Changes in biochemical composition of vacuoles isolated from Acer pseudoplatanus L. during cell culture

22

Biochimica et Biophysica Acta, 721 (1982) 22 29

Elsevier BiomedicalPress BBA 11052

C H A N G E S IN B I O C H E M I C A L C O M P O S I T I O N O F VACUOLES I S O L A T E D F R O M T A N U S L. DURING CELL CULTURE

A CEIl PSEUDOPLA

GILBERT ALIBERT, ANTOINE CARRASCO and ALAIN M. BOUDET Centre de Physiologic V~gbtale, Laboratoire Associb au C.N.R.S. No. 241, Universitb Paul Sabatier, 118 route de Narbonne. 31062 Toulouse Ckdex (France)

(Received March 8th, 1982) Key words: Vacuole," Protoplast; Cell fractionation," Cell cycle," (Acer pseudoplatanus)

Vacuoles were isolated from Acer pseudoplatanus cell suspension culture using a one-step procedure involving the lysis of the protoplast plasmalemma through a gradient of Ficoll containing DEAE-Dextran. The vacuole suspensions were slightly contaminated by other organelles (less than 5%) and the isolated vacuoles readily accumulated neutral red. Since a-mannosidase was located exclusively in the vacuoles it was used as a convenient marker. It was shown that the number of vacuoles per protoplast decreased as the cell aged. Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.

Introduction

Materials and Methods

The pioneering works of Matile's group [1,2] and Wagner and Siegelman [3], on the isolation of vacuoles, have opened new perspectives in the knowledge of this conspicuous cell compartment. Until now, most of the experiments on plants have been conducted with mature cells [4-7]. Such studies have provided useful data on the biochemical composition of the vacuoles but have given limited information on the dynamic changes which are likely to occur in these organelles. Plant cell suspension cultures offer suitable material for such a purpose: marked metabolic and physiological changes occur during the culture cycle over a relatively short period of time, in controlled and reproducible conditions. We describe here a procedure for the preparation of protoplasts and vacuoles from Sycamore cell suspension culture. In addition, we examine the changes in amino acids, organic acids and protein concentrations occurring in vacuoles during a cell culture cycle.

Cell suspension cultures o f S y c a m o r e

0167-4889/82/0000-0000/$02.75 © 1982 ElsevierBiomedical Press

The material used derives from an original strain kindly supplied by Professor J. Guern (C.N.R.S., Gif-sur-Yvette, France). The cells were grown on the liquid medium of Lamport [8] as modified by Lescure [9]: 20 ml cell suspension ( 8 - 9 . 1 0 5 cells/ml) were routinely transferred every 3 weeks into 250 ml new medium in 1-1 Erlenmeyer flasks and kept shaking at 150 rpm (New Brunswick Orbital shaker model G 10-21) in a continuous light of 60 W- m - z (Philips fluorescent TL 65 W) at 26°C. Samples were taken at different times after transfer to the new medium, corresponding to typical stages of growth: 4 days (end of the lag phase), 8 days (early exponential phase), 10 days (exponential phase) and 14 days (early stationary phase). Products a n d m e d i a Chemicals. Cellulase (Onozuka R

10) and

23 pectinase (Onozuka macerozyme R 10) from Kiuki Yakult M G F Co. Ltd., Japan; DEAE-Dextran and Ficoll 400 were purchased from Pharmacia (Uppsala, Sweden); Dextran sulphate, Mes (4morpholineethanesulphonic acid), D-mannitol and p-nitrophenyl a-D-mannoside from Sigma (Saint Louis, MO, U.S.A.); Tris from ICN (Hersham, Surrey, U.K.). Radiochemicals. [ 14C 1]Benzylamine-HC1 (14 Ci/mol) and [methyl-14C]nicotine dihydrochloride (10.1 Ci/mol) were from ICN; [6-3H]thymidine (45 Ci/mol) and [5,6-3H]uridine (40 Ci/mol) were from Amersham International (U.K.). Media. The following media were used: A, 25 mM Mes-tris buffer (pH 5.5) in 0.7 M mannitol; B, medium A at pH 6.5.

Methods Preparation and purification of protoplasts. The cells were harvested by filtration through a 35/~m nylon net and thoroughly rinsed with medium A. 6-8 g fresh weight were placed in a Petri dish (8-cm diameter) in 7 ml medium A containing 4% cellulase and 3% macerozyme. Digestion of cell walls was for 4 h at 36°C in a water bath, with agitation at 44 oscillations/min. The liberated protoplasts were filtered through a 25 ffm nylon net. The cells remaining on the filter were kept in 20 ml medium A, shaken and any additional released protoplasts were filtered off. The pooled protoplast suspensions were centrifuged for 1 min at 3900 X g (Martin Christ type UJ 15 centrifuge) and the pellets resuspended in 2 ml medium B. The protoplasts were purified for certain experiments by resuspending the previously obtained pellet in 2 ml 20% Ficoll in medium B. The suspension was introduced into the bottom of a 12 ml centrifuge tube (DuPont Instrument cat. 0019) and covered successively with 2 ml 5% Ficoll in medium B and 2 ml medium B. After 30 rain centrifugation at 1500Xg (Beckman type TJ6R centrifuge), the protoplasts which floated at the 20%/5% and 5%/0% Ficoll interfaces were harvested with a Pasteur pipette and diluted 5-fold with medium B. The suspension was then centrifuged again for 1 rain at 3900 X g and the highly purified protoplasts resuspended in medium B. Preparation of the vacuoles. The protocol previ-

ously described for the isolation of sweet clover vacuoles [10] was used with minor modifications. The protoplasts (1.5-2. 1 0 6 m l - l medium B) were centrifuged for 30 min at 1900 X g (Beckman TJ6R centrifuge) through a gradient containing (from the bottom to top): 2 ml 10% Ficoll, 2 ml dextran sulfate (1 m g . m l 1) in 5% Ficoll and 6 ml DEAE-Dextran (6 mg. m1-1) in 2.5% Ficoll, dissolved in medium B. The vacuoles at the 5%/10% Ficoll interface were sucked off with a Pasteur pipette. Vacuoles and protoplasts were examined by light microscopy and counted using a Fuschs Rosenthal haemocytometer.

[3H]Thymidine and [3H]uridine incorporation into nucleotides. 5-8 /xCi chosen radioactive precursor were injected aseptically through the cotton wool of the culture vial (this procedure avoids the deleterious effect of a gas shock which appeared to considerably reduce the protoplast yields). The incorporation was allowed to proceed for 14h, then the cells were harvested and the protoplasts and the vacuoles prepared as previously described. Aliquots of the protoplast or vacuole suspensions were diluted with an equal volume of 20% trichloracetic acid and sonicated for 3 x 20 s (MSE sonicator) in an ice-water bath. The sonicated suspensions were filtered through a Whatman No. 1 disk filter and the insoluble residue carefully rinsed with 20 ml methanol/chloroform/water (12:5:3, v/v) I11]. After drying in a dessicator, the filter disks were directly introduced into a scintillation vial and counted for radioactivity.

Incorporation of vacuolar probes into the protoplasts. Neutral red (0.5 or 1 . 1 0 - 1 0 - 4 g . m l - I ) , [ 14C]benzylamine or [14C]nicotine (5 #Ci each) were incubated separately with 10. 1 0 6 protoplasts in 2 ml medium B for 45 min. The protoplasts were then centrifuged for 1 rain at 3900 X g, resuspended in 2 ml medium B and centrifuged again, to eliminate the extraprotoplastic probe. After resuspending the pellet in medium B, 9.5 • 1 0 6 protoplasts were used as the source of vacuoles. The vacuoles and the remaining protoplasts (0.5- 1 0 6 protoplasts) were sonicated and either directly introduced into a scintillation vial for counting the radioactivity ([14C]benzylamine or [14C]nicotine assays) or centrifuged for 15 min at 10000 X g and the absorbance at 550 nm determined in the super-

24 natant solution (neutral red experiments). In all cases the results are expressed for 1 • 1 0 6 protoplasts or 1 • 1 0 6 vacuoles. Enzyme assays. Fumarase was estimated according to Cooper and Beevers [12], catalase according to Luck [13] and a-mannosidase following the method of Boiler and Kende [14]. Analyticalprocedures. Metabolite analysis, protoplasts and vacuoles were disrupted by addition of water and sonication. The homogenates were acidified to pH 4 and layered onto a Dowex 50 (H ÷ form) column. The organic acids which are not retained on the exchanger were identified by gas-liquid chromatography [16] and malate specifically quantified following the enzymatic procedure of Hohorst [15]. The cationic substances were eluted from the Dowex 50 with 1 M ammonia and the amino acids estimated by ion-exchange chromatography [17], using a Beckman amino acid analyser (model 119 BL). Protein determinations were carried out using the method of Bensadoun and Weinstein [18]. Radioactivity was measured by liquid scintillation spectrometry (Packard, model 2211), using Unisolve 1 (Koch light). Quenching was estimated by the dual channel technique. Results

Characteristics of the protoplasts and the vacuoles isolated from Sycamore cell suspension cultures Protoplasts could easily be obtained only when the cells were cultured under our strictly defined conditions. The yield of protoplasts per g fresh weight decreased drastically with the age of the culture (2, 1.1, 0.6, 0.3 and 0 protoplasts/g tissue for 8, 12, 18, 27 and 33 days, respectively). The protoplasts were as heterogenous in size as the original growing cells (Fig. 1). The crude preparations were contaminated with vacuoles and plastids (Fig. la) but could be highly purified by flotation on a Ficoll gradient (Fig. lb). The protoplasts readily absorbed neutral red and remained stable and viable (as shown by the Evans blue test) for at least 2 days when stored at 0-4°C in medium B. Using the rapid procedure devised for sweet clover [10], we were able to prepare purified vacuoles from Acer protoplasts. On a counting

Fig. 1. Protoplasts isolated from Sycamore cells (X440). a. Crude fraction: the suspension is mainly contaminated by plastics (thick arrows) and vacuoles (thin arrows), b. Purified fraction on Ficoll gradient. basis, the yield of vacuoles ranged from 60% for 4-day-old cells, to 25% for 15-day-old cells. A good vacuole yield was also obtained starting from protoplasts stored at 0-4°C up to a period of 48 h. The contamination of the vacuole preparations was checked by microscope examination (to detect the presence of protoplasts) and by estimation of specific e n z y m e m a r k e r s ( f u m a r a s e for mitochondria, catalase for microbodies). Additional tests were made using labelled precursors of nucleic acids in order to detect possible contamination by nuclei ([3H]thymidine assays) or ribosomes ([3H]uridine assays). Protoplasts were almost completely absent from the vacuole preparations (Fig. 2), while contamination by other organelles was 2-5% on a counting

25

Fig. 2. Isolated vacuoles (×440). The vacuoles are heterogeneous in size. No protoplast can be seen. Few vacuoles show adherences (thick arrows). Some of them contain precipitates (small arrows) while others appear empty (thin arrows).

basis (Table I). More than 95% of the isolated vacuoles could be stained by neutral red, showing that they retained their original acidity. When kept in the isolation medium (medium B containing Ficoll and dextran sulphate), the vacuoles appeared to be stable for at least 12h: only 2% lost after 12h; 29, 50 and 78% were lost after 24, 48 and 72 h, respectively. The concentration of the vacuoles in the collecting medium was usually low (1-2. 10 6 m1-1) and attempts to concentrate these suspensions were largely unsuccesful owing to the fragility of the organelles in the centrifugation steps. Determination of the number of vacuoles per protoplast Chemical probes, such as neutral red or some

amines, such as benzylamine or nicotine, easily cross the membrane and accumulate in the vacuoles [19-21]. We used these probes to determine the number of vacuoles per protoplast. Our results (Table II) show that the distribution ratio of the probes between the protoplast and the same number of the corresponding vacuoles ranges from 1.9 to 2.4. As nearly all the vacuoles are able to accumulate neutral red, the possibility of resealing of membranes during the isolation processes, leading to the production of extra vesicles, is to be ruled out. The measurement of et-mannosidase shows that the protoplast/vacuole ratio (P/V) activities are in good agreement with the results obtained with the probes. Taken together these data demonstrate that at this stage of development (7-day-old cells), one protoplast contains on average two vacuoles and that a-mannosidase is only located in vacuoles in Acer protoplasts and can, therefore, be used as a specific biochemical marker. The results (Table III) indicate that the number of vacuoles per protoplast changes along a culture cycle, starting from more than two vacuoles for 4-day-old cells, to one vacuole per protoplast in cells over 10-days-old (using a-mannosidase as a marker). Distribution of metabolites between the vacuolar and extravacuolar spaces of the cells Amino acids. The vacuolar amino acids represent 30-55% (Table IV) of the protoplast content, depending on the stage of development of the cells, the youngest having the highest amino acid vacuolar pool. All the protein amino acids are

TABLE I P U R I T Y C O N T R O L S OF T H E ISOLATED V A C U O L E S Contamination based on numeration of the organelles. Since cells contain variable number of vacuoles per protoplast, the actual contamination level of the preparations by extravacuolar organelles must be corrected by the corresponding factor (see Table III). Contamination

107 protoplasts

107 vacuoles

% in vacuolar fraction

Mitochondria (fumarase: AA n m 240. m i n - I ) Microbodies (catalase: AA n m 240- r a i n - t) Nuclei (DNA: dpm) Ribosomes (RNA: dpm)

0.130 1.500 401430 271640

0.006 0.058 22 400 6 190

4.6 3.8 5.6 2.3

26 TABLE II DISTRIBUTION OF PROBES AND a-MANNOSIDASE ACTIVITYBETWEEN PROTOPLASTSAND THE CORRESPONDING VACUOLES 7-day-old cells were used in these experiments. Neutral red (A at 550 rim)

Experiment 1 106 protoplasts (P) 106 vacuoles(V) P/V

Experiment 2 106 protoplasts (P) 10 6 vacuoles(V) P/V

(0.5.10 -4 g/ml)

(1.10 -4

0.455 0.180 2.4

0.840 0.400 2.1

0.610 0.270 2.1

[14C]benzylamine (dpm)

[14C]nicotine (dpm)

a-Mannosidase activity (AA at 450 nm.h 1)

15787 8 140 1.90

36040 18 191 1.98

1.646 0.961 1.72

encountered in the vacuolar sap; in contrast, the non-protein amino acid (y-aminobutyric acid) and the amides (asparagine, glutamine) are essentially extravacuolar. At the early stages of development, some amino acids are almost completely vacuolar (aspartate, isoleucine, tyrosine). Others are found 60-80% in vacuoles (leucine, phenylalanine, threonine, glycine, ornithine and arginine). Most of the amino acids which are present in high levels in vacuoles of the young cells (group I) decrease with the age of the culture. Conversely, a few amino acids (group II) first disappear and then accumulate again as the cells age.

TABLE III THE NUMBER OF VACUOLESPER PROTOPLASTWITH CELL AGE Cell age (in days)

a-Mannosidaseactivity (AA at 405 nm-h -1) in

plasts (P)

106 vacuoles (V)

0.903 1.031 0.791 1.199

0.408 0.661 0.769 1.122

10 6 p r o t o -

4 8 10 14

a-Mannosidase activity (AA at 450 nm.h- I)

Number of vacuoles per protoplasts (P/V) 2.2 1.56 1.02 1.06

g/ml)

Organic acids. Gas-liquid chromatography of vacuolar extracts showed the presence of lactate, glycerate, oxalate, succinate, fumarate, malate, citrate, shikimate, quinate and phosphoric acid in 10-day-old cells. For malate, enzymatic measurements show that the high malate content of young cells (Table V) is mainly due to vacuolar accumulation. The decrease in total malate from day 10, corresponds to a fall in the vacuolar pool (which was 74% for day 4 but only 21% for day 10), while the extravacuolar pool kept relatively constant (about 30 mmol/106 protoplasts). From day 10, malate accumulates again, but this time mainly in the cytoplasmic pool ( + 73 nmol at day 14). Proteins. The vacuolar proteins represent 1538% of the total protoplast proteins (Table VI). During growth, the relative proportion of the vacuolar to total proteins first increases and then decreases. In the late exponential phase, the vacuolar protein content decreases, even though the total proteins increase once more.

Discussion A one-step method for preparation and purification of vacuoles was recently devised for sweet clover mesophyll cells [10]. We demonstrate here that this method can be also applied, with minor modifications, to protoplasts isolated from Syca-

27 TABLE IV VARIATION OF T H E C O N T E N T OF SOLUBLE A M I N O ACIDS IN PROTOPLASTS A N D VACUOLES D U R I N G A CELL C U L T U R E CYCLE Cell age (days)

Amino acids in 106 protoplasts (nmol) 4

Aspartate - Serine Isoleucine Tyrosine Leucine Phenylalanine Ornithine Valine Alanine Threonine - Histidine - - 1 Lysine Arginine Glycine Proline Glutamate--~

8.1 21.7 16.3 10.5 27.9 20.1 8.7 25.4 94.6 17.9 6.8 23 17.1 22.9 16 69.6

-~ ~ ~ ] l

~ ~ ~

Total

8

406

GABA Glutamine Aspara~ne

45.8 22.1 8.2

% in vacuoles a

10

17.8 50.7 22 15.4 40.7 35.3 17.6 35.7 105 39 10.4 35 19.9 29.2 21.7 73 569

14

7.9 23.1 8.7 6.4 18.3 9.2 5.8 15.3 45.7 18.9 4.8 15.9 13.4 14.6 8.4 34.7

8

10

14

100 93 94 93 84 76 76 50 47 64 66 53 42 74 25 16

44 69 86 60 69 50 53 50 38 55 53 43 46 79 20 23

22 34 56 43 42 52 35 29 31 35 30 28 23 39 15 10

20 40 50 33 40 33 15 27 22 36 58 85 75 0 22

518

55

49

30

33

46 19 13

9 3 0

7 18 ll

13 7 0

9 0 0

19 43 23 14 43 32 5.8 35 99 38 9.8 26 9.8 26 20 75

251

136 18.8 14.3

4

18.5 13.6 2.7

Each value is corrected by the factor corresponding to the number of vacuoles per protoplast (see Table Ill).

more cell suspension cultures. The main advantages of this technique are the rapidity of the vacuole isolation and the good purity of the obtained vacuolar suspensions. On

the other hand, no changes in the osmolarity of the media occur during the isolation process, so that leakage of micromolecules from the vacuoles is reduced to a minimum. Finally, the organelles

TABLE V

TABLE VI

MALATE D I S T R I B U T I O N BETWEEN THE VACUOLAR A N D THE EXTRAVACUOLAR SPACES OF THE PROTOPLASTS D U R I N G A CELL C U L T U R E CYCLE

PROTEIN CONTENTS OF PROTOPLASTS AND VACUOLES AT D I F F E R E N T TIMES OF THE CELL CULT U R E CYCLE

Cell age (days)

Culture age (days)

#g proteins in 106 protoplasts

vacuoles a corresponding to 106 protoplasts

4 8 10 14

220 212 130 190

58.2 81.1 34.7 29.6

4 8 10 14

nmol malate in

% in vacuoles

106 protoplasts

vacuoles a corresponding to l06 protoplasts

133 70 34 111

99 37 7.1 38

a See footnote Table IV.

74 53 21 31

a See footnote Table IV.

vacuolar %

24 38 26.5 15.6

28 are quite stable with time when kept in the preparation medium. By comparison with the vacuolar/extravacuolar distribution of lipophilic probes, we have shown that a-mannosidase is exclusively intravacuolar in Acer protoplasts. Similar evidence has been obtained in tobacco cells [14], oats [24] and sweet clover [10]. Thus, a-mannosidase appears to be a general constituent of the vacuoles and we have used this enzyme as a vacuolar marker. We have shown that the number of vacuoles per protoplast varies with the age of the suspension culture, starting from more than two vacuoles per protoplast in young cells to one vacuole per protoplast, for cells in late exponential phase or in stationary phase. These results are in good accordance with the microscope observations of Nougar&te et al. [25] on Acer cells and confirm a progressive vacuolization of the cells with age [26]. The analysis of the vacuolar content confirms the presence of a vacuolar pool of amino acids as already demonstrated [10,23,27,28]. Whatever the developmental stage of the cells considered, basic amino acids (arginine, lysine, ornithine and histidine) never accumulate in the vacuole to the same extent as in yeast [29,30]. In addition, Acer vacuoles contain, as expected, a great variety of organic acids. Some of them, malate, succinate, citrate, oxalate and phosphate have been identified in vacuoles of other plants [23,31-33]. Using Acer cells, we demonstrated for the first time the vacuolar occurrence of lactate, glycerate, fumarate quinate and shikimate. Oxalate and quinate seem to be exclusively located within the vacuole while the vacuolar pools represent about 70% of the total shikimate, 40% of fumarate and phosphate and less than 30% of the other organic acids. We have focused our attention on the possible changes in metabolite concentrations occurring in vacuoles along a cell culture cycle. The total malate content of the cells varies considerably during growth, as described by Vanderhoven and Zr~d [34]. We demonstrate here that it is the vacuolar pool which is mainly concerned with these fluctuations. On the other hand, each amino acid exhibits a characteristic pattern of distribution between the vacuolar/extravacuolar spaces of the protoplast

depending on the age of the cells. In a general way, amino acid concentrations in the vacuoles decrease as the cells age; a similar pattern of variation can be drawn for proteins from day 8. Amino acids and proteins, stored during the first days of culture seem to be reused to provide cells with organic nitrogen mainly in the stationary phase when exogeneous nitrate is depleted (Fontaine S., Alibert G. and Boudet A.M., unpublished data).

Conclusion The results presented in this publication point out the dynamic status of the vacuolar system in plant cell suspension cultures. Thus, this organelle participates actively in the general 'economy' of the cell. The questions arising now concern the identification of the processes which allow the metabolites to flow into and out of the vacuoles, and the forces which trap the substances inside the vacuoles (since the metabolites do not flow from the isolated organelles even when the extravacuolar concentrations are zero). In this way, Acer pseudoplatanus cell suspension cultures provide a useful means of examining these problems.

Acknowledgements This research was supported by a grant from the 'Mission h la Recherche' to Gilbert Alibert and by C.N.R.S.L.A. No. 241.

References 1 Matile, Ph. (1966) Z. Naturforsch. 21 b, 871-878 2 Matile, Ph. (1978) Annu. Rev. Plant Physiol. 29, 193-213 3 Wagner, G. and Siegelman,H.W. (1975) Science 190, 12981299 4 Saunders, J.A. and Corm, E.E. (1978) Plant Physiol. 61, 154-157 5 Grob, K. and Matile, Ph. (1979) Plant Sci. Lett. 14, 327-335 6 Leigh, R.A. and Branton, B. (1976) Plant Physiol. 58, 656-662 7 Moskowitz, A.H. and Hrazdina, G. (1981) Plant Physiol. 68, 686-692 8 Lamport, D.T.A. (1964) Exp. Cell Res. 33, 195-206 9 Lescure, A.M. (1966) Physiol. Vrg. 4, 365-378 10 Boudet, A.M., Canut, H. and Alibert, G. (1981) Plant Physiol. 68, 1354-1358

29 11 Ferrari, T.E. and Widholm, J.M. (1973) Ann. Biochem. 56, 340-352 12 Cooper, T.G. and Beevers, H. (1969) J. Biol. Chem. 244, 3507-3513 13 Luck, H. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 886-888, Academic Press, New York 14 Boiler, T. and Kende, H. (1979) Plant Physiol. 63, 1123-1132 15 Hohorst, H.J. (1970) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. II, pp. 1544-1548, Verlag Chemie, Weinheim 16 De Smedt, P., Liddle, P.A.P., Cresto, B. and Bossard, A. (1979) Ann. Fals. Exp. Claim. 72, 633-642 17 Spackmann, D.H., Stein, W.H. and Moore, S. (1950) Anal. Chem. 30, 1190 18 Bensadoun, A. and Weinstein, D. (1976) Anal. Biochem. 70, 241-250 19 Langeron, M. (1949) Precis de microscopie (Brodard and Taupin, eds.), pp. 376-379, Masson, Paris 20 Kurkdjian, A. and Guern, J. (1981) Plant Physiol. 67, 953-957 21 Kurkdjian, A. (1982) Physiol. V6g. 20, 73-83

22 Butcher, H.C., Wagner, G.J. and Siegelman, H.W. (1977) Plant Physiol. 59, 1098-1103 23 Wagner, G.J. (1979) Plant Physiol. 64, 88-93 24 Martinoia, E., Hock, U. and Wiemken, A. (1981) Nature 289, 292-294 25 Nougar&te, A., Guern, J. and Grignon, C. (1968) C.R. Acad. Sci. Paris 266, 207-210 26 Sutton-Jones, B. and Street, H.E. (1968) J. Exp. Botany 19, 114-118 27 Nishimura, N. and Beevers, H. (1978) Plant Physiol. 62, 44-48 28 Sasse, F., Backs-Husemann, D. and Barz, W. (1979) Z. Naturforsch. 34c, 848-854 29 Wiemken, A. and Nurse, P. (1973) Planta 109, 293-306 30 Messenguy, F., Colin, D. and Ten Have J.P. (1980) Eur. J. Biochem. 108, 439-447 31 d'Auzac, J. and Lioret, C. (1974) Physiol. V6g. 12, 617-635 32 Buser, C. and Matile, Ph. (1977) Z. Pflanzenphysiol. 82, 462-466 33 Wagner, G.J. (1981) Plant Physiol. 67, 591-593 34 Vanderhoven, C. and Zr~d, J.P. (1978) Physiol. Plant. 43, 99-103