148

Biochimica et Biophysica Acta, 499 (1977) 148--155 © Elsevier/North-Holland Biomedical Press

BBA 28302

MELANIN BIOSYNTHESIS DURING D I F F E R E N T I A T I O N OF

PHYSAR UM POL YCEPHA L UM

I. C H E T

a and A. H U T T E R M A N N

b

a Department of Plant Pathology and Microbiology, The Hebrew University,Rehovot (Israel) and b Institut fiir Forstbotanik, G6ttingen University, G6ttingen (G.F.R.) (Received December 31st, 1976)

Summary Melanin synthesis in the m y x o m y c e t e Physarum polycephalum occurs during sporulation b u t n o t during spherule formation. Melanin-like pigment was extracted from spores. An almost identical substance of polyphenols was extracted from spherules and characterized by its ultraviolet and infrared absorbance spectra. Polyphenol oxidase activity in spherules was very low and showed only one weak isoenzyme band in isoelectric focusing polyacrylamide gels. A much higher activity, and an increasing number of isoenzymes, were detected in sporulating cultures after illumination during the differentiation process. The addition of melanin precursors resulted in the synthesis of brownish-yellow spherules, probably containing dopachrome, whereas the addition of polyphenol oxidase inhibitors resulted in yellow sporangia. The results indicate that melanin synthesis is probably only a stage in maturation b u t not an essential part of the morphogenetic process itself.

Introduction The m y x o m y c e t e Physarum polycephalum can be induced to form two different hard-walled forms, spores and spherules [1]. On macroscopic observation, the most striking difference between the two structures is the fact that spores are black whereas spherules are orange. The black pigment of the spores is melanin. This was first suggested by Ward and Havir [2] who found cresolase activity in Physarum extracts. Daniel [3,4] identified the black pigment as a melanin-protein complex. He also observed that starved plasmodia treated with N-ethylmaleimide either before or after induction with light form melanin pigments with or without phenolase substrates. McCormick et al. [5] characterAbbreviation: DOPA, dihydroxyphenylalanine; PPO, p o l y p h e n o l oxidase.

149

ized spore and spherule walls finding them similar except for the presence of approx. 15% melanin in the spore walls. The fact that spore and spherule walls have only quantitative differences in the same components, except for the melanin in the spores, yet have different morphological forms, suggests fine control of biosynthetic pathways [5]. This study deals with the enzymes and phenols involved in melanin synthesis in both types of differentiating cultures. This, in turn, can lead us to a better understanding of the control which regulates the differences between spherules and spores in the same isolate of P. polycephalum. Methods Axenic cultures of P. polycephalum were grown as a suspension of the microplasmodia in a semi-synthetic medium (N+C) [1]. Flasks containing 20 ml of liquid medium were shaken on-reciprocating shaker in a darkroom at 22 ° C. After 72 h the microplasmodia were centrifuged and then transferred to either the same medium, solidified for sporangia formation, a starvation medium (S+C) or a mannitol-supplemented synthetic medium [6] for spherule induction. Sporulating mutant CL-20 of P. polycephalum was kindly obtained from Dr. J. Dee and W. Grant, Leicester, U.K. Sporulation. Cultures were grown on N+C in the dark for 3--4 days and then illuminated for 4 h. The cultures were incubated again at 22°C and sporangia were observed 16--20 h later. Pigment extraction and phenol characterization. The dark pigment of the sporangia and phenols from the spherules were extracted b y 1 M KOH for 2 h at 100°C [7]. The ultraviolet spectra of the extracted pigments were determined with a recording U.V. Perkin-Elmer model 55 spectrophotometer. Infrared spectra were obtained with a Perkin-Elmer model 137 spectrophotometer b y using 2 mg of tested material pressed into a pellet with 100 mg of KBr. Quantitative phenol measurement was determined by FeC13/Fe(CN)3 reagent as a modification of the m e t h o d of Barton et al. [8] with catechol the standard phenol. Polyphenol oxidase activity. Various stages of differentiating cultures were homogenized in 0.1 M phosphate buffer, pH 6.0, with an Omnimixer (Sorvall) or Brown's Disintegrator. The temperature was not allowed to rise above 6°C. Cell debris were sedimented b y centrifugation at 40 000 X g for 30 min in a Sorvall RC-2B centrifuge at 4°C. Polyphenol oxidase activity was determined b y using 5 mM of L-dihydroxyphenylalanine (DOPA) as a substrate and 1 mg protein as an enzyme. Hallochrome production was measured at 475 nm in a Perkin-Elmer spectrophotometer at 30°C. An enzyme unit is defined as an absorbance change of 0.001/min under the above conditions [9]. P r o t e i n w a s determined b y the Folin-phenol reagent according to Lowry et al. [10]. Isoelectric focusing gel electrophoresis and isoenzyme identification. Isoelectric focusing [11] in 7.5% acrylamide gels was employed for protein separation using an L.K.B. (Sweden) ampholine carrier containing 40% ampholites with pH ranging from 3 to 10. The gels (8 cm long) were chemically polymerized b y K2S208. Protein samples, each containing 500 g protein, were applied in

150 10% sucrose under an ampholyte "protective layer". Electrophoresis was carried out at 5°C for 4 h, applying 1.5 mA per gel and gradually raising the voltage to 300--350 V. The pH gradient in the gels was determined by cutting gels without protein into 10-mm slices, soaking them in 2 ml distilled water for 1 h, and measuring the pH with a Radiometer (PHM62) pH meter. After electrophoresis, polyphenol oxidase (PPO) isoenzymes were tested according to Strafford and Galston [12]. Substrates for PPO were 3,4-dihydroxyphenylalanine. Catechol peroxidase isoenzymes were determined by immersing the gels in distilled water containing 0.02 M catechol for 30 min and then transferring them to 0.3% (v/v) H202. Stained gels were scanned by a linear transport unit built on a Techtron (Varian) spectrophotometer. Every experiment was repeated at least four times. Results

The nature of Physarum melanin-like pigment Phenols from spores and spherules were extracted by KOH. The extracted pigment from spores was black, whereas the one from spherules was brownishyellow. Neutralization of the dark pigment solution resulted in precipitation. The pigments from both structures were decolorized by oxidation with 1% NaOC1. They gave a positive reaction for phenols with FeC13/Fe(CN)3 reagent [8] and for polyphenols with FeC13 [13]. Using the Biuret method [14] the extracted pigment was found to contain proteins too. The absorption spectrum showed no peaks in the 400--700 nm range. Plotting the logarithm of absorbance versus wavelength gave a linear curve with a negative slope of --0.0034 for spores and --0.0036 for spherules. Only one deviation from linearity in the region of 370--400 nm appears in the spectrum of the spores (Fig. 1). The patterns of the infrared absorption spectrum of the extract from both sources are almost identical (Fig. 2). This spectrum which shows two typical peaks at 1400 and 140 cm -1 is similar to the infrared spectrum of similar preparations of melanins from other fungi [7,15]. Changes in phenols during sporulation Quantitative determination of the phenols during differentiation revealed that phenol content did not change significantly during spherulation, whereas very pronounced changes occurred during sporulation. Phenol content increased rapidly for 4 h after illumination, and then decreased during the formation of sporangia and their melanin synthesis (Fig. 3). Melanogenesis involves oxidation and polymerization o f phenols to polyphenols. Induction and inhibition of melanin synthesis Since spherules never synthesize melanin, an attempt was made to induce such synthesis. Dihydroxyphenylalanine (10 -3 M), Cu2÷and Zn2÷were added to spherulating cultures of Physarum. The mold was precipitated, phenols and polyphenols were extracted with 5 ml 1 M KOH at 100°C for 3 h and quantitatively determined. Results show that addition of precursors, such as DOPA, and bivalent cations

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F i g . 3. C h a n g e s i n p h e n o l c o n t e n t in s p o r u l a t i n g c u l t u r e s o f P. polycephaZum. F i g . 4. P o l y p h e n o l o x i d a s e a c t i v i t y in e x t r a c t s f r o m s p o r u l a t i n g (A) a n d s p h e r u l a t i n g (o) c u l t u x e s a n d g r o w i n g p l a s m o d i a (e).

152 TABLE I T H E E F F E C T O F M E L A N I N P R E C U R S O R S ON P O L Y P H E N O L C O N T E N T I N S P H E R U L E S Treatment

Polyphenol content (,ug/ml)

Control Cu 2+ + Z n 2+ DOPA D O P A + Cu 2+ D O P A + Cu 2+ + Zn 2+

5.2 6.1 9.5 12.0 21.2

essential for polyphenol oxidase activity, significantly increases polyphenol content in the spherules which indeed, became reddish-brown instead of the natural yellow pigment in the control (Table I). On the other hand, melanin inhibitors were added to the medium of sporulating cultures. Thioglycolic acid (10 -3 M), phenylthiourea (10 -3 M) and amino benzoic acid (10 -2 M) partially inhibited melanin synthesis. The most effective melanin inhibitor was found to be Na2 EDTA which at 2 - 1 0 -3 M partially inhibited melanogenesis and at concentrations higher than 5 • 10 -3 M completely blocked melanin synthesis. A

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Fig. 5. Gel s c a n n e r t r a c i n g of e l e c t r o p h o r e t i c p a t t e r n s o f p o l y P h e n o l o x i d a s e e x t r a c t e d f r o m sPo~lai~a_g cultures: A , g r o w i n g p | ~ o d i a ; B, p l a s m o d i a , 2 h a f t e r i l l u m i n a t i o n ; C0 p l u m o d i a , 5 h a f t e r i l l u m i n a t i o n ; D, plP-~m~odia~ 12 h a f t e r i l l u m i n a t i o n ; E, b r o w n i s h - y e l l o w spores; F, d a r k m a t u r e spores. Fig. 6. G e l s c a n n e r t r a c i n g o f e l e c t r o p h o r e t i c p a t t e r n s of p o l y p h e n o l o x i d a s e f r o m c u l t u r e s e x p o s e d t o 2 4 h i l l u m i n a t i o n w i t h o u t ( A ) o r in t h e p r e s e n c e o f (B) N a 2 E D T A .

153

This inhibition did not stop the morphogenetic process and only caused the formation of yellow sporangia. Upon addition of 3 • 10 -6 M Cu2÷to 3 • 10 -3 M Na~ EDTA-supplemented medium, melanin synthesis occurred and completely black sporangia appeared in the cultures.

Polyphenol oxidase (PPO) activity A kinetic study of polyphenol oxidase activity revealed a very active enzyme present in sporulating cultures whereas a very low activity was found in spherules (Fig. 4). Similarly, the activity of isoenzymes of polyphenol oxidase from spherulating cultures was very low and only one weak band was observed. In contrast, the activity of the isoenzymes extracted from sporulating plasmodia was very high. Fig. 3 reveals the increasing number of bands of PPO capable of oxidizing DOPA, which appear during illumination and before the completion of sporangia maturation. A significant decrease in isoenzymes was observed after melanin synthesis. In the presence of Na2 EDTA a very low activity and decreased number of isoenzymes were noted (Fig. 6).

Catechol peroxidase In a growing culture, only one defined band and a wide region of activity of catechol peroxidase could be detected on the polyacrylamide gels (Fig. 7). After illumination, the activity increased and more isoenzymes could be

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154

detected. The maximal number of isoenzymes, seven, was found 12 h after illumination, just before melanin synthesis took place. When melanogenesis was completed, the number of isoenzymes decreased to a similar situation which was detected in growing cultures (Fig. 7). Discussion

The formation of dark-brown or black pigment during the development of spores [16] or sclerotia [7,17] is a well-known phenomenon in fungi. P. polycephalum has two types of differentiation: sporulation and spherulation. Whereas spherule formation is :induced by starvation alone, for sporulation the starvation has to be followed by illumination [18]. McCormick et al. [5] analysed spore and spherule walls and found only quantitative differences between the two, except for the presence of melanin in the spores. Our data show iden: tical absorbance spectra of both the melanin extracted from spores and the phenols and polyphenols extracted from spherules. This may indicate that the spherules contain the substrates for melanin formation, although the pigment is not synthesized. Induction of pigment biosynthesis by DOPA, Cu:÷and Zn 2÷ resulted in brownish-yellow spherules containing increased amounts of phenols and a reddish-brown pfgment, probably dopachrome [19]. Enzymatic study revealed that spherulating cultures contain only traces of polyphenol oxidase activity and the enzyme appeared as only one band in the isoelectric focusing polyacrylamide gels. In contrast, sporulating cultures showed a very high activ;_ty of this enzyme which is directly involved in melanin synthesis [17]. Growing cultures showed only few isoenzymes, but, after illumination, new ones appeared increasing significantly towards sporulation and decreasing again after the completion o f the morphogenetic process. A similar effect was also observed when isoenzymes of catechol peroxidase were detected in the gels. It seems that inactive enzymes, present in the plasmodia, are activated by illumination and therefore, detected during sporulation. Chet and Henis [17,20] found that Na2 EDTA completely inhibited melanogenesis

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155

in sclerotia of Sclerotium rolfsii. Upon addition of Cu 2÷ the effect of the chelating agent was prevented since Cu 2÷ is essential for the activity of polyphenol oxidase. Our data show that an identical system is present in P: polycephalum and indeed, only yellow sporangia were formed in the presence of Na2 EDTA, but when Cu 2÷ were added, black sporangia could be observed. Similar effects were also achieved by other inhibitors, such as phenylthiourea [21]. Inhibition of melanogenesis did n o t interfere with initiation and formation of sporangia. Thus, it indicates that melanin production is involved in maturation Of sporangia rather than in their formation. On t h e basis of the res'ults obtained in this study it may be concluded that both types of structures are more similar to each other than it seems, and that they have some c o m m o n biochemical pathways and fine control during the early stages of differentiation (Fig. 8). Acknowledgements The work was supported by a grant from the Deutsche Forschungsgemeinschaft. We wish to thank Dr. J.W. Daniel for giving us access to unpublished material on this topic, and to Ms. R. Govrin for her excellent technical aqsi.~tance. References 1 Daniel, J.W. and Baldwin, H.A. (1963) in Methods in Cell Physiology (Prescott, D.M., ed.), Vol. I , pp. 9---41, A c a d e m i c Press, N e w York 2 Ward, J.W. and Haviz. E.A. (1957) Biochim. Biophys. A e t a 25, 440--442 3 Daniel, J.W. (1963) J. Cell Biol. 19, 18A 4 Daniel, J.W. (1966) in S y n c h r o n y Studies in Biosynthetic regulations (Cameron , I.L. and Padilla, G.M., eds.), pp. 117--152, A c a d e m i c Press, N e w York 5 McCormick, J~I., Blomquist, J.C. and Rusch, H.P. (1970) J. Bacteriol. 104, 1~19--1125 6 Chet, I. and Rusch, H.P. (1969) J. Bacteriol. 100, 673--678 7 Chet, I., Henls0 Y. and Mitchell, R. (1967) Can. J. Microbiol. 13, 137--141 8 Barton, G.M., Evans, R.S. ~ind Gardner, J.A.F. (1952) Nature 170, 2 ~ 9 Leonard, T.J. (1971) J. Bacteriol. 106, 162--167 10 Lowry, O.H., Rosebzough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 11 Wrigley, C. (1968) Scl. Tools 15, 17--23 12 Strafford, H.A. a n d Galston,~A.W. (1970) Plant Physiol. 46, 763--767 1 3 Menchez, J.R. and Heim, A.H. ( 1 9 6 2 ) J . Gen. Microbiol. 28, 665--670 14 'Gornall, A.G,, Bardawill, C.J. and David, M.M. (1949) J. Biol. Chem, 177, 751--766 1 5 Schaeffer, P. (1953) Arch. Biochem. Biophys. 47, 359--379 16 Kuo, M.J. and Alexander, M. (1967) J. Bacteriol. 94, 624--629 17 Chet, I. and Henis, Y. (1969) Soil Biol. Biochem. 1, 131--138 18 Rusch, H.P. (1969) Fed. Proe. 28, 1761--1770 19 Thomson, R.H. (1962) in Comparative Biochemistry (Florkin, M. and Mason, H.S.0 eds.), Vol. 3, PP. 727--753, A c a d e m i c Press, N e w York 20 Chet0 I. and Henis0 Y. (1968) J. Gen. Microbioi. 54, 231--236 21 Whittaker, J.R. (1966) Dev. Biol. 14, 1--7