LULA L. HILENSKI, PATRICIA L. WALNE, AND FRED SNYDER ..... In: B. C. Parker and R. M. Brown. Jr.. eds.. Contributions in Phycology. Allen. Press, Inc.
Plant Physiol. (1976) 57, 645-646
Aliphatic Chains of Esterified Lipids in Isolated Eyespots of Euglena gracilis var. bacillaris1 Received for publication October 17, 1975 and in revised form January 1, 1976
LULA L. HILENSKI, PATRICIA L. WALNE, AND FRED SNYDER Department of Botany, University of Tennessee, Knoxville, Tennessee 37916; Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37830 ABSTRACT
Isolted eyespot granules of Euglkna gracilis Klebs var. bacillaris Pnngsheim contained approximately 6% lipids (based on protein). Separation of the lipid extracts by thin layer chromatography revealed four major fractions: wax esters, triacylglycerols, free fatty acids, and phospholipids. Methanolysis of each fraction yielded between 27 and 29 different fatty adds ranging from 12:0 to 22:6. Acetates of the fatty alcohols of the wax fraction consisted of 11:0 to 18:0 carbon chains, with 14:0 being the major component; unsaturated alcohols were not detected.
Although analyses of lipids from euglenoid organisms have been reported (3-7, 9, 10), none has dealt exclusively with those of the eyespot. With the development of a technique for large scale isolation of eyespot granules from Euglena (1), the chemical properties of the eyespot can be investigated more readily. We report here one aspect of the biochemical study of eyespots-the characterization of glycerolipids and wax esters. MATERIALS AND METHODS Isolation of Eyespots and Extraction of Lipids. Axenic cultures of Euglena were grown and harvested and the eyespots isolated according to the methods of Bartlett et al. (1). Lipids were extracted from eyespot preparations by a procedure modified from Bligh and Dyer (2), in which the methanol contained 2% (v/v) acetic acid. Solvents were removed under vacuum using a rotary evaporator, and the lipid weight was determined on a Cahn G2 electrobalance. Chenical and Chromatographic Methods. Protein and phosphorous contents were determined by the procedures of Lowry et al. (8) and Rouser et al. (11), respectively. Neutral lipid and phospholipid standards for TLC were purchased from Supelco, Inc. The standards of the methyl esters of fatty acids were obtained from Applied Science Laboratories and of the fatty alcohols from Supelco, Inc. Neutral lipids were resolved on 250-,um layers of Silica Gel G in a solvent system of hexane-ether-acetic acid (80:20:1, v/v). Phospholipids were separated on layers of Silica Gel HR developed in chloroform-methanol-ammonium hydroxide (60:35:5, ' This investigation was supported in part by National Science Foundation Grant BMS 69-00229 A 04, in part by a University of Tennessee Faculty Research Grant, and in part by senior Fulbright-Hays and A.A.U.W. Postdoctoral Research Fellowships to P.L.W., and in part by the Energy Research and Development Administration. Contribution No. 459 from the Department of Botany, University of Tennessee, Knoxville. 645
v/v). Lipids were visualized by (a) spraying the plate with concentrated H2SO4, then charring for 20 min in an oven at 180 C or (b) spraying the plate with 0.1 % (w/v) dichlorofluorescein in ethanol, then viewing with UV light. We obtained lipid fractions by preparative TLC on 500-,um layers of Silica Gel G in the hexane-ether-acetic acid system described above. Components were recovered by scraping the silica gel directly into test tubes to which reagents for methylation or acetylation were added. Methyl ester derivatives of fatty acids were obtained using methanol containing 2% (v/v) concentrated H2SO4 at 100 C for 60 min. After the addition of water, methyl esters were extracted three times with hexane-ether (1: 1, v/v). Fatty alcohols, formed after methanolysis of the wax fraction, were acetylated with acetic anhydride in pyridine (4:1, v/v) for 60 min at 100 C. Water was added to the sample, and the acetates of the fatty alcohols were extracted three times using hexane-ether (2:1, v/v). Methyl esters of fatty acids were separated according to degree of saturation by chromatography on Silica Gel G layers impregnated with 12.5% (w/w) silver nitrate with development in a solvent system of hexane-ether (90:10, v/v). Components were recovered by scraping the silica gel into sintered glass funnels and eluting with 20% (v/v) methanol in ether. After the filtrate was added to hexane and washed with water, the hexaneether was evaporated, and methyl esters were dissolved in carbon disulfide for use in GLC. Acetates of the fatty alcohols were isolated on 500-,um Silica Gel G layers in the hexane-etheracetic acid system described above. Methyl esters of the fatty acids and acetates of the fatty alcohols were resolved on a Victoreen 4000 series gas-liquid chromatograph on columns (1.83 m x 3.18 mm) of 10% EGSSX coated on Gas-Chrom P, 100/120 mesh. Fatty acid methyl esters were analyzed at either 155 C or 185 C or by using temperature programming from 140 to 200 C (2 degrees/min); acetates of the fatty alcohols were separated at 175 C. Identification of the components was based on comparison of the retention times with known standards. RESULTS AND DISCUSSION The weight of lipids in the eyespot sample analyzed was 6.2% based on protein. Chromatographic separation of the extract in a neutral lipid solvent system revealed four major fractions. Fractions II and III had RF values identical to standards of triacylglycerols (RF = 0.54) and fatty acids (RF = 0.25), respectively. Methylation of fraction I yielded methyl esters of fatty acids and fatty alcohols (derived from wax esters with RF = 0.74), and a nonpolar unknown component corresponding to a hydrocarbon (RF = 0.77). The more polar fraction IV which remained at the origin contained phospholipids, but the amount of phospholipids varied in two different samples (13% and 3% of total lipids). Whether this variation reflects either differential banding of
646
HILENSKI, WALNE, AND SNYDER
Plant Physiol. Vol. 57, 1976
Table I. Percentages of Fatty Acids and Fatty Alcohols, Determined as Methyl Ester and Acetate Derivatives, in Eyespot Lipid Fractions Aliphatic1
Fraction2 II
III
IV
Methyl
Triacylglycerols Methyl
Fatty acids Methyl
Phospholipids Methyl
esters
esters
esters
esters
1.24
I Moieties
Wax esters
Acetate 11:0
0.35
12:0
4.95
2.43
1.74
1.31
13:0
20.90
10.33
4.82
3.89
1.49
14:0
34.65
17.27
13.66
10.39
7.47
15:0
15.82
2.43
2.52
2.51
3.19
16:0
15.50
9.12
9.67
12.88
16.45
16:1
2.34
4.84
3.18
4.36
16:2
0.63 0.70 0.24
0.23
0.10
0.77
0.34
0.86
1.49
0.45
0.86
1.55
6.88
3.10
5.20
3.11
18:1
4.05
8.92
10.91
9.81
18:2
1.91
2.39
2.85
3.27
18:3
3.16
1.70
20:2
2.48
6.21
5.10 4.93
4.20
20:3
0.91
1.42
1.21
3.36
20:4
8.86
10.51
8.41
8.83
22:2 or 20:5
8.93
9.44
7.20
5.45
2.00
3.50
5.10
17:0
17:1 18:0
1.57
22:4
3.81
24:1
3.92
2.39
24:2 or 22:5
4.96
8.02
7.17
6.68
22:6
3.89
6.38
6.20
6.51
1 Data were based on standards and 2 The
semi-logarithmic plots of retention times.
lipid fractions were isolated on Silia Gel
G in
solvent system of hexane-
ether-acetic acid (80:20:1, v/v). A blank in the table indicates that the components were not found in that
sample.
phospholipids in the sucrose gradient used in the isolation procedure or differential phospholipase activity in stored samples is under investigation. The phospholipids present in the eyespot sample may be components of membranes that surround eyespot granules (12, 13). Methyl ester derivatives of fatty acids from fractions I to IV and acetates of alcohols from fraction I are given in Table I. Korn (7) reported over 50 different fatty acids from light-grown cells of Euglena gracilis Z, and he obtained 51 fatty acids by further fractionation of an initial group of 29 constituents that were detected by GLC. The 29 initial constituents reported by Korn were similar, upon superficial examination, to the 27 to 29 fatty acids that we have detected thus far in the lipids of Euglena eyespots. Our data indicate that 14:0 is the major fatty alcohol in the wax esters of eyespots; it is also the major component in waxes from whole cells of Euglena grown on ethanol (5) and in the dark (9), and in waxes from mitochondria of etiolated Euglena (6).
The fatty acid composition of the wax component from eyespots is more complex than that reported previously (5, 9) for the wax esters obtained from extracts of whole cells of Euglena. In the latter, only fatty acids ranging from 12:0 to 18:0 were found, whereas we found the range to be from 12:0 to 22:6. In summary, our investigation revealed that eyespot granules contain wax esters, triacylglycerols, free fatty acids, and phospholipids containing a variety of fatty acids ranging from 12:0 to 22:6. Saturated fatty alcohols from 11:0 to 18:0, with a major component of 14:0, are present in the wax ester fraction.
Acknowledgments -We thank M. L. Blank for help with gas-liquid chromatography; A. Moehl for help with phospholipid analyses; E. A. Cress. P. S. Pagni. and N. Stephens for technical assistance.
LITERATURE CITED
BARTLETr, C. J., P. L. WALNE,
2.
3. 4.
5. 6.
7. 8.
9.
I(). 1 1.
12.
0. J. SCHWARZ, AND D. H. BROWN. 1972. Large scale isolation and purification of eyespot granules from Euglena gracilis var. bacillaris. Plant Physiol. 49: 881-885. BUGH, E. G. AND W. J. DYER. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-917. ERWIN, J. 1968. Lipid metabolism. In: D. W. Beutow, ed., The Biology of Euglena. Academic Press. New York. pp. 133-148. ERWIN. J. AND K. BLOCH. 1962. The x-linolenic acid contcnt of some photossnthetic microorganisms. Biochem. Biophys. Res. Commun. 9: 103-1018. GUEHLER. P. F.. L. PETERSON. H. M. TSCHIYA. AND R. M. DODSON. 1964. Composition of the wax formed by Euglena gracilis. Arch. Biochem. Biophys. 106: 294-298. KHAN. A. A. AND P. E. KOLATTUKUDY. 1973. Control of svnthesis and distribution of acyl moieties in etiolated Euglena gracilis. Biochemistry 12: 1939-1948. KORN, E. D. 1964. The fatty acids of Euglena gracilis. J. Lipid Res. 5: 352-362. LOWRY. 0. H.. N. J. ROSEBROUGH. A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. ROSENBERG. A. 1963. A comparison of lipid patterns in photosynthesizing cells of Euglena gracilis. Biochemistrs 2: 1148-1154. ROSENBERG, A. 1967. Euglena gracilis: a novel lipid energy reserve and arachidonic acid enrichment during fasting. Science 157: 1189-1191. ROUSER. G., A. N. SIAKOTOS. AND S. FLEISCHER. 1966. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorous analysis of spots. Lipids 1: 85-86. WALNE. P. L. 1971. Comparative ultrastructure of eyespots in selected euglenoid flagel-
lates. In: B. C. Parker and R. M. Brown. Jr.. eds.. Contributions
in
Phycology. Allen
Press, Inc., Lawrence, Kansas. pp. 107-120. 13. WALNE. P. L. AND H. J. ARNOIT. 1967. The comparative ultrastructure and possible functions of eyespots: Euglena granulata and Chlamsdomonas eugametos. Planta 77: 325353.
