Journal of Experimental Botany, Vol. 49, No. 319, pp. 163–170, February 1998
The function of guard cells does not require an intact array of cortical microtubules Sarah M. Assmann1 and Tobias I. Baskin2,3 1 Biology Department, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA 2 Division of Biological Sciences, 109 Tucker Hall, University of Missouri, Columbia, MO 65211–7400, USA Received 21 July 1997; Accepted 24 September 1997
Abstract The development of stomatal guard cells is known to require cortical microtubules; however, it is not known if microtubules are also required by mature guard cells for stomatal function. To study the role of microtubules in guard cell function, epidermal peels of Vicia faba were subjected to conditions known to open or close stomata in the presence or absence of microtubule inhibitors. To verify the action of the inhibitors, microtubules in appropriately treated epidermal peels were localized by cryofixation followed by freeze substitution and embedding in butyl-methyl methacrylate. Mature guard cells had a radial array of microtubules, focused toward the thick cell wall of the pore, and the appearance of this array was the same for stomata remaining closed in darkness or induced to open by light. Treatment of epidermal peels with 1 mM colchicine for 1 h depolymerized nearly all cortical microtubules. Measurements of stomatal aperture showed that neither 1 mM colchicine nor 20 mM taxol affected any of the responses tested: remaining closed in the dark, opening in response to light or fusicoccin, and closing in response to calcium and darkness. We conclude that intact microtubule arrays are not invariably required for guard cell function. Key words: Colchicine, cortical microtubules, cryofixation, guard cells, stomata, taxol, Vicia faba.
Introduction The development of stomatal guard cells is well known to involve cortical microtubules. In fact, developing guard cells have been a model system for studying how microtubules determine cell shape and control the deposition of
cellulose microfibrils. These studies have found that cortical microtubules are needed to produce a pair of kidney shaped cells surrounding an aperture, as well as to direct the asymmetric deposition of cellulose microfibrils in the cell wall, which enables the stoma to open and close in response to changes in osmotic potential (Palevitz, 1981; Sack, 1987). Once they mature, guard cells change shape to regulate the stomatal aperture. It is reasonable to ask whether these changes in shape require, or affect, the cortical microtubules. But even though the role of microtubules in guard cell development was recognized as important long ago, there has been little assessment of the role of cortical microtubules in guard cell function. The possible reasons why the role of microtubules in guard cell function has been little studied are both practical and conceptual. Practically, attempts to localize microtubules in mature stomata may have been frustrated by the thick cell wall, which hinders chemical fixation. Conceptually, microtubules are considered to orient microfibrils and specify cell shape; once guard cells reach maturity, their structure is plausibly sufficient to allow many rounds of opening and closing. However, microtubules have roles beyond shaping cells: the cytoskeleton, including microtubules, participates in signal transduction. For example, in animal cells, actin filaments terminate in special regions of the plasma membrane enriched in tyrosine receptor-kinases (Mochly-Rosen, 1995). For plant cells, analogous examples probably exist, although the evidence is fragmentary (Pickard, 1994). Of particular relevance to stomatal regulation is a recent demonstration that treating carrot cells with colchicine or oryzalin, which depolymerize microtubules, significantly increased the activity and open duration of channels permeable to calcium ( Thion et al., 1996). Calcium is one of the prime regulators of stomatal aperture. Treatment of epidermal peels with calcium inhibits stomatal opening and pro-
3 To whom correspondence should be addressed. Fax: +1 573 882 0123. E-mail:
[email protected] © Oxford University Press 1998
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motes stomatal closure (Schwartz, 1985; Mansfield et al., 1990), both of which effects appear to be mediated via elevation of cytosolic calcium (Gilroy et al., 1991). The activity of calcium channels in guard cells might, as in carrot cells, depend on microtubules. Thus, microtubules may be involved in the signal transduction required for guard cell function. A straightforward way to study the role of microtubules in guard cell function is through the use of inhibitors. Using such an approach, evidence has recently been obtained in support of the hypothesis that signal transduction for guard cell responses does involve the actin cytoskeleton ( Kim et al., 1995). The analogous hypothesis for the microtubule component of the guard cell cytoskeleton has been tested, to our knowledge, in only two previous reports, with conflicting results. In one report, colchicine inhibited light-dependent stomatal opening (Couot-Gastelier and Louguet, 1992); in the other, neither propyzamide, which depolymerizes microtubules similarly to colchicine, nor taxol, which stabilizes microtubules, affected the ability of stomata to close in response to abscisic acid (Jiang et al., 1996). As stomatal opening and stomatal closure are not simply the mechanistic reverse of one another(Assmann, 1993), it is possible that the above studies imply microtubules are required for opening but not for closing. However, these investigations used different species: Tradescantia virginiana for opening, and Vicia faba for closing, and each group stimulated opening or closing with only a single kind of treatment ( light or abscisic acid). Given the general lack of information concerning microtubule arrays of functioning guard cells, this study set out to characterize the microtubule arrays of mature guard cells of Vicia faba, and to test whether these arrays are involved in transducing either stomatal opening in response to light or fusicoccin, or stomatal closure in response to calcium and darkness.
Materials and methods Plant material Plants of Vicia faba L. were grown in growth chambers under a 10 h light (0.2 mmol m−2 s−1), 14 h dark regime, at 21/18 °C day/night. Plants were grown in three parts Metro-Mix 360 (Scotts-Sierra Horticultural Products, Marysville, OH, USA),1 part perlite (Schundler Co., Metuchen, NJ, USA), and were watered four times per week with quarter-strength Hoagland’s solution. All experiments used epidermal peels from the abaxial surface of young, fully expanded leaves of 3–4-week-old plants. Analysis of microtubules Epidermal peels were obtained and preincubated as described below to maximize stomatal closure. Peels were then transferred to incubation solution, consisting of 30 mM KCl, 0.1 mM CaCl , 10 mM MES, pH 6.1 (note: the pH of all solutions for 2 peels was adjusted with KOH ), and subjected to 1 h of darkness in the presence or absence of 1 mM colchicine. A third set of
peels was maintained in incubation solution for 2 h under white light (0.17–0.19 mmol m−2 s−1; GE ‘Brite Stiks’) in order to promote stomatal opening. Immediately following the termination of incubation, six epidermal peels from each treatment were cryofixed. At the same time, additional peels were visually inspected and it was confirmed that light had promoted stomatal opening. The protocol for preparing sections from peels for examination with light microscopy was essentially as described in Baskin et al. (1996). For handling, the peels were supported on tungsten wire loops coated with 1% Formvar, as described in Lancelle et al. (1986). For cryofixation, a wire loop was carefully brought up under a floating peel and the peel adhered to the Formvar. Then, most of the adhering buffer was wicked away and the sample immediately cryofixed by plunging into liquid propane held at −180 °C. Approximately 10–15 s elapsed between first adhering a peel to the loop and plunging. The plunging apparatus was constructed to minimize precooling of the samples prior to entry into the propane, accelerated samples to c. 5 m s−1 at entry, and propelled them through the cryogen for at least 4 cm. Samples were freeze substituted in freshly-opened acetone containing 1% acidified dimethoxypropane (Aldrich Chemical Corp. Milwaukee, WI. USA) to remove water ( Kaeser, 1989) for 48 h at −80 °C in a commercial freezer. Vials were then removed from the freezer and allowed to gradually warm to −4 °C over an 18 h period, and then to room temperature over 4–6 h. Temperatures were monitored with a thermocouple thermometer immersed in acetone. All steps after substitution were done at room temperature. Tissue, still maintained on the Formvar loops, was infiltrated with the following solutions (each with an incubation period of 1 h): 100% acetone; 25% resin: 75% acetone, 50% resin: 50% acetone; 75% resin: 25% acetone, 100% resin. A final infiltration with 100% resin was performed overnight. All infiltrations were performed on a rotator. Resin comprised 80% butyl-methacrylate, 20% methylmethacrylate (Aldrich), 0.5% benzoinethylether (Aldrich). The resin mixture was degassed with nitrogen for 20 min and stored at −20 °C. For embedding, the stem of the wire loop was severed and the loop portions were placed individually in capsules manufactured with flat bottoms ( TAAB Laboratories Ltd. Aldermaston, Berks, UK ). Resin was polymerized under long-wavelength UV light at 4 °C. Capsules were placed on a glass plate about 4 cm above an 8 W source for 20 h. Embedded wire was carefully cut out of the blocks prior to sectioning. Semi-thin sections (1.75 mm thick) were cut dry on an ultramicrotome, placed in droplets of water, and affixed to slides coated with 3-aminopropyltriethoxy silane (Angerer and Angerer, 1991) by heating briefly on a slide warmer for 2–5 min at 60 °C. Sections were extracted in acetone for 10 min and then immediately rehydrated in PBS containing 0.05% Tween-20 (PBS-Tween). Sections were rinsed (0.1% Tween-20 in PBS), incubated in primary antibody (2 h at 37 °C ), rinsed in PBSTween (3×10 min), incubated in secondary antibody (2 h at 37 °C ), rinsed in PBS-Tween (3×10 min), and mounted in an antifading reagent ( Vectashield; Vector Laboratories, Burlingame, CA). Antibodies were diluted and applied in 1% BSA, 0.1% azide and 0.05% Tween-20 in PBS. Antibodies used were as follows. Monoclonal anti-a-tubulin, raised against sea urchin axonemes (B-5-1-2, Sigma) was diluted 151000, and the secondary, goat anti-mouse Fab fragments conjugated with Cy3 (Jackson Immuno-Research Laboratories, West Grove, PA, USA), was diluted 15200. Fluorescence microscopy was performed with conventional epifluorescence
Microtubules in stomatal function 165 (Zeiss Axioplan), with fluorescence from Cy-3 observed through a standard rhodamine filter cube. 8–24 sections per peel, 4–6 peels per treatment were examined; each section contained numerous stomata. There were no detectable differences between peels from the same treatment. Stomatal movement assays For experiments on stomatal opening, bifoliate leaves were excised from plants and placed in distilled water under darkness for approximately 30 min before obtaining epidermal peels. Peels were floated cuticle side up on pre-incubation solution (1 mM CaCl , 10 mM MES, pH 6.1) for a minimum of 30 min. 2 This pre-incubation serves to randomize the peels and to promote stomatal closure (Schwartz et al., 1995). An assessment of baseline stomatal aperture was taken by measuring with an optical micrometer 15 stomatal apertures on each of three epidermal peels. Peels were then transferred to 4.5 cm diameter plastic Petri dishes containing an incubation solution consisting of 30 mM KCl, 0.1 mM CaCl , 10 mM MES, pH 6.1, either 2 alone, or with 1 mM colchicine or 20 mM taxol. For experiments with taxol, controls were incubated in the same concentration of DMSO (0.2%, v/v) as was added with the taxol. Dishes were placed in either white light (as described above) or darkness. Stomatal aperture measurements (15 apertures on each of three epidermal peels) were made for each treatment after 1, 2 and 3 h of incubation. For experiments testing the effect of 1 mM colchicine on stomatal opening induced by fusicoccin, fusicoccin was added to the incubation solution to a final concentration of 10 mM. All fusicoccin incubations were performed under darkness for 3.5 h, after which time stomatal apertures were quantified as described above. All experiments were replicated at least three times, and values shown represent means±SE. To induce open stomata whose closing responses could be assessed, biofoliate leaves were excised from plants and submerged abaxial side up in distilled water under white light (as described above) for approximately 2.5 h. Epidermal peels were then made and, for randomization, were floated cuticle side up in the incubation solution used for opening experiments. The entire peeling process required no more than 10 min. Peels were then transferred to a low-salt, high-calcium incubation solution (15 mM KCl, 0.5 mM CaCl , 10 mM MES, pH 6.1), 2 either alone, or with 1 mM colchicine, 20 mM taxol, or with the equivalent amount of DMSO. Stomatal apertures were measured as described above initially and following a 1 h incubation in darkness. The experiment was repeated three times. Chemicals Fusicoccin (Sigma) was prepared as a 1 mM aqueous stock solution, colchicine (Sigma) was prepared as a 100 mM aqueous stock, and taxol (Sigma) was prepared as a 10 mM stock in DMSO; all stocks were stored at −20 °C in darkness. The efficacy of the taxol stock was confirmed by its ability to coldstabilize bovine brain microtubules assembled in vitro.
Results Analysis of cortical microtubules To localize cortical microtubules in guard cells, epidermal peels of Vicia faba were cryofixed and, following freeze substitution in acetone, embedded in butyl-methylmethacrylate and microtubules were examined with indirect immunofluorescence in semi-thin sections.
Cryofixation was used because the thick-walled guard cells may be particularly difficult to fix with conventional, chemical fixatives. Although cryofixation at ambient pressure, as used here, may result in ice crystallizing in the interior of specimens even as thin as epidermal peels, which comprise only a single cell layer, it has been shown that the damage from these crystals is unresolvable through the light microscope, and preservation in cryofixed cells even with ice crystal damage is superior to that of aldehyde-fixed cells by several criteria (Baskin et al., 1996). The appearance of microtubule arrays was first compared in open and closed guard cells ( Fig. 1A–H ). The cortical microtubules were abundant and generally well organized, radiating outwards from the region of the cell closest to the pore. Despite the clear overall organization of the microtubules, divergent microtubules sometimes occurred. Comparing the structure of the cortical array between guard cells in dark and light treated peels, no consistent difference was seen (Fig. 1, compare A–D to E–H ). For the dark-treated peels, the images in Fig. 1(A–D) appear to show open stomata, but at the time of cryofixation the relatively closed status of dark-treated stomata was verified on companion peels to those frozen. The images in Fig. 1 do not provide a reliable indication of the size of the stomatal aperture in vivo. For example, the apertures shown in Fig. 1 are nearly double the maximal aperture for light treatment measured in peels (see below). The pore may have been distorted physically because of the hydrodynamics of freezing; but, on the other hand, the paradermal sections used are unlikely to have sampled the pore at its narrowest position. In a species like V. faba, the pore is narrowest at medial depths from the surface, but towards the mesophyll and the outside, the pore widens out (Pallas and Mollenhauer, 1972; Raschke, 1979). Therefore, at the focal plane used to measure stomatal apertures in vivo, a paradermal section will pass through the narrow part of the pore, cutting the guard cells more-or-less at mid-plane, and thus transecting cortical microtubules ( Fig. 1F, left-hand guard cell ). Whereas, to localize a portion of the cortical array in face view, as seen in Fig. 1, a paradermal section is required that passes near the top or bottom faces of the stoma, and which will therefore pass through a wider region of the pore. The absence of any apparent effects of colchicine or taxol on stomatal responses (see below) could indicate that intact microtubules are not required for stomatal responses; alternatively, negative results could indicate that these drugs failed to enter the guard cell cytosol. For guard cells in epidermal peels of V. faba, as used here, Jiang et al. (1996) have already obtained evidence that taxol entered the cells and stabilized microtubules, as expected. To see if colchicine depolymerized guard cell
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Fig. 1. Micrographs of cortical microtubules in guard cells of Vicia faba, localized with an anti-a-tubulin antibody in sections (1.75 mm thick) from epidermal peels that were cryofixed, freeze-substituted, and embedded in butyl-methyl-methacrylate. (A–D) Dark-treated peels; ( E–H ) light-treated peels; (I–L) dark-treated peels plus 1 mM colchicine. Note the well organized radial arrays in both dark and light treatments, and the presence of high background and few microtubules in the colchicine treatment. Magnification is the same for all panels: 1172×, scale bar=15 mm.
microtubules, microtubules were localized in epidermal peels treated with 1 mM colchicine for 1 h. Guard cells had brightly staining cytosol ( Fig. 1I–L), and in most images there were no microtubules detectable ( Fig. 1I,
J ). A few guard cells had some microtubules remaining ( Fig. 1K, L). Bright cytosolic staining as well as remnant microtubules are typical of plant cells treated with inhibitors that depolymerize microtubules (Cleary and
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Fig. 2. Effect of colchicine or taxol on stomatal aperture versus time for epidermal peels of Vicia faba. (A) Peels were incubated ±1 mM colchicine. (B) Peels were incubated ±20 mM taxol (or 0.2% DMSO for controls). Data are means of three replicate experiments ±SE. There was no significant effect of taxol or colchicine at any time.
Fig. 3. Effect of colchicine or taxol on stomatal aperture of epidermal peels of Vicia faba induced to open with 10 mM fusicoccin (A) or induced to close to with 0.5 mM CaCl in the dark (B). Baseline 2 represents the apertures measured immediately prior to treatment. The inductive stimulus was given alone (Control ), or with 1 mM colchicine, 0.2% DMSO, or 20 mM taxol, and apertures were measured after 3.5 h (A) or 1 h (B). Data are means of three replicate experiments ±SE. There was no significant effect of taxol or colchicine.
Hardham, 1988; Seagull, 1990; Baskin et al., 1994). This result shows that 1 h of incubation in colchicine was sufficient to depolymerize the great majority of cortical microtubules.
Thus, under all conditions assayed, in the presence of taxol or colchicine guard cells responded normally.
Epidermal peel experiments The requirement for microtubules in guard cell function was analysed by treating peels with 1 mM colchicine or 20 mM taxol and measuring stomatal apertures at various times thereafter. These doses typically are saturating for affecting microtubules in plant cells. Neither the rate nor the extent of stomatal opening in the light was significantly affected by colchicine (Fig. 2A) or by taxol ( Fig. 2B). In addition, neither drug promoted stomatal opening in the dark. Although there was modest stomatal opening in darkness, such opening has been reported previously and is attributed to the release of ‘backpressure’ on the guard cells when epidermal cells lose turgor as a result of injury from the peeling process ( Edwards et al., 1976; Klein et al., 1996). As an alternative to light, fusicoccin was used to drive stomatal opening and it was found that colchicine had no effect on this response with 5 mM (not shown) or 10 mM fusicoccin ( Fig. 3A). Finally, open stomata were closed by treatment with 0.5 mM CaCl and darkness, and again, significant 2 effects of colchicine or taxol were not found (Fig. 3B).
Discussion Microtubule organization in mature stomata In the mature guard cells of Vicia faba, as shown in the cryofixed sections, cortical microtubules beneath the paradermal walls are arranged in a radial array ( Fig. 1). The organization of microtubules in the mature guard cells of species other than grasses has not been extensively studied, but the radial arrangement seen here is consistent with electron micrographs of guard cells at late stages of development in the monocotyledon, onion (Palevitz and Hepler, 1976) and at maturity in the dicotyledons, pea (Singh and Srivastava, 1973) and bean (Galatis and Mitrakos, 1980). In these studies, guard cell apertures were not measured, but they were presumably open (or opening) because the methods describe harvesting greenhouse-grown plants. The radial arrangement of microtubules is also consistent with the radial arrangement of cellulose microfibrils known to be present in the guard cell walls (Sack, 1987). Interestingly, in V. faba, a radial arrangement has also been recently reported for actin filaments ( Kim et al., 1995), which indicates that guard
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cells share with several other plant cell types the co-alignment of microtubules and actin filaments ( Fukuda and Kobayashi, 1989; Lancelle and Hepler, 1991). When the plane of the section occasionally transected the epidermis, microtubule arrays were imaged interior to the dorsal or ventral guard cell walls. These arrays contained many more divergent microtubules than the radial arrays beneath paradermal walls, and sometimes appeared to be completely random (not shown). In dicotyledonous guard cells, others have noted divergent microtubules in similar planes (Singh and Srivastava, 1973; Galatis and Mitrakos, 1980). The dorsal and ventral arrays were not viewed frequently enough to determine if there was any difference between light and dark treatments. Influence of turgor pressure on microtubule organization It is of interest to compare microtubule organization between open and closed stomata because these states differ in turgor pressure. Although the absolute difference is not known with certainty, a 4 mm difference in aperture, as occurred here between light and dark treatments, is likely to reflect a turgor pressure change of around 0.8 MPa (Franks et al., 1995).Turgor pressure is predicted by a prominent model to affect microtubule organization significantly ( Williamson, 1990), but despite this there have been few studies that have examined the effects of different turgor pressures on the organization of the cortical array. Although treatment of mesophyll cells with one of several osmolytes, at greater than plasmolysing levels, completely depolymerized cortical microtubules (Bartolo and Carter, 1991), it is not clear what such an extreme treatment means physiologically. For less extreme changes, the results are conflicting: the orientation of microtubules was changed by treatment of pea epicotyls with levels of sucrose that were not quite plasmolysing ( Roberts et al., 1985), whereas the organization of microtubules was not changed by growing maize roots in a low water potential treatment that reduces turgor by 0.4 MPa (Liang et al., 1994). Similarly, for guard cells, we report here no conspicuous difference in the organization of microtubules over a physiological range of turgor change, i.e. between light and dark-treated guard cells (Fig. 1). Therefore, changes in turgor pressure (and osmolarity) within the range that prevails in vivo need not interfere with the organization of cortical microtubules. Cortical microtubules are known to be dynamic structures (Hush et al., 1994; Wymer and Lloyd, 1996). Even though microtubule organization was not affected by changing turgor pressure or osmolarity, either parameter might have affected microtubule dynamics. In T. virginiana, microtubules were present in opening stomata but were absent in closed stomata(Couot-Gastelier and
Louguet, 1992), and in stomata closed by exogenous abscisic acid, microtubule arrays were short and fragmented (Jiang et al., 1996). Although exogenous abscisic acid may have directly fragmented microtubules, this seems unlikely given that treatment of pea stems with this hormone enhances the stability of microtubules (Sakiyama and Shibaoka, 1990). Alternatively, the absent or fragmented microtubules reported previously in closed stomata could be explained if microtubules in closed stomata were more dynamic and hence more difficult to fix chemically than those of open stomata. Chemical fixation, as used by the above two groups, takes several minutes (see discussion in Baskin et al., 1996), a time that exceeds the half-life of cortical microtubules, as measured in several plant cell types (Hush et al., 1994; Wymer and Lloyd, 1996). Therefore, it is possible that cortical microtubules in closed stomata are destabilized compared to those of open stomata, perhaps as a consequence of the lowered turgor pressure. Images of dynamic cytoskeletal structures localized in chemically fixed preparations need to be interpreted with caution. Microtubules and signal transduction in guard cells Guard cells mediated stomatal opening in response to light or fusicoccin, and mediated stomatal closure in response to darkness and calcium, regardless of the presence of 1 mM colchicine. Colchicine at this concentration depolymerized most microtubules, as expected. It is a formal possibility that the remnant microtubules in the colchicine-treated cells were sufficient for the required signal transduction; however, this would be highly unusual because levels of depolymerization comparable to that of Fig. 1 inhibit if not abolish microtubulemediated responses, to our knowledge without exception. In addition, stomatal responses were not changed by taxol, which affects microtubules in the opposite manner, that is, by stabilizing them. That taxol stabilizes microtubules in epidermal peels of V. faba, as used here, has previously been shown (Jiang et al., 1996). From these results, we concluded that microtubule arrays are not involved in transducing these diverse opening and closing signals. These results are partially consistent with those of previous researchers. Jiang et al. (1996) stimulated closure of V. faba stomata in the light by application of 10 mM abscisic acid and, consistent with the results of the experiments here, the stomata closed regardless of whether microtubules had been depolymerized by propyzamide or stabilized by taxol. By contrast, light-dependent stomatal opening in T. virginiana was inhibited by colchicine (Couot-Gastelier and Louguet, 1992). However, the inhibition of opening by 1 mM colchicine required a 4 h pretreatment to become manifest, and when colchicine was removed, the guard cells recovered the ability to open
Microtubules in stomatal function 169 despite the fact that microtubules were still not observed. Therefore, the reported inhibition by colchicine could have resulted from a non-specific effect of this compound. Taken together, it appears that microtubules are required neither for stomatal opening in response to physical ( light) or chemical (fusicoccin) stimuli, nor for stomatal closure in response to physical (darkness) or chemical (abscisic acid, calcium) stimuli. These results are consistent with a recent report in which colchicine had no short-term effect on the activity of the potassium channels involved in uptake for stomatal opening: When colchicine was applied to Xenopus laevis oocytes expressing the KAT1 gene, which encodes these channels, no immediate effects on potassium currents were observed (Marten and Hoshi, 1997). Interestingly, the absence of a role for microtubule arrays is opposite to that reached for microfilament arrays by Kim et al. (1995). They observed that poisoning the microfilament system by either cytochalasin or phalloidin diminished stomatal responses. Thus, microfilaments may be the predominant cytoskeletal element involved in the signal transduction pathways of guard cells. Our finding no requirement for microtubules in guard cell function prompts us to ask: Why do guard cells maintain these costly structures? Only a subset of responses were examined and microtubules could be required for others. Additionally, guard cell function was only studied over the course of several hours, which is brief compared with the lifespan of a functioning stoma. It may be that continued deposition of oriented cellulose microfibrils, governed by oriented microtubules, is required to maintain appropriate wall structure in the face of repeated rounds of opening and closing. Therefore, a requirement for microtubules would only have been detected had peel responses been examined over a period of days, which experimentally is difficult if not impossible. Assessing any requirement for microtubules in guard cell physiology over the long term will require alternative approaches to epidermal peels; perhaps one such would be the identification and study of mutants with defective microtubule arrays.
Acknowledgements We thank Dr Jim Frazier, Department of Entomology, Penn State University for use of his plunge freeze apparatus (blueprints for which are available from him upon request), Dr Richard Cyr and Mr Rich Moore (PSU ) for performing the taxol experiments on bovine microtubules, and Ms Jan Wilson (MU ) for superb technical assistance. This research was supported by a tri-agency (NSF/DOE/USDA) ‘‘Cytonet’’ grant to SMA and TIB, and by NSF grant MCB-9316319 to SMA, and by a grant to TIB from the US Department of Energy (award No. 94ER20146), which does not constitute endorsement by that Department of views expressed herein.
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