Planta (Berl.)128, 143-148 (1976)
Planta 9 by Springer-Verlag 1976
The Control of Fruiting Body Formation in the Ascomycete Sordaria macrospora Auersw. by Arginine and Biotin: a Two-factor Analysis R. Molowitz, M. Bahn, and B. Hock* Arbeitsgruppe Biochemieder Morphogenese, Ruhr-Universit/~t Bochum, Postfach 2148, D-4630 Bochum Federal Republic of Germany
Summary. Fruiting body formation of Sordaria macrospora Auersw. is controlled by L-arginine and biotin
when the fungus is grown on a synthetic nutrient medium containing optimal concentrations of fructose, KNO3, KH2PO4, MgSO4, and ZnSO4. Arginine and biotin operate in very low concentrations which exclude unspecific nutrient effects. In spite of the complicated interactions of arginine and biotin which are shown qualitatively (Figs. 3 and 4a) and quantitatively (Figs. 2 and 4b), the following conclusions are reached: 1. In the absence of biotin, the development of Sordaria macrospora is blocked at the stage of small protoperithecia. The external addition of biotin (optimal concentration: 3-12 gg/1) allows the formation of fertile fruiting bodies. This effect cannot be imitated by arginine. The biotin effect is discussed in connection with stimulated R N A synthesis.-2. The developmental velocity is influenced by the external addition of arginine. Without arginine but at permissible biotin concentrations, the total life cycle takes about 10 days, in the presence of arginine (1 mM), however, about 6 days. - 3. The hyphal density, as well as the total number of fruiting bodies being produced, is controlled in a similar manner by biotin and arginine. The induction of fruiting body formation obviously takes place after the transgression of a critical hyphal density.
Introduction
Since the early investigations of G. Klebs (1900) on the reproduction of fungi, an enormous array of facts substantiates Klebs Law in its most general form: The conditions for vegetative growth and for repro* Author to whom inquiries should be sent.
duction are different; in lower organisms, environmental factors generally control the transition from growth to reproduction. In the ascomycete Sordaria macrospora, this principle is confirmed by the observation that starch agar only allows vegetative growth. The addition of a purified fraction from corn meal extract, however, induces the formation of fruiting bodies (Hock and Bahn, 1973). A further purification of the corn meal extract as well as a close examination of the general nutrient requirements of Sordaria macrospora have shown that fruiting body formation takes place in the presence of starch as carbon source, biotin, arginine, and the mineral salts KNO3, KH2PO4, MgSO4, and ZnSO4 (Bahn and Hock, 1974). If, however, either one of the five compounds starch, biotin, arginine, KNOa, or KH2PO4 is omitted only vegetative growth is observed. The presence of MgSO4 and ZnSO4 merely improves fruiting body formation under permissive conditions. Consequently, a multifactorial system has to be taken into account if morphogenesis of Sordaria macrospora is studied. Sordaria sharply differs from other systems where the lack of nutrients induces the formation of reproductive structures (e.g. Dictyostelium, Saprolegnia). In fact, the results gained with Sordaria contradict the more special statement of Klebs (1900, p. 161), that reproduction is initiated by factors which check growth. In Sordaria a more intensive branching of the hyphae can always be observed under conditions which permit fruiting body formation than under those which do not. The advantage of using a system of the Sordaria type for morphogenetic studies is obvious. In contrast to other fungal systems where the morphogenetic stimulus is provided by the removal of nutrients or by physical factors (e.g. changes of temperature, light intensity etc.), the stimulus provided by the addition of an exactly defined compound seems to guarantee
144
better chances for identifying the control mechanisms of morphogenesis since the fate of an added compound which can be traced in the cell should eventually lead to the crucial reaction(s). Since in Sordaria macrospora there are five compounds indispensable for fruiting body formation the question arises, which factor is closest to the crucial reaction(s)? Of course, it is possible to set up factorial experiments in which the contribution of all five, relevant compounds can be elicited. It can be foreseen, however, that in spite of an immense expenditure of experimental apparatus only a formalism will be gained which will be hardly interpretable in terms of causal relations. We follow a middle course between the very complex multifactorial analysis and the usual strategy, only varying the essential factors one at a time. In the present paper, several two-factor analyses are carried out with biotin and arginine as variables. Instead of starch, fructose is used, being a better defined carbon source. Concentrations of fructose and the mineral salts mentioned above are constantly held at a previously determined optimal level (Bahn and Hock, 1974), because no simple and straightforward relationship between these compounds and their morphogenetic control can be expected. In spite of the complicated interactions between biotin and arginine, it is shown that these two compounds control different processes participating in the morphogenesis of Sordaria macrospora. Material and Methods
R. Molowitz et al. : Fruiting Body Formation in Sordaria
Fig. 1. Determination of the n u m b e r of fruiting bodies. Fruiting bodies within the 4 perpendicular sectors comprising 10% of the total area were recorded
Table 1. N u m b e r of fruiting bodies recorded by the method shown in Fig. 1. In order to register the statistical reliability of the sampling method, the total counts of the plate (Fig. 1) were recorded a n d compared to the counts recorded by the model covering 10% of the total area. After each counting the model was turned through an angle of 9 ~ Total n u m b e r of fruiting bodies per plate: Mean obtained by the model covering 10% of the total area: Actual counts registered by the model: 846, 838, 827, 816, 832, 801,842, 824, 825,797 Standard deviation: Variation coefficient:
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Material The wild strain K * d from Sordaria macrospora Auersw. (collection Prof. Esser, Lehrstuhl f/ir Allgemeine Botanik, Ruhr-Universitfit Bochum) was used. Test cultures on starch agar, allowing only vegetative growth, were obtained according to Bahn and Hock (1974) and used for the inoculation of nutrient agar plates.
Nutrient Agar The following components were dissolved to 1 1 bidistilled water: 12 g fructose (p.A.), 2.2 g K N O 3 (p.A.), 1.0 g KH2PO4 (p.A.), 0.5 g MgSO4 (p.A.), 0.2 m g ZnSO4 (p.A.), 15 g agar (purest grade), arginine and biotin as indicated in the results. The p H was adjusted with K O H to 6.0. Biotin and agar were obtained from Serva, all other c o m p o u n d s from Merck. The media were sterilized by autoclaving for 30 min at 120 ~ C. N o differences in growth or formation of fruiting bodies could be observed if the ingredients were sterilized by sterile filtration. Sterile plastic petri dishes (9 cm diameter) were each filled with 20 ml nutrient agar.
Determination of the Number o f Fruiting Bodies The plates were photographed. The films were enlarged with a slide projector to a final plate diameter of 73 cm. Aided by a model outlining 10% of the total plate area by 4 perpendicular sectors with an angle of 9 degrees (Fig. 1), fruiting bodies with a m i n i m u m diameter of 200 g m (perithecia and transition stages from protoperithecia to perithecia) were counted manually. The total counts from all 4 sectors were subsequently multiplied by 10. The statistical reliability of the samples resembling a total of 10% from the plate area is indicated by Table 1. The results show that the error obtained by this sampling method is within acceptable limits. - F r o m each type of nutrient agar, 4 plates were counted. The calculations of the m e a n ~, the sample standard deviation s, and the s t a n d a r d deviation of the m e a n sx were carried out according to D o c u m e n t a Geigy (1960).
Determination of Dry Weight Cultures The plates were inoculated in the centre with small agar blocks (2 x 2 x 5 m m ) from 5-day old test cultures and incubated at 27 ~ C in the dark.
To separate the mycelium from the agar, the contents of each petri dish were emptied into a beaker containing 50 ml boiling water. The mycelium was collected with a Buchner funnel on a pre-weighed filter paper (Macherey,Nagel & Co.- Dfiren, type M N
R. Molowitz et al. : Fruiting Body Formation in Sordaria 617), washed 3 times with boiling water to remove any residues of agar, and dried for 20 h at 100 ~ C. After cooling for 4 h in a desiccator, the dry weight was determined.
145 10000
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Results
The life cycle of the monoecious and self-compatible ascomycete Sordaria rnacrospora Auersw. has been reviewed by Esser and Kuenen (1965) and Olive (1974). Within a few days after the cultures are started, ascogonia (female sexual organs) appear on the hyphae as hook or coil-shaped outgrowths from hyphae and become centres of perithecial development. Male sexual organs and conidia are not produced. The ascogonia become surrounded by sterile hyphae. At this stage, they are called protoperithecia. For the time being, the ascogonia contain one, at most two pairs of nuclei as deduced by genetic evidence (Esser and Straub, 1958). After passing through conjugated divisions, the nuclei migrate in pairs to the ascogenous hyphae which are produced by the ascogonia. The development of the asci with 8 ascospores follows. The formation of asci is preceded by a conspicuous enlargement of enveloping tissue forming the perithecial wall.
The ability of the fungus to change from vegetative growth to reproduction is the result of a combination of genetic competence and the effect of environmental factors. Whereas a considerable amount of genetic information has been made available, especially by Esser and Straub (1958), the contribution of environmental factors are poorly understood mainly because of the lack of defined synthetic nutrient media. With the aid of factorial analysis, Bahn and Hock (1974) compiled a suitable medium for Sordaria macrospora. The experiments communicated in this paper are carried out on this medium with the exception that fructose substitutes starch. In Fig. 2, the number of fruiting bodies as a function of biotin and arginine concentrations is shown for three different developmental stages. As fruiting bodies, perithecia and transition stages from protoperithecia to perithecia exceeding a diameter of 200 btm were recorded. For each of the 5 biotin concentrations which were selected for this experiment and ranged from 0.19 l.tg/1 to 3.0 pg/1, 11 different arginine concentrations were used in the range from 0 mM to 1.6 mM. Each individual experiment was carried out in 4 parallels. The curves were fitted by eye. Fig. 2 shows that the number of fruiting bodies as well as the velocity of fruiting body formation depends upon the concentrations of arginine and biotin. Without biotin, even after a long period of time, no fruiting bodies are produced. The threshold allowing fruiting body formation lies between 0.2 and 0.3 btg/1 biotin. A broad optimum is reached between 1.5 and 12 gg/1 biotin. (The curves above 3.0 btg/1 bio-
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tin, being very similar, are omitted in the diagrams). The time until the optimum is reached depends upon the age of the cultures and the arginine concentration. At day 7, the plateau is attained with ca. 1 mM arginine, at day 9, however, already with ca. 0.1 mM arginine. The most obvious effect of arginine, therefore, is an acceleration of fruiting body formation. With permissible concentrations of biotin, no fruiting bodies are found at day 5 if arginine is omitted from the nutrient medium. With the optimal arginine concentration, however, about 87% of the maximum number is reached at this stage. Four days later, the
146
R. Molowitz et al. : Fruiting Body Formation in Sordaria i
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differences between these two types of nutrient media are smaller but still significant. This means that fructose agar differs from starch agar used in preceding papers (Bahn and Hock, 1973; Bahn and Hock, 1974). The effect of arginine is only quantitative if fructose is used instead of starch. Fig. 3 shows the developmental stages reached at 5, 7, and 9 days after inoculation at different concentrations of arginine and biotin. In this figure, the acceleration of the development by arginine with respect to the different stages of fruiting body formation is shown at each level of biotin. At biotin concentrations of 0.75 gg/1 and lower, exclusively anomalous perithecia are produced containing either empty asci or asci with a few unripe spores. Only biotin concentrations of 1.5 pg/1 and higher allow the formation of normal perithecia. Cinematographic studies in our laboratory have indicated a close correlation between the density of hyphae and fruiting body production: If arginine is added to young cultures grown on the nutrient agar containing biotin but no arginine, an immediate increase of hyphal branching is observed followed by a fast perithecia formation. Under these conditions
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Fig. 4 a and b. Correlation between dry weight and fruiting body formation. (a) Influence of arginine (1 mM) and biotin (6 lxg/l) on fruiting body formation with respect to time. + and - indicate the presence or absence of the indicated compound. ~ mycelium, ~. small protoperithecia, t1~ protoperithecia, ~) perithecia without spores, ~)perithecia with unripe spores, (~ perithecia with ripe spores, ~ perithecia with ejected spores. (b) Influence of arginine (1 m M ) and biotin (6 gg/1) on the dry weight. Each point represents the mean of 4 independent experiments. The standard deviation of the mean is indicated, o - - o arginine +biotin, o .... o biotin, no arginine, e - - o arginine, no biotin, 9 --- 9 without arginine and biotin
the number of perithecia is far above the average. To find out whether this result can be generalized, the total mass of hyphae was determined by dry weight estimations from cultures grown for different lengths of time on nutrient agar with 4 combinations of arginine and biotin (Fig. 4b). The linear growth of the fungus is little influenced by biotin or arginine if applied separately whereas the combination of both compounds accelerates the linear growth about twofold (unpublished results). More pronounced effects are observed at the level of the hyphal density. At least during the first 3 days after inoculation, the mass of the mycelium is represented correctly by dry weight estimations. Later on, the contribution of perithecia and their precursors has to be considered. The curves shown in Fig. 4b confirm the correlation between the hyphal mass and the ability of perithecia production. The developmental stages reached at each point are indicated by Fig. 4a. Without biotin (irrespective of the arginine concentration), dry weights
R. Molowitz et al. : Fruiting Body Formation in Sordaria
remain at a low level. In this case, the stage of small protoperithecia cannot be transgressed. In the presence of biotin, however, even at the stage of small protoperithecia, the total mass is significantly higher. The correlation between mycelium mass and fruiting body production is also demonstrated by the comparison of cultures grown on biotin in the presence and absence of arginine. With the aminoacid, the development is accelerated. At the same time, an earlier increase of dry weight compared to the control was observed. In fact, differences in the dry weight are the earliest indication of arginine action. Arginine, on the other hand, also causes an early decline of the total mass resulting from autolysis.
Discussion
In this paper, a defined physiological control of fruiting body formation in Sordaria rnacrospora grown on a simple nutrient medium is described. Using the external concentrations of L-arginine and biotin as independent variables, two-factor analyses with respect to fruiting body formation are carried out. Based on these results, important prerequisites are given for the analysis of the mechanism effecting the transition from the vegetative to the reproductive phase. The results presented in this paper lead to the following conclusions: 1. Biotin controls the development succeeding early protoperithecial stages. Without biotin, the fungus is unable to produce perithecia. The number of perithecia is a function of the biotin concentration. The importance of biotin for fruiting body formation in the related species, Sordariafimicola, is already emphasized by Barnett and Lilly (1947). With the aid of some rough estimations of fruiting body numbers, comparable ranges of biotin requirements are found. In the case of Sordaria fimicola, however, biotin controls the formation of ascogonia. The function of biotin with respect to differentiation is not yet known. In any case, it must be assumed that Sordaria rnacrospora is able to synthesize just enough biotin to allow for vegetative growth. It is likely that a carboxylation reaction is limited by biotin and that the coenzyme is required in a mechanism taking part in fruiting body formation. possibly the synthesis of pyrimidine nucleotides requiring carbamyl phosphate as a precursor. As biotin functions as a prosthetic group of the carbamyl phosphate synthesis (Wellner et al. 1968) it is possible to construct the following sequence: Biotin application~synthesis of new RNA~induction of fruiting body formation. It is important that in Sordaria ~rni-
147
cola fruiting body formation can be selectively inhibited by 1 gM 5-fluorouracil without seriously inhibiting vegetative growth (Lindenmayer and Schoen, 1967). These experiments which have been confirmed by us for Sordaria rnacrospora (unpublished results) indicate that for fruiting body formation, the synthesis of new RNA is indeed necessary. In this respect, reports of Boeckx and Dakshinamurti (1975) on the action of biotin on R N A synthesis are of special interest: When biotin deficient rats receive a single injection of biotin a twofold increase in the incorporation, both in vivo and in vitro of precursors into nuclear RNA is observed as early as 2 h after the biotin treatment. The authors provide evidence that biotin is transported at an early stage to the nuclei. 2. The external application of L-arginine influences the velocity of the development cycle. This effect can be observed in the presence and absence of biotin (Fig. 4a). The developmental block at the stage of small protoperithecia, however, can be overcome only by biotin. Cultures grown on nutrient media containing biotin but no arginine reach the stage of perithecia ejecting spores within about 10 days, in the presence of both substances within about 6 days. The number of fruiting bodies is also considerably higher in the presence of arginine. Analogous conclusions for the action of arginine were drawn by Tinh and Griffiths (1974) for heterotrophic cultures of Chlorella (Emerson strain) grown on glucose. Here, arginine (2 mM) also accelerates the developmental cycle which is completed by the autospore production. The stimulatory effect of arginine is interpreted as an early attainment of a critical cell size depending on RNA synthesis. The authors assume an involvement of arginine in the mechanism controlling the onset of cell division. The participation of arginine in the control of morphogenesis seems to be a more general phenomenon. In other fungi (Podospora anserina, Polyporus ciliatus), fruiting body formation is also stimulated by arginine (unpublished results). 3. If Sordaria macrospora is grown on starch instead of fructose an intriguing problem appears from the observation that in this case both compounds biotin and arginine are obligatory for regular fruiting body formation (Bahn and Hock, 1973). Since the agar used in all our experiments does not contain any aminoacids it must be assumed that in the presence of fructose the fungus is able to produce levels of arginine or a related compound high enough to allow normal fruiting body formation. The delay of fruiting body formation in the absence of external arginine is then explained by a delayed synthesis of new enzymes of arginine biosynthesis. 4. A strong correlation between the total hyphal
148 mass (measured as dry weight) a n d the ability o f fruiting b o d y f o r m a t i o n is observed. I n fact, the addition o f any factor limiting fruiting b o d y f o r m a t i o n causes an immediate increase o f the h y p h a l mass at least any protoperithecia are produced. 24h before Obviously, there is a critical h y p h a l density which m u s t be reached if fruiting bodies are to be formed. Arginine and biotin influence this critical density in a complicated interaction. Fig. 4 b shows that the m a i n effect o f arginine is seen only in the presence but n o t in the absence o f biotin. A simple relation between these two c o m p o u n d s could be obscured by the possibility that after some delay S o r d a r i a synthesizes arginine or related molecules in the presence o f fructose. T h e a s s u m p t i o n o f a critical h y p h a l density is further supported by the observation (Bahn a n d H o c k , 1973) that any mechanical device enforcing a denser vegetative g r o w t h (e.g. a V-like obstacle) accelerates the fruiting b o d y formation. A causal relation between the increase o f the h y p h a l density and the fruiting b o d y f o r m a t i o n agrees with Klebs (1900, rule IV, p. 167) w h o considers g r o w t h as the first step and prerequisite o f reproduction because o f the longer nutrition phase during growth. H a w k e r (1967) argues on a similar basis if he assumes that perithecia are initiated only when a generally high level o f m e t a b o l i s m is maintained. A l t h o u g h it is t o o early to speculate a b o u t a detailed m e c h a n i s m o f fruiting b o d y induction, the effects o f arginine and biotin as described in this paper provide an experimental system suitable for further investigation. W e h o p e that the use o f m o r p h o l o g i c a l m u t a n t s together with biochemical investigations will provide a potent instrument for analysis o f the control mechanisms o f morphogenesis.
R. Molowitz etal.: Fruiting Body Formation in Sordaria We gratefully acknowledge the assistance of Mrs. U. Haberland as well as the financial support from the Deutsche Forschnngsgemeinschaft. We thank Mr. R.-A. Walk for valuable discussions.
References Bahn, M., Hock, B.: Morphogenese yon Sordaria: Die Induktion der Perithezienbildung. Ber. dtsch. Bot. Ges. 86, 309-311 (1973) Bahn, M., Hock, B.: Morphogenese von Sordaria: Induktion der Perithezienbildung dnrch Arginin. Ber. dtsch. Bot. Ges. 87, 433-442 (1974) Barnett, H.L., Lilly, B.G. : The effects of biotin upon the formation and development ofperithecia, asci, and ascospores by Sordaria fimicola Ces. and de Not. Amer. J. Bot. 34, 196~04 (1947) Boeckx, R.L., Dakshinamurti, K.: Effect of biotin on ribonucleic acid synthesis. Biochim. biophys. Acta (Amst.) 383, 282-289 (1975) Documenta Geigy. Wissenschaftliche Tabellen. J.R. Geigy A.G., 7. Aufl., Basel 1968 Esser, K., Kuenen, R.: Geuetik der Pilze. Berlin-Heidelberg-New York: Springer 1965 Esser, K., Straub, J. : Genetische Untersuchungen an Sordaria macrospora Auersw., Kompensation und Induktion bei genbedingten Entwicklungsdefekten. Z. Vererbungsl. 89, 729-746 (1958) Hawker, L.E. : The physiology of reproduction in fungi. Cambridge University Press 1957 Klebs, G.: Zur Physiologie der Fortpflanzung einiger Prize. III. Allgemeine Betrachtungen. Jb. Wiss. Bot. 35, 80 203 (1900) Lindenmayer, A., Schoen, H.F.: Selective effects of purine and pyrimidine analogues and of respiratory inhibitors on perithecial development and branching in Sordaria. Plant Physiol. 42, 1059-1070 (1967) Olive, L.S. : Sordaria. In: Handbook of genetics, pp, 553 561, Vol I. Ed: R.C. King. New York-London: Plenum Press 1974 Thinh. L.V., Griffiths, D.J. : The effect of L-arginine on the growth of heterotrophic cultures of the Emerson strain of Chlorella. I. Effects on cell growth, cell division, chloroplast development and nucleic acid synthesis. New Phytol. 73, 1087-1095 (1974) Wellner, V.P., Santos, J.L., Meister, A. : Carbamyl phosphate synthetase. A biotin enzyme. Biochemistry 7, 2848-2851 (1968) Received 10 September; accepted 1 October 1975
Planta 9 by Springer-Verlag 1976
The Control of Fruiting Body Formation in the Ascomycete Sordaria macrospora Auersw. by Arginine and Biotin: a Two-factor Analysis R. Molowitz, M. Bahn, and B. Hock* Arbeitsgruppe Biochemieder Morphogenese, Ruhr-Universit/~t Bochum, Postfach 2148, D-4630 Bochum Federal Republic of Germany
Summary. Fruiting body formation of Sordaria macrospora Auersw. is controlled by L-arginine and biotin
when the fungus is grown on a synthetic nutrient medium containing optimal concentrations of fructose, KNO3, KH2PO4, MgSO4, and ZnSO4. Arginine and biotin operate in very low concentrations which exclude unspecific nutrient effects. In spite of the complicated interactions of arginine and biotin which are shown qualitatively (Figs. 3 and 4a) and quantitatively (Figs. 2 and 4b), the following conclusions are reached: 1. In the absence of biotin, the development of Sordaria macrospora is blocked at the stage of small protoperithecia. The external addition of biotin (optimal concentration: 3-12 gg/1) allows the formation of fertile fruiting bodies. This effect cannot be imitated by arginine. The biotin effect is discussed in connection with stimulated R N A synthesis.-2. The developmental velocity is influenced by the external addition of arginine. Without arginine but at permissible biotin concentrations, the total life cycle takes about 10 days, in the presence of arginine (1 mM), however, about 6 days. - 3. The hyphal density, as well as the total number of fruiting bodies being produced, is controlled in a similar manner by biotin and arginine. The induction of fruiting body formation obviously takes place after the transgression of a critical hyphal density.
Introduction
Since the early investigations of G. Klebs (1900) on the reproduction of fungi, an enormous array of facts substantiates Klebs Law in its most general form: The conditions for vegetative growth and for repro* Author to whom inquiries should be sent.
duction are different; in lower organisms, environmental factors generally control the transition from growth to reproduction. In the ascomycete Sordaria macrospora, this principle is confirmed by the observation that starch agar only allows vegetative growth. The addition of a purified fraction from corn meal extract, however, induces the formation of fruiting bodies (Hock and Bahn, 1973). A further purification of the corn meal extract as well as a close examination of the general nutrient requirements of Sordaria macrospora have shown that fruiting body formation takes place in the presence of starch as carbon source, biotin, arginine, and the mineral salts KNO3, KH2PO4, MgSO4, and ZnSO4 (Bahn and Hock, 1974). If, however, either one of the five compounds starch, biotin, arginine, KNOa, or KH2PO4 is omitted only vegetative growth is observed. The presence of MgSO4 and ZnSO4 merely improves fruiting body formation under permissive conditions. Consequently, a multifactorial system has to be taken into account if morphogenesis of Sordaria macrospora is studied. Sordaria sharply differs from other systems where the lack of nutrients induces the formation of reproductive structures (e.g. Dictyostelium, Saprolegnia). In fact, the results gained with Sordaria contradict the more special statement of Klebs (1900, p. 161), that reproduction is initiated by factors which check growth. In Sordaria a more intensive branching of the hyphae can always be observed under conditions which permit fruiting body formation than under those which do not. The advantage of using a system of the Sordaria type for morphogenetic studies is obvious. In contrast to other fungal systems where the morphogenetic stimulus is provided by the removal of nutrients or by physical factors (e.g. changes of temperature, light intensity etc.), the stimulus provided by the addition of an exactly defined compound seems to guarantee
144
better chances for identifying the control mechanisms of morphogenesis since the fate of an added compound which can be traced in the cell should eventually lead to the crucial reaction(s). Since in Sordaria macrospora there are five compounds indispensable for fruiting body formation the question arises, which factor is closest to the crucial reaction(s)? Of course, it is possible to set up factorial experiments in which the contribution of all five, relevant compounds can be elicited. It can be foreseen, however, that in spite of an immense expenditure of experimental apparatus only a formalism will be gained which will be hardly interpretable in terms of causal relations. We follow a middle course between the very complex multifactorial analysis and the usual strategy, only varying the essential factors one at a time. In the present paper, several two-factor analyses are carried out with biotin and arginine as variables. Instead of starch, fructose is used, being a better defined carbon source. Concentrations of fructose and the mineral salts mentioned above are constantly held at a previously determined optimal level (Bahn and Hock, 1974), because no simple and straightforward relationship between these compounds and their morphogenetic control can be expected. In spite of the complicated interactions between biotin and arginine, it is shown that these two compounds control different processes participating in the morphogenesis of Sordaria macrospora. Material and Methods
R. Molowitz et al. : Fruiting Body Formation in Sordaria
Fig. 1. Determination of the n u m b e r of fruiting bodies. Fruiting bodies within the 4 perpendicular sectors comprising 10% of the total area were recorded
Table 1. N u m b e r of fruiting bodies recorded by the method shown in Fig. 1. In order to register the statistical reliability of the sampling method, the total counts of the plate (Fig. 1) were recorded a n d compared to the counts recorded by the model covering 10% of the total area. After each counting the model was turned through an angle of 9 ~ Total n u m b e r of fruiting bodies per plate: Mean obtained by the model covering 10% of the total area: Actual counts registered by the model: 846, 838, 827, 816, 832, 801,842, 824, 825,797 Standard deviation: Variation coefficient:
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Material The wild strain K * d from Sordaria macrospora Auersw. (collection Prof. Esser, Lehrstuhl f/ir Allgemeine Botanik, Ruhr-Universitfit Bochum) was used. Test cultures on starch agar, allowing only vegetative growth, were obtained according to Bahn and Hock (1974) and used for the inoculation of nutrient agar plates.
Nutrient Agar The following components were dissolved to 1 1 bidistilled water: 12 g fructose (p.A.), 2.2 g K N O 3 (p.A.), 1.0 g KH2PO4 (p.A.), 0.5 g MgSO4 (p.A.), 0.2 m g ZnSO4 (p.A.), 15 g agar (purest grade), arginine and biotin as indicated in the results. The p H was adjusted with K O H to 6.0. Biotin and agar were obtained from Serva, all other c o m p o u n d s from Merck. The media were sterilized by autoclaving for 30 min at 120 ~ C. N o differences in growth or formation of fruiting bodies could be observed if the ingredients were sterilized by sterile filtration. Sterile plastic petri dishes (9 cm diameter) were each filled with 20 ml nutrient agar.
Determination of the Number o f Fruiting Bodies The plates were photographed. The films were enlarged with a slide projector to a final plate diameter of 73 cm. Aided by a model outlining 10% of the total plate area by 4 perpendicular sectors with an angle of 9 degrees (Fig. 1), fruiting bodies with a m i n i m u m diameter of 200 g m (perithecia and transition stages from protoperithecia to perithecia) were counted manually. The total counts from all 4 sectors were subsequently multiplied by 10. The statistical reliability of the samples resembling a total of 10% from the plate area is indicated by Table 1. The results show that the error obtained by this sampling method is within acceptable limits. - F r o m each type of nutrient agar, 4 plates were counted. The calculations of the m e a n ~, the sample standard deviation s, and the s t a n d a r d deviation of the m e a n sx were carried out according to D o c u m e n t a Geigy (1960).
Determination of Dry Weight Cultures The plates were inoculated in the centre with small agar blocks (2 x 2 x 5 m m ) from 5-day old test cultures and incubated at 27 ~ C in the dark.
To separate the mycelium from the agar, the contents of each petri dish were emptied into a beaker containing 50 ml boiling water. The mycelium was collected with a Buchner funnel on a pre-weighed filter paper (Macherey,Nagel & Co.- Dfiren, type M N
R. Molowitz et al. : Fruiting Body Formation in Sordaria 617), washed 3 times with boiling water to remove any residues of agar, and dried for 20 h at 100 ~ C. After cooling for 4 h in a desiccator, the dry weight was determined.
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Results
The life cycle of the monoecious and self-compatible ascomycete Sordaria rnacrospora Auersw. has been reviewed by Esser and Kuenen (1965) and Olive (1974). Within a few days after the cultures are started, ascogonia (female sexual organs) appear on the hyphae as hook or coil-shaped outgrowths from hyphae and become centres of perithecial development. Male sexual organs and conidia are not produced. The ascogonia become surrounded by sterile hyphae. At this stage, they are called protoperithecia. For the time being, the ascogonia contain one, at most two pairs of nuclei as deduced by genetic evidence (Esser and Straub, 1958). After passing through conjugated divisions, the nuclei migrate in pairs to the ascogenous hyphae which are produced by the ascogonia. The development of the asci with 8 ascospores follows. The formation of asci is preceded by a conspicuous enlargement of enveloping tissue forming the perithecial wall.
The ability of the fungus to change from vegetative growth to reproduction is the result of a combination of genetic competence and the effect of environmental factors. Whereas a considerable amount of genetic information has been made available, especially by Esser and Straub (1958), the contribution of environmental factors are poorly understood mainly because of the lack of defined synthetic nutrient media. With the aid of factorial analysis, Bahn and Hock (1974) compiled a suitable medium for Sordaria macrospora. The experiments communicated in this paper are carried out on this medium with the exception that fructose substitutes starch. In Fig. 2, the number of fruiting bodies as a function of biotin and arginine concentrations is shown for three different developmental stages. As fruiting bodies, perithecia and transition stages from protoperithecia to perithecia exceeding a diameter of 200 btm were recorded. For each of the 5 biotin concentrations which were selected for this experiment and ranged from 0.19 l.tg/1 to 3.0 pg/1, 11 different arginine concentrations were used in the range from 0 mM to 1.6 mM. Each individual experiment was carried out in 4 parallels. The curves were fitted by eye. Fig. 2 shows that the number of fruiting bodies as well as the velocity of fruiting body formation depends upon the concentrations of arginine and biotin. Without biotin, even after a long period of time, no fruiting bodies are produced. The threshold allowing fruiting body formation lies between 0.2 and 0.3 btg/1 biotin. A broad optimum is reached between 1.5 and 12 gg/1 biotin. (The curves above 3.0 btg/1 bio-
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tin, being very similar, are omitted in the diagrams). The time until the optimum is reached depends upon the age of the cultures and the arginine concentration. At day 7, the plateau is attained with ca. 1 mM arginine, at day 9, however, already with ca. 0.1 mM arginine. The most obvious effect of arginine, therefore, is an acceleration of fruiting body formation. With permissible concentrations of biotin, no fruiting bodies are found at day 5 if arginine is omitted from the nutrient medium. With the optimal arginine concentration, however, about 87% of the maximum number is reached at this stage. Four days later, the
146
R. Molowitz et al. : Fruiting Body Formation in Sordaria i
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differences between these two types of nutrient media are smaller but still significant. This means that fructose agar differs from starch agar used in preceding papers (Bahn and Hock, 1973; Bahn and Hock, 1974). The effect of arginine is only quantitative if fructose is used instead of starch. Fig. 3 shows the developmental stages reached at 5, 7, and 9 days after inoculation at different concentrations of arginine and biotin. In this figure, the acceleration of the development by arginine with respect to the different stages of fruiting body formation is shown at each level of biotin. At biotin concentrations of 0.75 gg/1 and lower, exclusively anomalous perithecia are produced containing either empty asci or asci with a few unripe spores. Only biotin concentrations of 1.5 pg/1 and higher allow the formation of normal perithecia. Cinematographic studies in our laboratory have indicated a close correlation between the density of hyphae and fruiting body production: If arginine is added to young cultures grown on the nutrient agar containing biotin but no arginine, an immediate increase of hyphal branching is observed followed by a fast perithecia formation. Under these conditions
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the number of perithecia is far above the average. To find out whether this result can be generalized, the total mass of hyphae was determined by dry weight estimations from cultures grown for different lengths of time on nutrient agar with 4 combinations of arginine and biotin (Fig. 4b). The linear growth of the fungus is little influenced by biotin or arginine if applied separately whereas the combination of both compounds accelerates the linear growth about twofold (unpublished results). More pronounced effects are observed at the level of the hyphal density. At least during the first 3 days after inoculation, the mass of the mycelium is represented correctly by dry weight estimations. Later on, the contribution of perithecia and their precursors has to be considered. The curves shown in Fig. 4b confirm the correlation between the hyphal mass and the ability of perithecia production. The developmental stages reached at each point are indicated by Fig. 4a. Without biotin (irrespective of the arginine concentration), dry weights
R. Molowitz et al. : Fruiting Body Formation in Sordaria
remain at a low level. In this case, the stage of small protoperithecia cannot be transgressed. In the presence of biotin, however, even at the stage of small protoperithecia, the total mass is significantly higher. The correlation between mycelium mass and fruiting body production is also demonstrated by the comparison of cultures grown on biotin in the presence and absence of arginine. With the aminoacid, the development is accelerated. At the same time, an earlier increase of dry weight compared to the control was observed. In fact, differences in the dry weight are the earliest indication of arginine action. Arginine, on the other hand, also causes an early decline of the total mass resulting from autolysis.
Discussion
In this paper, a defined physiological control of fruiting body formation in Sordaria rnacrospora grown on a simple nutrient medium is described. Using the external concentrations of L-arginine and biotin as independent variables, two-factor analyses with respect to fruiting body formation are carried out. Based on these results, important prerequisites are given for the analysis of the mechanism effecting the transition from the vegetative to the reproductive phase. The results presented in this paper lead to the following conclusions: 1. Biotin controls the development succeeding early protoperithecial stages. Without biotin, the fungus is unable to produce perithecia. The number of perithecia is a function of the biotin concentration. The importance of biotin for fruiting body formation in the related species, Sordariafimicola, is already emphasized by Barnett and Lilly (1947). With the aid of some rough estimations of fruiting body numbers, comparable ranges of biotin requirements are found. In the case of Sordaria fimicola, however, biotin controls the formation of ascogonia. The function of biotin with respect to differentiation is not yet known. In any case, it must be assumed that Sordaria rnacrospora is able to synthesize just enough biotin to allow for vegetative growth. It is likely that a carboxylation reaction is limited by biotin and that the coenzyme is required in a mechanism taking part in fruiting body formation. possibly the synthesis of pyrimidine nucleotides requiring carbamyl phosphate as a precursor. As biotin functions as a prosthetic group of the carbamyl phosphate synthesis (Wellner et al. 1968) it is possible to construct the following sequence: Biotin application~synthesis of new RNA~induction of fruiting body formation. It is important that in Sordaria ~rni-
147
cola fruiting body formation can be selectively inhibited by 1 gM 5-fluorouracil without seriously inhibiting vegetative growth (Lindenmayer and Schoen, 1967). These experiments which have been confirmed by us for Sordaria rnacrospora (unpublished results) indicate that for fruiting body formation, the synthesis of new RNA is indeed necessary. In this respect, reports of Boeckx and Dakshinamurti (1975) on the action of biotin on R N A synthesis are of special interest: When biotin deficient rats receive a single injection of biotin a twofold increase in the incorporation, both in vivo and in vitro of precursors into nuclear RNA is observed as early as 2 h after the biotin treatment. The authors provide evidence that biotin is transported at an early stage to the nuclei. 2. The external application of L-arginine influences the velocity of the development cycle. This effect can be observed in the presence and absence of biotin (Fig. 4a). The developmental block at the stage of small protoperithecia, however, can be overcome only by biotin. Cultures grown on nutrient media containing biotin but no arginine reach the stage of perithecia ejecting spores within about 10 days, in the presence of both substances within about 6 days. The number of fruiting bodies is also considerably higher in the presence of arginine. Analogous conclusions for the action of arginine were drawn by Tinh and Griffiths (1974) for heterotrophic cultures of Chlorella (Emerson strain) grown on glucose. Here, arginine (2 mM) also accelerates the developmental cycle which is completed by the autospore production. The stimulatory effect of arginine is interpreted as an early attainment of a critical cell size depending on RNA synthesis. The authors assume an involvement of arginine in the mechanism controlling the onset of cell division. The participation of arginine in the control of morphogenesis seems to be a more general phenomenon. In other fungi (Podospora anserina, Polyporus ciliatus), fruiting body formation is also stimulated by arginine (unpublished results). 3. If Sordaria macrospora is grown on starch instead of fructose an intriguing problem appears from the observation that in this case both compounds biotin and arginine are obligatory for regular fruiting body formation (Bahn and Hock, 1973). Since the agar used in all our experiments does not contain any aminoacids it must be assumed that in the presence of fructose the fungus is able to produce levels of arginine or a related compound high enough to allow normal fruiting body formation. The delay of fruiting body formation in the absence of external arginine is then explained by a delayed synthesis of new enzymes of arginine biosynthesis. 4. A strong correlation between the total hyphal
148 mass (measured as dry weight) a n d the ability o f fruiting b o d y f o r m a t i o n is observed. I n fact, the addition o f any factor limiting fruiting b o d y f o r m a t i o n causes an immediate increase o f the h y p h a l mass at least any protoperithecia are produced. 24h before Obviously, there is a critical h y p h a l density which m u s t be reached if fruiting bodies are to be formed. Arginine and biotin influence this critical density in a complicated interaction. Fig. 4 b shows that the m a i n effect o f arginine is seen only in the presence but n o t in the absence o f biotin. A simple relation between these two c o m p o u n d s could be obscured by the possibility that after some delay S o r d a r i a synthesizes arginine or related molecules in the presence o f fructose. T h e a s s u m p t i o n o f a critical h y p h a l density is further supported by the observation (Bahn a n d H o c k , 1973) that any mechanical device enforcing a denser vegetative g r o w t h (e.g. a V-like obstacle) accelerates the fruiting b o d y formation. A causal relation between the increase o f the h y p h a l density and the fruiting b o d y f o r m a t i o n agrees with Klebs (1900, rule IV, p. 167) w h o considers g r o w t h as the first step and prerequisite o f reproduction because o f the longer nutrition phase during growth. H a w k e r (1967) argues on a similar basis if he assumes that perithecia are initiated only when a generally high level o f m e t a b o l i s m is maintained. A l t h o u g h it is t o o early to speculate a b o u t a detailed m e c h a n i s m o f fruiting b o d y induction, the effects o f arginine and biotin as described in this paper provide an experimental system suitable for further investigation. W e h o p e that the use o f m o r p h o l o g i c a l m u t a n t s together with biochemical investigations will provide a potent instrument for analysis o f the control mechanisms o f morphogenesis.
R. Molowitz etal.: Fruiting Body Formation in Sordaria We gratefully acknowledge the assistance of Mrs. U. Haberland as well as the financial support from the Deutsche Forschnngsgemeinschaft. We thank Mr. R.-A. Walk for valuable discussions.
References Bahn, M., Hock, B.: Morphogenese yon Sordaria: Die Induktion der Perithezienbildung. Ber. dtsch. Bot. Ges. 86, 309-311 (1973) Bahn, M., Hock, B.: Morphogenese von Sordaria: Induktion der Perithezienbildung dnrch Arginin. Ber. dtsch. Bot. Ges. 87, 433-442 (1974) Barnett, H.L., Lilly, B.G. : The effects of biotin upon the formation and development ofperithecia, asci, and ascospores by Sordaria fimicola Ces. and de Not. Amer. J. Bot. 34, 196~04 (1947) Boeckx, R.L., Dakshinamurti, K.: Effect of biotin on ribonucleic acid synthesis. Biochim. biophys. Acta (Amst.) 383, 282-289 (1975) Documenta Geigy. Wissenschaftliche Tabellen. J.R. Geigy A.G., 7. Aufl., Basel 1968 Esser, K., Kuenen, R.: Geuetik der Pilze. Berlin-Heidelberg-New York: Springer 1965 Esser, K., Straub, J. : Genetische Untersuchungen an Sordaria macrospora Auersw., Kompensation und Induktion bei genbedingten Entwicklungsdefekten. Z. Vererbungsl. 89, 729-746 (1958) Hawker, L.E. : The physiology of reproduction in fungi. Cambridge University Press 1957 Klebs, G.: Zur Physiologie der Fortpflanzung einiger Prize. III. Allgemeine Betrachtungen. Jb. Wiss. Bot. 35, 80 203 (1900) Lindenmayer, A., Schoen, H.F.: Selective effects of purine and pyrimidine analogues and of respiratory inhibitors on perithecial development and branching in Sordaria. Plant Physiol. 42, 1059-1070 (1967) Olive, L.S. : Sordaria. In: Handbook of genetics, pp, 553 561, Vol I. Ed: R.C. King. New York-London: Plenum Press 1974 Thinh. L.V., Griffiths, D.J. : The effect of L-arginine on the growth of heterotrophic cultures of the Emerson strain of Chlorella. I. Effects on cell growth, cell division, chloroplast development and nucleic acid synthesis. New Phytol. 73, 1087-1095 (1974) Wellner, V.P., Santos, J.L., Meister, A. : Carbamyl phosphate synthetase. A biotin enzyme. Biochemistry 7, 2848-2851 (1968) Received 10 September; accepted 1 October 1975
