Planta

Planta 149, 280-282 (1980)

9 by Springer-Verlag 1980

Mechanical Stabilization of Guard Cell Protoplasts of Viciafaba H. Schnabl 2, P. Scheurich 2, and U. Zimmermann 1 t 2

Arbeitsgruppe Membranforschung am Institut ffir Medizin der Kernforschungsanlage Jfilich, Postfach 1913, D-5170 J/ilich 1, and Institut ffir Botanik und Mikrobiologie der Technischen Universitfit, Arcisstra/3e 21, D-8000 M/inchen 2, Federal Republic of Germany

Abstract. Guard cell protoplasts of Vicia faba were immobilized in cross-linked Ca-alginate. No visible morphological changes were detected under the light microscope over a period of 14 days. The entrapped cells reacted normally to changes of the external osmolarity by shrinking and swelling. Addition of the calcium complexing agent, citrate, led to dissolution of the matrix. After reequilibration with Ca ions the released cells regained their ability to swell and shrink in response to external stress. The released protoplasts could be stained with the vital dye, neutral which was accumulated in the vacuoles. It should also be noted that the protoplasts can be transported when immobilized.

Key words: Alginate - Protoplasts (guard cells) - Stom a t a - Vicia.

Introduction

The mechanisms involved in stomatal movement and in water transport between guard, subsidiary, epidermal, and mesophyll cells are still not completely understood (Hsiao 1976; Schnabl and Ziegler 1977; Raschke 1979). In order to elucidate the underlying cellular processes, direct measurements of turgor pressure, of turgor-pressure-dependent processes, and of the relevant water transport, parameters are required on the cellular level. The miniaturized pressure probe described recently (Zimmermann 1978 ; Zimmermann and Steudle 1978; Htisken etal. 1978) has enabled the first direct measurements ever of some of these parameters to be made in the subsidiary, epidermal, and mesophyll cells of Tradescantia virginiana (Zimmermann et al. in press). Because of their kidneyshaped form and very small volume, stomata have so far been inaccessible to measurements of turgor

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pressure and related parameters. Very recently, Edwards and Meidner (1979) claimed to have measured the turgor pressure in stomata using the pressure probe technique. However, as pointed out by Zimmermann et al. (in press), the data are not reliable because of the artifacts involved in the measurements. Although in the future and with further improvements of the pressure probe technique turgor pressure measurements ought to be possible in stomata, a different method was chosen for the present to arrive at a description of the physical parameters (i.e., hydraulic conductivity, volumetric elastic modulus of the cell wall, pressure and coupling between water and solute flow) involved in stomatal movement. Experiments were carried out in isolated guard cell protoplasts of Vicia faba (Schnabl et al. 1978) which were stabilized in a polymeric matrix in order to allow a pressure gradient to be set up between the cell and the external solution. The mechanical stability of the polymeric matrix can be varied either by the degree of cross-linkage or by the use of various reactants (Klein and Wagner 1979). Since the protoplasts retain their spherical shape in the polymeric matrix, the introduction of microcapillaries with tip diameters of about 2 gm, such as those used for turgot pressure measurements with the modified pressure probe or for measurements of electrical potential difference, is facilitated. This spherical shape offers decisive advantages over analogous experiments on the stomata of intact leaves, where, in order to avoid leakages,, the microelectrodes of very small tip diameters (about 0.1 gm) can only be inserted at a certain angle because of the kidneyshaped form of the stomata (J. Lettau, University of Cambridge, U.K., personal communication). Experiments of this sort have proved to be extremely difficult. In the present communication we report on the experimental procedure employed to immobilize

H. Schnabl et al. : Mechanical Stabilization of Protoplasts

guard cell protoplasts of Vicia faba in an alginate matrix cross-linked with Ca 2+ ions. The Ca-alginate network has the advantage of permitting the cellular functions to be maintained over long periods of time while simultaneously providing the possibility of releasing the protoplasts after a given time by the dissolution of the matrix with the addition of Ca 2 + complexing agents.

Material and Methods Seeds of Viciafaba (Friihe Weil3keimlinge) were germinated and grown (Einheitserde ED 73, DIN 11540-80T) in a growth chamber at 20 ~ C (day) and 17~ C (night) with a photoperiod (15,000 lx, Osram, L 65 WJ20 R, hellweil3) of 12 h. The air humidity was 75% by day and 85% by night. The plants were watered three times a week. Leaves from approximately three-week-old plants were used for experimental purposes. The technique for the enzymic isolation of guard cell protoplasts and mesophyll protoplasts is described elsewhere in more detail (Schnabl et al. 1978). The protoplasts immobilized in the polymeric matrix were stored in a 0.6 M mannitol solution with 5 m M CaCI2. Na-alginate (trademark LD, Alginate Industries Ltd.) was supplied by courtesy of Prof. Klein, TU Braunschweig (see also Pilwat et al. in press).

Results

Na-alginate can be cross-linked in a few minutes with the addition of high concentrations (about 100 raM) of Ca 2+ ions. However, calcium concentrations of

281

this order lead to irreversible changes in the cell membrane. Lowering the Ca2+-concentration to physiological levels (about 5 raM) still results in cross-linkage but the time taken to complete the process becomes too long. Optimum reaction conditions for network formation without the loss of cellular functions and membrane transport properties are achieved using the following procedure in which high concentrations of calcium are still used for the cross-linking process, but the exposure time of the guard cell protoplasts to free Ca 2+ ions (i.e., not bound by the alginate) is kept relatively short. An alginate solution (10% w/w) in 0.5 M mannitol is mixed carefully at a ratio of 1:10 (packed cells to solution) with the protoplast suspension and spread onto a slide. This layer is then covered with a solution containing 100 mM CaC12 and 0.6 M mannitol and left to crosslink for one minute at room temperature. The crosslinked layer is rinsed thoroughly with a solution of 5 mM CaC12 in 0.6 M mannitol and stored at 4~ in the following medium: mannitol (510 mM), CaC12 (5mM), NaC1 (20mM), KC1 (10mM), KHCO3 (5 mM) and glucose (10 mM). In addition some samples were supplied with penicillin and streptomycin in order to suppress bacterial growth during storage. The effects of storing the cross-linked matrices were studied over a two-week period. Under the light microscope there were no visible morphological changes, as compared to freshly isolated guard cell protoplasts suspended in solution.

Fig. 1. A guard cell protoplast of ViciaJiTba embedded for 14days in the alginate matrix with a solution containing 0.51 M mannitol, 5 m M CaCI2, I0 mK KCI~ 20 m M NaC1, 5 mM KHCO3, 10 mM glucose and a mixture of antibiotics. The osmolarity was 640 mOsmol. Scale 1 cm = 5 ~am Fig. 2. Swelling of the guard cell protoplast shown in Fig. 1 was induced by adding a 0.5 M mannitol solution (photograph taken after 3 rain). Scale 1 cm = 5 lain Fig. 3. Shrinkage of the protoplast shown in Figs. 1 and 2 was induced in the presence of 0.8 M mannitol solution. Notice the remaining shape of the alginate matrix which surrounded the protoplast when incubated in the isotonic solution (640 mOsmol, Fig. 1). Scale 1 c m = 5 ~m Fig. 4. A guard cell protoplast of Vicia faba after storing for 14 days in the alginate matrix and subsequent releasing with a Na-citrate-buffer (20 mM ; pH 7.4, mannitol 0.8 M) for 2 h at room temperature. Scale 1 c m = 5 gm

282 The immobilized guard cell protoplast shown in Fig. 1 was stored for 14 days in the alginate matrix. Immobilized guard cell protoplasts can be sent over long distances by train without any apparent changes in morphology, m e m b r a n e integrity, or cellular functions (not shown). Thus, it is obvious that s t o m a t a protoplasts which are otherwise very fragile and cannot be transported in suspension can be subjected to transportation using this technique. In order to assess whether or not the protoplasts were intact, osmotic experiments were carried out using the light microscope. The osmotic behavior was tested by transferring guard cell protoplasts immobilized in a cross-linked matrix into hyper- and hypotonic media. After a one-minute exposure to 0.5 M mannitol, the guard cell protoplasts in the crosslinked matrix exhibit the same increase in volume as those protoplasts left free in suspension (Fig. 2). Because of the time taken for diffusion to occur in the cross-linked matrix the reaction time seems to be longer than for suspended protoplasts. The increase in volume is reversible. On transfer into 0.8 M mannitol the protoplasts shrink again, a process which is clearly indicated by the protoplasts coming away from the alginate coating as a result of the shrinking process (Fig. 3). The guard cell protoplasts m a y be freed from the matrix (Fig. 4) by allowing the Ca 2 + ions to complex for two hours with sodium citrate buffer (20 m M ; 0.8 M mannitol; p H 7.4) at r o o m temperature. As a result of the concomitant C a 2 + decrease within the membrane, the protoplasts exhibit a certain degree of fragility to osmotic stress and this leads to sudden bursting in hypotonic medium. After five minutes incubation in a solution containing 0.8 M mannitol and 5 m M CaC12, the original m e m b r a n e elasticity of the guard cell protoplasts is restored and with it their ability to swell and shrink in response to external osmotic stress.

Discussion The present study shows that guard cell protoplasts m a y be entrapped in and subsequently released from a cross-linked matrix without any apparent loss of cellular functions and m e m b r a n e integrity. This new technique has a number of possible applications. In this report it is demonstrated that immobilized guard

H. Schnabl et al.: Mechanical Stabilization of Protoplasts cell protoplasts can be stored for long periods of time and even transported. F r o m the osmotic experiments it can be concluded that pressure gradients in isolated immobilized guard cell protoplasts can be built up. This would,.in the future, allow for the study of pressure-related processes in stomata. Since the immobilization technique is generally applicable to other wall-less cells it would also be possible to study both m e m b r a n e transport and the function of other plant protoplasts, animal cells, t u m o r ceils, and lipid vesicles in the presence of a pressure gradient. We thus believe that the polymerization technique offers a new field for m e m b r a n e research. We would like to thank Prof. Dr. H. Ziegler, Technical University, Mtinchen, for stimulating discussions and Dr. D. Tomos, University of Wales, for reading the manuscript. This work was supported by a grant to U.Z. from the SFB 160 and by a grant, Schn 210, to H.S. from the DFG.

References Edwards, M., Meidner, H. (1979) Direct measurements of turgor pressure potentials J. Exp. Bot. 30, 829-837 Hsiao, T.C. (1976) Stomatal ion transport. In: Transport in plants II, Part B, Tissues and organs, pp. 195-221, Liittge, U., Pitman, M.G., eds. Springer, Berlin Heidelberg New York Htisken, D., Steudle, E., Zimmermann, U. (1978) Pressure probe technique for measuring water relations of cells in higher plants. Plant Physiol. 61,158 163 Klein, J., Wagner, F. (1979) Immobilizedwhole cells. In : DechemaMonographien, No. 82, pp. 142-164. Verlag Chemie, Weinheim Pilwat, G., Washausen, P., Klein, J., Zimmermann, U. (1980) Immobilization of cells. Z. Naturforsch. 35c, 352-356 Raschke, K. (1979) Movements of stomata. In: Physiology of movements; Encyclopedia of plant physiology, vol. VII, pp. 383-441, Haupt, W., Feinleib, M.E., eds. Springer, Berlin Heidelberg New York Schnabl, H., Bornman, Ch., Ziegler,H. (1978) Studies on isolated starch-containing (Vicia faba) and starch-deficient (Allium cepa) guard cell protoplasts. Planta 143, 33-39 Schnabl, H., Ziegler H. (1977) The mechanism of stomatal movement in Allium cepa L. Planta 136, 37-43 Zimmermann, U. (1978) Physics of turgor and osmoregulation. Annu. Rev. Plant Physiol. 29, 112-148 Zimmermann, U., Hiisken, D., Schulze, E.D. (in press) Direct turgor pressure measurements in individual leaf cells of Tradescantia virginiana. Planta Zimmermann, U., Steudle,E. (1978) Physical aspects of water relations of plant cells. Adv. Bot. Res. 6, 45 118 Received 8 December 1979; accepted 20 February 1980

Mechanical stabilization of guard cell protoplasts of Vicia faba.

Guard cell protoplasts of Vicia faba were immobilized in cross-linked Ca-alginate. No visible morphological changes were detected under the light micr...
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