Incorporation of [1-1 4C] Acetate into Lipids of Soybean Cell Suspensions 1 A.C. WILSON 2 and M. KATES, Department of Biochemistry, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5, and A.l. de la ROCHE, Research Branch, Agriculture Canada, Ottawa, Ontario, Canada K1A OC6 ABSTRACT

Suspension cultures of soybean cells incorporated [1-14C]acetate very rapidly into the fatty acid moieties of phospholipids and glycolipids when incubated at 26 C for up to 22 hr. The most rapidly labeled lipid was 3-sn-phosphatidylcholine, which contained 58% of the total fatty acid radioactivity after 16 rain; more than 75% of this label was found to be in the oleic acid of the phosphatidylch01ine. After longer periods of incubation, the proportion of 14C label decreased exponentially in phosphatidylcholine and increased markedly in an unidentified phospholipid (tentatively, bis-phosphatidic acid), di- and triacylglycerols, and glycolipids. The proportion of radioactivity in oleic acid also decreased exponentially, accompanied by increases in linoleic acid first and then in linolenic acid. Most of the labeled linolenic acid at 22 hr was found in the unidentified phospholipid, di- and triacylglyderols, and the glycolipid fraction. INTRODUCTION

Increased chilling resistance in seedlings has been associated with increased contents of unsaturated fatty acids, e.g., linoleic and linolenic acids, in membrane lipids (1,2). Studies in this laboratory on biochemical parameters selecting for increased cold tolerance in soybeans require a knowledge of the biosynthetic pathways for these polyunsaturated fatty acids. Use of plant cell culture techniques is of great potential value in such studies (3-5). Previous investigations of lipid metabolism in soybean suspension cultures have been mainly concerned with incorporation of [ 1 4 C ] acetate into fatty acids of total lipids (6,7) or incorporation of 14C-labeied fatty acids into lipid classes (7). The present communication reports on the kinetics of incorporation of [1-14C]acetate into individual lipids of soybean cell suspension cultures and the distribution of 14C among the fatty acids in the individual lipids. EXPERIMENTAL PROCEDURES Materials

[ 1-14C] Acetate (49 mCi/mmole), [ 1-I4C] linoleic acid, and [1-14C]linolenic acid were purchased from New England Nuclear, Lachine, Que. Radioactivity was determined in Aquasol liquid scintillation cocktail obtained from the same source. Gas chromatographic column 1Contributionno. 537, Ottawa Research Station, Agriculture Canada. A preliminary report was presented at the 20th International Conference on the Biochemistry of Lipids at Aberdeen, Scotland, September 1977. 2present address: Research Branch, Agriculture Canada, Ottawa, Ontario, Canada.

packings were obtained from Supelco, Bellefonte, PA. All solvents were of reagent grade and were distilled before use. Cell Culture

Suspension cultures of soybean (Glycine max (L) Merr. strain PI 189-950) were grown in the mineral salt medium described by Murashige and Skoog (8) using the vitamin and hormone supplements described for the B5 medium by Gamborg (9). The cultures (40 ml) were maintained at 26 C with shaking at 170 rpm in 125 ml Erlenmeyer flasks under fluorescent light (3000 lux) and were subcultured every 5-6 days. [1-14C] Acetate Incorporation

For long term labeling of cells, a 40-ml culture of 4-day-old cells was added to 500 ml of fresh medium and incubated for 4 days in a 2.8 liter Fernbach flask. One mCi of [1-14C] acetate was added and incubation continued for 66 hr. Cells were harvested by vacuum filtration, and lipids were extracted as described below. For kinetic studies, 50 /aCi of [1-14C] acetate was added to a 40oml culture of 4-dayold cells; 5-ml aliquots were removed after 10, 16, 45, 85 and 155 rain, and after 22 hr incubation at 26 C, and were extracted with 5 ml of boiling isopropanol (10,11); the mixture was centrifuged and the residue extracted twice with 5 ml of methanol-chloroform (2:1). The combined extracts were brought to dryness under reduced pressure and total lipids were extracted from the residue by the method of Bligh and Dyer as described elsewhere (10,11). The total lipids were dissolved in chloroform

504

LIPID SYNTHESIS IN CELL C U L T U R E 300

and aliquots were taken for thin layer chromatography (TLC) and determination of radioactivity.

200 E

Chromatographic Separations S e p a r a t i o n o f t h e lipid c l a s s e s w a s a c h i e v e d b y c h r o m a t o g r a p h y o n a c o l u m n o f silicic a c i d which was eluted with chloroform (neutral lipids), acetone (glycolipids), and methanol ( p h o s p h o l i p i d s ) as d e s c r i b e d e l s e w h e r e ( 1 0 ) . Each fraction was further separated by TLC on Silica G e l H w i t h c h l o r o f o r m - m e t h a n o l - 2 8 % ammonia (65:35:5, v/v) for phospholipids, chloroform-methanol (90:10, v/v) for gtycolipids, and petroleum ether (bp 60-70 C)-ethyl ether-acetic acid (80:20:1, v/v) for neutral lipidso T h e c o m p o n e n t s w e r e i d e n t i f i e d b y c o m parisOn of their mobilities with those of authentic lipid s t a n d a r d s a n d b y t h e u s e o f s p e c i f i c spray reagents (10). Two-dimensional TLC was performed with chloroform-methanol-28% ammonia (65:35:5, v/v) in the first dimension and chloroform-acetone-methanol-acetic acidw a t e r ( 1 0 : 4: 2: 2:1, v/v) i n t h e s e c o n d d i m e n s i o n . Spots were located by spraying the plates with a 0~ solution of 2',7'-dichlorofluorescein and viewing under ultraviolet light. Lipids were e l u t e d f r o m t h e silica gel w i t h c h l o r o f o r m methanol (1:1), the eluates were taken to

505

r~

l O0

u,! {r

50

a

2

4

6

8

10

DAYS A F T E R T R A N S F E R

FIG. 1. Growth curve o f soybean cells in suspension culture after transfer to fresh m e d i u m . Cells from each culture flask were harvested by filtration, washed, and dry weight was determined after 2 days freeze-drying foUowed b y 1 hr at 110 C. d r y n e s s u n d e r N 2 , a n d t h e r e s i d u e s w e r e diss o l v e d in c h l o r o f o r m . A l i q u o t s w e r e t a k e n f o r deacylation and 14C_counting"

Analysis of 14C-Labeled Fatty Acids T o t a l l a b e l e d l i p i d s w e r e s a p o n i f i e d b y refluxing with 90%-methanolic NaOH (0.3N) for 1 hr; sterols and other nonsaponifiables were e x t r a c t e d w i t h p e t r o l e u m e t h e r ( b p 4 0 - 6 0 C); t h e a l c o h o l i c p h a s e w a s a c i d i f i e d w i t h 6 N HC1,

TABLEI Lipid Analysis and Fatty Acid Composition of Individual Lipids (5-Day-Old Soybean Suspension) Lipid

Fatty acids (% by weight) a 18:0 18:1 18:2

% by weight

14:0"

16:0

Total lipid

100

tr

22.0

3.1

Phospholipids Unidentified ph ospholipid b Phosphatidylglycerol Ph osphatidylserine Ph osphatidylethanolamine Phosphatidylcholine Phosphatidylinositol Phosphatidic acid

0.2 6.8 trace 24.1 23.3 4.3 0.2

26.9 35.0 . . . . 24.1 18.4 31.4 26.4

6.5 4.0 . . 2.9 4.1 5.5 7.6

Glycolipids Monogalactosyl diacylglycerol Digalactosyl diacylglycerol Esterified steryl glycoside Steryl glycoside Cerebroside Neutral Lipids Triacylglycerol Free fatty acid Diacylglycerol Sterols Hydrocarbons Unidentified alcohols

3.8 7.6 3.6 2.8 1.0 13.1 0.1 0.4 7.1 0.6 1.0

0.9 --. . . ------- --

.

--3.6 --12.3 --37.3 . . . . . . . . . . . . . . . .

1.5 3.2 8.3 . . . .

5.5

.

18.2 10.0 . . . 14.4 16.0 10.3 13.6

.

18:3

18.9

50.9

23.8 22.5 . . . 30.7 26.5 22.9 23.9

22.5 28.2 28.0 35.1 30.0 28.5

5.0 7.0 6.0 8.5 16.1 11.7 . . . . . . . . . . . . . . . .

82.2 70.0 26.6

--2.9 1.0 11.3 24.6 3.6 21.6 5.7 14.8 17.5 2.4 25.1 6.8 23.8 19.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

60.2 36.8 22.8

aTrace a m o u n t s o f 15:0, 16:1, and a long chain fatty acid were also detected, but not 16:l-A3-trans or 16:3. hTentatively, bis-phosphatidic acid (see ref. 17). LIPIDS, VOL. 13, NO. 7

A.C. WILSON, M. KATES, AND A.I. de la ROCHE

506

::iiiiiii::i

;i:?

~

~i~i!~ !i!i!~iiiiiiiiiiiiil

methyl esters were identified by comparison of retention times and mobilities to those of standards (e.g., [I4C]linoleate, [14C] linoleate, etc.).

:i~i

9

!

......ii~ii~

~

Deacylation of Lipids

Individual lipids separated by TLC were deacylated as described elsewhere (10), and watersoluble phosphate esters and other products were identified by paper chromatography in phenol-water (100:38, w/v). .....

:

z ,., RESULTS AND DISCUSSION

. . . . . . . .

2

FIG. 2. Autoradiogram of a two-dimensional thin layer chromatogram of 14C-labeledtotal lipids of soybean suspension ceils (4-day-old cells labeled with [1-14C]acetate for 66 hr at 26 C). First dimension: chloroform-methanol-18% ammonia (65:35:5, v/v); second dimension: chloroform-acetone-methanolacetic acid-water (10:4:2:2:1, v/v). Abbreviations: NL: neutral lipid; SG, sterylglycoside; ESG, esterified sterylglycoside; CER, cerebroside; DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PA, phosphatidic acid; MGD, monogalactosyldiacylglycerol; DGD, digalactosyldiacylglycerol; PC, phosphatidylcholine;PS, phosphatidylserine; PI phosphatidylinositol; OR origin and the fatty acids were extracted with petroleum ether (10). Fatty acid methyl esters were prepared by treatment of the free acids with a freshly prepared solution of diazomethane (10). Fatty acid methyl esters from individual lipids separated by TLC were prepared directly by adding 2 ml of 2.5% methanolic-HC1 to the silica gel scraped from the plate and heating under reflux for 1 hr (10). Water (0.2 ml) was added, and the methyl esters were extracted with petroleum ether. Fatty acid methyl esters were analyzed by gas chromatography on a column of 5 % SP-2310 on 100/120 Chromosorb W (AW) (6 ft x 4 mm) at 190 C, using a flame ionization detector. The column was fitted with an effluent stream splitter, and radioactive methyl esters were either collected on cellulose filters (12) and counted in the scintill a t i o n cocktail described above, or were monitored continuously by a gas radioactivity counting system (Perkin-Elmer). Radioactive fatty acid methyl esters were also separated according to their degree of unsaturation by TLC on 10% AgNO3-Silica Gel H as described elsewhere (13). Radioactive areas were detected by a plate scanner (Varian) and scraped into scintillation vials for counting 14C. Fatty acid LIPIDS, VOL. 13, NO. 7

Soybean Cell Cultures

The growth rate of the soybean cells grown in suspension culture, as assessed by increase in dry weight (Fig. 1), was much greater (doubling time 48 hr) in the revised Murashige-Skoog medium (8) than that previously reported for cells grown in the B5 medium (6). The total lipid content of the cells averaged about 6% on a dry weight basis in agreement with previously reported values (3). The fatty acid composition (Table I) of the total lipids was characterized by a high concentration of linolenic acid and resembled most closely that of soybean leaves, as noted before by Tattrie and Veliky (14). The total lipids of 4-day-old soybean cell cultures contained about 59% phospholipids, 19% glycolipids, and 22% neutral lipids (Table I) in general agreement with the values reported by Stearns and Morton (6). A typical separation of these components is shown in Figure 2, and their quantitative analysis and fatty acid composition are given in Table I. The present results are in marked contrast to the low triacylglycerol, phospholipid, galactolipid, and sulfolipid contents and elevated sterol, sterol ester, sterol glycoside, and esterified sterol glycoside contents reported earlier for soybean suspension cultures (3). The differences may well be due to lipolysis occurring during extraction at room temperature with solvents such as chloroformmethanol or ether (3), but not with hot isopropanol as in the procedure used here (10,11), nor with boiling chloroform-methanol (6). Such lipolytic activity would result in preferential hydrolysis of the glycerolipids producing high contents of free fatty acids and resulting in artificially elevated contents of sterols and sterol glycosides. The fact that relatively low contents of free fatty acids, phosphatidic acid, and lyso-compounds were found in our culture extracts (Fig. 2, Table I) confirms the absence of significant phospholipase activity during the extraction procedure.

LIPID SYNTHESIS IN CELL CULTURE

507

TABLE II I n c o r p o r a t i o n of [ 14C] A c e t a t e into the Lipids of Soybean Cell

Suspension Cultures a Incubation Total radioactivity time in lipids 10 -3 x dpm (min) per ml culture 10 16 45 85 155 22.0 hr

167(6.0) b 649 (23.4) 698 (25.2) 716 (25.8) 709 (25.5) 611 (22.0)

Total PL 60 73 63 66 67 56

% Distribution of radioactivity in each classc Total NL Total GL TG+DG ST FA 6 4 4 4 s 21

5 4 5 6 10 8

1 1 3 8 9 13

H+A

2 3 12 5 3 1

26 15 13 11 6 1

aSuspensions (40 ml) of 4-day-old cells were incubated with 50 #Ci of [ 1-t4c] acetate; lipids were extracted and counted as.described in the text. bValues in parentheses are lipid 14C-activities expressed as percentages of added [ 14C ] acetate. CNeutral lipids (NL), glycolipids (GL), and phospholipids (PL) were separated by chromatography on a column of silicic acid eluted with chloroform, acetone, and methanol, respectively (10). Neutral lipids were further separated by preparative TLC into tri- and diacylglycerols (TG + DG), sterols (ST), free fatty acids (FA), hydrocarbons (H), and long chain alcohols (A). Values are expressed as % of radioactivity in t o t a l lipids. T h e lipid c o m p o s i t i o n o b s e r v e d h e r e ( T a b l e I) is similar t o t h a t r e p o r t e d for i m m a t u r e soyb e a n seeds (15) or for n o n p h o t o s y n t h e t i c p l a n t tissues generally (16). T h e m a j o r lipids were t h e phospholipids phosphatidylcholine (PC)and p h o s p h a t i d y l e t h a n o l a m i n e (PE) f o l l o w e d b y tria c y l g l y c e r o l , sterols, a n d digalactosyldiacylglycerol (DGD). Smaller amounts of m o n o g a l a c t o s y l d i a c y l g l y c e r o l (MGD), p h o s p h a tidylglycerol (PG), diphosphatidylglycerol (DPG), p h o s p h a t i d y l i n o s i t o l (PI), a n d esterified a n d u n e s t e r i f i e d steryl glycosides were also present. O u r c u l t u r e s also c o n t a i n e d a small a m o u n t of a n u n i d e n t i f i e d p h o s p h o l i p i d w i t h TLC mobilities similar t o t h o s e o f bisp h o s p h a t i d i c acid (bis-PA) previously i d e n t i f i e d in developing soybeans and suspension cultures of s o y b e a n cells (17). In c o n t r a s t t o earlier rep o r t s (3), s t e r o l esters were n o t d e t e c t e d in t h e c u l t u r e s e x a m i n e d here. [ 1-14C ] Acetate I ncorporation

I n c o r p o r a t i o n of [ 1 - 1 4 C ] a c e t a t e i n t o t o t a l lipids of s o y b e a n cell s u s p e n s i o n s was rapid u p t o 16 m i n a n d r e m a i n e d essentially c o n s t a n t at a level of a b o u t 25% of t h e a d d e d 14C up t o 22 h r (Table II). P h o s p h o l i p i d s a c c o u n t e d for m o s t o f t h e r a d i o a c t i v i t y ( 5 6 - 7 3 % ) in the lipids at all times. O f these, PC s h o w e d t h e m o s t rapid labeling, a m a x i m u m value b e i n g r e a c h e d at 16 m i n f o l l o w e d b y a r a p i d decrease in 14C cont e n t r e a c h i n g very low values at 22 h r (Fig~ 3). PE, PI, a n d PG were labeled slowly t o m a x i m a at 1.5 t o 2.6 hr, t h e r e a f t e r s h o w i n g decreases in radioactivity. In c o n t r a s t t h e u n i d e n t i f i e d p h o s p h o l i p i d (bis-PA) s h o w e d little labeling up t o 2.6 h r b u t at 22 h r was f o u n d t o c o n t a i n m o s t of t h e r a d i o a c t i v i t y p r e s e n t in t h e t o t a l lipids (Fig. 3).

/

PC PE PI+PG PX

\

~

4o

20

.o.

c/

\

~

~ A o D ..... 9

Zx

/

/

,o

/

-

"6

9

~o , ~ . ~ , " o.~5 0.25

0.5

I_7____1/ 1 2 T I M E , hours

~d 5

~o

zo

FIG. 3. Time course of incorporation of [1-14] acetate into major lipids of 4-day-old soybean suspension culture cells~ Soybean cells were incubated with [14C] acetate and sampled as described in the Experimental section of each of the times shown. Abbreviations: see legend to Figure 2. o PC; A, PE; o, PX; o, PI + PG; A, MGD + DGD. For convenience, time is represented on a logarithmic scale. Glycolipids c o n t a i n e d relatively low p r o p o r t i o n s ( < 5 % ) of 14C up t o 2.6 h r b u t t h e n showed a c o n s i d e r a b l e increase t o 21% a t 22 h r (Table II). Galactolipids (MGD + DGD) a c c o u n t e d for m o s t of t h e activity in t h e glycolipid f r a c t i o n (Fig. 3, T a b l e II), a n d n e u t r a l lipids a c c o u n t e d for 23-33% of t h e t o t a l lipid r a d i o a c t i v i t y at all t i m e s ( T a b l e II). Labeling of di- a n d t r i a c y l g l y c e r o l was rapid a n d r e m a i n e d relatively c o n s t a n t at a b o u t 10% u p t o 22 h r ( T a b l e II). Sterols, h o w e v e r , were labeled slowly at first a n d r e a c h e d a m a x i m u m o f 13% LIPIDS, VOL. 13, NO. 7

A.C. WILSON, M. KATES, A N D A.I. de la ROCHE

508

TABLE III Incorporation of [ 14C ] Acetate into Fatty Acids of Major Lipids of Soybean Suspension Cells a Time after addition o f [ 14C]acetate (min)

Total 14C in fatty acids 10 -3 x d p m per ml culture

16:0

Total

16 45 85 155 22.0 hr

490 535 546 548 522

29.1 28.5 25.9 26.3 14.4

60.4 52.6 45.8 34.1 8.2

8.9 16.7 22.8 28.2 24.6

1.6 2.2 5.5 11.4 52.8

PC

16 45 85 155 22.0 hr

289 227 215 182 13

19.8 21.5 21.9 23.1 19.6

76.2 60.7 51.4 42.9 11.9

3.0 16.0 23.4 28.2 18.0

1.0 1.8 3.2 5.8 50.5

PE

16 45 85 155 22.0 hr 16 45 85 155 22.0 br

89 97 121 169 16 5 16 18 17 277

36.8 37.1 38.4 36.5 30.6 53.8 42.4 40.1 35.7 27.6

57.1 52.2 44.9 38.5 15.4 40.9 48.3 43.7 45.1 8.0

5.2 8~ 13.7 19.4 24.0 3.9 8.0 12.9 15.0 24.5

1.0 1.8 3.0 5.6 29.9 1.3 1.2 3.3 4.2 40.0

TG+DG

16 45 85 155 22.0 hr

59 105 92 95 76

26.4 23.1 18.8 15.5 15.1

60.5 55.7 48.2 36.5 6.4

10.0 17,6 26.1 33.9 18.8

3.1 3.6 6.9 14.1 59.7

MDG+ DGD+ ESG

16 45 85 155 22.0 hr

18 21 25 31 109

37.7 36.6 27.4 21.9 26.7

38.2 39.0 34.2 26.8 11).6

11.3 16.7 23.9 26.9 15.3

12.9 8.7 14.4 24.4 47.4

Lipid b

PX

Distribution of [ 14C ] fatty acids in individual lipid c o m p o n e n t (%) 18:1 18:2

18:3

aThe percentage distribution of fatty acid radioactivity was obtained by gas liquid chromatography (GLC) and thin layer chromatography (TLC) analysis o f methyl esters prepared from the isolated lipids. Total fatty acid radioactivity in each lipid c o m p o n e n t was determined after saponification as described in the text. bAbbreviations: PC = phosphatidylcholine, PE = phosphatidylethanolamine, PX = unidentified phospholipid, TG = triacylglycerol, DG = diacylglycerol, MGD = monogalactosyldiacylglycerol, DGD = digalactosyldiacylglycerol, ESG = esterified sterolglycoside. o f t h e t o t a l lipid r a d i o a c t i v i t y a t 2 2 h r , w h i l e hydrocarbons and long chain alcohols containe d 1 5 - 2 0 % o f t h e t o t a l lipid 1 4 C a t e a r l y t i m e s , t h i s p r o p o r t i o n t h e n d e c r e a s i n g t o 1% a t 2 2 h r ( T a b l e II). The pattern of [14C]acetate labeling of g l y c e r o l i p i d s o b s e r v e d in t h e p r e s e n t s t u d i e s is similar to that reported previously for leaves (18-20). The effect of darkness and low tempera t u r e ( 1 6 C) o n t h e p a t t e r n o f l a b e l i n g o f t h e lipid classes was examined, but no significant differences were noticed, apart from the expected depression in the rate of labeling at the lower temperature.

Labeling of Fatty Acids

At all incubation times radioactivity was present m o s t l y i n p a l m i t i c ( 1 6 : 0), oleic ( 1 8 : 1 ) , linLIPIDS, VOL. 13, NO. 7

oleic (18:2), and linolenic (18:3) acids, no significant 14C being detected in myristic (14:0, palmitoleic (1 6 : 1 ) , trans-3-hexadecenoic ( 1 6 : 1), h e x a d e c a t r i e n o i c (16:3), or stearic ( 1 8 : 0 ) a c i d s . I n t h e t o t a l l i p i d s , 18:1 w a s labele d p r e f e r e n t i a l l y ( > 5 0 % ) at e a r l y i n c u b a t i o n times while relatively low percentages of 14C w e r e f o u n d in 1 8 : 2 a n d 1 8 : 3 ; 1 6 : 0 c o n t a i n e d about 30% of the 14C and this proportion decreased only slightly after longer incubation t i m e s ( T a b l e III)o 1 4 C - L a b e l i n g o f 1 8 : 1 d e c r e a s ed during the remaining incubation period while that of 18:2 increased reciprocally up to 2.6 hr and thereafter began to decrease. 1 4 C _ L a b e l i n g o f 1 8 : 3 i n c r e a s e d a f t e r 2 . 6 h r at a rate which appeared comparable to the comb i n e d r a t e s o f d e c r e a s e o f l a b e l i n g i n 18:1 a n d 18:2.

LIPID SYNTHESIS IN CELL CULTURE

509

Acetate 16:0~-ACP-~I 6:0-CoA 18:089 8:0-CoA""""'~ sn-3PA ) DG ) TG 18: I~-ACP---~I8:1-CoA"~acyl exchange 18: I-PC.-----~ 18:1 18:2PX

~ 18:3-PX

18:2!CoA-

acyl exchange) 18:!-PC/~ 118:1MGD-DGD

18:3!COA

acyl exchange)

18:3-PC

18:I-MGD-DGD

FIG. 4. Scheme for proposed mechanism of desaturation sequence of fatty acids in soybean cell cultures [adapted in part from Stumpf and Weber (5)]. Oleic acid of PC accounted for about 75% of the radioactivity in PC (Table III) corresponding to 44% of the total lipid fatty acid radioactivity at 16 min, thereafter decreasing to values of 12% and 0.3%, respectively, at 22 hr. The proportion of 14C in the 18:2 component of PC was very low at 16 rain but increased tenfold to a maximum at 2.6 hr, thereafter decreasing up to 22 hr (Table III). Only 1% of the 14C in PC was present in the 18:3 component at 16 rain, this value increasing slowly to 6.0% at 2.6 hr, and then to 50% at 22 hr (Table III); as a percentage of the total fatty acid 14C, the proportion at 22 hr was, however, only about 1%. The decrease in proportions of 14C in 18:1 and 18:2 of PC during the period from 2.6 hr to 22 hr was compensated for by increases in these acids in the unidentified phospholipid (bis-PA) and in the glycolipid fraction; the di- and triacylglycerol fraction, however, showed an increase only in the 18: 3 component (Table III). Steams and Morton (6) reported a low labeling rate of fatty acids from [14C]acetate in 21-day-old soybean cell cultures, but after a lag period of 30 rain to 1 hr, the percentage distribution of 14C among the fatty acids, with the exception of saturated acids, was very similar to that found here. Using 5-day-old cells, Stumpf and Weber (7) reported rapid incorporation of [14C]acetate in 18:1 and 18:2 acids but not into the 18:3 acid. However, conversion of [14C] oleate into 18:2 acid and [14C] linoleate into 18:3 was also observed. These results (7) as well as those of Morton and Steams (6) were suggestive of sequential desaturation of 18:1 -+ 18:2 ~ 18:3 acid. While the present results on labeling of the total fatty acids (Table III) are also consistent with such a sequential mechanism, the pattern of labeling of the fatty acids in the individual

lipids we observed suggests that a more complex desaturation sequence may be proposed (Fig. 4). In this sequence, labeled 18:l-PC rapidly accumulates by synthesis de novo from DG or by "acyl exchange" of unlabeled PC with labeled 18:I-CoA (21). The 18:1 of PC is then rapidly converted to 18:2 of PC during the first 2 hr of incubation, either by direct desaturation of 18:l-PC, as demonstrated previously with Chlorella chloroplasts (22) and pea-leaf micros o m e s (23) or by "acyl exchange" with 18:2-COA formed by desaturation of, 18: 1-CoA (7,24,25). Subsequently, both 18:1 and 18:2 of PC are then transferred to other glycerolipids, in particular the phospholipid PX (bis-PA) and the glycolipids (MGD + DGD). Linolenic acid accumulates in these lipids during the same period, and also to a limited extent in PC and PE (Table III). The 18:3-PC could be formed by desaturation of 18:2-PC or by "acyl exchange" with 18:3-COA, but the low amounts of labeled 18:3 detected in PC would tend to eliminate PC as a significant source of 18:3 for transfer to PX and MGD + DGD~ The accumulation of 18:3 in PC and the galactolipids may perhaps be explained by desaturation of 18:2 ~ 18:3 in situ in these components or by "acyl exchange" with 18:3-COA formed by desaturation of 18:2-COA (7). Pulse-chase experiments using [ 3H] glycerol and [14C]acetate in developing maize leaves (26) have confirmed that the 18:1 of PC is a major precursor of the 18:3 of galactolipids. The conversion of 18:1 -+ 18:2 has been associated with the endoplasmic reticulum of maize and pea-leaves, whereas desaturation of 18:2 ~ 18:3 has been proposed to occur in the chloroplasts (18,27). The mechanism of these desaturations and of the interorganelle lipid transfer still remains to be elucidated. Soybean LIPIDS, VOL. 13, NO. 7

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A.C. WILSON, M. KATES, AND A.I. de la ROCHE

suspension cultures may prove to be very useful systems in which to pursue these problems since advantage can be taken of existing methodology to produce protoplasts, allowing gentle cell breakage without damage to the intracellular organelles and minimizing cross-contamination and release of lytic enzymes. Furthermore, manipulation of the growth medium composition to promote differentiation might allow expression of cultivar differences in response to cold temperature stress. ACKNOWLEDGMENTS This work was supported by Grant A-5324 (MK) from the National Research Council of Canada, and Grant EMR-7603 (MK) from Agriculture Canada. We thank N. Long for growing the cultures. REFERENCES 1. Lyons, J.M., and J,K. Raison, Plant Physiol. 45:386 (1970). 2. Dogras, C.G., D.R. Dilley, and R.C. Herner, Plant Physiol 60:897 (1977). 3. Radwan, S.S., and H.Ko Mangold, in "Advances in Lipid Research," Vol. 14, Academic Press, New York, 1976, pp. 171-211. 4. Dix, P.J., and H.E. Street, Ann. Bot. 40:903 (1976). 5. Breidenbach, R.W, and A.J. Waring, Plant Physiol. 60:190 (1977). 6. Stearns, E.M., Jr., and W. T. Morton, Lipids 10:597 (1975). 7. Stumpf, P.K., and N. Weber, Lipids 12:120 (1977). 8. Murachige, T., and F. Skoog, Physiol. Plant 15:473 (1962).

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9. Gamborg, O.L., R.Ao Miller, and K. Ojima, Exp. Cell Res. 50:151 (1968). 10. Kates, M., in "Laboratory Techniques in Biochemistry and Molecular Biology," Edited by T.So Work and E. Work, North Holland Publishing Co., Amsterdam and London, 1972, pp. 350, 398-401, 436-441, 363-364, 526, 558-564. 11o de la Roche, A.I., C.J. Andrews, and M. Kates, Plant Physiol. 51:468 (1973). 12. Hammarstrand, K., J.M. Juntenen, and A.R. Hennes, Anal. Biochem. 27:172 (1969). 13. Pugh, E.L., and M. Kates, J. Biol. Chem. 252:68 (1977). 14. Tattrie, N~ and I.A. Veliky, Can. Jo Bot. 51:513 (1973). 15. Singh, H., and O.S. Privett, Lipids 5:692 (1970). 16. Kates, M., Adv. Lipid Res. 8:225 (1970). 17o Stearns, E.M., Jr., and W.T. Morton, Lipids 12:451 (1977). 18. Slack, C.R., and PoG. Roughan, Biochem. J. 152:217 (1975). 19o Roughan, P.G., Lipids 10:609 (1975). 20. Heinz, E., and JoL. Harwood, Hoppe-Seyler's, Z. Physiol. Chem. 358:897 (1977). 21o Shine, W.E., M. Mancha, and P.K. Sturnpf, Arch. Biochem. Biophys. 173:472 (1976). 22. Gurr, M.I., M.P. Robinson, and A.T. James, Eur. J. Biochem. 9:70 (1969). 23. Slack, C.R., P.G. Roughan, and J. Terpstra, Biochem. J. 155:71 (1976). 24. Vijay, I.K., and P.K. Stumpf, J. Biol. Chem. 247:360 (1972). 25. Abdelkader, A.B., A. Cherif, C. Demandra, and P Mazliak, Eur. J. Biochem. 32:155 (1973). 26. Slack, C.R~ P.G. Roughan, and N. Balasingham, Biochem. J. 162:289 (1977)o 27. Tremoli~res A., and P. Mazliak, Plant Sci. Lett. 2:193 (1974).

[Received February 2, 1978]