Lack of correlation between acid-induced respiration and germination inPhycomyces spores

EXPERIMENTAL

MYCOLOGY

8,

73-79 (1984)

Lack of Correlation between Acid-Induced Respiration Germination in Phycomyces Spores

an

MARC N. VERBEKE AND ANDRE J. VAN LAERE* Interdisciplinary Research Centre, Katholieke Universiteit Leuven, Campus Kortrijk, B-8500 Kortrijk, ar,d *Laboratory for Plantbiochemistry, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, 3030 Leuven (Heverlee), Be&urn Accepted for publication October 19, 1983 VERBEKE, M. N., AND VAN LAERE, A. J. 1984. Lack of correlation between acid-induced respiration and germination in Phycomyces spores. Experimental Mycology 8, 73-79. Solutions of pyruvic acid as well as acetic acid enhance spore respiration. However, a treatment with acetic acid activates the spores, but in solutions of pyruvic acid no activation is obtained. The enhancement of respiration does not suffice to induce the activation of germination. The concentration dependency of respiration and activation caused by acetic acid are also not identical. Acetic acid activates the spores at supra optimal concentrations that inhibit their respiration. Propionic acid and butyric acid are effective activators at much lower concentrations than acetic acid, although these substances are less useful as respiratory substrates. It is suggested that activation with monocarboxylic acids is not due to their role as a respiratory substrate. Some physicochemical effect of the activating acids appears likely. INDEX DESCRIPTORS: Phycornyces spores; dormancy; activation; respiratory metabolism; organic acids.

The constitutively dormant sporangiospores of Phycomyces blakesleeanus can be activated by several treatments, a brief heat treatment (Rudolph, 1960) or exposure to acetate (Borchert, 1962) being most reliable. Recently, a hypothesis suggesting that the metabolism of pyruvate by mitochondria would be the limiting factor keeping the spores in the dormant state was put forward, (Van Laere et al., 1980; Van Laere et al., 1982). An exogenous supply of acetate would circumvent this metabolic bottle neck, while heat activation would remove it. Indeed, pyruvate decarboxylation by isolated mitochondria was shown to be doubled by a prior heat shock of the spores. Moreover, the hypothesis would also explain why the spores could not be activated with pyruvate, the immediate precursor of acetate (Van Laere et al., 1980). However, real proof of this hypothesis has not been presented. The doubling of pyruvate decarboxylation after activation is also small in

comparison to the severalfold increase in 0, uptake by activated spores. Fur-t some additional findings indicate th only pyruvate metabolism, but also metabolic regulations are affected by shock. In spite of increased respirati ruvate was shown to accumulate in germinating spores (Van Laere et al., 4982; Rudolph et al., 1966) together with other glycolytic products such as glycerol (Delvaux, 1973) and lactate (Lurch, 1972). T~e~~f~~e the relation between activation and respiratory metabolism was further investigated. MATERIALS

AND METHODS

The experiments were done with the l+ strain of P. blakesleeanus Burgeff from the Halbsguth collection. Spores were grown and harvested as described by Van Assche et al. (1972). Oxygen uptake was measured with an oxygen probe (YSI 5331; c~nnec to a recorder) calibrated with water t had been saturated with air at the ternperature of the experiment. Spores (10 mg) 73 0147-5975184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any farm reserved.

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were preincubated in water at 20°C for 1 h before use. For measurements with activated spores, they were subjected to a heat shock (3 min, SK) just before the experiments. The (10 mg) spores were incubated in 10 ml test solution at the temperatures indicated and the amount of oxygen taken up in the next 10 min was determined. Due to the time dependence of uptake, and because the substrates occasionally influenced the state of the spores (activation or inhibition), oxygen consumption during the lo-min test period was not always linear. However, all time-dependent effects that may be relevant for the conclusions to be drawn are reported in the text. All figures representing oxygen uptake are drawn to the same scale. To estimate the capability of the spores to metabolize pyruvate, simple solutions of pyruvic acid were used. Uptake of this strong acid is only substantial at low pH values (Van Laere et al., 1980). Phycomyces spores are very resistant to low pH media (Van Assche et al., 1978). In our experiments 0.1 N HCl had only minor effects on spore respiration (not shown). Artifacts arising from acid taken up into the spores cannot be prevented by the use of buffering salts. On the other hand, cations have highly modulating effects on respiration as discussed further in text. Therefore it was preferable to use unbuffered solutions. The assay for pyruvate decarboxylation was done in stoppered 25ml Erlenmeyer flasks. The CO, was fixed on paper wicks impregnated with 0.05 ml of 4 N NaOH and hung on a hook fixed in the rubber stopper. Ten milligrams of air-dried spores were incubated in 2 ml of 50 mM unlabeled pyruvic acid, and about 0.5 &i of labeled substrate was added. After 2 h incubation at the indicated temperatures, the paper wicks were removed and the radioactivity was determined in a Beckman LS 9000 liquid scintillator. At each temperature an experimental vessel containing spores and a blank vessel without spores were run. The activity mea-

VAN

LAERE

surement from the blank vessel was subtracted from the activity measurement of the experimental vessel. Germination experiments and the determination of the germination percentages were performed essentially as reported in Van Assche et al. (1972). All experiments were repeated at least three times with consistent results. The values for oxygen consumption reported in the figures are the means of three replicates. For the experiments on germination induction only a typical experiment is reported. In the experiments of Fig. 6 the different acids were always studied in a single experiment. In this way the variability that could arise between successive experiments was avoided. These experiments were also repeated with consistent results. RESULTS

If a heat shock would remove a limitation of pyruvate utilization, one could expect that pyruvate (or its precursor, glucose) would enhance respiration much further when spores are brought to activating temperatures. The curve of the pyruvate- or glucose-dependent 0, uptake as a function of temperature would show a sigmoidal relationship, at least with dormant spores. With glucose as substrate a kind of sigmoidal relationship was obtained (see Fig. l), although the increase at 40°C (which is the minimal temperature at which germination is induced; see, e.g., Fig. 6) was not as sharp as would be expected if at that temperature a specific inhibition of the glucose metabolism would be removed. With pyruvic acid as substrate no sigmoidal relationship was found (see Fig. 2). However, with this strong acid, at higher temperatures which depend on the concentration of the acid, inhibitions become apparent. Therefore the capability of spores to metabolize pyruvate may be underestimated, especially at these higher temperatures. On the other hand, the results of Fig. 2 demonstrate that pyruvate metabolism is

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NH:, but are clearly not needed for germination induction. The role of various cations for respiration and germination will be reported elsewhere. To study the eventual limiting step in r-e: spiratory metabolism, spore respiration in pyruvic acid would be better corn with respiration in acetic acid so From such comparison (see Fig. 3) it can be seen that, at least at low pH, spore respiration can be enhanced almost to the same extent with both substrates. The eoncentration dependence is different. ences in permeability of the spores the two chemicals are probably inv With activated spores, respiratio ruvic acid (but also in acetic acid) that of dormant spores in that solution (Figs. 2 and 3). However, taking into account that endogenous respiration is also

FIG. I. Effect of exogenous glucose (1.5%) on the oxygen consumption of dormant and activated spores as a function of temperature, 0, Dormant spores in distilled water; q , dormant spores with glucose; 0, activated spores in water; q , activated spores with glucose.

also considerable in dormant spores. In fact, respiration of dormant spores in 0.03 and 0.05 M pyruvic acid at 30 to 35°C is comparable with the respiration rate of spores in 0.1 M Na-acetate, pH 6, at 30°C (0.75 bmol oxygen/l0 mg spores/l0 min; not shown in the figures). After pretreatment of spores in Na-acetate for 10 min about 80% of the spores germinated (see also Van Laere et al., 1980). In pyruvic acid, even after prolonged incubations under conditions where the respiration rate is not lower than in Na-acetate, no activation at all was obtained. It should be mentioned that in the presence of NH1; as counterion instead of Na+ , much more elevated respiration rates can be obtained with acetate (see Van Laere et al., 1980). Such elevated respiration rates are due to modulating effects of cations like

FIG. 2. Dependency of pyruvic acid-induced 0, uptake on temperature. Open symbols: dormant spores. 0, 0.03 M; Cl, 0.05 M; and a, 0.1 M pyruvic acid. A, Preactivated spores in 0.05 A4 pyruvic acid. The respiration of dormant spores in water at different temperatures is shown with a dotted line.

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IBGMOLAMTY

FIG. 3. Effects of different concentrations of pyruvic acid, propionic acid, and acetic acid on spore respiration. 0, Dormant spores in acetic acid; U, dormant spores in pyruvic acid; 0, activated spores in acetic acid; n , activated spores in pyruvic acid; A, propionic acid, dormant spores; A, spores pretreated in propionic acid, returned into water, respiration in water.

enhanced in activated spores (see also Fig. One could argue that the spores may not l), it cannot be concluded that the capa- become activated in the supra-optimal bility to utilize pyruvate as a substrate is acetic acid solutions but only after their respecifically affected by preactivation. turn to culture medium. However, spores That pyruvic acid is the substrate for the pretreated in 0.1 M acetic acid “behave” pyruvate-induced respiration is suggested as activated spores: in water their 0, upby the similar effect of temperature on the take is nearIy equal to (but does not exceed) pyruvate-induced O2 uptake (Fig. 2) and on that of heat-activated spores in water, and the decarboxylation of [14C]pyruvate shown in Fig. 4. The fact that azide (5 mA4) completely inhibited the effects of pyruvic acid (not shown) suggests that mitochondrial respiration is really involved. If pyruvate metabolism is not impaired in dormant spores, and if (at least at low pH) pyruvate can be used equally well as acetate as respiratory substrate, then why is it unable to induce spore germination? The logical answer is that acetate activation is not simply due to the role of acetate as a respiratory substrate. Consistent with that interpretation we found that the concentration dependency of respiration and activation caused by acetic acid are not identical (see Fig. 5). In fact, concentrated solutions FIG. 4. The temperature dependency of pyruvate deof acetic acid which even inhibit spore res- carboxylation by dormant Phycomyces spores. 0, piration are most effective on germination CO, production from [2-‘4Clpyruvate; I?, [1-‘4Cl induction. pyruvate.

Phycomyces SPORE GERMINATION

LOG MOLARITY

FIG. 5. A comparison of the effects of varying concentrations induction. 0, Respiration; cl, germination.

when glucose is supplied their respiration also rapidly increases to a level characteristic for activated spores in glucose, as in Fig. 1 (results not reported).

TEMPERATUREI’CI

FIG. 6. Temperature-dependent effects of different concentrations of acetic, propionic, and butyric acids on spore activation. 0, Acetic acid; 0, propionic acid; & butyric acid. . ~ *, 5 X 1O-4 M solutions; - - -, low3 M solutions; -, 5 x 1O-3 M solutions. The activation curve in water is presented by a simple straight line. The spores were treated 10 min in the solutions at different temperatures and transferred into culture medium.

of acetic acid on respiration

More evidence that acetate utilization as a substrate is not the basis of spore activation comes from experiments reporte Fig. 6. These results indicate that propionic and butyric acid effectively activate lower concentrations than acetic acid. T general interaction of temperature and aci is nearly additive with the three acids, so seemingly they operate by a common mechanism. If we assume that, like acetate, propionate acts as a respiratory substrate and is converted to acetate, it still cannot be understood why a 1QV3 M solution of propionic acid is more effective than a 5 x 10e3 M solution of acetic acid. In Fig. 3 it is demonstrated that propionate is not at all a good substrate for respiration. The effect of a 10T3 M solution on oxygen consumption is negligible. Nevertheless, with 10e3 M, and even with S x 10M4 M propionic acid, considerable germination induction is obtained (Fig. 6). In 5 x 10M3 and in 10P2 M solutio uptake is somewhat stimulated. when the spores pretreated in these solutions are returned to water, their oxygen consumption during the next 10 minutes does not decrease. A decrease would be expected if propionate is a substrate th utilized by the spores. Borchert (I962

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readily metabolized which induces spore germination. Propionic acid (and butyric acid), which is not a good substrate for respiration, is also more effective on spore activation than acetic acid, a better substrate for respiration. Our results with the monocarboxylic acids suggest that the efficiency of germiDISCUSSION nation induction increases with the innature of the acid. Recently Van Laere et al. (1982) sug- creasing hydrofobic gested that spore dormancy is due to a lim- Formic acid was indeed still less effective than acetic acid on spore activation (not itation in pyruvate utilization in dormant shown). Monocarboxylic acids therefore spores. Uptake of pyruvate by the mitomay interact with a hydrophobic activation chondria was considered to be the limiting factor. From our experiments the existence site (e.g., membranes) and cause activation of a limitation specifically due to pyruvate through a physicochemical effect, rather than play a role as a common metabolite. utilization can be doubted. We found that The fact that acetate could also induce under comparable conditions pyrnvic acid germination in the absence of oxygen and acetic acid stimulate respiration (Borchert, 1962) is consistent with a nonequally well. The acid-induced respiration and its dependency on the concentration of metabolic role of acetate. ‘The results obthe acid are not profoundly affected by heat tained with azide (Van Laere et al., 1980) are less conclusive. After pretreatment in preactivation of the spores. azide and acetate, spores do not germinate, In our experiments the normal permeability barrier toward pyruvic acid could be but it is not clear if the activation itself was circumvented, due to the high concentrainhibited. In some experiments we treated spores tions of undissociated molecules. However, the circumvention of the hypothetical per- in 2 x 10P3M acetic acid with 2 x IOh ii4 meability barrier to pyruvate utilization did azide solutions (25°C 20 min). When such spores were placed in germination medium not result in spore activation. Therefore, even if a limitation for pyruvate utilization they did not germinate. However, they also exists in dormant spores, it clearly could failed to do so, even after an additional heat not be the cause of spore dormancy. The shock (3 min, 50”(J), although heat activaproblem why p yruvic acid, while able to en- tion is not inhibited by azide (Van Laere et hance respiration, is still unable to induce al., 1980). Results obtained with azide spore germination (as acetic acid does), re- therefore cannot be safely interpreted. In conclusion, it seems that the evidence mains. The answer is that acetate activation against a metabolic role for acetic acid is is also not due to its role as a respiratory substrate. The different concentration de- stronger than the arguments for such a pendences for activation and respiration mechanism of action. argue against such a role. The fact that concentrated solutions of acetic acid that inACKNOWLEDGMENTS hibit spore respiration are most effective on thank Mr. Lievens J. for expert technical help. germination induction suggests that it is not A. We Van Laere gratefully acknowledges the receipt of the metabolized molecules, but precisely a grant from the Belgisch National Fonds voor Wetenthat fraction of the acetate that is not schappelijk Onderzoek.

demonstrated in an elegant way that propionate certainly does not only serve as a respiratory substrate, but also must have an effect on the utilization of endogenous reserves. The respiratory effects obtained with propionate thus seem to be a consequence of spore activation rather than its cause.

Phycomyces

comyces 415-428.

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and chemical activation of blakesleeanus spores. Exp. Mycof. 4:

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R. 1982. Pyruvate metabolism by mitochondria from dormant and activated Phycomyces blakesleeanus spores. J. Gen. Microbiof. 128: 15371545.