Fatty Acids of Spinach Chloroplasts

Abstract

The photosynthetic apparatus of higher plants consists of discrete cytoplasmic organelles, the chloro-plasts, which contain proteins, lipids, pigments, water. and other substances. Relatively little is known, apparently , concerning the composition of the lipid fraction of the chloroplast. Menke (8), studying chloro-plasts of spinach, Spiniacia oleracea, found that essentially all the lipids of the leaf were located in the chloroplasts. The chloroplasts had an average lipid content of 30.9 % of the dry weight, while the cyto-plasm had a lipid content of only 0.5 %. For this reason, a number of earlier studies of the lipid content and composition of green leaves as a whole are pertinent to the present problem. Chibnall and Channon (3), studying the fatty acids of leaves of cabbage, Brassica oleracea, found that approximately 10.7 % of the fatty acid fraction consisted of saturated acids, and 89.3 % of unsat-urated acids. The saturated fraction was composed of 70 % palmitic acid and 30 % stearic acid. The un-saturated fraction included large quantities of linoleic and linolenic acids. Speer et al. (13) examined the lipids of spinach leaves, finding that 53 % of the fatty acids occurred in the free form, with 47 % as glycer-ides. The saturated acids consisted chiefly of pal-mitic and stearic acids. Oleic acid made up 26.3 %, linoleic acid 34.7 %, and linolenic acid 12.7 % of the unsaturated lipid fraction. The fatty acids of the forage grasses Dactylis glomiierata and Lolimn perenne were examined by Smith and Chibnall (12). Saturated acids composed 1 1 % of the total in Dactylis and 12 % in Lolium; the corresponding figures for oleic acid were 16 and 22 %, for linoleic acid 31 and 26 %, and for linolenic acid 42 and 40 %, respectively. According to Chibnall (2), cabbage leaves contain 17.5 % glycerides, 12.3 % waxes, 4.5 % sterols, and 18.4 % phospholipids. Comparable figures for cocks-foot grass, Dactylis gloinerata, were 38.0 % glycer-ides, 23.5 % waxes, 3.0 % sterols, and 1.5 % phos-pholipids. Menke (9) isolated chloroplasts from spinach leaves by centrifugation; he found that their lipid content was approximately 37 %. The ether-soluble fraction of the chloroplasts is a complex mixture of pigments, fatty acids, glycerides, phosphatides, and sterols. Bot (1) isolated grana from chloroplasts of Lathyrius odoratuts and Spinacia oleracea. These were shown to have an ether-soluble fraction, excluding chlorophyll, of 18 to 30 % of the dry weight in sweet pea and 26 to 32 % in spinach. Comar (4) determined the lipid content of spinach chloroplasts as 34%. Menke and Jacob (10) extracted isolated chloro-plasts of spinach with ether or ether-alcohol mixtures. The extracts were fractionated into a larger acetone-soluble fraction, making up about 80 % of the total, and containing the pigments, triglycerides and sterols, and an acetone-insoluble fraction including phospha-tides and waxes. The pigment-free portion of the acetone-soluble fraction consisted of 5.1 % glycerol, 42.2 % fatty acids, 49.0 % triglycerides, and 2.1 % sterols. Sisakyan and Smirnov (11) found that fatty acids composed 57.8 % of the dry weight of the chloroplasts of sugar beet, 48.8 % in sunflower, and 60.0 % in red clover. Isolated chloroplasts of sunflower incorporated acetate-C14 into higher fatty acids in either light or darkness, but bean chloroplasts did not. In mano-metric experiments, chloroplasts of bean oxidized pal-mitic, oleic, linoleic, and linolenic acids. Therefore, the chloroplasts are metabolically active in both fatty acid synthesis and oxidation. Zill and Harmon (14) investigated the chloroplast lipids of spinach by the technique of chromatography on silicic acid columns. Of the crude whole spinach extract 24 % was represented by two monoglycerides, which were not specifically identified. Further studies of chloroplast lipids by these workers are in progress (personal communication). Debuch (5) has recently analyzed the fatty acid composition of chloroplasts of Antirrhiniom najus, a plastid mutant of this species, and Allium porrumn by means of gas chromatography. C16 fatty acids composed 10 to 20 % and C18 fatty acids composed 80 to 90 % of the total. Linolenic acid was the component present in greatest amount (44.1-71.3 % of the total), linoleic acid (15.0-29.2 %) and palmitic acid (9.5-19.7 %) were the other principal constituents. The studies reported in this paper were designed to determine the fatty acid composition of chloroplasts obtained from spinach leaves. Materials & Methods Fresh spinach leaves obtained from a local distributor were cut into small pieces and homogenized