Several authors have reported the isolation of guard cell protoplasts (GCPs) from leaves of Vicia faba L. for various purposes (Schnabl et al. 1978; Outlaw et al.
MIRCEN Journal, 1988, 4, 275-283
Rapid method for isolation and purification of protoplasts from epidermal tissue of Vicia faba L. leaves Laura P. Zanello
1,2,-x-, Nestor
R. Curvetto 1 and Francisco
J. Barrantes 2 (1) Centro de Recursos Naturales Renovables de la Zona Semi6rida and (2) Instituto de lnvestigaciones Bioquimicas de Bahia Blanca, Universidad Nacional deI Sur and Consejo Nacional de Investigaciones Cientificas y T~cnicag, 8000 Bahia Blanca, Argentina
Received 8 December 1987; revised 30 March 1988, accepted 15 April 1988.
Introduction
Several authors have reported the isolation of guard cell protoplasts (GCPs) from leaves of Vicia f a b a L. for various purposes (Schnabl et al. 1978; Outlaw et al. 1981; Scheurich & Zimmermann i981; Gotow et al. 1982; Schroeder et al. 1984; Sato 1985; Kottmeier & Schnabl 1986). However, there is only one report in the literature (Sato 1985) on the separation of epidermal cell protoplasts (ECPs) from foliar tissue of the same species. The procedure used by Sato (1985) consists of a three-step enzymatic digestion and a further purification of each foliar cellular type in a Percoll gradient, the whole procedure estimated to take about 6 h including tissue handling. Sato's main objective was to study lipid biosynthesis in foliar protoplasts of the species, for which purpose a considerable amount (0.2 to 1.3 • 106 cells) of protoplast material was needed. However, for morphological (Burgess et al. 1978; Schnabl et al. 1980), physical (Senn & Pilet 1981; Wolfe & Steponkus 1983) and cytochemical studies, less material is required, and the isolation procedure can be accordingly adapted. Furthermore, some experimental methods applied to isolated protoplasts pose very stringent requirements, one of the most important of these being the cleanness of the protoplast surface, for example the absence of cell wall remnants. One such method which requires absolute absence of cell wall remnants for perfect adhesion of a glass microelectrode to the protoplast plasmalemma, is the patch-clamp electrophysiological technique (Schroeder et al. 1984; Kado et al. 1986). In this communication we report a rapid and efficient method for the isolation and purification of viable ECPs and GCPs from leaves of Vicia faba. One operator can *To whom correspondence should be addressed at: Instituto de Investigaciones Bioquimicas de Bahia Blanca, C.C. 857, Camino Vecinal La Carridanga -Km 7, 8000 Bahia Blanca, Argentina (~) Oxford UniversityPress 1988
276
L. P. Zanello, N. R. Curvetto & F. J. Barrantes
accomplish this procedure in approximately 4 h. By simply increasing the initial amounts of plant tissue and digestion medium, the procedure is equally suitable for obtaining a greater quantity of protoplasts of either of the two cell types with the same yield and in almost the same time. The protoplasts obtained show no evidence of cell wall debris, thus fulfilling one of the aims of the present study,
Materials and methods
Plant material Seeds of Vicia faba L. var. major cv. 'sevillana' were germinated and soil-grown in pots in a greenhouse during March. They were subjected to temperatures ranging between 25 to 30~ (day) and 10 to 12~ (night). The plants were watered daily in order to prevent water stress. The 2nd to 4th pairs of fully expanded leaves of 3-weekold plants were harvested after storing the plants in darkness overnight. Protoplast isolation Six to eight leaves were collected and washed with distilled water. Abaxial epidermis was separated with forceps yielding about 10 cm 2 per leaf, as described by Weyers & Travis (1981), and floated in a basal medium on ice. The basal medium contained 0.6 i mannitol, 0.1% (w/v) bovine serum albumin, 100 p~M ascorbic acid, 0.1% (v/v) 2mercaptoethanol, and 1 m i CaC12, in 5 mM MES/KOH buffer, pH 5.8. Previous procedures for the isolation of protoplasts from epidermal tissue have included bovine serum albumin (Gotow et al. 1982; Shimazaki et al. 1982), or ascorbic acid (Outlaw et al. 1981) in the medium in order to improve viability and yield. After a pre-plasmolysis treatment for 30 min at 0~ in the same buffer, the peeled material was transferred to 5 ml of the first incubation medium, consisting of basal medium containing 0.5% (w/v) cellulase Y-C (Seishin Pharmaceutical Co., Tokyo, Japan) and 0.25% (w/v) pectinase (Sigma Chemical Co., St. Louis, MO, U.S.A.). The first incubation period was for 90 min at 22~ Thereafter, the material was gently drained to release the ECPs, removed from the digestion bath, and transferred to a second incubation medium consisting of 2% (w/v) cellulase Y-C and 0.1% (w/v) pectolyase Y-23 (Seishin Pharmaceutical Co.) in basal medium made up to 0.55 i in mannitol, for a further 90 min at 22~ The first incubation medium was subsequently filtered through Miracloth filters (mean pore diameter 100 p~m, Calbiochem, Behring Diagnostics, La Jolla, CA), and centrifuged at 500 • g for 10 min. The pellet, consisting of ECPs, mesophyll protoplasts, chloroplasts and debris, was gently resuspended in 5 ml basal medium made up to 30% (v/v) in Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden), transferred to a Babcock bottle, covered with 0.5 ml of 20% (v/v) Percoll in basal medium, and centrifuged at 800 x g for 5 min. Buoyant ECPs were recovered at the 20/30% interface by pipetting, washed in basal medium and pelleted by centrifugation at 500 • g for 10 min. Aliquots were taken for staining with neutral red, Evans blue, and Calcofluor, counting, measuring, and fixation for transmission electron microscopy. Samples from the bottom and middle of the remaining solution in the bottle were also taken for microscopic observation. In order to determine the existence of different populations of ECPs, purified ECPs were subjected to a further centrifugation step (800 • g, 5 min.) in a discontinuous
Isolation of Vicia faba protoplasts
277
Percoll density gradient consisting of 0, 10 and 30% (v/v) Percoll in basal medium containing 0.6 M mannitol. Samples from the 0/10% and 10/30% interfaces were taken for counting and cell diameter measurement. Once the second incubation period was concluded, the peeled material was gently shaken and washed in a small volume of new basal medium containing 0.55 U mannitol, in order to release the remaining GCPs. The second incubation medium and washing solution were then filtered through Miracloth, pooled, and centrifuged at 400 x g for 10 min. The pellet was gently resuspeuded in 0.5 to 1 ml of 10% (v/v) Percotl in basal medium containing 0.55 M mannitol. Purification of GCPs was performed in a discontinuous Percoll density gradient formed in haematocrit tubes, consisting of 1 cm layers of 10, 20, 40 and 80% (v/v) Percoll in basal medium containing 0.55 M mannitol, from top to bottom. After centrifugation at 400 x g for 10 min, the purified GCPs were found layered on the 40/80% boundary. Purified GCPs were also stained with neutral red, Evans blue and Calcofiuor, counted, measured, and prepared for transmission electron microscopy. The isolation procedure was run three times, starting with different plant material grown under the same environmental conditions.
Cell counting Cell number was determined for each protoplast type by using a Neubauer cell counting chamber, using the area corresponding to leucocyte counting. Four counts were taken in each experiment, and protoplast yield was calculated.
Cell measurement Cell diameters of protoplasts suspended in basal medium containing 0.6 M or 0.55 M mannitol were measured with an eyepiece micrometer. The mean diameter of each protoplast type - ECPs and GCPs obtained at the 20/30% and 40/80% Percoll interfaces - was calculated over 30 measurements. The mean diameter of each EPC population - obtained at the 0/10% and 10/30% Percoll interfaces - was calculated over 100 measurements.
Viability tests Protoplast viability was determined according to the general procedures proposed by several authors (Larkin 1976; Wagner et al. 1978). One drop of protoplast suspension was mixed with a drop of 0.1% (w/v) neutral red or 0.1% (w/v) Evans Blue in 0.6 M or 0.55 M mannitol, and the percentage of viable protoplasts determined using a Reichert microscope at a magnification of 125.
Assessment of complete cell wall digestion One drop of protoplast suspension was mixed with a drop of 0.1% (w/v) Calcofiuor (Fluorescent Brightner 28, Sigma Chemical Co., St. Louis, MO, U.S.A.) in 0.6 M Or 0.55 r~ mannitol. Microscope observations were performed in a Nikon episcopicfluorescence microscope (Nikon Optiphot XF-EF), using 365 nm excitation and 420 nm emission.
278
L. P. ZaneUo, N. R. Curvetto & F. J. Barrantes
Transmission electron microscopy ECP and GCP pellets were resuspended in 4% (w/v) glutaraldehyde in 100 mM NazHPOa/NaH2PO4 buffer, pH 7.2, made up to 0.4 M mannitol for ECPs and 0.31 ~a for GCPs, for 2h at 0~ (Roland 1978). After fixation, the cells were gently centrifuged. The fixed pellets were washed three times with Na2HPO4/NaH2PO4 buffer for 10 min each time. They were then fixed with 1% (w/v) OsO4 in 50 mM Na2HPO4/NaH2PO4 buffer for 2 h at 0~ This was followed by four 10-min washes with Na2HPO4/NaHaPO4 buffer and double distilled water, dehydration in ethanol series, and finally embedding in Spurt low-viscosity medium. Ultrathin sections were cut with an LKB Ultratome V ultramicrotome. Conventional double post-staining of the sections was done with uranyl acetate and lead citrate. Specimens were examined with a Jeol JSM-100 CXII electron microscope and photographed at 2,700 to 67,000 magnification on Agfa-Gevaert film. Results and discussion
ECP isolation
Aliquots of ECPs recovered at the 20/30% Percoll interface (Fig. la) indicated a recovery of about t.32 • I05 elements/mt. This represents a yield of 930 protoptasts/ cm 2 epidermal tissue, about 6% of potential protoplast production calculated by considering about 75 stomata/mm 2 epidermal tissue and an ECP/GCP ratio of 3:1 (Curvetto 1986). The ECP preparation was 100% pure as judged by microscopic observation; no mesophyll cell protoplasts were found at the 20/30% interface. Diameter measurement of these cells gave values of 37.8 + 10.7 p~m. Though individual cells had diameters from 16 to 60 ~m, 70% of them were within the narrower 28 to 48 ~m range. Rapid accumulation of neutral red in the vacuoles and exclusion of the non-permeating dye Evans blue revealed about 90% viability. Calcofluor staining revealed no cellulosic remnants on the surface of ECPs, confirming the total digestion of cell walls. Electron microscope observations confirmed the integrity of the plasmalemma and tonoplast (Figs. lb, c). Samples from the middle and bottom of Babcock bottles showed that debris, chloroplasts, and mesophyll protoplasts were released with the first incubation. As in the case reported by Sato (1985), ECPs also separated in two populations of 'light' and 'heavy' cells when subjected to discontinuous density gradient centrifugation in Percoll. They were recovered from the 0/10% and 10/30% interfaces, with a relative abundance of 60% and 40% respectively. A relatively small quantity of ECPs of low density was found on the 0% Percoll layer; these protoplasts required centrifugal forces higher than 800 • g for pelleting. The reason for the occurrence of ECPs of different densities remains unclear as yet. Estimates of the mean diameter of each fraction (Fig. 2) did not indicate any significant statistical difference (p < 0.05). From these results it follows that Sato's proposal that each type of cell has a different diameter does not seem to be warranted. The difference in density of our protoplasts with respect of those of Sato (1985) may reside in the variety of broad bean used and environmental growth conditions. The time required for ECP isolation using this procedure including peeling, incubation, purification and washing was only 130 to 140 min. This duration compares very favourably with those of other reported procedures (Schnabl et al. 1978; Sato
25
2O
o ~
15
--+
~.
+~ 10
\
~+"
5//+,/,/ C,
12
16
\ \
~
""'%20
24
28
32
36
40
44
48
52
56
60
Cell diameter ~um)
Fig. 2 Diameters of the two populations of ECPs separated by centrifugation in a Percoll gradient. Mean cell diameters (+ S.D., n = 100) of light (11) and heavy (+) protoplasts are 31.88 + 8.52 Ixm and 32.16 _+ 8.39 ~m, respectively. See text for details.
Fig. 3 Guard cell protoplasts isolated from the abaxial epidermis of Viciafaba leaves, a: light micrograph of clumped GCPs stained with Neutral Red 0.1%; b, c: electron micrographs of one GCP showing the well preserved cytoplasmic structures. Ch: chloroplast; M: mitochondrion; N: nucleus; Pro: plasma membrane; V: vacuole.
280
L. P. Zanello, N. R. Curvetto & F. J. Barrantes
Fig. I Epidermal cell protoplasts isolated from the abaxial epidermis of Vicia faba leaves, a: light micrograph of a drop of purified ECPs; b, c: electron micrographs of one ECP showing its large vacuole containing several osmophyllic lipid globules (c), and a detail of the well preserved plasma membrane, tonoplast and organelles (b). C: cytoplasm; Lg: lipid globule; Pm: plasma membrane; T: tonoplast; V: vacuole.
1985). This time saving procedure has the added advantage of avoiding unnecessarily prolonged exposure of protoplasts to the enzyme solution.
GCP isolation Counts of G C P s found at the 40/80% Percoll interface (Fig. 3a) indicated a recovery of 3.7 • 105 elements/ml, corresponding to a yield of 2,200 protoplasts/cm 2 foliar area, about 10% of potential protoplast production. GCPs were smaller in size than ECPs,
Isolation of Vicia faba protoplasts
281
with a mean diameter of 16.2 + 1.9 txm. About 95% of GCPs rapidly accumulated neutral red in their vacuoles (Fig. 3a), and excluded Evans blue from the cytoplasm. The ultrastructure of the well preserved GCPs corresponds to the description given by Allaway & Setterfield (1972) for guard cells in foliar tissue of Vicia faba (Fig. 3b, c). Samples from the remaining interfaces of the discontinuous Percoll density gradient were taken for light microscope observation, GCPs were also present at the 20/40% Percoll interface and at the bottom of the tubes, but in lesser quantities than those recovered from the 40/80% boundary, and with a high degree of contamination, On the other hand, GCPs at the 40/80% boundary were 100% pure. A considerable proportion of GCPs were found clumped in groups of up to a hundred (Fig. 3a). This was not an indication of incomplete digestion of the cell walls, as Schnabl et al. (1978) supposed. The absence of cellulose microfibrils was checked by staining with Calcofluor. We think that clumping, which was not present among ECPs, could be the result of a neutralization of surface charges, a possibility which is currently under study. With the procedure described here we were able to obtain good preparations of purified ECPs and GCPs in a reasonably short time, with only one operator and using small amounts of enzymatic media. In our laboratory we have run other previously documented protocols for the same purpose, but preparations fulfilling all the quality criteria reported here were seldom obtained.
Acknowledgements We are grateful to Eng. Silvia E. Delmastro for her technical assistance in the preparation of specimens for transmission electron microscopy.
References ALLAWAY,W. G. & SETrERFIELD, G. 1972 Ultrastructural observations on guard cells of Vicia
faba and Alliurn porrum. Canadian Journal of Botany 50, 1405-1414. BURGESS, J., LINSTEAD,P. J. & BONSALL,V. E. 1978 Observations on the time course of wall development at the surface of isolated protoplasts. Planta 139, 85-91. CURVErro, N, R. 1986 Thesis. Universidad Nacional del Sur, Bahia Blanca, Argentina, Gorow, K., KONDO, N. & SYONO, K. 1982 Effect of CO2 on volume change of guard cell protoplasts from Vicia faba L. Plant and Cell Physiology 23, 1063-1070. KADO, R, T., KURKDJIAN, A. & TAKEDA, K. 1986 Transport mechamsms in plant cell membranes: An application for the patch-clamp technique. Physiologie Vegetale 24, 227-244. KO~MEIER, C. & SCHNABL,H. 1986 The Km-value of phosphoenolpyruvate carboxylase as an " indicator of the swelling state of guard cell protoplasts. Plant Sciences 43,213-217. LARKIN, P. J. 1976 Purification and viability determinations of plant protoplasts. Planta 128, 213-216. OUTLAW, W. H., MAYNE, B. C., ZENGER, V. E. & MANCHESTER,J. 1981 Presence of both photosystems in guard cells of Vicia faba L., Plant Physiology 67, 12-16. ROLAND, J. C. 1978 In: Electron microscopy and cytochemistry of plant cells. Hall JL, ed. pp. 1-62. Elsevier/North-Holland Biomedical Press. SATO~N. 1985 Lipid biosynthesis in epidermal, guard and mesophyll cell protoplasts from leaves of Vicia faba L. Plant and Cell Physiology 26, 805-811. SCHEURICH, P. & ZIMMERMANN, U. 1981 Electrically stimulated fusion of different plant cell protoplasts. Mesophyll cell and guard cell protoplasts of Viciafaba L. Plant Physiology ~7, 849-853. SCHNABL,H., BORNMAN,C. H. & ZIEGLER,H. 1978 Studies on isolated starch-containing (Vicia faba) and starch-deficient (AUium cepa) guard cell protoplasts. Planta 143, 33-39.
282
L. P. Zanello, N. R. Curvetto & F. J. Barrantes
SCHNABL, H., VIENKEN, J. & ZIMMERMANN, U. 1980 Regular arrays of intramembranous particles in the plasmalemma of guard cell and mesophyll cell protoplasts of Vicia faba. Planta 148, 231-237. SCHROEDER,J. I., HEDRICH, R. & FERNANDEZ,J. M. 1984 Potassium-selective single channels in guard cell protoplasts of Vicia faba. Nature (London) 312, 361-362. SENN, A. & PILET, P. E. 1981 Electrophoretic mobility, zeta potential and surface charges of maize root protoplsts. Z. Pflanzenphysiologie 102, 19-32. SHIMAZAKI,K., GOTOW,K. & KONDO, N. 1982 Photosynthetic properties of guard cell protoplasts from Vicia faba L. Plant and Cell Physiology 23, 871-879. WAGNER, G. J., BUTCHER, H. C. & SIEGELMAN,H. W. 1978 Plant protoplasts. A useful tool for plant research and student instruction. BioScience 28, 95-101. WEYERS, J. D. B. & TRAVIS, A. J. 1981 Selection and preparation of leaf epidermis for experiments on stomatal physiology. Journal of Experimental Botany 32, 837-850. WOLFE, J. & STEPONKUS,P. L. 1983 Mechanical properties of the plasma membrane of isolated plant protoplasts. Plant Physiology 71, 276-285.
Summary A method has been developed for isolating and purifying epidermal and guard cell protoplasts (ECPs and GCPs) from leaves of Viciafaba L. This method has three essential characteristics: 1) requires only small quantities of initial plant tissue; 2) is rapid, being based on a two-step enzymatic digestion and purification by discontinuous density gradient centrifugation using Percoll, and 3) gives a high viability of purified protoplasts. The procedure yielded about 6% ECPs and 10% GCPs on the basis of their occurrence on epidermal foliar tissue, the final suspension of protoplasts being 100% pure. Cell viability was assessed by the ability of each protoplast type to accumulate neutral red in their vacuoles. Values of 90% and 95% were obtained for ECPs and GCPs respectively. The complete lack of cell wall after enzymatic treatment was checked at the light microscope level by the absence of Calcofluor fluorescent staining of cellulosic material. Representative counts for purified ECPs and GCPs obtained at the interfaces of 20/30% and 40/80% Percoll gradients were 1.32 • 10s and 3.7 x 105 elements/ ml, which represents a yield of 930 and 2,200 protoplasts/cm 2 of epidermal tissue respectively. The integrity of the plasma membrane and organelles after the isolation procedures was confirmed by transmission electron microscopy and by the ability of protoplasts to exclude Evans blue.
R6sum6 M(thode rapide pour l'isolernent et la purification de protoplastes du tissu (pidermique des feuilIes de Vicia faba L. Une m6thode a 6t6 d6velopp6e pour l'isolement et la purification de proto-plastes 6pidermiques (ECPs) et de cellules de garde (GCPs) de feuilles de Viciafaba L. Cette m6thode pr6sente trois caract6ristiques essentielles: 1) Elle ne requiert que de petites quantit6s du tissu v6g6tal originel; 2) elle est rapide, car elle se base sur une digestion enzymatique en deux 6tapes et une purification dans un gradient discontinu de densit6 utilisant le Percoll, et 3) elle fournit une forte proportion de protoplastes purifi6s bien vivants. La proc6dure fournit environ 6% d'ECPs et 10% de GCPs sur la base de leur pr6sence dans le tissu foliaire 6pidermique et d'une suspension pure a 100% de protoplastes. La viabilit6 des cellules a 6t6 test6e par la capacit6 de chaque type de protoplaste d'accumuler de rouge neutre darts ses vacuoles. On a obtenu des valeurs de 90% et de 95% respectivement pour les ECPs et les GCPs. L'absence totale de parDi cellulaire apr~s le traitement enzymatique a 6t6 v6rifide au microscope optique par l'absence de fluorescence apr6s coloration du mat6riel cellulosique par le calcofluor. Des comptages typiques pour les ECPs et les GCPs purifi6s obtenus aux interfaces 20/30% et 40/80% des gradients de Percoll ont donn6 1.32 x 105 et 3.7 x 105 616ments/mt, ce qui repr6sente des rendements respectifs de 930 et de 2200 protoplastes par cm 2 de tissu 6pidermique. L'intdgrit6
Isolation of Vicia f a b a protoplasts
283
de la membrane plasmique et des organites apres les procedures d'isolement a 6t6 confirmee par microscopie electronique ~ transmission et par la papacite de protoplastes d'exclure le bleu d'Evans.
Resumen
Mdtodo rdpido de aislamiento y purificaci6n de protoplastos a partir de tefido epidermico de hojas de Vicia faba L. Se ha desarrollado un metodo para el aislamiento y purificaci6n de protoplastos de celulas epidermicas y celulas guardianas (PCEs y PCGs) de hojas de Viciafaba L. Este metodo posee tres caracteristicas esenciales: 1) solamente requiere pequefias cantidades de tejido vegetal inicial; 2) rapidez, en base a una digesti6n enzim~ttica de s61o dos etapas y centrifugaci6n en gradiente discontinuo de Percolt, y 3) la elevada viabilidad de los protoplastos purificados. Este metodo permiti6 obtener ca. 6% de PCEs y 10% de PCGs sobre la base de su ocurrencia en el tejido epidermico foliar, con una pureza eel 100% para las suspensiones finales de protoplastos. Se determin6 la viabilidad de cada tipo celular por su habilidad de acumular rojo neutro en sus vacuolas, obteniendose valores de 90% y 95% para PCEs y PCGs respectivamente. Se determine la ausencia total de pared celular despues del tratamiento enzim~ltico mediante microscopia de fluorescencia con Calcofluor, especffico para sustancias celul6sicas. El recuento de PCEs y PCGs purificados - obtenidos en las interfases 20/30% y 40/80% del gradiente de Percoll - fue de 1,32 • 105 y 3,7 x 105 elementos/ml, 1o cual represent6 an rendimiento de 930 y 2200 protoplastosi cm 2 de tejido epiddrmico respectivamente. La integridad de la membrana plasmfitica y de las organelas rue confirmada por microscopia electrdnica de transmisidn y pot la habilidad de los protoplastos de excluir azul de Evans.
