This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution4.0 International License.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschungin Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:Creative Commons Namensnennung 4.0 Lizenz.

CONTROL OF FATTY ACID AND LIPID BIOSYNTHESIS 5 3

Control of Fatty Acid and Lipid Biosynthesis in Euglena gracilis by Ammonia, Light and DCMU

P . POHL a n d H . WAGNER

Institut für Pharmazeutische Arzneimittellehre der Universität München

(Z. Naturforsch. 27 b, 53—61 [1972] ; received October 9, 1971, revised November 2, 1971)

When Euglena gracilis was grown under white (fluorescent) light in media containing high concentrations of ammonium chloride (more than 0.005%), the main lipids synthesized were mono-galactosyl diglyceride, digalactosyl diglyceride, phosphatidyl glycerol, sulfolipid and the all eis 47,10-16:2, 47,10,13-16:3, Zl4,7,10,13-16:4, A9,12-18:2 and A9,12,15-18:3 fatty acids. At low levels of ammonium (less than 0.002%) these compounds were produced only in small amounts, while neutral lipids, phosphatidyl choline, phosphatidyl ethanolamine and the 14:0, 16:0 and 16:1 fatty acids predominated.

When Euglena gracilis was grown in white light in the presence of dichlorophenyldimethylurea (DCMU) or in the dark, fatty acid and lipid biosyntheses followed the same pattern as in white light at low levels of ammonium. Similar results were obtained when nitrate served as the only nitrogen source in the light and in the dark.

The results indicate that in Euglena gracilis there are a light independent and a light and ammonium dependent pathway of fatty acid biosynthesis. Both pathways seem to be in association with specific lipids.

Green algae 1 - 8 and the phytoflagellates Euglena gracilis9-1' and Ochromonas danica18 synthesize predominantly unsaturated C16 and C18 fatty acids with 1—4 double bonds. Lesser amounts of un-saturated C20 and C2, fatty acids are found. Red and brown algae 3' 7 mainly produce unsaturated C18 and C20 fatty acids and only small quantities of C16 fatty acids with one or two double bonds. They lack the 16:3 and 16:4 fatty acids which are charac-teristic constituents of the green algae and phyto-flagellates. In an investigation19 carried out on various green algae we observed that the biosynthe-sis of these compounds depends on the ammonium or nitrate level in the nutrient medium. The low nitrogen content of seawater could be responsible for their absence in red and brown algae. The bio-synthesis of these unsaturated fatty acids is in-fluenced by l ight1 2 - 1 4 '1 6 . Since they are predomi-nantly found in galactolipids 13 '14 ' 16 '17 we were interested to study the influence of light and of the nitrogen content of the nutrient medium on both fatty acid and lipid biosynthesis. Euglena gracilis was chosen for our experiments because it can be

Requests for reprints should be sent to Dr. PETER POHL, Institut für Pharmazeutische Arzneimittellehre der Univer-sität, D-8000 München 2, Karl-Str. 29, Germany.

Abbreviations: GLC: gas liquid chromatography TLC: thin layer chromatography; DCMU: (3-(3,4-dichlorophenyl)-1,1-dimethyl urea; Card.: cardiolipin; DGDG: digalacto-syl diglyceride; MGDG: monogalactosyl diglyceride; PC: phosphatidyl choline; PE: phosphatidyl ethanolamine; P G : phosphatidyl glycerol; SL: sulfolipid; NL 1 (see Figs. 2 and 3) : neutral lipids (mainly wax esters and tri-glycerides) ; NL 2 : neutral lipids (mainly diglycerides).

grown in the dark and in the light and contains polyunsaturated C16 , C18 , C20 and C22 fatty acids 10.

Material and Methods Euglena gracilis Klebs (strain Z, leg. De Saedeler)

was obtained as an agar culture from the Pflanzenphy-siologisches Institut, Universität Göttingen, Nikolaus-berger Weg.

Nutrient medium The medium of CRAMER and MYERS 20 was used in a

modified form: K2HP04: 1,3 g/1; KH.,P04: 1 g/1; Na-citrate-11 H.,0: 0,8 g/1; MgS04-7H20: 0,2 g/1; CaCl,-2H,0: 0,02 g/1; Fe2(S04)3-6 H20: 3 mg/1; MnCL• 4 H20: 1,8 mg/1; Co(N03)2-6 H20: 1,3 mg/1; ZnS04 • 7 H.,0: 0,4 mg/1; Na2Mo04-2 H20: 0,2 mg/1; CuS04 • 5 H20: 0,02 mg/1; Thiamine-HCl: 0,05 mg/1; Vit B12: 0,0005 mg/1; NH4C1: 0,005 - 0,5 g/1.

The cultures were grown free of bacteria to the stationary phase of growth under continuous shaking at 25°. The optical density of the cell suspensions was measured with an Eppendorf spectrophotometer at 587 nm.

G r o w t h u n d e r w h i t e l i g h t Seven cultures of 500 ml were grown in erlenmeyer

flasks, the content of NH4C1 in the medium ranging from 0,0005 to 0,02 per cent. Time of growth: 15 — 22 days. Three cultures were grown with KN03 (0,001 - 0,03%) for 20 days.

G r o w t h in the d a r k Five cultures of 500 ml were grown for 10 days at

varying contents of NH4C1 (0,001-0,02%) and with adition of 0,1% of Na-acetate. Two cultures were grown in the dark, the medium containing 0,05% NH4C1 and 0,1% Na-acetate. After reaching the stationary phase of growth (10 days), one culture was subsequently

5 4 P. POHL AND H. WAGNER

grown in the light for 20 days with 0,35 g DCMU [3-(3,4-dichlorophenyl)-1,1-dimethyl urea] added to the medium, giving a 3 x l O _ 3 M concentration. The other culture was grown without DCMU in the light.

L i p i d e x t r a c t i o n After reaching the stationary phase of growth the

cells were centrifuged, freeze dried, weighed, finely ground with sea sand and extracted 4 times for 2 hours with 25 ml of chloroform —methanol (1 : 2, v/v) under nitrogen and continuous shaking at room temperature. The solution of total lipids was filtered and the main part of the solvent removed under a stream of nitrogen. The lipids were then transferred to a weighed test tube, concentrated to dryness under nitrogen and dried to a constant weight in a vacuum desiccator. Fol-lowing this the lipids were taken up in chloroform — methanol (1 : 2, v/v) to give a standard lipid solution of exactly 5 mg of total lipids per x ml of solvent.

Thin layer chromatography of lipids Solvent system: Acetone —benzene —water (91 —

30 — 8) 23. For good separation it was essential to use column-distilled solvents. The TLC-chambers contained paper saturated with solvents. Thin layer plates

A slurry of 4 g silicagel HF 254 Merck in 10 ml water was used for preparing one plate (12x20 cm2). Thickness: 0,25 mm. The plates were activated for 2 hours at 120° and thereafter stored under vacuum in a desiccator over P205 . Detection od lipids

The total lipids were applied to the TLC-plates in a line by means of a soft brush. After TLC the lipids were sprayed with an alkaline solution of Rhodamin 6G (equal volumes of 0,006% Rhodamin 6G in water and of 8% NaOH24) and visualized under UV-light, (366 nm).

Preparation of fatty acid methyl esters The fatty acid methyl esters were obtained as de-

scribed previously 19> 23 by transmethylation of the total lipids or of the TLC lipid zones with sodium methy-late. Gas chromatography

Model Packard, a) 20% Reoplex 400 on chromosorb WS (45 -60 mesh), column 3 m x 4 m m ; b) 15% EGSS-X on gaschrom P (100 —120 mesh), column 2mx5mm; column temperature: 190°; flow rate: 60ml argon/min.; amounts injected: ca. 0,01 mg.

Chlorophyll determinations These determinations were carried out according to

the method of ARNON 25.

Results The data of Fig. 1 show that Euglena grown in

white light at low contents of ammonium in the

nutrient medium (0,0005-0,002%) mainly synthe-sized the 14:0, 16:0 and 16:1 fatty acids and only low amounts of the 16:2, 16:3, 16:4, 18:2 and 18:3 compounds. With increasing contents of NH4C1 this fatty acid composition changed signifi-cantly. The 14:0, 16:0 and 16:1 as well as the 12:0, 18:0, 18:1, 20:4 and 20:5 compounds de-creased and the 16:2, 16:3, 16:4, 18:2 and 18:3 acids increased. In fact the absolute amounts of all the five latter compounds increased continuously. The decrease of the 16:2 and 18:3 fatty acids at NH4C1 levels higher than 0,005% (see Fig. 1) was only relative and due to the enormous increase of the 16:4 fatty acid.

These results are consistent with our former obser-vations 19 on various green algae. Only oleic acid showed some difference as its percentage remained rather constant and did not increase so significantly at low levels of ammonium as it did in the green algae.

Fig. 2 demonstrates that under white light at low concentrations of ammonium Euglena mainly pro-duced neutral lipids and phospholipids (PC, PE). MGDG, DGDG, PG and SL were present only in small amounts. In agreement with the fatty acid compositions shown in Fig. 1 there seemed to be a limit at about 0,003% NH4C1 in the medium. At concentrations below this limit Euglena produced only trace amounts of MGDG, DGDG, PG, SL and of polyunsaturated C16 and C18 fatty acids. These lipids and fatty acids, however, predominated at NH4C1 levels higher than 0.005 per cent.

The above increase of unsaturated fatty acid and of lipid biosynthesis at high levels of ammonium was paralleled by an increase of chlorophyll forma-tion from 0.02% of the dry weights (at 0.005% NH4C1) to about 2.9% (at 0.02% NH4C1) (Fig. 1).

Figs. 3/A and 3/B show the component fatty acids derived from aliquot amounts of the individual lipids of Euglena grown at 0.005% NH4C1. These data demonstrate that also in the light at sufficient-ly high levels of ammonium, the neutral lipids, PC and PE mainly contained the 14:0, 16:0, 18:0, 16:1, 18:1, 18:4, 20:4 and 20:5 fatty acids. The 16:2, 16:3, 16:4, 18:2 and 18:3 fatty acids were found predominantly in MGDG and DGDG.

In contrast to our observations wih the green algae, Euglena gracilis grown in white light used nitrate only poorly as a nitrogen source. The dry weight was about 20% of that obtained from cul-

CONTROL OF FATTY ACID AND LIPID BIOSYNTHESIS 5 5

O O.OOI Fig. 1. Influence of NH4® on fatty acid biosynthesis in Euglena gracilis (growth under the white light and at varying

contents of NH4C1 in the nutrient medium).

— Front

Card. S S 79

MCDG

— NL 1

— NL 2

— MGDG

— Card

— DGDG

— SL — PG — PE — PC + Lyso—PC — Start

A B C D

Fig. 2. TLC of total lipids (0.5 mg each) of Euglena gracilis (Growth under white light and at varying contents of NH4C1 in the nutrient medium). A) 0.001%; B) 0.002%; C) 0.005%;

D) 0.01% NH4C1. Fig. 3/A

5 6 P. POHL AND H. WAGNER

Fig- 3/B Fig. 3/A and 3 /B: Quantitative distribution of fatty acids in the individual lipids of Euglena gracilis. (Growth under white light at 0.005% NH4C1 in the nutrient medium.) GLC (Reo-plex) of aliquot amounts of the fatty acid methyl esters of the individual lipids. 1 = 12 :0 ; 2 = 14 :0 ; 3 = 1 5 : 0 ( ? ) ; 4 = 16 :0 ; 5 = 16 :1 ; 5a = trans3-16:l; 6 = 16 :2 ; 7 = 16 :3 ; 8 = 16:4 + 18 :0 ; 9 = 18 :1 ; 10 = 18:2 ; 11 - 18 :3 ; 12 =

18 :4 ; 13 = 20 :4 ; 14 = 20:5.

tures grown at equivalent levels of NH4C1. Even at high concentrations of KN03 the unsaturated C1G

am C18 acids were present only in small amounts (see Table 1). The 14:0 and 16:0 were the main fatty acids produced. The chlorophyll content of these cultures remained at a low level of about 0.05% of the dry weights. Neutral lipids and phospholipids (PC, PE) were the main lipids produced. MGDG, DGDG, PG and SL were present only in trace amounts. Similar results were obtained when Euglena was grown with KN03 in the dark (un-published) .

When grown in the dark (Table 2) at varying contents of NH4C1, all samples had a very similar fatty acid composition. It resembled the one at growth in white light at very low contents of am-monium: All samples primarily contained the saturated 14:0 and 16:0 fatty acids and only low quantities of unsaturated 16:2, 16:3, 16:4, 18:2 and 18:3 fatty acids. Small amounts of 18:4, 20:4, 20:5 and 22:6 fatty acids could be detected in all samples. No chlorophyll was produced in the dark.

Fig. 4 demonstrates the lipid composition of Euglena gracilis grown in the dark at varying levels

Fattv acids Content of KN0 3 in the medium (% of total fatty acids) 0.003% 0,01% 0,03%

1.6 3.1 16.2 27.4 29.7 23.2

9.7 8.0 1.3 1.0 tr tr 0.5 0.4 1.4 1.1 8.1 7.8 4.6 4.0 4.8 3.8 3.5 3.2 2.2 1.7

6.2 5.3

3.2 3.1 0.2 0.2 7.0 6.7

0.046 0.050

Table 1. Influence of nitrate on fatty acid biosynthesis in Euglena gracilis. (Growth under white light and at varying

contents of KN0 3 in the nutrient medium.)

of NH4C1. MGDG, DGDG, PG and SL were synthe-sized only in low amounts, whereas the neutral lipids, PC and PE predominated. No increase of MGDG, DGDG, PG and SL at high levels of NH4C1 could be observed.

— Front

— NL 1

— NL 2

— MGDG

— C a r d

— DGDG

— SL

— PG

— PE

— PC + Lyso—PC — Start

Fig. 4. TLC of total lipids (0.5 mg each) of Euglena gracilis. (Growth in the dark and at varying contents of NH4C1 in the nutrient medium). A) 0.001%; B) 0.005%; C) 0.05% NH4C1; D) Standard sample (lipid extract of a light grown green

alga).

12:0 1.3 14:0 15.9 16:0 28.6

47-16:1 10,5 47,10-16:2 1.2

47.10.13-16:3 tr 44,7,10,13-16:4 0.6

18:0 1.3 J9 -18 : l 8.9

Zl9,12-18:2 4.1 Zl 9.12.15-18:3 4.2

/J6.9,12,15-18:4 4.1 20:0 2.2

45,8,11,14} Zl8,11.14,\1\

zJ5,8,11,14,17-20:5 2.5 44,7,10,13,16,19-22:6 0.3

unknown 8.2 Chlorophyll content (% of dry weight) 0.048

CONTROL OF FATTY ACID AND LIPID BIOSYNTHESIS 5 7

Two cultures were grown in the dark with the medium containing 0.05% NH4C1 and 0.1% Na-ace-

Fatty acids * Content of NH4C1 in the medium y/o Ui lUliU

fatty acids) 0.001% 0.002% 0.005% 0.01% 0.02%

12:0 2.2 1.7 1.4 2.0 2.0 14:0 30.0 27.9 22.8 26.1 24.0 16:0 14.6 11.2 15.1 12.4 14.4 16:1 4.4 3.1 4.1 3.7 4.0 16:2 4.6 6.8 5.9 5.7 5.6 16:3 0.9 1.0 1.8 1.6 1.6 16:4 tr tr tr tr tr 18:0 1.3 1.7 5.0 1.6 1.8 18:1 3.1 3.7 7.3 6.5 7.6 18:2 1.3 2.0 1.8 2.8 2.0 18:3 1.9 0.7 0.9 0.8 0.8 18:4 3.6 4.8 3.2 3.7 4.0 20:0 tr tr tr tr tr 20:4 7.1 8.2 11.0 11.4 11.2 20:5 6.2 6.8 6.8 8.2 8.0 22:6 0.9 0.8 0.6 0.7 0.8 unknown 17.7 20.4 12.8 13.4 12.0

Table 2. Fatty acids in Euglena gracilis. (Growth in the dark at 0.1% Na-acetate and at varying contents of NH4C1 in

the nutrient medium.) * Structures see Table 1.

täte. After reaching the stationary phase of growth (10 days) one culture was grown in the light for 20 days under addition of DCMU which blocks photosynthesis from the moment of addition. The other one was grown in light without DCMU.

Fatty acids * Growth in Subsequent Subsequent (% of total the dark growth in light growth in light

fatty acids) ( + D C M U ) without DCMU

12:0 tr tr tr 14:0 9.2 42.8 3.4 16:0 27.6 32.7 7.5 16:1 9.1 10.2 4.9 16:2 1.1 tr 25.8 16:3 tr tr 4.4 16:4 tr tr 14.4 18:0 1.2 2.0 2.4 18:1 24.1 12.3 1.9 18:2 1.9 tr 8.5 18:3 1.8 tr 22.5 18:4 3.4 tr tr 20:0 tr tr tr 20:4 9.2 tr 1.3 20:5 1.5 tr 2.8 22:6 0.3 tr tr

Chlorophyll-content (% of — — 4.1 dry weight)

Table 3. Influence of DCMU on fatty acid biosynthesis in Euglena gracilis. (Growth in the dark to stationary phase (10 days), then in light (20 days) with DCMU added to the

medium.) * Structures see Table 1.

Table 3 shows the differences between these two cul-tures. The cells grown with DCMU remained colour-less. The dry weight did not change. The saturated 14:0 and 16:0 fatty acids increased from about 37% in the dark to 75 percent. No MGDG, and only trace amounts of DGDG, PG, SL could be detected by TLC. The main lipids were neutral lipids, PC and PE.

Discussion

The fatty acids and lipids mentioned in this paper have already been reported to occur in Euglena gra-cilis 9~17. The above data demonstrate that in Euglena gracilis ammonium as well as light are re-quired for the biosynthesis of four lipids (MGDG, DGDG, PG and SL) and of the unsaturated 16:2, 16:3, 16:4, 18:2 and 18:3 fatty acids. The strain of Euglena gracilis used for our experiments needed a minimum content of about 0.003% NH4C1 in the nutrient medium. The green algae previously in-vestigated by us 19 could be grown at about 0.0003% NH4C1. This indicates that Euglena needed approxi-mately ten times as much NH4C1 as the green algae. Euglena gracilis grew very poorly when nitrate was used as the only nitrogen source. Under these con-ditions it produced only low amounts of SL, PG, MGDG, DGDG and of polyunsaturated C16 and C18

fatty acids. This indicates that for the biosynthesis of these compounds ammonium and not nitrate is essential. In contrast to the green algae 19, the capa-city of Euglena gracilis to reduce nitrate to ammo-nium seemed to be minimal. It remains to be in-vestigated wThether this is common to all species of Euglena *.

No effect on fatty acid biosynthesis was observed when the content of MgS04 and K2HP04 in the medium was increased or when NaCl and MgClo was added. The addition of (NH4 )2HP04 , (NH4)2S04 or urea, however, had the same effect as NH4C1 (unpublished results). No experiments were carried out with RbCl and CsCl.

Particularly the 16:4 fatty acid seems to depend on sufficient levels of ammonium and on good light conditions. In our experiments this fatty acid was the most "unstable" of all unsaturated fatty acids

* This observation was confirmed with another strain of Euglena gracilis and with Euglena mutabilis, both ob-tained from the Culture Collection of Indiana, Blooming-ton, USA.

5 8 P. POHL AND H. W A G N E R

investigated, because its biosynthesis was diminished at first and most significantly when the conditions of growth became suboptimal, i. e. at low concentra-tions of ammonium in the medium and under un-sufficient light conditions, but also at high tem-peratures and unsufficient oxygen supply (un-published) .

Our results concerning the dependence of un-saturated fatty acid biosynthesis on white light are in agreement with those of ROSENBERG 1 2 - 1 4 who observed that in Euglena gracilis the content of un-saturated C16 and C18 fatty acids increased upon illumination; C O N S T A N T O P O U L O S and BLOCH 1 0

achieved the same effect by increasing the light intensity. Similar results have been obtained by NICHOLS 26 with Chlorella vulgaris.

Our experiments show that in Euglena gracilis the biosynthesis of these unsaturated fatty acids as well as of SL, PG, MGDG and DGDG are influenced by light. Furthermore, under optimal light condi-tions, they are controlled by the ammonium content of the medium and by DCMU. Because of the simi-

* NAGAI and BLOCH 28 have considered two more pathways of fatty acid biosynthesis in photoauxotroph Euglena gra-cilis: a) 10 : 0 - > 3-10 : 1 5-12 : 1 - > 7-14 : 1 9-16 : 1 — 1 1 - 1 8 : 1 and b) 12 : 0 - > 3-12 : 1 - > 5-14 : 1 —> 7-16 : 1 - > 9-18 : 1. There are at present, however, no

lar influence of the above three factors on lipid and fatty acid biosynthesis it was necessary to do light experiments which were similar to some of those done formerly n - 1 4 . In Scheme 1 a model of fatly acid biosynthesis in Euglena gracilis is proposed. This model tries to correlate our observations and will be the basis for more detailed studies.

The similar effect of the above three factors on unsaturated fatty acid biosynthesis suggests that the 16:2, 16:3, 16:4, 18:2 and 18:3 fatty acids form a special group or branch in fatty acid bio-synthesis. Therefore two pathways (A and B) of fatty acid biosynthesis in Euglena gracilis are pro-posed in Scheme 1. Both pathways seem to be as-sociated with specific lipids. Pathway A goes from acetate to the polyunsaturated C1 8 , C20 and C22

fatty acids. No 16:2, 16:3 and 16:4 fatty acids, but small quantities of 18:2 and 18:3 fatty acids seem to be synthesized via this pathway. Similar pathways have already been proposed 10 and descri-bed n ' 1 7 in Euglena gracilis and in Acantha-moeba 27. This pathway seems to be independent of

(MGDG DGDG)

reports about the lipids associated with these pathways. As for this reason it is difficult to say whether they are part of pathway A or B they have been ommited in the above Schcme.

5 -74 . , ? ^ A ^(PC.PE)

C2

t • 6 C2 14:0 1

16:0

7-16:1 ? J8:0

(Neutral Heids . 9 ' ' \ ' 2 0 : 0

phospholipids ) | ^ 9,12-18:2

9,12,15-18:3 11,14-20:2

i 6,9,12,15-18:4 11,14,17-20:3 8,11,14-20:3

8,11, 14,17-20^4^ 5,8,11,14-20:4 I - I 5,8,11,14,17-20:5 C^

A | (Polyenoic fatty acids) C22 (Polyenoic fatty acids)

Pathway A (Independent of light)

(Via 16:0 in SL and Jr ans 3-16:1 in PG 7 )

4,7,10,13-16:4 9,12,15-18:3

Pathway B (Dependent on light and ammonlun

Scheme 1. Proposed pathways of fatty acid biosynthesis in Euglena gracilis

CONTROL OF FATTY ACID AND LIPID BIOSYNTHESIS 5 9

light. It appears to be used in the light at low concentrations of ammonium and in the dark. The fatty acids of pathway A are mainly located in neu-tral lipids (wax esters, triglycerides) and phospho-lipids (PC, PE) (see Fig. 3) .

In addition to the pathway A already described a new pathway B is proposed in Scheme 1. It leads to the unsaturated 16:2, 16:3, 16:4 and to the major part of the 18:2 and 18:3 fatty acids. This pathway seems to require light and a minimum amount of ammonium in the medium and is blocked by DCMU.

In our experiments the biosynthesis of SL, PG, MGDG and DGDG was influenced in a similar way to the unsaturated C16 and C18 fatty acids: It also required light and a minimum amount of ammo-nium in the medium, and was blocked by DCMU, too. These lipids as well as the unsaturated C16 and C18 fatty acids are characteristic constituents of chloroplasts 24' 2 9 _ 3 2 . In agreement with the findings of other authors 13 '14 ' 17> 26, MGDG and DGDG con-tained the major part of the 16:2, 16:3, 16:4, 18:2 and 18:3 fatty acids. So pathway B seems to be as-sociated with these galactolipids, SL and PG.

SL was unique in containing high amounts of palmitic acid 1 7 ' 2 6 ' 3 2 _ 3 5 , PG was characterized by the tr3-16:l fatty acid 2 6 , 3 6~4 1 . This acid is syn-thesized from palmitic acid in Chlorella vulgaris, the synthesis being directly light dependent42. This compound is also hydrogenated to give again pal-mitic acid in the same organism43. The functions of both SL and PG in fatty acid biosynthesis are still unknown. Possibly they play a role in a kind of fatty acid transfer of palmitic acid from neutral or phospholipids via a palmitic acid in SL and a trans?>-\6:\ acid in PG to the unsaturated C16 and C18 fatty acids in MGDG and DGDG under the in-fluence of light. Experiments are under way to de-monstrate whether or not such a transfer exists in Euglena gracilis.

All of the lipids associated with the system of fatty acid biosynthesis proposed here seem to have special functions because they are all characterized by their fatty acid compositions (see Fig. 3 and Table 4) . This is in agreement with the data given by other authors 17, 26> 33) 38.

There seem to be small quantities of 18:2 and 18:3 acids synthesized via pathway A. This can be concluded from the occurence of minor quantities of these compounds in neutral lipids and phospho-

lipids and from their presence even wdien Euglena was grown in the light at low levels of ammonium and in the dark. This would explain why under these conditions the biosynthesis of the 18:2 and 18:3 fatty acids was not so much affected as that of the 16:2, 16:3 and 16:4 compounds. During our ex-periments always a-Iinolenic acid (9,12,15-18:3), but no significant amounts of y-linolenic acid (6,9,12-18:3) could be detected. The 12:0, 14:0, 16:0, 18:0, 16:1 and 18:1 fatty acids were present in low amounts in all of the lipids if not particularly mentioned in Table 4.

Lipid Typical fatty acids *

Neutral lipids 14:0, 16:0, 18:1, 18:4, 20:4, 20:5, 22:6 PC 14:0, 16:0, 18:1, 18:4, 20:4, 20:5, 22:6 PE 14:0, 16:0, 18:1, 18:4, 20:4, 20:5 PG 16:0, trans3-16:l, cis7-16:l SL 16:0, 16:1 MGDG 16:2, 16:3, 16:4, 18:2, 18:3 DGDG 16:2, 16:3, 16:4, 18:2, 18:3

Table 4. Typical fatty acids in the individual lipids of Euglena gracilis. * If not particularly mentioned here, all of the above lipids also contain low amounts of 12:0, 14:0,

16:0, 18:0, 16:1 and 18:1 fatty acids.

M G D G and D G D G seem to have somewhat dif-ferent functions because M G D G was characterized by high amounts of the 16:4 and 18:3 acids and D G D G by a higher percentage of the 16:2 acid. NICHOLS 44>45 was even able to subdivide M G D G

from Chlorella vulgaris into five species which all differed in their fatty acid compositions. There is some evidence given by our experiments that the biosynthesis of M G D G is more sensitive to poor conditions of growth than that of D G D G . At de-creasing concentrations of ammonium M G D G

seemed to decrease more than D G D G (Fig. 2) . The before mentioned "instability" of the 16:4 fatty acid might therefore be due to the fact that the major part of this compounds is located in M G D G .

The increase of unsaturated fatty acid biosyn-thesis at high levels of N H 4 C 1 (Fig. 1) might give an explanation for the recent observations of K A N A -ZAWA and coworkers 46. They reportet that addition of N H 4 C 1 to dark grown cultures of Chlorella pyre-noidosa and of N H 4 C 1 and K N O A to light grown cultures caused a decrease of 14C flow into sucrose and an increase into amino and K r e b s cycle acids.

Under the conditions described here oleic acid seemed to be more associated with pathway A. This

6 0 P. POHL AND H. W A G N E R

compound was mainly located in PC and PE and only to a small extend in M G D G and D G D G . This confirms the observations of GURR 47 who found that in Chlorella vulgaris oleic acid is preferably bound to PC.

There is the question whether the pathways pro-posed above might also be found in terrestrial plants. NEWMAN and WALLACE48'49 reported that low levels of nitrogen caused an increase of the 16:0 and a decrease of the 18:3 fatty acids in the chloroplasts of several higher plants, which agrees with our findings in Euglena gracilis. According to our present knowledge50-52, however, terrestrial plants seem to lack the 16:2 and 16:3 fatty acids. There are only two reports about the occurrence of the 7,10,13-16:3 fatty acid in the leaves and chloro-plasts of spinach 53' 54. Possibly in terrestrial plants these compounds are only present in very low amounts as intermediates in the biosynthesis of the predominating unsaturated C18 fatty acids *. NAGAI 5 5

demonstrated that spinach chloroplasts can convert stearyl-ACP, but not stearyl-CoA and palmityl-ACP into oleic acid when ferredoxin is added.

The light dependence of the unsaturated C16 and C18 fatty acids is of further importance as it gives an explanation for the phenomenon of the "essen-tial fatty acids" in animals. These fatty acids (Al, 10-16:2, Al, 10,13-16:3, A4,7,10,13-16:4, A9,12-18:2, A9,12,15-18:3 and biosynthetically related acids) are fatty acids of the linoleic and lino-lenic fatty acid type and cannot be synthesized by animals 5 6 _ 6 4 . Presumably this is due to the light dependence of the biosynthesis of these compounds. Animals have to take up the essential fatty acids from plant food. It is in agreement with this fact that SL, PG, MGDG and DGDG are not reported to occur in animals.

During our experiments the biosynthesis of chlorophylls was always influenced similarly to that of the unsaturated C16 and C18 fatty acids and of

* While this paper was in progress, G. R. JAMIESON and E. H. REID reported the occurrence of 16:3 in a multitude of angiosperms (Phytochemistry 10, 1837 [1971]) .

1 E . KLENK, D . EBERHAGEN, a n d H . P . K O O F , H o p p e - S e y l e r ' s Z. physiol. Chem. 334, 44 [1963],

2 R . G . A C K M A N , P . M . J A N G A A R D , R . J . HOYLE, a n d H . BROCKERHOFF, J. Fisheries Res. Board Canada 21, 747 [1964].

3 H . W A G N E R a n d P . P O H L , B i o c h e m . Z . 3 4 1 , 4 7 6 [ 1 9 6 5 ] , 4 P. M. WILLIAMS, J. Fisheries Res. Board Canada 22, 1107

[1965]. 5 H . W A G N E R a n d P . P O H L , P h y t o c h e m i s t r y 5 , 9 0 3 [ 1 9 6 6 ] .

SL, P G , M G D G and D G D G : in the dark and at low levels of ammonium in the light, no or very little chlorophyll was produced, wrhereas large quantities were synthesized in the light at high levels of am-monium (see Fig. 1). The influence of these factors on chlorophyll biosynthesis is known 6 5 - 6 7 . In chloroplast membranes the chlorophylls, SL, PG, M G D G and D G D G are associated with one an-other 24' 30. In Euglena gracilis var. bacillaris 68 the formation of chloroplast lamellae and of chloro-phylls seems to be correlated. GOLDBERG and OHAD 6 9 '7 0 reported the biosynthesis of constant molar proportions of pigments, total glycolipids and polyunsaturated fatty acids in the chloroplasts of Chlamydomonas when membrane synthesis pro-ceeded at a constant rate. These reports indicate that possibly the biosynthesis of chlorophylls as well as that of the above lipids and unsaturated fattv acids is a function of chloroplast development.

Our experiments and those of other authors n ' 1 3 ' 14,16,17 s j 1 0 w that the biosynthesis of chloroplast lipids and fatty acids requires light. To date, how-ever, only the formation of the trans-3-16:1 fatty acid has been shown to be directly light depen-dent 42. So it remains unknown whether the biosvn-thesis of the other compounds is directly or in-directly controlled by light. Furthermore we do not know whether there is an interdependence between chlorophylls and the chloroplast lipids and fatty acids, or whether these compounds depend separa-tely on an unknown substance or system which in itself requires ammonium and light. There is much evidence that the mitochondria and chloroplasts of Euglena71-75 and of algae and higher plants76-89

have their own species of DNA and RNA and their own genetic system which are independent of the nucleus. The above phenomena are possibly some-how related to this fact.

The Deutsche Forschungsgemeinschaft is given thanks for the support of this work.

6 L. CHUECAS and J. R. RILEY, J. Marine biol. Assoc. United Kingdom 46, 153 [1966].

7 R . G . A C K M A N , C . S . T O C H E R , a n d J . M C L A C H L A N , J . Fisheries Res. Board Canada 25, 1603 [1968].

8 P . P O H L , H . W A G N E R , a n d T . PASSIG, P h y t o c h e m i s t r y 7 , 1565 [1968],

9 J . E R W I N a n d K . B L O C H , B i o c h e m . Z . 3 3 8 , 4 9 6 [ 1 9 6 3 ] . 10 E. D. KORN, J. Lipid Res. 5, 352 [1964]. 1 1 D . H U L A N I C K A , J . E R W I N , a n d K . B L O C H , J . b i o l . C h e m i s t r y

239, 2778 [1964]. 1 2 A . ROSENBERG a n d M . PECKER, B i o c h e m i s t r y 3 , 2 5 4

[1964].

CONTROL OF FATTY ACID AND LIPID BIOSYNTHESIS 61

1 3 A . ROSENBERG, J . GOUAUX, a n d P . MILCH, J . L i p i d R e s . 7, 733 [1966],

1 4 A . ROSENBERG a n d J . G O U A U X , J . L i p i d R e s . 8 , 8 0 [1967].

1 5 J . NAGAI a n d K . BLOCH, J . b i o l . C h e m i s t r y 2 4 2 , 3 5 7 [1967].

1 6 G . CONSTANTOPOULOS a n d K . BLOCH, , J . b i o l . C h e m i s t r y 242, 3538 [1967].

1 7 B . W . NICHOLS a n d R . S . APPLEBY, P h y t o c h e m i s t r y 8 , 1 9 0 7 [1969],

1 8 T . H . HAINES, S . AARONSSON, J . L . GELLERMANN, a n d H . SCHLENK, N a t u r e [ L o n d o n ] 1 9 4 , 1 2 8 2 [ 1 9 6 2 ] .

1 9 P . P O H L , T . PASSIG, a n d H . W A G N E R , P h y t o c h e m i s t r y 1 0 , 1505 [1971].

2 0 M . CRAMER a n d J . MYERS, A r c h . M i k r o b i o l . 1 7 , 3 8 4 [1952].

2 1 H . M Ö H R a n d G . SCHOSER. P l a n t a 5 3 , 1 [ 1 9 5 9 ] . 2 2 H . M Ö H R a n d E . VAN NES, Z . B o t . 5 1 , 1 [ 1 9 6 3 ] . 2 3 P . POHL, H . GLASL, a n d H . W A G N E R , J . C h r o m a t o g r . [ A m -

sterdam] 49,488 [1970]. 2 4 C . F . ALLEN, P . G O O D , H . F . DAVIES, P . CHISUM, a n d S . D .

FOWLER, J . A m e r . O i l C h e m i s t s ' S o c . 4 3 , 2 2 3 [ 1 9 6 6 ] . 23 D. I. ARNON, Plant Physiol. 24 ,1 [1949]. 26 B. W. NICHOLS, Biochim. biophysica Acta [Amsterdam]

106, 274 [1965]. 27 E. D. KORN, J. biol. Chemistry 239, 396 [1964], 28 J. NAGAI and K. BLOCH, J. biol. Chemistry 240, PC 3702

[1965]. 29 A. A. BENSON, Natl. Res. Council Publ. Nr. 1145 [1963]. 3 0 A . A . BENSON, J. A m e r . O i l C h e m i s t s ' S o c . 4 3 , 2 6 5 [ 1 9 6 6 ] . 3 1 T . WEIER a n d A . A . BENSON, A m e r . J . B o t . 5 4 , 3 8 9 [ 1 9 6 7 ] . 32 B. W. NICHOLS, Lipids 3, 354 [1968]. 3 3 B . W . NICHOLS. A . T . JAMES, a n d J . BREUER, B i o c h e m . J .

104,486 [1967]. 3 4 J . S . O ' B R I E N a n d A . A . BENSON, J . L i p i d R e s . 5 , 4 3 2

[1964]. 35 A. RADUNZ, Hoppe-Seyler's Z. Physiol. Chem. 350, 411

[ 1 9 6 9 ] . 3 6 F . HAVERKATE, J . DE GIER, a n d L . L . M . V A N DEENEN,

E x p e r i e n t i a [ B a s e l ] 2 0 , 5 1 1 [ 1 9 6 4 ] , 3 7 R . O . WEENINCK a n d F . B . SHORLAND, B i o c h i m . b i o p h y -

sica Acta [Amsterdam] 84 ,613 [1964]. 3 8 C . F . ALLEN, P . G O O D , H . F . DAVIES, a n d S . D . FOWLER,

Biochem. biophysic. Res. Commun. 15, 424 [1964] . 3 9 F . HAVERKATE a n d L . L . M . VAN DEENEN, B i o c h i m . b i o -

physica Acta [Amsterdam] 106, 78 [1965] 40 B. W. NICHOLS, Phytochemistry 4, 769 [1965]. 4 1 C . F . ALLEN, O . HIRAYAMA, a n d P . G O O D , T h e B i o c h e m i -

stry of Chloroplasts, p. 195 (T. W. GOODWIN), Acad. Press, London and New York 1966.

4 2 B . W . NICHOLS. P . HARRIS, a n d A . T . JAMES, B i o c h e m . biophysic. Res. Commun. 21, 473 [1965].

4 3 C . T . BARTELS, A . T . JAMES, a n d B . W . NICHOLS, E u r . J . Biochem. 3, 7 [1967].

4 4 B . W . NICHOLS a n d R . MOORHOUSE, L i p i d s 4 , 3 1 1 [ 1 9 6 9 ] . 45 S. SAFFORD and B. W. NICHOLS, Biochim. biophysica Acta

[Amsterdam] 210,57 [1970]. 4 6 T . K A N A Z A W A , K . K A N A Z A W A , M . R . K I R K , a n d J . A .

BASSHAM, Plant Cell Physiol. 11, 445 [1970]. 4 7 M . I . G U R R , M . P . ROBINSON, a n d A . T . JAMES, E u r . J .

Biochem. 9, 70 [1969]. 4 8 J . W . WALLACE a n d D . W . NEWMAN, P h y t o c h e m i s t r y 4 , 4 3

[1966]. 49 D. W. NEWMAN, Plant Physiol. 41, 328 [1966]. 5 0 I I . SCHLENK a n d J . L . GELLERMAN, J . A m e r . O i l C h e m i s t s '

Soc. 42,504 [1965]. 51 A. RADUNZ, Physochemistry 6, 399 [1967]. 52 P. POHL and H. WAGNER, Fette, Seifen, Anstrichmittel, in

press. 5 3 F . T . W O L F , J. G . CONIGLIO, a n d J . T . DAVIS , P l a n t P h y -

siol. 37 ,83 [1962].

54 A. RADUNZ, Hoppe-Seyler's Z. Physiol. Chem. 343, 294 [1966].

5 5 J . NAGAI a n d K . BLOCH, J . b i o l . C h e m i s t r y 2 4 1 , 1 9 2 5 [1966],

5 6 J . F . M E A D , G . STEINBERG, a n d D . R . H O W T O N , J . b i o l . Chemistry 205, 683 [1953],

57 P. D. KLEIN and R. M. JOHNSON, Arch. Biochem. Biophy-sics 48, 380 [1954].

58 E. KLENK, Hoppe-Seyler's Z. Physiol. Chem. 302, 268 [1955].

5 9 G . STEINBERG, W . H . SLATON, D . R . H O W T O N , a n d J . F . MEAD, J. biol. Chemistry 220, 257 [1956].

60 J. F. MEAD, J. biol. Chemistry 227, 1025 [1957]. 61 A. J. FULCO, J. biol. Chemistry 234, 1411 [1959]. 6 2 J . F . M E A D , M . K A Y A M A , a n d R . REISER, J . A m e r . O i l C h e -

mists' Soc. 37,438 [I960] . 6 3 F . LEUPOLD a n d G . KREMER, H o p p e - S e y l e r ' s Z . P h y s i o l .

Chem. 318, 251 [I960] . 6 4 E . KLENK a n d G . KREMER, H o p p e - S e y l e r ' s Z . P h y s i o l .

Chem. 320, 111 [I960] . 65 F. C. CZYGAN, Arch. Mikrobiol. 61, 81 [1968]. 66 F. C. CZYGAN, Arch. Mikrobiol. 62, 209 [1968], 6 7 M . NISHIMURA a n d H . HUZISIGE, J . B i o c h e m . 4 6 , 2 2 5

[1959], 0 8 A . I . STERN, J . A . SCHIFF, a n d H . T . EPSTEIN, P l a n t P h y -

siol. 39, 220 [1964]. 69 I. GOLDBERG and I. OHAD, J. cellular Biol. 44, 563 [1970]. 70 I. GOLDBERG and I. OHAD, J. cellular Biol. 44, 572 [1970]. 7 1 L . LEFF, M . MANDEL, H . T . EPSTEIN, a n d J . A . SCHIFF,

Biochem. Biophys. Res. Commun. 13, 126 [1963]. 72 D. S. RAY and P. C. HANAWALT, J. molecular Biol. 9, 812

[1964]. 7 3 G . BRAWERMAN a n d J . M . EISENSTADT, B i o c h i m . b i o p h y -

sica Acta [Amsterdam] 91,477 [1964]. 7 4 M . H . ZELDIN a n d J . A . SCHIFF, P l a n t P h y s i o l . 4 2 , 9 2 2

[1967], 7 5 R . M . SMILLIE, D . GRAHAM, M . R . DWYER, A . GRIEVE, a n d

N. F. TOBIN, Biochem. biophysic. Res. Commun. 28, 604 [1967].

76 H. Ris and W. PLAUT, J. cellular Biol. 13, 383 [1962]. 7 7 E . H . L . CHUN, M . H . V A U G H A N , a n d A . R I C H , J . m o l e c u -

lar Biol. 7 ,130 [1963], 7 8 A . GIBOR a n d S . GRANICK, S c i e n c e [ W a s h i n g t o n ] 1 4 5 ,

890 [1964], 7 9 D . J . L . LUCK a n d E . REICH, P r o c . na t . A c a d . S e i . U S A

52,931 [1964]. 80 D. C. SHEPHARD, Biochim. biophysica Acta [Amsterdam]

108, 635 [1965]. 81 T. IWAMURA, Progress in Nucleic Acid Research and Mole-

c u l a r B i o l o g y , V o l . 5 , (J . N . DAVIDSON a n d W . E . C O H N ) , Acad. Press, New York and London 1966.

8 2 K . K . TEWARI a n d S . G . WILDMAN, S c i e n c e [ W a s h i n g t o n ] 153, 1269 [1966],

83 B. R. GREEN and M. P. GORDON, Biochim. biophysica Acta [Amsterdam] 145,378 [1967].

84 H. G. RUPPEL, Z. Naturforsch. 22 b, 1068 [1967]. 8 5 H . G . SCHWEIGER, W . L . DILLARD, A . GIBOR, a n d S . BER-

GER, Protoplasma 64,1 [1967]. 8 6 T . BISALPUTRA a n d A . A . BISALPUTRA, J . U l t r a s t r u c t . R e s .

17, 14 [1967], 8 7 E . BALTUS, J . E . EDSTROEM, M . JANOWSKI, J . H A N O C Q -

QUERTIER, R . TENCER, a n d J . BRÄCHET, P r o c . na t . A c a d . Sei. USA 59, 406 [1967].

88 S. GRANICK and A. GIBOR, Progress in Nucleic Acid Research and Molecular Biology, Vol. 6, (J. N. DAVIDSON and W. E. COHN) , Acad. Press, New York and London 1967.

89 S. P. GIBBS, J. Cell Sei. 3, 327 [1968].