biosynthesis and desaturation of the different diacylglycerol moieties in higher plants

Review

Biosynthesis and Desaturation of the Different Diacylglycerol Moieties in Higher Plants

MARGRIT FRENTZEN

Institut fur Allgemeine Botanik, Ohnhorststr. 18,2000 Hamburg 52

Received January 6, 1986 . Accepted January 12, 1986

1. Introduction

Glycerolipids of higher plants carry predominantly C16- and Cis-acyl groups which are distributed in a characteristic pattern between the two positions of the glycerol backbone (Heinz 1977). According to these positionally specific distributions of the fatty acids, glycerolipids can be divided into two groups (Fig. 1). One group is exclusively esterified with C l6-fatty acids at the G2 position while the C-1 position contains C IS- and to a lesser extent Cwacyl groups. Since this distribution corresponds to the typical fatty acid pattern of glycerolipids from cyanobacteria, it is called prokaryotic.

The other group carries Cis-fatty acids at both positions whereas C l6-acyl groups are excluded from the C-2 position. This eukaryotic pattern is characteristic of glycerolipids from extraplastidic membranes.

In the glycerolipids of plastidial membranes both types of fatty acid distribution are found. In all plants, plastidial phosphatidylglycerol shows a prokaryotic fatty acid pattern, while phosphatidylcholine from the same membrane system exhibits a eukaryotic pattern. The glycolipids monogalactosyl-, digalactosyl-, and sulfoquinovosyl-diacylglycerol, which are the major membrane lipids of plastids, possess both types of fatty acid patterns but the ratio of prokaryotic to eukaryotic glycolipids varies significantly in different plants (Heinz 1977). For instance, the fatty acid distribution of monogalactosyldiacylglycerol is nearly all prokaryotic in Anthriscus; in Spi· nacia the ratio of the two patterns is about 1, while in Cucurbita or Vicia monogalactosyldiacylglycerol is eukaryotic. Apiaceae and Chenopodiaceae belong to the group of plant families which contain not only linolenic acid but also hexadecatrienoic acid. These plants are termed 16: 3 plants and are opposed to the 18: 3 plants

Abbreviation list: ACP: acyl carrier protein; Cho: choline; CoA: coenzyme A; DAG: diacylglycerol; DGD: digalactosyldiacylglycerol; Gal: galactose; Gro: glycerol; G3P: glycerol 3-phosphate; LP A: lysophosphatidic acid; MGD: monogalactosyldiacylglycerol; P A: phosphatidic acid; PC: phosphatidylcholine; PG: phosphatidylglycerol; SQD: sulfoquinovosyldiacylglycerol; TAG: triacylglycerol. Fatty acids are denoted by number of carbon atoms and double bonds.

J. Plant Physiol. Vol. 124. pp. 193-209 (1986)

194 MARGRIT FRENTZEN

® @ Fig. 1: Positionally specific distribution of the C16- and Cis-acyl groups in the diacylglycerol moieties with a) prokaryotic and b) eukaryotic fatty acid patterns.

such as Cucurbitaceae or Fabaceae which contain only CIS: 3 as trienoic fatty acid. Generally, in 16: 3 plants the proportion of prokaryotic glycerolipids is distinctly higher than in 18: 3 plants. This is because plastids of 16: 3 plants contain in addition to prokaryotic phosphatidylglycerol prokaryotic glycolipids whereas in 18: 3 plants this fatty acid pattern has only been conserved in the plastidial phosphatidylglycerol.

As recently shown, the biosynthesis of prokaryotic and eukaryotic diacylglycerol moieties occurs in different subcellular compartments. The formation of the prokaryotic diacylglycerol backbones is completely catalyzed by enzymic activities from plastids whereas the nucleocytoplasmic part of the cell, mainly the endoplasmic reticulum (ER), is the site of the construction of the eukaryotic molecules.

2. Biosynthesis and desaturation of diacylglycerol moieties with prokaryotic fatty acid patterns

The prokaryotic biosynthesis of monogalactosyldiacylglycerol and phosphatidylglycerol has largely been elucidated by the following investigations: - Subplastidiallocalization of the enzymic activities (Andrews and Keegstra 1983,

Andrews and Mudd 1985, Andrews et al. 1985, Block et al. 1983 a, b, Cline and Keegstra 1983, Dome et al. 1982 a);

- in vitro labelling experiments with isolated chloroplasts from different 16: 3- and 18:3-plants (Gardiner and Roughan 1983, Gardiner et al. 1984a, Heinz and Roughan 1983);

- characterization of the plastidial glycerophosphate acyltransferase system (Frentzen et al. 1983).

According to the positional and fatty acid specificities of the glycerophosphate and monoacylglycerophosphate acyltransferase, phosphatidic acid with a prokaryotic pattern is formed in the envelope (Fig. 2, reactions 1, 2). This phosphatidic acid serves as substrate for the subsequent biosynthesis of monogalactosyldiacylglycerol as well as phosphatidylglycerol (Fig. 2, reactions 3, 4 and 7 - 9). Divalent cations and pH-values may be involved in the controlled channelling of phosphatidic acid into the alternative reaction sequences (Andrews and Mudd 1985, Block et al. 1983 b, Joyard and Douce 1979, Roughan 1985, Sparace and Mudd 1982).

Monogalactosyldiacylglycerol is used as precursor for the biosynthesis of digalactosyldiacylglycerol (Heinz 1977). It can react with another UDP-galactose to form digalactosyldiacylglycerol (Fig. 2, reaction 5). However, this reaction step has not directly been demonstrated to occur in plastids. The observed galactosylation of monogalactosyldiacylglycerol can be explained by the galactolipid: galactolipid galactosyltransferase (Fig. 2, reaction 6) the presence of which is well documented in plastids (van Besouw and Wintermans 1978, 1979, Heemskerk et al. 1983, 1985). This enzyme transfers a galactose from one monogalactosyldiacylglycerol to another. In contrast

J Plant Physiol. Vol. 124. pp. 193-209 (1986)

Diacylglycerol moities 195

ENVELOPE STROMA ENVELOPE CYTOPLASMA E R

16 {18 1l6) PGro

~ { 18 1l6) fnlty acids 16

16:3 plnnts PGroP

and ~ ; 18:3 plonts

16{18(16) COP

f 16{~8(16) {~8(16) 1 { 18{16.18

PCha

..f."" ¢

18{16.18

¢ 16: 3plonts ~ 16:J plonts 16{181l6)

and 18{16.18

{ '" 18: 3 plants

"] 6cp ~ 6

16{18116) 18{16.18 Gol2 Gal2

Fig. 2: Prokaryotic and eukaryotic pathway of the biosynthesis of glycerolipids from chloroplasts (according to the references given in the text). 1 and 12 = acyl-ACP (CoA): glycerol 3-phosphate acyltransferase; 2 and 13 = acyl-ACP (CoA): l-acylglycerol 3-phosphate acyltransferase; 3 and 14 = phosphatidic acid phosphatase; 4 = UDP-galactose: diacylglycerol galactosyltransferase; 5 = UDP-galactose: monogalactosyldiacylglycerol galactosyltransferase; 6 = monogalactosyldiacylglycerol: monogalactosyldiacylglycerol galactosyltransferase; 7 = CTP: phosphatidic acid cytidyltransferase; 8 = CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase; 9 = phosphatidylglycerophosphate phosphatase; 10 = acyl-ACP hydrolase; 11 = acyl-CoA synthetase; 15 = CDP-choline: diacylglycerol cholinephosphotransferase; 16 = acylCoA: lysophosphatidylcholine acyltransferase; PL TP = phospholipid transfer protein.

to recent reports (Heemskerk et al. 1985), this enzyme activity could be localized in the outer envelope membrane as demonstrated by experiments with thermolysinetreated chloroplasts (Dome et al. 1982 a, b) as well as by results of optimized enzyme assays with subplastidial fractions (Heemskerk et al. unpublished). Whether this intergalactosyl transferase is involved in the biosynthesis of prokaryotic digalactosyldiacylglycerol, has not been elucidated.

The prokaryotic diacylglycerol is probably used not only for the biosynthesis of galactolipids, but also for that of sulfolipid (Haas et al. 1980). As was recently reported (Kleppinger-Sparace et aI. 1985), chloroplasts are able to synthesize the polar headgroup autonomously, and UDP-sulfoquinovose perhaps serves as sugar donor. But the exact pathway has not yet been clarified.

J. Plant Physio/. VoL 124. pp. 193-209 (1986)


% 40

20

40

20

40

20

16:0 16:1 16:1 t 16:2 16:3 18:0 18: 1 18:2 18:3

wild - type

mutant ]81

mutant ] B 2

Fig. 3: Fatty acid composition of lipids from leaves of wild-type and mutant Arabidopsis tha· Liana plants (drawn from results of Browse et al. 1984).

Consequently, the primary products of de novo biosynthesis in chloroplasts are glycerolipids which predominantly carry oleoyl groups at position 1 while position 2 is specifically esterified with palmitoyl residues. It is most likely that these molecular species are then used as substrate for the desaturation of the esterified acyl groups (Roughan and Slack 1982, 1984). However, all attempts to demonstrate such a desaturation in vitro using subplastidial fractions as enzyme source have not been successful.

Browse et al. (1984, 1985) undertook a different approach to characterize the plastidial desaturase systems by their experiments with mutants from A rabidopsis thaliana. The analyses of the fatty acid composition of the leaf lipids from different mutants revealed interesting results (Fig_ 3). The wild-type as well as the mutants JB1 and JB2 contain almost identical C I6I'CI8 ratios. The fatty acid pattern of JB1, however, shows reduced levels of both C18 : 3 and C16 : 3 in comparison to the wild-type and a correspondingly higher percentage of C18 : 2 and C I6 : 2• The mutant JB2 also contains reduced levels of polyenoic acids but in this mutant cis-.6-9 monoenoic acids show compensating increases. Again the patterns of the C18- as well as the C l6-fatty acids change in the same way. These results clearly imply that the desaturase activities af-

! Plant PhysioL. VoL. 124. pp. 193 - 209 (1986)


M G D D G D S 0. D P G %

60

40 wild- type

20

60

40 ::~: mutant ]8 1

20 W

60

40 mutant ]8 2

20 ...

M G D D G D S 0. D P G

Fig.4: Percentage of Cis-fatty acids in individual lipids from leaves of wild-type and mutant A rabidopsis plants (drawn from results of Browse et al. 1984).

fected by the mutations inJB1 andJB2 can act on both Cw and Cs-acyl groups. The authors conclude from these results that the site for the insertion of a new double bond is determined from the methyl end of the chain. In chloroplasts the biosynthesis of dienoic and trienoic acids is, therefore, catalyzed by n-6 and n-3 desaturase activities. Furthermore, the observed accumulation of cis monoenoic acids instead of n-9, n-3 dienoic acids in JB2 indicates that a double bond in n-3 can only be inserted if


198 MARGRIT FRENTzEN

M60 060 P6 18:1

1

18: 1 18: 1 16: 0 16:01 16:01

Gal IGoll2 ®-Gro

1 MGD spec, A 7c 1 n-91 desolurose

1 PG spec,A 31ln-131 desolurose

18:1

1

18: 1 16: 1 16:111

Gal ®-Gro

n-6 desolurose

18:2

1

18: 2 18: 2 16: 2 16:01 16:111

Gal IGoll2 ®-Gro

; j .-, _not,." : 18:3

1

18: 3 18: 3 16:3 i6:01 16:111

Gal IGol12 ®-Gro

Fig. 5: Scheme of the desaturation of prokaryotic glycerolipids in chloroplasts (according to the references given in the text).

a double bond in n-6 is already present. The fatty acid analysis of individual lipids from chloroplasts of wild-type and mutants (Fig. 4) revealed that the changes in the fatty acid pattern, shown in Fig. 3, are not only observed'in a defined lipid, but also in all lipids which can be synthesized by plastids. In the mutants these lipids have reduced levels of Cs: 3 and a correspondingly higher proportion of CIS: 2 or CIS: I, respectively.

Hence chloroplasts possess n-6 and n-3 desaturase activities which do not show any pronounced specificities with respect to the chain length of the acyl groups, the position of the glycerol backbone, in which fatty acids are esterified, and the lipid head group. But the enzymes specifically oxidize acyl groups with double bonds at either n-9 or n-9 and n-6. The still significant amount of polyenoic acids in both mutants may be due to the desaturases having only been changed in their activities, or to the presence of more than one n-6 and n-3 desaturase in plastids. Fig. 5 summarizes the desaturation of glycerolipids with a prokaryotic fatty acid pattern in chloroplasts. According to in vivo and in vitro labelling experiments (Heinz and Roughan 1983, Roughan 1975, Siebertz and Heinz 1977, Siebertz et al. 1980, Williams and Khan 1982, Williams et al. 1976) besides the n-6 and n-3 desaturase activities, the chloroplasts must possess at least two further desaturase activities which display both lipid and positional specificities. One should exclusively use phosphatidylglycerol as a substrate and specifically introduce the .:l-3-trans double bond into the C16 : 0 esterified at the C-2 position. Mutants of Arabidopsis which lack only this desaturase activity,



have been described (Browse et al. 1985). Interestingly, these mutants do not reveal any ultrastructural or functional differences of the chloroplast when compared to the wild-type (Browse et al. 1985, McCourt et al. 1985). Therefore the authors conclude that the role of .::1-3-trans C16 : l-phosphatidylglycerol is more subtle than previously proposed (Dubacq and Tremolieres 1983) although in vitro .::1-3-trans C16 : rphosphatidylglycerol stabilizes light harvesting chlorophyll-alb protein oligomer (McCourt et al. 1985, Remy et al. 1982, 1984). The other desaturase activity has to possess a specificity for monogalactosyldiacylglycerol and the palmitoyl group esterified to position 2. As shown by comparative investigations of Siebertz and Heinz (unpublished), high activities of this enzyme can only be detected in young developing leaves. The desaturation of C16 :0 in monogalactosyldiacylglycerol results in species with n-9 acyl groups at both positions and only these monoenoic acyl groups can be further oxidized by the n-6 and subsequently by the n-3 desaturases, so that C18:3/C16:3 species, typical for 16: 3-plants, are formed. On the other hand digalactosyldiacylglycerol and phosphatidylglycerol only carry n-9 acyl groups for the sequential desaturation at the C-1 position. This is also true for sulfoquinovosyldiacylglycerol not shown in Fig. 5. Therefore within this group of lipids C18:3/C16:0 and C18:3/C16:1 species, respectively, are the main products (Fig. 5). As mentioned previously, direct evidence of the different desaturase activities has not been established.

In contrast to the prokaryotic glycerolipids, the eukaryotic species cannot be synthesized within the chloroplasts, but must be imported from the nucleocytoplasmic compartment, probably the ER.

According to in vivo double labelling experiments, the whole diacylglycerol backbone of phosphatidylcholine formed in the microsomal membranes is incorporated into the eukaryotic glycolipids of the plastids (Ohnishi and Yamada 1980, Slack et al. 1977).

3. Biosynthesis and desaturation of diacylglycerol moieties with eukaryotic fatty acid patterns

The ER is the primary site of the biosynthesis of eukaryotic glycerolipids in the extraplastidic part of the cell, but not the exclusive one, since mitochondria and dictyosomes are also capable of de novo biosynthesis (Moore 1982, Sauer and Robinson 1985). However, the biosynthetic capacity of mitochondria is confined to a small part of its membrane lipids, mainly phosphatidylglycerol and cardiolipin (Moore 1982). Furthermore it is doubtful whether the biosynthetic capacity observed in isolated dictyosomes (Sauer and Robinson 1985) play a role under in vivo conditions (Robinson et al. unpublished results). Consequently the activities of the ER are relevant to the biogenesis of the entire membrane system of cells. However, the microsomal system from photosynthetically active tissue is less well characterized than that from developing oil seeds. Since phosphatidylcholine has a decisive metabolic role not only in the biosynthesis of eukaryotic glycolipids in leaves, but also in the biosynthesis of polyunsaturated triacylglycerols in developing seeds, the system from oil seeds is described first.

]. Plant Physiol. Vol. 124. pp. 193-209 (1986)


3.1. Developing oil seeds

The microsomal fraction from oil seeds possesses all the necessary enzyme activities for de novo biosynthesis of triacylglycerol, namely glycerol 3-phosphate acyltransferase, monoacylglycerol 3-phosphate acyltransferase, phosphatidic acid phosphatase and diacylglycerol acyltransferase. The glycerol 3-phosphate acyltransferase specifically catalyzes the acylation of position 1 (Ichihara 1984) and preferably directs palmitoyl groups to this position (Ichihara 1984, Griffiths et al. 1985), while the second acyltransferase displays a high specificity for unsaturated C l8-acyl residues with a preference for C 18 : 2 (Griffiths et al. 1985, Stobart et al. 1983). Eukaryotic fatty acid patterns are thus established by these specificities and selectivities of the microsomal glycerophosphate acyltransferase system. On the other hand, the diacylglycerol acyltransferase does not show a pronounced fatty acid specifity (Shine et al. 1976), so that the fatty acid composition at the C-3 position of triacylglycerol may only be governed by the relative proportions of different acyl-CoA thioesters available to the enzyme. In addition to these enzymic activities, the microsomal fractions show high

GJP

lPA

PA

COP-choline ,--------. ±CMP

{C16:0.C18:1

OAG -pool C18: 1

CoA

{C16, C16

TAu C18 C16, C16

PC-pool QcyHoA pool

Fig. 6: Biosynthetic scheme of polyunsaturated triacylglycerols in developing oil seeds (according to the references given in the text). 1 = glycerol3-phosphate acyltransferase; 2 = l-acylglycerol3-phosphate acyltransferase; 3 = phosphatidic acid phosphatase; 4 = diacylglycerol acyltransferase; 5 = diacylglycerol cholinephosphotransferase; 6 = C 18 : I-phosphatidylcholine desaturase; 7 = C 18 : 2-phosphatidylcholine desaturase; 8 = lysophosphatidylcholine acyltransferase.



cholinephosphotransferase activity which catalyzes not only the forward but also the backward reaction and which nonspecifically uses the different molecular species (Slack et al. 1983, 1985).

The phosphatidylcholine formed in the microsomes is used as a substrate for the sequential desaturation of the oleoyl groups esterified at the C-1 as well as the C-2 position of the glycerol backbone (Slack et al. 1979, Rochester and Bishop 1984, Stobart and Stymne 1985). Furthermore, the microsomal fraction catalyzes reversible lysophosphatidylcholine acyltransferase reaction. As shown by Stymne and coworkers (Stobart et al. 1983, Stymne et al. 1983, Stymne and Stobart 1984 a, b, 1985), this acyltransferase exclusively attacks the C-2 position of phosphatidylcholine, possesses a high specificity for unsaturated CIs-fatty acids and slightly prefers CIS: I. Hence this enzyme preserves the eukaryotic fatty acid pattern but effects an acyl exchange between acyl-CoA and position 2 of phosphatidylcholine by the combined backward and foreward reactions (Stymne and Stobart 1984 a).

As summarized in Fig. 6, the biosynthesis pathway of triacylglycerol via phosphatidylcholine makes polyunsaturated CIs-fatty acids available for oil synthesis.

On the one hand, polyunsaturated fatty acids can be channelled to triacylglycerol by the reverse reaction of cholinephosphotransferase (Fig. 6, reaction 5). By this pathway the whole diacylglycerol moiety of phosphatidylcholine is incorporated into triacylglycerol.

On the other hand, polyunsaturated fatty acids as CoA-thioesters are provided by

Chloroplast

C16: 0- ACP .......... ~

[lB:1- ACP--t

{lpB:1

{lB:l

16: 0 P

G3P

LPA

PA

ER

{16:0.1B:l

18: 1 P

Fig. 7: Positional and fatty acid specificities or selectivities of the glycerol3-phosphate acyltransferase system from chloroplasts and ER membranes (Frentzen et al. 1983, 1984 and unpublished results). Solid lines symbolize the thioesters preferred by the acyltransferases.

J Plant Physiol. Vol. 124. pp. 193-209 (1986)


the activity of the lysophosphatidylcholine acyltransferase. This combined forward and backward reaction alters the acyl-CoA mixture exported from the plastids, resulting in a decrease of CIS: 1- and an increase of CIS: r or CIS: 3-groups corresponding to the fatty acid selectivity of the acyltransferase and the fatty acid composition at position 2 of phosphatidylcholine, respectively. Thus polyunsaturated Cls-CoA thioesters are available for the diacylglycerol acyltransferase activity. Furthermore, the acyl-CoA pool also provides the glycerophosphate acyltransferase system with substrates, so that CIS :2- or C IS :3-groups can initially be channelled to diacylglycerol in the course of its de novo biosynthesis. The degree of such channelling strongly depends upon the activity of the lysophosphatidylcholine acyltransferase, and this activity can only alter the acyl-CoA pool decisively if the microsomal membranes possess highly active desaturases so that polyunsaturated Cis-acyl groups are rapidly formed in phosphatidylcholine. According to recently published data the contribution of this pathway depends upon the oil seed species and the stage of seed development (Stymne and Stobart 1985, Stobart and Stymne 1985, Griffiths et al. 1985).

In summary, acyl exchange coupled to the diacylglycerol phosphatidylcholine equilibration gives rise to a continuous enrichment of the glycerol backbone with polyunsaturated fatty acids. Since the experiments to elucidate this complex pathway were carried out with microsomal fractions, the precise location of some of these enzymatic activities needs further investigation.

3.2. Photosynthetically active tissue

As mentioned earlier, phosphatidylcholine also plays a central role in photosynthetically active tissue in the biosynthesis of eukaryotic lipids, for example the plastidial glycolipids.

Following the elucidation of the prokaryotic pathway in leaf chloroplasts, Frentzen et al. (1984) began investigating de novo biosynthesis of phosphatidylcholine in leaf. Initial experiments were designed to determine whether the microsomal glycerophosphate acyltransferase system as well as the plastidial one controls the characteristic distributions of the glycerolipid fatty acids in these membranes.

Separation of post mitochondrial fractions from spinach leaf homogenates on different sucrose density gradients revealed that most of the microsomal glycerophosphate and monoacylglycerophosphate acyltransferase activities are localized in the ER membranes (Frentzen et al. 1984, and unpublished results). The positional and fatty acid specificities of the two microsomal acyltransferases were compared to those of the plastidial system (Fig.7). The microsomal glycerol 3-phosphate acyltransferase possesses a high positional specificity by exclusively directing acyl groups to the C-1 position of the glycerol backbone, such specificity was also observed for the plastidial enzyme activity. But in contrast to the plastidial enzyme, the microsomal acyltransferase preferably utilizes palmitoyl groups, showing an opposite although less pronounced fatty acid selectivity (Frentzen et al. 1983, 1984).

The observed differences are even more significant for the l-acylglycerol 3-phosphate acyltransferases from the two compartments: While the plastidial enzyme



specifically uses palmitoyl groups, the microsomal acyltransferase preferentially directs oleoyl groups to position 2 of the glycerol backbone (Frentzen et al. 1983, 1984). The low levels of palmitate incorporation may be due to contamination with plastidial enzymes (Frentzen et al. unpublished results).

In conclusion, the microsomal acyltransferase system from spinach leaves shows very similar specificities and selectivities to the corresponding enzymic activities from developing oil seeds (Ichihara 1984, Griffiths et al. 1985, Fig. 6).

Consequently the different subcellular glycerol 3-phosphate acyltransferase systems play a decisive role in determining the characteristic fatty acid patterns: The plastidial enzymes establish the prokaryotic pattern and the microsomal system, the eukaryotic pattern.

The phosphatidic acid formed in ER membranes serves as a precursor for the biosynthesis of the different phospholipids which can subsequently be used for the linoleic and linolenic acid biosynthesis.

In contrast to observations with subplastidial fractions, investigations with microsomal membranes conclusively demonstrate a desaturation of acyl groups esterified to phosphatidylcholine. This desaturation occurs in membrane fractions not only from developing oil seeds, as mentioned previously, but also from photosynthetically active tissue.

Murphy et al. (1983 a, b, 1984 a, b, 1985 b) succeeded in demonstrating a desaturation of oleoyl groups esterified to phosphatidylcholine during in vitro labelling experiments with microsomal fractions from young developing pea leaves. Incubations with labelled oleoyl-CoA resulted in a rapid incorporation of C 18 : I into phosphatidylcholine. This acylation was stimulated by exogenously added lysophosphatidylcholine, indicating that the reaction is catalyzed by a lysophosphatidylcholine acyltransferase (Murphy et al. 1984 a, b). Whether the acyltransferase from leaves catalyzes not only the forward but also the backward reaction, as described above for the enzymic activity from developing oil seeds, has not yet been elucidated.

In an O 2 and NADH dependent reaction the oleoyl groups esterified to phosphatidylcholine were desaturated to C I8 :2• From the specific radioactivities determined for the oleoyl and linoleoyl groups of the microsomal phosphatidylcholine the authors concluded that a functional association of the acyltransferase and the desaturase exists. Accordingly the phosphatidylcholine formed by the activity of the acyltransferase does not mix with the bulk membrane pool of phosphatidylcholine, but is directly channelled to the desaturase.

Positional analyses of the phosphatidylcholine labelled during in vitro experiments revealed surprising results (Murphy et al. 1985 b). As was observed for the acylation of lysophosphatidylcholine in developing oil seeds oleoyl groups were preferably esterified to the C-2 position in pea leaf tissue. In contrast to seed microsomes, however, the C 18 : I-labelling at position 2 decreased with time while a continuous increase of labelled oleoyl residues was observed at the C-1 position. After 35 min about 40 % of the total radioactivity was found at position 1. A continuous increase was also detected in the labelling of C 18 : 2, both in position 2 and in position 1. The authors speculated that the appearance of labelled acyl groups in position 1 is due to acyltransfer

J. Plant Physiol. Vol. 124. pp. 193 - 209 (1986)


from the C-2 position via deacylation/reacylation and/or acyl exchange, although they did not exclude a direct acylation and subsequent desaturation of oleoyl residues at position 1. However, molecular species analysis of phosphatidylcholine labelled under desaturating and nondesaturating conditions clearly revealed that C I6 :0, C,s :oI CIS: I species are the major substrates for the oleoyl phosphatidylcholine desaturase. Consequently, in contrast to microsomal fractions from developing oil seeds, in leaf microsomes the desaturation of oleoyl groups occurs preferably or even exclusively at the C-2 position of phosphatidylcholine (Murphy et al. 1985 b). It is possible that in the near future data from purified and reconstituted desaturases of microsomal membranes will be available since Kader and coworkers (Galle et al. 1984, Bonnerot et al. 1985) have succeeded in purifying two components of the desaturase complex, NADH-cytochrome bs reductase and cytochrome bs.

The results concerning the eukaryotic pathway in the ER membranes of leaves are summarized in Fig. 2. As depicted in the scheme, the partially desaturated phosphatidylcholine is incorporated into the bulk membrane pool and then transferred to other membrane systems.

As was concluded from in vivo double labelling experiments (see preceding) and directly demonstrated by in vitro experiments (Ohnishi and Yamada 1982, Dubacq et al. 1984), eukaryotic phosphatidylcholine is transported from the ER to the chloroplasts probably by the action of phospholipid transfer proteins. In the chloroplast the phosphatidylcholine is integrated into the envelope membrane (Miquel et al. 1984) and its diacylglycerol moiety incorporated into monogalactosyldiacylglycerol (Ohnishi and Yamada 1982, Dubacq et al. 1984). The reactions catabolizing phosphatidylcholine to diacylglycerol are not known.

As in the prokaryotic pathway eukaryotic monogalactosyldiacylglycerol serves as precursor for the synthesis of digalactosyldiacylglycerol. According to the fatty acid patterns of the eukaryotic glycolipids, CI~CIS monogalactosyldiacylglycerol species should be preferentially galactosylated. Furthermore, the enzyme catalyzing the formation of sulfoquinovosyldiacylglycerol from diacylglycerol should possess an even more pronounced selectivity for C,~C,s-species.

In vivo labelling experiments have shown that chloroplasts import different molecular species for the biosynthesis of eukaryotic glycolipids in which linoleoyl groups usually dominate (Williams et al. 1983, Heinz and Roughan 1983). The desaturation to the characteristic CIS: 3/CIS : 3- and C16 : oIC,s : 3-species again occurs in the chloroplasts, possibly by the activity of the rather unspecific n-6 and n-3 desaturases.

The existence of rather unspecific n-3 desaturases was indicated by experiments with the pyridazinon herbicide San 9785 (Kahn et al. 1979, Lem and Williams 1981, Willemot et al. 1982, Davis and Harwood 1983). Unless the plants metabolize the herbicide to inactive compounds (Murphy et al. 1985 a), the treatment with San 9785 results in a decreased level of trienoic acids and a correspondingly higher amount of dienoic acids. This change in the fatty acid patterns is observed both in the prokaryotic glycerolipids and in the eukaryotic glycolipids of the membrane systems of chloroplasts.

Since the activity of a linoleoyl-CoA ,::l,s-desaturase has been demonstrated to occur

J. Plant Plrysiol. Vol. 124. pp. 193-209 (1986)


in the thylakoids of pea chloroplasts (Grechkin et al. 1984), thioester dependent desaturation in combination with transacylation reactions cannot be excluded as a possible mechanism for glycerolipid desaturation.

The biosynthesis capacities of the two pathways of glycerolipid synthesis must be well balanced to result in the different levels of prokaryotic and eukaryotic glycerolipids in the membrane system of chloroplasts of the various plants.

The ability to form prokaryotic glycolipids is decisively controlled by the activity of the plastidial phosphatidic acid phosphatase. In vitro labelling experiments with isolated chloroplasts from different 16: 3- and 18: 3-plants indicate that the phosphatase activity is highly correlated with the amount of hexadecatrienoic acid: The lower the content of C 16 : 3 the lower the activity of phosphatidic acid phosphatase (Gardiner and Roughan 1983, Gardiner et al. 1984 a, Heinz and Roughan 1983). Furthermore, 18: 3-plants convert proportionally much less prokaryotic diacylglycerol to monogalactosyldiacylglycerol in comparison to 16: 3-plants (Gardiner et al. 1984 a, b, Heinz and Roughan 1983). This is probably due to the different subplastidiallocation of the diacylglycerol galactosyltransferase. In the 16: 3-plant spinach this enzyme is located in the inner envelope membrane (Dome et al. 1982 a) and the 18: 3-plant pea in the outer membrane (Cline and Keegstra 1983).

The biosynthetic capacities of both pathways are also regulated by the concentrations of glycerol 3-phosphate. As shown by both in vitro and in vivo labelling experiments (Gardiner et al. 1982), elevated cellular concentrations of glycero13-phosphate in leaves of 16: 3 and 18: 3-plants have no effect on the total incorporation of acetate into lipids, but significantly stimulate the biosynthesis of prokaryotic glycerolipids with an concomitant decrease in the labelling of eukaryotic glycerolipids. In this regulation the different affinities of the glycerol 3-phosphate acyltransferase for the acyl acceptor may play a decisive role.

These initial results indicate a complex regulatory mechanism for the biosynthesis of glycerolipids in plastids.

Acknowledgements

The author wishes to thank Professor Dr. E. Heinz for helpful discussions and Dr. J. Andrews for critically reading the manuscript. Investigations carried out in the author's laboratory were supported by the Deutsche Forschungsgemeinschaft.

References

ANDREWS, J. and K. KEEGSTRA: Acyl-CoA synthetase is located in the outer membrane of pea chloroplast envelopes. Plant Physiol. 72,735-740 (1983).

ANDREWS, J. and J. B. MUDD: Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization. Plant Physiol. 79, 259-265 (1985).

ANDREWS, J., J. B. OHLROGGE, and K. KEEGSTRA: Final step of phosphatidic acid synthesis in pea chloroplasts occurs in the inner envelope membrane. Plant Physiol. 78, 459-465 (1985).

BLOCK, M. A., A.-J. DORNE, J. JOYARD, and R. DOUCE: The acyl-CoA synthetase and acyl-CoA thioesterase are located on the outer and inner membrane of the chloroplast envelope respectively. FEBS Lett. 153, 377 -381 (1983 a).

- - - - The phosphatidic acid phosphatase of the chloroplast envelope is located on the inner envelope membrane. FEBS Lett. 164, 111-115 (1983 b).



BONNEROT, c., A. M. GALLE, A. JOLLiOT, and J.-c. KADER: Purification and properties of plant cytochrome bs• Biochem. J. 226, 331-334 (1985).

BROWSE, J., P. MCCOURT, and C. R. SOMMERVILLE: A mutant of Arabidopsis lacking a chloroplast-specific lipid. Science 227, 763-765 (1985).

BROWSE, J. A., C. R. SOMMERVILLE, and P. J. MCCOURT: Glycerolipid metabolism in leaves: New information from Arabidopsis mutants. In: SIEGENTHALER, P.-A. and W. EICHENBERGER (eds.): Structure, Function and Metabolism of Plant Lipids, 167 -170, Elsevier Science Publishers, Amsterdam (1984).

CLINE, K. and K. KEEGSTRA: Galactosyltransferases involved in galactolipid biosynthesis are located in the outer membrane of pea chloroplast envelopes. Plant Physiol. 71, 366-372 (1983).

DAVIES, A. o. and J. L. HARWOOD: Effect of substituted pyridazinones on chloroplast structure and lipid metabolism in greening barley leaves. J. Exp. Botany 34, 1089-1100 (1983).

DORNE, A.-J., M. A. BLOCK, J. J OYARD, and R. DOUCE: Studies on the localization of enzymes involved in galactolipid metabolism in chloroplast envelope membranes. In: WINTERMANS, J. F. G. M. and P. J. C. KUIPER (eds.): Biochemistry and Metabolism of Plant Lipids, 153 -164, Elsevier Biomedical Press, Amsterdam (1982 a).

- - - - The galactolipid: galactolipid galactosyltransferase is located on the outer surface of the outer membrane of the chloroplast envelope. FEBS Lett. 145, 30 - 34 ( 1982 b).

DUBACQ, J. P., M. MIQUEL, D. DRAPIER, A. TRfMOuERES, and J. C. KADER: Some aspects of the role of chloroplast envelope membranes in lipid metabolism. In: SIEGENTHALER, P.-A. and W. EICHENBERGER (eds.): Structure, Function and Metabolism of Plant Lipids, 311-314, Elsevier Science Publishers, Amsterdam (1984).

DUBACQ, J. P. and A. TRfMOUERES: Occurrence and function'of phosphatidylglycerol containing ~3-trans-hexadecenoic acid in photosynthetic lamellae. Physiol. Veg. 21, 293 - 312 (1983).

FRENTZEN, M., W. HAREs, and A. SCHIBURR: Properties of the microsomal glycerol 3-P and monoacylglycerol 3-P acyltransferase from leaves. In: SIEGENTHALER, P.-A. and W. EICHENBERGER (eds.): Structure, Function and Metabolism of Plant Lipids, 105-110, Elsevier Science Publishers, Amsterdam (1984).

FRENTZEN, M., E. HEINz, T. A. McKEoN, and P. K. STUMPF: Specificities and selectivities of glycerol-3-phosphate acyltransferase and monoacylglycerol-3-phosphate acyltransferase from pea and spinach chloroplasts. Eur. J. Biochem. 129, 629-636 (1983).

GALLE, A. M., C. BONNEROT, A. JOLUOT, andJ.-C. KADER: Purification of a NADH-ferricyanide reductase from plant microsomal membranes with zwitterionic detergent. Biochem. Biophys. Res. Commun. 122,1201-1205 (1984).

GARDINER, S. E., E. HEINZ, and P. G. ROUGHAN: Rates and products of long chain fatty acid synthesis from [1-14C]acetate in chloroplasts isolated from leaves of 16: 3 and 18: 3 plants. Plant Physiol. 74, 890-896 (1984 a).

GARDINER, S. E. and P. G. ROUGHAN: Relationship between fatty-acyl composition of diacylgalactosylglycerol and turnover of chloroplast phosphatidate. Biochem. J. 210, 949-952 (1983).

GARDINER, S. E., P. G. ROUGHAN, and J. BROWSE: Glycerolipid labelling kinetics in isolated intact chloroplasts. Biochem. J. 224, 637 -643 (1984 b).

GARDINER, S. E., P. G. ROUGHAN, and C. R. SLACK: Manipulating the incorporation of [1)4C} acetate into different leaf glycerolipids in several plant species. Plant Physiol. 70, 1316-1320 (1982).

GRECHKIN, A. N., T. E. GAFAROVA, and J. A. TARCHEVSKY: Linoleate ~Is-desaturase activity of pea leaf chloroplasts is localized in thylakoids. In: SIEGENTHALER, P.-A. and W. EICHENBERGER (eds.): Structure, Function and Metabolism of Plant Lipids, 51- 54, Elsevier Science Publishers, Amsterdam (1984).



GRIFFITHS, G., A. K. STOBART, and S. STYMNE: The acylation of sn-glycerol3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctonus L.) seed. Biochem. J. 230, 379-388 (1985).

HAAs, R., H. P. SIEBERTZ, K. WRAGE, and E. HEINz: Localization of sulfolipid labelling within cells and chloroplasts. Planta 148, 238-244 (1980).

HEEMSKERK., J. W., G. BOGEMANN, and J. F. G. M. WINTERMANS: Turnover of galactolipids incorporated into chloroplast envelopes. An assay for galactolipid: galactolipid galactosyltransferase. Biochem. Biophys. Acta 754,181-189 (1983).

- - - Spinach chloroplast: localization of enzymes involved in galactolipid metabolism. Biochim. Biophys. Acta 835, 212-220 (1985).

HEINZ, E.: Enzymatic reactions in galactolipid biosynthesis. In: TEVINI, M. and H. K. LiCHTENTHALER (eds.): Lipids and Lipid Polymers in Higher Plants, 102-120, Springer-Verlag, Berlin (1977).

HEINZ, E. and P. G. ROUGHAN: Similarities and differences in lipid metabolism of chloroplasts isolated from 18: 3 and 16: 3 plants. Plant Physiol. 72,273-279 (1983).

!cHIHARA, K.: sn-glycerol-3-phosphate acyltransferase in a particulate fraction from maturing safflower seeds. Arch. Biochem. Biophys. 232, 685-698 (1984).

JOYARD, J. and R. DOUCE: Characterization of phosphatidate phosphohydrolase activity associated with chloroplast envelope membranes. FEBS Lett. 102, 147 -150 (1979).

KHAN, M.-V., N. W. LEM, K. R. CHANDORKAR, andJ. P. WILLIAMS: Effects of substituted pyridazinones (San 6706, San 9774, San 9785) on glycerolipids and their associated fatty acids in leaves of Vicia faba and Hordeum vulgare. Plant Physiol. 64, 300-305 (1979).

KLEpPINGER-SPARACE, K. F., J. B. MUDD, and D. G. BISHOP: Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants: The incorporation of 35S04 by intact chloroplasts. Arch. Biochern. Biophys. 240,859-865 (1985).

LEM, N. W. and J. P. WILLIAMS: Desaturation of fatty acids associated with monogalactosyl diacylglycerol: The effects of San 6706 and San 9785. Plant Physiol. 68, 944-949 (1981).

MCCOURT, P., J. BROWSE, J. WATSON, C. J. ARNTZEN, and C. R. SOMERVILLE: Analysis of photosynthetic antenna function in a mutant of Arabidopsis thaliana (L.) lacking trans-hexadecenoic acid. Plant Physiol. 78, 853-858 (1985).

MIQUEL, M., M. A. BLOCK, J. JOYARD, A.-J. DORNE, J.-P. DUBACQ, J.-c. KADER, and R. DOUCE: Protein-mediated transfer of phosphatidylcholine from liposomes in spinach chloroplast envelope. In: SIEGENTHALER, P.-A. and W. EICHENBERGER (eds.): Structure, Function and Metabolism of Plant Lipids, 295-298, Elsevier Science Publishers, Amsterdam (1984).

MOORE, T. S. Jr.: Phospholipid biosynthesis. Ann. Rev. Plant Physiol. 33,235-259 (1982). MuRPHY, D. J., J. L. HARWOOD, K. A. LEE, F. ROBERTO, P. K. STUMPF, and J. B. ST. JOHN: Differ

ential responses of a range of photosynthetic tissues to a substituted pyridazinone, Sandoz 9785. Specific effects on fatty acid desaturation. Phytochem. 24, 1923 -1929 (1985 a).

MuRPHY, D. J., K. D. MUKHERJEE, and E. LATZKO: Lipid metabolism in microsomal fraction from photosynthetic tissue. Effects of catalase and hydrogen peroxide on oleate desaturation. Biochem. J. 213, 249-252 (1983 a).

- - - Oleate metabolism in microsomes from developing leaves of Pisum sativum L. Planta 161,249-254 (1984 a}.

MuRPHY, D. J., K. D. MUKHERJEE, and I. E. WOODROW: Functional association of a monoacylglycerophosphocholine acyltransferase and the oleoylglycerophosphocholine desaturase in microsomes from developing leaves. Eur. J. Biochem. 139, 373 - 379 (1984 b).

MuRPHY, D. J., I. E. WOODROW, and K. D. MUKHERJEE: Substrate specificities of the enzymes of the oleate desaturase system from photosynthetic tissue. Biochem. J. 225, 267 -270 (1985 b).

MuRPHY, D. J., I. E. WOODROW, E. LATZKO, and K. D. MUKHERJEE: Solubilisation of oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. The oleate desaturase system of pea leaf microsomes. FEBS Lett. 162, 442-446 (1983 b).



OHNISHI, J. and M. YAMADA: Glycerolipid synthesis in A vena leaves during greening of etiolated seedlings II. a-Linolenic acid synthesis. Plant & Cell Physiol. 21, 1607-1618 (1980).

- - Glycerolipid synthesis in Avena leaves during greening of etiolated seedlings ill. Synthesis of a-linoleoyl-monogalactosyl diacylglycerol from liposomallinoleoyl-phosphatidylcholine by Avena plastids in the presence of phosphatidylcholine-exchange protein. Plant & Cell Physiol. 23, 767 -773 (1982).

REMy, R., A. TRfMouEREs, F. AMBARD-BRETTEVIllE: Formation of oligomeric light harvesting chlorophyll alb protein by interaction between its monomeric form and liposomes. Photobiochem. Photobiophys. 7, 267 -276 (1984).

REMy, R., A. TRfMouEREs, J. C. DuvAL, F. AMBARD-BRETTEVILLE, J. P. DUBAcQ: Study of the supramolecular organisation of light harvesting chlorophyll protein (LHCP). FEBS Lett. 137, 271-275 (1982).

ROCHESTER, C. P. and D. G. BISHOP: The role of lysophosphatidylcholine in lipid synthesis by developing sunflower (Helianthus annuus L.) seed microsomes. Arch. Biochem. Biophys. 232,249-258 (1984).

ROUGHAN, P. G.: Phosphatidyl choline: donor of 18-carbon unsaturated fatty acids for glycerolipid biosynthesis. Lipids 10, 609-614 (1975).

- Cytidine triphosphate-dependent, acyl-CoA-independent synthesis of phosphatidylglycerol by chloroplasts isolated from spinach and pea. Biochim. Biophys. Acta 835,527 -532 (1985).

ROUGHAN, P. G. and C. R. SLACK: Cellular organization of glycerolipid metabolism. Ann. Rev. Plant Physiol. 33,97 -132 (1982).

ROUGHAN, G. and R. SLACK: Glycerolipid synthesis in leaves. Tms 9, 383-386 (1984). SAUER, A. and D. G. ROBINSON: Subcellular localization of enzymes involved in lecithin biosyn

thesis in maize roots. J. Exp. Botany 36, 1257 - 1266 (1985). SHINE, W. E., M. MANCHA, and P. K. STUMPF: Fat metabolism in higher plants differential incor

poration of acyl-coenzymes A and acyl-acyl carrier proteins into plant microsomal lipids. Arch. Biochem. Biophys. 173, 472-479 (1976).

SIEBERTZ, H. P. and E. HEINZ: Labelling experiments on the origin of hexa- and octadecatrienoic acids in galactolipids from leaves. Z. Naturforsch. 32c, 193-205 (1977).

SIEBERTZ, H. P., E. HEINZ, J. JOYARD, and R. DOUCE: Labelling in vivo and in vitro of molecular species of lipids from chloroplast envelopes and thylakoids. Eur. J. Biochem. 108, 177 -185 (1980).

SLACK, C. R., L. C. CAMPBELL, J. A. BROWSE, and P. G. ROUGHAN: Some evidence for the reversibility of the cholinephosphotransferase-catalysed reaction in developing linseed cotyledons in vivo. Biochim. Biophys. Acta 754, 10-20 (1983).

SLACK, C. R., P. G. ROUGHAN, and N. BALASINGHAM: Labelling studies in vivo on the metabolism of the acyl and glycerol moieties of the glycerolipids in the developing maize leaf. Biochern. J. 162, 289-296 (1977).

SLACK, C. R., P. G. ROUGHAN, and J. BROWSE: Evidence for on oleoylphosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower seeds. Biochem. J. 179, 649-656 (1979).

SLACK, C. R., P. G. ROUGHAN, J. A. BROWSE, and S. E. GARDINER: Some properties of cholinephosphotransferase from developing safflower cotyledons. Biochim. Biophys. Acta 833, 438-448 (1985).

SPARACE, S. A. and B. MUDD: Phosphatidylglycerol synthesis in spinach chloroplasts: Characterization of the newly synthesized molecule. Plant Physiol. 70, 1260-1264 (1982).

STOBART, A. K. and S. STYMNE: The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower. Planta 163, 119-125 (1985).

STOBART, A., S. STYMNE, and G. GLAD: The synthesis of linoleate and phosphatidic acid and its relationship to oil production in the microsomes of developing seeds of safflower (Carthamus tinctorius, L. var. Gila). Biochim. Biophys. Acta 754, 292-297 (1983).



STYMNE, S. and A. K. STOBART: Evidence for the reversibility of the acyl-CoA: lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver. Biochem. J. 223, 305-314 (1984 a).

- - The biosynthesis of triacylglycerols in microsomal preparations of developing cotyledons of sunflower (Helianthus annuus L.). Biochem. J. 220, 481-488 (1984 b).

- - Oil synthesis in vitro in microsomal membranes from developing cotyledons of Linum usitatissimum L. Planta 164, 101-104 (1985).

STYMNE, S., A. K. STOBART, and G. GLAD: The role of the acyl-CoA pool in the synthesis of polyunsaturated 18-carbon fatty acids and triacylglycerol production in the microsomes of developing safflower seeds. Biochim. Biophys. Acta 752, 198-208 (1983).

VAN BESOUW, A. and J. F. G. M. WINTERMANS: Galactolipid formation in chloroplast envelopes I. Evidence for two mechanisms in galactosylation. Biochim. Biophys. Acta 529, 44-53 (1978).

- - The synthesis of galactosyldiacylglycerols by chloroplast envelopes. FEBS Lett. 102, 33-37 (1979).

WILLEMOT, c., C. R. SLACK, J. BROWSE, and P. G. ROUGHAN: Effect of BASF 13-338, a substituted pyridazinone, on lipid metabolism in leaf tissue of spinach, pea linseed and wheat. Plant Physiol. 70, 78-81 (1982).

WILLIAMS, J. P. and M. U. KHAN: Lipid biosynthesis in Brassica napus leaves I. 14C-Labelling kinetics of the fatty acids of the major glycerolipids. Biochim. Biophys. Acta 713, 177 -184 (1982).

WILLIAMS, J. P., M. U. KHAN, and K. MITCHELL: Galactolipid biosynthesis in leaves of 16: 3- and 18: 3-plants. In: THOMSON, W. W., J. B. MUDD and M. GmBS (eds.): Biosynthesis and Function of Plant Lipids, 28 - 39. Waverly Press, Baltimore, Maryland (1983).

WILLIAMS, J. P., G. R. WATSON, and S. P. K. LEUNG: Galactolipid synthesis in Vicia faba leaves II. Formation and desaturation of long chain fatty acids in phosphatidylcholine, phosphatidylglycerol and the galactolipids. Plant Physiol. 57, 179-184 (1976).


biosynthesis and desaturation of the different diacylglycerol moieties in higher plants

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