The Galactolipid, Phospholipid, Fatty Acid Composition ... · sulfolipid from the total phospholipid, using either acetone-acetic acid-water or toluene-ethyl acetate-ethanol, and

Plant Physiol. (1974) 53, 496-502

The Galactolipid, Phospholipid, and Fatty Acid Composition ofthe Chloroplast Envelope Membranes of Vicia faba L.'

Received for publication September 17, 1973 and in revised form November 19, 1973

R. 0. MACKENDER2 AND RACHEL M. LEECHDepartment of Biology, University of York, Heslington, York, England

ABSTRACT

The galactolipid, phospholipid, and fatty acid composition ofchloroplast envelope membrane fractions isolated from leavesof Vicia faba L. has been determined. The major lipids in thisfraction are: monogalactosyldiglyceride, 29%; digalactosyldi-glyceride, 32%; phosphatidylcholine, 30%; and phosphatidyl-glycerol 9%. The lipid composition of the chloroplast envelopemembranes is qualitatively eimilar to that of the lamellar mem-branes isolated from the same plastids, but the proportion ofeach lipid present is very different. The total galactolipid tototal phospholipid ratio was 1.6 :1 in the envelope and 11.1 : 1in the lamellae. The monogalactosyldiglyceride-digalactosyl-diglyceride ratio was 0.9 : 1 in the envelope and 2.4 : 1 in thelamellae. Both membranes lack phosphatidylethanolamine.

Linolenic acid is the major fatty acid in the envelope lipidsrepresenting 63% of the total fatty acid, whereas in the lamellaeit represents 83%. The same fatty acids are present in both theenvelope and lamellar lipids except the trans-A'-hexadecenoicacid, which is confined to the lamellar lipids, particularly thephospholipid fraction.A quantitative comparison of the lipid and fatty acid composi-

tions of the envelope with those of mitochondrial and micro-somal fractions indicates that the chloroplast envelope has acomposition intermediate between that of the chloroplast lamel-lae and these extrachloroplastic membranes.

Although the ultrastructure of the chloroplast envelope hasbeen examined in leaf cells (11, 19, 37), and its permeabilityproperties studied both in the whole leaf (13, 14, 32) and re-cently in isolated chloroplasts (5, 15-18, 22, 29, 36, 38), noth-ing is known of its chemical composition. Chemical character-ization of the envelope membranes must be carried out onisolated membranes, and methods for the isolation (26) andpurification (27) of chloroplast envelope membranes have al-ready been published. These methods yield membrane frac-tions in which 85 to 90% of the membranes originate from thechloroplast double envelope. The lipid composition of thesemembrane fractions has now been investigated, and the fattyacid composition of the total lipid and of some of the individuallipids was determined. The present paper reports details of

' This work was supported by the Science Research Council,Grant B/SR/8692 and by an Imperial Chemical Industries fellow-ship to R.O.M.

2Present address: Department of Botany, The Queen's Univer-sity, Belfast, N. Ireland.

these determinations together with analyses of other cell mem-branes (chloroplast lamellae, mitochondria, and microsomes)isolated from the same leaves of Vicia faba L.

MATERIALS AND METHODS

Subcellular Fractions. These were isolated from leaves of19- to 21-day-old plants of Vicia faba L. var. Dwarf WhiteFan, grown as described previously, and placed in the darkfor 66 to 72 hr before harvesting (26).The leaves (30-40 g) were homogenized at full speed for

3 and then 5 sec in an Atomix blender with 180 ml of ice-coldmedium A: 53 mm Na2HPO,/KH2PO4 buffer containing 500mM D(-)sorbitol, 10 mM MgC12 and 10 mm EDTA-Na2, pH7.3. The resulting homogenate was filtered through four layersof cotton organdy and eight layers of 25 ,um mesh nylon.Chloroplasts and chloroplast envelope membranes (pellet P4)were isolated from this filtrate by a modification of previousmethods (26) as shown in Figure 1. Chloroplasts were preparedby sedimentation at 3,000g for 15 sec, and the resuspended pel-let was then layered onto a 10-ml (4 cm) band of 400 mm buff-ered sucrose and centrifuged at 5OOg for 12 sec. This secondcentrifugation reduced the mitochondrial and bacterial con-tamination by 60% and removed the majority of broken plas-tids (25).

Isolation of Chloroplast Envelopes. For the isolation of thechloroplast envelopes, the chloroplasts were ruptured osmoti-cally by incubation in buffered 10 mM MgC12. Each washedchloroplast pellet was resuspended in 10 ml of grinding me-dium from which the sorbitol had been omitted (medium E),and was incubated at 0 C for 10 min. The suspensions werehomogenized in a TenBroeck ground-glass homogenizer byraising and lowering the plunger rapidly three times. The ho-mogenized suspension was diluted with 20 ml of incubationmedium and centrifuged at 3,000g for 10 min. Floating mate-rial was removed and the supernatant decanted and recentri-fuged at 20,000g for 30 min. The pellet from this spin con-tained the chloroplast envelope membranes. The crude envelopefraction was washed once by resuspending in 20 ml of mediumE (medium A minus sorbitol) and recentrifuging at 20,000g for15 min.The suspensions were observed by phase contrast microscopy

at each stage during the isolation procedure. Initially, the in-tact chloroplasts were opaque and shining in appearance, butafter incubation the suspensions contained many chloroplastswith grayish halos. In each of these chloroplasts the lamellarsystem was eccentric within the grayish balloon-like structure.The grayish structures were pinched off free of lamellae andresembled red blood cell ghosts. These ghosts were seen to bederived from the chloroplast envelopes, and increased greatlyin number after homogenization. The later stages in the proce-dure were designed for the collection of the ghosts free from

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CHLOROPLAST ENVELOPE LIPIDS

Filtered leaf homogenate (in Medium A)

3,OOOg for 15 sec.

Pellet P1 (crude chloroplasts)

500g for 12 min.

thro' 4O0mM sucrose.

Pellet P2 (intact chloroplasts)

Incubated in Medium E

Supernatant Sl

3,000g for 5 min.

4'Supernatant S2

20,000g f}r 30 min.

4'homogenized

3,000g for 10 min.

lwPellet

Pellet SP2

3,000g for 5 min.

Supernatant S3

l00,OOOg for lhr.

Supernatant

500g for 5 min

(repeated I timeg

-0 20,000g for 30 min.

s)

Pellet

20,000g for 15 min.

Pellet P4ZVEIOPEi

,\ ;~~~~4Supernatant S4

20,000g for 30 min.

'"MITOCHONDRIAL ,FRACTION"

Pellet SP4

lOO,OOOg for lhr.

4,

Pellet SP5MICTIOMALFRAOTI0Nt

FIG. 1. Flow diagram showing the preparation of subcellular fractions from leaves of Vicia faba L. All pellets were resuspended in medium E(see text) except pellet P1 which was resuspended in medium A.

lamellae membranes, and the final envelope fraction containednumerous ghosts of varying sizes.

Isolation of Chloroplast Lamellae. The chloroplast lamellaefraction (pellet P3) was prepared by washing the fraction byresuspending it in 30 ml of medium E, homogenizing gently ina TenBroeck ground glass homogenizer and centrifuging at5OOg for 5 min.

Isolation of Mitochondrial and Microsomal Fractions. Themitochondrial and microsomal fractions were isolated from thesupernatant S1 following recentrifugation at 3,000g for 5 minto remove most of the remaining chloroplasts and lamellarmembranes. The details of the fractionation are shown inFigure 1. The supernatant S2 was decanted and recentrifugedat 20,000g for 30 min to give pellet SP2 and supernatant S3.The pellet SP2 was resuspended in 30 ml of medium E and re-centrifuged at 3,000g for 5 min; the resulting pellet was dis-carded and the supernatant was recentrifuged at 20,000g for30 min. The final pellet was designated the mitochondrial frac-tion. The supernatant S. was centrifuged at 100,OOOg for 1 hrto give pellet SP4. This pellet was washed once by resuspendingit in 30 ml medium E and recentrifuging at 100,000g for 1 hr.The pellet obtained was designated the microsomal fraction.

The mitochondrial and microsomal fractions have been identi-fied on the basis of the centrifugal forces required to sedimentthem. The mitochondrial fraction was previously shown tohave a high succinic dehydrogenase activity (25).

After isolation each cellular fraction was resuspended inmedium E. All manipulations were carried out between 0and 4 C.

LIPID ANALYSIS

Extraction. Lipids were extracted in dim light by shaking thefractions with approximately 50 times their volume of chloro-form-methanol (2:1 v/v). The extracts were washed overnightat 4 C in the dark with 20% of their volume of 100 mM NaCl(9) under N2. After washing, the (lower) chloroform phase ofthe extract was removed and evaporated to dryness at 30 Cunder vacuum. The dried extract was taken up in chloroformand re-evaporated twice more. The extracts were stored dryunder N2 at -25 C in the dark.

Chzromatography. Individual galactolipids and phospholipidswere separated from the bulk lipid extract by two-dimensionalTLC on H or HR grade silica gel (Merck) using chloroform-

Plant Physiol. Vol. 53, 1974 497

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MACKENDER AND LEECH

methanol-water (65:25:4, v/v/v) in the first dimension andacetone-acetic acid-water (100:2:1, v/v/v) in the second di-mension (10). The total phospholipid fraction was separatedfrom the neutral lipids and the galactolipids by one-dimensionalTLC using either acetone-acetic acid-water (100:2:1, v/v/v) ortoluene-ethyl acetate-ethanol (2:1:1, v/v/v) as solvent. Lipidsfor quantitation were located with iodine vapor which wasallowed to evaporate before analysis. Lipids for fatty acidanalysis were located by spraying with 0.2% 2',7'-dichloro-fluorescein in 50% ethanol and visualized under UV light.

Lipid Estimation. Galactolipids were estimated as galactose(in the presence of absorbent) by the phenol-sulfuric acidmethod of Roughan and Batt (33). Galactose in 2% (w/v)phenol in water was used as the standard.

Phospholipids were estimated (in the presence of absorbent)as Pi by the method of Bartlett (4) following their digestionby 72% perchloric acid. KH2PO4 was used as the standard.Areas of absorbent equivalent in size to those of the lipid spotswere used as blanks; this was found to be particularly necessaryfor the accurate determination of the galactolipids.

Fatty acid analysis. Fatty acid methyl esters of the totallipid and of individual lipids were prepared with boron tri-fluoride in methanol (28). It was impossible to separate thesulfolipid from the total phospholipid, using either acetone-acetic acid-water or toluene-ethyl acetate-ethanol, and it wastherefore part of the spot remaining at the origin which wasused in these analyses. The methyl esters were purified beforegas-liquid chromatography by TLC using petroleum ether(60-80 b.p.)-diethyl ether, 4:1 (v/v) as solvent. The methylesters which were visualized with dichlorofluorescein wereidentified by reference to a standard of methyl oleate. Theywere eluted with diethyl ether and analyzed in a Hewlett Pack-ard S750 on columns of 10% polyethylene glycoladipate onhigh performance chromosorb (80-100 mesh) at 200 C. Thecarrier gas was argon with a flow rate of 50 ml/ min.

Chlorophyll. Chlorophyll was determined in 80% acetoneextracts containing small quantities of chloroform (<2% byvolume) by the method of Arnon (2).

RESULTS

All the subcellular fractions contained fragments of chloro-plast lamellae. The analyses (except for the total fatty acidcomposition of the total lipid extracts) have therefore beencorrected according to the following equation:

L = y - cx

where L = the actual quantity of lipid in the fraction aftercorrection (,tmoles); y = the total quantity of lipid in the frac-tion before correction (jumoles); c = the Chl content of thefraction (,tg); x = the concentration of the lipid in the lamellarmembranes (,umoles/,ug Chl).

The assumption has been made that all the Chl in the cellis confined to the lamellar membranes. Any Chl present inother cell fractions will therefore be due to the presence oflamellar fragments. The presence of the associated lamellarlipids in the total lipid extracts of each cell fraction is correctedfor by using the correction equation.The results of the analyses of chloroplast envelope and

lamellar membranes are shown in Table I. For comparisonwith the composition of other membranes, the data in TableII give the number of moles of each lipid per 1000 moles oflipid analyzed. Qualitatively the acyl lipids of both types ofchloroplast membrane are identical but the proportions inwhich they are present are very different. The galactolipids arethe major lipids in both membrane fractions, but whereas they

Table I. Galactolipid and Phospholipid Composition ofSubcellular Fractions Isolated from Leaves of

Vicia faba L.The analyses of the chloroplast envelope, mitochondrial, and

microsomal fractions have all been corrected for lamellar con-tamination as described in the text, and are the mean of the num-ber of determinations shown in parentheses.

PhospholipidsAnalyzed

Subcellular GL/PLi MGDG/ Phos-Fraction DGDG Phospha- Phospha- Phatdy-

tidyl- tidyl- ethanodl-choline glycerol etamnel

miolar ratios mioles %Chloropast 1.60 00.29 0.90 z 0.16 77 22 trace2

envelope (6)Chloroplast 11.10 A 0.49 2.40 i 0.14 34 66 trace2

lamellae (5)Mitochondrial (3) 0.30 i 0.03 0.80 z 0.04 57 10 33Microsomal (2) 0.40 4z 0.08 0.40 4z 0.04 67 13 20

1 PL = phosphate remaining at the origin after one dimensional TLC of bulklipid extract in either acetone-acetic acid-water (100:2:1 v/v/v) or toluene-ethyl-acetate-ethanol (2: 1: 1 v,'v/v).

2 <0.6%/7o.

Table II. Lipid Composition of Subcellular Fractionis Isolatedfrom Leaves of Vicia faba L.

The data in this tableTable I.

have been recalculated from those in

LipidSubcellular Fraction

MGDG DGDG PC PG PE

moles/1000 moles of lipid analyzed

Chloroplast envelope 291 324 296 89 0Chloroplast lamellae 654 262 28 55 0Mitochondrial 102 128 435 78 257Microsomal 82 204 476 96 142

represent 61% of the envelope lipids, they constitute 92% ofthe lamellar lipid. There is also relatively more DGDG3 in theenvelope than in the lamellae, and this is reflected in theMGDG/DGDG ratio which is 0.9:1 in the envelope and2.4: 1 in the lamellae.

Phosphatidylcholine and phosphatidylglycerol are the twomajor phospholipids in both membranes, but whereas PC isthe major phospholipid in the envelope (77%), PG is the majorphospholipid in the lamellae (66%). There were also three mi-nor phospholipids in both fractions. Phosphatidylethanolaminewhich was the only one of these to be positively identified,was only present in trace amounts (<0.6%) in both fractions.Another of these spots, the same size as PE ran in the positionof phosphatidylinositol.The fatty acid composition of the total lipid extract and of

the MGDG, the DGDG, and the PL plus SL of all four sub-cellular fractions are shown in Tables III and IV. The fattyacids of the envelope and lamellar lipids are qualitatively simi-lar except that the trans-A5-hexadecenoic acid was only foundin the lamellar lipids. Quantitatively, however, the fatty acid

'Abbreviations: DGDG: digalactosyldiglyceride; GL: galactolip-ids (MGDG + DGDG); MGDG: monogalactosyldiglyceride; PL:phospholipids; SL: sulfolipid; PC: phosphatidylcholine; PG: phos-phatidylglycerol; PI: phosphatidylinositol; PE: phosphatidylethanol-amine.

498 Plant Physiol. Vol. 53, 1974




compositions of the two membranes are rather different. Lino-lenic acid (18:3) is the major fatty acid in the lipids of bothmembranes, but there is always a much lower proportion inthe envelope than in the lamellae; this is reflected in the lowerunsaturated to saturated fatty acid ratios in the envelope.The composition of the chloroplast envelope membrane also

differs from the extrachloroplastic membranes in which thephospholipids are the major class of lipid. For comparison withthe envelope lipids, only PC, PG and PE were measured indi-vidually in the mitochondrial and microsomal fractions. Therewere two other minor phospholipids present in both fractions,one of which ran in the position of phosphatidylinositol. Inthese fractions the phospholipids are respectively 77% and71% of the total lipid. PC is the major single lipid in bothfractions accounting for 43.5% and 47.6%, respectively, ofthe total lipid. PE is the other major phospholipid in boththese fractions. PG is also present in them in small but signifi-cant proportions (Tables I and II). The digalactosyl diglycerideis the major GL in these fractions. This is reflected in theMGDG/DGDG ratio which is 0.8:1 in the mitochondrialfraction and 0.4:1 in the microsomal fraction. Linolenic acidis the major fatty acid in the total lipid, the MGDG, and theDGDG, but not the PL + SL of both fractions; it is, however,proportionally very much less in these lipids than in those ofthe chloroplast membranes. This decrease is reflected in lowratios between the unsaturated and the saturated fatty acids.Linolenic acid (18:2), the major fatty acid of the PL + SLfraction, and palmitate are the other major fatty acids present

Table III. Fatty Acid Compositioni of Total Lipid Extractedfrom Subcellular Fractions Isolated from Vicia faba L.

The results are the means of duplicates of the number ofanalyses shown in parentheses.

Subcellular Fraction 16:0 16:1 16:lA3t 18:0 18:1 18:2 18:3 TFA!SFAi

moles %GChloroplast envelope (3) 13.3 1.8 0.0 4.31 5.8 11.5|63.3 4.7:1Chloroplast lamellae (3) 5.9 0.0 1.2 1.6 3.2 5.282.9 12.3:1Mitochondrial fraction (1) 19.3 1.66 0.0 3.9 5 .831.1 37. 3 3.3:1Microsomal fraction (1) 19.8 1.1 0.0 4.8 6 .1 27.640.7 3.1:1

5UFA = unsaturated fatty acids = 16:1; 16:LA3t; 18:1; 18:2; 18:3. SFA =saturated fatty acids = 16:0; 18:0.

(Tables III and IV). There was no trace of the trans-A3-hex-adecenoic acid in any of the lipids of either extrachloroplasticmembrane fraction. It is possible that there were traces ofarachidic acid present, but with the analytical system used itwas not always readily distinguishable; it has therefore beenomitted from all the calculations.

DISCUSSION

The primary aim of the work presented in this paper wasthe determination of the lipid and fatty acid composition ofchloroplast envelope membrane preparations isolated fromVicia faba L. In order to determine whether a quantitative or aqualitative diversity or both exist between lipids of differentmembranes in the same organelle, and between different or-ganelles of the same cell, the composition of the chloroplastenvelope membranes has been compared with those of othercellular membranes isolated from the same leaves.The establishment of the identity of the envelope fraction

has been described under "Materials and Methods" and else-where (25). When examined in the electron microscope, thepellet of the membrane fraction (20,000g) showed some grada-tion (26). Near the top, the pellet consisted of an almost puremembrane fraction of single and double membrane-boundvesicles 0.1 to 5.0 ,um in diameter. The larger double mem-

brane-bound vesicles are almost certainly double chloroplastenvelopes in which both outer and inner envelope membranesare still present. The smaller double and the single membranevesicles would appear to be vesiculated pieces of outer or

inner envelope membranes or both. The appearance of theenvelope fraction is illustrated in Figure 2. Toward the bottomof the pellet some mitochondria and a few granal membranestacks were also present. On a protein basis the major con-taminants are mitochondria (about 4%) and lamellar mem-branes (about 10%). All the lipid analyses for the chloroplastenvelope fraction have therefore been corrected for lamellarcontamination and the values in the tables gives the composi-tion of a membrane fraction in which roughly 95% of themembranes are from the chloroplast envelope. The envelopefraction is composed of closed vesicles up to 5 /-m in diameter,at least 20% of which are limited by a single membrane. Be-cause it is impossible to identify these single membranes as

either inner or outer envelope membranes, the analyses mustbe regarded as an average for the two envelope membranes.

Table IV. Fatty Acid Compositionz of the Moniogalactosyl and Digalactosyl Diglycerides and of the Total Phospholipid + Sulfolipid,Extracted from Subcellular Fractions Isolated from Leaves of Vicia faba L.

The fatty acid composition of lipids separated by one-dimensional TLC using toluene ethyl acetate-ethanol (2:1:1 v/v/v). The analy-ses have been corrected for lamellar contamination as described in the text, and are the means of duplicates of the number of analysesshown in parentheses.

Subcellular Fraction Lipid 16:0 16:1 16: lA3t 18:0 18:1 18:2 18:3 UFA/SFA

moles %

Chloroplast envelope (3) MGDG 10.3 2.0 0.0 5.6 10.9 8.9 62.1 5.2Chloroplast lamellae (3) 1.1 0.0 0.2 0.5 1.4 2.9 93.8 61.5Mitochondrial fraction (1) 11.8 1.8 0.0 12.0 25.1 3.3 45.9 3.2Microsomal fraction (1) 5.5 2.0 0.0 3.3 6.6 17.3 64.9 10.4Chloroplast envelope (3) DGDG 8.9 0.7 0.0 3.4 2.5 3.4 80.9 7.1Chloroplast lamellae (3) 4.2 0.4 0.0 1.8 2.2 1.7 89.7 15.7Mitochondrial fraction (1) 16.4 0.9 0.0 5.8 5.8 14.3 56.8 3.5Microsomal fraction (1) 14.2 1.5 0.0 6.0 6.0 16.3 56.0 3.9Chloroplast envelope (3) PL + SL 18.1 3.9 0.0 3.0 6.0 24.7 44.2 3.7Chloroplast lamellae (3) 18.5 0.0 7.3 2.9 4.2 14.2 53.0 3.7Mitochondrial fraction (1) 21.9 0.8 0.0 3.8 6.9 40.3 25.9 2.9Microsomal fraction (1) 23.1 1.8 0.0 4.8 8.7 33.7 27.6 2.6

Plant Physiol. Vol. 53, 1974 499



MACKENDER AND LEECH

I.i..

B..i

It .I

,Si:.4:I.l+

r

I..0...

N

..

1

.:.

.kl:~~~~~~~~~t.,~~~~~~~~~~~~~~~~~~0

N.-

*I.*s ... ...

.'-

.'1

Cs*

--9 -.

*KN,.he

*x ^:

_!f<---vA2;titi a/ ,C

.

f ,q. XT os

*:;. z

4,. d

:... Byq ;ss fto

A

.C

FIG. 2. Electron micrograph of the chloroplast envelope membrane fraction (X 75,000). The pellet was fixed in 2.5%o glutaraldehyde in 50 mmorthophosphate buffer, pH 7.3, and postfixed in %l,, osmium tetroxide (30 min each at 0 C). Dehydration was through acetone and embedding inEpon. Sections were poststained in lead citrate (23). (Photo published with permission of W. W. Thomson.)

500 Plant Physiol. Vol. 53, 1974

.-e,

s. i:M.. q.

Nill.

-" "": ....

.,p;'t




The composition of these envelope preparations (extractedimmediately after isolation) are essentially the same as thosereported previously (27) for freeze-dried samples, except thatthe freshly isolated preparations have lower GL/PL ratiosand more PC. Phospholipid breakdown (indicated by thepresence in the freeze dried samples of phosphatidic acid) isnot apparent in the extracts of the freshly isolated material.The fact that the fatty acid composition of the total lipid calcu-lated from the GL/PL and MGDG/DGDG ratios (Table I)and also the fatty acid compositions of these individual lipids(Table IV) agree with the experimentally determined fatty acidcomposition indicates that both the lipid and fatty acid analysesare substantially correct (Table V).The major differences in the lipid composition of chloroplast

envelopes and chloroplast lamellae undoubtedly reflect theirfunctional differences. In the envelope there is an increase inthe proportion of phospholipid, a decrease in the proportionof MGDG relative to DGDG, and a very much higher PC con-tent.

The fatty acid composition of the envelope lipids are quali-tatively similar to those of the lamellar lipids, with the excep-tion of the trans-A3-hexadecenoic acid which is absent, eventhough PG is present in the membrane. Linolenic acid is themajor fatty acid of all the envelope lipids and all the lamellarlipids, but proportionally much less is present in the envelope,particularly in the MGDG. The high proportion of 18:3 in thephospholipid fraction may well be a reflection of its high PCcontent, inasmuch as chloroplastic PC in spinach has beenshown to contain much more 18:3 than PC isolated from amicrosomal fraction of the same leaves (6). The significance ofmembrane fatty acid composition in membrane function is stilluncertain. However, a high proportion of unsaturated fattyacids in a membrane has been shown to confer stability on themembrane (12). Unsaturated fatty acids also have a lowertransition temperature than the equivalent chain length satu-rated fatty acid. Thus a membrane like the chloroplast en-velope, with a high proportion of unsaturated fatty acids,may be expected to be stable but more mobile over a widertemperature range.

Whether the individual membranes of the envelope havedifferent lipid compositions is still to be determined. It hasbeen suggested from studies with the electron microscope thatthe lamellar membranes develop from invaginations of theinner envelope membrane (21). Unless there are large differ-ences in the composition of the two envelope membranes anysuch developmental hypothesis must now also account for theapparent differences in the lipid composition of the envelopeand lamellar membranes.The analyses of the lamellar membranes and of the mito-

Table V. Comparison of the Calculated a,td ExperimenitallyDeterminied Fatty Acid Compositioni of the Total LipidExtracted from Subcellular Fractionts Isolated from

Leaves of Vicia faba L.The moles% 16:0, 18:2, and 18:3 in the total lipid (calculated)

were calculated from the data in Tables II and IV.

16:0 18:2 18:3Subcellular Fraction

Caic. Expt. Calc. Expt. Calc. Expt.

moles %0Chloroplast envelope 12.0 13.3 13.2 11.5 60.9 63.3Chloroplast lamellae 3.3 5.9 3.6 5.2 88.4 82.9Mitochondrial 20.2 19.3 32.9 31.1 32.0 37.3Microsomal 19.5 19.8 28.6 27.6 36.2 40.7

Table VI. Comparison of some of the Lipid And Fatty AcidCharacteristics of Subcellular Membranies Isolated from

Leaves of Vicia faba L.A plus indicates similarity between fractions.

Lipid Characteristic

~~~oC 0~~~~~~~~~~Subcellular Fraction iA0 A A

Microsomal00action+ 0 00 a - a..-

-.4 < A A <

Chloroplast lamellae + + + +Chloroplast envelope + + + + + + + +Mitochondrial + + + +

fractionMicrosomal fraction + + + +

chondrial and the microsomal fractions are similar to those re-ported for these membranes by other workers (1, 3, 6, 7, 20, 24,30, 35). There are minor differences which are to be expectedwhen comparing analyses from different species. A comparisonof the lipid and fatty acid composition of these subcellularfractions shows that the chloroplast envelope has character-istics in common with every other subcellular fraction (TableVI). The relatively high galactolipid content, the high propor-tion of PG (% of PL), the absence of PE, and the relativelyhigh proportion of 18:3 in the total lipid are all characteristicsshared with the lamellae. The low MGDG/DGDG ratio, thehigh PC content, the DGDG as the major galactolipid, theabsence of the trans-A3-hexadecenoic acid, and the high propor-tion of palmitate in the total lipid are characteristics sharedwith the mitochondrial and microsomal fractions. This appar-ently intermediate character of the envelope lipid compositionraises some interesting questions about the site(s) of synthesisof the envelope membrane components.The comparisons made here between the lipid composition

of the different cellular membranes do little to advance ourunderstanding of any relationship which exists between thecomposition and function of a membrane. However, the appar-ent correlation which exists in a number of other cell mem-branes (8, 31, 34), between a high phospholipid content andthe presence of specific transport processes is also found in thechloroplast envelope; the inner envelope membrane is knownto have specific transport properties (16, 17).

Several points of interest about cellular membrane composi-tion have emerged from these analyses. The lipid compositionof all the membranes are qualitatively similar with the excep-tion of PE. Because this was found in both chloroplast mem-brane fractions in only very small quantities (<0.5%) andcould be accounted for as mitochondrial contamination, weconclude that it is confined to the extrachloroplastic mem-branes. The MGDG, the DGDG, and PG were present in allthe membrane fractions analysed and therefore are not spe-cifically chloroplast lipids. Furthermore the proportion of PG(as a percentage of the total lipid) is greater in the envelope(8.9%), the microsomes (9.6%), and the mitochondria (7.8%)than it is in the lamellae (5.5%) (Table II). All the lipids in allthe membranes contain the same fatty acids with the exceptionof the trans-A3-hexadecenoic acid which is only found in thelamellae.We conclude from our analyses that the major differences in

acyl lipid composition between leaf cell membranes with widelydiffering function are quantitative rather than qualitative.

Acknowledgmenzt-We are most grateful to W. W. Thomson for permission topublish Figure 2.

501Plant Physiol. Vol. 53, 1974



502 MACKENDEF

LITERATURE CITED

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2. ARNON, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenol-oxidase in Beta vulgaris. Plant Physiol. 24: 1-15.

3. BAILE, J. B., S. F. YANG, AN-D A. A. BENSON. 1966. Lipids in plant mito-chondria. Fed. Proc. 25: 405.

4. BARTLETT, G. R. 1959. Phosphorus assay in column chromatography. J. Biol.Chem. 234: 466-468.

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The Galactolipid, Phospholipid, Fatty Acid Composition ... · sulfolipid from the total phospholipid, using either acetone-acetic acid-water or toluene-ethyl acetate-ethanol, and