Cerulenin-Induced Modification of Structural and Photosynthetic Characteristics in Chlorella

Institute of Biophysics and Institute of Comparative Animal Physiology, University of Szeged; Institute of Biochemistry, Biological Research Center, Szeged, Hungary

Cerulenin-Induced Modification of Structural and Photosynthetic Characteristics in Chlorella T. HERCZEG, E. LEHOCZKI, T. FARKAS, I. ROJIK and L. SZALAY With 4 figures Received January 17, 1979 . Accepted February 3, 1979

Summary Electron micrographs, fluorescence and fluorescence excitation spectra (at 77 OK) were taken, fatty acid and pigment analysis, cell division and oxygen evolution (at 30 0c) were studied in synchronous cultures of Chlorella pyrenoidosa with and without cerulenin. After 72 h treatment with 22.5,uM cerulenin several structural and functional changes were observed. The most apparent structural changes: plasmolysis, fragmentation of the pyrenoid grains, alterations in the organization of the chloroplast. Functional changes are: inhibition of cell division (from 15 ,uM to higher concentrations of cerulenin) and inhibition of DCMUeffect on photosystem-2 activity. Inhibition of cell division could be partially restored by exogenous fatty acids. These changes can be correlated with the effect of cerulenin on the fatty acid composition and on the pigment system. The fatty acid content decreases from 556 to 428,ug/mg dry weight and the ratio of saturated to unsaturated fatty acids increases from 0.61 to 0.96 in cerulenin-treated samples. The chlorophyll content remains the same, whereas the carotenoid content increases from 1.50 to 2.31 nM/l07 cell upon cerulenin treatment. Conclusions: cerulenin is a powerful specific inhibitor of fatty acid biosynthesis in algal cells; fatty acids are required to maintain the integrity of pyrenoid grains and the capacity for cell division and cerulenin affects the photosynthetic apparatus: the composition of the pigment system and the function of both photosystems.

Key words: cerulenin, inhibition of lipid biosynthesis, chemical modification of membranes, oxygen evolution, fluorescence, cell division, Chlorella.

Introduction

The lipids are important structural and functional elements of chloroplast membranes. For study of their roles a number of methods are used such as fatty acid supplementation, treatment with inhibitors or lipolytic enzymes, in vitro membrane modification. One of the most promising methods that can be used under in vivo circumstances, i.e. in studying intact organisms, is to modify its fatty acid composition. Cerulenin is an antibiotic produced by the fungus Cephalosporium caerulens, which is a specific inhibitor of fatty acid biosynthesis in bacteria (VANCE et aI., 1972;

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et aI., 1973), fungi (NOMURA et aI., 1972; OHNO et aI., 1974; NICKERSON 1978) and higher plants (JAWORSKI et aI., 1974; ApPLEBY, 1974; WARING and LATIES, 1977) without influencing the catabolism of fatty acids or other biochemical processes directly. Therefore, cerulenin is believed to be a very effective biochemical tool for studying not only fatty acid biosynthesis but also the structures and functions of photosynthetic membranes of algae (LEHOCZKI et aI., 1979). In this paper the effects of cerulenin on the structure and photosynthetic parameters of Chiorella pyrenoidosa are discussed in an attempt to clarify the role of fatty acids in the photosynthetic' membranes, with emphasis on the photosynthetic activity. D'AGNOLO

and

LEASTMAN,

Material and Methods Culturing Chlorella pyrenoidosa (strain Emerson) was cultured in Tamiya medium (TAMIYA et a!., 1953) at 25°C and synchronized by using a 16 h light/8 h dark regime, according to the method of AACH (1952). The mean time of cell division was 18.5 h, and the standard deviation of the rate of cell division was 1.14 h. The variation coefficient characteristic of the rate of synchronization (HAGAR, 1972) was 0.061. The light intensity was 10 W /m 2 • The cultures were aerated continuously with atmospheric air. All experiments were carried out after the third light/ dark period. Electron microscopy The cells were fixed in glutaraldehyde according to HAYAT (1972), postfixed with OS04 and embedded in Durcupan-araldite. Sections were picked up on grids, and stained with uranyl acetate and lead citrate. Lipid extraction and determination of fatty acids The cells collected by centrifugation were vortexed in the presence of 10 ml chloroform: methanol (2: 1) solution and the extract was processed according to FOLCH et a!. (1957). The chloroform phase was adjusted set to 5 ml, and aliquots were taken for further analysis. Fatty acid methyl esters were prepared by trans esterification of 1 ml aliquots of the extract containing 17: 0 as internal standard. The separation was performed using a JEOL JGC 1100 gas chromatograph equipped with a flame ionization detector. The 12 ft X 3 mm stainless steel column was filled with Sp-2340 (Supelco). Identification was made with the aid of calibrated standards, and quantitation by a triangulation technique. Spectroscopic meamrements The absorption spectra were measured at room temperature with an SF-18 recording spectrophotometer equipped with an integrating sphere. The prompt fluorescence (excited at 480 nm) and fluorescence excitation spectra at 77 OK (observed at 730 nm) of whole cells were obtained with a Perkin-Elmer MPF-44 A spectrofluorimeter. In both cases the reabsorption was less than 5 0/0. The pigment content were determined according to FRENCH (1960). Oxygen evolution The oxygen-evolving actIvltles were measured polarographically with a Clark electrode at 30°C. Cells were suspended in the growth medium and adjusted to a density of 10,ug

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ChUml cell. The suspensions were illuminated by saturating light. DCMU was added in 10,uM final concentration to dark-adapted cultures.

Chemical treatments Cerulenin (Makor Chemicals Ltd., Israel) was added to the Chiarella cultures within 3 h after the beginning of the light period, when the cells were in the D" state (LORENZEN, 1964). The cerulenin concentration was 22.5 ,uM, except in the experiments shown in Fig. 1. All properties of cells were investigated after 72 h cerulenin treatment. In some cases fatty ~"cids (Sigma, 99 % pure), in a concentration of 100 ,ug/ml in the form of K+ salt solution, were added to the medium simultaneously with cerulenin.

Results

Cell multiplication Cerulenin caused a concentration-dependent inhibition of cell multiplication. Fifty percent inhibition was achieved with about 2.5 pM cerulenin; an almost complete inhibition required more than 15-20,uM (Fig. 1).

J'-

4

E 3

x

"'0

~

.a E ::J c:

=;

u

o

5 10 154045

c (inJ..LMlFig. 1: The dependence of cell density in Chiarella suspensions on the concentration of cerulenin after 72 h cerulenin treatment. AWAYA et al. (1975) and OHNO et al. (1975) found that the decrease of the cell multiplication of Escherichia coli and Saccharomyces cerevisiae due to cerulenin was partly restored by exogenous fatty acids. We observed a similar phenomenon in ChIarella cultures. If the rate of cell division without any treatment is regarded as 100 Ufo, it will be zero per cent in cultures with 22.5 pM cerulenin after 72 h. However, on the addition of 100 ,ug/ml of either stearic, oleic, linoleic or linolenic acid in the form of their potassium salts, simultaneously with cerulenin, the rate of cell division becomes 33, 58, 59 and 52 Ufo, respectively.

Cell structure The most characteristic organelles of Chiarella cells grown under normal conditions are the pyrenoid grains separated by a double membrane (GIBBS, 1962) and Z. PJlanzenphysial. Bd. 94. S. 55-64. 1979.

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T. HERCZEG, E. LEHOCZKI, T. FARKAS,!. ROJIK and L. SZALAY

the cup-shaped chloroplast (ALBERTS SON and LEYON, 1954) with membranes running nearly parallel. The pyrenoid grains of cerulenin-treated cells are in pieces, the thylakoids are disordered, and the cell wall is detached from the cytoplasm (Fig. 2 B). A statistical comparison of 800 cells showed that these alterations could be observed in more than 80 Ofo of the cells, while more than 90 Ofo of the untreated control cells displayed a normal electron microscopic structure.

Fatty acid composition Current views (OMURA, 1976) on the mode of action of cerulenin invoke the formation of a covalent complex with the fatty acid synthetase. Consequently, cerulenin inhibition should be highly irreversible. The inhibiting effect of cerulenin on fatty acid biosynthesis is shown in Table 1. Not only was the absolute amount of fatty acids reduced by cerulenin treatment, but the fatty acid composition was also changed. The ratios of saturated to unsaturated fatty acids in untreated and cerulenin-treated cells were 0.61 and 0.96, respectively. The increase of this ratio depends slightly on minor changes in the experimental conditions (the age of cultures, temperature, etc.).

Pigment composition Cerulenin treatment had practically no influence on the chlorophyll content of the cells; this is why no difference can be found in the red region of absorption Table 1: Fatty acid composition, amount of fatty acids and the ratio of saturated to unsaturated fatty acids in intact and 22.5,uM cerulenin-treated Chlorella cells after 72 h treatment. Fatty acid: ratio of numbers of C-atoms to double bonds

Amount of fatty acid in Ofo Control Cerulenintreated 35.0

16: 0 16: 1 16: 3 18 : 0 18 : 1 18 : 2 20: 0 18 : 3

31.1 4.3 5.5 2.6 19.8 22.3 4.2 10.2

3.7 4.3 18.5 2.5 9.8 20.8

Total

100.0

100.0

Fatty acid content ,ug fatty acid mg dry weight

556

428

Ratio of saturated to unsaturated fatty acids Z. Pjlanzenphysiol. Bd. 94. S. 55-64. 1979.

0.61

5.4

0.96

Cerulenin induced modifications in Chlorella

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Fig. 2: Electron micrographs of intact (magnifications: A - X24,000, B - X64,000) and cerulenin-treated (magnifications: C - X 18,500, D - X 64,000) Chlorella cells after 72 h treatment with 22.5 ,uM cerulenin. (CP: chloroplast, M: chloroplast membrane, P: pyrenoid grain, CW: cell wall).

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T. HERCZEG, E. LEHOCZKI, T. FARKAS, 1. ROJIK and L. SZALAY

Table 2: Pigment content and pigment content r::ttios of intact and 22.5,uM cerulenintreated Chiarella cells after 72 h treatment. Pigment content in nM/107 cell Pigments

Control cultures

Cerulenintreated cult.

Chlorophy ll-a Chlorophyll-b Carotenoids

5.07 1.66 1.50

5.43 1.76 2.31

3.05

3.08

4.48

3.11

Pigment ratios Chl-a/Chl-b Chl-a + Chl-b Carotenoids

spectra of cerulenin-treated and untreated whole cells. The amount of carotenes, however, increased in cerulenin-treated cells (Table 2). Fluorescence properties

Cerulenin treatment markedly influenced the distribution of excitation energy among chlorophyll forms; the long-wave F 724 band of fluorescence of cerulenintreated cells became more intense than that of the control, while the bands at shorter waves, F 700 and F 687, were reduced (Fig. 3). Since the carotenoid content is considerable higher in cerulenin-treated cells the increase in the intensity of the F 724 band might be ascribed to a more efficient energy transfer from the carotenoid to the chlorophylls. However, the fluorescence excitation spectrum of the F 724 band

~

~CII

a: 650

700 ?'(nm)-

750

Fig. 3: Normalized fluorescence spectra of intact (solid line) and 22.5,uM cerulenin-treated (broken line) Chiarella suspensions at 77 oK after 72 h treatment; normalization to the unit area, exciting wavelength 430 nm.

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~

~

&

400

500 600 1\ (nm)-

700

Fig. 4: Relative fluorescence excitation spectra of intact (solid line) and 22.5,uM cerulenintreated (broken line) Chiorella suspensions at 77 oK after 72 h treatment, observation at 730 nm.

(Fig. 4) contradicts this assumption. With observation at F 724 band, the intensities of fluorescence of cerulenin-treated and untreated cells showed significant differences only in the excitation regions around 430 nm and in the red above 600 nm characteristic of chlorophyll molecules. No difference was found around 500 nm, the region mainly associated with the carotenoids. Cerulenin treatment therefore induces changes in the energy distribution among the chlorophyll molecules, and there is no alteration in the energy transfer from carotenoids to chlorophylls.

Oxygen evolution In spite of the profound changes in cell structure and composition, the net oxygen evolution after cerulenin treatment did not alter essentially (Table 3). In control cultures the oxygen evolution was inhibited by IOI1M DCMU, as expected. In cerulenin-treated cultures, however, only a very slight DCMU-inhibition of net oxygen evolution appeared, in spite of the absence of any artificial electron Table 3: Effect of cerulenin-, DCMU- and (cerulenin + DCMU)-treatment on the photosynthetic oxygen evolution in Chiorella suspensions at 30°C after 72 h treatment. Chlorella

Dark respiration

Oxygen evolution in light

Net photosynthetic 02-evolution

in 11M 02/nM ChI. hour Control Cerulenin-treated DCMU-treated Cerulenin + DCMU treated

0.106 0.100 0.234

0.512 0.569 in traces 0.415

0.618 0.687 0.649

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T.

HERCZEG,

E.

LEHOCZKI,

T.

FARKAS,!. ROJIK

and 1.

SZALAY

acceptor. Table 3 shows that the DCMU-insensitivity is a complex phenomenon: DCMU reduced the apparent oxygen evolution by about 30 %, but the increased dark respiration fully compensated it. Discussion

Since cerulenin, a potent inhibitor of fatty acid biosynthesis in ChIarella (Table 1) inhibits the cell division as well (Fig. 1) fatty acid synthesis is obviously required for cell multiplication to occur. This is, however, not surprising if we consider that intense membrane genesis must take place during cell division and lipids are important constituents. The partial restoration of cell division by fatty acid supplementation corroborates this statement. Though the chemical composition of pyrenoid grains is not known (GIBBS, 1962; ARNTZEN and BRIANTAIS, 1975), since fatty acids are required to maintain the integrity of pyrenoid grains lipids should play an important role >in the pyrenoid formation. As for the lipids in the chloroplast membranes the electron micrographs confirm their basic importance in membrane formation (BENSON, 1971). Changes in the saturated to unsaturated fatty acid ratio in algae grown in the presence of cerulenin suggest modification in the phase bahaviour of membranes (CHAPMAN, 1975) but the type of membranes affected remains to be mvestigated. The increased carotenoid content of cerulenin-treated ChIarella (Table 2) might be a consequence of a shift of biosynthetic processes from the synthesis of fatty acid to that of carotenoids as a partial compensation for the fatty acid deficiency by production of carotenoids of a lipid character. It is understandable that cerulenin treatment does not influence the chlorophyll content (Table 2): chlorophylls are mainly bound to proteins (THORNBER, 1975) and only a small proportion are linked to lipids directly (ROSENBERG, 1967). It is generally accepted that the excitation energy flow between the two photosystems is regulated by chloroplast membranes (MURATA, 1969; MYERS, 1971), and that structural changes are accompanied by fluorescence changes (BURKE et aI., 1978). The existence of (at least three) fluorescent chlorophyll-a components of ChIarella at low temperature (GOEDHEER, 1972) led to the finding of a correlation between the photosystems and the fluorescence of these components: the fluorescence bands F 687 and F 700 can be attributed to photosystem-2, and F 724 to photosystem-l (PAGAGEORGIOU, 1975). According to SATOH and BUTLER (1978), the increase of F 735 in the fluorescence of spinach chloroplasts is accompanied by a decrease of the yield of photo oxidation of P-700, the reaction center of photosystem-l, and therefore fluorescence and photochemistry are in competition at low temperature. Similarly, the increase of F 724 in cerulenin-treated ChIarella at low temperature (Fig. 3) may be in correlation with a decreased photochemical activity of photosystem-l. Since photochemistry as expected physically and chemically should not depend on temperature, the same should apply at any temperature. Consequently, fatty acid Z. P/lanzenphysiol. Bd. 94. S. 55-64. 1979.

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deficiency and changes in the fatty acid composition caused by cerulenin treatment lead to a decrease of photosystem-l activity. In contrast to the profound changes in structure and in several photosynthetic characteristics; no change is found in the activity of photosystem-2; after cerulenin treatment the net photosynthetic oxygen evolution (together with dark respiration and oxygen evolution in light) is virtually unchanged (Table 3). Surprisingly, however, cerulenin causes a DCMU-insensitivity of photosystem-2 even in the absence of any artificial acceptor (Table 3). This means that the effect of cerulenin also leads to conformational changes of the membranes, so that the site of DCMU-attack becomes inaccessible after cerulenin treatment, or a new pathway for oxygen evolution is opened (LEHOCZKI et al., 1979). Acknowledgements We are grateful to Dr. S. OMURA (Tokyo, Japan) for a cerulenin sample used in preliminary experiments, and to Dr. J. LAVOREL (GiflYvette, France) for the Chiorella strain.

References AACH, H. G.: Arch. Mikrobiol. 17,213 (1952). ALBERTSSON, P. A. and H. LEYON: Exp. Cell Research 7, 288 (1954). ApPLEBY, R. S. Phytochemistry 13, 2745 (1974). ARNTZEN, CH. J. and J. M. BRIANTAIS: In: GOVINDJEE (Ed.): Bioenergetics of Photosynthesis, pp. 51. Academic Press, London, 1975. AWAYA, ]., T. OHNO, H. OHNO, and S. OMURA: Biochem. Biophys. Acta 409, 267 (1975). BENSON, A. A.: In: M. GIBBS (Ed.): Structure and Function of Chloroplasts, pp. 129. Springer-Verlag, Berlin, 1971. BURKE, ]. J., C. L. DITTO, and C. J. ARNTZEN: Arch. Biochem. Biophys. 187, 252 (1978). CHAPMAN, D.: Quart. Rev. Biophys. 8, 185 (1975). D'AGNOLO, G., 1. S. ROSENFELD, J. AWAYA, S. OMURA, and P. R. VAGELOS: Biochem. Biophys. Acta 326,155 (1973). FOLCH, J., M. LEES, and G. H. SLOANE STANLEY: J. BioI. Chem. 226, 497 (1957). FRENCH, C. S.: In: W. RUHLAND (Ed.): Encyclopedia of Plant Physiology, 5/1, pp. 259. Springer-Verlag, Heidelberg, 1960. GIBBS, S. P.: ]. Ultrastruct. Res. 7, 418 (1962). - J. Ultrastruct. Res. 7,247 (1962). HAGAR, W. G.: In: Carnegie Institution Year Book, Stanford, California, 72, pp. 388, 1972. HAYAT, M. A.: Basic Electron Microscopy Techniques, pp. 1. Van Nostrand Company, New York,1972. JAWORSKI, J. G., E. E. GOLDSCHMIDT, and P. L. STUMPF: Arch. Biochem. Biophys. 163, 769 (1974). LEHOCZKI, E., T. HERCZEG, and L. SZALAY: Biochim. Biophys. Acta 545, 376 (1979). LORENZEN, H.: In: E. ZENTHEN (Ed.): Synchrony in cell division and growth, pp. 571. Wiley (Interscience), New York, 1964. MURATA, N.: Biochim. Biophys. Acta 189, 171 (1969). MYERS, J.: Ann. Rev. Plant Physiol. 22, 289 (1971). NICKERSON, K. W. and E. LEASTMAN: Exp. Mycology 2, 26 (1978). NOMURA, S., T. HORIUCHI, T. HATA, and S. OMURA: J. Biochem. (Tokyo) 71, 783 (1972).

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OHNO, T., T. KESADO, J. AWAYA, and S. OMURA: Biochem. Biophys. Res. Comm. 57, 1119 (1974). OHNO, T., J. AWAYA, and S. OMURA: Antimicrob. Agents and Chemother. 9, 42 (1976). OMURA, S.: Bacterio!' Rev. 40, 681 (1976). PAPAGEORGIOU, G.: In: GOVINDJEE (Ed.): Bioenergetics of Photosynthesis, pp. 319. Academic Press, New York, 1975. ROSENBERG, A.: Science 157, 1191 (1967). TAMIYA, H., E. HASE, K. SHIBATA, A. MITUYA, T. IWAMURA, T. NIHEI, and T. SASA: In: J. S. BURLEW (Ed.): Algal culture from Laboratory to Pilot Plant, pp.204. Carnegie Institution of Washington Publication 600, Washington, D. c., 1953. VANCE, D., 1. GOLDBERG, O. MITSUHASHI, K. BLOCH, S. OMURA, and S. NOMURA: Biochem. Biophys. Res. Comm. 48, 649 (1972). WARING, A. J. and G. G. LATIES: Plant. Physio!. 60, 11 (1977). Dr. T. HERCZEG, Dr. E. LEHOCZKI and Dr. L. SZALAY, Institute of Biophysics, J6zsef Attila University, 6722 Szeged, Egyetem u. 2., Hungary. Dr. T. FARKAS, Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Odesszai krt. 62., Hungary. Dr. 1. ROJIK, Institute of Comparative Animal Physiology, J6zsef Attila University, 6726 Szeged, Kozepfasor 52, Hungary.

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