Biochem. Physiol. Pflanzen 180, 645-653 (1985)

The Physiology of Grapevine (V it is vinifera L.) Protoplasts Isolated from Green and Senescing Leaves

L. F. DE FILIPPIS and H. ZIEGLER Institllt fiir Botanik der Technisehen Universitiit, ~Iiinchen,

Federal Republic of Germany

Key Term Index: protoplasts, respiration, photosynthesis, efflux of metabolites; Vi tis vinifera

Summary

~Iesophyll protoplasts were isolated from 3 cllltivars of grapevines (l'itis t'/:nifera L.). Various digestion enzymes were tested, and only 3 cl'llulases Wl're fountl to release protoplasts

from leaf slices. Individually, even these 3 digestion enzymes produce low numbl'rs of protoplasts, but when all 3 were used together, up to 106 protoplasts/ml/g tissue were obtained. ~Iesophyll proto­plasts from grel'n leaves were found to bp OVl'r 90% intact, small in tliaml'ter (X = 10.4,um), carried out high rates of respiration and photosynthesis (as measured by O2 exehange); and were found to fix 14C02 • Rates of photosynthesis were generally higher than in intact plants, but within the same order of magnitude as other mesophyll protoplasts. Protoplasts from senpsring leans were also over 90 % intact, significantly larger in diameter (X = 15.8,um), carried out lower rates of respiration, but had signifieantly lower rates of photosynthesis. Sem'seing protoplasts had grl'ater rates of efflux of metabolites than the green protoplasts.

Introduction

Isolated plant cells (protoplasts), especially from photosynthetic tissue (mesophyll) have now been used to study the physiology, biochemistry and totipotency of plants. The preparation of intact viable protoplasts with the ability to regenerate into callus and plantlets, has with a few exceptions been virtually restricted to the Solanacean family (CREPY et al. 1982). The inability to even produce protoplasts from some species is a serious shortcoming in the utilization of protoplasts for applied plant physiology and breeding purposes. Intact protoplasts, or for that matter intact chloroplasts and mitochondria from the European Grapevine (Vitis vinifera 1.) have been difficult to achieve. Even with recent improved methods, removal of membranes is often unavoid­ably accompanied by the loss of some essential components of the respiratory and photo­synthetic electron transport chain.

A few reports successfully isolating protoplasts from grapevine have been published by HASLER et al. (1982, 1983). These protoplasts were intact, they were able to fix 14COZ in a similar manner to intact plants, and under the electron microscope appeared to keep their structural integrity. Their isolation method used leaf slices infiltrated under reduced pressure, and the medium contained polyethylene gl~'col and high con­centrations of salts such as CaCI2, MgClz and KCI. Grapevine protoplasts have also been isolated from peeled (and therefore detached) berry skin, with the aid of a complex

646 L. F. DE FILIPPIS and H. ZIEGLER

protoplast medium and a very long incubation period in a digestion enzyme (BROWN and COOMBE 1984). It is now clear that the biochemistry and division of protoplasts are adversely affected by storage under a high concentration of osmoticum, addition of high concentration of salts (e.g. CaCI2) and long periods of incubation in digestion enzymes (KAISER and HEBER 1983; WAKASA et al. 1984).

We report here on a simple and quick method of isolating intact protoplasts from leaves of 3 different cultivars of grapevines grown under glasshouse conditions. Proto­plasts were isolated from green and senescing leaves, and their physiology compared.

Materials and Methods

Plants and Isolation of Protoplasts

Potted grapevine plants (Vilis villifera L. cv Baeehus, Contessa and GA 47-42) were kept in growth cabinets at 20°C and under continuous illumination (100 W . m-2). The oldest plants were 6 months old, and fully expanded leaves were always used.

Leaves of grapevines were detached and wounded (punctured) with a needle to ensure plasmolysis whieh helped to rell'ase a greater number of protoplasts (BOKELMANN and ROEST 1983). Leaves were then cut into segments (1 mm2) and incubated in 0.6 M mannitol, 1 mM CaCI2, 0.5 % bovine serum albumin (BSA), 1) ml\I MES-KOH (pH 5.6) and various concentrations of digestion enzymes (HAMPP and ZIEGLER 1980). Only 3 eellulases wen' found to be effl'etivc, each from a different species of fungus:

Cellulysin (Trichoderma viride) (Calbioehem); Cellulase (Aspergillus niger) (Sigma); Cellulase (Penicillium funiculosurn) (Sigma).

The resulting protoplast suspension was filtered through a nylon net (lOO.um), and purified by floating twice on a suerose-mannitol gradient eontaining 0.75 M sucrose and 1 mM CaCl2 oVl'rlayed with 1 ml buffered medium containing 0.5 mannitol, 1 mM CaCI2 and IS mM MES-KOH (pH 6.0). The protoplasts WPTe eollected and resuspended in a medium containing 0.4 M sorbitol, 1 mM CaCI2, 5 mM KHC03 and 25 mM Tricine (pH 8.0); a bicarbonate medium suitable for O2 eledrodc measure­ments and HC02 fixation (DE FILIPPIS et a!. 1980). Protoplasts werl' stored on ice (dark) in the bi­carbonate medium, and used within one hour of isolation.

Oxygen Exchange alld [14 CJ CO2 Fixation

Respiratory oxygen uptake or photosynthetic oxygen evolution of cells in the light (120 W . m-2) was measured with a Clark-type O2 electrode at 20°C as described by DELIEU and WALKER (1972). The rates of CO2 fixation were determined at 20°C by introducing 5 mM [HC] KHC03 (0.5,uCi . ,umol-1 ) III the dark or light (120 W . m-2) as deseribed by DE FILIPPIS et a!. (1980).

Efflux of I{ alld HC-Fixed Products from Cells

To measur~ th~ release of labelled fixation products, washed protopla~ts were allowed to fix HC02 at 20°C for 15 min as described above. For K efflux, cells were allowed to take up the normal levels of K from th~ medium. In both cases, cells were washed 3 times with fresh bicarbonate medium (carbon efflux) or K-dcfieient medium (K efflux) and gently resuspended. After incubation for another 30 min (exchange of HC·labl'lIed compounds or K), aliquots wer~ centrifuged through 50,u1 silicon oil AR80 (60 s, 19,000· g, 20°C; Beckman, microfuge 152). Protoplast cells were at the bottom and the cell-free supernatant collceted. K inside the cells was released by heating in a boiling water bath, and the K content of both cl'll·free supernatant and extract~d pellet was determined using a flame photometer (DE FILIPPIS and HAMPP 1980). HC·labelled compounds in the supl'rnatant and the pellet after centrifugal filtration were determined as described above.

Protoplasts from Green and Senescing Grapevines 647

Protoplast Numbers, rital Staining, Protein alld Chlorophyll

Protoplast numbl'rs Wl'fe counted with an improved double Neubauer haema('ytometer (Assistent), and their diameters measnred with a graduated micrometer scalp. Protein was measured by the naph­thalene blue-black procedure according to BIUMIIALL et al. (1969) and chlorophyll as deseribed by ARNON (1949). Suffieipnt 0.3 % Neutral Red (pH 7.0) (Merek) was added to bring the final concentra­tion to 0.06 % or sufficient 0.5 % Evans Blue (pH 7.0) (Serva) for a final concentration of 0.25 %. Eaeh dye was in 0.5 :\f mannitol and protoplasts were stained for no longer than 1 h.

Results and Discussion

'Cell Wall Degrading Enzymes and Protopla8is Optimum numbers of protoplasts were achieved using between 0.5 and 0.75 g leaf

material per 5 ml of enzyme mixture, and in fact using more tissue per ml of enzyme appeared to inhibit protoplast production. Wounding the tissue was not essential for production of protoplasts as observed by BOKELlIfAXX and ROEST (1983) but helped greatl~- to increase protoplast numbers. This was probabl~r due to better plasmolysis and also by increasing the cut surface area of tissue in contact with the digestion en­zymes.

When investigating different digestion enzymes commonly used to produce proto­plasts, only the enzyme cellul~-sin by itself produced protoplasts. But the numbers produced were low (Fig. 1), and independent of enzyme concentration (up to 4%).

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648 L. F. DE FILIPPIS and H. ZIEGLER

Various other cellulases, pectinases, hemicellulases and commercial mixtures of cellu­lases, pectinases, and proteases were found to produce no protoplasts. It was then decided to investigate various combinations of cellulase enzymes from different companies and also extracted from different fungal species. Only 3 cellulases were found to be effective, each from a different species of fungus as outlined in Materials and Methods.

The surprise finding was that the combination of the three cellulases produced a synergistic effect with numbers of protoplasts extracted far in excess of numbers ex­pected from the individual combinations of enzymes (Fig. 1). The digestion time was quite critical and most protoplasts were achieved between 2 and 2.5 h of incubation, by 3, 4 and 6 h less and less protoplasts were obtained (Fig. 1). These results point out three important features: Firstly, that various digestion enzymes commonly used to produce protoplasts are possibly not pure, and over a long exposure time various small quantities of proteases and lipases (impurities) are detrimental to protoplast membranes, as also reported previously (DE FILIPPIS and HAMPP 1980). In fact VAN DER VALK (1984) has found high protease activity in most commercial mixtures of digestion en­zymes. Their possible destructive effect on membranes is acknowledged, especially with time and elevated temperatures. This may explain the lower number of intact protoplasts extracted after 3 h of incubation. Secondly, it may be possible that cellulases extracted from different fungi act in different ways and at different sites on the same plant organ. This is a possible explanation of the strong synergistic effect. It is clear that in some plants a combination of digestion enzymes may be successful where single enzymes were not. Thirdly, and not surprisingly only cellulase enzymes were effective, other digestion enzymes although at times effecting good tissue digestion released no intact proto­plasts. This suggests that digestion and release of intact protoplasts from tissue are not necessarily related.

Intactness and Diameter of Protoplasts All protoplasts isolated were over 90 % intact (Table 1) as assessed by the exclusion

of Evans Blue (a dye indicating intact plasmalemma) and accumulation of Neutral

Table 1. Respiration, photosynthesis and intactness of grapevine protoplastsisolated from green and senescing leaves. The valueE are the range obtained from the means of 2 replicated with 5 parallels in each treatment, or the means of at least 100 randomly selected protoplasts viewed under the haemacytometer. 106

green protoplasts correspond to 10.8 ,ug chlorophyll or 341,ug of protein; 106 senescing protoplasts correspond to 3.1 flg chlorophyll or 286,ug of protein

Protoplast Type

Green

Sencscing

Intact Cells (%)

Evans Blue Neutral Red

92-95

92-94

91-97

96-98

O2 Exchange (,umol/h/1 OS cells)

Respira­tion

Photosynthe­sis

0.11-0.19 0.89-1.03

0.08-0.15 0.17-0.20

14C02 Fixation (,umol/h/106 cells)

1.05-1-27

0.22-0.29

Protoplasts from Green and Senescing Grapevines 649

Red (a dye indicating intact tonoplast). Protoplasts isolated from green leaves appear to be similar to isolated protoplasts as described by other authors (HASLER et al. 1983) but smaller in diameter (X = 10.4,um), and different to protoplasts from the berry skin (Fig. 2). The latter cells having larger vacuoles and fewer chloroplasts, and also being larger than the mesophyll protoplasts as detailed below:

Mesophyll protoplasts: 8-32,um (HASLER et al. 1983) Berry skin protoplasts: 50-60,um (BROWN and COOMBE 1984)

On the other hand protoplasts extracted from senescing leaves have significantly greater protoplast diameters (X = 15.8,um), have fewer chloroplasts and larger vacu­oles; thus resembling more closely berry skin protoplasts (Fig. 3). The difference in

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PROTOPLAST DIAMETER (~m) PROTOPLAST DIAMETER (~m)

Fig. 2. Size distribution of grapevine protoplasts. A: Protoplasts from green leaves (X = 10.38,um; SD = 3.46; SE = 0.20; n = 315), B: Protoplasts from senescing leaves (X = 15.81,um; SD = 3.61; SE = 0.20; n = 317).

Fig. 3. Typical protoplasts from grapevines viewed in a haemacytometer. A: Protoplasts from green leaves, B: Protoplasts from sencscing leaves. Bar represents 15,um.

650 L. F. DE FILIPPIS and H. ZIEGLER

protoplast size may only reflect that the plants used for this study were young, with leaves only about half the final size they attain in the field. It remains to be tested later this summer whether the same isolation method is effective in extracting large numbers of intact protoplasts from mature grapevine leaves grown in the field. The problem with mature, field grown leaves may be the higher concentrations of phenolics and tan­nins.

Photosynthesis and Respiration of Protoplasts Protoplasts isolated in large numbers after 2 h of incubation in the three cellulase

enzymes have rates of respiration and photosynthesis (Table 1) in the same order of magnitude as other intact protoplasts from Ca plants (HAMPP and GOLLER 1983; KAISER

and HEBER 1983). These photosynthetic rates however are higher than those pre­viously reported for isolated protoplasts from grapevines (HASLER et al. 1982) and intact leaves (MALIPIERO et al. 1981). It is not surprising that protoplasts in a high bicarbonate buffered medium have high rates of photosynthesis, especially when exposed to uniform high light intensities (120 W . m-2), with little or no shading of light such as is present in intact leaves. It is also well known that high inorganic salt content in the medium (especially CaCI2) does inhibit photosynthetic oxygen evolution (KAISER

and HEBER 1983). Naturally varietal differences and the physiological state of the rootlings may be other factors influencing photosynthetic rates.

Not surprisingly protoplasts isolated from senescing leaves have decreased rates of photosynthesis, with respiration not so strongly inhibited. Even when photosynthesis is expressed per mg chlorophyll, photosynthetic rates are lowered in senescing material. The data obtained from protoplast is therefore consistent with data previously reported with intact plants and also using other plant species (RHODES 1980).

Efflux and Protoplast Technology In Table 2 results are given that show the efflux of labeled fixation products and the

major accumulated inorganic cation, potassium from green protoplasts. Less than 10% of accumulated K and 14C-Iabelled products are transported out of protoplasts, in­dicating that at least 90% of cells are intact. The 10% lost could be accounted for solely

Table 2. Efflux of K and 14e-Iabeled fixation products froln green grapevine protoplasts over 30 min. The values are the means of 2 replicates with 5 parallels each. 1()6 protoplasts correspond to 12.3 fig chlorophyll or 354 fig of protein

Efflux 14C (flmolj106 cells) K (nmolj1OS cells) Conditions inside outside % released inside outside % released

Dark O°C 0.82 0.07 7.9 2.02 0.19 8.6

20°C 0.85 0.08 8.6 2.03 0.20 9.0

Light O°C 0.89 0.09 9.2 2.02 0.20 9.0

20°C 0.86 0.10 10.4 2.05 0.21 9.3

Protoplasts from Green and Senescing Grapevines 651

Table 3. Efflux of K alld 14 (;-/abeled fixation products froll! sellescing grapevine Plotop/asts over 30 min. The values are the means of 2 replicates with 5 parallels each. 106 protoplasts correspond to 2.9 pg chlorophyll or 271 pg of protein

Efflux HC (pmol/1OS eells) K (nmol/106 rells) Conditions inside outside % released inside outside % released

Dark O°C 0.23 0.04 14.8 1.85 0.32 14.7

20°C 0.21 0.05 19.2 1.89 0.36 16.0

Light o °C 0.22 0.05 18.5 1.94 0.40 17.1

20°C 0.25 0.07 21.9 1.96 0.46 19.0

by broken protoplasts, which according to the dye indicators used amount to about 10% of cells. In Table 3 results of a similar experiment are given, but now protoplasts from senescing leaves are used. These protoplasts now have rates of efflux of K and 14C-Iabelled products twice that of green protoplasts. This cannot be explained by more protoplasts not being intact, and reflects a greater permeability of senescing cell mem­branes to ions and organic compounds.

This permeability change is consistent with some reports detailing metabolic turn­over and transport from the leaf to other parts of the plant, as the process of senescence continues (RHODES 1980). It is therefore important for the carbohydrate and ionic balance of the grapevine that leaves be left on the plant for as long as possible. It is well known that organisms possess the ability to maintain high internal levels of K (DE FILIPPIS and HAlVIPP 1980). This is achieved by an energy requiring transport mechanism located in, or on the cell membrane. Therefore any plasmalemma permeability is due to some damage to these transport systems and loss of K or macromolecules. Especially so K efflux, which is an extremely sensitive indicator of membrane damage even when visually membranes appear intact.

Successful isolation of intact functioning protoplasts is only the first, but an im­portant step in the use of protoplast technology for the genetic improvement of crop plats. In fact to date there have been few reports on intact protoplast isolation from horticultutally important plants (especially hard-wood species). The relative short time needed to produce large numbers of protoplasts, their intactness and viability in a medium of low osmotic potential makes biochemical and physiological investigations relevant. It should also enable, with more certainty, the next step in protoplast tech­nology, i.e. regeneration of protoplasts into a callus (W AKASA et al. 1984). This is the last and final step to be achieved in the complete cycle from isolated protoplasts to plantlet production in grapevines, because the tissue culture techniques needed for plantlet production from stem tip meristem callus is already well known (BARLASS and SKENE 1980).

Naturally other techniques involved in genetic manipulation of plants can be de­veloped, such as protoplast fusion which can be observed and affected under the light

652 L. F. DE FILIPPIS and H. ZIEGLER

microscope (VIENKEN et al. 1983), selection of protoclonal variants and mutants, and interaction between evolutionary divergent species.

Acknowledgements

The authors gratefully acknowledge the donation of grapevine plants by Prof. M. G. ALLEWELDT and Dr. R. RUHL, helpful suggestions by Dr. D. GAFF, and financial support from the Alexander von Humboldt-Stiftung (LDF).

References

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BARLASS, M., and SKENE, K. G. M.: Studies on the fragmented shoot apex of grapevine. I. The regenerative capacity of leaf primordial fragments. J. Exptl. Bot. 31, 483-488 (1980).

BOKELMANN, G. S., and ROEST, S.: Plant regeneration from protoplasts of potato (Solanum tuberosum cv. BINTJE). Z. Pflanzenphysiol. 109, 259-265 (1983).

BRAMHALL, S., NOACK, N., Wu, M., and LOEWENBERG, J. R.: A simple colorimetric method for determination of protein. Anal. Biochem. 31, 146-148 (1969).

BROWN, S. C. and COOMBE B. G.: Solute accumulation of grape pericarp cells. II. Studies with protoplasts and isolated vacuoles. Biochem. Physiol. Pflanzen 1'19, 157-171 (1984).

CREPY, L., CHUPEAU, M.-C., and CHUPEAU, Y.: The isolation and culture of leaf protoplasts of Cichorium intybus and their regeneration into plants. Z. Pflanzenphysiol. 10'1, 123-131 (1982).

DE FILIPPIS, L. F., and HAMPP, R.: An improved method of measuring photosynthetic electron transport in Euglena under in vi vo conditions. Arch. Microbiol. 126, 237-243 (1980).

DE FILIPPIS, L. F., HAMPP, R., and ZIEGLER, H.: Protoplasts as a means of studying chloroplast development in vitro. Plant Physiol. 66,1-7 (1980).

DELIEU, T., and WALKER, D. A.: An improved cathode for the measurement of photosynthetic oxygen evolution by isolated chloroplasts. New Phytol. 71, 201-225 (1972).

HAMPP, R., and GOLLER, M.: Compartmentation of labeled fixation products in intact mesophyll protoplasts from Avena sativa L. after in-situ inhibition of the chloroplast phosphate translocator. Planta 159, 314-321 (1983).

HAMPP, R., and ZIEGLER, H.: On the use of oat protoplasts to study chloroplast development. Planta 14'1,485-494 (1980).

HASLER, l\L, RUFFNER, H. P., and RAST, D. M.: High yield isolation of grape leaf protoplasts as an instrument in physiological research. Experientia 38, 564-565 (1982).

HASLER, l\L, RUFFNER, H. P., and RAST, D. M.: Ultrastructure of grape leaf protoplasts in com­parison with the source tissue. Vi tis 22, 193-201 (1983).

KAISER, G., and HEBER, D.: Photosynthesis of leaf cell protoplasts and permeability of the plasma­lemma to some solutes. Planta 15'1, 462-470 (1983).

MALIPIERO, V., RUFFNER, H. P., and RAST, D. M.: Photorespiration and malate formation in grape leaves. Z. Pflanzenphysiol. 104, 243-251 (1981).

RHODES, l\L J. C.: Respiration and senescence of plant organs. In: DAVIES, D. D. (ed.): The bio chemistry of plants - a comprehensive treatise, Vol. 2, 419-462 (1980).

VAN DER VALK, H. C. P. M.: Determination of proteases in isolated washed protoplasts: Inactivation of proteases in cell wall-degrading enzyme mixtures used in protoplast isolation. Plant Sci. Lett. 36,201-204 (1984).

Protoplasts from Green and Senescing Grapevines 653

VIENKEN, J., ZIMMERMANN, D., GANSER, R., and HAMPP, R.: Vesicle formation during electro-fusion of mesophyll protoplasts of Kalanchoe daigremoniialla. Planta 11)7,331-335 (1983).

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Received May 30, 1984; accepted June 24, 1985

Author's address: Prof. Dr. H. ZIEGLER, Lehrstuhl fiir Botanik, Institut fiir Botanik and Mikro­biologie, Technische Dniversitat Miinchen, ArcisstraBc 21, D - 8000 Miinchen 2; L. F. DE FILIPPIS, School of Agriculture, Riverina-Murray Institute of Higher Education, P. O. Box 588, Wagga Wagga NSW 2650, Australia.

Biochem. Physiol. Pflanzen 180, 653-654 (1985)

Buchbesprechung

RUDOLF HAGEMANN: Allgemeine Genctik. 542 Seiten, 201 Abbildungen, 37 Tabellen. VEB Gustav Fisrher Verlag, Jena 1984. Preis: Broschur DDR 25,- M; Ausland 33,- DM.

Die Zahl der deutsch- und englischsprachigen Lehrbiicher der Genetik hat in den letzten Jahren stark zugenommen. In der ~Iehrzahl dieser Biicher liegt der Schwerpunkt der Darstellung verstand­licherweise auf dem Sektor der Molekulargenetik. Dies hat zur Folge, daB den verschiedenen Diszi­plinen der klassischen Genetik nur wenig, haufig zu wenig Beachtung geschenkt wird. Oftmals gilt dies auch fiir diejenigen Bereiche, in dencn intensive Forschungsarbeit betrieben wird und die -wenn auch "klassisch" - durehaus nieht im Sinne von iiberholt aufzufassen sind. Je intensiver aber molekulargenetische Probleme an Eukaryonten bearbeitet werden, umso notwendiger wird ein soli des Wissen auf dem Gesamtgebiet der Genetik.

43 Biochem. Physiol. Pflanzen, Bd. 180