Lipid biosynthesis in developing and germinating soybean cotyledons

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Lipid Biosynthesis The Formation

of Palmitate

176, 53-62 (1976)

in Developing and Germinating Cotyledons and Stearate by Chopped Preparations

R. J. PORRA Division

ofPlantZndustry,

AND

Soybean

Tissue and Supernatant

P. K. STUMPF’

Commonwealth Scientific and Industrial Research Organization, Canberra City, A.C.T. 2601, Australia Received

December

P.O. Box1600,

30, 1975

Chopped tissue from developing soybean cotyledons incorporated [l-‘%]acetate into palmitate, stearate, oleate, and linoleate, but with germinating cotyledons much less [lYJlacetate was incorporated and the principal labeled products were palmitate, stearate, and oleate. When supernatant fractions from developing cotyledons were incubated with [l-‘4C]acetate or [2-%]malonate the principal labeled products were palmitate and stearate. Supernatant fractions from germinating seed incorporated [P-“Clmalonate into palmitate and also into short chain fatty acids including decanoate, laurate, and myristate. Supernatants from developing cotyledons required acyl carrier protein (ACP), ATP, CoA, and reduced pyridine nucleotides for maximal rates of incorporation of either [l-‘4C]acetate or [2-‘%]malonate into palmitate and stearate. The de novo fatty acid synthetase which converts acetyl- and malonyl-ACP’s to palmityl ACP was active in supernatant fractions from both young and old developing cotyledons. The elongation system, converting palmityl ACP to stearyl ACP, was more active in supernatants from younger than from older developing cotyledons. In experiments with chopped tissue the elongation system appeared equally active throughout the development process. These results are consistent with the view that the de novo and elongation systems are separate entities and that the elongation system in older cotyledons is less stable to the methods used to prepare supernatant fractions.

In recent years a number of investigators has studied lipid biosynthesis in developing seeds including safflower (1, 2), castor bean (3, 4), Crumbe (51, rape (61, and both developing and germinating soybean (7-13); Appelqvist (14) has reviewed the considerable literature on this subject. Although much has been done to elucidate the mechanisms involved in fatty acid biosynthesis, many questions remain. These include the role of ACP2 and CoA deriva1 Permanent address: Department of Biochemistry and Biophysics, University of California, Davis, Calif. 95616. 2 Abbreviations used: ACP, acyl carrier protein; DAF, days after flowering; DAG, days after germination; glc, gas-liquid chromatography; tic, thinlayer chromatography. 53 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

tives in the biosynthesis of fatty acids, the mechanism of the desaturation and hydroxylation reactions by which fatty acids can be further modified, the nature of the processes controlling the biosynthesis of C,, and C,, fatty acids, and the capacity of both developing and germinating seeds to synthesize fatty acids. However, the principal reason for the present studies, as decribed more fully in the discussion section, was the work of previous investigators (79) which suggested that the cofactor requirement and hence the mechanism of’ fatty acid biosynthesis in soybean cotyledons were different from that observed in other oil seeds (see Ref. (14)). In this paper we investigated the capacity of the de nova fatty acid synthetase

54

PORRA

AND

system (15) in chopped tissue and supernatant fractions from soybean cotyledons to incorporate [lJ4C]acetate and [2J4Clmalonate into palmitate and its subsequent conversion by the elongation system (15) to stearate. In particular, the effects of both development and germination upon the de nouo and elongation systems were examined. It is clearly demonstrated that ACP, ATP, CoA, and reduced pyridine nucleotides are essential for maximal rates of incorporation of labeled acetate or malonate into palmitate and stearate and that the mechanism of fatty acid biosynthesis in soybean cotyledons is not significantly different from that observed in other oil seeds. EXPERIMENTAL Reagents and chemicals. [l-14Clacetate (58.1 mCi/ mmol) and [2-‘Qmalonate (I7 mCi/mmol) were supplied by The Radiochemical Centre, Amersham. ACP was prepared from Escherichia coli by the method of Majerus et al. (161; the purification was taken to the (NH&SO, fractionation and acid precipitation step and the ACP thus produced is about 10% pure. We thank Professor F. Gibson, Department of Biochemistry, Institute of Advanced Studies, Australian National University, Canberra, for providing E. coli cytosol and are also grateful to Dr. Ward Shine, Department of Biochemistry and Biophysics, University of California, Davis, for gifts of E. coli ACP. Solutions used contained approximately 1 pg of ACP/pl. An approximately 1.5% (w/v) solution of diazomethane in diethyl ether was prepared from Nmethyl-N-nitroso-p-toluenesulfonamide (Diazald) supplied by the Aldrich Chemical Co., Milwaukee, Wis. Growth of soybean plants. Grant-strain soybeans (Glycine max var. Merr.) were grown, one plant per pot, in a mixture of equal parts vermiculite and perlite. The pots were moistened once a day with a nutrient solution containing (in milligrams/liter): Ca(N0,,),.4H,O (9501, (NH,)H,PO, (1201, KNO,, (1601, MgSO,.7H,O (4901, H,BO,, (0.6), MnC1,.4H,O (0.41, ZnSO,.7H,O (0.091, CuS0,.5H,O (0.051, H9Mo0, (0.02), Co(NO,,),.GH~O (0.0251, FeS04.7H,0 chelated with EDTA (24.9), and sufficient NaOH to give pH 5.8. The plants were grown in 8 h of natural light at 25°C (dayY2O”C (night) until the first flower bud appeared (approximately 4 weeks). Then the pots were transferred to artificially lit cabinets maintained at 25°C (day)lBO”C (night) and illuminated for 8 h per day with incandescent and fluorescent lamps (210 fc; 365 PEinsteins rner s-l between

STUMPF 400 and 700 nm). Each flower bud was tagged with date of appearance. Germination conditions. To determine the effect of germination on fatty acid synthesis, seeds were suspended in water and vigorously aerated for 4 h and then grown in the dark in moist vermiculite. At the required time after germination the seeds were harvested and the remnants of testa removed. To diminish bacterial contamination the seeds were soaked for 10 min in a dilute sodium hypochlorite solution and then washed for 15 min with running tap water. Soybean cotyledon preparations. Soybean seeds were picked at various times after flowering or germination, the testa were removed, and the cotyledons (4 g) were chopped finely with a razor blade before homogenizing with 8.0 ml of 0.1 M Tricine buffer (pH 8.0) containing 1.0 mM mercaptoethanol in an Ultra-Turrax homogenizer (Janke & Kunkel KG, IKA-WERK, Staufen i. Breisgau) for 30 s at 75% of line voltage. The homogenate was then filtered through buffer-moistened Miracloth (Calbiochem) and was centrifuged for 30 min at 28,OOOgat 4°C. The precipitate was discarded and the supernatant fraction contained approximately 15 mg/ml of protein as estimated by the method of Lowry et al. (17) using bovine serum albumin as standard. Chopped tissue preparations were made by chopping cotyledons from which the testa had been removed (0.5 g) to approximatley l-mm cubes with a razor blade and suspending them in 0.05 M K-phosphate buffer (1.0 ml; pH 7.5). Assay of incorporation of ll-‘4CIacetate or L2‘4Clmalonate into fatty acids by soluble cotyledon fractions. Supernatant fraction (0.25 ml) was incubated aerobically for 30 and 60 min at 22°C in the presence of (in micromoles): CoA (0.21, ATP (2.51, NADH (0.4), NADPH (0.41, MgCl, (0.51, MnCIZ (0.5), KHCO,, (30), [l-“Clacetate (0.25; 1 &il, bovine serum albumin (0.5 mg), ACP (25 pg), mercaptoethanol (0.51, and Tricine-HCl buffer, pH 7.8 (250); final volume 1 ml. When 12-‘4Clmalonate (0.4; 1 &i) replaced acetate as substrate, KHCO, was omitted and the soybean extract used was increased to 0.5 ml. All reactions were stopped by the addition of 8 N NaOH (0.2 ml). The labeled free fatty acids formed by heating the alkaline mixture at 80°C for 30 min were extracted as follows. The hot solution was chilled, mixed with concentrated HCl (0.2 ml) and shaken with CHCl,CH,OH reagent (2:1, v/v; 2.0 ml). After centrifugation to separate the phases, the bottom CHCl, layer was removed with a Pasteur pipet and shaken vigorously with 1 N CH,COOH (2 ml) when labeled acetate was used as substrate, or with 0.1 N malonic acid (2 ml) when labeled malonate was used as substrate. After centrifugation, the bottom CHCI:, layer was removed and evaporated to dryness at 80°C under a stream of N,. The lipid residue was

FATTY

ACID

BIOSYNTHESIS

dissolved in 1.0 ml of petroleum ether (60-80°C) and an aliquot (0.1 ml) was counted by the usual procedures in 0.5% (w/v) Z,&diphenyloxazole (PPO) in toluene in a Packard Tri-carb liquid scintillation spectrometer with a counting efficiency of 75% for 14C. In assays with cell-free preparations an incorporation of 6680 and 4170 cpm per total assay mixture corresponded to an incorporation of 1 nmol of acetate and malonate, respectively. In assays with chopped tissue, an incoporation of 96,750 cpm corresponded to an incorporation of 1 nmol of acetate. To prepare methyl esters for glc analyses the remainder of the petroleum ether solution (0.9 ml) was evaporated to dryness under N, and treated with diazomethane. Assay of incorporation of [l-‘4Clacetate into fatty acid by chopped cotyledon tissue. Chopped tissue prepared as described above and containing 0.5 g fresh weight of tissue was incubated aerobically at 22°C for 2 and 4 h with (in micromoles): KHCO:, (30) and [l-Wlacetate (0.086; 5 $i); the final volume was 1.2 ml. The reaction was stopped by the addition of 1 N H,SO, (0.1 ml). To extract lipids, CHCl,,-CH,OH reagent (6 ml) was added and, after 12 h at room temperature, the defatted tissue was removed by filtration and the residue washed once with CHCl,-CH,OH reagent (2.0 ml). The pooled CHCl,-CH,OH washes were evaporated to dryness at 80°C under a stream of N,. The lipid residue was redissolved in 1 ml of petroleum ether (SO-80°C) and an aliquot (0.1 ml) was evaporated to dryness before counting in 0.5% (w/v) PPO in toluene as described above. To prepare methyl esters for glc analysis, the remainder of the petroleum ether solution (0.9 ml) was evaporated to dryness under N, and treated with diazomethane. Estimation of total lipid content of soybean cotyledons. Finely chopped cotyledons (1 g) were extracted with CHCl,-CH,OH reagent (20 ml) by homogenizing for 30 s at 75% of line voltage in an Ultra-Turrax homogenizer. After 1 h at room temperature, the suspension was filtered and the filtrate shaken with 1% (w/v) NaCl (5 ml). After centrifugation the bottom CHCl, layer was transferred to a preweighed tube and the CHCl, removed at 80°C under a stream of N, until a constant weight, that of the total lipid, was obtained. To obtain methyl esters of the fatty acids in the total lipid fraction for glc analysis, the residue was dissolved in 8 N KOH (0.2 ml) and 50% aqueous methanol (1 ml) and heated at 80°C for 30 min. After treatment with concentrated HCl (0.2 ml) and extraction with CHCl,-CH,OH reagent (2 ml), the bottom CHCl, layer was evaporated to dryness and the residue esteritied with diazomethane. Preparation of fatty acid methyl esters with diazomethane and glc analysis. The residues of free fatty

55

IN SOYBEANS

acids obtained above were dissolved in CH,OH (two drops) and 1 ml of a solution (approximately 1.5%, w/v) of diazomethane in diethyl ether. After 30 min in an ice bath the excess diazomethane and solvents were removed at room temperature under a stream of N?. The residue was dissolved in petroleum ether (30 ~1) prior to injection into the radio-glc system described by Stumpf and Boardman (18). For identification of W-labeled shorter-chain CC,,,-C,,) fatty acids produced by supernatants of germinating seed, the oven temperature was reduced from 178 to 143°C. Hydrogenation procedure. The chain length of an unusual (16:l) fatty acid, formed from [Wlacetate by a bacterial contaminant of chopped germinating cotyledon tissue was determined by identification of the product of hydrogenation as palmitic acid (16:O). The reduction was carried out by reducing the acid in the presence of H, (40 psi) and 5% palladium oxide on charcoal at room temperature for 1 h in a Parr hydrogenator (Parr Instrument Co. Inc., Moline, Ill.). Ozonolysis procedure. The position of the double bond in the 16:l fatty acid was located by identifying the products of reductive ozonolysis by glc (19). Ozonolysis was carried out with a Micro-ozonizer (Supelco Inc., Bellefonte, Pa.). RESULTS

I. Experiments Cotyledons

AND

DISCUSSION

with Developing

Soybean

The effect of seed development on [l-‘4Clacetate incorporation into fatty acids by chopped cotyledon tissue. The results in

Table I show the increase in cotyledon fresh weight, total endogenous lipid, and changes in the composition of the fatty acid component as the soybean seed matures. The proportions of palmitate remained approximately constant from 15 to 50 days after flowering (DAF) even though the total endogenous lipid increased 6.5 fold on a per-seed basis or 2.3-fold on a fresh weight basis. Thus, to maintain this constant ratio between palmitate and Cl8 fatty acids considerable synthesis of palmitate (16:O) by the de novo system must have occurred, matched by a corresponding increase in activity by the elongation system. Of the C,, acids, the proportion of stearate (l&O) and oleate (l&l) remained approximately constant but the proportion of linoleate (X3:2) increased while that of linolenate (183) decreased. As expected, the incorporation of [l-

56

PORRA

AND TABLE

THE

DAF

15 20 25 30 40 50

Dried seed

EFFECT

OF COTYLEDON

Fresh weight w seed (g)” 0.09 0.14 0.12 0.23 0.23 0.28 0.25

DEVELOPMENT

STUMPF I

ON FRESH WEIGHT SOYBEAN SEE@

Total endogenous lipid

AND ENDOGENOUS

Proportion

(mgi seed)

(w/g fresh wtl

16:0

2.9 6.5 6.5 14.6 16.1 19.6 28.5

31 46.5 54.5 63.5 70 70 114.0

20 25 14 20 14 24 16

LIPID

CONTENT

OF

of fatty

acid in endogenous (70)

lipid

18:O

18:l

189

l&3:3

3

22 17 18 14 20 15 16

40 44 53 55 53 55 62

r 5 1 3 1 3

” Seeds were harvested on the cited day after flowering (DAF). The procedure used to determine endogenous lipid and individual fatty acids are described in the experimental section, b Only the cotyledons were weighed. ( t < 1%.

15 14 10 10

10 5 3

the total

14Clacetate into fatty acids by chopped cot- supernatant fractions. The effect of subyledon tissue increased as seed matura- strate concentration on the incorporation tion proceeded (Table II). Consistent with of [lJ4C]acetate and [2-14C]malonate into the changes in endogenous lipid (Table I), fatty acids by a cotyledon supernatant the extent of 14Cincorporation into palmi- fraction was studied: Maximal incorporation of label was obtained with substrate tate was relatively constant throughout the maturation period and little if any concentrations of 250 nmol/ml (acetate) stearate accumulated at any stage (Table and 400 nmol/ml (malonate). Preliminary II). Synthesis of [14Cllinoleate from ll- studies showed that the incorporation of 14C]acetate appears to be slower than the both labeled substrates was linear with synthesis of [14Cloleate because, in gen- time for approximately 2 h when assayed eral, the 4-h incubations showed more as described in the experimental section. [14Clacetate flowing into [14Cllinoleate The incorporation of [1-‘4C]acetate inthan after 2 h, while the [‘4Cloleate pool creased linearly with an increase in supersize remained relatively constant (Table natant fraction up to 0.25 ml and the incorThere was no synthesis of poration of [2-14C!]malonate increased linII). [14Cllinolenate from [1J4Clacetate. early up to at least 0.5 ml of supernatant. The intracellular distribution of the soy- Preliminary studies also showed that the bean cotyledon fatty acid synthetase sys- rate of the incorporation of [l-14Clacetate into fatty acids was variable while that of tem. Plastid, mitochondrial, microsomal, and soluble cytoplasmic fractions were [2J4C]malonate was relatively constant; it prepared from cotyledons (35 DAF) by the has been shown by McKenzie et al. (21) differential centrifugation technique of that the activity of the acetyl-CoA carboxLord et al. (20) and assayed for [2- ylase of soybean cotyledon is low and vari14C]malonate incorporation into fatty acids able. Consequently, [2-14Clmalonate was used as substrate in subsequent experi(see Experimental). Only the supernatant fraction had activity, and the activity ob- ments with supernatant fractions. When served was equivalent to that observed in malonate was used as substrate, acetate the crude homgenate. Thus all subsequent was not added as “primer” for fatty acid experiments were carried out on crude su- synthesis because sufficient acetyl-CoA for pernatant fractions prepared as described this purpose is generated by malonyl-CoA in the experimental section. decarboxylase (22). Cofactor requirements. For some time The incorporation of [2-‘4Clmalonate now it has been postulated (23) that the into long chain fatty acids by cotyledon

FATTY

ACID

BIOSYNTHESIS

57

IN SOYBEANS

TABLE II. THE EFFECTOF DEVELOPMENTON THE INCORPORATION OF 11-14CIA~~~~~~ INTO FATTY ACIDS BY CHOPPEDCOTYLEDONTISSUE” DAF

Total [lJ4Clacetate incorporation into fatt acids per h by cx opped tissue (nmol/ seed)

(nmol/g fresh wt)

Distribution

Incuba2

of 14C into newly

16:0

formed fatty

acids (%)

18:O

18:l

18:2

18:3

Fit;

15

0.25

2.9

2 4

28 33

8 9

43 37

21 21

0 0

20

0.33

2.4

2 4

15 21

P

57 56

28

0

t

23

0

25

0.30

2.5

2 4

18 16

t t

67 52

15 32

0 0

30

1.94

a.4

2 4

15 17

t t

59 53

26 30

0 0

40

2.75

12.0

2 4

20 13

t t

61 45

19 42

0 0

50

2.16

7.7

2 4

16 20

t t

66 56

18 24

0 0

a Chopped cotyledon tissue (0.5 g), prepared from seeds harvested on the cited DAF, was assayed as described in the experimental section for [l-14C]acetate incorporation into fatty acids. Fresh weight per seed and total endogenous lipid contents are given in Table I. b t < 1%.

intermediates of fatty acid biosynthesis in plants are thioester derivatives of ACP, a small polypeptide containing a 4-phosphopantotheine moiety. Figure 1 clearly shows that the addition of ACP stimulated

I 4 2

0

25 is 50 ACYL CARRIER PROTEIN CONCENTRATlON i(rg/ml)

FIG. 1. Effect of ACP Wlmalonate incorporation natant fraction (0.5 ml) “Clmalonate incorporation tion) in the presence of O-75 of ACP/fil)

concentration on [2into fatty acids. Superwas assayed for [2(see experimental sec~1 of ACP solution (1 pg

the incorporation of [2-14C]malonate into fatty acids by cotyledon supernatant fractions. At least 75 pg of ACP was necessary for maximal incorporation and was approximately equivalent to an eightfold stimulation of activity. As ACP is not readily available, only 25 pg was used in subsequent experiments: This was found to be a satisfactory routine assay procedure for not only was there a five- to sixfold stimulation of [2-14Clmalonate incorporation but, in addition, linear time curves and linear enzyme- and substrate-concentration curves were obtained as described in the previous sections. We showed that CoA, ATP, and NADPH/NADH were also essential for maximal activity: The optimum concentration of each cofactor is shown in the assay conditions described in the experimental section. A more pronounced stimulation of fatty acid biosynthesis by NADPH/NADH was obtained when the cotyledon supernatant was dialyzed to remove endogenous pyridine nucleotides and with this dialyzed superna-

58

PORRA

AND

STUMPF

tant it was found that NADPH and NADH ACP but without these additions the optiwere both equally effective as the reduc- mum for [14C]oleate formation from [2tant in fatty acid biosynthesis. However, i4C]malonate was pH 7.4 (Fig. 2). This inan investigation of the pyridine nucleotide dicates that the pH optima of other comporequirement of fatty acid biosynthesis (15) nents of the fatty acid synthetase system showed that NADPH was the preferred occur at pH values higher than pH 6.0. reductant for the elongation system but a Indeed, little palmitate or stearate, both of mixture of both was required for the de which are precursors of oleate, was formed novo system. Hence a mixture was used in at pH 6.0 (Fig. 2). The pH optima for [‘4C]palmitate and [14Clstearate formation all routine assays. were about pH 8.0 and 7.6, respectively. The effect of pH on [Z-“K’lmalonate inThe effect of seed development on the corporation into various fatty acids. Figinto fatty ure 2 indicates that pH 7.8 was optimal for incorporation of [2-“Clmalonate [2-‘4Clmalonate incorporation into total acids by cotyledon supernatant fractions. The pattern of incorporation of labeled malfatty acids by a cotyledon supernatant fraction. The principal labeled fatty acids onate into fatty acids by cell-free, supernaformed were palmitic and stearic acids. tant fractions during seed development Unlike the experiments described in the (Table III) was different from that obfollowing paper (24), no ferredoxin or ferTABLE III redoxin:NADP reductase was added to the THE INCORPORATION OF [2-'"CIMALONATE INTO reaction mixture so that only small FATTY ACIDS BY THE SUPERNATANT FRACTION OF amounts of [14Cloleate were detected. No DEVELOPING COTYLEDONE? [14C]linoleate or [14C]linolenate formation DAF Total [2was ever observed. In the presence offer“Clmalonate Distribution of ‘VI! into incorporation redoxin and ferredoxin:NADP reductase, newly formed fatty acids (%I into fatty acids it was shown (24) that pH 6.0 was optimal per h by supernatant for the conversion of stearyl ACP to oleyl

FIG. 2. The pa-activity curve for malonate incorporation into fatty acids. A supernatant fraction was prepared as described in the experimental section but the tissue was homogenized in distilled H,O. This supernatant fraction (0.5 ml) was assayed for 12-Wlmalonate incorporation into fatty acid (see experimental section) but the final pH’s of the reaction mixtures (pH 6.4-7.7) were adjusted to the cited pH with K-phosphate buffer, and reaction mixtures (pH 7.8-8.5) were adjusted with Tricine buffer.

(nmoli seed)

(nmoli g fresh wt)

Incubation period (min)

l&O

18:0

18:l

15

4.8

52.4

30 60

43 33

57 67

0 0

20

3.4

24.3

30 60

52 48

41 44

7 8

25

3.7

24.6

30 60

40 45

53 36

19

7

30

7.2

24.0

30 60

59 38

41 62

0 0

40

5.0

16.8

30 60

77 83

23 17

0 0

50

4.2

15.0

30 60

83 57

17 43

0 0

n Supernatant fractions (0.5 ml) from seeds harvested on the cited DAF were assayed for 30 and 60 min at 22°C for [2-14C1malonate incorporation into fatty acids. Procedures for the isolation and determination of individual and total ‘C-labeled fatty acids are described in the experimental section. Fresh weight per seed and total endogenous lipid contents are given in Table I.

FATTY

ACID

BIOSYNTHESIS

served when intact cells, i.e., chopped tissues, were incubated with labeled acetate (Table II). First, there was no increase in incorporation of [14Clmalonate during seed development. Indeed, on a per-seed basis the activity was almost constant while on a fresh weight basis there was actually a decrease in activity as the seed matured. Second, the incorporation of label into oleate was small even though it was shown in the following paper (24) that stearyl ACP desaturase was active throughout the development period. The low incorporation into oleate can probably be attributed to the omission of ferredoxin and ferredoxin:NADP reductase from the assay mixture and to the differences in pH optima for the fatty acid synthetase system (pH 7.8) and for stearyl ACP desaturase (pH 6.0). Third, and in marked contrast to the results of Rinne (8) and Rinne and Canvin (9), no incorporation of label into linoleate or linolenate was ever observed. The pattern of incorporation of [214C!]malonate into newly formed fatty acids by supernatants (Table III) shows a loss of activity of the elongation system as the seed matures. No such loss is observed when chopped tissue preparations were incubated with labeled acetate (Table II). Thus it would appear that the elongation system becomes more labile as seed develTABLE

opment progresses and is more prone to inactivation by the procedures used to make cell-free preparations. II. Experiments with Germinating Soybean Cotyledons The effect of germination on [l‘4CIacetate incorporation into fatty acids by chopped cotyledon preparations. During the first 6 days of germination of dried seed from our seed stocks, the cotyledon fresh weight and endogenous lipid content remained much the same as indicated in the “Dried seed” entry in Table I. The only fatty acids detected were palmitate, stearate, oleate, linoleate, and linolenate as indicated in the above-mentioned entry; no shorter-chain fatty acids such as decanoate (ClO:O), laurate (C12:0), and myristate (C14:O) were detected (cf. Table V). During germination, chopped cotyledon tissue incorporated [14Clacetate into fatty acids (Table IV), principally palmitate and oleate, but incorporation was generally lower than observed in developing tissue (Table II) where labeled linoleate also accumulated. No incorporation of label into shorter-chain fatty acids was observed (cf Table V). Seeds for these germination studies had to be soaked in hypochlorite solutions to inhibit growth of bacterial IV

THE EFFECT OF GERMINATION ON THE INCORPORATION OF [1-14C]A~~~~~~ TISSUE” DAG

1

Total [1J4CIacetate incorporation into fatty acids per h by chopped tissue

59

IN SOYBEANS

Distribution

INTO FATTY ACIDS BY CHOPPED

of 14C into newly

(nmoll seed)

(nmol/g fresh wt)

Incubation period (h)

16:0

18:O

0.57

1.20

2 4

23 22

formed fatty

acids (%I

18:l

182

183

t

77 78

0 0

0 0

P

3

0.87

1.82

2 4

71 71

t t

29 29

0 0

0 0

6

0.40

1.09

2 4

60 68

10 6

30 26

0 0

0 0

a Chopped cotyledon tissue (0.5 g) was prepared (DAG) and assayed for [l-14CIacetate incorporation

from seeds harvested into fatty acids.

on the cited day after germination

60

PORRA

AND

STUMPF

TABLE

V

THE INCORPORATION OF [Z-WIMALONATE INTO FATTY ACIDS BY THE SUPERNATANT FRACTION OF GERMINATING SOYBEANS” DAG

1 6

Fresh weight per seedb (g)

Total [‘Qmalonate incorporation into fatty acids per h by supernatant fraction

Distribution

of 14C into newly formed fatty

acids (%)

(nmoll seed)

(nmol/g fresh wt)

lo:o

120

14:o

16:0

l&O

18:l

2.03 1.69

4.42 3.92

16 30

23 23

33 c

29 47

0 0

0 0

0.46 0.43

(1Cotyledon supernatant fractions (0.5 ml), prepared (see Experimental) from seeds harvested cited DAG. were assaved for 60 min at 22°C for i2-‘4Clmalonate incorporation. * Only the cotyledons were weighed. c t < 1%.

contaminants which could, during the assay of chopped tissue, incorporate [l14C]acetate into palmitoleate (16:l n-7) which was identified as described in the experimental section. The effect of germination ration of [2-‘+Z’lmalonate by cotyledon supernatant

on the incorpointo fatty acids fractions. Con-

sistent with the decreased incorporation of [lJ4C[acetate into fatty acids by chopped germinating cotyledons (Table IV), the incorporation of [2-‘*C]malonate by supernatants from germinating cotyledons (Table V) was much lower than that observed with supernatants derived from developing cotyledons (Table III). The distribution of 14Clabel in the fatty acids formed from [2J4C]malonate (Table

on the

V) showed the accumulation of decanoate, laurate, and myristate as well as palmitate. The only fatty acid to accumulate when the de nouo system is functioning normally is the final product, palmitate. The reason for the accumulation of intermediate fatty acids is not known. In an attempt to solve this problem fresh and boiled supernatants from germinating cotyledons were incubated with a supernatant from developing cotyledons (Table VI). None of these mixed incubations resulted in the accumulation of 14C-labeled short chain fatty acids. This observation would suggest that deacylases in the germinating tissue are not interrupting the synthesis of palmitate by the de nouo system by hydrolyzing intermediate short

TABLE VI THE INHIBITORY EFFECT OF A SUPERNATANT FRACTION FROM GERMINATING SOYBEANS ON [2-14ClMALONATE INCORPORATION BY A SUPERNATANT FRACTION FROM DEVELOPING SOYBEANS” Expt No.

1 2 3

Supernatant

fraction

J-Day germinating g-Day germinating 30-Day developing

from

seeds (A) seeds (B) seeds (C!)

Malonate incorporation (nmol) 0.71 0 5.88

Inhibition (%I

-

Distribution of ‘*C in newly formed fatty acids (%I 14:o

16:0

18:O

19 0

66 29

15 71

Total of 1 + 3 Total of 2 + 3

6.59 5.88

0 0

4 5

AiC A (boiled)

+ C

4.19 2.68

36 59

0 0

60 52

40 48

6 7

B+C B (boiled)

+ C

1.28 2.84

78 52

0 0

81 61

19 39

(LSupernatant fractions (0.5 ml) from germinating and developing soybeans were incubated for 1 h at 22°C as described in the experimental section; however, the final volume ofthe incubation mixture was 1.5 ml.

FATTY

ACID

BIOSYNTHESIS

chain acyl ACP derivatives; further, direct assays revealed that there was less deacylase activity in the supernatant fractions of germinating seeds than from developing seeds. Since p-oxidation enzymes cannot react with acyl ACP derivatives, these short chain acids cannot be a product of this degradative pathway. The results, however, do not preclude the possibility that the de nouo system after germination is so unstable to cell disruption that the resulting enzyme complex has decreased affinity for short chain acyl ACP derivatives. In this case the released short chain derivatives could be metabolized to palmitate and stearate by the supernatant fraction from the developing seeds. The results (Table VI), however, indicate that the supernatants from the germinating seeds contain a heat-stable inhibitor. The nature of this inhibition is not clear. III.

Concluding

Remarks

Much of the general discussion of the relationship of these results to the biosynthesis of palmitate, stearate, and oleate is presented in the discussion section of the following paper by Stumpf and Porra (24). There are, nonetheless, several results which, because they are inconsistent with previous reports, are discussed here. First, it was reported by Inkpen and Quackenbush (7) that 14C-labeled laurate, myristate, palmitate, and stearate could be converted by a cell-free preparation of soybean cotyledons to [14Cloleic acid with the addition of only ATP, CoA, NADPH, MgC12, and MnSO,. This is noteworthy not only because ferredoxin and ferredoxin:NADP reductase were not added (cf. Ref. (24)) but more especially because no ACP was added. That ACP was not required has led to the proposition (14) that fatty acid synthesis in soybean cotyledon is unusual. In this paper, however, it has been clearly demonstrated that ACP is essential for maximal rates of fatty acid synthesis in soybean supernatants. Second, unlike Rinne (8) and Rinne and Canvin (9) we were unable to demonstrate [14C]linoleate or [14C]linolenate formation from llJ4Clacetate or [2-14Clmalonate by cell-free soybean cotyledon preparations. The finding of these labeled unsaturated

IN SOYBEANS

61

fatty acids (8, 9) is surprising since these investigators did not add ACP, ferredoxin or ferredoxin:NADP reductase which have been shown in the next paper (24) to be necessary for maximal rates of oleate formation. Our failure to detect [14Cllinoleate or [14C]linolenate is difficult to explain since our method of detection was by radioglc (see Experimental), which is generally regarded as the preferred method (25); the other investigators (8, 9) used an AgNO,tic procedure to separate, detect, and identify 14C-labeled fatty acids. ACKNOWLEDGMENTS We thank Mrs. Lidia Calis for skilled technical assistance. This research was supported in part by NSF Grant 37 880-X administered by P.K.S. REFERENCES 1. MCMAHON, V. AND STUMPF, P. K. (1966) Plant Physiol 41, 148-156. 2. STUMPF, P. K. (1975) J. Amer. Oil. Chem. Sot., 52, 484A-490A. 3. NAKAMURA, Y., AND YAMADA, M. (1974) Plant Cell Physiol. 15, 37-48. 4. YAMADA, M., USAMI, Q., AND NAKAJIMA, K. (1974) Plant Cell Physiol. 15, 49-58. 5. GURR, M. I., BLADES, J., APPLEBY, R. S., SMITH, C. G., ROBINSON, M. P., AND NICHOLLS, B. W. (1974) Eur. J. Biochem. 43, 281-290. 6. NORTON, G., AND HARRIS, J. F. (1975) Planta (Berlin) 123, 163-174. 7. INKPEN, J. A., AND QUACKENBUSH, F. W. (1969) Lipids 4, 539-543. 8. RINNE, R. W. (1969) Plant Physiol 44, 89-94. 9. RINNE, R. W., AND CANVIN, D. T. (1971) Plant Cell Physiol. 12, 387-393. 10. RINNE, R. W., AND CANVIN, D. T. (1971) Plant Cell Physiol. 12, 395-403. 11. RUBEL, A., RINNE, R. W., AND CANVIN, D. T. (1972) Crop Sci. 12, 739-741. 12. NELSON, D. R., AND RINNE, R. W. (1975) Plant Physiol. 55, 69-72. 13. HARWOOD, J. L. (1975) Phytochemistry 14, 19851990. 14. APPLEQVIST, L-hi. (1975) in Recent Advances in the Chemistry and Biochemistry of Plant Lipids (Galliard, T., and Mercer, E. I., eds.), pp. 247-286, Academic Press, New York. 15. JAWORSKI, J. G., GOLDSCHMIDT, E. E., AND STUMPF, P. K. (1974) Arch. Biochem. Biophys. 163, 769-776. 16. MAJERUS, P. W., ALBERTS, A. W., AND VAGELOS, P. R. (1969) Methods Enzymol. 14, 43-50. 17. LOUTRY, 0. W., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275.

62

PORRA

AND

18. STUMPF, P. K., AND BOARDMAN, N. K. (1970) J. Biol. Chem. 245, 2579-2587. 19. STEIN, R. A., AND NICOLAIDES, N. (1962) J. Lipid Res. 3, 476-470. 20. LORD, J. M., KAGAWA, T., AND BEEVERS, H. (1972) Proc. Nat. Acad. Sci. USA 69, 24292432. 21. MCKENZIE, E. A., KENNEDY, I. R., AND LEES, E. M. (1975) Proc. Aust. Biochem. Sot. 8, 45. 22. HATCH, M. L., AND STUMPF, P. K. (1962) Plant

STLJMPF Physiol. 37, 121-126. 23. HUANG, K. P., AND STUMPF, P. K. (1971) Arch. Biochem. Biophys. 143, 412-427. 24. STUMPF, P. K., AND PORRA, R. J. (1976) Arch. B&hem. Biophys. 176, 63. 25. KATES, M. (1972) in Techniques of Lipidology: Isolation, Analysis and Identification of Lipids, (T. S. Work, and E. Work, eds.), p. 446, North-Holland, Amsterdam.