Plant Physiol. (1980) 66, 666-6710032-0889/80/66/0666/06/$00.50/0

In Vivo Pathway of Oleate and Linoleate Desaturation inDeveloping Cotyledons of Cucumis sativus L. Seedlings'

Received for publication February 29, 1980 and in revised form May 29, 1980

DENIS J. MURPHY AND PAUL K. STUMPFDepartment of Biochemistry and Biophysics, University of Calhfornia, Davis, California 95616

ABSTRACT

Exogenous 1-14CIoleic acid and 11-'4Cllinoleic acid were taken up andesterified to complex lipids by greening cucumber (Cucumis sativus L.)cotyledons. Both 14C-labeled fatty acids were initially esterified to phos-phatidylcholine prior to eventual accumulation in triacylglycerols andgalactolipids. Kinetic data suggest that esterification occurs prior to de-saturation and that phosphatidylcholine is the initial site of both 14CI-oleate and 11-14Cllinoleate esterification and of II-'4Cloleate desaturationto 11-_4Cllinoleate. 11-14CILinoleic acid was esterified more rapidly thanI'4Cloleic acid and its desaturation product, 11-14CIa-linolenate, occurredmainly on monogalactosyl diacylglycerol, although some was also observedon the other major acyl lipids, including phosphatidylcholine.

The biosynthesis of polyunsaturated fatty acids by plant tisuesremains an area of controversy some 20 years after the originaldemonstration by Smirnov (22), shortly thereafter confirmed byMudd and McManus (7) and by Stumpf and James (23), thatisolated chloroplasts could incorporate ["Clacetate into long-chainfatty acids. The activities of the de novo fatty acid synthetase,palmitoyl-ACP elongase and stearoyl-ACP desaturase have beenwell-characterized (4, 5, 24) and have recently been shown, in thecase of mature spinach leaves, to be exclusively confined to thechloroplast (14). Isolated spinach chloroplasts are also capable ofsynthesizing oleic acid from photosynthetically fixed CO2 and ofincorporating the fatty acid into galactolipids (6, 8-10).

In contrast, repeated attempts to observe the subsequent con-

version of oleic acid to polyunsaturated fatty acids in isolatedchloroplasts have proved unsuccessful. This has led to the proposalthat there is an extrachloroplastic site for the desaturation ofoleateto the polyunsaturated fatty acids that constitute the bulk of leaffatty acids (21, 27). In vivo-labeling kinetics from 14CO2and [14C]acetate are consistent with an acyl-carrier role by pC2during oleate desaturation (12, 13), but definitive in vitro data are

lacking (15, 17, 18, 21). Meanwhile, the nature of the linoleatedesaturase remains unknown.

In vitro studies have been hampered by the low rates of desat-uration found in cell-free systems, and even in some intact tissues.We have recently demonstrated that etiolated cucumber cotyle-dons respond to illumination with an induction of the enzyme

'This work was supported in part by Grant 2R01 GM 19213-08 fromthe National Institute of General Medical Sciences.

' Abbreviations: PC, phosphatidylcholine; DG, diacylglycerol; DGD,digalactosyl diacylglycerol; FFA, free fatty acid; MG, monoacylglycerol;MGD, monogalactosyl diacylglycerol; PA, phosphatidic acid; PE, phos-phatidylethanolamine; PG, phosphatidylglycerol; SQD, sulfoquinovosyldiacylglycerol; TG, triacylglycerol.

activities responsible for oleate and linoleate desaturation (11).Exogenous "C-labeled fatty acids are rapidly acylated by thistissue and their effective bypassing of the fatty acid synthetaserenders them far more efficient substrates for studying desatura-tion than either "CO2 or [1-'4Clacetate.We have previously noted that etiolated tissue will preferentially

oxidatively degrade exogenous fatty acids (12), whereas greeningtissue will rapidly acylate and then desaturate long chain unsatu-rates. In this study we have followed the acylation, desaturation,and interconversion of ["4CJoleic acid and [1-'4C]linoleic acid inorder to elucidate the mode of their metabolism in a developingphotosynthetic tissue.

MATERIALS AND METHODS

Plant material was grown and incubated with "C-labeled sub-strates under conditions similar to those described in the previouspaper (12).

Lipid Analysis. Reactions were terminated by immersion of thecotyledons in hexane-isopropyl alcohol (3:2, v/v) followed byhomogenization of the tissue in a TenBroek ground glass homog-enizer until it was completely solubilized, with the exception of asmall amount ofwhite fibrous residue. This residue was repeatedlyextracted in hexane-isopropyl alcohol (3:2, v/v) and the extractswere combined. Aqueous contaminants from the extracts wereremoved by partitioning against 50% of their volume of 6%aqueous Na2SO4. The organic solvent phase containing lipid wasremoved under a stream of N2 and the lipid residue was redis-solved in a small volume of chloroform. Aliquots of the total lipidmixture were removed for analysis for fatty acids by GLC.

Total lipid mixtures were resolved by two-stage single dimen-sional TLC. The neutral lipids were initially separated in petro-leum ether-diethyl ether-acetic acid (70:30:1, v/v), which resolvedthe mixture into bands containing triacylglycerol (RF = 0.62),FFA (RF = 0.46), 1,3-DG (RF = 0.26), 1,2-DG (RF = 0.20), MG(RF = 0.08), and polar lipids (RF = 0.02). The polar lipid bandwas removed from the plate, eluted with chloroform:methyl alco-hol-formic acid-H20 (97:97:4:2, v/v), and concentrated under N2.Polar lipids were then resolved in chloroform-methyl alcohol-acetic acid-H20 (85:15:10:3.5, v/v) into bands containing MGD(RF = 0.91), PA (RF = 0.79), PE (RF = 0.59), DGD (RF = 0.62),PG (RF = 0.35), PC (RF = 0.31), and SQD (RF = 0.21). The lipidbands, which were clearly resolved from each other, were locatedby means of nondestructive spray reagents, such as iodine ordichlorofluorescein, and 14C label was localized with a Packardmodel 7201 radiochromatogram scanner or by autoradiography(1 1).

Quantitative radioactivity determinations were made by elutinglipid bands as previously described and adding aliquots of theeluate to a counting vial. After removal of the elution solvents,the samples were taken up in a PCS (Phase Combining System,Amersham/Searle)-xylene (2:1, v/v) cocktail and counted in aBeckman LS 230 liquid scintillation counter.

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PATHWAY OF POLYUNSATURATE BIOSYNTHESIS

Methyl esters of total lipid mixtures or separated lipid bandswere prepared by alkaline hydrolysis in KOH followed by esteri-fication with diazomethane as previously described (11). Alter-natively, the separated lipid bands were scraped directly into tubescontaining 5 ml of either methyl alcohol-H2SO4 (20:1, v/v) forpolar lipids or benzene-methyl alcohol-H2SO4 (20:10:1, v/v) forneutral lipids. Boiling chips were added and the mixture wasrefluxed at 110 C for 4 h. After cooling, 7 ml distilled H20 wasadded to each tube and the methyl esters were extracted twice in4 ml petroleum ether. The ether extracts were washed with 2 mldistilled H20 and concentrated under N2 for separation on a 10%DEGS-PA (Supelco, Bellefonte, Pa.) 0.4-cm diameter x 1.83 mcolumn at 170 C. Radioactivity of the column effluent was mon-itored with a Nuclear-Chicago Biospan model 4998 radioactivitydetector.

Lipids and fatty acids were determined quantitatively on aPackard model 417 GLC with a flame-ionization detector and a0.4-cm diameter x 1.83 m column packed with 10%1o EGSS run at180 C column using C170 as an internal standard.

RESULTS

Glycerolipid and Fatty Acid Composition. In order to quantitatethe data from the fatty acid-labeling studies, the absolute glycer-olipid compositions of the principal lipid components of germi-nating cucumber cotyledons were determined. Table I shows theamounts of the principal glycerolipids of 7-day-old etiolated cot-yledons compared with similar tissue illuminated for 16 h. Themajor lipids in the dark-grown cotyledons were PC, TG, and PE,which together accounted for over 60%o of the total glycerolipids.Following a 16-h illumination, the absolute amounts of theselipids decreased only slightly. However, since the amount of totallipid increased by 29%, the relative proportion of PC, TG, and PEdeclined by 14% from 60 to 46% of the total. At the same time,after greening, the principal chloroplast lipids that constitute thematrix of the photosynthetic membranes, ie. MGD, DGD, SQD,and PG, increased both in relative and absolute amounts.The fatty acid compositions of the principal glycerolipids are

given in Table II. The fatty acids of the etiolated cotyledons wererelatively deficient in 18:3, which is the major fatty acid of thephotosynthetic membrane systems, but which accounted for only4% of the total in the etiolated tissue. After 16 h illumination, theproportion of 18:3 rose to 19% of the total, whereas the amount(mg g-1 fresh weight) of total acyl residues rose by 22%, whichindicates that a rapid synthesis of 18:3 was taking place. Thenewly synthesized 18:3 mainly accumulated in the galactolipidsMGD and DGD and in the chloroplast phospholipid PG. Rela-tively low amounts of 18:3 were found in the other principalchloroplast lipid, SQD. The increase in net 18:3 levels was accom-

Table I. Quantitative Acyl Lipid Composition of 7-day-old CucumberCotyledons

Lipid Greened for Etiolated16 hmg lipid g % mg lipid g' %

fresh wt fresh wtMG 0.38 1.5 0.25 1.2DG 1.50 5.8 1.43 7.1FFA 0.81 3.1 0.77 3.8TG 2.93 11.3 3.33 16.5SQD 2.43 9.4 1.59 7.8PC 6.97 26.7 6.76 33.6PG 3.96 15.3 1.76 8.7PE 2.09 8.1 2.13 10.6MGD 3.17 12.2 1.40 6.9DGD 1.72 6.6 0.74 3.7Total 25.96 100.0 20.16 99.9

Table 11. Fatty Acid Composition of the Gh'cerolipids of 7-day-oldCucumber Cotyledons

Total Fatty Acid

Lipid Greened for 16 h Etiolated

16:0 18:0 18:1 18:2 18:3 16:0 18:0 18:1 18:2 18:31)

MG 28.1 10.6 13.1 37.7 9.7 42.5 13.3 13.3 18.5 12.4DG 16.5 12.6 18.5 50.6 1.8 13.7 17.6 25.6 40.3 2.8FFA 24.6 4.0 14.9 43.6 12.9 23.5 9.8 15.9 40.5 10.2TG 15.6 11.3 19.3 53.1 0.7 20.9 11.9 19.7 46.3 1.2SQD 33.7 16.9 11.8 23.7 13.9 20.2 11.2 16.8 47.6 4.2PC 36.3 13.0 7.6 31.8 11.2 40.4 9.4 12.3 35.1 2.8PG 28.7 12.1 5.5 11.9 41.6 40.7 20.5 9.1 16.5 13.1DGD 44.0 4.7 2.4 29.4 19.5 61.1 5.6 6.4 20.0 6.9PE 24.6 12.4 9.3 39.4 14.3 41.4 6.2 10.8 37.5 4.1MGD 22.4 6.8 12.7 9.1 49.2 63.3 5.4 8.3 23.1 8.6

a 100 x 18:3 acyl residues/total acyl residues: 18.7.h 100 x 18:3 acyl residues/total acyl residues: 3.8.

-1

4'C_Oleate

9 r

7 \s

6

5

3 6 12 24 32Time (hr)

FIG. 1. Distribution of "C radioactivity from [1-'4Cloleic acid in theglycerolipids of greening cucumber cotyledons. Only the principal labeledlipid classes are shown. Duplicate batches of cotyledon pairs (about 100mg) were each incubated with I ,uCi [1-'4CIoleic acid (about 17 nmol)applied in 10 j.I ethylene glycol monoethyl ether solution. This was allowedto dry and the cotyledons were placed in 2 ml 0.1 M phosphate buffer (pH6.5). Incubations were performed under aerobic conditions at 25 C andfluorescent white light of intensity (quantum flux) <200 [LE m-2 s-'. Theincubations were halted after 3 to 32 h by extraction of the plant materialinto hexane-isopropyl alcohol (2:1, v/v). Results are expressed as averagevalues from at least two experiments.

panied by a decline in the amount of 18:2 in the tissue, indicatingthat desaturation of the endogenous 18:2 pool was the principalmethod of generating 18:3 early in the greening process.

Incorporation of 11-"4CIOleic Acid into Lipids by GreeningCucumber Cotyledons. Exogenous [1-'4C]oleic acid was esterifiedto complex lipids at a rapid rate for the first 6 h of the incubationsand at a constant but slower rate for the next 26 h (Fig. 1). PC wasthe most rapidly labeled lipid from [1-'4C]oleate. The 14C labelaccumulated in this lipid over the first 6 h but then declined until>50%o of the label had been lost after 32 h. Although by far thegreatest flux of label was through PC, it was also observed thatPE and, to a lesser extent DG, exhibited similar labeling kineticsfrom [1-_4C]oleic acid. The principal end product in which "Clabel accumulated was TG.

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MURPHY AND STUMPF

"C-labeled Fatty Acid Composition of Glycerolipids Labeledfrom 1I-"4ClOleic Acid. The fatty acid-labeling patternsof the principal glycerolipids from greening tissue incubated with[1-'4CJoleic acid were determined to elucidate possible lipid sitesfor desaturation to polyunsaturates. The results of the analysis ofthe eight major labeled lipids are shown (Fig. 2). Although free[1-'4C]oleic acid steadily declined during the incubations, no morethan trace levels of nonesterified desaturation products were everfound. The most interesting pattern was provided by PC, in whichthe majority of the added [1-'4C]oleate initially accumulated. [1-'4C]Oleoyl-PC rapidly declined with a rise in [1-"CJlinoleoyl-PC.The decline in the amount of oleate in PC after 6 h was notbalanced by the appearance of equivalent amounts of linoleateand a-linolenate in the same lipid. The data would suggest thatlinoleate was probably the major acyl species to be lost from thePC pool. PE, PA, and DG showed a similar loss of "C, althoughmuch less than did PC. The initial accumulated acyl group wasoleate with linoleate then being lost from the lipid. The principalend product of ['4C]acyl accumulation was the TG pool. Here, allthree major C18 fatty acids were taken up with almost equalfacility in an apparently nonspecific manner. This contrasts withthe major galactolipid in the tissue, MGD, which preferentiallytook up a-linolenic acid, presumably from other complex lipiddonors.

Incorporation of 11-"4CILinoleic Acid into Complex Lipids. Ex-ogenous [11-4C]linoleic acid was esterified to complex lipids farmore rapidly than was [1-_4C]oleic acid (Fig. 3), and its esterifi-cation was virtually completed within 6 h. The primary lipidacceptor for linoleate was once again PC, although the phospho-lipid accumulated 14C for 12 h before showing a net decline. Theefflux of 14C from PC was more rapid than in the [1-_4CJoleic acidincubations (Fig. 3), with 40% of the label being lost between 12and 32 h. An apparent flux of 14C was also observed in PE andDG, but this was far less dramatic than that seen in PC. TG andMGD accumulated 14C at almost equal rates for the first 12 h,after which MGD became more heavily labeled.

14C-labeled Fatty Acid Composition of Glycerolipids Labeledfrom 11-4CiLinoleic Acid. The rapid decline in '4C-labeled freelinoleic acid was not accompanied by an increase in free ["4CIa-linolenic acid (Fig. 4), and virtually all of the ['4C]a-linolenate

Lipid labelling patterns from (1 4C) oleate

TG -1FFA

formed was found esterified to complex lipids. PC accumulated['4CJlinoleate more rapidly than the other glyceroli?ids, and theacyl species lost from PC seemed to be mainly [1- 4C]linoleate,but a slight decline of ['4C]a-linolenate labeling occurred. Verylittle a-linolenate was found in DG; ['4CJlinoleate was initiallyesterified to DG and subsequently transferred to other glycerolip-ids. Both TG and MGD accumulated significant [14CJlinolenatederived from ['4CJlinoleate.

Specific Radioactivities of Glycerolipids Labeled from I1-"4Cl-Oleic Acid. Since the pool sizes of the principal "C-labeledglycerolipids differ markedly (Table I), it is necessary to allow forsuch differences when considering the extent to which each glyc-erolipid pool is labeled. In Figure 5, the specific radioactivities ofthe acyl moieties of the five main glycerolipids after incubationwith [1-'4C]oleic acid are plotted. The data give a more accuratepicture of the flux of '4C label through each of the glycerolipidsthan is possible by plotting only dpm in each lipid (ie. Fig. 1).After 6 h, PC is the most heavily labeled lipid. The data suggesta flux of "C through PC and PE, followed by an accumulation of"C in MGD and TG. After taking into account the pool sizes ofMGD and TG present in the incubated tissue, it was concludedthat the MGD pool was consistently labeled to a higher specificradioactivity than the TG pool. The data in Figure 5 describe onlythe flux of total '4C in each lipid after [I -'4CJoleic acid incubations,but it has already been shown that this "C can be further separatedinto the desaturation products of [1-_4Cjoleic acid. The specificradioactivities of [1-_4C]oleate, [1-14CJlinoleate, and [1-14C]a-lino-lenate from PC and MGD are plotted in Figure 6. These twolipids were chosen since they are the major sites of 14C flux andaccumulation, respectively. The specific activity of ['4CJoleate roserapidly in PC but then declined. The specific activity of[1_-4C]linoleate formed from [14C]oleate rose in both PC andMGD for most of the time points.

Specific Radioactivities of Lipids Labeled from [1-4CILinoleicAcid. The flux of 14C through DG, PC, MGD, or TG was seeneven more clearly when the substrate was ['4C]linoleic acid (Fig.7). Both DG and PC are transiently labeled, but MGD and TGare the sites of 14C accumulation. We also observed that thespecific radioactivities of the [1-_4CJlinoleoyl species of DG, PC,and MGD were similar, whereas [1-_4C]linoleoyl-MGD was

DGo

PA

3 6 12 24 32 3 6 12 24 32 3 6 12 24 32 3 6 12 24 32 3 6 12 24 s2Time (hr) Time hr

FIG. 2. Fatty acid-labeling patterns of the principal glycerolipid classes of greening cucumber cotyledons incubated with [1-'4CJoleic acid. Lipidswere separated by two-stage, one-dimensional TLC in petroleum ether-diethyl ether-acetic acid (80:20:1, v/v) followed by chloroform-methanol-aceticacid-H20 (85:15:10:35, v/v). Fatty acid classes were resolved using a radio-GLC containing a 10%1o DEGS-PA column.

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PATHWAY OF POLYUNSATURATE BIOSYNTHESIS

Time (hr)

FIG. 3. Distribution of '4C radioactivity from [1-'4CJlinoleic acid in theglycerolipids of greening cucumber cotyledons. Only the principal labeledlipid classes are shown. Incubations were performed as described in thelegend to Figure 1.

3 C 12 24 32

Timt (Hr)

FIG. 5. Time course of the specific radioactivity of the acyl residues ofthe principal labeled lipids from greening cucumber cotyledons incubatedwith [11-4CJoleic acid.

S 1 2 2 32 3TG 1 G PE

2 18:2

18:3~~~~~~8360 2 24 32 3 6 12 24 2 36 2 24 32

Time (hr)

FIG. 4. Fatty acid labeling patterns of the principal glycerolipid classesof greening cucumber cotyledons incubated with Il1-_4CJlinoleic acid.

labeled to a higher specific radioactivity than was [1-_4C]linole-noyl-PC. Since the specific radioactivity of linolenoyl-MGD wasalways higher than that of linolenoyl-PC, it is unlikely that a-linolenate is transferred from PC to MGD. This is similar to thedata from the [1-_4C]oleic acid treatments (Fig. 6) in suggesting atransfer of linoleoyl residues from PC to MGD, with the finaldesaturation to a-linolenate occurring on the galactolipid.

DISCUSSION

A role for PC as the substrate for oleate desaturation in aphotosynthetic tissue was originally suggested by James' group ( 1,13) following studies using the unicellular alga Chlorella vulgaris.There is now substantial in vivo evidence from [14C]acetate and[14C]glycerol 3-P feeding studies that PC is also the major substrateof oleate desaturation in the leaves of higher plants (3, 15-18, 21).More recently, several groups of investigators have described PCacyltransferase activity in a number of developing seed systems(19, 20, 25, 28) and it has even been proposed that the majorpathway of triacylglycerol biosynthesis in developing cotyledons

3 C 12 24 32Time Ibri

FIG. 6. Time course of the specific radioactivities of the (top) oleoyl,(middle) linoleoyl and (bottom) linolenoyl residues of PC and MGD fromgreening cucumber cotyledons incubated with [1-'4Cloleic acid.

12Tim (NrO

FIG. 7. Time course of the specific radioactivity of the acyl residues ofthe principal labeled lipids from greening cucumber cotyledons incubatedwith [I-'4Cllinoleic acid.

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670 MURPHY A

occurs via PC (19). These findings were not unequivocally con-firmed in this study since, although the major flux of '4C from [1-'4C]oleic acid was indeed through PC and this lipid containedhigh levels of ['4C]linoleate derived from [14C]oleate, the triac-ylglycerol pool accumulated mono-, di- and trienoic Cis fatty acidsin an apparently nonspecific manner. The greening cucumbercotyledons represented a tissue whose metabolism was in theprocess of changing from an heterotrophic to an autotrophicmode. It is possible that the triacylglycerol pathway that operatesin maturing cotyledons and mainly accumulates linoleoyl residues(20) is not operating under these conditions and that the triacyl-glycerol pool simply accumulates whatever acyl residues are avail-able.The rate of a-linolenate formation from linoleate was similar

whether the original substrate was [1_14C]oleic acid or [1_14C]_linoleic acid (data not shown). This implies that the two '4C-labeled fatty acids were taken up into the tissue at similar ratesand that the rate of acylation, which was much higher for[1_-4C]linoleic acid, was not a limiting factor in their eventualdesaturation. The total rate of exogenous linoleate desaturation toa-linolenate was 2 to 3 nmol g-' fresh weight h-1 at a substrateconcentration of 20 nmol 14C-labeled fatty acid per 100 mg tissue.Since the greening cotyledons used in this study contained 0.8 to1.0 mg Chl g--' fresh weight, the overall rate of linoleate desatu-ration was of the order of 2.5 to 3.5 nmol fatty acid desaturatedmg-' Chl h-'. These data are only rough approximations of therates of desaturation in this particular greening tissue. However,assuming that the entire pool of linoleate in the cotyledon wereavailable for desaturation, then the maximal rate would be about2 [smol desaturated mg-' Chl h-'. Since so much of the fatty acidin the cotyledon is linoleate (>50%), much of it must be inacces-sible to desaturation, performing either a structural or storage role.However, these figures of 2.5 nmol to 2.0 jtmol mg-' Chl h-1 doset lower and upper limits on the potential for a-linolenate bio-synthesis from linoleate in this particular developing tissue. Sim-ilar studies on 14Co0 uptake into the lipids of developing spinachleaves indicated that the maximal rate of linoleate desaturation toa-linolenate in this tissue was 35 nmol mg- I Chl h-' (D. J.Murphy, unpublished data).When the specific radioactivities of the principal labeled lipids

are taken into account, it is apparent that diacylglycerol wasinitially the most rapidly labeled lipid. Furthermore, a-linolenateonly accumulated in DG very slowly, whereas there was a muchmore rapid flux of both oleate and linoleate through the lipid.These data support the proposal that oleate- and linoleate-con-taining DGs may serve as precursors for PC and MGD, respec-tively, but they do not support the proposal, based on ['4C]acetatefeeding studies, that dilinolenin is the precursor of MGD (27).The specific activities of the individual acyl residues of PC andMGD may suggest that PC accumulates oleate and desaturates itto linoleate and that a transfer of acyl groups exists betweenlinoleoyl-PC and linoleoyl-MGD with the final desaturation toa-linolenate occurring on the MGD molecule.The data are subsequently in agreement with ['4C]acetate-la-

beling studies in pea, wheat, and barley leaves (26) and, althoughcucumber cotyledons accumulated somewhat more a-linolenate intheir PC than these other species, the specific activity of a-linole-noyl-MGD was always greater than that of a-linolenoyl-PC, whichprecludes a role for PC as the donor of a-linolenate as suggestedby Roughan (15). The reason for such a relatively high [ 4C]a-linolenate content in cucumber PC as compared with parallelstudies on maize leaves (2) may be the preponderance of PC ingreening cucumber cotyledons (27% of the total glycerolipid) ascompared with maize leaves (6% total Flycerolipids). The absenceof any significant accumulation of [ 4C]oleate by galactolipids(Figs. 2 and 6) contrasts with the relatively high levels of["Cloleate and even [''Clpalmitate following ["4Clacetate labeling

ND STUMPF Plant Physiol. Vol. 66, 1980

in a true-leaf system (26). In such tissues, galactolipids form 65 to80% of the total glycerolipid. It may be that the large pool ofgalactolipids simply acquires relatively saturated fatty acids bynonspecific acyl exchange and that their desaturation can proceedonly relatively slowly since the normal substrates are other lipids(e.g. PC). It is notable that the relatively small but growinggalactolipid pool in greening cucumber cotyledons only becomeslabeled to any great extent with polyunsaturated fatty acids. Thefeeding to plant tissues of relatively large quantities of exogenoussubstrates such as ["C]acetate and '4C-labeled fatty acids doubtlessimposes a constraint on the metabolism of the tissue which mayproduce artifacts. Thus, the "-,cumulation of ["4C]linolenate in thePC of a hi§h-PC tissue (cucumber cotyledons) and the accumu-lation of [' C]palmitate and ['4CJoleate in the galactolipids of ahigh-galactolipid tissue (true leaves), when the endogenous fattyacid contents of these lipid are not consistent with such a process,may well be such an artifact of active acyl transferases acting onthe newly labeled lipid pools.

Acknowledgments The authors wish to thank Ms. Billie Gabriel and Ms. GaleDunham for their assistance in the preparation of this manuscript.

LITERATURE CITED

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2. HAWKE JC, PK STUMPF 1980 The incorporation of oleic and linoleic acids andtheir desaturation products into the glycerolipids of maize leaves. Plant Physiol65: 1027- 1030

3. HEINz E 1977 Enzymatic reactions in galactolipid biosynthesis. In M Tevini, HKLichtenthaler, eds, Lipids and Lipid Polymers in Higher Plants. Springer-Verlag, Berlin, pp 102-120

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5. JAWORSKI JG, PK STUMPF. 1974 Properties of a soluble stearyl-acyl carrierprotein desaturase from maturing Carthamus tinctorius. Arch Biochem Biophys162: 158-165

6. McKEE JWA, JC HAWKE 1978 The incorporation of '4C-bicarbonate and 4C02into the constituent fatty acids of monogalactosyldiacylglycerol by spinachchloroplasts and leaves. FEBS Lett 94: 273-276

7. MUDD JB. TT MCMANUS 1962 Metabolism of acetate by cell-free preparationsfrom spinach leaves. J Biol Chem 237: 2057-2063

8. McKEE JWA, JC HAWKE 1979 The incorporation of ['4Clacetate into theconstituent fatty acids of monogalactosyldiglyceride by isolated spinach chlo-roplasts. Arch Biochem Biophys 197: 322-332

9. MURPHY DJ, RM LEECH 1977 Lipid biosynthesis from '4C-bicarbonate, 12-'4Clpyruvate, and [1-'4Clacetate during photosynthesis by isolated spinachchloroplasts. FEBS Lett 77: 164-168

10. MURPHY DJ,RM LEECH 1978 The pathway of '4C-bicarbonate incorporated intolipid in isolated photosynthesising spinach chloroplasts. FEBS Lett 88: 192-196

11. MURPHY DJ. PK SrUMPF 1979 Light-dependent induction of polyunsaturatedfatty acid biosynthesis in greening cucumber cotyledons. Plant Physiol 63: 328-335

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13. NICHOLs BW, AT JAMES, J BREUER 1967 Interrelationships between fatty acidbiosynthesis and acyl-lipid synthesis in Chlorella vulgaris. Biochem J 104: 486-496

14. OHLROGGE JB. DN KUHN. PK STUMPF 1979 Subcellular localization of acylcarrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad SciUSA 76: 1194-1198

15. ROUGHAN PG 1970 Turnover of the glycerolipids of pumpkin leaves. BiochemJ 117: 1-8

16. ROUGHAN PG 1975 Phosphatidylcholine: donor of 18-carbon unsaturated fattyacids for glycerolipid biosynthesis. Lipids 10: 609-614

17. SLACK CR, PG ROUGHAN 1975 The kinetics of incorporation in vivo of['4CJacetate and 4CO. into the fatty acids of glycerolipids in developing leaves.Biochem J 152: 217-228

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Plant Physiol. Vol. 66, 1980

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