Biosynthesis of γ-linolenic acid in developing seeds of borgae (Borago officinalis L.)

52

Biochirnica et Biophysica Acta, 1158 (1993) 52-58 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/$06.00

BBAGEN 23837

Biosynthesis of y-linolenic acid in developing seeds of borage ( Borago officinalis L.) A . M . G a l l e a, M . J o s e p h b, C. D e m a n d r e b, p. G u e r c h e a, j . p . D u b a c q c, A . O u r s e l b, P. M a z l i a k b G . P e l l e t i e r a a n d J . C . K a d e r b a Laboratoire de Biologie Cellulaire, LN.R.4., Versailles Cedex (France) and b Laboratoire des Biomembranes V£g~tales, C.N.R.S., U.R.A. 1180, Paris (France) and c Laboratoire des biomembranes et Surfaces Cellulaires V~g~tales, C.N.R.S., U.R.A. 311, G.D.R. 1002, Ecole Normale SupHieure, Paris (France)

(Received 30 March 1993)

Key words: 3,-Linolenicacid; A6-Desaturase; Seeds; Membrane; Acylation; Borago of'ficinalis

A6-desaturation of [14C]linoleoyl-CoA or [14C]oleoyl-CoA leading to the synthesis of 3,-linolenic acid was studied in vitro with microsomal fractions from developing seeds of Borago of-ficinalis.Time course of the reaction, effects of protein and precursor concentrations and nucleotide requirements were examined. These parameters allowed us to improve the in vitro A6-desaturation assay. We observed that the precursors were acylated mainly in phosphatidylcholine, diacylglycerol and triacylglycerol, and then desaturated. NADH was absolutely required when [14C]oleoyl.CoA was the precursor, but not when [14C]linoleoyl-CoAwas the precursor although it stimulated the reaction. The in vitro A6-desaturase activity was found mainly in phosphatidylcholine, associated with enriched endoplasmic reticulum membranes (ER) from embryos. No activity was observed in ER from seed coat or seedling. During maturation of the seeds, A6-desaturase reached its highest activity 14 to 16 days after pollination.

Introduction In sharp contrast to animals, only few plants contain 3,-linolenic acid (octadeca-A6,9,12-trienoic acid) as triunsaturated fatty acid; the major triunsaturated fatty acid in plants is usually a-linolenic acid (octadecaD9,12,15-trienoic acid). The sources of plant 7-1inolenate are the seed oils of the common borage (Borago officinalis L.) [1], evening primerose (Oenothera biennis L.) [2,3] and the fruits of black currants (Ribes nigrum L.) [4], in which 3,-linolenic acid represents 26%, 9% and 19% of total fatty acid weight, respectively. Gamma-linolenic acid is also found in algae and cyanobacteria [5,6], fungi [7,8] and protozoa [9]. In animals, 3,-linolenic acid is synthesized from linoleic acid by a zl6-desaturase. The activity of this membranous enzymatic complex has been extensively

Correspondance to: A.M. Gaile, Laboratoire des Biomembranes Vdg6tales, 4 place Jussieu, T53 3ame 6tage case 154, 75252 Paris Cedex 05, France. Abbreviations: DAG, diacylglycerol;HPLC, high performance liquid chromatography; MGDG, monogalactosyldiacylglycerol;NL, neutral lipids; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, polar lipids; TAG, triacylglycerol;TLC, thin layer chromatography

studied; it is affected by certain diets [10-13] or drugs [14]. Catalase, bovine serum albumin and N A D H are necessary for in vitro A6-desaturase activity [15,16]. Solubilization of A6-desaturase from rat liver microsomes was achieved by Triton X100 or sodium deoxycholate [17], then the enzyme was purified and partially characterized [18]. The formation of ~/-linolenic acid by this za6-desaturase was further optimized by removal of the y-linolenic formed [19]. Cytochrome b 5 was proved to be the electron donor for the A6-desaturase of rat liver microsomes [20]. Although y-linolenic acid is synthesized by animals, this synthesis tends to decrease with, either the age of the subject or several pathologies. It is sometimes necessary to supply this deficiency by adding y-linolenic extracted from plants into the diets. This is the reason why it appears of interest to study y-linolenic acid synthesis in the seeds in which it accumulates. In plants, polyunsaturated fatty acids are synthesized by successive desaturations first, of oleic acid to linoleic acid (octadeca -,~9,12- dienoic acid) by a /112desaturase and secondly, to a- or y-linolenic acids by A15- or za6-desaturases, respectively. The A12-desaturase is localized in higher plants either in the endoplasmic reticulum membranes [21-24] or in chloroplasts [25]; it also has been found in fungi [26]

53 and in some protozoa [27]. In the endoplasmic reticulum membranes, the A12-desaturase is included in an enzymatic complex where cytochrome b 5 and NADHcytochrome b 5 reductase (EC 1.6.2.2) are also implied [28]. Concerning the A15-desaturase, in vivo and in vitro studies of a-linolenic acid synthesis have brought evidence for a dual localization of the A15-desaturase complex involved in this synthesis. Working with microsomes of developing linseed cotyledons, Browse and Slack [29] showed an active metabolisation of linoleoyl-phosphatidylcholine to a-linolenoyl-phosphatidylcholine, essentially on the fatty acid esterified on carbon 2 of the sn-glycerol backbones. On the other hand, a A15-1inoleoyl-desaturase was evidenced in chloroplasts [30,31] and more recently at the chloroplast envelope level [25]. This chloroplastidial-desaturase was efficient on MGDG molecules and seemed restricted to linoleoyl residues acylated on carbon 1 of the sn-glycerol backbones [32]. In contrast, A6-desaturase has been evidenced only in microsomal membranes. However the enzyme has not been yet isolated from plants. The biosynthesis of y-linolenate in microsomal membranes of B. officinalis cotyledons was studied for the first time by Stymne and Stobart [33] and Griffiths et al. [34]. Microsomes of developing cotyledons were shown efficient in the synthesis of y-linolenate from linoleoyl-CoA exogenously supplied in vitro to the cotyledons. Styrene and Stobart [33] have supposed that an equilibrium between PC and DAG was observed during the synthesis of TAG. Hence, they argue that PC could be the precursor for the synthesis of storage lipids [35,36]. To assert these previous results obtained from in vivo and in vitro experiments and to offer a more powerful test for y-linolenate synthesis with the aim of further isolating the enzyme concerned, we have undertaken a complete in vitro study of y-linolenate and oleate desaturations with B. officinalis seeds microsomes The characteristics and the improvement of the in vitro A6-desaturation assay were further studied in the present work. The in vitro A6-desaturase activity of microsomal membranes from different seed tissues and the changes of activity occurring along the maturation of B. of-ficinalis seeds were also examined. Materials and methods

Chemicals [1-14C]linoleoyl-CoA (sp. radioactivity 2 GBq/mmol) was purchased from NEN Dupont de Nemours (USA). [1-14C]oleoyl-CoA (sp. radioactivity 2GBq/mmol), Catalase (thymol-free, 10000-25000 units/rag protein), bovine serum albumin (BSA, fatty acid free), CoA-SH, NADH and lipid standards for chromatography were purchased from Sigma.

Biological material B. of-ficinalis L. (common borage) seeds were obtained from the Institut National de Recherche Agronomique (I.N.R.A., Versailles, France). The flowers were hand pollinated and the developping seeds were harvested from 8 days after pollination to mature seeds.

Preparation of microsomal membranes All manipulations during the isolation procedure were performed at 4°C. Developing seeds were homogenized (10 g of seeds to 150 ml of extraction medium) in 70 mM phosphate buffer, pH 7.2 containing 0.33 M sucrose, 5 mM cysteine chloride, 1,000 units of catalase/ml of extraction medium and 1% of BSA. The homogenate was filtered through a double layer of Miracloth and centrifuged at 15000 x g for 20 min. The supernatant was then centrifuged at 100000 x g for 90 min. The resulting microsomal pellet was resuspended in 70 mM phosphate buffer (pH 7.2) containing 0.33 M sucrose. The protein concentration in this microsomal suspension was determined according to Lowry et al. [37], with BSA as standard protein. Endoplasmic reticulum membranes from embryos were enriched by centrifugation on sucrose gradient, according to Sturm et al. [38].

Enzyme assays Microsomal assays were carried out at 30°C with constant shaking, in eomplete darkness and air. The incubation mixtures contained 10 mg BSA, 1,000 units of catalase, 200 nmol of CoA, 0.33 M sucrose, 4 nmol [1-14C]linoleoyl-CoA, cofactors and microsomal membranes in 70 mM phosphate buffer (pH 7.2). The final volume of the reaction mixture was 1 ml. The efficiencies of the A6 and A12-desaturases were calculated as the ratios: ([y-14C]linolenic acid)/([14C]linoleic acid + [y-14C]linolenic acid) and ([14C]linoleic a c i d + [ y 14C]linolenic acid)/([14C]oleic acid + [14C]linoleic acid + [y-14C]linolenic acid) in percent, respectively.

Analytical procedures Incubations were stopped by addition of boiling methanol and lipids were extracted according to Bligh and Dyer [39]. Separations of lipid classes were performed on silica gel plates using petroleum ether-diethylether-acetic acid (70: 30: 0.4, v/v) [27] for' the neutral lipids, or chloroform-acetone-methanol-acetic acid-water (50 : 20 : 10 : 10 : 5, v/v) [40] for polar lipids. Lipids were detected by the fluorescence of primuline under UV light at 365 nm [41]. Each spot of lipid was scraped off from the plate, and transmethylated according to Metcalfe and Schmitz [42] or Carreau and Dubacq [43]. The fatty acids methyl esters were extracted in pentane and further separated according to their number of double bonds [27] on TLC plates

54 impregnated with silver nitrate by immersion of the bottom-half of the plate in a 8% mgNO 3 in ethanol/ water, 9/1 by volume solution for 30 s. For non-radioactive experiments, the fatty acid composition was analyzed by gas-chromatography (GC) in a Varian 3300 gas-chromatograph fitteded with a glass capillary column filled with Carbowax 20 M (0.32 mm i.d. x 50 m). For radioactive experiments, 14C-labelled spots were detected by autoradiography, scraped off from the plate, the radioactivity was counted by scintillation in permafluor. Counting was further corrected for quenching. The fatty acids methyl esters were also separated by radio-gas-liquid-chromatography (RGC) on a Carbowax capillary column (0.53 mm i.d. × 25 m) and the radioactivity was measured in a continuous flow apparatus (Flow-one/beta GCR, Radiomatic Instruments). Methyl esters of 14C-labelled y- and a-linolenic acid were authentified by radio-gas-liquid-chromatography. Such analysis of labelled fatty acids was not possible with HPLC or silver nitrate impregnated TLC because y and a isomers were not separated by these methods. Results and discussion

Lipid composition of B. officinalis seeds microsomes The fatty acid compositions of the major glycerolipids found in the microsomes from whole seeds, embryos and green seed coats are given in Table I. Palmitic acid was present in embryos and seed coats

TABLE I Fatty acid composition (weight %) of the major lipid classes of microsomes prepared from whole B. officinalis seeds, embryos or seed coats. Each value is an average of four independant determinations. The analysis of fatty acids were carried out by gas-chromatography. Fatty acids were identified by reference to chemical standards from Sigma Cie. (see the 'Materials and methods' section). The standard deviations for each measurement was less than 0.5%. Lipid classes

Origin

Fatty acid (weight %) 16:0 16:1

18:0 18:1 18:2 718:3 a18:3 18:4

PC

seed 20 embryo 19 coat 18

1 1 3

9 7 5

19 9 29

40 45 36

8 18 2

3 1 6

Trace 1

PE

seed 27 embryo 25 coat 29

2 3 5

11 7 4

11 5 14

43 51 41

5 9 2

DAG

seed 18 embryo 16 coat 25

1 2 4

12 5 8

18 11 14

38 46 36

11 20 8

2 Trace 5

TAG

seed 14 embryo 13 coat 19

0.5 Trace 1

7 6 7

23 20 26

37 40 33

18 21 12

0.5 2

1 Trace Trace 5 -

Trace -

with an appreciable percentage (13 to 29%), whatever the lipid classes considered. Traces of palmitoleic acid were also detected. Stearic acid was present with similar percentages (7-12%) in all tissues and all lipid classes. The amount of oleic acid was higher in neutral lipids than in polar lipids and higher in microsomes from seed coats than from other tissues. Linoleic acid was the most abundant fatty acid in all lipid classes. Its percentage was always higher in embryos than in seed coats. A relative abundancy of y-linolenic acid ranging from 5 to 21% and the presence of a-linolenic acid (1 to 6%) were also observed, y-Linolenic acid was largely present in embryos (9 to 21%), especially in PC, DAG and TAG, while a-linolenic acid was concentrated in seed coat tissues. In embryos, PE was the lipid class which has the highest linoleic acid content but the lowest content in y-linolenic acid. This result suggested that the za6-desaturase was less effective upon this lipid. It did not support the hypothesis of the origin of y-linolenic acid in PE by a desaturase activity in situ, but much more a transacylation reaction from PC to PE. Octadecatetraenoic acid was detected only as traces. The fatty acid compositions of seeds were mainly due to averages of the compositions of embryos and seed coats. It is important to note that in whole seeds, DAG and PC had similar acyl compositions with major palmitic, oleic and linoleic acids and a noticeable percentage of y-linolenic acid. In embryos and seed coats, DAG and TAG have similar fatty acid compositions although a higher fatty acid amount of oleic acid are found in TAG than in DAG. These results concerning the main fatty acid composition of B. officinatis seeds are in good agreement with those of Stymne and Stobart [33] and Griffiths et al. [34], but the percentage of oleic acid in microsomes of embryos found in the present work is lower. Moreover, these authors did not report any trace of a-linolenic and palmitoleic acid in cotyledons.

Improvement of the in vitro A6-desaturase assay To follow in vitro the biosynthesis of y-linolenic acid, it is necessary to supply B. officinalis microsomes with [t4C]linoleoyl-CoA. The experimental assay of in vitro a6-desaturation with direct addition of the radioactive precursor to microsomal membranes, used in the present work, was a simplification of the procedure of Stymne and Stobart [33] which labelled seed cotyledons in vivo from [t4C]linoleoyl-CoA, previous to the isolation of microsomes. It was thus of interest to determine the different parameters controlling this A6-desaturase activity in vitro. Microsomes from 14-16 days old seeds (see later, the influence of seed maturation stage) were selected to optimize the incubation conditions for /t6-desaturase activity.

55 labelled pmma-linoienk acid

Nucleotide requirements [14C]oleoyl-CoA and [14C]linoleoyl-CoA were used as precursors in two sets of experiments carried out in order to evaluate the efficiency of NADH as electron donor in the course of desaturation activities. In the absence of NADH, no desaturation occurred when [14C]oleoyl-CoA was provided to microsomal membranes. In contrast, when [14C]linoleoyl-CoA was used as a precursor, traces of labelled y-linolenic acid were obtained. Thus A6-desaturase was still active with the very low concentrations of endogenous NADH. When [14C]oleoyl-CoA and NADH were added to microsoreal membranes (Fig. 1A), the radioactivity of [14C]oleoyl-CoA was recovered both in linoleic and y-linolenic acids. The efficiencies (see definition in 'Materials and methods') of the two successive A12 and A6-desaturations were 27% and 21%, respectively. The same NADH concentration stimulated strongly the A6-desaturase and 17% of the total radioactivity was recovered in y-linolenic acid with [~4C]linoleoylCoA as a substrate (Fig. 1B).

Determination of optimal incubation parameters For further physiological experiments, it was interesting (in regard of our conditions of detection by RGC), to be at the optimum of in vitro biosynthesis of

1

3

6

A

1

6

3

B

5 5

4;

4,

I

250

6

7

0

20

40

60

0

20

40

60

retention time(min)

Fig. I. Radio-gas-liquid chromatograms of fatty acids from P C after in vitro desaturation in presence of N A D H . The precursor was respectively: IA- [14C]oleoyl-CoA, IB- [14C]linoleoyl-CoA. B. officina//s microsomes (1 m g of embryo proteins) were incubated with 4 nmol of precursor for 90 min. The chromatograms shown are representative of a typical experiment. For incubation conditions and analytical procedure, see the 'Materials and methods' section. 1, Palmitic acid; 2, Palmitoleic acid; 3, Heptadecanoic acid (as standard); 4, Stearic acid; 5, Oleic acid; 6, Linoleic acid; 7, -/-linolenic acid 8, a-linolenicacid.

(pma)

inkUed Iinak* acid ( p ~ ) 1200.

2A

211

,ooi ."~

30

2ooi

10 0 50

100

150 incubation lime (rain)

incubation time (Irnin)

% of gamma.linolenic acid 20

inbened ratty acid (pmoO 800-

2D

2C

500. 400i ~0.

....m....... ,N .M'"N"'" ..w o

0~

30

,0

~

1~

incubation tinw (rain)

,o

O~ 0

. I

.

. 2

. 3

4

mg of embryo proteins

Fig. 2. Effects of incubation time and microsomal protein concentrations on the kinetics of polar and neutral lipids during in vitro A6-desaturation. B. officinalis seed microsomes were prepared, incubated and analyzed as described in 'Materials and methods'. (A) Effect of incubation time on acylation reactions: the amount of [14C]linoleoyl-CoA and microsomal proteins were 4 nmol and 1 mg per assay, respectively. Results are expressed in pmol of linoleic acid acylated in PC, DAG and TAG. (B) Effect of incubation time on desaturation reactions: incubation were performed as in (A) Results are expressed in pmol of y-linolenic acid biosynthesized in PC, DAG and TAG. -<3-, PC; - • - , DAG; - • -, TAG. (C) Effect of incubation time on A6 and A12 desaturation reactions: the precursor was [14C]oleoyl-CoA (4 nmol) and the amount of microsomal proteins was 1 mg. Results are expressed in pmol of radioactive fatty acids recovered in PC. - - I - - , Oleic acid; . . . . × . . . . , Linoleic acid; - ~ - , y-linolenic acid. (D) Effect of protein concentration: the precursor was [14C]linoleoyl-CoA (4 nmol) for an incubation time of 90 min. Results are expressed as the efficiencies of y-linolenic acid synthesis (%) in PC ( - o - ) . All results are the mean of two independant experiments.

y-linolenic acid, in order to get maximal labelling in y-linolenic acid and reproducible results. First, a kinetic study of the 3,-linolenic acid biosynthesis using 1 mg of embryo proteins was performed with [14C]linoleoyl-CoA as precursor. The recovery of [14C]linoleic acid was analyzed especially in PC and NL. Under these conditions (Fig. 2A), the acylation of the [14C]linoleic acid in PC started rapidly and reached a plateau at 30 min. In DAG and TAG, the acylation rate was lower and after 60 rain was stable throughout the incubation time. The in vitro biosynthesis of y-linolenic acid in PC increased rapidly and was linear for 90 min (Fig. 2B). In DAG and TAG, the labelled y-linolenic acid was detectable after few minutes of incubation. DAG appeared a little more labelled than TAG. A second set of experiments was carried out with [14C]oleoyl-CoA as precursor (Fig. 2C). As in the first experiment, the acylation of PC by the precursor was

56 rapid and, as for the A6-desaturation, the A12-desaturation proceeded immediately after addition of the precursor, and reached its highest activity after 60-120 min of incubation. In these conditions of time (90 min), an increase in concentration of embryo microsomal proteins from 0.25 to 4 rag. ml - t of incubation mixture resulted in a significant and almost linear increase of radioactive 3'-linolenic acid in vitro synthesis up to 1 mg. Then, a plateau was observed from 1 rag. ml-1 to 4 mg. ml-1 (Fig. 2D). Using 1 mg of embryo proteins for an incubation time of 90 min, three concentrations of [~4C]linoleoylCoA were assayed: 1, 2 and 4 nmol- ml-1 of incubation mixture. It is interesting to note that these concentrations are much lower than those used by Stymne and Stobart [33]. The percentages of ([ 3'-14C]linolenoyl-CoA)/([ t4C]linoleoyl-CoA + [ 3'- 14C]linolenoylCoA) recovered were respectively 12%, 15% and 20% (not shown). From these experiments, we concluded that the best conditions of incubation to obtain the maximum of radioactivity in 3'-linolenic acid were: 1 mg of embryo proteins, 4 nmol. ml-1 of [14C]oleoyl-CoA or [14C]linoleoyl-CoA, presence of NADH and 90 min of incubation time.

Recovery of [3"-14C]linolenic acid in various lipid classes The lipid classes were examined during in vitro synthesis of 3'-linolenic acid experiments. The acylation of linoleic acid occured mainly in PC, TAG and DAG (Fig. 3A) while PE, PI and PA were less acylated. Analysis showed a larger acylation of linoleic acid in TAG than in DAG which confirmed the hypothesis that the precursor was also used for the last acylation during the formation of TAG. The radioactive 3,-linolenic acid synthesized in vitro was recovered mostly in PC and DAG (Fig. 3B). [3'-14C]linolenate was present at low levels in TAG, PE, PA and PI. By comparing [14C]linoleate and [3'-14C]linolenate levels in DAG and TAG, it can be concluded that the major fatty acid in the position 3 of sn-glycerol of TAG would be preferentially linoleate. This analysis of the A6-desaturase activity shows that the formation of DAG and TAG probably originates from PC which is the major substrate of d6-desaturase [34]. In contrast, the lower content in 3'-linolenic acid in PE suggests that it was less used in the formation of storage lipids. Taking in account these results, all further analysis were performed on PC or NL. In vitro A6-desaturase activity and tissue specificity Embryo and seed coats of developing seeds contain respectively 30% and 70% of total seed proteins. In embryo microsomes the a6-desaturase activity (21%) was found identical to that of whole seed microsomes

distributicm oflahell~l linol¢~cacid in the lipid ~ (1~~) SO 40 210 20 IO 0

PC TAG DAG PE

PA

PI

lipid ch~se~ di~trtbatimt dlabelled gamw~-Iinolenic add in the lipid cl~ees (in %) 60. SO. 40. 30. 20. 10 O.

PC TAG DAG rE

PA

PI

lipid classes

Fig. 3. Synthesis of phospholipids and neutral lipids after in vitro acylation and A6-desaturation in B. officinalis microsomes. Results are expressed as percentage of radioactivity recovered in linoleic acid (A) and in y-linolenic acid (B). B. microsomes (1 mg of embryo proteins) were incubated with [14C]linoleoyl-CoA (4 nmol) for 90 min. For experimental details see 'Materials and methods'. For each experiment, results are the m e a n of two independant determinations.

(19%). Further experiments were carried out on microsomes from whole seeds of various ages. The biosynthesis of 3,-linolenic acid was also observed with endoplasmic reticulum membranes from embryo microsomes and confirmed the hypothesis that A6-desaturase was an enzyme linked to endoplasmic reticulum membranes. Using the enriched endoplasmic reticulum preparations, the quantity of proteins necessary to detect y-linolenic acid synthesis by RGC could be lowered to 250/~g/ml of incubation mixture instead of 1 mg. Radioactive 3,-linolenic acid was never obtained from the green seed coats although these tissues contain 2% to 12% of this acid depending on the lipid considered. Further experiments would be required to show whether the A6-desaturase was inhibited or if the optimal conditions for in vitro desaturation in seed coats were different. No radioactive a-linolenic was recovered after in vitro desaturation in any microsomes from seed coats, embryos or whole seeds. These results suggest that A6-desaturase in seed coats and A15-desaturase in embryos or coats of seeds are not active at least in vitro. The formation of 3,-linolenic

57 total rad~activity e¢

pnlm41mle~ add (ped)

*d~ mdtmcevay (p,~) ,,~ 6~

.°"

tt . . . . . . . . . .

O

=-

......

,t ........

,&

""

• ..........

~

Conclusions

8O

o.....

u

......

8/10 1 2 / 1 4 14/16 20 stage of maturing l e d s (days)

8/10 12114 14/16 20 stalgeof maturing seeds (days)

Fig. 4. Analysis, at different stage of the maturing B. officinalis seeds, of the radioactivity (pmol) recovered from the precursor ([14Cllinoleoyl-CoA, 4 nmol) added (A) sum of linoleic and y-linoienic acids in pmol, and (B) y-linolenic acid alone in pmol. B. officinalis microsomes (1 mg of microsomai proteins) were incubated with [14C]linoleoyi-CoA (4 nmol) for 90 rain. For other experimental details see Materials and Methods. Results on each stage are the mean of two independant experiments. - r a - , PC; . . . . • . . . . , PE; x - , PA; - - • - -, PI; - - - • - - - , N L .

acid from labelled [14C]linoleoyl-CoA was never observed in vitro with young seedling microsomes.

Variations of in vitro A6-desaturase activity during maturation of seeds The experiments described above devoted to improve the in vitro desaturation tests, were performed with seeds harwested 15-20 days after pollination according to Stymne and Stobart [33]. It was thus of interest to have more informations on the evolution of the in vitro biosynthesis of y-linolenic acid in seeds during maturation. In order to carry out in vitro experiments with microsomes from seeds at different stages of growth, flowers were hand pollinated, identified and the resulting seeds were respectively harvested from 8 to 20 days. Microsomal membranes were prepared from these seeds and, after incubation with [14C]linoleoyl-CoA for 90 rain, the total labelling of fatty acids (linoleate + y-linolenate) from phospholipids and neutral lipids was determined in order to estimate the acylation activity (Fig. 4A). In the earliest stages (8-10 days), the acylation proceeded preferentially in PC, then, at later stages, NL were the most labelled lipids in agreement with the accumulation of storage lipids in seeds. Up to day 14-16, PE, PA and PI labelling was low and stable. When the seed coat began to turn black (around day 20), the labelling of these phospholipid classes sharply decreased while high levels of acylation still occurred in NL and PC. The in vitro A6-desaturase activity was also considered (Fig. 4B). Whatever the developmental stage, the labelled y-linolenate was recovered mainly in PC, NL and at a lower level in PE. In mature seeds (not shown), a lower acylase activity was detected, and the A6-desaturase was less efficient. These results show that the A6-desaturation reached its highest level in seeds at 14-16 days after pollination, mainly in PC, NL and PE.

The fatty acid composition of micro~omes from whole seeds, embryos and coats in B. officinalis seeds was examined, y-Linolenic acid was found abundant in embryos. This contrast with green seed coats where low amount of y-linolenic acid was present but where a-linolenic acid was more important. These results were confirmed by the localization of a A6-desaturase in embryos and the absence of this activity in seed coats, further evidenced in vitro with microsomal membranes. As suggested by previous works, A6-desaturase belongs to an enzymatic complex. To reach its highest efficiency, the desaturase requires that the other proteins of the complex (cytochrome b 5 and NADH-cytochrome b 5 reductase) are in suitable conditions to function. The presence of acyltransferases is also required to esterify the precursor ([ 14C]linoleoyl-CoA) on lipids prior to desaturation. In the present experiments, the highest level of acylation was obtained in shorter times (10 min) than the highest level of A6-desaturation (90 rain). The decrease of labelled linoleic acid in PC and the increase of labelled y-linolenic acid synthesis were complementary, which suggest a substrate/product relation. From these in vitro experiments, it was concluded that a high proportion of molecules needed to be acylated before the optimum of A6-desaturase activity could be observed. Acylation and desaturation are tightly bound to allow labelled y-linolenic biosynthesis. When oleic acid was the substrate two successive desaturations were necessary for the biosynthesis of y-linolenic acid. In this case, a lag phase was observed. It could be correlated to the accessibility of the new biosynthesized substrate ([14C]linoleate) to the second desaturase. NADH has been proved to be an essential electron donor for efficient in vitro A12 and A6-desaturations. However, in contrast with Styrene and Stobart [33], we could observe traces of y-linolenic acid in absence of any exogenous NADH. The original method used for fatty acid analysis by decreasing the detection limit could explain this difference. It was accepted from several studies [23] that PC was the substrate for the formation of DAG and TAG. The experiments reported above show that with the in vitro biosynthesis of y-linolenic acid, PC was the most interesting lipid class and that the evolution of desaturation was quite similar in PC and NL. It argues for the synthesis of NL by hydrolysis of the polar head of PC molecule, and a third acylation by linoleic acid preferentially. In agreement with Stymne and Stobart [33] negligible contribution of PE for TAG formation was evidenced. Finally, from this present work, it is clear that B. officinalis is highly suitable for the in vitro study of

58

A6-desaturase, especially in enriched endoplasmic reticulum membranes from embryo at the 14-16 days stage after pollination. Further experiments will consider the molecular species of lipids involved in the A6-desaturation reaction and, finally aim to the purification and characterization of the A6-desaturase. References 1 Whipkey, A., Simon, J.E. and Janick, J. (1988) J. Am. Oil Chem. Soc. 65, 979-984. 2 Mukherjee, K.D. and Kiewitt, I. (1987) J. Agric. Food Chem. 35, 1009-1012. 3 Yaniv, Z., Ranen, C., Levy, A. and Paleviteh, D. (1989) J. Exp. Bot. 40, 609-613. 4 Traitler, H., Winter, H., Richli, U. and Ingenbleek, Y. (1984) Lipids 19, 923-928. 5 Nichols, B.W. and Wood, B.J.B. (1967) Lipids 3, 46-50. 6 Cohen, Z., Didi, S. and Heimer, Y.M. (1992) Plant Physiol. 98, 569-572. 7 Fukuda, H. and Morikawa, H. (1987) Appl. Microb. and Biotech. 27, 15-20. 8 Lindberg, A.M. and Hansson, L. (1991) Appl. Microbiol. Biotechnol. 36, 26-28. 9 Conner, R.L., Burtness, B. and Fergusson, K.A. (1984) Lipids 19, 285-288. 10 Garg, M.L., Sebokova, E., Thomson, A.B.R. and Clandinin, M.T. (1988) Biochem. J. 249, 351-356. 11 Mahfouz, M.M. and Kummerow, F.A. (1989) Lipids 24, 727-732. 12 Leikin, A.I. and Brenner, R.R. (1989) Biochim. Biophys. Acta 1005, 187-191. 13 Ulmann, L., Poisson, J.P., Blond, J.P. and B6zard, J. (1991) Biochim. Biophys. Acta 1086, 230-236. 14 Kawashima, Y., Musoh, K. and Kozuka, H. (1990) J. Biol. Chem. 265, 9170-9175. 15 Jeffcoat, R., Dunton, A.P. and James, A.T. (1978) Biochim. Biophys. Acta 528, 28-35. 16 Okayasu, T., Ono, T., Shinijima, K. and Imai, Y. (1977) Lipids 12, 267-271. 17 Okayasu, T., Nagao, M. and Imai, Y. (1979) FEBS Lett. 104, 241-243.

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