The role of water in the activation ofPhycomyces blakesleeanus sporangiospores

EXPERIMENTALMYCOLOGY

10, 190-

195 (1986)

The Role of Water in the Activation of Phycomyces blakesleeanus Sporangiospores MARC N. VERBEKE AND ANDRE J. VAN LAERE~ Interdisciplinary

Research Centre, Katholieke Universiteit Leuven, Campus Kortrijk, B-8500 Kortrijk, Belgium Accepted for publication March 13, 1986

VERBEKE, M. N., AND VAN LAERE, A. J. 1986. The role of water in the activation of Phycomyces blakesleeanus sporangiospores. Experimental Mycology 10, 190-195. Dormant Phycomyces spores have a low water content of only about 50%. After heat activation their water content increases and the osmotic potential decreases. Water uptake is mainly due to increased cell wall extensibility. The enhancement of spore respiration during heating, or after an activating heat shock, is not mainly due to an increasing water content. Nevertheless respiration depends on the water potential of the spores. The inhibition caused by decreasing the water potential osmotically is compensated by increasing the temperature, with about 10°C per osmolal increase in the medium. The activation process also depends on the water potential, but the shift of the activation temperature is only 2°C per osmolal increase in the medium. This indicates that water plays a role in activation which is different from its role in respiration. From these and earlier findings it is suggested that during activation some spore compounds have to become hydrated. o 1986 Academic Press, Inc.

INDEX

DESCRIPTORS:

Phycomyces; spore; heat activation; germination;

water potential; spo-

rangiospore; temperature.

One of the most favored methods used to break the constitutive dormancy of Phycomyces blakesleeanus sporangiospores is a short heat shock (e.g., 3 min at SO’C) (Halbsguth and Rudolph, 1959). During the heat shock water has to be present, since dry spores cannot be activated by heat (Halbsguth and Rudolph, 1959; Van Assche et al., 1977; Verbeke et al., 1981). Little attention has been paid to the role of water during activation even though there is substantial evidence that water plays a role in the activation process. Calorimetric experiments have shown that a hydration phenomenon occurs during heat activation of the spores (Verbeke et al., 1981). Moreover, one of the earliest detectable morphological effects of heat activation is spore swelling due to water uptake (Van Laere et al., 1980a). Phycomyces spores can also be r Present address: Laboratory for Plant Biochemistry, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, 3030 Leuven (Heverlee), Belgium. 190 0147-5975186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

activated by solutions of monocarboxylic acids, such as acetic acid and propionic acid (Sommer and Halbsguth, 1957; Borchert, 1962). These monocarboxylic acids do not function as chemical metabolites as suggested earlier (Van Laere et al., 1982), but instead appear to operate through a physicochemical mechanism (Verbeke and Van Laere, 1984). Again, water could play an important role in this physicochemical activation. Indeed the results reported by Verbeke and Van Laere (1984) indicate that a lyotropic anion series exists for the efficiency of this organic acid activation. The varying efficiencies of different ions on biochemical systems are often related to their hydration effects (e.g., on proteins). Therefore, the activating effects of anions (and probably cations such as K+ and NH:; see Pambor, 1979; Delvaux, 1973; Van Laere et al., 1980b) could be due to hydration effects in the spores. The changed protein denaturation spectrum, an immediate result of activation with heat or chemicals

Phycomyces

SPORE

GERMINATION:

(Van Cauwelaert and Verbeke, 1979), could also be the result of their changed hydration. In order to clarify the role of water and hydration in dormancy and activation we investigated the effect of lowered water potential on the water content and on the activation and respiration of the spores. MATERIALS

AND

METHODS

Spores of the I+ strain of Phycomyces b2akesleeanus Burgeff (ATCC 56533) were grown and harvested as described by Van Assche et al. (1972). For activation, spores were suspended in water or NaCl solutions and heated 3 min at the indicated temperature. Then they were rapidly cooled, centrifuged, and resuspended in germination medium. Germination medium and microscopic determination of percentage germination were as reported previously by Van Assche et al. (1972), except that Sfluorouracil (5 x 1W4 M) was added to inhibit germ tube formation. The activation experiments were repeated at least three times with similar results. In Figs. 1 and 2 the results of a self-contained experiment are presented. Oxygen uptake was measured with an oxygen probe (YSI 5331) as reported earlier (Verbeke and Van Laere, 1984). For the measurement of respiration, activated spores were subjected to a heat shock (3 min, 50°C in water) just before the experi-

0

LO

50

60

70

TPC I

FIG. 1. Temperature-dependent spore different NaCl concentrations: 0, water; 1 m; *, 2 m; q , 3 m; A, 4 m.

activation in q , 0.5 m: 8,

ROLE

OF WATER

ment. During the IO-min measuring period no important time-dependent changes were found. The total amount of oxygen taken up by the spores during the IO-min peri is shown in the figures. Oxygen consu tion values are means of three replicates. For the determination of water uptake as a function of water potential, absut 10 mg of dry dormant or preactivated (3 min, SK in water, dried by air stream) spores in small aluminum pans was e~~i~ibrat~d at 25°C in plastic boxes containing NaCl solutions of different molality (m). experiments indicated that a tion was insufficient, but for ~~c~bati~~ times between 48 and 72 h no s~g~ifica~t~y different water contents we 48 h the spores were weighed, at 120°C and weighed again 0.1 mg with a Cahn 20 balance. The amount of wat was calculated per 108 mg dry material. The mean results of three replicates are presented in Fig. 3. The standard deviations are not shown, because clearly exceed the size of the s water content was not measu mersion in the solutions, in order to avoid interstitial water. Nevertheless control experiments showed that the water content after uptake from the vapor phase did not appreciably differ from that of spores equilibrated in liquid. In the control experiments, the interstitial water was removed by filtration. The results concerning water uptake, activation, and respiration are presented as a function of the molal NaCl concentration. The results cannot be plotted as a ~~~cti~~ of the water potential, because spore lots were treated at different temperatures (and water potential depends on te eraturej ~ By using molal concentrations consistently, results from different experiments can. at least roughly, be compared c~~v~~i~~t~~. RESULTS

If water is actively involved

in spore ac-

192

VERBEKEANDVANLAERE

time mln

FIG. 2. Germination induction (%) as a function of the time period in which the spores are heated at 39°C: 0, in water; Fl, in 0.1 m NaCl.

tivation, we might expect that the water content would affect the activation temperature. Spores with different water contents were obtained by incubating them in solutions of nonpermeant solutes such as NaCl. The spores were suspended in the solutions at the different temperatures for 3 min, rapidly cooled, and resuspended in germination medium. Figure 1 shows the percentage of germination as a function of the pretreatment temperature in NaCl solutions of different strength. The activation curves are shifted toward higher temperature with increasing concentration, by some 2°C per osmolal unit. With the highest NaCl concentrations, the maximal degree of germination decreases. This is due to the fact that at the high temperature where spores would be activated in those solutions, killing of the spores starts. Indeed, the nongerminated spores after such treatments also did not germinate when they were placed under conditions where all viable cells germinate (e.g., suspension in nutrient medium with 10 mM ammonium acetate). The effects of lower concentrations of NaCl were tested in a more sensitive system, where germination induction was measured as a function of the time that the spores had been pretreated at 39°C (see Fig. 2). Under these conditions 0.1 m NaCl clearly reduced the activation of the spores. Similar results were observed with other nonpermeant osmotic agents such as sucrose or CaCl, (not shown).

A small decrease in the water content (see Fig. 3) thus seems to affect the’germination induction. In solutions of permeant solutes no decrease in the water content of the spores is to be expected. Solutions of the rapidly permeating ethylene glycol in fact did not increase the activation temperature. On the contrary, 8 and 10 m solutions of ethylene glycol shifted the activation curves to lower temperatures, but important fractions of the spores also lost viability (results not shown). Clearly, the effects of ethylene glycol on the spores were not related to the water content. The water content of the spores as a function of the NaCl concentration in the medium is shown in Fig. 3. An important finding is that 100 mg dry dormant spores takes up only 100 mg water at saturation over water. This estimate (water content 50%) is appreciably lower than the 80% value reported by Furch (1978). We confirmed our (50%) estimate by two other methods: (1) by weighing water-imbibed spores and removing the interstitial water by filtration; (2) by estimating the interstitial water by measuring the radioactivity which could be washed out from spore

FIG. 3. Water uptake (mg) per 100 mg completely dry, dormant (0) and activated (0) spores in 48 h, as a function of the molal NaCl concentration of the solution present in the moisture chambers.

Phycomyces

SPORE

GERMINATION:

mNoCl

FIG. 4. Oxygen consumption by 10 mg activated (open symbols) or dormant (filled symbols) spores during a IO-min period at different temperatures as a function of the NaCl concentration: A, 20°C: W, 25°C: El, 30°C; 0,35”C; 0,4o”C; 0,5o”c.

cakes after incubation in [3H]thymidine of known radioactivity (dpm/ml). In the control experiments, after correction for the “free” water, the water content of dormant spores never exceeded 50%. Preactivated spores, similarly equilibrated over NaCl solutions lower than 1 m, take up more water than dormant spores (Fig. 3). Probably as a result of increased water uptake, spores swell during early germination (Van kaere et al., 1980a). The increased water uptake after preactivation seems to be due to the increased cell wall extensibility. Indeed, by taking up more water, activated spores have a less negative osmotic potential (7~) than dormant spores (see below). Mence, at equilibrium with the same weak NaCl solution (water potential ), the pressure potential (P) in activated spores is less positive than in dormant spores (9 = T + P). The relation between the components of the water potential (T, P) can be measured by replotting the data of Fig. 3 according to Tyree and Hammel (1972). In such plots, the amount of water lost by spores when they are removed from water to NaCl solu-

ROLE

OF WATER

893

tions is shown as an inverse function of the NaCI concentration. A straight line is thus obtained at high NaCl co~centrati~ns~ where the spores are plasmolyze turgor is absent (data not shown). Wit results from Fig. 3, the straight line staree at 0.8 m with activated spores an NaCl with dormant spores. Cle vated spores can take up more dormant spores before turgor is built up (75 and 47 mg, respectively, Fig. 3). Extrapolation of the straight line revealed the osmotic potential at saturation over water ammel, 1972). The extrare 0.33 m (NaCl) for activated spores and 0.68 m for dormant spores, demonstrating the diluti spore contents as the preactivat swell. Dilution of the cytoplasm might be the cause of the swelling of rn~toc~~~~~~a and nuclei after activation, reported by Pambor (1979) an chondria of dor shrunken structure (Grove, I976) and the respiratory rate of dormant spores is low(Fig. 4). Therefore we studied the r~~at~o~ship between respiration and water content. The spores were s solutions of different str uptake was measured for 1 results (Fig. 4) it is clear ratory rate was further crease in the water potentia vated spores, the respiratory rate was only inhibited at increased N&l con~e~trati~~~~ and in a temperature-dependent rna~~er~ At 30°C inhibition began at 1.5 m NaCl, at and at 50°C snly at 2.5 rrr C respiration was about halfmaximal in 1.3 m NaCl, but at 50°C halfmaximal respiration was induced by a 3 M NaCl solution. These approaches indicate that a temperature rise of aboout 10°C compensated for each osmolal increase (0.5 m NaCl) in the medium. With g~rrn~~a~~o~ induction a temperature rise of only 2°C sufficed to compensate for a 1 osmolal increase (see Fig. 1). The interaction of temperature and water potential for respiration

194

VERBEKEANDVANLAERE

of dormant spores is difficult to measure. At low temperatures the respiratory rates and the effects of NaCl on respiration are small (Fig. 4), and at higher temperatures the spores would not remain dormant (Figs. 1 and 2). Nevertheless, the results with dormant spores indicated that dormant and activated spores also have different respiratory rates in solutions where they have no different water content (in 1.5, 2, and 3 m NaCl, Fig. 4). DISCUSSION

Spores, with extremely low water content and in the absence of turgor, can still be activated by heat. In fact, it is not the water content as such that determines the activation temperature. The activation temperature increases almost linearly with the osmolality of the medium (Fig. 1). The water content is not linearly related to the osmolality (Fig. 3). Together the results indicate that spore activation depends on the water potential, which is a measure of the free energy of the solvent in the spores. Activation by heat is at least in part due to the increasing free energy of the spore water at higher temperature. The free energy of the water in dormant spores is very low: equivalent to 0.6 m NaCl solution at saturation. The water content in dormant spores is also low. A water content of 50% indeed is very low, especially taking into account the large concentration in the spores of hydrophilic compounds able to bind part of the water (e.g., trehalose up to 35% of the dry weight; Rudolph and Ochsen, 1969). Activated spores swell due to the increased cell wall extensibility, as suggested earlier from indirect evidence (Verbeke and Van Laere, 1982). As a result, during early germination the spore contents are diluted (deduced from Fig. 3). The increasing water content might play a role by increasing metabolic activities in activated spores (e.g., protein and nucleic acid synthesis; Van Assche and Carlier,

1973). The dependency of metabolism on the water content has been studied in detail by Clegg (1978) for Artemia salina cysts. However, the dramatic increase in respiratory rate, an almost immediate effect of activating temperatures (Verbeke and Van Laere, 1984), is not simply due to an (eventually) increased water content. In preactivated spores, water content can be held at the same level as in dormant spores by incubation in concentrated NaCl solutions (Fig. 3). Nevertheless, their respiratory rates in such solutions clearly exceed that of dormant spores (Fig. 4). The respiratory rates of activated and of dormant spores does depend on the water (potential). This is not unexpected because the spore water has to allow diffusion of respiratory substrates and intermediates. For respiration water could be just a filler, as suggested by Clegg (1978). The role of the water for the activation process certainly differs from its role for respiration. This appears from the finding that the free energy of the solvent molecules (affected by temperature and osmotically) does not influence both processes in the same way. The question remains: What kind of work has to be performed during spore activation? As indicated in the introduction, during activation a hydration process occurs (Verbeke et al., 1981). A tempting explanation for present and earlier findings is, therefore, that activation would involve hydration of some essential spore components. Indeed, “solubilization” depends on the free energy of the solvent. Therefore the activation temperature would behave as a colligative property (Fig. 1). Chemicals that affect the solvent properties (like D,O, higher alcohols, amphiphiles , glycerol, ethylene glycol, discussed in Tanford, 1980) could therefore, also affect activation as known (Verbeke et al., 1981; Thevelein et al., 1979; Verbeke and Van Laere, 1984) by their effect on the solvent in the spores. This suggests that it might be interesting to study the spore water during and after acti-

Phycomyces

vation with techniques tric spectroscopy.

SPORE GERMINATION:

like NMR or dielec-

A. Van Laere gratefully acknowledges the receipt of a grant from the Belgisch Nationaal Fonds voor Wetenschappehjk Gnderzoek. REFERENCES BORCHERT, R. 1962. Ueber die Azetat-Aktivierung der Sporangiosporen von Phycomyces blakesleeanus. Beitr. Biol. Pflanz. 38: 31-61. CLEGG, J. S. 1978. Hydration-dependent metabolic transitions and the state of cellular water in Artemia cysts. In Dry Biological Systems (J. H. Crowe and J. S. Clegg, Eds.), pp 117-153. Academic Press, New York. DELVAUX, E. 1973. Some aspects of germination induction in Phycomyces blakesleeaaus by an ammonium-acetate pretreatment. Arch. Mikrobiol. 88: 273-284. FURCH, B. 1978. On the water potential, osmotical pressure and velocity of water uptake of dormant sporangiospores of Phycomyces blakesleeanus. 2. 88: 269-272.

FIJRCH, B. 1981. Spore germination: Heat mediated events. In The Fungal Spore: Morphogenetic Controls (G. Turian and H. R. Hohl, Eds.), pp. 413-433. Academic Press, New York. GROVE, S. N. 1976. Form and function of the zygomycete spore. In The Fungal Spore (D. J. Weber and W. M. Hess, Eds.), pp. 559-592. Wiley, New York. HALBSGUTH, W., AND RUDOLPH, H. 1959. Untersuchungen tiber die Warme-Aktivierung der Sporangiosporen von Phycomyces blakesleeanus. Arch.

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143-153. RUDOLPH, H., AND OCHSEN, B. 1969. TrehaloseUmsatz warmeaktivierter Sporen von Phycomyces blakesleeanus. VI. Beitrag zur Kausalanalyse der W&rmeaktivierung von Pilzsporen. Arch. Mikrobiol. 65: 163-171. SOMMER, L., AND HALBSGUTH, W. 1957. Grundlegende Versuche zur Keimungsphysiologie von Pilzsporen. Forschungsber. Wirtsch. Verkehrminist. Nordrhein-Westfalen 411: l-85.

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Wiley. New York. THEVELEIN, J. M., VAN ASSCHE, J. A., CARLIER, A. R., AND HEREMANS. K. 1979. Heat activation of Phycomyces blakesleeanus spores: Thermodynamics and effect of alcohols, furfural and high pressure. J. Bacterial. 139: 478-485. TYREE, M. T., AND H-MEL, H. T. 1972. The measurement of the turgor pressure and the water relations of plants by the pressure-bomb technique. J. tion ofMicelles

ACKNOWLEDGMENT

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VAN Asscw~, 9. A., CASUER, A. R., AND DE KEERSMAEKER, H. 1. 1972. Trehalase activity in dormant and activated spores of Phycomyces blakesleeanus. Planta 103: 327-333. VAN ASSCHE, J. A.: CARLIER, A. R., AND VAN TIEGFIEM, L. L. C. 1977. The effect of gamma radiation on breaking of dormancy in Phycomyces spores. Arch. Mikrobiol. 113: 95-97. VAN CAUWELAERT, E H., AND VERBEKE, M.N. 1979. Differential scanning, calorimetric observations concerning the activation mechanism of Phycomyces Commun.

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VAN LAERE, A. J., VAN ASSCHB, 9. A., AND CARLIER, A. R. 1980b. Metabolism and chemical acfivation of Phycomyces blakesleeanus spores. Exp. Mycol.

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VAN LAERE, A. Jo, VAN DEN BOSCH: R. R., AND CARLIER, A. R. 1982. Pyruvate metabolism by mitochondria from dormant and activated Phycomyces blakesleeanus spores. 9. Gen. Microbio!. 128: 1.537-1545. VERBEKE, M. N., VAN CAUWELAERT, E PI., AND JADOT, R. 1981. Calorimetric aspects of the heat activation of spores of Phycomyces blakesieeanus. Biochem.

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VERBEKE, M. N., AND VAN LAERE, A.J. 1982. On the role of the spore cell wall in the activation of Phycomyces blakesleeamrs spores. Exp. Mycol. 6: 313-320. VERBEKE, M. N., AND VAN LAEIRE, A. J. 1984. Lack of correlation between acid-induced respiration and germination in Phycomyces spores. .#Zxp.Mycol. 8: 73-79.