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Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum Blackwell Publishing Ltd

George Komis1, Basil Galatis1, Hartmut Quader2, Dia Galanopoulou3 and Panagiotis Apostolakos1 1Department

of Botany, Faculty of Biology, University of Athens, Athens 15784, Greece; 2Biocenter Klein Flottbek, University of Hamburg, D-22609

Hamburg, Germany; 3Laboratory of Biochemistry, Faculty of Chemistry, University of Athens, Athens, 157 71, Greece

Summary Author for correspondence: Basil Galatis Tel: +003 210 7274646 Fax: +003 210 7274702 Email: [email protected] Received: 17 September 2007 Accepted: 2 December 2007

• The role of phosphoinositide-specific phospholipase C (PI-PLC) signaling in the macrotubule-dependent protoplast volume regulation in plasmolyzed root cells of Triticum turgidum was investigated. • At the onset of hyperosmotic stress, PI-PLC activation was documented. Inhibition of PI-PLC activity by U73122 blocked tubulin macrotubule formation in plasmolyzed cells and their protoplast volume regulatory mechanism. In neomycin-treated plasmolyzed cells, macrotubule formation and protoplast volume regulation were not affected. In these cells the PI-PLC pathway is down-regulated as neomycin sequesters the PI-PLC substrate, 4,5-diphosphate-phosphatidyl inositol (PtdInsP2). These phenomena were unaffected by R59022, an inhibitor of phosphatidic acic (PA) production via the PLC pathway. • Taxol, a microtubule (MT) stabilizer, inhibited the hyperosmotic activation of PI-PLC, but oryzalin, which disorganized MTs, triggered PI-PLC activity. Taxol prevented macrotubule formation and inhibited the mechanism regulating the volume of the plasmolyzed protoplast. Neomycin partly relieved some of the taxol effects. • These data suggest that PtdInspP2 turnover via PI-PLC assists macrotubule formation and activation of the mechanism regulating the plasmolyzed protoplast volume; and the massive disorganization of MTs that is carried out at the onset of hyperosmotic treatment triggers the activation of this mechanism. Key words: phospholipase C, protoplast volume regulation, Triticum turgidum, tubulin macrotubules. New Phytologist (2008) 178: 267–282 © The Authors (2008). Journal compilation © New Phytologist (2008) doi: 10.1111/j.1469-8137.2007.02363.x

Introduction Whole plants express protoplast volume regulatory mechanisms, measurable in plasmolyzed cells. Early events of plasmolyzed protoplast volume homeostasis include global rearrangements of either the actin filament (AF) or microtubule (MT) cytoskeleton, somehow controlling the protoplast hydraulic conductivity (Komis et al., 2002a,b, 2003). In plasmolyzed root cells of Triticum turgidum, protoplast volume regulation is mediated by tubulin macrotubules promptly

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formed de novo, downstream of the hyperosmotically induced MAP kinase activation (Komis et al., 2002b, 2004; Lu et al., 2007). Abiotic stress including hyperosmolarity elicits diverse signaling cascades, including the generation and recycling of diverse bioactive phospholipid species and the crucial involvement of phospholipase C and D (PLC and PLD) species in the above phenomena (Cote et al., 1996; Pical et al., 1999; Takahashi et al., 2001; Meijer & Munnik, 2003; Zonia & Munnik, 2004; Wang, 2006).

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Phosphoinositide-specific PLC (PI-PLC) cleaves 4,5-diphosphate-phosphatidyl inositol (PtdInsP2) to diacylglycerol (DAG) and 1,4,5-inositol triphosphate (InsP3; DeWald et al., 2001; Rhee, 2001). Diacylglycerol becomes rapidly phosphorylated by DAG kinase (DAGK) to phosphatidic acic (PA), thus propagating PA signaling commenced by the catalytic activity of PLD (Testerink & Munnik, 2005). 1,4,5-Inositol triphosphate is released to the cytoplasm, mediating calcium release from intracellular stores (Meijer & Munnik, 2003; Wang, 2004; Monteiro et al., 2005). 4,5-Diphosphate-phosphatidyl inositol itself interacts with specific domains in lipid-binding proteins controlling their subcellular localization and/or activation. For this purpose, pathways leading to PtdInsP2 synthesis are closely coupled to its breakdown (Pical et al., 1999; DeWald et al., 2001). Phosphatidic acic produced by PLD or PLC/DAGK acts either directly as a secondary messenger or becomes phosphorylated to the plant-specific lipid, diacylglycerol pyrophosphate, by PA kinase (van Schooten et al., 2006). Hyperosmotic stress triggers these pathways in plant cells (Testerink & Munnik, 2005; Wang, 2006). In T. turgidum root cells, the hyperosmotic induction of PA synthesis by PLD is essential for the activation of a hyperosmotically induced MAPK cascade, the concomitant tubulin cytoskeleton remodeling and finally the expression of plasmolyzed protoplast volume regulation (Komis et al., 2006). Here, the probable role was investigated of PI-PLC-related signaling pathways in the hyperosmotic response and the expression of protoplast volume regulation in root cells of T. turgidum, studying the effects of neomycin, U73122, U73343, R59022, 1,2-dioctanoyl glycerol (DOG), phorbol 12-myristate 13-acetate (PMA), oryzalin and taxol on plasmolyzed root cells. Neomycin forms complexes with PtdInsP2, inhibiting the interactions of the latter with lipidbinding proteins as well as the cleavage by PI-PLC (Gabev et al., 1989). U73122 inhibits the catalytic activity of PIPLC (Bleasdale et al., 1989), leading to sustained PtdInsP2 concentrations (Saul et al., 2004), while U73343 is a minimally active analogue of U73122 (Bleasdale et al., 1989). R59022 inhibits DAG kinase activity (de Chaffoy de Courcelles, 1990) and the accumulation of PA through the PLC/DAGK pathway (Testerink & Munnik, 2005). Phorbol 12-myristate 13-acetate is a cell-permeable DAG analogue that stimulates DAG-related processes (Baudouin et al., 2002; Komis et al., 2004), while DOG is a readily permeable DAG (Larsen & Wolniak, 1990). Taxol stabilizes MTs and stimulates their de novo formation at subcritical tubulin concentrations (Panteris et al., 1995), while oryzalin disintegrates tubulin polymers (Komis et al., 2002b). The PI-PLC inhibitors described are routinely used to investigate the role of phosphoinositide turnover in cellular responses against hyperosmotic stress (Einspahr et al., 1988; Pical et al., 1999; Takahashi et al., 2001; Zonia & Munnik, 2004).

New Phytologist (2008) 178: 267–282

Materials and Methods Plant material and treatments Wheat (Triticum turgidum L. cv. Athos) seedlings, 36 – 48 h old, were used throughout. Seedlings were immersed in hyperosmotic solutions in the presence or absence of neomycin, U73122, U73343, R59022, taxol, oryzalin, PMA, DOG or combinations. Hyperosmotic exposure was carried out in 1 m aqueous mannitol solutions for up to 60 min. For inhibitor studies, roots where first incubated in the appropriate inhibitor aqueous solution for 2 h and subsequently in 1 m mannitol solutions containing neomycin, U73122, U73343, R59022, taxol, oryzalin, PMA, DOG, or combinations at the designated concentrations for up to 120 min. Neomycin sulfate (Sigma, St Louis, MA, USA) was diluted in water or 1 m mannitol from a 10 mm aqueous stock solution to working concentrations of 10, 20, 50, and 100 µm. U73122 and U73343 (Calbiochem, Darmstadt, Germany) were dissolved in chloroform and aliquoted in appropriate quantities. Chloroform was evaporated under a stream of argon and the residue was dissolved in DMSO to yield stock solutions of 10 mm which were diluted in water or aqueous 1 m mannitol to yield working concentrations of 10, 20, 50 and 100 µm. For most experiments conducted here, U73122 and U73343 were applied at 50 µm. Taxol (10 mm; Sigma) or R59022 (10 mm; Calbiochem) DMSO stocks were diluted in water or aqueous 1 m mannitol to 20 and 50 µm, respectively. PMA and DOG (Sigma) were used at 10 µm diluted from 10 mm stock solutions in DMSO. Oryzalin was prepared as a 10 mm stock solution in anhydrous acetone, which was appropriately diluted in water or aqueous 1 m mannitol to 20 µm. Living cell examination Living plasmolyzed rhizodermal cells were monitored by differential interference contrast (DIC) optics and photographed through a Zeiss Axiocam MRc5 (Zeiss, Oberkochen, Germany). The mean volume of plasmolyzed convex protoplasts was estimated according to Höffler’s equation, assuming a cylindrical protoplast shape, and statistical analysis was conducted as previously described (Komis et al., 2002b). Protoplast volume estimation in the affected plasmolyzed root tips took into account viable cells using a series of criteria applicable to DIC optics, including the ability of protoplasts to re-expand following deplasmolysis. For viability testing, roots treated as described were briefly stained with a mixture of propidium iodide (PI; 10 µg ml−1)/ fluorescein diacetate (FDA; 10 µg ml−1) (both from Sigma) to discriminate living and dead cells with confocal laser scanning microscope (CLSM) imaging, as previously described (Komis et al., 2006).

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Tubulin immunolabeling and morphometric assessment of the tubulin polymer content Roots were processed for tubulin immunolabeling as previously described (Komis et al., 2002b, 2006). Fluorescently labeled specimens were visualized through a Zeiss Axiocam MRc5 coupled to a Zeiss Axioplan microscope or by CLSM (TCS 4D, Leica Microsystems, Bensheim, Germany). To assess differences in the relative tubulin polymer content, digital images of fluorescently labeled cells from different treatments were processed as described in Komis et al. (2002b), using an algorithm designed for Image Color Gauge version 0.1, by N. Apostolakos (Isaac Newton Group of Telescopes, La Palma, Spain). Electron microscopy and morphometric analysis of tubulin polymers Root tips were processed for transmission electron microscopy (TEM) as described previously (Komis et al., 2002b, 2006). Measurement of tubulin polymer outer diameter (OD) was carried out using the CorelDraw Graphics Suite X3 (Corel Suite, Pantone) dimension tool on TEM micrographs calibrated with a grated replica grid as before (Komis et al., 2001, 2002b). Determination of PI-PLC activity Assessment of PI-PLC activity was carried out in roots following the previously mentioned treatments according to standard protocols (Melin et al., 1987; Pical et al., 1992; Cho et al., 1995). In brief, roots were homogenized in 50 mm HEPES, pH 7.3, 250 mm sucrose, 10 mm DTT, 0.01% Triton X-100 and protease inhibitors cocktail (Sigma). The extract was clarified at 5600 g at 4°C for 10 min and protein content was determined by Lowry assay. Ten micrograms of total protein were mixed with 25 µl of 50 mm Tris-HCl, pH 6.5, and 5 µl of a Ca2+/EDTA solution to yield 10 µm of free Ca2+ (Homma & Emori, 1997). The reaction was initiated by adding 10 µl of substrate (3H-PtdInsP2, 12 000 cpm and 3 nmol of cold PtdInsP2 purified from a phosphoinositide mixture (Sigma) dispersed in 0.01% sodium deoxycholate) and carried out for 15 min at 37°C. The reaction was quenched by the addition of 1 ml of ice-cold chloroform : methanol (2 : 1) and 250 µl of ice-cold 1 N HCl. Phases were separated at 2000 g (2 min; room temperature) and 300 µl of the upper-acid/methanolic phase containing InsP3 produced by the enzymatic activity were drawn and mixed with 10 ml of dioxan-based scintillation cocktail. Scintillation counting was carried out in a Wallac 1209 Racbetta counter (Pharmacia, Surrey, UK). Cpm measurements were converted to nmol mgprotein–1 min–1 and finally results were plotted as percentages of control.

Results Hyperosmotic stress induces PI-PLC activation in T. turgidum roots The early hyperosmotic activation of PI-PLCs was confirmed by directly assaying PI-PLC activity in hyperosmotically treated T. turgidum root extracts. PI-PLC is rapidly activated, as treatment with 1 m mannitol triggers PI-PLC activation to above basal values at 1 min postexposure, peaking at 3 min (peak activity of 0.29 ± 0.08 nmol mgprotein–1 min–1, n = 3, Fig. 1a). At this time, PI-PLC activity increased by approx. 150% with respect to the basal values. Subsequently, PI-PLC activity declines over a 10 min period following immersion to mannitol (Fig. 1a). In our case, it is interesting that the PI-PLC activation time course coincides with the time course of the expression of the protoplast volume regulatory mechanism, which is completed within the first 5 min after exposure (Komis et al., 2002b, 2006). Figure 1b depicts the effects of U73122 and neomycin on the peak hyperosmotically induced PI-PLC activity. Thus, in roots pretreated with 50 µm U73122 or 100 µm neomycin for 2 h and exposed for 3 min to 1 m mannitol in the presence of either 50 µm U73122 or 100 µm neomycin, the hyperosmotically induced PI-PLC activity is reduced by approx. 40 and 30%, respectively (Fig. 1b). Notably, neither U73122 nor neomycin affects the basal PI-PLC activity, which is detected at isotonic conditions (Fig. 1b). These results justify the use of U73122 and neomycin against the hyperosmotically induced PI-PLC activation, showing furthermore that the inhibitory effect is restricted to hypertonic conditions. Microtubule dynamics modulate PI-PLC activity in T. turgidum roots At the onset of hyperosmotic treatment, MTs of T. turgidum root cells are massively depolymerized and eventually macrotubules are formed to near completion at 5 min postexposure (Komis et al., 2002b, 2004, 2006). PI-PLC activity follows the opposite time course (Fig. 1a), peaking when MT depolymerization is complete and declining as macrotubule formation progresses. In order to test the role of tubulin polymer dynamics per se at the onset of PI-PLC activity, two specific MT poisons were used: oryzalin, which induces MT depolymerization, and taxol, which stabilizes MTs. Oryzalin at 20 µm induces the activation of PI-PLC in the absence of hyperosmotic conditions (Fig. 1c). Additionally, oryzalin has no effect in the hyperosmotic activation of PI-PLC (Fig. 1c). It is noteworthy that the oryzalin-induced PI-PLC activity levels approximate peak values observed with the hyperosmotic treatment (Fig. 1c). By contrast, taxol has no effect on PI-PLC activity under isotonic conditions, but suppresses the hyperosmotic activation of PI-PLC activity to nearly basal values (Fig. 1c).

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Fig. 1 (a–c) Graphical representation of phosphoinositide-specific phospholipase C (PI-PLC) enzymatic analysis. All bars represent mean % of control ± SE of two to three independent experiments per case. Every experiment was measured in duplicate. (a) Time course of PI-PLC activation following exposure of roots of Triticum turgidum to 1 M mannitol (Man., n = 3). (b) Effects of PI-PLC inhibitors on the isotonic or hyperosmotic PI-PLC activity. Treatments: (1) 1 M mannitol, 3 min (n = 3); (2) 50 µM U73122, 2 h (n = 2); (3) 50 µM U73122, 2 h then 1 M mannitol plus 50 µM U73122, 3 min (n = 2); (4) 100 µM neomycin, 2 h (n = 2); (5) 100 µM neomycin, 2 h then 1 M mannitol plus 100 µM neomycin, 3 min (n = 2). ∗, P < 0.05 compared with bar (1). (c) Effects of oryzalin and taxol on the isotonic or hyperosmotic PI-PLC activity. Treatments: (1) 1 M mannitol, 3 min (n = 3); (2) 20 µM oryzalin, 10 min (n = 3); (3) 20 µM oryzalin, 2 h, then 1 M mannitol plus 20 µM oryzalin, 3 min (n = 2); (4) 20 µM taxol, 10 min (n = 2); (5) 20 µM taxol, 2 h, then 1 M mannitol plus 20 µM taxol, 3 min (n = 2). *, P < 0.05 (2 vs control); †, P < 0.05 (5 vs 1).

Neomycin and U73122 have opposite effects on the size of the plasmolyzed protoplast Examination of living rhizodermal cells confirmed that plasmolysis in 1 m mannitol concluded within 5 min postexposure, while in most cells the protoplast assumes a convex form (Fig. 2a). The mean protoplast volume of such cells was estimated at 31 650 ± 937 µm3 (Fig. 3a; see also Komis et al., 2002b). PI/FDA staining revealed that root cells remain viable in the hyperosmotic conditions over long periods (Supplementary material, Fig. S1a).


Monitoring of living rhizodermal cells pretreated with 100 µm neomycin for 2 h and plasmolyzed along with 100 µm neomycin showed that protoplasts initially shrink over a period of 5 –10 min, and subsequently swell, reaching equilibrium (Fig. 2b). In the vast majority of these cells, the plasmolyzed protoplast is convex (Fig. 2b) The plasmolyzed protoplast volume of neomycin-treated rhizodermal cells equilibrates at approx. 30 min postexposure to the osmoticum, showing a dose response against neomycin from 10 to 100 µm (Fig. 3b). At 100 µm of neomycin, the mean plasmolyzed protoplast volume was estimated at 37 750 ± 1113 µm3,


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Fig. 2 (a–f) Representative differential interference contrast (DIC, a–c, e, f) or transmission electron microscopy (TEM) (d) images of Triticum turgidum cells plasmolyzed in the absence (a) or presence (b–f) of phosphoinositide-specific phospholipase C (PI-PLC) modulators. Asterisks denote the plasmolyzed protoplast. Bars, 10 µm (a–c, e, f); 5 µm (d). (a1, a2) Time-lapse imaging of a living rhizodermal cell plasmolyzing in 1 M mannitol. Instants at 5 (a1) and 30 (a2) min show that the protoplast volume remains constant through time. (b1, b2) Time-lapse imaging of a living rhizodermal cell pretreated with 100 µM neomycin for 2 h and plasmolyzed in 1 M mannitol supplemented with 100 µM neomycin. Instants at 5 (b1) and 60 (b2) min show the initial protoplast shrinkage and its gradual recuperation over time. (c) Living rhizodermal cell pretreated with 50 µM U73122 for 2 h and plasmolyzed in 1 M mannitol plus 50 µM U73122 for 30 min. Comparison of the protoplast size against the overall cell size (crosswall-to-crosswall distance) shows that U73122 elicits a steep decrease of the plasmolyzed protoplast volume. (d) Low-magnification TEM overview of root cells pretreated with 50 µM U73122 for 2 h and plasmolyzed for 30 min in 1 M mannitol supplemented with 50 µM U73122. Note the intense protoplast volume shrinkage and opacity, indicating mass water outflow. (e) Living rhizodermal cell pretreated with 50 µM U73343 for 2 h and plasmolyzed in 1 M mannitol plus 50 µM U73343 for 30 min. The presence of U73343 in the plasmolyticum does not cause the intense shrinkage observed in the case of U73122 (cf. Fig. 2a2,c). (f) Living rhizodermal cell pretreated with 50 µM R59022 for 2 h and plasmolyzed for 30 min in 1 M mannitol plus 50 µM R59022 (cf. Fig. 2a2).

thus 19% higher than that of cells plasmolyzed in plain osmoticum (Fig. 3a). In all concentrations tested, neomycin was nontoxic under isotonic conditions (Fig. S1b) but became more toxic under hypertonic conditions, in a dose-dependent manner (Fig. S1c,d; cf. Fig. S1a). U73122 is not cytotoxic at concentrations of 10 and 50 µm. More than 80% of the cells remain alive even in prolonged hyperosmotic treatment (Table 1; see also Fig. S1e). Besides, cells plasmolyzed for 3, 5, 10, 20 and 30 min in 1 m mannitol plus 50 µm U73122 retain their capacity to deplasmolyze. However, in prolonged times of deplasmolysis, a large number of these cells become necrotized. Moreover,

the U73122-treated nonplasmolyzed cells remain alive for a long time (Fig. S1f ). Root cells exposed to 50 µm U73122 for 2 h before and up to 30 min during the hyperosmotic treatment exhibited a markedly different response from that of plasmolyzed cells exposed to neomycin. As visualized by DIC optics or TEM, the protoplast of U73122-treated plasmolyzed rhizodermal (Fig. 2c) or parenchymal (Fig. 2d) cells become markedly shrunk. Time-lapse monitoring of living rhizodermal U73122-treated cells subjected to plasmolysis showed that protoplast shrinkage is completed within 5 min postexposure, as in the case of neat mannitol. In these cells, the protoplast



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Fig. 3 (a) Histograms depicting the effects of phosphoinositide-specific phospholipase C (PI-PLC) modulators on the mean protoplast volume of plasmolyzed rhizodermal cells of Triticum turgidum. Treatments: (1) 1 M mannitol, 30 min; (2) 100 µM neomycin, 2 h, and 1 M mannitol plus 100 µM neomycin, 30 min; (3) 50 µM U73122, 2 h, and 1 M mannitol plus 50 µM U73122, 30 min; (4) 50 µM U73343, 2 h, and 1 M mannitol plus 50 µM U73343, 30 min; (5) 50 µM R59022, 2 h, and 1 M mannitol plus 50 µM R59022, 30 min. *, P < 0.05; **, P < 0.001, compared with bar (1). (b) The dose response of the plasmolyzed rhizodermal protoplast volume against increasing neomycin concentrations. Treatments: (1) 1 M mannitol, 30 min; (2) 10 µM neomycin, 2 h, and 1 M mannitol plus 10 µM neomycin, 30 min; (3) 20 µM neomycin, 2 h, and 1 M mannitol plus 20 µM neomycin, 30 min; (4) 50 µM neomycin, 2 h, and 1 M mannitol plus 50 µM neomycin, 30 min; (5) 100 µM neomycin, 2 h, and 1 M mannitol plus 100 µM neomycin, 30 min. *, P < 0.05, compared with bar (1).

Table 1 Percentage of living cells in root tips of Triticum turgidum that were treated for different time periods with 1 M mannitol supplemented with U73122 at different concentrations Treatments

Living cells

Dead cells

% living cells

10 µM U73122, 2 h + 1 M mannitol plus 10 µM U73122, 30 min 10 µM U73122, 2 h + 1 M mannitol plus 10 µM U73122, 2 h 50 µM U73122, 2 h + 1 M mannitol plus 50 µM U73122, 30 min 50 µM U73122, 2 h + 1 M mannitol plus 50 µM U73122, 2 h

846 637 706 481

54 79 143 107

94% 88.96% 83.15% 81.8%

Results of every experiment correspond to confocal laser scanning microscope (CLSM) study of 10 different root tips stained with PI/FDA.

size was profoundly smaller compared with cells plasmolyzed in plain or neomycin-supplemented osmoticum, being 17 730 ± 1175 µm3, thus 41% smaller than untreated plasmolyzed cells (Fig. 3a), similar to oryzalin-treated plasmolyzed cells, which depletes tubulin polymers (Komis et al., 2002b).


Study of the course and magnitude of plasmolysis, and the viability of root cells exposed to a wide range of U73343 concentrations, revealed no defects (Fig. 2e, cf. Fig. 2a; Fig. S1g, cf. Fig. S1a), suggesting that the effects of U73122 should be rather specific and not unspecifically cytotoxic. At


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Fig. 4 Histograms showing the effects of taxol alone or along with phosphoinositide-specific phospholipase C (PI-PLC) modulators on the protoplast volume of plasmolyzed rhizodermal cells of Triticum turgidum. Treatments: (1) 1 M mannitol, 30 min; (2) 20 µM taxol, 2 h, and 1 M mannitol plus 20 µM taxol, 30 min; (3) 20 µM taxol and 100 µM neomycin, 2 h, and 1 M mannitol plus 20 µM taxol and 100 µM neomycin, 30 min; (4) 50 µM U73122, 2 h, and 1 M mannitol plus 50 µM U73122, 30 min; (5) 20 µM taxol and 50 µM U73122, 2 h, and 1 M mannitol plus 20 µM taxol and 50 µM U73122, 30 min; (6) 20 µM taxol, 2 h, and 1 M mannitol plus 20 µM taxol and 50 µM U73122, 30 min. *, P < 0.05 (3 vs 2); **, P < 0.001 (6 vs 5); †, P < 0.05 (2, 4, 5 vs 1).

50 µm of U73343, the mean plasmolyzed protoplast volume was estimated at 30 716 ± 856 µm3, similar to the mean protoplast volume of rhizodermal cells plasmolyzed in mannitol alone (Fig. 3a). DAGK inhibition does not interfere with plasmolyzed protoplast volume regulation Root cells treated with 50 µm R59022 for 2 h before and up to 30 min during the hyperosmotic treatment did not display quantifiable changes in the time course and the extent of plasmolysis (Fig. 2f; cf. Fig. 2a). The average volume of plasmolyzed, convex, rhizodermal protoplasts was estimated at 30 850 ± 1100 µm3, essentially similar to that of cells plasmolyzed in mannitol alone (31 650 ± 937 µm3; Fig. 3a). Likewise, their viability was unaffected by R59022 in either isotonic or hypertonic conditions (Fig. S1h). The effects of DAG depletion as a function of PtdInsP2 turnover blocking was probed by plasmolyzing cells in 1 m mannitol, which was supplemented with either 10 µm DOG or PMA. In either case, plasmolysis proceeded with no observable differences compared with untreated plasmolyzed cells (Fig. S2a1,a2,b1,b2, cf. Fig. 2a; Fig. S2a3,b3, cf. Fig. S1a), suggesting that DAG should not have an important role in the plasmolyzed protoplast volume regulation or the osmotic tolerance of root cells. Taxol has adverse effects on protoplast volume regulation during plasmolysis and counterbalances the effects of PI-PLC modulators Taxol disturbs protoplast volume regulation in plasmolyzed cells As the biochemical analysis of PI-PLC activity showed that taxol inhibits the hyperosmotically induced activation of PI-PLCs (Fig. 1c), it was further investigated whether MT

prestabilization by taxol could affect the course of plasmolyzed protoplast volume regulation and the effect of U73122 or neomycin in this process. In roots pretreated for 2 h with, and plasmolyzed for 30 min in the presence of, 20 µm taxol, the mean plasmolyzed protoplast volume was estimated at 17 725 ± 525 µm3, diminished by 37% against cells plasmolyzed in mannitol alone (Fig. 4), almost to the size of cells plasmolyzed in the presence of oryzalin (Komis et al., 2002b). The cells plasmolyzed in the presence of taxol display prolonged plasmolysis and constitutive reduction of the protoplast volume (Fig. 5a, cf. Fig. 2a). These findings favor the hypothesis that the early MT disorganization (Komis et al., 2002b) is essential for activating the macrotubule-dependent protoplast volume regulatory mechanism. Neomycin opposes the effects of taxol in plasmolyzed cells In cells pretreated with 20 µm taxol for 2 h and plasmolyzed in taxol-supplemented mannitol for up to 30 min, the further addition of 100 µm neomycin, resulted in the gradual, slow but definite increase of the plasmolyzed protoplast volume (Fig. 5b1–b6; cf. Fig. 5a1–a6). The mean final plasmolyzed protoplast volume in these cells was 19 975 ± 527 µm3, thus by 11% higher than the mean protoplast volume of cells pretreated with and plasmolyzed in the presence of 20 µm taxol alone (Fig. 4). These observations suggest that neomycin partially reversed the effects of taxol. Taxol partially relieves the effects of U73122 on plasmolyzed cells Addition of 20 µm taxol together with 50 µm U73122 for 2 h before and 30 min after the onset of plasmolysis did not rescue the hyperosmotic behavior of cells. The mean protoplast volume of rhizodermal cells with convex protoplasts was 18 140 ± 1308 µm3, similar to that of cells plasmolyzed in the presence of U73122 only (Fig. 4). When,



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Fig. 5 Time-lapse differential interference contrast (DIC) demonstration of the effects of taxol (a1–a6) and their reversal by neomycin (b1–b6) on Triticum turgidum protoplast volume regulation under hyperosmotic conditions. Bar, 10 µm, (a1–b6). (a1–a6) Continuous protoplast retraction, as visualized at defined intervals, over a 30 min period, of a living rhizodermal cell pretreated for 2 h with 20 µM taxol and plasmolyzed in 1 M mannitol plus 20 µM taxol. The protoplast volume is not stabilized over time and equilibrates at suboptimal amounts compared with neat mannitol treatment (cf. Fig. 2a1, a2). (b1–b6) Plasmolyzing rhizodermal cell in successive instants. The cell was pretreated with 20 µM taxol for 2 h and subsequently with 1 M mannitol plus 20 µM taxol for 5 min (b1–b3). Afterwards, the plasmolytic medium was supplemented with 100 µM neomycin (instants to the right of the black line separating b3 and b4) and the cell was observed for a total of 30 min after the onset of plasmolysis (b4 through b6). The addition of neomycin inhibits the taxol-induced protoplast shrinkage (b4–b6; cf. a4–a6) and furthermore promotes the gradual increase of the protoplast volume (b4–b6; cf. b1–b3).

U73122 was omitted from the preconditioning solution, the presence of taxol in the plasmolyticum (1 m mannitol plus 50 µm U73122) antagonized the effects of U73122, resulting in improved protoplast volume regulation. In this case, the mean plasmolyzed protoplast volume was 25 461 ± 1347 µm3, thus 44% higher than that of plasmolyzed cells exposed to U73122 alone (17 730 ± 1175 µm3; Fig. 4). The hyperosmotic behavior of root cells in the presence of PI-PLC modulators correlates with the tubulin cytoskeleton remodeling Neomycin induces the excessive formation of tubulin polymers Root cells exposed to acute hyperosmolarity bear a wealth of highly organized tubulin polymers (Fig. 6a; Komis et al., 2002b), bearing a larger mean OD compared


with that of MTs. In the present study, OD measurements of MTs showed that it ranged between 19 and 25 nm (mean diameter 20.67 ± 0.13 nm) with only 31 out of 365 (8.5%) MTs displaying 25 nm OD. In root cells plasmolyzed in mannitol, 97% (264 out of 273) of the total tubulin polymers measured had an OD between 26 and 38 nm (mean OD, 30.97 ± 0.15 nm), while the remaining 3% (nine out of 273) were classified as MTs (mean OD, 22.34 ± 0.51 nm). Presumably, MTs are massively substituted by tubulin macrotubules (Table 2) within 5 min postexposure (Komis et al., 2006). Cells pretreated for 2 h with, and plasmolyzed for 30 min in the presence of, 100 µm neomycin, displayed intensely fluorescing tubulin strands, forming cortical arrays similar to those found in untreated plasmolyzed cells (Fig. 6b; cf. Fig. 6a). Digital densitometric analysis showed that the tubulin polymer


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Fig. 6 Effects of phosphoinositide-specific phospholipase C (PI-PLC) modulators on the hyperosmotically induced tubulin polymer reorganization as visualized by confocal laser scanning microscope (CLSM) of tubulin-immunolabeled plasmolyzed root cells of Triticum turgidum. Bar, 5 µm (a–f). (a) Cell treated with 1 M mannitol for 30 min, overpopulated with prominent cortical tubulin strands. (b) Cell pretreated with 100 µM neomycin for 2 h and plasmolyzed in 1 M mannitol plus 100 µM neomycin for 30 min. Tubulin polymers appear entangled and occasionally form thick bundles (arrows). (c, d) Cells pretreated with 50 µM U73122 for 2 h and plasmolyzed in 1 M mannitol plus 50 µM U73122 for 30 min. Tubulin fluorescence appears in dot- (c) or rod-like (d) formations dispersed throughout. N, nucleus. (e) Cell pretreated with 50 µM U73343 for 2 h and plasmolyzed in 1 M mannitol plus 50 µM U73343 for 30 min. Cortical tubulin strands are formed as in neat mannitol treatment (cf. Fig. 6a). (f) Cell pretreated with 50 µM R59022 for 2 h and plasmolyzed in 1 M mannitol plus 50 µM R59022 for 30 min, displaying a wealthy and well-organized network of cortical tubulin strands.

Table 2 Occurrence of tubulin polymers Treatment

Microtubule

Macrotubule

Control Mannitol, 30 min Neomycin, 2 h + neomycin/mannitol, 5 min Neomycin, 2 h + neomycin/mannitol, 30 min U73122, 2 h + U73122/mannitol, 30 min R59022, 2 h + R59022/mannitol, 30 min Taxol, 2 h + taxol/mannitol, 5 min Taxol, 2 h + taxol/mannitol, 30 min Taxol, 2 h + taxol/neomycin/mannitol, 30 min Ttaxol/U73122, 2 h + taxol/U73122/mannitol, 30 min Taxol, 2 h + taxol/U73122/mannitol, 30 min

+++ (100%) + (3%) ++ (85%) + (39%) − + (12%) +++ NA ++ (89%) + (39%) − + (17%)

− ++ (97%) + (15%) ++ (61%) − ++ (88%) − NA + (11%) ++ (61%) − ++ (83%)

+++, prevalence; ++, dominance; +, occurrence; −, absence. NA, not applicable: insufficient polymers for measurements.

content in such cells was increased by 20% compared with cells plasmolyzed in the absence of neomycin (Fig. 7). In root cells treated with 100 µm neomycin for 2 h under isotonic conditions, the cortical tubulin polymer organization was essentially the same as that of cortical MTs of control cells (Fig. S3a,b).

Neomycin-treated plasmolyzed cells bear mixed populations of MTs and macrotubules, with macrotubules gradually prevailing, as assessed by tubulin polymer OD measurements. In cells pretreated with, and plasmolyzed in the presence of, neomycin for 5 min, MTs (mean OD, 22.46 ± 0.11 nm) constitute 85% of the total polymers measured (316 out of



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macrotubules (148 out of 170 measured) with a mean OD of 32.8 ± 0.4 nm, and the remaining 12% (22 out of 170 measured) were MTs, having a mean OD of 24.1 ± 0.3 nm (Table 2). Tubulin cytoskeleton organization in taxol-treated plasmolyzed cells

Fig. 7 Histograms depicting the tubulin polymer content in Triticum turgidum root cells plasmolyzed in mannitol (1) or in mannitol plus neomycin (2). Treatments: (1) 1 M mannitol, 30 min; (2) 100 µM neomycin, 2 h, and 1 M mannitol plus 100 µM neomycin, 30 min. *, P < 0.05, (2 vs 1).

372), while the remaining 15% (56 out of 372) were macrotubules (mean OD, 28.15 ± 0.23 nm). At 30 min exposure to the neomycin-supplemented plasmolyticum, 39% of the total tubulin polymers were MTs (161 out of 411), having a mean OD 23.73 ± 0.13 nm and the remaining 61% (250 out of 411) were macrotubules (mean OD 29.83 ± 0.19 nm; Table 2). It is probable that neomycin preserves a vast number of pre-existing MTs, while allowing the course of macrotubule formation to proceed accordingly. As a consequence, a wealth of tubulin polymers occupying the cortical cytoplasm was observed by TEM (Fig. S4a). U73122 inhibits the formation of tubulin macrotubules Tubulin immunolabeling in U73122-treated cells for 2 h before and 30 min after the onset of plasmolysis showed the complete depletion of tubulin polymers. In these cells, tubulin immunofluorescent spots or rods were randomly dispersed throughout the cytoplasm (Fig. 6c,d). TEM examination confirmed the absence of MTs or macrotubules in these cells (Table 2). The effects of U73122 should be rather specific, as treatment with U73343 had no effect on macrotubule formation in plasmolyzed cells (Fig. 6e, cf. Fig. 6a,c,d). Moreover, U73122 is ineffective against cortical MTs of nonplasmolyzed cells (Fig. S3c, cf. Fig. S3a). R59022 does not affect the formation of tubulin macrotubules Cells treated for 2 h before and plasmolyzed for 30 min along with R59022 possessed cortical tubulin polymer arrays similar to those of untreated plasmolyzed cells (Fig. 6f; cf. Fig. 6a). R59022-treated plasmolyzed interphase cells displayed cortical tubulin polymers that were 88%


Neomycin antagonizes the effects of taxol on hyperosmotically induced tubulin cytoskeleton remodeling In nonplasmolyzed T. turgidum roots treated with 20 µm taxol, the interphase cells display wealthy cortical MT arrays (Fig. S3d) with a higher MT density than in control interphase cells, while dividing cells show abnormal spindles (Panteris et al., 1995). In root cells preincubated for 2 h with 20 µm taxol and plasmolyzed in mannitol plus 20 µm taxol for up to 60 min, the tubulin cytoskeleton responded in a time-dependent manner. Immunofluorescence studies of the above cells revealed the gradual disorganization of the tubulin cytoskeleton. At 1 to 5 min postexposure, the cortical cytoplasm is traversed by tubulin strands thinner than those of cells plasmolyzed in neat mannitol (Fig. 8b, cf. Fig. 8a). These probably represent residual MTs that initially displayed organization patterns similar to those of taxol-treated nonplasmolyzed cells (Fig. 8b, cf. Fig. S3d), but progressively became more diffuse and disorganized. Tubulin polymer disorganization is more prominent at later intervals, peaking at 20 min postexposure (Fig. 8c, cf. Fig. 8b). The formation of cortical tubulin polymers resumes slowly, becoming evident at 30 – 60 min following the onset of plasmolysis, although not reaching the amounts of cells plasmolyzed in neat mannitol (Fig. 8d,e, cf. Fig. 8a). Taxol-treated cells plasmolyzed for up to 5 min bear sparse cortical MTs (Table 2). In such cells, exposed to the plasmolyticum for 30 min, 89% of tubulin polymers (753 out of 846 counted) were identified as MTs (mean OD, 22.06 ± 0.06 nm), while the remaining 11% (93 out of 846 measured polymers) were macrotubules (mean OD, 26.79 ± 0.12 nm; Table 2). These findings suggest that the addition of taxol in the plasmolyticum largely inhibits the MT-to-macrotubule transition. The smaller mean OD of macrotubules in taxoltreated plasmolyzed cells (26.79 ± 0.12 nm), compared with that of macrotubules formed in cells plasmolyzed in mannitol alone (30.97 ± 0.15 nm) can be attributed to the action of taxol itself. Taxol induces the formation of tubulin polymers with a smaller OD and protofilament number, either in vitro (Diaz et al., 1998) or in vivo (Mogensen & Tucker, 1990). In addition, in taxol-treated, nonplasmolyzed root cells of T. turgidum, only 2.5% of the total MTs measured (10 out of 390) had a mean OD of approx. 25 nm, whereas in control cells, such MTs represented 8.5% of the total MT population, which was a statistically significant difference (P < 0.05). The inhibition of macrotubule formation in taxol-treated plasmolyzed cells was best depicted in dividing cells, where fluorescence was localized on mass and amorphous tubulin


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Fig. 8 Effects of taxol on the hyperosmotically induced tubulin polymer organization and their gradual reversal by the coapplication of neomycin as visualized by tubulin immunolabeling. Bar, 10 µm, (a–o). (a) Triticum turgidum root cortex cell subjected to 1 M mannitol treatment for 30 min. Note the abundance and organization of cortical tubulin polymers. (b–e) Time-dependent effects of taxol in the hyperosmotically induced formation of tubulin polymers in root cortex cells. Cells were pretreated with 20 µM taxol for 2 h and subjected to 1 M mannitol plus 20 µM taxol for up to 60 min. Representative cells were sampled after 5 (b), 20 (c), 30 (d) and 60 min (e). Note the gradual disorganization of cortical tubulin polymers progressing from 5 to 20 min (b, c; cf. a) and their gradual recuperation from 30 to 60 min postexposure (d, e; cf. b, c). (f) Mitotic root cell plasmolyzed in 1 M mannitol for 30 min. Inset: chromosomes after Hoechst 33258 staining. (g–j) Progressive disintegration of mitotic spindle in root cells pretreated with 20 µM taxol for 2 h and plasmolyzed in 1 M mannitol plus 20 µM taxol for up to 60 min. Cells were sampled after 5 (g), 20 (h), 30 (i) and 60 min postexposure (j). The mitotic spindle gradually disintegrates to amorphous perichromosomal tubulin accumulations. This phenomenon starts at 5 min (g), becomes evident at 20 min (h), peaks at 30 min (i), but progressively is reinstated during the next 30 min (j). Insets: respective chromosome fluorescent images after Hoechst 33258 staining. (k–m) Time-dependent response of the tubulin cytoskeleton of root cortex cells pretreated with 20 µM taxol for 2 h and plasmolyzed in 1 M mannitol plus 20 µM taxol and 100 µM neomycin for up to 30 min. Although cortical tubulin polymers appear short, dispersed and malorganized at 5 min postexposure (k), their abundance and organization progressively resume, being evident at 20 min (l) and completed at 30 min (m) (k–m; cf. b–d). (n) Total depletion of tubulin polymers in a root cortex cell pretreated with 50 µM U73122 plus 20 µM taxol for 2 h and plasmolyzed for 30 min in 1 M mannitol supplemented with 50 µM U73122 and 20 µM taxol. (o) Malorganized but wealthy tubulin polymers in a root cortex cell pretreated with 20 µM taxol for 2 h and plasmolyzed for 30 min in 1 M mannitol supplemented with 50 µM U73122 plus 20 µM taxol.



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perichromosomal accumulations (Fig. 8g–i) and not to wellorganized macrotubule bundles substituting for kinetochore MTs, in hyperosmotically treated mitotic cells (Fig. 8g –i, cf. Fig. 8f; Komis et al., 2001, 2002b). Additionally, the partial recovery of the tubulin cytoskeleton in the taxol-treated plasmolyzed cells during prolonged hyperosmotic treatment was clear in dividing cells (Fig. 8j). The addition of neomycin to the previous treatment accelerated the hyperosmotically induced macrotubule formation (Fig. 8k–m, cf. Fig. 8b – d). Cells pretreated with taxol for 2 h and plasmolyzed in the presence of neomycin and taxol for up to 20 min displayed well-organized cortical tubulin strands (Fig. 8k,l, cf. Fig. 8b,c). Those remaining in the above plasmolyticum for 30 min exhibited almost the same tubulin polymer organization found in cells plasmolyzed in mannitol alone or in mannitol plus neomycin (Fig. 8m, cf. Figs 6a,b, 8a). TEM examination showed that these cells display excessive, well-organized bundles of tubulin polymers, adjacent to the plasmalemma (Fig. S4b). In a total of 1074 tubulin polymers measured, 652 (61%) were macrotubules (mean OD, 28.63 ± 0.09 nm), while the remaining 422 (39%) were identified as MTs (mean OD, 23.72 ± 0.07 nm, Table 2). These data show that neomycin reverses the effects of taxol on remodelling of the tubulin cytoskeleton in plasmolyzed cells. Taxol rescues macrotubule formation in the presence of U73122 Taxol treatment alongside U73122 for 2 h before and 30 min after the onset of plasmolysis did not prevent tubulin polymer depletion. These cells lack tubulin polymers (Fig. 8n), an event confirmed with TEM examination (Table 2). When roots were preconditioned with 20 µm taxol for 2 h and subjected to osmoticum supplemented with 20 µm taxol and 50 µm U73122 for 30 min, tubulin immunolabeling revealed an increased number of cells bearing recognizable tubulin polymers. These form cortical strands (Fig. 8o). In these cells 83% of tubulin polymers measured (218 out of 262 polymers) were at the size range of macrotubules (mean OD 31.7 ± 0.2 nm), while the remaining 17% were MTs (mean OD 24.2 ± 0.2 nm, Table 2). The occurrence of macrotubules in these cells (Fig. S4c) suggests that taxol antagonized the effects of U73122 against macrotubule formation.

Discussion PI-PLC activity is required for the onset of protoplast volume regulation in plasmolyzed root cells The biochemical assays carried out in T. turgidum root extracts demonstrated that acute hyperosmolarity triggers the rapid and transient activation of undefined PI-PLC species (Fig. 1a). The time course of the hyperosmotically induced PI-PLC activity is in agreement with several previous studies (Einspahr et al., 1988; Cho et al., 1993; Pical et al., 1999; DeWald et al.,


2001; Takahashi et al., 2001). Treatment with neomycin and U73122 PI-PLC inhibitors resulted in the partial inhibition of the hyperosmotically induced PI-PLC activity (Fig. 1b), confirming previous results (Takahashi et al., 2001). Although the specificity of U73122 effects has been disputed (Mogami et al., 1997), it nonetheless clearly bears a PI-PLC inhibitory action in plasmolyzed roots of T. turgidum. The rapid PtdInsP2 production and turnover by PI-PLCs is well documented in plants experiencing hyperosmolarity (Einspahr et al., 1988; Pical et al., 1999; DeWald et al., 2001; Takahashi et al., 2001; Meijer & Munnik, 2003). Several other studies have also implicated PI-PLC activity in turgorregulated phenomena, including pollen tube emergence and growth (Zonia & Munnik, 2004), stomatal guard cell function (Hunt et al., 2003; Mills et al., 2004), and hormonally induced mesophyll protoplast volume changes (Kolla et al., 2004). Data from the present study show that PtdInsP2 turnover, through the hyperosmotic activation of PI-PLC species, is required for the expression of protoplast volume regulatory mechanisms in plasmolyzed T. turgidum root cells. This view is supported from the findings that the PI-PLC inhibition by U73122 (Fig. 1b) induced the abrupt reduction of the final mean protoplast volume compared with cells plasmolyzed in neat mannitol. As exemplified by Saul et al. (2004) (see also Takahashi et al., 2001), U73122 exposure should result in excessive PtdInsP2 accumulation under hyperosmotic stress, thereby suggesting that sustained elevated PtdInsP2 concentrations may suppress protoplast volume regulation in plasmolyzed T. turgidum root cells. This view is supported by the finding that neomycin treatment, which sequesters PtdInsP2 (Gabev et al., 1989), potentiated protoplast volume regulation, bringing equilibrium at higher values than those measured for cells plasmolyzed in neat mannitol. In the present work, neomycin was used at superstoichiometric concentrations over PtdInsP2. Thus, it is likely that it effectively sequesters the PtdInsP2 produced under hyperosmolarity, which is not the case in U73122-treated plasmolyzed cells, where PI-PLC inhibition should result in the overaccumulation of active PtdInsP2. Although the neomycin-mediated PtdInsP2 sequestration reduces the extent of plasmolyzed protoplast volume reduction, it does not promote long-term survival of cells under stress, suggesting that any imbalance in PtdInsP2 turnover is deleterious under hyperosmotic conditions. The effects of neomycin in this case might be related to the induction of tubulin polymer turnover that is necessary for macrotubule formation. The latter is required for the activation of the plasmolyzed protoplast volume regulation mechanism (Komis et al., 2002b, 2004, 2006). PtdInsP2 turnover mediates tubulin cytoskeleton remodeling in plasmolyzed cells Data presented here revealed that the U73122-induced impairment of protoplast volume regulation under


Research

hyperosmotic stress is related to the inhibition of tubulin polymer formation (Table 2). The same is observed when tubulin polymers are artificially depleted by the use of oryzalin (Komis et al., 2002b). It is noteworthy that either oryzalin or U73122 treatments apparently resulted in the same extent of protoplast volume reduction (this study; Komis et al., 2002b). These findings favor the hypothesis that overaccumulation of PtdInsP2 as an effect of U73122 treatment has similar results to those of oryzalin, thus targeting the mechanism of hyperosmotically induced macrotubule formation. The effects of excessive PtdInsP2 concentrations against tubulin polymer formation might be direct or indirect. As in U73122-treated plasmolyzed cells PI-PLC activity is inhibited (Fig. 1b) and PtdInsP2 is not cleaved to DAG or InsP3, it may be supposed that tubulin polymer disorganization is related to the absence of the signaling PtdInsP2 derivatives described earlier. Probably, the low concentrations of InsP3 resulting from the U73122 treatment may impair cytoplasmic calcium buffering necessary for tubulin polymer formation (for discussion, see Komis et al., 2001, 2002b). Mogami et al. (1997) showed that U73122 could promote the release of calcium from intracellular stores independently of its PI-PLC inhibitory effect, thus disturbing cytoplasmic calcium homeostasis. The contribution of PA to the formation of tubulin macrotubules in plasmolyzed cells of T. turgidum was shown previously (Komis et al., 2006). Relative absence of DAG might contribute to the absence of macrotubules in U73122treated plasmolyzed cells, owing to impaired PA production through the DAG/DAGK pathway (Meijer & Munnik, 2003; Testerink & Munnik, 2005). This, however, is not likely for the following reasons: (i) R59022, a specific DAGK inhibitor, does not affect tubulin polymer formation (Table 2) and the behavior of cells under hyperosmotic stress; (ii) treatment with the cell-permeable DAG analogues DOG or PMA was also ineffective (Figs S2, S3e,f ). Therefore, it might be postulated that in T. turgidum plasmolyzed root cells, the contribution of PA produced through the DAG/DAGK pathway to macrotubule formation and the expression of protoplast volume regulation is negligible. It seems that PLD-mediated PA production suffices for the formation of macrotubules in this system (Komis et al., 2006). The effects of PtdInsP2 against macrotubule formation are likely the result of the PtdInsP2 overaccumulation itself, in U73122-treated plasmolyzed cells, as PtdInsP2 was shown to possess potent MT-depolymerizing capacity (Popova et al., 1997, 2002; Chang et al., 2005). Furthermore, conditions stimulating PtdInsP2 production in animal cells, such as carbachol treatment (Popova et al., 2002), are associated with transient MT disassembly. This view is further supported by the observed effects of neomycin in plasmolyzed cells, whereby PtdInsP2 concentrations should rise, but PtdInsP2 should also be unavailable for protein–lipid interactions (Gabev et al., 1989). In such cells, MTs and numerous

macrotubules were found (Table 2), while a marked increase in the mean protoplast volume has been found, consistent with the increased tubulin polymer number. Therefore, the neomycin-mediated inactivation of PtdInsP2 that is induced under hyperosmotic stress seems to enable the persistence of MTs without interfering, at least qualitatively, in the hyperosmotically induced macrotubule formation (Table 2). MT disassembly triggers the activation of protoplast volume regulation in plasmolyzed cells Unexpectedly, the biochemical analysis of PI-PLC activity in T. turgidum root extracts following treatment with the MT drugs oryzalin and taxol showed that these treatments affected PI-PLC activation. Oryzalin, a potent MT disruptor triggered PI-PLC activity under isotonic conditions without affecting the hyperosmotic activation of PI-PLC (Fig. 1c). Besides, taxol, a promoter of tubulin polymerization, quenched the hyperosmotically induced PI-PLC activity (Fig. 1c). These results imply the existence of an interrelation between tubulin cytoskeleton dynamics and PI-PLC regulation. Microtubule depolymerization occurs at the onset of plasmolysis (Komis et al., 2002b, 2006) and probably correlates with the switching on of the PI-PLC activity. The root cells exposed to mannitol for 1 min display a few malorganized MTs. At this time, PI-PLC activity is well above basal values (Fig. 1a). As MT depolymerization progresses, so does the PI-PLC activity, which peaked at 3 min postexposure when root cells are depleted of MTs. These results suggest a possible link between the mechanism of PI-PLC activation and the hyperosmotically induced tubulin polymer turnover. Oryzalin triggers the rapid and quantitative MT depolymerization, mimicking the hyperosmotically induced MT disassembly, thus artificially inducing the PI-PLC activation. By contrast, taxol prevented the hyperosmotically induced MT disassembly (Fig. 8b,c; Table 2), hindering indirectly the activation of PI-PLC. Similar effects of taxol have been observed in neuroblastoma SK-N-SH cells (Popova & Rasenick, 2003). Also unexpected was the effect of taxol in the plasmolyzed protoplast volume regulation. Since in T. turgidum root cells the osmotic tolerance and adjustment of the protoplast volume seem to be dependent on the occurrence of cortical tubulin polymers (Komis et al., 2002b, 2006), the artificial stabilization of MTs should, or at least is expected to, have a positive effect. However, the mean protoplast volume of cells treated with and plasmolyzed in the presence of taxol was dramatically diminished, reaching almost the amount resulting from tubulin polymer depletion by the aid of oryzalin (Komis et al., 2002b). Interestingly, the inhibition of the mechanism regulating the protoplast volume in taxol-treated plasmolyzed cells is related to the inhibition of PI-PLC activation (Fig. 1c) and macrotubule formation (Table 2). Although tubulin polymer



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formation recuperates in time, the vast majority of them are MTs (Table 2), evidently unable to exert regulation on protoplast volume. Following this discussion, the inhibition of macrotubule formation in those cells could be explained, assuming the existence of elevated PtdInsP2 as a result of PI-PLC impairment (Fig. 1c). In this case, the effects of taxol may be specified to certain PI-PLC isoforms, the regulation of which is tightly coupled to MT dynamics (Popova et al., 1997, 2002). To this extent, PLC-γ1 of NIH3T3 cells was found to interact directly with β-tubulin (Chang et al., 2005). PtdInsP2 interacted with β-tubulin, inhibiting its polymerization but enforcing the association of β-tubulin with pleckstrin homology (PH) domains of PLCγ1 triggering its activity. As PLCγ1 activity increases, PtdInsP2 is cleaved, allowing the polymerization of tubulin into MTs. Although there are no PH-domain PI-PLC in higher plants (MuellerRoeber & Pical, 2002), it may be suggested that there exist putative indirect functional interactions between PI-PLC–PtdInsP2 signaling and the MT cytoskeleton, as implied by the effects of taxol and oryzalin on PI-PLC activity. This interesting possibility awaits further substantiation. The fact that neomycin partially relieves the taxol effects in the plasmolyzed cells, allowing macrotubule formation (Table 2), suggests that in T. turgidum, PtdInsP2, PI-PLC activity and tubulin polymers are functionally integrated. Although speculative, it could be suggested that the rapid and massive MT disorganization observed at the onset of the hyperosmotic treatment (Komis et al., 2001, 2002b) is related to the activation of PI-PLC. The activation of PI-PLC allows PtdInsP2 cleavage, permissive to macrotubule formation and the triggering of the plasmolyzed protoplast volume regulatory mechanism. The capacity of MTs to act as components of signal transduction pathways has been also documented in alfalfa cells. In these, oryzalin treatment artificially turns on the activation of HAMK (heat-shock-activated mitogen-activated protein kinase) and SAMK (stress-activated mitogen-activated protein kinase), mimicking the effects of heat and cold stress, respectively (Sangwan et al., 2002). It is also interesting that recently, Lu et al. (2007) provided a link between MT dynamics and hyperosmotically induced ABA water stressrelated signaling, further substantiating the probable role of cortical tubulin polymers in the perception of and reaction to water stress (Komis et al., 2002b; Wasteneys, 2004). Conclusions In conclusion, the data presented, in addition to previous data (Komis et al., 2006), support the view that PLD and PI-PLC signaling pathways are distinctly involved in the activation and expression of protoplast volume regulation in plasmolyzed root cells of T. turgidum. Under hyperosmotic stress, the PI-PLC pathway ensures that the hyperosmotic increase of PtdInsP2 concentration will be transient, while the PLD pathway controls the vast PA production, phenomena of


primary importance for macrotubule formation and activation of the volume regulatory mechanism of the plasmolyzed protoplast.

Acknowledgements We appreciate all the help received by Mr N. Apostolakos (Issac Newton Group of Telescopes, La Palma, Canary Islands, Spain), Mrs Elke Woelken (Biocenter Klein Flottbek, Hamburg, Germany) and Dr Eleftheria Letsiou (Laboratory of Biochemistry, Faculty of Chemistry, University of Athens). The present study was financed by grants from State Scholarship Foundation of Greece and DAAD (project ‘IKYDA 2002’), the Hellenic Ministry of Education and Religious Affairs, the EU (project ‘Pythagoras I’) and the University of Athens (project ‘Kapodistrias’).

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Supplementary Material The following supplementary material is available for this article online: Fig. S1 Confocal laser scanning microscope (CLSM) assessment of hyperosmotic tolerance and viability of root cells exposed to phosphoinositide-specific phospholipase C (PI-PLC) modulators, after PI/FDA dual staining under isotonic or hyperosmotic conditions. Fig. S2 1,2-dioctanoyl glycerol (DOG, 10 µm) or 10 µm phorbol 12-myristate 13-acetate (PMA) treatment for 2 h before and 30 min during plasmolysis in 1 m mannitol does not affect the time course of plasmolysis and the viability of root cells against acute hypertonicity. Fig. S3 Confocal laser scanning microscope (CLSM) or epifluorescent visualization of the cortical tubulin polymer



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organization in control root cells and root cells incubated in the presence of phosphoinositide-specific phospholipase C (PI-PLC) modulators under isotonic or hyperosmotic conditions.

This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/ j.1469-8137.2007.02363.x (This link will take you to the article abstract).

Fig. S4 Transmission electron microscopy visualization of cortical tubulin polymers formed under acute hyperosmolarity in the presence of phosphoinositide-specific phospholipase C (PI-PLC) modulators.

Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the journal at New Phytologist Central Office.

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