germination, pollen grains were hydrated overnight in a moist chamber at room temperature and then suspended in BK medium (Brewbaker and Kwack, 1963) containing 15% sucrose in petri dishes (0.2±0.4 mg pollen/ml BK medium) at room temperature under gentle oscillation. Controls were represented by pollen grains cultured in BK medium containing 15% sucrose without the addition of herbicides. The distribution of microtubules was investigated by immuno¯uorescence techniques on samples collected, respectively, after 4 and 23 h of germination. Immunogold investigations were carried out on samples collected after 4 h of germination. Three hours after germination, herbicides were added to the culture medium and the eects of herbicide treatment were tested by immuno¯uorescence techniques after 1 and 20 h of subsequent culturing. Immunogold labelling and electron microscopy observations were carried out on samples collected after 1 h of herbicide treatment. 2.2. Herbicides Herbicides used in the test belong to dierent classes of chemical compounds able to aect a large number of plants. They were selected among those mainly used in central Italy and particularly in the countryside around the town of Viterbo. All herbicides, tested at ®eld concentration according to the indications of the suppliers (Muccinelli, 1998),
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were added to the petri dishes containing pollen tubes growing in BK medium: . Illoxan (methyl (RS)-2-[4-(2,4-dichlorophenoxy) phenoxy] propionate) from Roussel-Hoechst Agrovet. Final concentration: 2.73 mg/ml. . Banvel 21S (3,6-dichloro-o-anisic acid) from Siapa. Final concentration: 0.35 mg/ml. . Tre¯an (2,6-dinitro-N,N-dipropyl-4-tri¯uoromethylaniline) from DowElanco. Final concentration: 1.53 mg /ml. . Ronstar [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphennyl)-1,3,4-oxadiazol-2(3H)-one] from RhonePoulenc Agro. Final concentration: 0.85 mg/ml. . Roundup 400 [N-(phosphonomethyl) glicine] from Monsanto Italia. Final concentration: 3.44 mg/ml.
2.3. Fluorescence techniques For immuno¯uorescence (IF) investigations, we used the procedure described by Del Casino et al. (1993). Brie¯y, pollen tubes were ®xed in fresh 3% PFA prepared in PM (50 mm PIPES pH 6.9, 1 mm ethylene glycol-bis(baminoethyl ether) N,N,N0 , N0 -tetraacetic acid (EGTA), and 0.5 mm MgCl2) containing 15% sucrose for 30 min. After three washes in PM, samples were incubated for 10 min in the dark in 2% cellulysin (Calbiochem, CA, USA). Following three washes, the samples were post®xed in cold acetone ( 20 C) for 5 min and rinsed in Tris-buered saline (TBS), pH 7.2. After two washes in buer, a monoclonal antibody to the a-tubulin subunit (Amersham, UK) diluted 1:200 was applied to the samples for 1 h at 37 C. Pollen tubes were rinsed in three changes of the buer and samples incubated in second antibody ¯uorescein isothiocyanate (FITC)-conjugated goat antimouse (Cappel, ICN, Germany) diluted 1:400 for 1 h. Subsequently, samples were rinsed three times in buer and mounted on slides in 5% n-propyl gallate in glycerol to minimise fading of the ¯uorescent conjugate. Staining of nucleic acids was carried out with propidium iodide (Molecular Probes, Inc. Eugene, OR, USA) diluted 1:7000 and added to samples immediately before observation at the microscope. Depending on samples, optical sections (0.8±1 mm each) were collected by a Leica confocal laser scanning microscope model 40TD. Images were printed by an Epson Stylus Colour 800 printer. In control experiments, immuno¯uorescence observations were carried out after 4 and 23 h of pollen culture, respectively. In herbicide-treated samples, immuno¯uorescence investigations were carried out after 1 and 20 h of herbicide treatment, respectively. 2.4. Immunogold labelling For immunoelectron microscopy (IEM) observations, we followed the procedure described by Tirlapur et al.
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(1995). Pollen tubes were ®xed with a mixture of 2.5% glutaraldehyde+1.6% paraformaldehyde in 0.1 m phosphate buer, pH 6.9 for 20 min at room temperature. After rinsing in the same buer for 10 min, samples were dehydrated in a graded ethanol series and embedded in medium grade LR White resin (Multilab Supplies, Surrey, UK). The resin was polymerized in tightly capped gelatine capsules for 48 h at 50 C. Ultrathin sections were obtained using a Reichert Ultracut ultramicrotome with a diamond knife, and collected on nickel grids. For immunogold staining (IGS) non-speci®c antigens were blocked with 0.5% bovine serum albumin (BSA) in 0.05 m Tris±HCl buer pH 7.6 for 15 min. Sections were incubated for 3 h in a moist chamber with a primary antibody to the a tubulin subunit (Amersham) diluted 1:500 in 0.05 m Tris±HCl buer, pH 7.6 containing 0.2% BSA. The grids were washed in 0.05 m Tris±HCl, pH 7.6 for 20 min and then in 0.05 m Tris±HCl, pH 7.6 containing 0.1% BSA for 10 min. Sections were incubated with a secondary goat anti-mouse antibody conjugated to 10 nm gold particles (Aurion, Wageningen, The Netherlands), diluted 1:20 in 0.02 m Tris±HCl buer, pH 8.2. After rinsing in 0.05 m Tris±HCl buer containing 0.1% BSA for 10 min and in 0.05 m Tris±HCl buer for 20 min, the grids were washed three times with distilled water (for 5 min). Sections were subsequently stained with uranyl acetate and lead citrate and observed with a Jeol JEM EX II transmission electron microscope at 120 kV. Primary antibody was omitted in control sections. In control experiments, immunogold labelling observations were carried out after 4 h of pollen culture, whereas in herbicide-treated materials analyses were accomplished after 3 h of uninhibited growth and 1 h of herbicide treatment. 3. Results 3.1. Control: 4 h of germination in BK medium After 4 h of culture in control conditions, around 60% of pollen grains were germinated. The generative cell was generally present within the tube. IF techniques allowed the selective staining of microtubules within both the pollen tube and the generative cell (GC). Pollen tube microtubules were distributed along the tube and located for the most part in the peripheral region of the tube cytoplasm (Plate 1a). Within the GC (Plate 1b1), bundles of microtubules formed a basket-like structure and, as shown by a cross-section of the same GC (Plate 1b2), microtubules located in the peripheral region of the cytoplasm with a regular distribution. At IEM level GC, as shown by a cross-section (Plate 1c), is located within the pollen tube and surrounded by
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a wall. Into the GC, the nucleus comprises a large part of the cell volume. Gold particles were observed both in the pollen tube cytoplasm and within the GC. In the pollen tube, gold particles are frequently located in the peripheral region of the tube cytoplasm (data not shown). At higher magni®cation, as shown by crosssection (Plate 1d), groups of gold particles speci®cally located within the GC cytoplasm. 3.2. Control: 23 h of germination in BK medium After 23 h of culture, very long pollen tubes were present in the samples. In some cases, the GC stretched its spindle-like shape, suggesting that the division into two sperm cells was occurring. Some sperm cells were also observed. When investigated at IF level, a microtubular apparatus was still present within the tube (data not shown), the organisation and distribution of which resembled that observed in the 4-h cultured pollen tube. Within the elongated GC, a basket-like structure of microtubules matching that present within the GC after 4 h of culture was observed (Plate 1e).
3.3. Pollen tubes grown in the presence of herbicides All the herbicides tested had an eect on the vegetative cell microtubules. At IF level, ¯uorescent spots were distributed in the pollen tube cytoplasm (Plate 1f), whereas, at IEM level, gold particles were randomly distributed in the pollen tube cytoplasm (data not shown). Herbicides aected the GC microtubules in dierent ways. Results for individual herbicides are reported below. 3.3.1. Illoxan Pollen cultured for 3 h in BK medium and for one additional hour after Illoxan treatment was clearly aected by the herbicide. Compared with the equivalent control, pollen tubes were shorter and the GC round up and increased in volume. IF Ð Within the GC, microtubules were depolymerised and a diused ¯uorescence was present. In some cases short ®laments were observed (Plate 2a). IEM Ð In comparison with the control, only gold particles were present in the cytoplasm. In contrast to the control, label was present in the nucleus (Plate 2b).
Plate 1. (a) Microtubules in the vegetative cell of the pollen tube. Single microtubules are arranged along the longitudinal axis of the tube (380). (b1) Microtubules in the generative cell. Within the generative cell bundles of microtubules form a basket-like structure (470). (b2) Cross-section of the same GC. Microtubules bundles locate in the peripheral region of the GC cytoplasm and are distributed regularly (470). (c) TEM micrograph with immunogold label for tubulin. Cross-section of pollen tube. VCC, vegetative cell cytoplasm; W, GC wall; GCC, generative cell cytoplasm; N, nucleus (23,000). (d) Groups of gold particles are located in the generative cell cytoplasmic lobes (GCC). W, wall; N, nucleus (100,000). (e) GC after 23 h of culture showing cell division. Microtubules still maintain their basket-like structure within the spindle-like shaped GC (450). (f) Pollen tube microtubules aected by herbicide treatment: depolymerised tubulin is identi®ed as bright spots. All tested herbicides provided similar staining (420).
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Twenty hours of Illoxan treatment aected the growth of the tubes, since they were shorter with respect to the equivalent control. IF Ð the immunostaining pattern con®rmed what was already observed after 1 h of Illoxan treatment. The herbicide strongly aected the GC microtubules and a diuse ¯uorescence was present (Plate 2c). In a few cases short ®laments were observed. As shown by double staining with propidium iodide, the nuclear material was distributed in the entire cell volume (Plate 2d). 3.3.2. Banvel 21S Pollen tubes germinated for 1 h in the presence of Banvel 21S showed similar length to those in control conditions. The GC substantially preserved the spindlelike shape, although a small increase in volume was sometimes observed. IF Ð The GC microtubular apparatus was partially aected. Microtubules formed the characteristic basket-like structure; however, some ¯uorescent spots were clearly observed, thus suggesting
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the presence of both polymerised and depolymerised tubulin (Plate 3a). IEM Ð at EM level, the GC morphology was very similar to the control material. Many gold particles were present in the cytoplasmic lobes (Plate 3b). Pollen tubes germinated for 20 h in the presence of Banvel 21S were shorter than the pollen tubes of the equivalent control. IF Ð The elongated spindle-like shape of the GC resembled that observed in the control. Within the GC, only ¯uorescent spots were observed (Plate 3c). Double staining by propidium iodide showed nuclear material ®lling a large part but not all of the GC volume (Plate 3d). 3.3.3. Tre¯an In comparison with the equivalent control, pollen tubes germinated for 1 h in BK medium containing Tre¯an were shorter. The generative cell changed its shape and increased in volume. IF Ð within the GC (Plate 4a) only a diuse ¯uorescence and some brighter
Plate 2. (a) Pollen tubes cultured for one hour in presence of Illoxan labeled with anti-tubulin. A diuse ¯uorescence and few short microtubule ®laments are present within the generative cell (480). (b) TEM micrograph with immunogold label for tubulin. Few gold particles are present both in the cytoplasm and in the nucleus (arrow). C, cytoplasm; N, nucleus; (85,000). (c) Twenty h of Illoxan treatment. Within the generative cell microtubules are completely depolymerised (480). (d) Propidium iodide staining of the same GC as in (c) Nuclear material is distributed in the entire cell volume (480).
Plate 3. (a) Pollen tubes cultured for 1 h in the presence of Banvel 21S. Microtubules maintain the characteristic basket-like structure within the generative cell (480). (b) TEM micrograph with immunogold label for tubulin. Samples treated with Banvel 21S were very similar to the control. C, cytoplasm; N, nucleus; W, GC wall (50,000). (c) After 20 h of treatment with Banvel 21S, the GC still preserves its spindle-like shape; the GC microtubular apparatus appeared depolymerised and ¯uorescence spots were observed (300). (d) The nuclear material does not ®ll the entire cellular volume as seen after the staining with propidium iodide (300).
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spots were observed, thus con®rming the depolymerisation of microtubules. IEM Ð at IEM level, gold particles localised in the GC cytoplasm (Plate 4b). After 20 h of germination in BK medium containing Tre¯an, pollen tubes were shorter when compared with the control. IF Ð the GC maintained the shape already observed in the pollen germinated for 1 h in BK medium containing Tre¯an. No microtubules in the pollen tube were observed whereas a diuse ¯uorescence staining within the GC con®rmed the complete depolymerisation of microtubules (Plate 4c). Propidium iodide staining showed the nuclear material as widely diused in the entire volume of the cell (Plate 4d). 3.3.4. Ronstar In comparison with the equivalent control, pollen tubes grown for 1 h in BK medium containing Ronstar were shorter. The GC did not substantially increase in volume and maintained the spindle-like shape. IF Ð short microtubules showed a random distribution within the GC; some ¯uorescent spots were also detected (Plate 5a). IEM Ð clusters of gold particles were observed within the cytoplasmic lobes (Plate 5b).
After 20 h of incubation in BK medium containing Ronstar, pollen tubes were shorter in comparison with the control. The GC increased in volume while preserving the spindle-like shape. IF Ð within the GC, microtubules were not detected and ¯uorescence spots of varying size were observed in the peripheral region of the GC cytoplasm (Plate 5c). As shown by propidium iodide staining, the nuclear material was not distributed in the entire GC volume (Plate 5d). 3.3.5. Roundup 400 In comparison with the control, pollen tubes germinated for 1 h in BK medium containing Roundup 400 were shorter. The GC increased in volume and preserved the spindle-like shape. IF Ð within the GC, a diuse ¯uorescent staining mainly localised in the peripheral region of the cytoplasm. No microtubules were observed (Plate 6a). IEM Ð At IEM level gold particles were detected within the GC cytoplasm (Plate 6b). In comparison with the control, after 20 h of incubation in BK medium containing Roundup 400, pollen tubes were shorter. The GC increased in volume and changed their characteristic spindle-like shape to an
Plate 4. (a) 1 h of treatment with Tre¯an. The GC has rounded up and only some brighter spots were observed after labeling for tubulin (470). (b) At IEM level, gold particles are localised in the GC cytoplasm. C, cytoplasm; N, nucleus; W, GC wall (60,000). (c) A diuse ¯uorescence was present in the GC of pollen tube grown for 20 h in the presence of Tre¯an con®rming a complete depolymerisation of MTs (470). (d) Propidium iodide staining showed the nuclear material to be widely diused in the entire volume of the cell (470).
Plate 5. (a) After 1 h of Ronstar treatment short microtubules showed a random distribution (470). (b) Clusters of gold particles were observed within the cytoplasmic lobes. C, cytoplasm; N, nucleus; W, GC wall (80,000). (c) Twenty h after treatment only ¯uorescent spots were detected in the peripheral region of the GC cytoplasm (470). (d) As shown by propidium iodide staining, the nuclear material was not distributed in the entire GC volume (470).
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Plate 6. (a) After 1 h of Rondup 400 treatment in the peripheral region of the GC cell a diuse ¯uorescence was present (470). (b) At IEM level gold particles were detected within the GC cytoplasm. C, cytoplasm; N, nucleus; W, GC wall (75,000). (c) After 20 h of treatment within the GC, no microtubules were found and a diuse ¯uorescent staining was detected (470). (d) As shown by propidium iodide, the nuclear material was distributed in the entire cell volume (470).
elongated cylinder-like shape. IF Ð Within the GC, no microtubules were found and a diuse ¯uorescent staining was detected. In particular, an intense staining was observed in the central region of the GC (Plate 6c). As shown by propidium iodide staining, the nuclear material was distributed in the entire cell volume (Plate 6d). 4. Discussion In previous experiments (unpublished results), pollen grains, which were directly cultured in herbicide-containing medium, did not germinate or germinated at very low percentage. Here we cultured the pollen grains for 3 h before adding the herbicides; after 3 h of culturing, as shown in controls, pollen tubes elongated for hundreds of micrometers and the microtubular cytoskeleton of both the vegetative and generative cell was fully organised (for review see Geitmann and Emons, 2000). The examined herbicides exert their toxicological eects on dierent compartments and structures of the cell, depending on their chemical nature (as described by suppliers information). Our results clearly show that many of the tested herbicides aect the pollen tube growth. Therefore it is possible that the pollen tube microtubular apparatus does not represent the main target for the toxicological action of herbicides. However, the chemical action of herbicides on microtubules is consistent and reproducible and it is clearly revealed at both IF and IEM level. Within the tube, the vegetative cell microtubules are already depolymerised after 1 h of herbicide treatment, thus con®rming a lower resistance of this population of microtubules to chemical stress conditions (Pierson and Cresti, 1992). In the absence of microtubules, the pollen tubes continue to grow, thus con®rming that microtubules do not have a key role in the pollen tube growth process (AÊstrom et al., 1995; Cai et al., 1997). However, the pollen tube length is reduced when compared with the controls.
The generative cell and its microtubular cytoskeleton show dierent behaviour after treatment with each herbicide. Depending on their eects on the generative cell microtubules, we can divide the examined herbicides into three dierent groups. One group consisted of Illoxan, Tre¯an and Roundup 400, a second includes Ronstar, and the third consisted of Banvel 21S. Although there exist some minor dierences among the individual components, all herbicides of the ®rst group aect the GC and its microtubular cytoskeleton in a consistent manner. After 1 h of culturing in the presence of herbicides, the GC changes its shape and the depolymerisation of microtubules is prominent. We can conclude that the chemical activity of these herbicides occurs within a short time and is dramatic. The increase in volume and the presence of nuclear material dispersed in the entire cell volume after 20 h of culturing in the presence of herbicides suggests a physiological alteration of cytoplasmic components and damages of the nuclear envelope. In particular, this is con®rmed by Illoxan treatment: as shown by gold particles, tubulin molecules result in the nuclear zone probably mixed to nuclear materials. The chemical eect of Ronstar is not immediate and after 1 h of culturing in the presence of the herbicide, the GC preserves the spindle-like shape and the microtubular cytoskeleton is only partially aected. Microtubules are still present and, as shown by the gold particles speci®cally clustered into the cytoplasmic lobes, they maintain their characteristic organisation in bundles within the cytoplasm. After 20 h of culturing in the presence of Ronstar, the GC is signi®cantly aected. The cell increases in volume and microtubules are not evident within the GC. The ¯uorescence is not diuse; intense ¯uorescent spots speci®cally localised in the peripheral region of the GC cytoplasm suggest the presence of depolymerised tubulin. The spreading of nuclear material into the GC but not in the entire cell volume suggests that the structural integrity of cellular compartments is preserved.
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Banvel 21S hardly aects the pollen tubes. However, the pollen tube length is more similar to the control than that of pollen tubes grown in the presence of other herbicides. After 1 h of herbicide treatment, the GC essentially maintains the spindle-like shape although a slight increase of the cell volume is observed. The GC microtubular apparatus is partially aected and a basketlike structure of microtubules is still present. The occurrence of ¯uorescent spots suggests that a process of microtubule depolymerisation takes place. After 20 h of herbicide treatment, the GC does not seem particularly aected. The GC has an elongated shape resembling that of dividing GCs (see control, Plate 1e). Microtubules are not present, whereas ¯uorescent spots suggest the presence of depolymerised tubulin mainly in the peripheral region of the GC cytoplasm. The herbicide does not induce the diusion of nuclear material in the entire volume of the cell, thus con®rming the integrity of the nuclear envelope and a less disruptive action of Banvel 21S in comparison with the other herbicides. In terms of structural and biochemical properties of the pollen tube microtubules, these data con®rm the presence of dierent patterns of microtubules within the pollen tube and the GC. It has been already shown that dierent tubulin isoforms are present in both the microtubular apparatus of pollen tubes and of GCs and that they are localised dierently (AÊstrom, 1992; Del Casino et al., 1993). It is likely that the dierent resistance of microtubules to the stress conditions induced by herbicides and the characteristic staining patterns could be dependent on speci®c biochemical properties related to dierent tubulin isoforms. Herbicide resistance is also generally related to dierence in the amino acid composition of tubulins, since spontaneous mutation in tubulin genes can confer resistance to dinitroaniline herbicides (Anthony et al., 1998). From a general point of view, it is evident that many of the examined herbicides aected also the GC wall, although the particular embedding resin did not allow detailed investigations at ultrastructural level to be carried out. In some cases, the nuclear membrane seems to be aected, thus con®rming a general chemical action of the herbicides.
(Kristen and Kappler, 1995), have also been introduced (for a review see Kristen, 1997) in addition to speci®c assays on animal cells cultures. The pollen tube proved to be a highly sensitive indicator for the toxicity assessment of herbicides and synthetic compounds because the germination and elongation of pollen tubes is aected by treatment with these molecules. Data shown in this work con®rm that the pollen tube is a good model for toxicological tests and, in addition, introduce the study of its microtubular apparatus as an additional parameter for a deeper evaluation of the toxicological activity of herbicides. Further, results reported here also represent matters for general consideration. In terms of agriculture and environment, the evidence of consistent biological damage induced by herbicides on in vitro growing pollen tubes raises questions concerning their role in the higher plant fertilisation process. In fact, it could be possible that they reduce the level of pollen fertility and consequently the number of fertilised eggs in non-target plants. In addition, since some plants could be more naturally resistant to herbicides than others, they could contribute to plant selection in the area aected by the herbicide treatment. In terms of human health, in recent times herbicides, in addition to their use in agriculture, have been applied to plants growing within towns and cities and as a consequence the number of people interacting with such chemicals has greatly increased. It is evident that the use of herbicides must be carefully evaluated and planned by administrators, and scienti®c research should supply tools for a wider and complete evaluation and planning. With this in view, the results reported here can represent a further contribution. Acknowledgements The authors wish to thank Professor Li Yi-Qin of the Department of Biological Sciences and Biotechnology of Tsinghua University, Beijing, China, for helpful suggestions during the preparation of the manuscript.
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