Ca2+ fluxes in developing Trichoderma viride mycelium

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Ca2+ fluxes in developing Trichoderma viride mycelium Martin Šimkovic, Svetlana Kryštofová, and L’udovít Varecka

Abstract: The properties of both Ca2+ influx and efflux in the mycelium during the life cycle of Trichoderma viride were studied by means of 45Ca2+ and by X-ray fluorescence spectroscopy measurements. The properties of the 45Ca2+ influx and effluxes indicate that they are mediated by different transport systems. The Ca2+ influx could be mediated by an electrogenic Ca2+/nH+ antiport, or by an Ca2+ uniport system. Both Ca2+ influx and efflux were stimulated by the uncouplers (and the treatment leading to the suppression of energy metabolism) and by azalomycin F, an antifungal agent. Salicylate stimulated the Ca2+ efflux, but inhibited the Ca2+ influx. In the isolated preparation of crude vacuolar/ mitochondrial fraction, salicylate induced the Ca2+ release, as did A23187. Azalomycin F moderately released Ca2+ from the microsomal fraction. On the other hand, uncouplers did not release Ca2+ from the isolated organelles, but inhibited to a different extent the ATP-dependent and -independent Ca2+ influx. The results could be explained in terms of the capacitative Ca2+ influx mechanism. The rate of 45Ca2+ influx, or of the 40Ca2+ content, was maximal after about 30 h of submerged cultivation, and then decreased. The results show that loading of internal Ca2+ stores occurs in the early stages of the development of mycelium only, and the Ca2+ influx mechanism is developmentally down-regulated, being almost nonexistent during its later stages. In older mycelium, growth seems to be autonomous of the extracellular Ca2+ until the onset of conidiation. Key words: Trichoderma viride, development, Ca2+ influx, Ca2+ efflux, salicylate, uncoupler, azalomycin F. Résumé : Nous avons cherché à caractériser l’influx et l’efflux du Ca2+ dans le mycélium au cours du cycle de reproduction de Trichoderma viride à l’aide du 45Ca2+ et de la spectroscopie en fluorescence à rayons X. L’influx et l’efflux du 45Ca2+ ont semblé contrôlés par différents systèmes de transport. L’influx du Ca2+ pourrait être contrôlé par un système antiport Ca2+/nH+ électrogène ou par un système uniport Ca2+. L’influx et l’efflux du Ca2+ étaient tous deux stimulés par des découpleurs (et le traitement menant à la suppression du métabolisme énergétique) et par un antifongique l’azalomycine F. Le salicylate a stimulé l’efflux mais inhibé l’influx du Ca2+. Dans une préparation d’extrait brut de vacuoles et de mitochondries, le salicylate a provoqué une libération modérée du Ca2+ comme A23187. L’azalomycine F a causé une libération modérée du Ca2+ par la fraction microsomale. D’autre part, les découpleurs n’ont pas causé de libération de Ca2+ par des organelles isolées mais ils ont inhibé à différents degrés l’influx du Ca2+ dépendant et indépendant de l’ATP. Les résultats pourraient s’expliquer en fonction d’un mécanisme capacitif d’influx du Ca2+. La vitesse d’influx du 45Ca2+ ou le contenu en 40Ca2+ atteignait son maximum après environ 30 h en culture submergée et diminuait par la suite. Les résultats démontrent que l’accumulation des réserves internes de Ca2+ se produisait seulement dans les premiers stades du développement du mycélium et que le mécanisme d’influx du Ca2+ subissait une régulation à la baisse à mesure que le développement se poursuivait et qu’il devenait presque silencieux dans les derniers stades. Chez les mycéliums plus âgés, la croissance du mycélium a semblé indépendante du Ca2+ extracellulaire jusqu’au début de l’étape de la formation des conidies. Mots clés : Trichoderma viride, développement, influx du Ca2+, efflux du Ca2+, salicylate, découpleur, azalomycine F. [Traduit par la Rédaction]

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Abbreviations: A23187, calcimycin; AZA, azalomycin F; Cz-D +/-YA, Czapek-Dox medium with/without yeast autolysate; DBP, dibutylphtalate; DCCD, 1,3-dicyclohexylcarbodiimide; DES, diethylstilbestrol; DOP, dioctylphtalate; drm, dry mass; EDTA, ethylene diaminetetraacetic acid; EGTA, ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid; FCCP, carbonyl cyanide ptrifluoromethoxyphenylhydrazone; Hepes, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; Mes, 2-(Nmorpholino)ethanesulfonic acid; Mops, 3-(N-morpholino)propanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; Q10, temperature quotient; SA, salicylic acid; TCA, trichloroacetic acid; TCS, 3,3′,4′,5-tetrachlorosalicylanilide; Tris, tris(hydroxymethylamino)methane. Received August 18, 1999. Revision received November 17, 1999. Accepted November 23, 1999. M. Šimkovic, S. Kryštofová, and L. Varecka.1 Department of Biochemistry and Microbiology, Slovak University of Technology, Radlinského 9, 81102-Bratislava, Slovak Republic. 1

Author to whom all correspondence should be addressed (e-mail: [email protected]).

Can. J. Microbiol. 46: 312–324 (2000)

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Introduction Knowledge of Ca2+ homeostasis is an important aspect of physiology of filamentous fungi. Calcium has been shown to be involved in the control of conidiation (Pitt and Barnes 1993; Roncal et al. 1993), conidia germination (Sato 1994; Rivera-Rodriguez and Rodriguez-Del Valle 1992), zoospore germination (Donaldson and Deacon 1992), hyphal branching (Reissig and Kinney 1983; Hudecová et al. 1994; Grinberg and Heath 1997), hyphal tip growth (Schmid and Harold 1988; Jackson and Heath 1993, for review; Garrill et al. 1993; Levina et al. 1995), dimorphism (Muthukumar and Nickerson 1984; Alsina and Rodriguez-Del Valle 1984; Gadd and Brunton 1992; Mendoza et al. 1993; Karuppayil and Szaniszlo 1997), phospholipid biosynthesis (Giri et al. 1994), phytoparasitism (Elad and Kirshner 1992), and cell cycle regulation (Luc et al. 1992, 1993). Unlike Saccharomyces cerevisiae, where Ca2+ homeostasis has been described in detail at the biochemical and genetic levels (Cunningham and Fink 1994, for review), progress has not been achieved in the study of Ca2+ homeostasis in filamentous fungi. Nevertheless, inward Ca2+ transport was demonstrated in Ophiostoma ulmi (Gadd and Brunton 1992), Penicillium notatum (Pitt and Barnes 1993), and in Trichoderma viride (Kryštofová et al. 1995). The Ca2+-ATPase and the ATP-dependent transport has been observed in Neurospora crassa inside-out plasma membrane vesicles (Stroobant and Scarborough 1979), and the ATP-dependent Ca2+ transport has been observed in Ustilago maydis vesicles (Hernandez et al. 1994). On the other hand, data about the Ca2+ efflux from the intact mycelium has not yet been published. There are data available about Ca2+ accumulation within fungal organelles. In Neurospora crassa, evidence about the vacuolar Ca2+ accumulation was obtained (Cornelius and Nakashima 1987; Miller et al. 1990), and its release by inositol-1,4,5 tris phosphate was documented (Cornelius et al. 1989). Similar molecular and transport characteristics in other fungi are not known. The study of inward Ca2+ transport is of special importance for the understanding of overall Ca2+ homeostasis. It is known that filamentous fungi and yeasts can grow at very low Ca2+ concentrations in the presence of EGTA. Although the interpretation of these experiments is not straightforward (see Youatt 1993, for review; 1994), along with the above mentioned data, they indicate the role of intracellular stores in supporting vegetative growth in fungi. In our previous paper (Šimkovi et al. 1997) we found that the Ca2+ influx into the vegetative Trichoderma viride mycelium is a function of its age. In this manuscript, we present data which describe the properties of both Ca2+ influx and efflux in T. viride developing vegetative mycelium, and provide evidence for the interplay between the extracellular Ca2+ and intracellular Ca2+ stores in sustaining vegetative growth and conidiation.

Materials and methods Strain Trichoderma viride strain CCM F-534 from the Czechoslovak Collection of Microorganisms, T.G. Masaryk University, Brno, Czech Republic, was used.

313 45

Ca2+ influx measurement

Suspensions of hyphae from submerged cultures were used for these experiments. Two hundred millilitres of liquid Czapek-Dox medium containing 0.5% (w/v) yeast autolysate (Cz-D plusYA) in 500 mL flasks were inoculated with Trichoderma viride conidia, and the suspension was cultivated on a rotary shaker (4 Hz) for 20 h at 27°C. The mycelial suspension was concentrated by vacuum filtration on a nylon net, and washed with 200 mL of 0.15 M NaCl. The filtration and washing was repeated three times and, finally, the mycelium was suspended in 3% (w/v) sucrose containing 25 mM Tris-HCl, pH 7.4, to a final volume of 70 mL. The mycelial suspension was completely homogeneous. It was kept at ambient temperature, and immediately used for the experiments. Aliquots of the suspension were incubated with 45Ca2+ (final concentration 0.5 mM, specific activity about 1000 cpm/nmol) for 1 h, or the time as indicated in Figs. 1, 5, 7, and 10–12, at 25°C. Subsequently, 1 mL aliquots were withdrawn and filtered on a membrane filter using a vacuum filtration apparatus and a Whatman GF/A glass microfiber filter. The pellet was washed with 2 × 4.5 mL 0.15 M NaCl containing 10 mM EDTA-Tris, pH 7.4, and was taken for liquid scintillation counting.

Submerged cultivation and radiolabeling of mycelium

Cultivation was carried out as above in the presence of 1 µCi (1 Ci = 37 GBq) of 45CaCl2 per 1 mL medium. The 20-h-old mycelium with incorporated radioactivity was harvested by centrifugation at 6000 × g for 5 min. The cell pellet was resuspended in Cz-D minus YA, containing 1 mM EDTA, and again centrifuged as above. This step was repeated thrice. The remaining EDTA in mycelium was removed by washing three times under the same conditions with Cz-D minus YA only. The mycelial pellet was diluted with Cz-D minus YA to a final concentration of approximately 4 mg of mycelium (dry mass) per millilitre. Only homogenous mycelial suspensions were taken for experiments. All experiments were carried out at 25°C, unless otherwise indicated in Figs. 2A and 5C. 45

Ca2+ efflux measurement

The radioactively-labelled suspension was incubated under desired conditions (usually at 25°C) in the presence of tested substances. Aliquots (150 µL) of the suspension of hyphae were withdrawn at times between 0 and 120 min, and added to 50 µL of 50 mM EDTA-Tris (pH 7.4), which was layered on 150 µL of dibutylphtalate (DBP) and dioctylphtalate (DOP) in a ratio of 2:1 in an Eppendorf tube, and spun for 3 min at 6000 × g. The aqueous layer from the top of the DBP:DOP phase was taken directly for liquid scitillation counting. The organic phase was removed, and the pellet on the bottom of the Eppendorf tube was treated with 10% (w/v) TCA and 10 mM La3+ for 30 min, and finally, centrifuged. The supernatant was taken for LSC. The results are expressed as averages of triplicates ± standard deviation of a representative of three experiments. 40

Ca2+ content of mycelium and conidia

The content of Ca2+ in the mycelium was measured by means of X-ray fluorescence spectroscopy (X-MET 920, Outokumpu Instruments, Finland). The samples of mycelium and (or) conidia were prepared for the measurements as follows. (i) A 50 mL suspension of submerged mycelium was filtered through the pre-weighed paper filter and washed with 200 mL of 0.15 M NaCl containing 10 mM Tris-EDTA, pH 7.4. The mycelium was dried and its mass was estimated by weighing. Filters with mycelium were placed into aluminum oxide crucibles and heated for 1 h at 600°C. The ash was extracted with 3 mL of 5% HNO3 (v/v) and used for the measurements. (ii) Aerial mycelium was cultivated on a paper disc put on the surface of Cz-D+YA agar at 25°C for the times indi© 2000 NRC Canada

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Fig. 1. (A) 45Ca2+ influx over time. Results are representative of 3 experiments. (B) Dependence 45Ca2+ influx on pH, and effect of TCS. pHo was set to indicated values with 25 mM Na-Hepes, Na-Mops, Na-Mes, and Tris buffers. Values were corrected for the radioactivity retained at the time zero. Concentration of TCS was 30 µM (0.5% (v/v) methanol in control). Results are representative of two experiments.

cated in Figs. 3 and 4. The paper disc was treated as described in (i). The results are expressed as averages of triplicates ± standard deviation of a representative of three experiments.

Subcellular fractionation The isolation of organelles was based on sedimentation through sucrose density gradients (Cornelius and Nakashima 1987), with several modifications. After growing for 20 h in the dark at 27°C on a rotary shaker, the mycelia were harvested by filtration through a nylon net and washed with 0.15 M NaCl. The mycelium was resuspended in 50 mL of ice cold homogenization medium (0.8 M mannitol; 25 mM Tris-HCl pH 7.4; 1 mM EDTA; 0.1 mM ATP; 1 mM PMSF; 1 mM benzamidine), mixed with glass beads and disrupted on a Polytron homogenizer for 5 × 1 min. The homogenate was centrifuged for 20 min at 1000 × g to remove cell debris and glass beads, and the resulting supernatant was spun down for 30 min at 14 000 × g. The resulting pellet was used as a mitochondrial/vacuolar fraction, and the supernatant as a microsomal one. All the above steps were carried out at 4°C.

Determination of

45

Ca2+ influx by isolated cell fractions

This was measured in a reaction mixture containing the membrane fraction (protein concentration about 1 mg·mL–1), 0.8 M sorbitol (or mannitol), 25 mM Tris-HCl (pH 7.4), 1 mM Mg2+ and 1 mM ATP. The influx was initiated by adding 45CaCl2 to a final concentration of 50 µM with specific activity of about 10 000 cpm·nmol–1. At time intervals following the start of influx, a 100 µL sample of the assay mixture was removed, and the membrane fractions were collected by vacuum filtration on the Millipore nitrocellulose membrane filters (pore size 0.6 µm). The filter was then rapidly washed with 2 × 4.5 mL aliquots of buffer containing 0.3 M sucrose and 10 mM EDTA-Tris (pH 7.4), and taken for liquid scintillation counting. The results are expressed as the average of duplicates ± standard error of a representative of the number of experiments indicated in the legends to Figs. 10 and 11.

Protein determination Proteins were determined by the method of Lowry (Smith 1992) with bovine serum albumin as standard.

Chemicals The chemicals used were from the following sources: Radionuclide 45CaCl2 from Radiochemical Centre, Amersham, U.K.; eth-

ylene glycol bis (2-aminoethylether)-N,N,N′,N′- tetraacetic acid (EGTA) and dimethylsulphoxide (DMSO) were from Merck, Darmstadt, Germany; mannitol was from Serva, Heidelberg, Germany; benzamidine, dicyclohexylcarbodiimide, diethylstilbestrol, phenylmethanesulfonyl fluoride (PMSF), and carbonyl cyanide ptrifluoromethoxy-phenylhydrazone (FCCP) from Sigma, St. Louis, Mo.; valinomycin, monensin A, and A23187 from Calbiochem, Luzern, Switzerland; 3,3′,4′,5-tetrachlorosalicylanilide (TCS) from Eastman-Kodak, Rochester, N.Y.; NaVO3 from Reachim, Moscow, Russia; Tris-(hydroxymethylamino) methane and agar were purchased from Medika, Bratislava, Slovakia. Azalomycin F was kindly provided by Prof. Dr. V. Betina, University of Trnava, Slovakia, mucidin by Prof. Dr.J. Šubík, Comenius University, Bratislava, Slovakia, and dioctylphtalate by Dr. Ivan Hudec, Department of Plastics, Slovak University of Technology, Bratislava. All other reagents were purchased from Lachema, Brno, Czech Republic.

Results Ca2+ influx Basal observations 45 Ca2+ accumulated in the submerged Trichoderma viride mycelium with t1/2 = 20 min, and the steady-state was reached within about 45 min (Fig. 1A). The extent of the influx in the steady-state varied between 0.022 ± 0.008 nmol·min–1·mgdrm–1 (n = 6). The accumulation was also pH and temperature dependent. This pH dependence indicated that the 45Ca2+ influx was only slightly increased between pH 6.5 and 7.2. Further increase in pH (up to pH 8.3) was accompanied with an increase in the 45Ca2+ influx which was maximal at pH about 8.0 (Fig. 1B). The temperature dependence of the Ca2+ influx was rather complex. Between 15 and 28°C, the influx was inhibited up to 28°C upon increasing the temperature. Between 28 and 42°C, the 45Ca2+ influx increased, and the maximal value was obtained at 42°C (Fig. 2A). The Q10 value for this temperature interval was larger than 2, and the activation energy of the influx of 95 ± 15 kJ/mol can be calculated from the Arrhenius plot (not shown). The 45Ca2+ influx was also dependent on the external Ca2+ concentration. © 2000 NRC Canada

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Fig. 2. (A) The effect of temperature on the 45Ca2+ influx. Results are representative of 2 experiments. Values were corrected for radioactivity retained at time zero. Concentration of TCS was 30 µM (0.5% (v/v) methanol in control). (B) Dependence of Ca2+ on the external 45Ca2+ concentration. Results are representative of two experiments. Values were corrected for radioactivity retained at time zero.

Table 1. The effect of inhibitors on

45

Ca2+ influx. 45

Exp. No.

Inhibitors

Concentration (mM)

Ca2+ influx (nmol Ca2+·min·mgdrm–1)

1 1 1 1 2 2 3 3 3 4 4 5 5 6 6 7 7 8 8

None NaCN Mucidine NaN3 None 2-Desoxyglucose + monoiodacetate None NaVO3 NaVO3 None TCS None Valinomycin None Monensin A None Azalomycin F None Salicylic acid

0 10 0.1 mg·mL–1 10 0 20 + 10 0 1 10 0 0.03 0 0.02 mg·mL–1 0 0.02 mg·mL–1 0 0.01 mg·mL–1 0 8

0.0162 0.0343 0.0196 0.0225 0.010 0.016 0.00263 0.0035 0.0062 0.0168 0.0519 0.0079 0.0106 0.0094 0.0112 0.0198 0.112 0.025 0.018

It was found to be saturable with respect to Ca2+ with KM(Ca2+) = 2.1 ± 0.2 mM, although a minor linear component of the 45 Ca2+ influx was revealed (Fig. 2B). The effect of energy conversion inhibitors and ionophore on 45Ca2+ influx The 45Ca2+ influx was only slightly sensitive to treatments known to inhibit energy-conversion processes. Transport does not seem to be tightly coupled with oxidative metabolism, because the addition of uncouplers (up to 30 µM TCS, or 30 µM FCCP), respiratory inhibitors (up to 100 µg·mL–1 mucidin, 10 mM cyanide), and F0F1-ATPase (up to 10 mM azide) stimulated the 45Ca2+ influx to various degrees (Table 1). The effect of the uncouplers under various conditions

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0005 0.0001 0.0022 0.0016 0.002 0.005 0.0003 0.0002 0.0002 0.007 0.004 0.003 0.002 0.002 0.004 0.003 0.003 0.006 0.005

45

Ca2+ influx (% of control) 100.0 211.2 120.6 138.9 100 160 100 133.1 235.1 100.0 308.9 100.0 134.9 100.0 119.6 100 565 100 72

(pH, temperature) is shown in the Figs. 1 and 2. The substitution of sucrose with 2-deoxyglucose (20 mM) did not change the 45Ca2+ influx (not shown), nor did the simultaneous presence of 2-deoxyglucose and monoiodoacetate (20 and 10 mM, respectively) (Table 1). Valinomycin, a K+ specific electrogenic ionophore (30 µM), and monensin A, an electroneutral Na+(K+)/H+ ionophore (30 µM), stimulated the 45Ca2+ influx by about 35 and 20%, respectively (Table 1). Two other agents were found to influence the 45Ca2+ influx. Millimolar concentrations of salicylate, an agent with powerful and multiple effects in plants (Kawano et al. 1998) and a Ca2+ homeostasis perturbing agent in yeasts (Mori et al. 1998), slightly inhibited 45Ca2+ influx (Table 1). Azalomycin F, a compound with an inhibitory effect on the © 2000 NRC Canada

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Fig. 3. (A) The 45Ca2+ influx in submerged mycelium during its development. Mycelium of age indicated was taken for measurement of 45Ca2+ content and dry mass. Values of influx were corrected for radioactivity retained at time zero. Results are representative of three experiments. (B) The 45Ca2+ content in submerged mycelium during its development. Mycelium of age indicated was taken for measurement of 40Ca2+ content and dry mass. Results are representative of three experiments.

growth and conidiation of T. viride (S. Kryštofová, unpublished observation), significantly stimulated the 45Ca2+ influx (Table 1). Developmental changes The extent of the 45Ca2+ accumulation dramatically changed, depending on the age of the submerged vegetative mycelium. While the total influx of 45Ca2+ increased to its maximum in the first 30 h of cultivation, further cultivation was accompanied by a steep decrease. And although the mass of mycelium increased steadily up to the end of cultivation (about 72 h), the specific influx (nmol Ca2+·min–1·mgdrm–1) decreased continuously (Fig. 3A). The findings obtained in this experiment were corroborated by the absolute Ca2+ measurement using X-ray-induced fluorescence spectrometry. It was found that the maximum Ca2+ content of hyphae was attained after about 90 h, and the older mycelium, although its mass increased up to about 120 h, did not increase the Ca2+ content (Fig. 3B). The specific Ca2+ content (µmol·gdrm–1) monophasically decreased with increasing the mycelium age (Fig. 3B). It should be noted that the Ca2+ loading is not due to the limitations in the external Ca2+ content, as the experiment was performed under saturation conditions with respect to Ca2+; less than 10% of total external Ca2+ was accumulated in hyphae and (or) bound to their surface (not shown). Similar results were obtained when the 40Ca2+ content of the T. viride dark-grown aerial mycelium was measured (Fig. 4), although in this case the data were collected only from large (older) colonies. In agreement with results from the submerged mycelium, the increment of the Ca2+ content was low, although the increase of the mycelial mass

Fig. 4. The 40Ca2+ content in the aerial mycelium during its development. The cultivation of aerial mycelium in the dark and measurement of mycelial mass and 40Ca2+ content was performed as described in Materials and methods. The arrow shows the time over which conidia appeared, as observed under safe red light illumination. Results are representative of two experiments.

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Fig. 5. (A) 45Ca2+ efflux over time. The concentration of FCCP was 30 µM (0.5% (v/v) methanol in control). Results are representative of 3 experiments. (B) pHo in relation to 45Ca2+ efflux. Conditions are similar to those depicted in Fig. 1B, but the effect of the uncoupler was not tested. Results are representative of two experiments. (C) 45Ca2+ efflux and temperature dependance. Values were corrected for radioactivity at time zero. Concentration of FCCP was 30 µM (0.5% (v/v) methanol in control). Results are representative of two experiments.

was substantial, and the specific Ca2+ content decreased. At the same time, the experiment was devoted to studying changes in the Ca2+ content of aerial mycelium under conditions which favoured the initiation of starvation-induced conidiation. Before the onset of conidia formation, both mycelial mass and total mycelial Ca2+ transiently decreased. However, the formation of conidia was accompanied by a return of mass, and the Ca2+ content was in agreement with our previous 45Ca2+ autoradiography observations (Šimkovi et al. 1997). The specific Ca2+ content of mycelium did not change significantly during or after conidia formation (Fig. 4). Similar results were obtained when the light pulse was used to trigger conidiation (not shown). The above results suggest that the ontogenesis of the submerged mycelium starts with the loading of internal Ca2+ stores, which supply the organism for its later stages. The steady-state level of Ca2+ in the Ca2+ stores seems to be maintained by keeping the Ca2+ stores completely filled. Nevertheless, the possibility exists that activity of the putative, ontogenetically delayed, Ca2+ efflux pathway is involved in maintaining the steady-state Ca2+ levels, as shown in Figs. 1–4. Therefore, the properties of the putative Ca2+ efflux pathway were studied. Ca2+ efflux Basal observations and effects of inhibitors Submerged mycelium, when cultivated in the presence of 45 Ca2+ (see Materials and methods), and washed with medium containing 10 mM EDTA (or EGTA), retained radioactivity to the extent about 5000 ± 800 cpm·mgdrm–1. The analytical Ca2+ concentration in the medium, as determined by X-ray-induced fluorescence spectroscopy, was 12.25 mM. Assuming that the isotopic equilibrium was attained during the cultivation, the average Ca2+ concentration of 17.7 ± 2.9 nmol Ca2+·mgdrm–1 (n = 12) was calculated. This value is not in agreement with the direct measurement of the Ca2+ content by X-ray-induced fluorescence spectroscopy, where the average value of 453.3 ± 56.1 nmol Ca2+·mgdrm–1 (n = 3) was obtained. This difference seems to be due to the low degree of extraction of radioactivity from the precipitated mycelium. The alkaline extraction increased the recovery of

radioactivity, but introduced significant experimental errors (unpublished). Now, a systematic method is used to determine the quantitative release of 45Ca2+ from the mycelium. The incubation of the 45Ca2+-labelled mycelium in the cultivation medium without external radioactivity enabled us to study the properties of the Ca2+ efflux. The efflux of radioactivity had a hyperbolic time-course (Fig. 5A), and reached the steady-state after approximately 4–5 h at 25°C (not shown). The extent of the Ca2+ efflux varied markedly between individual experiments. Its average value was 0.0116 ± 0.0010 nmol·min–1·mgdrm–1 (n = 4). The Ca2+ efflux was influenced by extracellular pH (pHo), which varied in range between 4 and 8 (Fig. 5B). Upon increase of pHo, the rate of the 45Ca2+ efflux decreased monophasically, and the decrease between pH 4 and 8 was about 40% (i.e., about 10% 䉭pHo). Temperatures between 15°C and 37°C positively influenced the rate of the 45Ca2+ efflux (Fig. 5C); the Q10 for the efflux was 1.67. The 45Ca2+ efflux was not significantly affected by the addition of 40Ca2+, or monovalent cations (Na+, K+, 100 mM) (Table 2). Cu2+ (100 µM) (Table 2) and a surplus of EGTA slightly inhibited the 45Ca2+ efflux. In order to characterize the mechanisms underlying the Ca2+ efflux, the effects of several inhibitors of transport systems possibly involved in the outward Ca2+ transport were tested. Their effects are summarized in the Table 2. It was found that inhibitors of P-type ATPases, vanadate (up to 10 mM), diethylstilbestrol (100 µM), and dicyclohexyl carbodiimide (100 µM) exerted a minor inhibitory effect on the Ca2+ efflux. The inhibitory effect of vanadate significantly inreased in the presence of uncoupler. The inhibitors of V-and F-type ATPases, such as erythrosine B (70 µM), azide (2 mM), and nitrate (100 mM) showed similarly small inhibitory effects on the 45Ca2+ efflux (Table 2). The values of the 45Ca2+ efflux did not decrease steeply with age of mycelium in accordance with Ca2+ influx (Fig. 6). Ca2+ mobilization and intracellular stores The small extent of the Ca2+ efflux and the small effects of inhibitors on the Ca2+ efflux demonstrated above suggest that the majority of hyphal Ca2+ is stored in intracellular © 2000 NRC Canada

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Can. J. Microbiol. Vol. 46, 2000 Table 2. The effects of selected inhibitors on

45

Ca2+ efflux from the submerged mycelium. 45

Exp. No.

Inhibitors

Concentration (mM)

Ca2+ efflux (nmol Ca2+·min–1·mgdrm–1)

1 1 2 2 3 3 3 4 4 5 5 6 6 6 7 7 8 8 9 9 10 10 10

None – control 40 Ca2+ ions None Cu2+ ions None K+ ions Na+ ions None EGTA None DCCD Control (+FCCP) DCCD + FCCP DES + FCCP None Vanadate Control (+FCCP) Vanadate + FCCP None Erytrosine B None NaNO3 NaN3

0 1 0 0.1 0 100 100 0 5 0 0.1 0.03 0.1 + 0.03 0.1 + 0.03 0 10 0.03 10 + 0.03 0 0.1 0 100 2

0.0099 0.0108 0.0177 0.0244 0.0116 0.0113 0.0114 0.0149 0.0119 0.0117 0.0095 0.0209 0.0281 0.030 0.0116 0.0105 0.0224 0.0159 0.0155 0.0134 0.0139 0.0113 0.0104

organelles. The release of Ca2+ from the putative stores is expected to increase the cytoplasmic Ca2+ concentration, and Ca2+ in turn stimulates the Ca2+ efflux mechanism. Several compounds were found to stimulate the 45Ca2+ efflux. The addition of a Ca2+-ionophore A23187 (10 µg·mL–1) significantly stimulated the Ca2+ efflux (Fig. 7A). Similarly, uncouplers TCS and FCCP, in concentrations of 30 µM, showed a stimulatory effect on the 45Ca2+ efflux (Fig. 7B). Salicylate, up to 7mM (Fig. 8A), and azalomycin F, up to 30 µg·mL–1 (Fig. 8B), stimulated the 45Ca2+ efflux to an extent comparable to that induced by uncouplers. The similarity of effects of all substances suggested that the release of Ca2+ from organelles precedes the efflux from cells. This notion was experimentally tested below. The 45Ca2+ fluxes into and from intracellular stores The first approach in addressing this problem was based on the study the interaction of agents found to stimulate the 45 Ca2+ efflux from the intact mycelium. Saturated concentrations of these agents were used for these experiments in order to obtain maximal responses. Salicylate (7 mM) was able to exert the release of Ca2+ when added after azalomycin F (30 µg·mL–1) or TCS (30 µM). However, when the addition of salicylate preceded that of azalomycin F, or TCS, the last substances were unable to exert the release of Ca2+ (Figs. 9A, B). Thus, salicylate-induced Ca2+ release also involves components released by the uncoupler and azalomycin F. The effect of salicylic acid was mimicked by the addition of butyric acid at millimolar concentrations (not shown). On the other hand, the effects of TCS and azalomycin F on the 45Ca2+ release were additive (Fig. 9C).

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0009 0.0011 0.0012 0.0029 0.0011 0.0011 0.0007 0.0010 0.0024 0.0010 0.0008 0.0018 0.0047 0.0077 0.0001 0.0007 0.0012 0.0005 0.0025 0.0016 0.0025 0.0019 0.0019

45Ca2+ efflux (% of control) 100 108.2 100 137.8 100 97.1 97.5 100 78.6 100 81.1 100 134.4 143.5 100 90.5 100 70.1 100 86.5 100 81.1 74.7

Fig. 6. The 45Ca2+ efflux from submerged mycelium during its development. Ages indicated were taken for the measurement of 45 Ca2+ efflux in the presence of TCS (30 µM), under control conditions between 0 and 60 min, and measurement of dry mass. Values of influx were corrected for radioactivity retained at time zero. Results are representative of two experiments.

When a crude mitochondrial/vacuolar preparation was isolated and used as a model (Cornelius and Nakashima 1987), both Ca2+ influx and release could be studied. The preparation could be loaded with 45Ca2+ in both ATP-dependent and ATP-independent manners. The former exceeded about 3 times the ATP-independent influx (Fig. 10A). The ATP© 2000 NRC Canada

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Fig. 7. The effects of A23187 (A) and uncouplers (FCCP and TCS)(B) on the 45Ca2+ efflux from the submerged mycelium. The concentration of A23187 was 30 µg·mL–1, and that of uncouplers was 30 µM. In controls, the same volume of 0.5% (v/v) methanol was added.

Fig. 8. The effects of salicylate (A) and azalomycin F (B) on the 45Ca2+ efflux from the submerged mycelium, measured between 0 and 120 min. Values of efflux were corrected for radioactivity retained at time zero, and were calculated by least-square method. In controls, the same volume of 0.5% (v/v) methanol was added. Results are epresentative of two experiments.

independent transport was stimulated by FCCP (about 70% at 30 µM FCCP) (Fig. 10A), and, approximately to the same extent, by salicylate (1 mM). Azalomycin F (30 µg·mL–1) had no effect on the ATP-independent 45Ca2+ transport (Fig. 10A). The ATP-dependent 45Ca2+ influx (Fig. 10B) was influenced by these agents in a different way. Uncouplers inhibited the influx by about 40% (Fig. 10B), whereas the effect of azalomycin F was even more pronounced. Salicylate exerted an inhibitory effect on the extent, but not on the initial rate, of the ATP-dependent transport. These agents, when applied in the same concentrations, also influenced the 45Ca2+ influx by the microsomal fraction, which was obtained as a supernatant after spinning down the vacuolar and mitochondrial fraction (Figs. 11A, B). These effects were different from those observed in the vacuolar/ mitochondrial fraction. Uncouplers stimulated the influx without ATP, but had no effect on influx in the presence of ATP. Azalomycin F stimulated the ATP-independent influx, but inhibited the ATP-dependent one. In the absence of ATP,

salicylate inhibited the initial rate of the Ca2+ influx, but increased the amount of the influx (Fig. 11A). However, in the presence of ATP (Fig. 11B), no effect of salicylate was observed. The release of the label was studied when uncoupler (30 µM), azalomycin F (30 µg·mL–1), and salicylate (1 mM), or A23187 (10 µg·mL–1), were added at the above concentrations to a suspension previously pre-loaded with 45Ca2+ in ATP-dependent manner (Fig. 12). A23187 exerted the most pronounced effect in both vacuolar/mitochondrial and microsomal fractions. Salicylate induced a larger release of 45 Ca2+ in the vacuolar/mitochondrial fraction than in the microsomal fraction. However, azalomycin F induced 45Ca2+ release in the microsomal fraction only, and TCS, surprisingly, did not induce 45Ca2+ release in either preparation (Figs. 12A, B). We made several attempts to separate the mycelial homogenate into individual fractions, by means of ultracentrifugation on discontinous and continous sucrose gradients. The migration of individual organelles was monitored with © 2000 NRC Canada

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Fig. 9. The effects of TCS, azalomycin F, and salicylate on the 45Ca2+ efflux. (A) Left panel: Azalomycin F (30 µg·mL–1) was added to the suspension pre-labelled with 45Ca2+, and the 45Ca2+ efflux was measured. After 45 min (arrow), salicylic acid (7 mM) was added and the measurement was continued. The same volume of 0.5% (v/v) methanol was added to the control. Right panel: the order of addition of salicylic acid and azalomycin F was reversed. (B) Left panel: TCS (30 µM) was added to the suspension pre-labelled with 45 Ca2+ and the 45Ca2+ efflux was measured. After 45 min (arrow), salicylic acid (7 mM) was added, and the measurement was continued. The same volume of 0.5% (v/v) methanol was added to the control. Right panel: the order of addition of salicylic acid and TCS was reversed. (C) Left panel: TCS (30 µM) was added to the suspension pre-labelled with 45Ca2+, and the 45Ca2+ efflux was measured. After 45 min (arrow), azalomycin F (30 µg·mL–1) was added, and the measurement was continued. The same volume of 0.5% (v/v) methanol was added to the control. Right panel: the order of addition of TCS and azalomycin F was reversed.

Fig. 10. The effects of uncouplers, salicylate, and azalomycin F on the ATP-independent (A) and ATP-dependent (B) 45Ca2+ influx by the vacuolar/mitochondrial fraction of the homogenate from submerged mycelium. The concentration of FCCP was 30 µM, and that of azalomycin F was 30 µg·mL–1. The concentration of salicylic acid was 1 mM. The values were corrected for those of ATP-independent Ca2+ influx.

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Fig. 11. The effects of uncouplers, salicylate, and azalomycin F on the ATP-independent (A) and ATP-dependent (B) 45Ca2+ influx by the microsomal fraction of the homogenate from submerged mycelium. The concentrations of additions were as in Fig. 10. In (B), the values were corrected for those of ATP-independent Ca2+ influx.

Fig. 12. The effects of uncoupler, salicylate, and azalomycin F on the 45Ca2+ release from the vacuolar/mitochondrial (A) and the microsomal fraction (B) pre-loaded with 45Ca2+ in ATP-dependent manner. The concentrations of additions were as in Fig. 10. In B, the values were corrected for those of ATP-independent Ca2+ influx.

marker enzymes. However, the purity of individual fractions (mitochondria, vacuoles, golgi apparatus, endoplasmic reticulum and plasma membranes) was not sufficient (not shown), and we did not measure Ca2+ movements in these preparations.

Discussion If Ca2+ exerts physiologically relevant effects in filamentous fungi, their membranes should be equipped with transport systems capable of conveying Ca2+ in both directions across the plasma and intracellular membranes. It could be supposed that ontogenesis of the mycelium from germinating conidia is accompanied by Ca2+ influx from the medium, because it is improbable that the Ca2+content of conidia is sufficient to supply the Ca2+ needs of the mycelium, as well

as those of the next generation of conidia. In addition, the passive Ca2+ transport into the cytoplasm is known to play an active role in triggering physiological events. That is why the inward Ca2+ transport from the cell exterior is expected to have a central role in the communication of cells with their environment. An important question is whether 45Ca2+ influx is mediated by a transport protein (a carrier). Of five essential criteria necessary to prove the catalytic character of Ca2+ influx, only two could be regarded as unambiguous. Those criteria are the saturability (Fig. 2B) and inhibition of transport with Ca2+ congeners, or substrate analogues, such as Sr2+, Ba2+, or Mg2+; or inorganic Ca2+ antagonists, such as Cd2+, or Co2+ (Kryštofová et al. 1995). The effect of temperature on Ca2+ transport would yield a Q10 of more than 1.03 if transport is due to more than just simple diffusion, and thus indi© 2000 NRC Canada

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cating that the process is catalyzed by a carrier; we can therefore conclude that between 20°C and 25°C, the observed Ca2+ influx is mediated by a process of simple diffusion (Fig. 2A). This conclusion, however, would be an oversimplification, because at temperatures above 28°C, the 45 Ca2+ influx increased when Q10 was larger than 2. Furthermore, other results (Figs. 3–12) show that the influx of Ca2+ is related to the subsequent intracellular Ca2+ translocation. Therefore, the complex temperature dependence probably reflects that steady state Ca2+ influx is established by the activity of several transport systems with different Q10 values, which translocate Ca2+ in both (opposite) directions. Other criteria for establishing the presence of a Ca2+ carrier in the T. viride membrane, such as the existence of specific Ca2+ transport inhibitors, or the specificity of the putative Ca2+ transport system, have not been met. In the course of our study, several tens of candidate compounds were tested, but no effective inhibitor of the Ca2+ influx was found (unpublished). The specificity of the divalent and (or) trivalent ion transport is currently being investigated in our laboratory. As to the question of the driving force of the Ca2+ influx, the manipulation of Na+ and K+ content in the medium does not significantly change influx (Table 1). This suggests that the alkali metal cations, and their gradients, are not part of the driving force. Valinomycin, the electrogenic K+-specific ionophore which presumably increased the membrane potential, stimulated the 45Ca2+ influx by about 25% (Table 1). This indicates that the putative Ca2+ influx system is influenced by the membrane potential. The stimulatory effects of uncouplers (TCS, FCCP) (Figs. 1, 2; Table 1) are not in accordance with the notion that the H+ gradient is a component of the driving force for Ca2+ influx, because the dissipation of proton gradients is expected to inhibit the Ca2+ influx. Thus, the effect of uncouplers on the Ca2+ influx, and the stimulation of the Ca2+ influx in alkaline pH together with the effect of valinomycin (Fig. 1B), could be explained by a model of the electrogenic Ca2+/nH+ (n < 2) antiporter. It is also possible that the putative transport system is a Ca2+ uniporter, since the stimulatory effect of alkaline pH (Fig. 1B) is due to the removal of Ca2+-inhibiting H+ from the putative transport system. In any case, the osmotic gradient of Ca2+ should also contribute to the driving force. H+/ Ca2+ antiporters were found in fungal (Dunn et al. 1994) and plant (Schumaker and Sze 1986; Hirschi 1996) vacuolar membranes and in bacteria (Matsushita et al. 1986; Ivey et al. 1993), but we have not found data indicating its presence in the fungal plasma membrane. It was found (Figs. 5–9; Table 2) that the Ca2+ efflux occurs simultaneously with the Ca2+ influx, but with efflux properties which are markedly different from those of Ca2+ influx. The temperature (Fig. 5C) dependence and the Q10 of the process suggest that it is mediated by a Ca2+ transport protein which could not be identified. The differences in the pH dependencies of Ca2+ influx and Ca2+ efflux (Fig. 1B and Fig. 5B) indicate that the transport systems conveying Ca2+ across the plasma membrane in opposite directions are not identical. Using data obtained with Neurospora crassa (Stroobant and Scarborough 1979) or Ustilago maydis (Hernandez et al. 1994), the transport system involved in the Ca2+ efflux might be the Ca2+-ATPase. In our study, only


moderate effects of inhibitors were observed (Table 2). The P-ATPase inhibitors, such as DCCD and vanadate, had little effect. The effect of vanadate in the presence of an uncoupler (see below), could support the involvement of the Ca2+-ATPase. Remarkably, the Ca2+ efflux was stimulated by the presence of uncoupler (Fig. 8). This effect excludes the role of H+ electrochemical potential as the driving force of the Ca2+ efflux across the cytoplasmic membrane. It could be explained by the release of Ca2+ from intracellular Ca2+ stores, and subsequent increased availability for the Ca2+-extruding transport systems which have, in general, a KM(Ca) in the range 0.1–1.0 µM. The increase of the cytoplasmic Ca2+ concentration could explain the increase in the inhibitory effect of vanadate in the presence of an uncoupler. The effects of inhibitors of V-and F-type ATPase inhibitors, which moderately inhibited the Ca2+ efflux (Table 2), could not be explained by our data. These inhibitors probably exert multiple, or non-specific, effects on the transport systems in T. viride, and their target should be determined only by independent experimental analysis. The study of agents which were supposed to release Ca2+ from intracellular organelles brought results which, in part, support the notion that increased Ca2+ availability is the cause of the increased Ca2+ efflux. This is certainly possible for salicylate and azalomycin F (Figs. 10–12). Uncouplers induced, surprisingly, no Ca2+ release from the mitochondrial/vacuolar fraction, in contrast with the results obtained in yeast vacuoles (Ohsumi and Anraku 1983). It is possible that the releasable Ca2+ was lost from the mitochondrial part of this fraction during the isolation procedure, which could explain the absence of a significant uncoupler-induced Ca2+ efflux from this fraction. Thus, the stimulatory effect of uncouplers on the Ca2+ efflux in our experiments could be explained only by their differential effects on the ATPdependent and -independent Ca2+ influx in the mitochondrial/vacuolar fraction (Fig. 10). The membranes from the Golgi apparatus do not seem to be involved in the effect of uncouplers, because the Ca2+ influx by Golgi membranes (in yeasts) is not sensitive to the uncoupler (Sorin et al. 1997). We were able to confirm these results for the ATP-dependent Ca2+ influx (Fig. 11). On the other hand, the additive effects of TCS and azalomycin F (Fig. 9) indicate that they are acting on different membranes. It seems that azalomycin F releases Ca2+ from either the Golgi apparatus or endoplasmic reticulum membranes, but not from mitochondrial/vacuolar membranes (Figs. 11–12). However, both these agents release only a fraction of total potentially released Ca2+ by salicylate (Fig. 12). Salicylate seems to potentially be a universal Ca2+ releasing agent from intracellular Ca2+ stores. The stimulatory effects of agents known to release Ca2+ from intracellular stores can be explained in terms of capacitative Ca2+ influx, which has been coined for animal (Penner et al. 1993; Parekh and Penner 1997; Putney 1997, for review) and recently also for yeast (Csutora et al. 1999) cells. Such a mechanism fully explains the stimulatory effects of TCS, and azalomycin F, but not of salicylate on the 45 Ca2+ influx and efflux. Salicylate, which inhibited the Ca2+ influx (Table 1) despite its Ca2+ releasing action (Fig. 12), could become a useful tool in the study of the capacitative Ca2+ influx. Whether the effects of monensin or valinomycin © 2000 NRC Canada

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could be explained in terms of capacitative Ca2+ transport, remains to be established. The data from the ontogenetic experiments show that the specific Ca2+ influx to the submerged mycelium decreases with its age (Figs. 3–4). This can be explained either by the delayed ontogenetic appearance (or activation) of a putative Ca2+ efflux transport system, or by the supposition that filling up a putative Ca2+ store precedes the maturation of the submerged vegetative mycelium. The activity of the 45Ca2+ efflux, however, did not increase during the aging of the vegetative mycelium (Fig. 6). Therefore, the store-filling model seems to be more appropriate for the explanation of decreased Ca2+ influx in the aged vegetative mycelium. The above results suggest that several pools participate in the creation of Ca2+ stores and, after their saturation, the fungus behaves autonomously with respect to external Ca2+ during vegetative development. Nevertheless, conidia formation represents a kind of discontinuity in the Ca2+ homeostasis outlined above. This process is accompanied by the decrease of mycelial mass and total Ca2+ content (Fig. 4), which is followed by an increase in these parameters (Fig. 4). Thus, it seems to be due to the ability to take up Ca2+ from the extracellular environment during conidia formation, rather than the redistribution of cell Ca2+ during this stage of the Trichoderma life cycle. The corresponding Ca2+ transport systems and signalling pathways have yet to be discovered.

Acknowledgements This work was supported Slovak Grant Agency VEGA (Grant No. 1/4203/97). The authors express thanks to Dr. Ondrej urilla (Laboratory of the Main Customs Office, Bratislava) for the measurement of calcium by X-ray fluorescence spectrometry.

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