Lipid Composition and Fatty Acid Synthesis in the Blue Green Alga Anabaena doliolum

HiOl'hem, Physiol. Pflanzen 174, G85-G90 (1979)

Lipid Composition and Fatty Acid Synthesis in the Blue Green Alga Anabaena doliolum P. S. SUKHIJA, MANl\WHAN SINGH and I. S. BHATIA Dep;utment of Biochemistry, Punjab Agricultural University, Ludhiana, India Key Term Index: lipids, fatty acid synthesis; Anabaena dalialum.

Summary The nature of polar lipids and fatty acid biosynthesis was studied in Anabaena dolialurn, a blue green alga. The polar lipid fraction consisted of MGDG, DGDG, PG, SG, pigments and sulpholipids. In addition, one unidentified lipid was also present. CoA, ATP, G-G-P and F-1,6 di-P stimulated the fatty acid synthesis in the algal cells. Exogenous supply of ADP, Pi, NADH was without such a stimulatory effect. Light was an essential pre-requisite for fatty acid biosynthesis. Supply of citrate and isoeitrate to the algal cells reduced the fatty acid and lipid biosynthesis.

Introduction Anabaena dalialurn is a blue green alga which possesses a photo autotrophic mode of growth. This alga is filamentous, colony forming, heterocystous and can fix atmospheric nitrogen. Much work has been done on fatty acid biosynthesis in higher plants (YAMADA and NAKAMURA 1975). However, no report was available on the aspect in this particular alga. The present investigation was, therefore, undertaken to study incorporation of acetate into fatty acids and lipids of the isolated algal cells.

Material and Methods The alga, Anabaena doliolulI! Bhardwaja (iVostacaceae), was maintained in ALLuN and AI\NON (1955) combined nitrogen free medium with 1.5 per cent agar, when required. The illumination was provided by 100 Wand 60 W incandescent bulbs and two 40 W fluorescent tubes fitted at a distance of 35 cm. The average light intensity at the surface of culture flasks varied between 1,500 and 2,000 lux. The cultures were illuminated daily for 20 h followed by a 4 h exposure to diffused day light. The material of the requisite age of the alga was harvested by centrifuging the alga from the medium at 2,000 g for 10 min. To make the organism free from the culture medium, w;lshings with distilled water were given. Abbreviations: DTT, dithiothreitol; CoA, coenzyme A; ATP, adenosine triphosphate; ADP, adenosine disphosphate; F -l,G-di-P, fructose-1,G diphosphate; G-G- P, glucose-G-phosphate, Tricine (N-tris-hydroxymethyl) methyl glycine; cpm, counts per minute; NADH, nicotinamide adenine dinucleotide (redueed); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); MGDG, monogalactosyl diglyceride; DGDG, digalactosyldiglyceride; PG, phosphatidyl glycerol; SG, sterol glycoside.

686

P. S. SUKHIJA, :\1. SINGH and 1. S. B HATU

Methods Totallipids were extracted by the method of FOLCH et a1. (1957). Polar and non-polar lipid fractions were obtained by the solvent partition methods of NICHOLS (1964). Separation and identification {If various polar lipid components on thin layer chromatography (T. L. C.) plates of the algal oil was done as reported by SUKHIJA and Bn.uLI. (1970). The chlorophyll content was estimated by the method of ARNON (1949). Isolation of intact Anabaena cells and acetale incorporation studies The intact cells were isolated by the method of GLl.QUlNLI. et a1. (1974). The isolated cells were examined for their structural integrity under phase contrast microscope (Olympus: Model P. A. BC-25). About 90 % of the cells were intact. Acetate ineorporation in 7 days old alga was studied by the method described by KAXNANGARA and STUMPF (19i:!). The norm,tl reaction mixture contained the following compounds in ,umoles: Tricine 100, sorbitol 100, NaHCO a 60, K 2 HP04 100, DTT 2.5, ATP 4.0, CoA 1.0, 0.08% Triton 100. The mixture also contained sodium I-HC-acetate O.:.! ml (2,uCi) and Anabaena cells containing 100,ug chlorophyll. The final volume was adjusted to:.! ml with a pH value of 8. This incubation media served as a contrc! mixtnre for fatty acid synthesis. To this mixture some additional cofactors/compounds were added in order to see their effeet on fatty acid synthesis. The reaction was allowed to run at 30 DC for 60 min under a light of 1.500-2.000 lux intensity. The reaction was stopped by adding 0.1 ml of 10 N H 2 S0 4 and 6 ml chloroform: methanol (1: 1, v/v) mixture, and dried over ,tnhydrous sodium sulphate. After flushing with the nitrogen, the lipid extract was evaporated to dryness on a water b:'.th maintained at 60 cC. The dried sample was taken up in 5 ml of chloroform and 1/5 of it was put in a glass seintillation vial. After evaporation of the solvent under an infra red lamp, the radioactivity was measured in u. liquid seintillation spectrometer (Paekard, U.S.A. ~rodel 3330) with Bray's solution (ilK\, Y 1960). The remaining chloroform fraction containing lipids was saponified by methylated KOH, acidifed and the liberated fatty acids were recovered by the method described by RIL\TU et al. (1978). Radioactiyit~· of fatty acids was measured by scintillation spectrometer as already deseribed.

Results and Discussion

Lipid composition The alga found was to contain 11.3 to 17.8 per cent total lipids (Table 1). This amount increased with the age of the culture and attained a maximum value on the 7 days old culture and thereafter showed a regular decline. At all stages of growth, the polar lipids formed the major fraction of the total lipids and the amount of non-polar lipids remained almost constant.

Identification of lipids The polar lipid fraction of the total lipids of Anabaena doliolum were resolved by one dimensional thin layer chromatography. The basic solvent system gave a reasonably good separation with slight overlapping among some of the spots. Seven polar components were found to be present. The results are reported in Table 2. Spot 4 and 7 gave a positive test with lX-naphthol (JACIN and MISHKIN 1965) and bezidine periodate (CIFONELLI and SMITH 1954) reagent and were thus identified as digalactosyl diglyceride and monogalactosyl diglyceride, respectively. The presence of these lipids has already been reported in Anabaen(t variabilis and Anacystis nidulans (NICHOLS et al. 1965 and NICHOLS and "WOOD 1968). Positive test with benzidine periodate reagent as suggested

Lipid Composition and Fatty Acid Synthesis in Allabaena doliolulII

687

Table 1. Lipid composition of Anabaena doliolum during growth. (g/100 g dry wt) Age (days)

Total lipids

Polar lipids

Non-polar lipids

Fatty ,tcids

4 7 10 14

12.5 17.8 13.9 11.3

10.2 15.G 11.3 8.3

2.3 2.2 2.1 2.0

4.4 7.1 5.3 4.0

Table 2. Teutalirc identification of t"arious polar lipid components of Anabaena doliolum ~

.:

co

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;:

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;

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:..

Z 0

P-

if.2

1 2 3 4 5 G 7

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0.24 0.37 0.01 0.07 0.70 0.89 0.9G

:;. ~

c..

.:

:..ao

.....,

.S ',....,

~ .: ~

+(Y) +(Y) +(Y) +(Y) +(Y) +(Y) +(Y)

+ Positive re,tction - ~egatiye reaetion

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Tentative identification

0

=<

LC,

+(W) +(B)

+(Pn)

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+(Pn) Y = Pu = Pu-r = W = = B

+(W)

Unidentified? S nlpholipid Phosphatidylglyeerol Digalactosyl diglyceride Sterol glycoside Pigments i\Ionogalactosyl diglyceride

yellow colonr ag,tinst white baekground Purple colour against white baekground Purple red colour ag,tinst white background White colour against blue background Blue colour against white baekground

by LEPAGE (1964) indicated that spot 2 was a sulpholipid. Spot 3 gave a positive test with modified spray reagent for phospholipids. On the bais of reported RF values (NICHOLS and WOOD 1968 and NICHOLS et al. 1965), it was identified as phosphatidyl glycerol. Spot 5 gave a positive Lieberman Burchard reaction (HEFTMAN et al. 1966) an was thus tentatively identified as some sterol containing lipid. On the bais of Rf values, it was specified as a sterol glycoside. Spot 1 did not give positive test with any spray reagent and thus remains unidentified. Spot 6, by its physical appearance, was of the pigments. This spot gave a long yellow streak which may be due to the carotenoids and other pigments.

Acetate incorporation studies in Anabaena The data presented in Tables 3 and 4 are the average of two determinantions. The results given in Table 3 show that about % of the radioactivity was incorporatee in the total lipid and only % into fatty acids {'yen though the fatty acids are the major

688

P.

S. SUKHlJA,

M.

SINGH

and 1.

S. BHATIA

Table 3. Cofactorsrequirements for acetate incorporation in lipids and fatty acids of intact cells of Anabaena doliolurn Additional cofactors: ADP, Pi (5ttmol each), F-l, 5-di-P, NADH, NADPH, G-6-P (2.5 ttmol ea.ch) Light intensity = 1,500-2,000 lux Re
Control -ATP -CoA -ATP + ADP -ATP + ADP + Pi +DADH +NADPH +G-6-P +NADH + NADPH + G-6-P F-l,6-di-P Control (dark)

cpm [l-14 el-acetate incorporated/l00 ttg Total lipids

Fatty acids

3,480 2,110 1,880 2,740 2,970 3,390 3,410 4,350 4,420 3,730 430

1,540 710 580 760 870 1,490 1,420 1,850 1,910 1,650 Not determined

components of the true fat. The incorporation in the fatty acid fraction was somewhat lower than expected, possibly, because a good part of the label might htwe gone into the pigments which arc present in large amounts in the total lipids. The removal of CoA and ATP from the control resulted in a decrease of the acetate incorporation into the fatty acid fraction and hence total lipids. This points to the ability of these compounds to penetrate the cell membranes. Evidence is available which indicates that these compounds can penetrate chloroplast membrane of the higher plants (HABER and SANTARIUS 1965). Addition of ADP or ADP + Pi did not equal the effect of added ATP presumably because ADP cannot enter the cell membrane. It has been reported (HEBER 1974) that plant chloroplast in analogy to mitochondri,1 contain a specific translocation which facilitates exchange transfer of external ATP with other adenylates. Addition of G-6-P which probably serves as a source of NADPH and acetyl CoA as reported by MUDD and McMANUS (1965) in chloroplasts, increased the fatty acid biosynthesis. F-1,6-di-P enhanced the incorporation of acetate probably through its conversion to G-6-P. Both ~ADH and XADPH are known to be reducing agents in fatty acid biosynthesis in higher plants (YAMADA and NAKAMURA 1975) and, expectedly, their supply to the cell should enhance fatty acid biosyntheis. The absence of any stimulatory effect of exogenous NADH or NADPH has been attributed to their inability to enter the intact cells as has also been observed by YAMADA and NAKA::IIURA (1975). The stimulation for fatty acid synthesis sho'wn by a mixture of G-6-P + HADP+ + NADPH is primarily due to G-6-P and not added NADPH or NADP+. The observed reduced rate of acetate incorporation in the dark is in keeping with the well established vital role of light in the biosYlIthesis of fatty acids in the chloroplasts (BROOKS and STUMPF 1965). The data on the effect of isocitrate and citrate are shown in Table 4. Both these compounds inhibited fatty acid biosynthesis but the effect was more marked with iso-

i'. Lipid Composition and Fatty Acid Synthesis in Anabaena doliolwil

689

Table 4. Effect of citrate and isocitTlite Ott acetate incorporation rate itt lipids and fatty acids ot Anabaena doliolum cells. Component added {mM)

cpm of [1-14C]-acetate inorporatedj 100,ug Chl/h Lipids

F,ttty acids

Isocitrate 0.01 0.02 0.03 D.Of> 0.10 .control

3,120 2,490 2,060 1,970 1,730 3,680

0.01 0.02 D.03 0.05 0.10 .control

3,240 2,810 3,180 3,380 3,040 3,580

1,680 1,0GO 1,020 890 760 1,860

Citrate 1,740 1,320 1,660 1,G50 1,590 1,860

-citrate. The per cent inhibition increased with the increased concentration. These results support the findings of REINSTEIN and STmIPF (1969) in wheat germ. The compounds perhaps exerted their inhibitory effect by feed back mechanism as suggested by REINSTEIN and STUMPF (1969). References ALLEX, M. B., and ARl\ON, D. 1.: Studies on nitrogen fixing blue green algae. 1. Growth and nitrogen fixation by Altabael/a cylindrica. Plant Physiology. 30, 366-372 (1955). ARNON, D. 1.: Copper enzymes in isolated ehloroplasts. Polyp enol oxidase in Beta vulgaris. Plant Physiol. 24, 1-5 (1949). BIIATU, 1. S., AIILJA, K. L., and SGKHlU, P. S.: Changes in the aetivity of acetyl CoA earboxyhlse in germinating and ripening sunflower seeds. Physiol. Plant. 44, 141-144 (1978). BRAY, G. A.: A simple efficient liquid scintillator for counting aqueous solutions in a liqpuid scintillation eounter. Anal. Biochem. 1, 279-286 (1960). J~ROOKS, J. D., and STl:MPF, P. K.: A soluble fatty acid synthesis system from lettllec chloroplasts. Biochem. Biophys. Acta. 98, 21i3-216 (1965) . .cIFONELLI, J., and S.HTII, F.: Detection of carbohydmtes and other carbohydmte compounds by p,tper chromatogmphy. Anal. Chem. 26, 1132-1134 (1904). }'OLCII, J., LEES, :.\1., and SLOAXE-STAl\LEY, G. H.: A simple method for isolation and purific,ltion of total lipids from animal tissues. J. BioI. Chem. 226, 497-509 (1967). GIAQrIXT_\, R. T .. SEDIOX, B. R., BEltING, C. L., and DILLEY, R. A.: Inhibition of coupling fador activity of chloroplast membntne by diazonium compounds. J. BioI. Chem. 249, 2873-2878 (1974). HABER, U. V., and SAXTARlUS, K. A.: Compartmentation ,Ind reduction of pyridine nue/cotides in relation to photosynthesis. Biochem. Biophys. Acta. 109, 390-408 (1966). I-[EBER, U.: l\Iet,lbolite exchange between chloroplasts and cytoplasm. Ann. Rev. Plant Physiol. 25, 393-421 (1974).

690 P. S. SUKHlJA, .M. SINGH and I. S. BHATIA, Lipid Composition and Fatty Acid Synthesis etc. HEFHIAN, E., Ko, S. '1'., and BENNET, R. D.: Response of steroids to sulphuric acid in thin layer chromatography. J. Chromatog. 21, 490-494 (1966). HEINSTEIi'i, P. F., and STUMPF, P. K.: Fat metabolism in higher plants. XXXVII. Properties of wheat germ acetyl CoA carboxylase. J. BioI. Chem. 244, 0374-5381 (1969). JACIN, H., and MISHKIN: Separation of carbohydrates on borate impregnated silica gel G plate. J. Chromatog. 18, 170-173 (1965). KANNANGARA, C. G., and STUMPF, P. K.: Fat metabolism in higher plants. The biosynthesis of polyunsaturated fatty acids by isolated spinach chloroplasts. Arch. Biochem. Biophys. 148, 414-424 (1972). LEPAGE, M.: The separation and identificatiun of plant phospholipids and glycolipids by two dimen- , sional TLC. J. Chromatog. 13, 99-103 (1964). MUDD, J. B., and Mc}IANUS, T. T.: Relationship of the synthesis of lipid and \vater soluble compounds by chlorplasts. Phmt PhysioL 40, 340-B44 (1960). ~ICHOLS, B. \Y.: The separation of lipids by thin layer chromatography. Lab. Pntc. 13, 299-305 (196-1). - HARRIS, R. Y., and J.UIES, ..\. '1'.: The lipid metabolism of blue green algae. Bioehem. Biophys. Res. Conllnun. 20, 256-262 (1960). - and WOOD, B. J. D.: Xe\\" glycolipids specific to X2 ·fixing blue green algae. Nature 217,767-768 (1968). SlCKHIJA, P. S., and BHATIA, I. S.: Lipids of t,lramira (Eruca sath-a) and linseed (Limoll usitah"ssi1111(111) Tentative indentification of various lipid components and their fatty acid make· up. Indian J. Hiochem. i, 271-7-1 (1970). YAMADA, M., and XAK.UIURA, Y.: Fatty acid synthesis by spinach chloroplasts II. The pathway from PGA to fatty arids. Plant & Cell PhysioL 16, 151-162 (1975).

Reeeired •.\ugust 15, 1978, ill rel·ised form January 29, 1979. Author's address: P. S. SCKIIIJA, Department of Biochemistry, Punjab Agricultnral University Ludhiana, India.