Phosphatidyl Choline" Donor of 18-Carbon Unsaturated Fatty Acids for Glycerolipid Biosynthesis P.G. ROUGHAN, Plant Physiology Division, D.S.I.R., Private Bag, Palrnerston North, New Zealand ABSTRACT

A specific role for PC in the desaturation of oleate to linoleate in Chlorella has been suggested (5), and recent evidence indicates a direct desaturation of PC bound oleate in fungal microsomes (6-9). Thus far, only the earlier work on pumpkin has indicated an apparent net flux of fatty acids through PC and into other glycerolipids. This communication reports in more detail on the kinetics of labeling of PC in intact pumpkin leaf, and on the transfer of fatty acids from PC to other glycerolipids within the leaf. The phenomenon has been observed in leaves of other species, and when using different methods of introducing the radioactive precursor into the leaves.

Kinetics of radiocarbon incorporation into the phosphatidyl choline of pumpkin leaf fed 1-14C-acetate at low light intensity were strongly suggestive of lipid bound fatty acids acting as substrates for desaturase enzymes. After pulse labeling in direct sunlight with the same precursor, phosphatidyl choline and phosphatidyl glycerol contained up to 90% of t o t a l glycerolipid radiocarbon at the shortest sampling times. Subsequent loss of radiocarbon from phosphatidyl choline and a corresponding gain in other glycerolipids is taken to indicate a net flow of long chain fatty acids through phosphatidyl choline and into other lipids. It is proposed that there may be 2 separate synthetases in leaf tissue, one producing oleic and the other palmitic acids as their end products. Oleic acid is transferred almost exclusively to phosphatidyl choline, where it is further desaturated to linoleic and linolenic acids before being made available for the biosynthesis of other lipids. Palmitic acid is transferred mainly to phosphatidyl glycerol, where it is desaturated to trans-A3-hexadecenoic acid.

METHODS

INTRODUCTION

F r o m an earlier study ( l ) on the incorporation of tA4C-acetate into pumpkin leaf lipids and the subsequent metabolism of fatty acids bound to various glycerolipids, it was suggested that the phospholipid, phosphatidyl choline (PC), played an important role in the mechanism of long chain fatty acid desaturation in leaves. It appeared possible from those results that PC bound fatty acids were, in fact, the substrates for the enzymes which desaturated oleic to linoleic and linolenic acids in leaves (2) in a manner analogous to the role of phosphatidyl ethanotamine (PE) bound oleate in the synthesis of methyl stearic acid (3) and cyclopropane fatty acids (4) in bacteria. It was further suggested from the pumpkin leaf study that the polyunsaturated fatty acids formed on PC were subsequently transferred to other glycerolipids, particularly monogalactosyl diglyceride (MDG), within the cell (1).

Pumpkin plants (Cucurbiter pepo, culfivar 'Queensland Blue') were grown in the glasshouse between September and November, or were grown and used in the field between January and March. Spinach (Spinacea oleracea), sorghum (Sorghum bicolor), and sunflower (Helianthus anuus) were glasshouse grown in Spring, and sunflower was also grown and used in the field in Summer. The radioactive precursor, 1-14C-acetate, was fed either by painting 1-2 ml (50-100/~c) of a 1-2 mM solution evenly over the surface of attached leaves with a camel hair brush, or via the cut petiole of detached leaves. To enable measurement of a time course o f 1-14C-acetate incorporation into leaf lipids, the rate of uptake of the precursor applied to the leaf surface was reduced by shading the leaf. Sampling of leaf material, extraction, separation, and counting of lipids and preparation of glycerolipid fatty acids were essentially as described previously ( I , I 0 ) . Nonpolar glycolipid and phospholipid classes were prepared from larger scale extractions by the silicic acid-acetone column chromatographic technique (11). Phosphatidyl choline was isolated from phospho/ipid fractions by semipreparative, thin layer chromatography (TLC). F a t t y acid moieties of PC were isolated, hydrogenated, and degraded b y the method of Harris, et al., (12). F a t t y acid methyl esters were analyzed for concentration and radioactivity using 200 x 0.4 cm columns of 15% ethylene glycol sue-

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TABLE I Time Course of 14C Incorporation from 1-14C-acetate into Pumpkin Leaf Phosphatidyl Choline under Low Light a Total fatty acid 14C (%)

Time after labeling (rain)

PC -14C (dpm x 10"3/sample)

16:0

107 217 400 900

7 8 7 7

15 30 60 120

18:0

18:1

18:2

6 6

86 61 53 31

31 34 47

18:3

7

as0 #c of 1-14C-acetate was applied to the leaf surface in a shaded area of a glasshouse. The light intensity was one-tenth of direct sunlight in the glasshouse. TABLE II Transfer of Radiocarbon from Phosphatidyl Choline to Other Glycerolipids after Pulse Labeling of Pumpkin Leaf with 1-14C-Acetatea Glycerolipid b PC PG MGD PE PI DGD SL Totals

cpm x 10-4/g fresh leaf 0.5 hr 2 hr 62 11.8 3.2 6.1 2.5 0.3 >0.1 86.0

48 12.4 11.4 8.8 2.8 0.8 >0.1 84.2

Net change -14 + 0,6 + 8.2 + 2.7 + 0.3 + 0.5 0

a l 0 0 # c (2 /amole) of 1-14C-acetate in 2 ml was applied evenly over tile surface of a large leaf on a field grown plant and in full sunlight. One-half of the leaf was removed 0.5 hr after and the other half 2 hr after applying the precursor. Uniformity of the application is indicated by similarity in total glycerolipid radiocarbon from each half leaf. Nonpolar lipids accounted for about 12% of the total lipid radiocarbon at both sampling times. b p c = phosphatidyl choline; PG = phosphatidyl glycerol; MGD= monogalactosyl diglyceride; PE= phosphatidyl ethanolamine; PI = phosphatidyl inositol; DGD = digalactosyl diglyceride; SL = sulpholipid. c i n a t e o n C h r o m a s o r b W in a n A e r o g r a p h M o d e l 2 0 0 gas c h r o m a t o g r a p h f i t t e d w i t h a thermal conductivity detector, and coupled to a N u c l e a r Chicago Biospan r a d i o a c t i v i t y d e t e c t o r . In later e x p e r i m e n t s , similar c o l u m n s were e m p l o y e d in a Y a n a c o G-80 gas c h r o m a t o g r a p h f i t t e d w i t h f l a m e i o n i z a t i o n d e t e c t o r s a n d efflue n t s t r e a m splitters. M e t h y l esters were coll e c t e d in 75 x 0.4 c m glass t u b e s m o i s t e n e d w i t h x y l e n e , a n d were f l u s h e d i n t o vials for scintillation counting. RESULTS

S h a d e d p u m p k i n leaf (light i n t e n s i t y onet e n t h o f direct s u n l i g h t in t h e glasshouse) incorp o r a t e d 50 /~mole of 1-14C-acetate i n t o PC at LIPIDS, VOL. 10, NO. 10

a linear rate f o r 2 h r ( T a b l e I). O n r a d i o a u t o graphs o f t o t a l lipids s e p a r a t e d b y TLC, PC was t h e first s p o t t o appear, a n d was, b y far, t h e s t r o n g e s t s p o t at all times. P h o s p h a t i d y l glycerol a n d n o n p o l a r lipids, i.e., t h e c h r o m a t o graphic f r o n t , were t h e n e x t m o s t s t r o n g l y labeled lipids, b u t , b y 2 h r PE, p h o s p h a t i d y l i n o s i t o l (PI) a n d MGD also c o n t a i n e d d e t e c t able r a d i o a c t i v i t y . D e n s i t o m e t r i c scans of radioa u t o g r a p h s s h o w e d t h a t at 15 min, 80% o f t h e r a d i o c a r b o n i n c o r p o r a t e d i n t o t h e t o t a l lipid was in PC, a n d 20% was in t h e n o n p o l a r lipid. By 2 hr, this h a d c h a n g e d t o 60% in PC, 15% in PG, 1 5% in n o n p o l a r lipid, a n d t h e r e m a i n d e r d i s t r i b u t e d a m o n g MGD, PE, a n d PI. N e i t h e r digalactosyl diglyceride ( D G D ) n o r s u l p h o l i p i d (SL) c o n t a i n e d r a d i o a c t i v i t y d e t e c t a b l e b y this method. Oleic acid was t h e first f a t t y acid o f PC t o i n c o r p o r a t e label, a n d was, i n fact, labeled a l m o s t to e q u i l i b r i u m b y 2 hr. Linoleic acid labeling w i t h i n PC initially lagged b e h i n d t h a t o f oleic acid, b u t t h e n a c c e l e r a t e d a n d e x c e e d e d t h a t o f oleic acid w i t h i n t h e 2 h r ( T a b l e I). In graphical f o r m , t h e s e d a t a p r o d u c e d curves t y p i c a l o f a p r e c u r s o r - p r o d u c t r e l a t i o n s h i p for t h e r e a c t i o n PC-oleate + PC-linoleate, a n d t h e s u m o f t h e r a d i o a c t i v i t y i n oleic plus linoleic acids i n c r e a s e d l i n e a r l y over t h e 2 hr. Incorp o r a t i o n i n t o PC b o u n d p a l m i t i c a n d stearic acids was linear w i t h t i m e a n d a n o r d e r o f magn i t u d e slower t h a n t h e initial r a t e for oleate. Significant labeling of the a-linolenate moiety o f PC was n o t o b s e r v e d u n t i l a f t e r 2 hr. Leaves of field g r o w n p u m p k i n i n c o r p o r a t e d 1 0 0 / a c (2 # m o l e ) of 1-14C-acetat e i n t o lipids in < 30 m i n in full s u n l i g h t ( T a b l e II). W h e n t h e 2 halves o f t h e s e leaves were e x a m i n e d at 0.5 a n d 2 h r a f t e r this pulse labeling, a clear i n d i c a t i o n was o b t a i n e d o f t r a n s f e r of r a d i o c a r b o n f r o m PC t o o t h e r lipids, especially MGD, even i n this relatively s h o r t p e r i o d ( T a b l e II). T h e r e was n o change in t h e labeling o f t h e n o n p o l a r lipid

PC AND FATTY ACID METABOLISM

61 1

TABLE IIl Concentrations and Specific Radioaetivities of Oleic and Linoleie Acids in Nonpolar Lipids Compared with Phosphatidyl Choline of Pumpkin Leaf* Specific radioactivity Concentration

(dpm#zg fatty acid)

Otg/g fresh wt leaf)

0.5 hr

2.0 hr

Nonpolar lipid

18:1 18:2 Phosphatidyl choline 18:1

5.4 7.0 25 100

18:2

2,800 1,460

2,540 1,915

15,000 3,000

3,900 2,800

*See Table II. TABLE IV

Relative Specific Radioactivities of Fragments Prepared from Phosphatidyl Choline-bound Oleic and Linoleic Acids Isolated from Sunflower Leaf Labeled 20 min with 1-14C-acetatea

Degradation product

12:0

13:0

14:0

Fatty Acids 15:0

16:0

17:0

18:0

97

86

90

88

86

106

102

NMb

22

20

22

33

36

41

Relative Specific Activity Phosphatidyl choline18:1

Phosphatidyl choline18:2

apC-oleate was spiked after isolation and prior tm hydrogenation and a-oxidation with 250 #g of unlabeled methyl stearate, while PC-linoleate was taken through the whole procedure (1 I) unfortified. The precursor was applied to the leaf surface of glasshouse grown plants in full sunlight. bNM = not measured. fractions which contained ca. 12% of the total lipid radioactivity. Specific radioactivities of the oleic and linoleic acids of the nonpolar lipids were compared with those in PC to determine whether labeling of the nonpolar lipids could have preceded labeling of PC. In spite of 5-fold and 14-fold higher concentrations of oleic and linoleic acids, respectively, PC fatty acids had 5- and 2-fold higher specific radioactivities, respectively, at the earliest sampling times (Table III). It seems unlike/y, therefore, that labeling of nonpolar fatty acids preceded labeling in PC fatty acids. The 18-carbon fatty acids of PC were formed by de novo synthesis rather than by elongation of existing 16-carbon chains. Degradation, by a-oxidation (12) of oleic and linoleic acid moieties of PC isolated from sunflower leaf 20 man after applying 1-14C-acetate to the leaf surface in full sunlight, showed that oleic acid was uniformly labeled (Table IV), while linoleic acid was labeled more heavily toward the carboxyl end of the chain. This latter pattern of labeling might be expected in oleic acid at

much shorter sampling times, because the first labeled product to emerge from the oleic acid synthetase after 1-14C-acetate application would be labeled exclusively in the C-1 position. Although this apparent involvement of PC in polyunsaturated fatty acid metabolism was most marked in leaves of rapidly growing pumpkin plants, experiments with attached and detached leaves of other species confirmed these findings (Table V). PC was always the most heavily labeled glycerolipid at the earliest sampling times, and a very high proportion of the radiocarbon in PC was associated with the oteic and linoleic acid moieties (Table V). The second most strongly labeled glycerolipid was PG, and this was usually, though not always, labeled in the palmitic and trans-A3-hexadecenoic acid moieties (Table V). These 2 lipids accounted for 70-90% of the total radioactivity recovered in the leaf glycerolipids. PC radioactivity invariably declined with time relative to total lipid radioactivity after pulse feeding 1-14C-acetate, whether by surface application LIPIDS, VOL. 10, NO. 10

P.G. ROUGHAN

612

TABLE V Incorporation of 1-14C-acetate into Glycerolipids and their Constituent Fatty Acids of Different Leaves

Leaf

Gl',~erolipidc

Hr after feec:,~g

Total glycerolipid-14C (%)

Spinacha

PC PG PC PG MGD PC PG MGD PC PG MGD PC PG

2.5 2.5 1 1 1 0.75 0.75 0.75 0.33 0.33 0.33 1 1

41 30 45 25 15 50 26 11 63 20 5 67 20

Sorghuma Sunflowera Sunflowera Pumpkin b

Total fatty acid 14C (%) 16:0 + 16:1 13 73 16 46 41 3 44 13 14 65 29 10 71

18:1 + 18:2 86 27 84 54 59 93 53 86 82 33 69 89 29

aLeaf fed by petiole uptake of 1-14C-acetate; spinach in subdued daylight, sorghum and sunflower in full sun. bLeaf fed by surface application of the precursor in full sunlight. cpc = Phosphatidyl choline; PG = phosphatidyl glycerol; MGD = monogalactosyl diglyceride. TABLE VI Turnover of Phosphatidyi Choline (PC) Bound and Phosphatidyl Glycerol (PG) Bound Fatty Acids after Pulse-feeding 1-14C-Acetate to a Leaf of a Slowly Growing Pumpkin Plant a Hr after labeling

16:0

Total fatty acid 14C (%) 16:1 18:0 18:1 18:2

18:3

PC 0.5

14

0

0

1

19

0

0

29 37 49 52

0 0 0 0

81

6 20 44 92 PG 0.5

4 3 12 12

42 32 14 4 0 0

44 46 39 27 18 15

0 14 21 21 21

4

1

75

0 5

0 0

19 20

0 0

0 0

6 20 44 92

72 65 38 23

10 18 46 54

0 0 0 0

18 17 16 19

0 0 0 0

0 0 0 0

aAt 0.5 hr, PC contributed 52% and PG 34% of total glycerolipid radiocarbon. After 44 hr, these figures were 27 and 33%, respectively. At 44 hr, monogalactosyl diglyceride (MGD) contained 16% of glycerolipid radiocarbon and ct-linoleic acid 65% of MDG fatty acid radioactivity. The 1-14C-acetate was fed by application to the leaf surface in full sunlight. o r b y p e t i o l e u p t a k e . In c o n t r a s t , t h e p r o p o r tion o f r a d i o a c t i v i t y in o t h e r glycerolipids increased w i t h t i m e , t h o u g h t o t a l lipid radioc a r b o n r e m a i n e d fairly c o n s t a n t . The relative i n c o r p o r a t i o n o f 1-14C-acetate LIPIDS, VOL. 10, NO. 10

i n t o PC c o m p a r e d w i t h PG of p u m p k i n leaf was n o t c o n s t a n t , but a p p e a r e d t o vary w i t h growing c o n d i t i o n s o f t h e plants. F o r reasons outl i n e d earlier (1), large leaves h a d b e e n i n d u c e d o n small p o t g r o w n plants b y r e p e a t e d l y rem o v i n g t h e growing p o i n t s . It was f o u n d , however, t h a t t h e s e leaves i n c o r p o r a t e d p r o p o r t i o n a t e l y m o r e 1-14C-acetate i n t o PG c o m p a r e d w i t h PC t h a n did leaves o f field g r o w n plants used in the earlier s t u d y (1). The PC/PG-14C ratio was f o u n d to be 1.5-2.2 for leaves o f p o t g r o w n plants labeled at m i d d a y , and a ratio o f 3.3-3.7 for t h e same material labeled at 8 am. The ratio was c o n s i s t e n t l y 5-6 in leaves o n t h e large field g r o w n plants labeled b e t w e e n 9-11 am. Because t h e bulk o f t h e r a d i o c a r b o n in PG was in p a l m i t i c acid, and b e c a u s e leaves o f t h e slowly g r o w i n g plants w o u l d be e x p e c t e d to c o n t a i n high e n d o g e n o u s levels o f p h o t o s y n t h a t e , t h e s e results suggest t h a t 1-14C-acetate m a y be biased m o r e or less t o w a r d t h e s y n t h e s i s o f p a l m i t i c acid t h a n oleic acid, d e p e n d i n g o n t h e n u t r i t i o n a l status o f t h e leaf. It was also a p p a r e n t t h a t w h e n the PC]PG-14C ratio was l o w , the 16-carbon f a t t y acids c o n t a i n e d prop o r t i o n a l l y m o r e o f t h e t o t a l f a t t y acid radioc a r b o n w i t h i n PG t h a n w h e n this r a t i o was high (Tables V and VI). In c o n t r a s t , h o w e v e r , t h e r e were n o instances w h e n t h e 16-carbon f a t t y acids o f PC c o n t a i n e d > 16% o f t h e t o t a l PC f a t t y acid r a d i o c a r b o n at t h e earlier sampling times. A loss o f 18-carbon f a t t y acids f r o m PC was

PC AND FATTY ACID METABOLISM reflected in the steady increase in the proportional labeling of palmitic acid in PC in a pulse chase type of experiment (Table VI). During the chase, the specific radioactivities of oleic and linoleic fell quite rapidly, while that of palmitic acid remained constant. On the other hand, the specific radioactivity of palmitic acid of PG declined steadily, while that of trans-A 3hexadecenoic acid within PG steadily increased (Table VI). There was no further metabolism of PG bound oleic acid. This experiment also serves to illustrate the relatively specific insertion of palmitic acid into PG and oleic acid into PC.

DISCUSSION The kinetics of 1-14C-acetate incorporation into oleic acid of PC, and the subsequent movement of the label from esterified oleic to linoleic acids strongly suggest that PC b o u n d oleate was the direct precursor of PC bound linoleate, i.e., oleic acid was desaturated while esterified to PC. The alternatives are either that oleate was split off the phosphatide, desaturated while attached to the desaturase enzyme, and reattached to the phosphatide (5), or that the oleate was split off the phosphatide and desaturated as the Coenzyme A derivative ( 1 3 ) b e fore reattachment to the phosphatide. In either case, after reattachment, the linoleate again must have been detached either for further desaturation to a-linolenate or for transfer of the acyl group to other lipids. If, as seems likely, the desaturation of oleyl-PC proceeds as far as a-linolenyl-PC, then the latter mechanisms would require 3 separate attachings and detachings of PC-acyl groups to complete the process of desaturation. It probably would be more favorable energetically to perform the desaturation on the PC bound fatty acids. Good evidence for such a direct desaturation of oleyl-PC has been obtained with yeast microsomes (7). Recently, it has been proposed that linolenate formation in leaves proceeds by elongation of a hexadecatrienoic acid (14,15), but direct desaturation of linoleate to linolenate has been demonstrated in plants (16-18). In Penicillium chrysogenum, although both pathways may operate concurrently, the route of linolenate synthesis under aerobic conditions is predominantly via linoleate (19). In young maize leaves, linolenate appears to be synthesized by direct desaturation of linoleate, rather than by chain elongation of a hexadecatrienoic acid (20). a-Linolenic acid is the predominant fatty acid of green leaves, and is the major end

613

l=•--]l=,-Oley•-]l•

Linoiiyt-PC ~

~

PalmitoyI-PG ~

Linolenyl-PC

t-~3-16:1-PG

FIG. 1. Scheme to account for proposed interrelationships between long chain fatty acids and glycerolipids after feeding 1-I4C-acetate to intact leaves. Heavy arrows indicate major and light arrows minor flow paths of long chain fatty acids. product of 1A4C-acetate incorporation into leaves (1). Most of this a-linolenate is esterified to the chloroplast glycerolipids MGD and DGD, but in every pumpkin glycerolipid, with the exception of PG, it is the major component (1). Rates of a-linolenate biosynthesis, however, are quite slow, relative to those of oleate and linoleate, as judged by incorporation of 1-14Cacetate (2). While the evidence for further metabolism of linoleate to linolenate while esterified to PC is not nearly as convincing as that for oleate to linoleate, it is a logical extension of the hypothesis that this should occur. Tile failure of a-linolenate to accumulate in PC could be explained by a transfer, almost immediately u p o n formation, of the labeled fatty acid to other lipids. It was clearly shown by tong term studies of glycerolipid interrelationships in attached pumpkin leaves that the 18-carbon fatty acids were lost from PC (1), and has been amply confirmed in the present study. In every instance, the counts in PC dedined with time after pulse labeling with 1-14C-acetate, and the specific radioactivity of esterified palmitate remained unchanged, while that of oleate plus linoleate plus linolenate steadily declined. At the same time, the specific radioactivities of these fatty acids in other glycerolipids were increasing with time, even though the counts in total lipids did not change and all available 1-14C-acetate had long since been fixed. Thus, a transfer of 18-carbon fatty acids from PC to other glycerolipids appears certain. The behavior of the fatty acid moieties of PG was in sharp contrast to that of the acyl groups of PC. At the earliest times after pulse labeling, only palmitate and oleate of PG were significantly radioactive. In some cases there was no further movement of label from oleate even after 92 hr, and the only change that took place was a decrease in labeling of palmitate accompanying an almost equivalent increase in labeling of trans-Zk3-hexadecenoic acid. This is quite consistent with the suggestion, based on different data (21), that this unique fatty acid L1PIDS, VOL. 10, NO. 10

P.G. ROUGHAN

614

was s y n t h e s i z e d f r o m p r e c u r s o r w h i l e esterified t o PG. This a p p a r e n t specific i n s e r t i o n o f d i f f e r e n t f a t t y acids i n t o t h e d i f f e r e n t p h o s p h a t i d e s , a n d t h e variable r a t i o o f P C / P G r a d i o c a r b o n in leaves growing under different conditions, p r o m p t s t h e suggestion t h a t t h e r e m a y be 2 s e p a r a t e f a t t y acid s y n t h e t a s e s in leaf tissue. O n e o f t h e s e p r o d u c e s p a l m i t i c acid as its e n d p r o d u c t , a n d this is t r a n s f e r r e d a l m o s t exclusively to PG, while t h e o t h e r p r o d u c e s oleate as its e n d p r o d u c t , a n d this is t r a n s f e r r e d a l m o s t exlusively t o PC (Fig. 1). D e s a t u r a t i o n of t h e b o u n d acyl groups t h e n t a k e s place. F i n a l l y , t r a n s f e r o f t h e acyl g r o u p s f r o m PC t o o t h e r n e w l y s y n t h e s i z e d lipids c o m p l e t e s t h e picture. D i f f e r e n t rates o f s y n t h e s i s f r o m 1-14C-acetate o f palrnitate relative t o oleate ( a f f e c t i n g t h e P C / P G r a d i o c a r b o n r a t i o ) could be a result o f d i f f e r e n t s t i m u l a t i o n s or i n h i b i t i o n s o f e i t h e r s y n t h e t a s e i n leaves f r o m slowly growing c o m p a r e d w i t h rapidly growing plants. T h e o t h e r possibility is t h a t oleate is f o r m e d s i m p l y b y c h a i n e l o n g a t i o n of p a l m i t a t e prod u c e d b y a single s y n t h e t a s e , a n d this is foll o w e d b y d e s a t u r a t i o n of t h e s t e a r a t e (22). H o w e v e r , it is w o r t h c o n s i d e r i n g t h a t in a u t o t r o p h i c a l l y g r o w n Euglena gracilis, t h e r e are at least 2 s y n t h e t a s e s capable o f de n o v o s y n t h e s i s of l o n g c h a i n f a t t y a d d s , a n d t h a t t h e p r e d o m i n a n t p r o d u c t s o f t h e s e s y n t h e t a s e s are palmirate a n d stearate, r e s p e c t i v e l y (23). Also, in d e v e l o p i n g s o y b e a n c o t y l e d o n s , it was concluded t h a t oleate was n o t f o r m e d f r o m palmit a t e or s t e a r a t e , b u t was s y n t h e s i z e d in parallel w i t h these s a t u r a t e d f a t t y acids (24). ACKNOWLEDGMENT Part of this work was carried out while the author was on study leave at the Division of Plant Industry, The Commonwealth Scientific and Industrial Research Organization, Canberra. The Commonwealth Scientific and Industrial Research Organization provided finan-

LIPIDS, VOL. 10, NO. 10

cial assistance. N.K. Boardman made arrangements for the study leave and provided working facilities. REFERENCES I. Roughan, P.G., Biochem. J. I17:I (1970). 2. James, A.T., Biochirm Biophys. Acta 70:9 (1963). 3. Akamatsu, Y., and J.H. Law, J. Biol. Chem.

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