Planta

Planta (1988) 176: 506-512

9 Springer-u

1988

Role of plastidial acyl-acyl carrier protein: Glycerol 3-phosphate acyltransferase and acyl-acyl carrier protein hydrolase in channelling the acyl flux through the prokaryotic and eukaryotic pathway Imma L~hden and Margrit Frentzen* Institut fiir Allgemeine Botanik, Universitfit Hamburg, Ohnhorststrasse 18, D-2000 Hamburg 52, Federal Republic of Germany

Abstract. In order to investigate whether the relative activities of the plastidial acyl-acyl carrier protein (ACP):glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and acyl-ACP hydrolase play a role in controlling the acyl flux through the prokaryotic and eukaryotic pathway, we determined these enzymic activities in stroma fractions from 16:3- and 18:3-plants using glycerol 3-phosphate and labelled acyl-ACP as substrates. Several factors were examined which might influence the activities within plastids, such as leaf development, salts at physiological concentrations, stroma pH and substrates available to the enzymes. An appreciable alteration of the two enzymic activities was only observed with changes in the pH and substrate concentration, especially the concentration of glycerol 3phosphate. An increase in pH from 7 to 8 resulted in a decreased ratio of acyltransferase versus hydrolase activity in stroma fractions from both pea (Pisurn sativum L.) and spinach (Spinacia oleracea L.), whereas exogenously added glycerol 3-phosphate, which only influenced the acyltransferase, raised this ratio. On the other hand, the relative activities of the two enzymes stayed rather constant at oleoyl-ACP concentrations between I and 2 gM not only when it was offered alone but also in a mixture with palmitoyl-ACP. At pH 8, the stroma pH of illuminated chloroplasts, and at physiologically relevant substrate concentrations we observed clear differences between the 16: 3plants spinach and mustard (Sinapis alba ssp. alba L.) and the 18:3-plants pea and maize (Zea mays L.). In accordance with the different proportions of prokaryotic glycerolipids in the two groups of * To whom correspondence should be addressed Abbreviations: ACP=acyl carrier protein; Tricine=N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine;fatty acids are denoted by number of carbon atoms and double bonds

plants, pea and maize showed distinctly lower ratios of acyltransferase versus hydrolase activity than spinach and mustard. Consequently the relative activities of the plastidial glycerol 3-phosphate acyltransferase and acyl-ACP hydrolase can play a decisive role in controlling the acyl flux through the different pathways at least in these plants. Key words: Acyl-acyl carrier protein:glycerol 3phosphate acyltransferase - Acyl-acyl carrier protein hydrolase - Eukaryotic pathway (acyl flux) - Prokaryotic pathway (acyl flux)

Introduction

Glycerolipids of the plastidial membrane systems possess prokaryotic fatty-acid distributions, in which the C-2 position of the glycerol backbone is exclusively esterified with C16-fatty acids, as well as eukaryotic distributions, in which position 2 only carries Cls-fatty acids. However, the ratio of the two patterns varies substantially in different plants (Heinz 1977). Generally, in 16: 3-plants the proportion of prokaryotic glycerolipids is distinctly higher than in 18 : 3-plants, since in 18 : 3-plants this fatty-acid pattern is almost exclusively conserved in the plastidial phosphatidylglycerol whereas 16:3-plants contain additionally a greater or lesser amount of prokaryotic glycolipids. It is generally accepted that de-novo biosynthesis of the plastidial glycerolipids occurs via two different pathways (Roughan and Slack 1982). Glycerolipids with prokaryotic fatty-acid patterns are entirely formed within plastids, whereas the nucleocytoplasmic part of the cell, mainly the endoplasmic reticulum, is the site of the construction of the eukaryotic diacylglycerol species. The eukaryotic pathway requires the export of long-chain

I. L6hden and M. Frentzen: Acyltransferase versus hydrolase f a t t y acids f r o m the plastids to the e n d o p l a s m i c r e t i c u l u m a n d the i m p o r t o f e u k a r y o t i c diacylglycerol moieties f r o m the e n d o p l a s m i c r e t i c u l u m to the plastids. Since the p r o p o r t i o n o f p r o k a r y o t i c a n d e u k a r y o t i c glycerolipids varies distinctly f r o m p l a n t to plant, the b i o s y n t h e s i s rates o f b o t h p a t h w a y s m u s t c o r r e s p o n d i n g l y be well b a l a n c e d a n d c o n t r o l l e d . T h e d a t a so far available a l r e a d y indicate c o m plex r e g u l a t o r y m e c h a n i s m s b o t h at the level o f e n z y m i c activities, s u c h as the plastidial p h o s p h a t i d a t e p h o s p h a t a s e ( G a r d i n e r a n d R o u g h a n 1983; H e i n z a n d R o u g h a n 1983; G a r d i n e r et al. 1984), a n d at the level o f s u b s t r a t e c o n c e n t r a t i o n s , n a m e l y glycerol 3 - p h o s p h a t e ( G a r d i n e r et al. 1982) a n d acyl-acyl carrier p r o t e i n ( A C P ) i s o f o r m s ( G u e r r a et al. 1986). F u r t h e r m o r e , the flux o f acyl g r o u p s t h r o u g h the t w o p a t h w a y s c o u l d be directly c o n trolled b y the relative activities o f the t w o plastidial enzymes, acyl-ACP hydrolase, which hydrolyses the A C P - t h i o e s t e r s to free f a t t y acids a n d A C P a n d a c y l - A C P : glycerol 3 - p h o s p h a t e a c y l t r a n s f e r ase, w h i c h directs the acyl g r o u p s f r o m the A C P thioesters to the C-1 p o s i t i o n o f glycerol 3 - p h o s p h a t e . H e n c e , in plastids, b o t h activities c o m p e t e f o r the p r o d u c t s o f f a t t y - a c i d b i o s y n t h e s i s (Shine e t a l . 1976; O h l r o g g e et al. 1978; F r e n t z e n e t a l . 1983), w h e r e the acyl g r o u p s are d i r e c t e d to the e u k a r y o t i c p a t h w a y b y the activity o f the h y d r o l a s e a n d to the p r o k a r y o t i c o n e b y the activity o f the a c y l t r a n s f e r a s e . To investigate this possible regulat o r y m e c h a n i s m , we h a v e d e t e r m i n e d the activities o f these t w o plastidial e n z y m e s in p l a n t s w h i c h c o n t a i n different levels o f p r o - a n d e u k a r y o t i c glycerolipids.

Materials and methods Chemicals'. [1-14C]Palmitic acid (2.15GBq-mmol 1), [l14C]oleic acid (2.07 GBq-mmol-1), [1 ~4C]stearic acid (2.22 GBq.mmol- 1), sn-[U- 14C]glycerol 3-phosphate (6.29 GBq.mmol 1), [1-14C]palmitoyl-CoA (1.92 GBq. retool-1), [1 14C]oleoyl_CoA (2.07 GBq.mmol 1), and uridine diphosphate-D-[U-14C]galactose (5.74 GBq.mmol-1) were purchased from Amersham Buchler (Braunschweig, FRG). [114C]Palmitoyl-ACP, [1-14C]oleoyl-ACP and [l-~4C]stearoylACP were synthesized according to Rock and Garwin (1979) from acyl carrier protein (Calbiochem, La Jolla, Cal., USA) and the corresponding labelled fatty acids by acyl-ACP synthetase purified from Escherichia eoli (Rock and Cronan 1979). The purification of acyl-ACP was carried out as described before (Rock and Garwin 1979). Other biochemicals came from Sigma (Mfinchen, FRG) and Merck (Darmstadt, FRG). Plant materials. Spinach (Spinacia oleraeea L., vat. subito) was grown hydroponically as described by Andrews and Heinz (1987). Maize (Zea mays L. cv. Zuckermais) and pea (Pisum sativum L. cv. Progress No. 9) seedlings were cultivated in a mixture of vermiculite and perlite in a growth chamber at 11 h

507 light (4500 lx) with day/night temperatures of 20~ C/17 ~ C and a relative humidity of 50%. Tobacco (Nicotiana tabaeum L.) and mustard (Sinapis alba ssp. alba L. cv. Arda Gelbsent) were grown in a greenhouse. Preparation of stroma and envelope membranes. Intact chloroplasts were isolated from freshly harvested plant material, purified on Percoll gradients and separated from residual Percoll as described before (Bertrams et al. 1981), using the resuspension medium according to Lilley et al. (1975). For the preparation of maize chloroplasts, 0.2% bovine serum albumin (BSA; w/v) and 2 mM ascorbic acid were added (Jenkins and Russ 1984) to the medium. After breaking the chloroplasts osmotically in 10 mM 3-(N-morpholino)propanesulfonic acid(Mops)NaOH, pH 7.6, 5 mM dithioerythritol, the thylakoids and envelope membranes were separated from soluble proteins by differential centrifugation (5 min at 40000.g and 1 h at 150000.g). After separating low-molecular-weight substances from the resulting supernatant by gelfiltration on a Sephadex G-25 column (Pharmacia, Freiburg, FRG) the stroma fi-actions were stored in portions under argon at - 2 0 ~ C. Under these conditions the enzymes were stable for at least 7 d. Envelope membranes were isolated from purified chloroplasts according to Joyard and Douce (1976). E n z y m e assays Glycerol 3-phosphate acyltransferase. The activity of the acyltransferase was routinely determined in a standard assay as described before (Bertrams and Heinz 1981). Briefly, the 80-1al assays consisted of 0.25 M Mops-NaOH, pH 7.4, 4.8 mg BSA, 0,24 mM palmitoyl-CoA, up to 30 I~g stroma protein and 2,0 mM sn-[U-14C]glycerol 3-phosphate (0.23 GBq.mmol-1). Reactions were terminated and lipophilic products were extracted after 10 rain at 24~ C according to Hajra (1974). Acyl-acyl carrier protein (ACP) hydrolase. The standard reaction mixture was composed of 80 mM N-[2-hydroxy-l,lbis(hydroxymethyl)ethyl]glycine (Tricine)-NaOH, pH 8.6; 1.2 gM [1-14C]oleoyl-ACP (2.07 GBq.mmo1-1) and up to 3 ~tg stroma protein in a total volume of 50 gl. The reaction was allowed to proceed for 5 rain at 24~ C after which time it was terminated and free fatty acids were extracted as outlined by Ohlrogge et al. (1978). The activities of acyl-ACP hydrolase and glycerol 3-phosphate acyltransferase were measured simultaneously in 80-gl assays, containing 80 mM Tricine-NaOH, pH 8.2, 0.3 mM glycerol 3-phosphate, 0.5-3 gg protein and 1.2 ~tM [1-14C]oleoylACP (2.07 GBq.mmol ~), unless stated otherwise. After a 5rain incubation at 24~ C the reactions were terminated and the lipophilic products were extracted and separated by thin-layer chromatography (Bertrams and Heinz 1981). The analysis of fatty acids was carried out as described before (Bertrams and Heinz 1981). Uridine 5'-diphosphate-galactose- diacylglycerol galactosyltransferase. The activity was determined according to Douce (1974), but the procedure was slightly modified. The 50-gl assays consisted of 50ram Tricine-NaOH, pH 7.8, 2.5 mM MgClz, 16.5 ~tM uridine 5'-diphosphate-[U-14C]galactose (5.74 GBq.mmo1-1) and up to 20 gg enzyme protein. After 15 rain at 24~ C the reactions were terminated and the products extracted in the same way as the acyltransferase assays. Acyl-CoA hydrolase. The reaction mixture consisted of 50 mM Trieine-NaOH, pH 8.6, 3.8 gM [l-14C]palmitoy1-CoA

508

Spinacia

(1.92 GBq. m m o l - 1 ) and up to 20 gg protein in a total volume of 50 lal. Incubations lasted for 5 rain at 24 ~ C and were terminated and extracted in the same way as the acyl-ACP hydrolase assays.

v= Results and discussion

Assay conditions. For rapid and independent determinations of the activities of acyl-ACP hydrolase and glycerol 3-phosphate acyltransferase, standard assays were carried out in which labelled oleoylACP was used as a substrate for the hydrolase and labelled glycerol 3-phosphate and palmitoylCoA were used as substrates for the acyltransferase. Under these conditions the acylation of glycerol 3-phosphate was proportional to the stroma protein added up to 40 ~g and the hydrolysis of acyl-ACP up to 3 ~tg. The analysis of the reaction products showed that, in the acyltransferase assay, l-acylglycerol 3-phosphate and, in the hydrolase assay, free fatty acids were formed as sole lipophilic compounds. In order to determine the ratio of acyltransferase versus hydrolase activity both activities were measured simultaneously in assays containing labelled acyl-ACP and unlabelled glycerol 3-phosphate. The addition of glycerol 3-phosphate did not alter the activity of the hydrolase. On the other hand, J-acylglycerol 3-phosphate was only formed when glycerol 3-phosphate was added exogenously, indicating that the stroma fractions used in the experiments were free of endogenous acyl acceptor. The formation rate of both free fatty acids and J-acylglycerol 3-phosphate was constant for at least 5 min and up to 3 ~tg stroma protein. Recoveries of the enzymic activities and subplastidial localization. In stroma fractions prepared from purified chloroplasts of different plants the recovery was about 80% of the total plastidial activity of both acyl-ACP hydrolase and glycerol 3-phosphate acyltransferase, whereas neither diacylglycerol galactosyltransferase nor acyl-CoA hydrolase activities could be detected. Hence, the isolation procedure did not alter the ratio of acyltransferase versus hydrolase activity, and the stroma fractions were not contaminated by envelope membranes. In accordance with previous data (Bertrams and Heinz 1976; Shine et al. 1976; Joyard and Douce 1977; Ohlrogge etal. 1978; Joyard and Stumpf 1980, 1981; McKeon and Stumpf 1982; Douce et al. 1987) the results further demonstrate that both enzymes behaved like soluble proteins and that the hydrolase possessed a pronounced speci-

1'5i~

I. L6hden and M. Frentzen: Acyltransferase versus hydrolase Pisum

-1,0

1,0

7

"0,

i

6.8

i

i

7,2

7,5 pH

i

8,0

i

8.&

i

8.8

i

7,2

i

7.6 pH

i

8.0

i

8,/,

Fig. 1. Formation of l-oleoylglycerol 3-phosphate ( o - o ) and oleic acid (A-A) by stroma fractions of spinach and pea chloroplasts from labelled oleoyl-ACP and glycerol 3-phosphate as a function of the pH. Incubation media were buffered with a mixture of Mops and Tricine, 0.1 M each

ficity for ACP thioesters, since CoA thioesters were not used as substrates. On the other hand, envelope membranes isolated from the same chloroplast preparations efficiently hydrolysed palmitoyl-CoA and to a lesser extent oleoyl-CoA but neither palmitoyl-ACP nor oleoyl-ACP. Consequently, these and other results (Shine et al. J 976; Ohlrogge et al. 1978; Joyard and Stumpf 1980, 1981; McKeon and Stumpf 1982) disprove the assumption that ACPthioesters are the physiological substrates for the hydrolase bound to the envelope membranes (Mudd etal. 1986, 1987; Roughan and Slack 1982).

Effect of different ions on the enzymic activities. Figure 1 shows the activities of both acyl-ACP hydrolase and glycerol 3-phosphate acyltransferase as a function of the pH of the reaction mixture. The glycerol 3-phosphate acyltransferase exhibited a rather broad pH optimum in the buffer system used for these experiments, whereas the acyl-ACP hydrolase showed highest activities at alkaline pH values, in agreement with previous data (Ohlrogge et al. 1978). Consequently, in stroma fractions from both pea and spinach chloroplasts the ratio of acyltransferase versus hydrolase activity decreased substantially with increasing pH. As shown by acetate-labelling experiments with isolated chloroplasts, long-chain acyl-ACP thioesters are formed in illuminated organelles while in the dark their concentrations immediately drop to almost zero (Soll and Roughan 1982). In vivo the plastidial enzymes are thus provided with substrates when the pH of the stroma is about 8. Therefore, the determinations of enzymic activities in the stroma fractions were carried out at this pH value.

I. L6hden and M. Frentzen: Acyltransferase versus hydrolase

(o~}

(o) ~

~. 0,8-

~ 0

-0,8

v=

509 Table 1. Fatty-acid specificity of the plastidial glycerol 3-phosphate acyltransferase and acyl-ACP hydrolase. The activities determined with palmitoyl(16 : 0)-ACP and stearoyl(18 : 0)-ACP are given as a percentage of the activities determined with oleoyl(18 : 1)-ACP, - = not determined

E

Reaction product

7E

Enzyme source

-0,4

g, 0,4-

Acyl-ACP 16:0

18:0

18:1

activity (%)

age 0f leaves Fig. 2. Activities of glycerol 3-phosphate acyltransferase (e e) and acyl-ACP hydrolase (A-A), as well as the corresponding ratio of acyltransferase versus hydrolase ( D - u ) , in stroma fractions of spinach chloroplasts according to the age of leaves from which the organetles had been isolated. Abscissa: 1 = young pale greenish leaves up to a length of 5 cm; 2 = green leaves of 5-9 can in length; 3 = fully developed dark-green leaves of 9-18 cm in length

Despite the pH-dependent variation of the ratio of glycerol 3-phosphate acyltransferase versus acyl-ACP hydrolase activity the 16:3-plant spinach showed distinctly higher ratios than the 18:3plant pea at all pH values tested. On the other hand, the light-dependent increase in the plastidial Mg 2+ concentration (Portis and Heldt 1976) did not seem to play a role in controlling the two enzymic activities since neither hydrolase nor acyltransferase activity was altered by the addition of MgC!2 at physiological concentrations. Furthermore, KC1 and CaC12 at concentrations of 140 mM and 30 raM, respectively, determined in chloroplasts (Demmig and Gimmler 1983 ; Kreimer et al. 1985; Robinson 1986) did not influence the ratio of the two activities.

Effect of leafage on the enzymic activities. To determine whether the ratio of acyltransferase versus hydrolase activity varies within plants according to the age of leaves, stroma fractions were isolated from spinach leaves at different stages of development. The specific activities of both acyltransferase and hydrolase were higher in stroma fractions of young pale-greenish spinach leaves than in those of dark-green fully developed ones (Fig. 2). However, since both activities decreased to almost the same extent the ratio of the two activities stayed fairly constant (Fig. 2). Similar results were obtained when such experiments were carried out with pea seedlings up to an age of 16 d after sowing, whereas in older seedlings the ratio of acyl-

Free fatty acids

Spinacia Pisum Nicotiana

0.8 1.4 1.6

1-Acylglycerol 3-phosphate

Spinaeia Pisum Nicotiana

23.7 64.0 73.7

2.5 4.8 -

100 100 100

-

100 100 100

transferase versus hydrolase activity slightly increased.

Dependence of the enzymic activities on different acyl-A CP thioesters. According to previous investigations, the acyl-ACP hydrolases from avocado mesocarp (Ohlrogge et al. 1978), Carthamus seeds (McKeon and Stumpf 1982), as well as spinach leaves (Shine et al. 1976) possess a very high specificity for oleoyl-ACP in comparison to palmitoyland stearoyl-ACP. We could confirm this specificity not only with stroma fractions from spinach but also with those from tobacco and pea leaves (Table 1). In comparison with the hydrolase, the fatty-acid specificity of the plastidial glycerol 3phosphate acyltransferase was less pronounced (Table 1), as described before (Frentzen etal. 1983). Thus, offering saturated acyl-ACP thioesters instead of oleoyl-ACP resulted in a complete alteration in the ratio of acyltransferase versus hydrolase activities. In vivo, however, the enzymes are provided with a mixture of saturated and monounsaturated acyl-ACP thioesters (Soll and Roughan 1982; Roughan 1986). Therefore, we investigated whether the activities of both acyltransferase and hydrolase were influenced when an equimolar mixture of palmitoyl- and oleoyl-ACP, instead of oleoyl-ACP alone, was used as substrate. As shown in Table 2, an appreciable alteration of the enzymic activities was not observed when oleoyl-ACP was offered alone or in a mixture with palmitoyl-ACP. Furthermore, fatty-acid analysis of the reaction products showed that the hydrolase as well as the acyltransferase preferentially utilized oleoyl-ACP (Table 2). These results are consistent with the high fatty-acid specificity of the hydrolase (Shine et al. 1976; Ohlrogge et al. 1978; McKeon and Stumpf 1982) and the high fatty-acid selectivi-

510

I. L6hden and M. Frentzen: Acyltransferase versus hydrolase

acyltransferase and acyl-ACP hydrolase using oleoyl-ACP (1.2 gM) or an equimolar mixture of palmitoyl- and oleoylACP (2.4 laM) as acyl donors. The percentage of 18:1 in the reaction products formed from the acyl-ACP mixture is given in parenthesis Reaction product

Enzyme source

1,51

Spinacia

1,5-1

=- 0,5 7-

Acyl-ACP 18:t

/

Pisum

T a b l e 2. Comparison of the activities of glycerol 3-phosphate

0,5

16:0/18:t

(nmol. min- t. rag- i protein)

E E

1-Acylglycerol 3-phosphate

Pisum Spinaeia

0.18 0.73

0.17 0,82

(88) (90)

Free fatty acid

Pisum Spinacia

1.18 1.24

0.90 1.02

(86) (91)

ty of the acyltransferase from pea and spinach (Bertrams and Heinz 1981 ; Frentzen et al. 1983; Douady and Dubacq 1987). Since the oleoyl-ACPdependent activities of the enzymes were hardly influenced by the addition of palmitoyl-ACP, the hydrolysis and acylation rates determined with oleoyl-ACP could be used directly for the estimation of the relative activities of the two enzymes. Influence of substrate concentrations on the two enzymic activities. Determination of the dependence

of glycerol 3-phosphate acyltransferase and acylACP hydrolase activities on the oleoyl-ACP concentration clearly showed differences between 16: 3- and 18: 3-plants (Fig. 3). In accordance with the different levels of prokaryotic glycerolipids in the two groups of plants, pea and maize showed distinctly lower ratios of acyltransferase versus hydrolase activities than spinach and mustard where the ratio came close to unity. As determined so far with the stroma fractions from pea and spinach, these results were not altered when the experiments were carried out with acyl-ACP mixtures instead of oleoyl-ACP alone. Furthermore, the clear difference in the glycerol 3-phosphate acyltransferase activities from pea and spinach was also observed when the glycerol 3-phosphate concentration was varied (Fig. 4), while the hydrolase activities from both plants were similar (Fig. 3). The calculation of the ratio of the two enzymic activities as a function of the substrate concentration showed that in all plants the ratio slightly decreased with increasing oleoyl-ACP concentrations. However, in the range from I to 2 gM oleoyl-ACP, which can be assumed to be physiologically relevant (Soll and Roughan 1982; Roughan 1986), the ratio stayed rather constant. On the

Sinopis

lea

0,&"

0,2-

0,1-

i

i

I

i

oleoyl-ACP (,oM)

Fig. 3. Dependence of glycerol 3-phosphate acyttransferase ( o - o ) and acyl-ACP hydrolase (zx-zx) activities in stroma fractions from different plants on the concentration of oleoyl-ACP

oa

~- 0,3E

L= 0.2E EH

0,I-

o',1

0',2

o',3

glycerol 3- phosphate(rnl4)

o',5

Fig. 4. Formation of l-oleoylglycerol 3-phosphate by stroma fractions of spinach ( o - o ) and pea ( o - o ) chloroplasts as a function of the concentration of glycerol 3-phosphate

other hand, glycerol 3-phosphate influenced the acyltransferase but not the hydrolase activity, and therefore the ratio of the enzymic activities within plastids. According to the data shown in Fig. 4, the acyltransferase from pea reached substrate saturation at lower concentrations than the spinach enzyme. The apparent K m values for glycerol 3-phospate in the presence of oleoyl-ACP were 14 gM for the pea enzyme but 75 gM for the enzyme from spinach, whereas the corresponding Vmax value was fourfold higher for the spinach than for the pea acyltransferase. Although these experiments were

I. L6hden and M. Frentzen: Acyltransferase versus hydrolase

carried out with a suboptimal oleoyl-ACP concentration of 1.2 ~tM, the apparent K m value determined for the spinach enzyme was of the same magnitude as the value of 31 pM previously determined with the purified enzyme (Frentzen et al. 1983). The kinetic constants may be compared with stroma concentrations of glycerol 3-phosphate in spinach chloroplasts oscillating between 140 ~tM in the light and 220 pM in darkness (Sauer and Heise 1984), while values about sevenfold higher have been reported for Amaranthus chloroplasts (Cronan and Roughan 1987). These data indicate that in vivo the glycerol 3-phosphate acyltransferase from pea, in contrast to that from spinach, is saturated with substrate. Therefore, variations in the glycerol 3-phosphate concentration should have a more pronounced effect on the biosynthetic rate of prokaryotic glycerolipids in spinach than in pea chloroplasts. A stimulation of the biosynthesis rate of prokaryotic glycerolipids with a concomitant decrease in the formation of eukaryotic ones by elevated cellular concentrations of glycerol 3-phosphate has been shown by both in-vitro and in-vivo labelling experiments (Gardiner et al. 1982). In summary, the observed differences in the ratios of glycerol 3-phosphate acyltransferase versus hydrolase activities in the two 18:3-plants pea and maize and the two 16:3-plants spinach and mustard demonstrate that these enzymes can play a decisive role in controlling the acyl flux through the prokaryotic and eukaryotic pathway. The ratio of the two enzymic activities can be influenced to a certain degree by the substrate concentrations available in plastids, especially by that of glycerol 3-phosphate. As shown so far for enzyme fractions from spinach, the activities of glycerol 3-phosphate acyltransferase and acyl-ACP hydrolase can also be controlled at the level of gene expression of ACP isoforms (Guerra et al. 1986). Consequently, these results give further evidence for the existence of complex mechanisms for the regulation of the biosynthesis of plastidial glycerolipids. Moreover, our first experiments with stroma fractions from the 16:3-plant tobacco, showing ratios of acyltransferase versus hydrolase activities as low as those from pea, indicate that the different regulatory mechanisms may be of varying importance, or that the biosynthesis of the plastidial glycerolipids may be controlled differently in various plants. Further experiments are in progress to solve this problem. We would like to thank Professor Ernst Heinz (Institut f/Jr Allgemeine Botanik, Universitfit Hamburg, FRG) for helpful

511 discussions and critically reading the manuscript. This research was supported by the Deutsche Forschungsgemeinschaft.

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