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

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

Plant Physiol. (1974) 53, 496-502

The Galactolipid, Phospholipid, and Fatty Composition of the 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. LEECH Department of , University of York, Heslington, York, England

ABSTRACT these determinations together with analyses of other mem- branes (chloroplast lamellae, mitochondria, and microsomes) The galactolipid, phospholipid, and composition of isolated from the same leaves of Vicia faba L. chloroplast envelope membrane fractions isolated from leaves of Vicia faba L. has been determined. The major in this fraction are: monogalactosyldiglyceride, 29%; digalactosyldi- MATERIALS AND METHODS , 32%; , 30%; and phosphatidyl- Subcellular Fractions. These were isolated from leaves of 9%. The composition of the chloroplast envelope 19- to 21-day-old plants of Vicia faba L. var. Dwarf White membranes is qualitatively eimilar to that of the lamellar mem- Fan, grown as described previously, and placed in the dark branes isolated from the same plastids, but the proportion of for 66 to 72 hr before harvesting (26). each lipid present is very different. The total galactolipid to The leaves (30-40 g) were homogenized at full speed for total phospholipid ratio was 1.6 :1 in the envelope and 11.1 : 1 3 and then 5 sec in an Atomix blender with 180 ml of ice-cold in the lamellae. The monogalactosyldiglyceride-digalactosyl- medium A: 53 mm Na2HPO,/KH2PO4 buffer containing 500 ratio was 0.9 : 1 in the envelope and 2.4 : 1 in the mM D(-)sorbitol, 10 mM MgC12 and 10 mm EDTA-Na2, pH lamellae. Both membranes lack . 7.3. The resulting homogenate was filtered through four layers Linolenic acid is the major fatty acid in the envelope lipids of cotton organdy and eight layers of 25 ,um mesh nylon. representing 63 % of the total fatty acid, whereas in the lamellae Chloroplasts and chloroplast envelope membranes (pellet P4) it represents 83%. The same fatty are present in both the were isolated from this filtrate by a modification of previous envelope and lamellar lipids except the trans-A'-hexadecenoic methods (26) as shown in Figure 1. Chloroplasts were prepared acid, which is confined to the lamellar lipids, particularly the by sedimentation at 3,000g for 15 sec, and the resuspended pel- phospholipid fraction. let was then layered onto a 10-ml (4 cm) band of 400 mm buff- A quantitative comparison of the lipid and fatty acid composi- ered sucrose and centrifuged at 5OOg for 12 sec. This second tions of the envelope with those of mitochondrial and micro- centrifugation reduced the mitochondrial and bacterial con- somal fractions indicates that the chloroplast envelope has a tamination by 60% and removed the majority of broken plas- composition intermediate between that of the chloroplast lamel- tids (25). lae and these extrachloroplastic membranes. Isolation of Chloroplast Envelopes. For the isolation of the chloroplast envelopes, the chloroplasts were ruptured osmoti- cally by incubation in buffered 10 mM MgC12. Each washed chloroplast 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 were homogenized in a TenBroeck ground-glass homogenizer by Although the ultrastructure of the chloroplast envelope has raising and lowering the plunger rapidly three times. The ho- been examined in leaf cells (11, 19, 37), and its permeability mogenized suspension was diluted with 20 ml of incubation properties studied both in the whole leaf (13, 14, 32) and re- medium and centrifuged at 3,000g for 10 min. Floating mate- cently in isolated chloroplasts (5, 15-18, 22, 29, 36, 38), noth- rial was removed and the supernatant decanted and recentri- ing is known of its chemical composition. Chemical character- fuged at 20,000g for 30 min. The pellet from this spin con- ization of the envelope membranes must be carried out on tained the chloroplast envelope membranes. The crude envelope isolated membranes, and methods for the isolation (26) and fraction was washed once by resuspending in 20 ml of medium purification (27) of chloroplast envelope membranes have al- E (medium A minus sorbitol) and recentrifuging at 20,000g for ready been published. These methods yield membrane frac- 15 min. tions in which 85 to 90% of the membranes originate from the The suspensions were observed by phase contrast microscopy chloroplast double envelope. The lipid composition of these at each stage during the isolation procedure. Initially, the in- membrane fractions has now been investigated, and the fatty tact chloroplasts were opaque and shining in appearance, but acid composition of the total lipid and of some of the individual after incubation the suspensions contained many chloroplasts lipids was determined. The present paper reports details of with grayish halos. In each of these chloroplasts the lamellar system was eccentric within the grayish balloon-like structure. The grayish structures were pinched off free of lamellae and ' This work was supported by the Science Research Council, These ghosts were seen to be Grant B/SR/8692 and by an Imperial Chemical Industries fellow- resembled ghosts. ship to R.O.M. derived from the chloroplast envelopes, and increased greatly 2Present address: Department of Botany, The Queen's Univer- in number after homogenization. The later stages in the proce- sity, Belfast, N. Ireland. dure were designed for the collection of the ghosts free from 496 Plant Physiol. Vol. 53, 1974 CHLOROPLAST ENVELOPE LIPIDS 497

Filtered leaf homogenate (in Medium A)

3,OOOg for 15 sec.

Pellet P1 (crude chloroplasts) Supernatant Sl 500g for 12 min. 3,000g for 5 min. thro' 4O0mM sucrose. 4' Supernatant S2 Pellet P2 (intact chloroplasts) 20,000g f}r 30 min. Incubated in Medium E homogenized 4' 3,000g for 10 min. Pellet SP2 Supernatant S3 3,000g for 5 min. l00,OOOg for lhr. lw Pellet Supernatant ,\ 500g for 5 min -0 20,000g for 30 min. ;~~~~4 Supernatant S4 (repeated I timegs) 20,000g for 30 min.

Pellet Pellet SP4 20,000g for 15 min. lOO,OOOg for lhr.

4, Pellet P4 Pellet SP5 ZVEIOPEi '"MITOCHONDRIAL , MICTIOMAL FRACTION" FRAOTI0Nt 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 contained The mitochondrial and microsomal fractions have been identi- numerous ghosts of varying sizes. fied on the basis of the centrifugal forces required to sediment Isolation of Chloroplast Lamellae. The chloroplast lamellae them. The mitochondrial fraction was previously shown to fraction (pellet P3) was prepared by washing the fraction by have a high succinic dehydrogenase activity (25). resuspending it in 30 ml of medium E, homogenizing gently in After isolation each cellular fraction was resuspended in a TenBroeck ground glass homogenizer and centrifuging at medium E. All manipulations were carried out between 0 5OOg for 5 min. and 4 C. Isolation of Mitochondrial and Microsomal Fractions. The mitochondrial and microsomal fractions were isolated from the LIPID ANALYSIS supernatant S1 following recentrifugation at 3,000g for 5 min to remove most of the remaining chloroplasts and lamellar Extraction. Lipids were extracted in dim light by shaking the membranes. The details of the fractionation are shown in fractions with approximately 50 times their volume of chloro- Figure 1. The supernatant S2 was decanted and recentrifuged form-methanol (2:1 v/v). The extracts were washed overnight at 20,000g for 30 min to give pellet SP2 and supernatant S3. at 4 C in the dark with 20% of their volume of 100 mM NaCl The pellet SP2 was resuspended in 30 ml of medium E and re- (9) under N2. After washing, the (lower) chloroform phase of centrifuged at 3,000g for 5 min; the resulting pellet was dis- the extract was removed and evaporated to dryness at 30 C carded and the supernatant was recentrifuged at 20,000g for under vacuum. The dried extract was taken up in chloroform 30 min. The final pellet was designated the mitochondrial frac- and re-evaporated twice more. The extracts were stored dry tion. The supernatant S. was centrifuged at 100,OOOg for 1 hr under N2 at -25 C in the dark. to give pellet SP4. This pellet was washed once by resuspending Chzromatography. Individual galactolipids and phospholipids it in 30 ml medium E and recentrifuging at 100,000g for 1 hr. were separated from the bulk lipid extract by two-dimensional The pellet obtained was designated the microsomal fraction. TLC on H or HR grade silica gel (Merck) using chloroform- 498 MACKENDER AND LEECH Plant Physiol. Vol. 53, 1974 methanol-water (65:25:4, v/v/v) in the first dimension and Table I. Galactolipid and Phospholipid Composition of acetone-acetic acid-water (100:2:1, v/v/v) in the second di- Subcellular Fractions Isolated from Leaves of mension (10). The total phospholipid fraction was separated Vicia faba L. from the neutral lipids and the galactolipids by one-dimensional The analyses of the chloroplast envelope, mitochondrial, and TLC using either acetone-acetic acid-water (100:2:1, v/v/v) or microsomal fractions have all been corrected for lamellar con- toluene-ethyl acetate-ethanol (2:1:1, v/v/v) as solvent. Lipids tamination as described in the text, and are the mean of the num- for quantitation were located with iodine vapor which was ber of determinations shown in parentheses. allowed to evaporate before analysis. Lipids for fatty acid analysis were located by spraying with 0.2% 2',7'-dichloro- Phospholipids fluorescein in 50% ethanol and visualized under UV light. Analyzed Lipid Estimation. Galactolipids were estimated as galactose Subcellular GL/PLi MGDG/ Phos- Fraction DGDG Phospha- Phospha- Phatdy- (in the presence of absorbent) by the phenol-sulfuric acid tidyl- tidyl- ethanodl- method of Roughan and Batt (33). Galactose in 2% (w/v) glycerol etamnel phenol in water was used as the standard. Phospholipids were estimated (in the presence of absorbent) miolar ratios mioles % as Pi by the method of Bartlett (4) following their digestion Chloropast 1.60 0.290 0.90 z 0.16 77 22 trace2 envelope (6) by 72% perchloric acid. KH2PO4 was used as the standard. Chloroplast 11.10 A 0.49 2.40 i 0.14 34 66 trace2 Areas of absorbent equivalent in size to those of the lipid spots lamellae (5) were used as blanks; this was found to be particularly necessary Mitochondrial (3) 0.30 i 0.03 0.80 z 0.04 57 10 33 for the accurate determination of the galactolipids. Microsomal (2) 0.40 4z 0.08 0.40 4z 0.04 67 13 20 Fatty acid analysis. Fatty acid methyl esters of the total 1 PL = phosphate remaining at the origin after one dimensional TLC of bulk lipid and of individual lipids were prepared with boron tri- lipid extract in either acetone-acetic acid-water (100:2:1 v/v/v) or toluene-ethyl- fluoride in methanol (28). It was impossible to separate the acetate-ethanol (2: 1: 1 v,'v/v). sulfolipid from the total phospholipid, using either acetone- 2 <0.6%/7o. acetic acid-water or toluene-ethyl acetate-ethanol, and it was therefore part of the spot remaining at the origin which was Table II. Lipid Composition of Subcellular Fractionis Isolated used in these analyses. The methyl esters were purified before from Leaves of Vicia faba L. gas-liquid chromatography by TLC using petroleum ether The data in this table have been recalculated from those in (60-80 b.p.)-diethyl ether, 4:1 (v/v) as solvent. The methyl Table I. esters which were visualized with dichlorofluorescein were identified by reference to a standard of methyl oleate. They Lipid were eluted with diethyl ether and analyzed in a Hewlett Pack- Subcellular Fraction ard S750 on columns of 10% polyethylene glycoladipate on MGDG DGDG PC PG PE high performance chromosorb (80-100 mesh) at 200 C. The moles/1000 moles of lipid analyzed carrier gas was argon with a flow rate of 50 ml/ min. Chloroplast envelope 291 324 296 89 0 Chlorophyll. Chlorophyll was determined in 80% acetone Chloroplast lamellae 654 262 28 55 0 extracts containing small quantities of chloroform (<2% by Mitochondrial 102 128 435 78 257 volume) by the method of Arnon (2). Microsomal 82 204 476 96 142 RESULTS represent 61% of the envelope lipids, they constitute 92% of All the subcellular fractions contained fragments of chloro- the lamellar lipid. There is also relatively more DGDG3 in the plast lamellae. The analyses (except for the total fatty acid envelope than in the lamellae, and this is reflected in the composition of the total lipid extracts) have therefore been MGDG/DGDG ratio which is 0.9:1 in the envelope and corrected according to the following equation: 2.4: 1 in the lamellae. = - Phosphatidylcholine and are the two L y cx major phospholipids in both membranes, but whereas PC is where L = the actual quantity of lipid in the fraction after the major phospholipid in the envelope (77%), PG is the major correction (,tmoles); y = the total quantity of lipid in the frac- phospholipid in the lamellae (66%). There were also three mi- tion before correction (jumoles); c = the Chl content of the nor phospholipids in both fractions. Phosphatidylethanolamine fraction (,tg); x = the concentration of the lipid in the lamellar which was the only one of these to be positively identified, membranes (,umoles/,ug Chl). was only present in trace amounts (<0.6%) in both fractions. The assumption has been made that all the Chl in the cell Another of these spots, the same size as PE ran in the position is confined to the lamellar membranes. Any Chl present in of . other cell fractions will therefore be due to the presence of The fatty acid composition of the total lipid extract and of lamellar fragments. The presence of the associated lamellar the MGDG, the DGDG, and the PL plus SL of all four sub- lipids in the total lipid extracts of each cell fraction is corrected cellular fractions are shown in Tables III and IV. The fatty for by using the correction equation. acids of the envelope and lamellar lipids are qualitatively simi- The results of the analyses of chloroplast envelope and lar except that the trans-A5-hexadecenoic acid was only found lamellar membranes are shown in Table I. For comparison in the lamellar lipids. Quantitatively, however, the fatty acid with the composition of other membranes, the data in Table II give the number of moles of each lipid per 1000 moles of lipid analyzed. Qualitatively the lipids 'Abbreviations: DGDG: digalactosyldiglyceride; GL: galactolip- acyl of both types of ids (MGDG + DGDG); MGDG: monogalactosyldiglyceride; PL: chloroplast membrane are identical but the proportions in phospholipids; SL: sulfolipid; PC: phosphatidylcholine; PG: phos- which they are present are very different. The galactolipids are phatidylglycerol; PI: phosphatidylinositol; PE: phosphatidylethanol- the major lipids in both membrane fractions, but whereas they amine. Plant Physiol. Vol. 53, 1974 CHLOROPLAST ENVELOPE LIPIDS 499 compositions of the two membranes are rather different. Lino- (Tables III and IV). There was no trace of the trans-A3-hex- lenic acid (18:3) is the major fatty acid in the lipids of both adecenoic acid in any of the lipids of either extrachloroplastic membranes, but there is always a much lower proportion in membrane fraction. It is possible that there were traces of the envelope than in the lamellae; this is reflected in the lower arachidic acid present, but with the analytical system used it unsaturated to saturated fatty acid ratios in the envelope. was not always readily distinguishable; it has therefore been The composition of the chloroplast envelope membrane also omitted from all the calculations. differs from the extrachloroplastic membranes in which the phospholipids are the major class of lipid. For comparison with DISCUSSION the envelope lipids, only PC, PG and PE were measured indi- vidually in the mitochondrial and microsomal fractions. There The primary aim of the work presented in this paper was were two other minor phospholipids present in both fractions, the determination of the lipid and fatty acid composition of one of which ran in the position of phosphatidylinositol. In chloroplast envelope membrane preparations isolated from these fractions the phospholipids are respectively 77% and Vicia faba L. In order to determine whether a quantitative or a 71% of the total lipid. PC is the major single lipid in both qualitative diversity or both exist between lipids of different fractions accounting for 43.5% and 47.6%, respectively, of membranes in the same organelle, and between different or- the total lipid. PE is the other major phospholipid in both ganelles of the same cell, the composition of the chloroplast these fractions. PG is also present in them in small but signifi- envelope membranes has been compared with those of other cant proportions (Tables I and II). The digalactosyl diglyceride cellular membranes isolated from the same leaves. is the major GL in these fractions. This is reflected in the The establishment of the identity of the envelope fraction MGDG/DGDG ratio which is 0.8:1 in the mitochondrial has been described under "Materials and Methods" and else- fraction and 0.4:1 in the microsomal fraction. Linolenic acid where (25). When examined in the electron microscope, the is the major fatty acid in the total lipid, the MGDG, and the pellet of the membrane fraction (20,000g) showed some grada- DGDG, but not the PL + SL of both fractions; it is, however, tion (26). Near the top, the pellet consisted of an almost pure proportionally very much less in these lipids than in those of membrane fraction of single and double membrane-bound the chloroplast membranes. This decrease is reflected in low vesicles 0.1 to 5.0 ,um in diameter. The larger double mem- ratios between the unsaturated and the saturated fatty brane-bound vesicles are almost certainly double chloroplast acids. envelopes in which both outer and inner envelope Linolenic acid (18:2), the major fatty acid of the PL SL membranes + are still present. The smaller double and the single membrane fraction, and palmitate are the other major fatty acids present vesicles would appear to be vesiculated pieces of outer or inner envelope membranes or both. The appearance of the Table III. Fatty Acid Compositioni of Total Lipid Extracted envelope fraction is illustrated in Figure 2. Toward the bottom from Subcellular Fractions Isolated from Vicia faba L. of the pellet some mitochondria and a few granal membrane The results are the means of duplicates of the number of stacks were also present. On a basis the major con- analyses shown in parentheses. taminants are mitochondria (about 4%) and lamellar mem- branes (about 10%). All the lipid analyses for the chloroplast Subcellular Fraction 16:0 16:1 16:lA3t 18:0 18:1 18:2 18:3 TFA! envelope fraction have therefore been corrected for lamellar SFAi contamination and the values in the tables gives the composi- moles %G tion of a membrane fraction in which roughly 95% of the Chloroplast envelope (3) 13.3 1.8 0.0 4.31 5.8 11.5|63.3 4.7:1 membranes are from the chloroplast envelope. The envelope Chloroplast lamellae (3) 5.9 0.0 1.2 1.6 3.2 5.282.9 12.3:1 fraction is composed of closed vesicles up to 5 /-m in diameter, Mitochondrial fraction (1) 19.3 1.66 0.0 3.9 5 .831.1 37. 3 3.3:1 at least 20% of which are limited by a single membrane. Be- Microsomal fraction (1) 19.8 1.1 0.0 4.8 6 .1 27.640.7 3.1:1 cause it is impossible to identify these single membranes as 5UFA = unsaturated fatty acids = 16:1; 16:LA3t; 18:1; 18:2; 18:3. SFA = either inner or outer envelope membranes, the analyses must saturated fatty acids = 16:0; 18:0. be regarded as an average for the two envelope membranes.

Table IV. Fatty Acid Compositionz of the Moniogalactosyl and Digalactosyl 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 analyses shown 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 5.2 Chloroplast lamellae (3) 1.1 0.0 0.2 0.5 1.4 2.9 93.8 61.5 Mitochondrial fraction (1) 11.8 1.8 0.0 12.0 25.1 3.3 45.9 3.2 Microsomal fraction (1) 5.5 2.0 0.0 3.3 6.6 17.3 64.9 10.4 Chloroplast envelope (3) DGDG 8.9 0.7 0.0 3.4 2.5 3.4 80.9 7.1 Chloroplast lamellae (3) 4.2 0.4 0.0 1.8 2.2 1.7 89.7 15.7 Mitochondrial fraction (1) 16.4 0.9 0.0 5.8 5.8 14.3 56.8 3.5 Microsomal fraction (1) 14.2 1.5 0.0 6.0 6.0 16.3 56.0 3.9 Chloroplast envelope (3) PL + SL 18.1 3.9 0.0 3.0 6.0 24.7 44.2 3.7 Chloroplast lamellae (3) 18.5 0.0 7.3 2.9 4.2 14.2 53.0 3.7 Mitochondrial fraction (1) 21.9 0.8 0.0 3.8 6.9 40.3 25.9 2.9 Microsomal fraction (1) 23.1 1.8 0.0 4.8 8.7 33.7 27.6 2.6 500 MACKENDER AND LEECH Plant Physiol. Vol. 53, 1974

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FIG. 2. Electron micrograph of the chloroplast envelope membrane fraction (X 75,000). The pellet was fixed in 2.5%o glutaraldehyde in 50 mm orthophosphate buffer, pH 7.3, and postfixed in %l,, osmium tetroxide2;titi(30 min eachaat 0 C). Dehydration was through acetone and embedding in Epon. Sections were poststained in lead...citrate (23). Byq(Photo published;sswith permissionf of W. W. Thomson.) Plant Physiol. Vol. 53, 1974 CHLOROPLAST ENVELOPE LIPIDS 501 The composition of these envelope preparations (extracted Table VI. Comparison of some of the Lipid And Fatty Acid immediately after isolation) are essentially the same as those Characteristics of Subcellular Membranies Isolated from reported previously (27) for freeze-dried samples, except that Leaves of Vicia faba L. the freshly isolated preparations have lower GL/PL ratios A plus indicates similarity between fractions. and more PC. Phospholipid breakdown (indicated by the presence in the freeze dried samples of ) is Lipid Characteristic not apparent in the extracts of the freshly isolated material. The fact that the fatty acid composition of the total lipid calcu- ~~~oC 0~~~~~~~~~~ lated from the GL/PL and MGDG/DGDG ratios (Table I) Subcellular Fraction iA0 A A and also the fatty acid compositions of these individual lipids 0 - a - a.. (Table IV) agree with the experimentally determined fatty acid Microsomal00action+ 0 0 composition indicates that both the lipid and fatty acid analyses .4- < A A < are substantially correct (Table V). Chloroplast lamellae + + + + The major differences in the lipid composition of chloroplast Chloroplast envelope + + + + + + + + envelopes and chloroplast lamellae undoubtedly reflect their Mitochondrial + + + + functional differences. In the envelope there is an increase in fraction the proportion of phospholipid, a decrease in the proportion Microsomal fraction + + + + of MGDG relative to DGDG, and a very much higher PC con- tent. chondrial and the microsomal fractions are similar to those re- The fatty acid composition of the envelope lipids are quali- ported for these membranes by other workers (1, 3, 6, 7, 20, 24, tatively similar to those of the lamellar lipids, with the excep- 30, 35). There are minor differences which are to be expected tion of the trans-A3-hexadecenoic acid which is absent, even when comparing analyses from different species. A comparison though PG is present in the membrane. Linolenic acid is the of the lipid and fatty acid composition of these subcellular major fatty acid of all the envelope lipids and all the lamellar fractions shows that the chloroplast envelope has character- lipids, but proportionally much less is present in the envelope, istics in common with every other subcellular fraction (Table particularly in the MGDG. The high proportion of 18:3 in the VI). The relatively high galactolipid content, the high propor- phospholipid fraction may well be a reflection of its high PC tion of PG (% of PL), the absence of PE, and the relatively content, inasmuch as chloroplastic PC in spinach has been high proportion of 18:3 in the total lipid are all characteristics shown to contain much more 18:3 than PC isolated from a shared with the lamellae. The low MGDG/DGDG ratio, the microsomal fraction of the same leaves (6). The significance of high PC content, the DGDG as the major galactolipid, the membrane fatty acid composition in membrane function is still absence of the trans-A3-hexadecenoic acid, and the high propor- uncertain. However, a high proportion of unsaturated fatty tion of palmitate in the total lipid are characteristics shared acids in a membrane has been shown to confer stability on the with the mitochondrial and microsomal fractions. This appar- membrane (12). Unsaturated fatty acids also have a lower ently intermediate character of the envelope lipid composition transition temperature than the equivalent chain length satu- raises some interesting questions about the site(s) of synthesis rated fatty acid. Thus a membrane like the chloroplast en- of the envelope membrane components. velope, with a high proportion of unsaturated fatty acids, The comparisons made here between the lipid composition may be expected to be stable but more mobile over a wider of the different cellular membranes do little to advance our temperature range. understanding of any relationship which exists between the Whether the individual membranes of the envelope have composition and function of a membrane. However, the appar- different lipid compositions is still to be determined. It has ent correlation which exists in a number of other cell mem- been suggested from studies with the electron microscope that branes (8, 31, 34), between a high phospholipid content and the lamellar membranes develop from invaginations of the the presence of specific transport processes is also found in the inner envelope membrane (21). Unless there are large differ- chloroplast envelope; the inner envelope membrane is known ences in the composition of the two envelope membranes any to have specific transport properties (16, 17). such developmental hypothesis must now also account for the Several points of interest about cellular membrane composi- apparent differences in the lipid composition of the envelope tion have emerged from these analyses. The lipid composition and lamellar membranes. of all the membranes are qualitatively similar with the excep- The analyses of the lamellar membranes and of the mito- tion of PE. Because this was found in both chloroplast mem- brane fractions in only very small quantities (<0.5%) and Table V. Comparison of the Calculated a,td Experimenitally could be accounted for as mitochondrial contamination, we Determinied Fatty Acid Compositioni of the Total Lipid conclude that it is confined to the extrachloroplastic mem- Extracted from Subcellular Fractionts Isolated from branes. The MGDG, the DGDG, and PG were present in all Leaves of Vicia faba L. the membrane fractions analysed and therefore are not spe- The moles% 16:0, 18:2, and 18:3 in the total lipid (calculated) cifically chloroplast lipids. Furthermore the proportion of PG were calculated from the data in Tables II and IV. (as a percentage of the total lipid) is greater in the envelope (8.9%), the microsomes (9.6%), and the mitochondria (7.8%) 16:0 18:2 18:3 than it is in the lamellae (5.5%) (Table II). All the lipids in all Subcellular Fraction the membranes contain the same fatty acids with the exception Caic. Expt. Calc. Expt. Calc. Expt. of the trans-A3-hexadecenoic acid which is only found in the lamellae. moles %0 We conclude from our analyses that the major differences in Chloroplast envelope 12.0 13.3 13.2 11.5 60.9 63.3 acyl lipid composition between leaf cell membranes with widely Chloroplast lamellae 3.3 5.9 3.6 5.2 88.4 82.9 differing function are quantitative rather than qualitative. Mitochondrial 20.2 19.3 32.9 31.1 32.0 37.3 Microsomal 19.5 19.8 28.6 27.6 36.2 40.7 Acknowledgmenzt-We are most grateful to W. W. Thomson for permission to publish Figure 2. 502 MACKENDEFPIR AND LEECH Plant Physiol. Vol. 53, 1974

LITERATURE CITED 20. KATES, M. 1970. Plant phospholipids and . Advan. Lipid Res. 8: 225-265. 1. ABDELKADER, A. B., A. M. CATESSON, P. MAZLIAK, A. THIBAUDIN, AND A. 21. KIRK, J. T. 0. AND R. A. E. TINNEY-BASSETT. 1967. The Plastids. W. H. TRE-MOLIERES. 1968. Renouvellement des lipides dans diverses fractions cellu- Freeman and Company, London. laires veg6tales. Bull. Soc. Frang. Physiol. Veget. 14: 323-349. 22. KRAUSE, G. H. 1971. Indirekter ATP-transport zwischen Chloroplasten und 2. ARNON, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenol- Zytoplasma wahrend der Photosynthese. Z. Pflanzenphysiol. 65: 13-23. oxidase in Beta vulgaris. Plant Physiol. 24: 1-15. 23. LEESE, B. M., R. M. LEECH, AND W. W. THOMSON. 1971. Isolation of plastids 3. BAILE, J. B., S. F. YANG, AN-D A. A. BENSON. 1966. Lipids in plant mito- from different regions of developing maize leaves. Second International chondria. Fed. Proc. 25: 405. Congress on Photosynthesis, Stresa. 2: 1485-1494. 4. BARTLETT, G. R. 1959. assay in column chromatography. J. Biol. 24. LURSSEN, K. 1970. Volumen, Trockengewicht und Zusammensetzung von Chem. 234: 466-468. Etioplasten und ihren Ergrunungsstadien. Z. Naturforsch. 25b: 1113-1119. 5. BASSHAM, J. A., M. KIRK, AND R. G. JENSEN. 1968. Photosynthesis by isolated 25. MACKENDER, R. 0. 1971. Studies on the isolation and composition of chloro. chloroplasts. I. Diffusion of labelled photosynthetic intermediates be- plast envelope membranes. Ph.D. thesis. York, England. tween isolated chloroplasts and suspending medium. Biochim. Biophys. 26. MACKENDER, R. 0. AND R. M. LEECH. 1970. Isolation of chloroplast envelop( Acta 153: 211-218. membranes. Nature 228: 1347-1349. 6. DEVOR, K. A. AND J. B. MUDD. 1971. Structural analysis of phosphatidyl- 27. NIACKENDER, R. 0. AND R. M. LEECH. 1971. The isolation and characterization choline of plant tissue. J. Lipid Res. 12: 396-402. of plastid envelope membranes. Second International Congress in Photo- 7. DOuCE, R., T. GUILLOT-SALOMON, C. LANCE, AND MN. SIGNOL. 1968. Etude synthesis, Stresa. 2: 1431-1440. comparee de la composition en phospholipides de mitochondries et de chloro- 28. MORRISON, W. R. AND L. Ml. SMITH. 1964. Preparation of fatty acid methyl- plastes isol6s de quelques tissus v6g6teux. Bull. Soc. Franc. Physiol. Veget. esters and dimethylacetals from lipids with boron trifluoride. J. Lipid Res. 14: 351-373. 5: 600-608. 8. EMMELOTT, P., C. J. Bos, E. L. BENEDETTI, AND P. H. RUMKE. 1964. Studies 29. NOBEL, P. S. AND C. T. WAN-G. 1970. permeability of pea chloro- on plasma membranes. 1. Chemical composition and enzyme content of plasts as measured by osmotically determined reflection coefficients. Biochim. plasma membranes isolated from rat liver. Biochim. Biophys. Acta 90: Biophys. Acta 211: 79-87. 126-145. 30. ONGTJN. A., W. W. THOMSON, AND J. B. MIUDD. 1968. Lipid composition of 9. FOLSCH. J., M. LEES, AND G. H. SLOANE STANLEY. 1957. A simple method chloroplasts isolated by aqueous and nonaqueous techniques. J. Lipid Res. for the isolation and purification of total lipids from animal tissues. 9: 409-415. J. Biol. Chem. 266: 497-509. 31. PARSoNS, D. F., G. R. WILLIAMS, W. THOMPSON, D. AND B. CHANCE. 10. GARDNER, H. W. 1968. Preparative isolation of and WILSON, monogalactosyl digalacto- 1967. Improvements in the procedure for purification of mitochondrial syl diglycerides by thin layer chromatography. J. Res. 9: Lipid 139-141. outer and inner membrane. Comparison of the outer membrane with smooth 11. GIBBS, S. P. 1962. The ultrastructure of the of Ultra- chloroplasts algae. J. . In: E. Quagliarello, S. Papa and E. C. Slatyer, eds., struct. Res. 7: 418-435. Mitochondrial Structure and 12. GUARNIERI, NI. AND R. MI. JOHNSON. The essential Compartmentation. P. Adriatica Editrice, 1970. fatty acids. Advan. Bari. pp. 29-73. Lipid Res. 8: 155-167. 13. HEBER, U. W. AND K. A. SANTARIUJS. 1965. Compartmentation and reduction 32. ROBERTS, G. R., A. J. KEYS, AND C. P. WHITTINGHAM. 1970. The transport of pyridine nucleotides in relation to photosynthesis. Biochim. Biophys. of photosynthetic products from chloroplasts of tobacco leaves. J. Exp. Bot. Acta 109: 390-408. 21: 683-692. 14. HEBER, U. WV. AND J. WILLENBRINK. 1964. Sites of synthesis and transport of 33. ROUGHAN, P. G. AN-D R. D. BATT. 1968. Quantitative analysis of sulpholipid photosynthetic products within the leaf cell. Biochim. Biophys. Acta 82: (sulphoquinovosyl diglyceride) and galactolipids (monogalactosyl and di- 313-324. galactosyl diglycerides) in plant tissue. Anal. Biochem. 22: 74-88. 15. HELDT, H. W. 1969. Adenine nucleotide translocation in spinach chloroplasts. 34. ROuSER, G., G. J. NELSON, S. FLEISCHER, AND G. SIMON. 1968. Lipid com- FEBS Lett. 5: 11-14. position of animal cell membranes, organelles and organs. In: D. Chapman, 16. HELDT, H. W. AND L. RAPLEY. 1970. Unspecific permeation and specific ed., Biological Membranes, Academic Press, New York. pp. 5-69. uptake of substances in spinach chloroplasts. FEBS Lett. 7: 139-142. 35. SCHWERTNER, H. A. AND J. B. BAILE. 1973. Lipid composition of plant mito- 17. HELDT, H. W. AND L. RAPLEY. 1970. Specific transport of inorganic phosphate, chondria and chloroplasts. J. Lipid Res. 14: 235-242. 3-phosphoglycerate, and dihydroxyacetone phosphate, and of dicarboxylates 36. STOCKING, C. R. AND S. LARSEN. 1969. T chloroplast-cyloplasmic shuttle and across the inner membrane of spinach chloroplasts. FEBS Lett. 10: 143-148. the reduction of extraplastid NAD. Biochem. Biophys. Res. Commun., 37: 18. HELDT, H. W. AND F. SAUER. 1971. The inner membrane of the chloroplast 278-282. envelope as the site of specific metabolite transport. Biochim. Biophys. Acta 37. WEIER, T. E. AND W. W. THOmSON. 1962. Mlembranes of mesophyll cells of 234: 83-91. Nicotiana rustica and Phaseolus vulgaris with particular reference to the 19. HESLOP-HARRISON, J. 1963. Structure and morphogenesis of lamellar systems chloroplast. Amer. J. Bot. 49: 807-820. in grana-containing chloroplasts. 1. Membrane structure and lamellar 38. WERDEN, K. AND H. W. HELDT. 1972. Accumulation of bicarbonate in intact architecture. Planta 60: 243-260. chloroplasts following a pH gradient. Biochim. Biophys. Acta 283: 430-441.

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