Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum
2008; Wiley; Volume: 178; Issue: 2 Linguagem: Inglês
10.1111/j.1469-8137.2007.02363.x
ISSN1469-8137
AutoresGeorge Komis, B. Galatis, H. Quader, Dia Galanopoulou, P. Apostolakos,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoNew PhytologistVolume 178, Issue 2 p. 267-282 Free Access Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum George Komis, George Komis Department of Botany, Faculty of Biology, University of Athens, Athens 15784, Greece;Search for more papers by this authorBasil Galatis, Basil Galatis Department of Botany, Faculty of Biology, University of Athens, Athens 15784, Greece;Search for more papers by this authorHartmut Quader, Hartmut Quader Biocenter Klein Flottbek, University of Hamburg, D-22609 Hamburg, Germany;Search for more papers by this authorDia Galanopoulou, Dia Galanopoulou Laboratory of Biochemistry, Faculty of Chemistry, University of Athens, Athens, 157 71, GreeceSearch for more papers by this authorPanagiotis Apostolakos, Panagiotis Apostolakos Department of Botany, Faculty of Biology, University of Athens, Athens 15784, Greece;Search for more papers by this author George Komis, George Komis Department of Botany, Faculty of Biology, University of Athens, Athens 15784, Greece;Search for more papers by this authorBasil Galatis, Basil Galatis Department of Botany, Faculty of Biology, University of Athens, Athens 15784, Greece;Search for more papers by this authorHartmut Quader, Hartmut Quader Biocenter Klein Flottbek, University of Hamburg, D-22609 Hamburg, Germany;Search for more papers by this authorDia Galanopoulou, Dia Galanopoulou Laboratory of Biochemistry, Faculty of Chemistry, University of Athens, Athens, 157 71, GreeceSearch for more papers by this authorPanagiotis Apostolakos, Panagiotis Apostolakos Department of Botany, Faculty of Biology, University of Athens, Athens 15784, Greece;Search for more papers by this author First published: 24 January 2008 https://doi.org/10.1111/j.1469-8137.2007.02363.xCitations: 14 Author for correspondence: Basil GalatisTel:+003 210 7274646Fax:+003 210 7274702Email: bgalatis@biol.uoa.gr AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary • 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. 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 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). 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 lipid-binding proteins as well as the cleavage by PI-PLC (Gabev et al., 1989). U73122 inhibits the catalytic activity of PI-PLC (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). 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). 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 1Open in figure viewerPowerPoint (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). 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 mm 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). 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). Figure 2Open in figure viewerPowerPoint (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). Figure 3Open in figure viewerPowerPoint (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). 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, 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). 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 846 54 94% 10 µm U73122, 2 h + 1 m mannitol plus 10 µm U73122, 2 h 637 79 88.96% 50 µm U73122, 2 h + 1 m mannitol plus 50 µm U73122, 30 min 706 143 83.15% 50 µm U73122, 2 h + 1 m mannitol plus 50 µm U73122, 2 h 481 107 81.8% Results of every experiment correspond to confocal laser scanning microscope (CLSM) study of 10 different root tips stained with PI/FDA. 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 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 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. Figure 4Open in figure viewerPowerPoint 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). Figure 5Open in figure viewerPowerPoint 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). 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, 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
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