Article
Reference
Developmental Changes in the
45Ca
2+Uptake by Trichoderma viride Mycelium
ŠIMKOVIČ, M., et al.
Abstract
The rate of the ⁴⁵Ca²⁺ uptake by the submerged Trichoderma viride mycelium increased with the age of the culture from 6 h until a maximum which was reached at about 30 h, and then decreased until the uptake was virtually zero. The decrease in the rate of the ⁴⁵Ca²⁺ uptake was accompanied by an increase of mycelial mass. The uptake rate could not be reactivated upon substituting the medium for a fresh one, without or with dilution of the mycelium. The results suggest that the rate of ⁴⁵Ca²⁺ uptake reflects the biological age of the submerged culture. The surface-cultivated mycelium took up ⁴⁵Ca²⁺ proportionally with time. The autoradiography of colonies showed that ⁴⁵Ca²⁺ was distributed homogeneously throughout the mycelium during vegetative growth while conidiation was accompanied by a massive accumulation of ⁴⁵Ca²⁺ in conidia.
ŠIMKOVIČ, M., et al . Developmental Changes in the
45Ca
2+Uptake by Trichoderma viride Mycelium. Folia Microbiologica , 1997, vol. 42, no. 2, p. 125-129
DOI : 10.1007/BF02898720
Available at:
http://archive-ouverte.unige.ch/unige:123249
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Folia Microbiol. 42 (2), 125-129 (1997)
Developmental Changes in the 45Ca 2 + Uptake by Trichoclerma viride Mycelium
M. ~IMKOVIg a, R. ORTEGA-PEREZ b, G. TURIAN h, V. BETINA a, D. HUDECOVA a and E. VARE~KA a*
aDepartment of Biochemistry and Microbiology, Slovak Technical Universi~ Bratislava, Slovalda bLaboratoire de Microbioiogie Generale, University of Geneva, Geneva, Switzerland
Received October ~ 1996 Revised version November 8~ 1996
ABSTRACT. The rate of the 45Ca2+ uptake by the submerged Tr/choderma v/r/de mycelium increased with the age of the culture from 6 h until a maximum which was reached at about 30 h, and then decreased until the uptake was virtually zero. The decrease in the rate of the 45Ca2+ uptake was accompanied by an increase of mycelial mass. The uptake rate could not be re- activated upon substituting the medium for a fresh one, without or with dilution of the mycelium. The results suggest that the rate of 45Ca2+ uptake reflects the biological age of the submerged culture. The surface-cultivated mycelium took up 45Ca2+
proportionally with time. The autoradiography of colonies showed that 45Ca2 + was distributed homogeneously throughout the mycelium during vegetative growth while conidiation was accompanied by a massive accumulation of 45Ca2 + in conidia.
Ca 2 + homeostasis is known to play an important role in animal, plant and microbial cells in controlling basic physiological processes. In fungi the knowledge of the Ca 2+ homeostasis and the cor- responding transport and regulatory mechanisms has lately been developing (see O'Day 1990, Youatt 1993, for reviews) and the literature data indicate that Ca 2§ ions may play an important role in the morphogenesis and differentiation of filamentous fungi (Muthukumar et al. 1991; Reissig and Kinney 1983; Pitt and Poole 1984; Pitt and Barnes 1993; Muthukumar and Nickerson 1984; Muthukumar et al.
1991; Dicker and Turian 1990; Robson et al. 1991a,b; Gadd and Brunton 1992), especially in the growth of hyphal tips (see Jackson and Heath 1993, for review). The cellular constituents important in main- taining the Ca 2§ homeostasis were found to be vacuoles (Eilam et al. 1985; Cornelius and Nakashima 1987; Duma et al. 1994). Transport systems involved in Ca 2§ homeostasis were identified in the oomycete Saprolegnia ferax (Garrill et al. 1993) and in yeasts (Rudolph et al. 1989), and genetical tools were developed that may lead to the identification of Ca 2+ influx pathway in yeasts (Beeler et al. 1994;
Fu et al. 1994). In this paper we present observations describing developmental changes in Ca 2 § uptake by Trichoderma viride.
M A T E R I A L S A N D M E T H O D S
Strain. Trichoderma viride, strain CCM F-534 from the Czechoslovak Collection o f Micro- organism o f the Masaryk University, Brno (Czech Republic).
Cultivation
Submerged cultivation. Two hundred mL of liquid Czapek-Dox medium in 500-mL flasks containing 0.5 % yeast autolysate was inoculated by T. viride conidia (106/mL) and the suspension was cultivated in the dark on a rotary shaker (4 Hz) at 25 ~ for the time indicated in the Figures.
Surface cultivation. The fungus was grown in Petri dishes (120 mm diameter) on Czapek-Dox agar containing 0.5 % ( W / V ) yeast autolysate at 25 ~ The plates (in duplicate) were inoculated by filter paper disks (5 mm diameter) soaked in a suspension of conidia in 0.02 % Tween 20 ( V / V ) (106 conidia per disk), or with vegetative mycelium overgrowing the disks, placed on the agar plate cov- ered with cellophane sheet under red light illumination and incubated in the dark at 25 ~ All inspec- tions of plates and measurements of colonies were done under the red light in order to prevent photo- conidiation. If conidiation was to be induced, the plates with 36 h old colonies were illuminated by a 10-min pulse of white light (fluorescent lamp, 1.2 klx) and then incubated in the dark for 1 d.
Controls without illumination were treated in parallel.
*Corresponding author.
126 M. ~IMKOVI(~ et al. Vol. 42
45Ca2 + uptake
Submerged cultures. Aliquots of the suspension of hyphae from submerged cultures were used for the experiments. 2.5 mL aliquots were withdrawn from the culture by means of micropipette tips with cut-off ends under sterile conditions and incubated for 0 or I h with 45CAC12 (final concentration 0.55 retool/L) (specific activity about 375 cpm/umol) without stirring. The mycelial suspension had to show no apparent inhomogeneities and was carefully stirred before withdrawing the aliquots. Subse- quently, I mL aliquots were filtered on a membrane filter using a vacuum filtration apparatus and a Millipore nitrocellulose filter (pore diameter 0.65 ~tm). The pellet was washed with 2 • 4.5 mL Cza- p e k - D o x medium containing 10 mmol/L EDTA (pH 7.0) and was taken for liquid scintillation count- ing. In parallel, 2 - 1 0 mL aliquots were filtered under the same conditions for the determination of mycelial mass. The results are expressed as average of duplicates _+ standard error of a representative of three experiments.
Surface culture. The fungus was cultivated on Czapek-Dox agar containing 0.5 % yeast autolysate and covered with filter paper disks for 40 h in the dark at 25 ~ transferred to agar medium containing 45Ca 2+ (75-150 kBq) under red illumination (time 0), and then incubated again at 25 ~ At time intervals between 5 and 45 h duplicates of disks were withdrawn from the medium, the mycelium was flushed with Czapek-Dox medium containing 10 mmol/L EDTA onto the Millipore membrane filtration funnel, washed with 2 • 10 mL of Czapek-Dox medium and taken directly for liquid scintillation counting. After counting the mycelial precipitate was filtered through a nylon mesh in order to remove the scintillation cocktail, and rinsed with pure dioxane. The precipitate was trans- ferred to an Eppendorf tube, covered with I mL 0.1 mol/L NaOH and proteins were extracted by incubation at 70 *C for i h. The rests of mycelia were removed by centrifugation and the supernatants were used for protein determination with the Lowry reagent. The results are expressed as an average of duplicates _+ standard error of a representative of three experiments.
Autoradiography of colonies. Paper disks (90 mm diameter) were inoculated with 5 ~tL of a suspension containing about 5 • 108 conidia per mL soaked into 5 mm disks, placed in the center of the disks and cultivated on radioactive Czapek-Dox agar containing 45Ca 2+ (75-150 kBq) for 28 h in the dark (control under a circadian illumination). Part of the disks were illuminated by white fluores- cent light (1.2 klx for 10 rain) and the cultivation in the dark continued for another 1 d. The growth was stopped by transferring the disks onto the Biichner funnel and washing them with 100 mL of Cza- p e k - D o x medium containing 10 mmol/L EDTA. They were then immediately placed on a pre-heated vacuum polyacrylamide gel dryer, covered with polyethylene sheet and dried at 95 *C for 10 mill. The protective polyethylene sheet was removed and dried disks were exposed to the Medix Rapid X-ray film (Foma, Hradec Kr~ilov6, Czech Republic). The exposure time varied from 3 to 28 d. After the autoradiography the colonies were visualized by staining with Coomassie Brilliant Blue R-250 accord- hag to the procedure used for polyacrylamide gel staining and destaining.
Determination of mycelial mass. Two mL aliquots of the suspension were intensively drained on a membrane filter to determine the wet mass, dried at 90 ~ to a constant mass and weighed.
Chemicals. 45CAC12 from Radiochemical Centre, Amersham (UK); ethylenediaminetetraacetic acid (EDTA) from Merck, Darmstadt (Germany); all other reagents we purchased from Lachema, Brno (Czech Republic).
RESULTS A N D DISCUSSION
The measurement of 45Ca2+ uptake was started after 10-15 h of cultivation when hyphae from the conidial inoculum appeared in our conditions and the mycelial mass could be reliably esti- mated. Aliquots of the mycelium took up 45Ca2+ in accordance with observations published earlier (Krygtofov~i et al. 1995). The radioactivity incorporated into the mycelium after 1 h incubation with the radionnclide rose to a maximal value which was obtained after about 30 h of cultivation and then progressively decreased (Fig. 1A) until the end of cultivation (usually 65 h but in one experiment 120 h). The decrease of the 45Ca2 + uptake continued in spite of the expected increase in mycelial mass (Fig. 1B) which reached the steady-state value after about 70 h of cultivation in our conditions. The expression of 45Ca2+ uptake in nmol Ca 2+ per h per mg of mycelial mass (i.e. as the specific uptake) showed that its time course decreases monophasically with time (Fig. 1C) until virtually zero values were observed. The difference in the time courses of the radioactivity accumulation and the mycelial growth shows that 45Ca2 § accumulation occurs only at an early stage of the mycelial development. It is
1997 DEVELOPMENTAL CHANGES IN THE 45Ca2+ UPTAKE BY T. viride MYCELIUM 127
possible that the intraceUular Ca 2+ storage sites are filled with Ca 2+ at this stage, to be later utiliTed for the forthcoming growth, or that the Ca 2§ emux pathway is activated in the aged myceGum, mim- icking a decrease in Ca 2§ influx. The mechanism of the 45Ca2+ uptake down-regulation is currently investigated in our laboratory. Whatever the mechanism, however, this observation impGes that the rate of the 45Ca2+ uptake by the submerged mycelium could represent a measure of its biological age.
6 45Ca2.
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. 2
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Fig. 1. Changes in 45Ca 2+ uptake (A, C) and in mycelial mass (B) during growth of T. viride submerged mycelium; A: 45Ca2+
uptake, epm/h x 10-3; aliquots of submerged culture were withdrawn at times indicated and the uptake was measured for 0 (closed squares) and 1 h (open squares); 13: mycelial mass (dry mass, rag) from aliquots withdrawn for 45Ca2 + uptake measure- ments; C: specific 45Ca2+ uptake, nmol mg -1 h - l ; results from A and B are expressed as nmol Ca2+/mg dry mass assuming an isotopic equilibrium between the medium and the mycelium.
In order to test the above assumption, two experiments were made. First, the Ca 2+ uptake was measured in a 60-h-old mycelium which was subsequently transferred from the cultivation medium to an equal volume of a fresh medium and both Ca 2§ uptake and mycelial mass were measured after 6 and 15 h of cultivation as above. Second, 60-h-old mycelium was transferred to a larger volume of the fresh medium so that the density of the suspension decreased four times and the cultivation continued as above. The 45Ca2 § uptake did not increase despite of the substantial increase in mycelial mass in both experiments
(not shown).
Thus, neither the exhaustion of nutrients nor the contact inhibition are responsible for the decrease and/or the arrest of the Ca 2§ uptake during the submerged cultivation, and the possibility that the rate of the Ca 2 § uptake is an age-related parameter is still not disproved.I I I I
6 0
30 120 45Ca2.
9 0
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O 10 20 30 40 5 0 O h
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Fig. 2. Changes in 45Ca2+ uptake (A) and mycelial mass (13) during growth of T. viride aerial mycelium;
45 2+
A: 45Ca2 + uptake, epm x 10-3; colonies were withdrawn from an agar medium containing Ca at time indicated and the incorporated radioactivity was measured as described in Materials and Methods; 13: the protein content (rag) of samples treated in A.
The cultivation of the fungus on agar medium showed that aerial mycelium took up 45Ca2+
proportionally to time up to 60 h (Fig. 2). After this period the formation of conidia started
(not
shown)
and further measurement would yield ambiguous results. These experiments showed that the128 M. ~IMKOVI(~ eta/. Vol. 42
rate of the 45Ca2+ uptake in the aerial mycelium closely follows the mycelial mass increase during the development, at variance with results obtained in the submerged myeelium.
1 2 3 4
Fig. 3. Distribution of radioactivity in aerial mycelium grown on a medium containing 45Ca 2+ under various illumination regimes; top: photographs of Coomassie BdUiant Blue stained colonies. The photograph of the dark-cultivated mycelium showed the occassional appearance of starvation-induced conidia which have their counterparts on the autoradiograph; / -- circadian illumination (52 h cultivation time, 2 -- 5 h after illumination (33 h cultivation time), 3 -- 24 h after illumination (52 h cultivation time), 4 -- dark (52 h cultivation time); bottom: negative images of autoradiograms in the same order from the left as top. The time of exposure 28 d.
In order to test changes in 45Ca2+ uptake during a prolonged surface cultivation leading to the formation of conidia and to avoid the ambiguity of the bulk radioactivity measurement, a method of in situ radioactivity detection was developed using the autoradiography of vacuum- and heat-dried colonies for the study of Ca 2§ movements during growth and conidiation. It was found that the radio- activity is homogeneously distributed throughout the colony before the conidia are formed (Fig. 3). The formation of conidia was accompanied by a massive accumulation of radioactivity which was co-local- ized with conidia formed upon illumination or in the aged mycelium (Fig. 3), suggesting that Ca 2 + is an abundant constituent of conidia, similarly as it is a constituent of bacterial spores (Rosen 1982, for review). It should be mentioned at this point that the colonies were extensively washed with EDTA- containing medium. This procedure removes divalent ions from both mycelial and conidial surfaces so that the remaining radioactivity revealed in autoradiograms represented the internal cell a n d / o r coni- dial constituents. The question can be raised whether the Ca 2 + deposits in conidia may represent the Ca 2§ supply for their germination. This possibility was studied by means of conidia harvested from an agar medium supplemented with 45Ca2+. Their germination was accompanied by a slow release of radioactivity (Fig. 4) between 1 - 8 h of cultivation, i.e., before the nascent hyphae perforated the coni- dial wall. This suggests that at least part of the conidial Ca 2+ deposits has a solely structural role which becomes superfluous during germination. As for the putative role of Ca 2§ in the process of metabolic regulation during germination, it should be defined by different experimental approaches.
1997 DEVELOPMENTAL CHANGES IN THE 45Ca2+ UPTAKE BY T. viride MYCELIUM 1 ~
Fig. 4. Release of 45Ca 2+ (cpm x 10 -3) during the germination of conidia grown up in the presence of 45Ca2+. The micro- organism was cultivated on Czapek-Dox agar containing 75 kBq/mL of 45Ca2+ until conidiated. The conidia were har- vested, washed with radioactivity-free Czapek-Dox medium and adjusted to the concentration 107/mL. The conidial suspension was cultivated at 28 ~ on a rotary shaker (4 Hz). At times indi- cated, 0.5 mL aliquots were taken and centrifuged through a dibutylphthalate layer. Radioactivity in the supernatant was assayed by liquid scintillation counting.
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This work was supported by the Swiss National Science Foundation (joint Swiss-Slovak project 75LPJ041485) and Slovak Grant Agency (grant no. 1/1158/93).
REFERENCES
BEELER T., GABLE ]C, ZHAO C., DUNN T.: A novel protein, CSG2p, is required for Ca 2+ regulation in Saccharomyces cerevisiae.
J.Biol.Chem. 269, 7279-7284 (1994).
CORNELIUS G., NAKASmMA H.: Vacuoles play a decisive role in calcium homeostasis in Neurospora crassa. ZGen.Microbioi. 133, 2341-2347 (1987).
DICKER J.W., TURIAIq G.: Calcium deficiencies and apical hyperbranching in wild-type and "frost" and "spray" morphological mutants of Neurospora crassa. Z Gen.Microbiol. 136, 1413-1420 (1990).
DUNN T., GAnLE IC, BEELER T.: Regulation of cellular C-a 2+ by yeast vacuoles. ZBiol.Chem. 269, 7273-7278 (1994).
EtLAM Y., LAVl H., GRossowrrz N.: Cytoplasmic Ca2+ homeostasis maintained by a vacuolar Ca 2+ transport system in yeast Saccharomyces cerevisiae. Z Gen.Microbiol. 131, 623-629 (1985).
Fu D., BEELER T., DUNS T.: Sequence, mapping and disruption of CCC1, a gene that cross-complements the Ca2+ sensitive phenotype of csgl mutants. Yeast 10, 515-521 (1994).
GADD G.M., BRUIn'ON A.H.: Calcium involvement in dimorphism of Ophiostoma ulmi, the Dutch elm disease fungus, and characterization of calcium uptake by yeast cells and germ tubes. ZGen.Microbiol. 138, 1561-1571 (1992).
GARRILL A., JACKSON S.L., LEg' 1L1L, HEATH I.B.: Ion channels and tip growth stretch activated channels generate Ca 2 + gradi- ent for hyphal growth. Eur.Cell.BioL 60, 358-365 (1993).
JACKSON S.L., HEATH I.B.: Roles of calcium ions in hyphal tip growth. MicrobioLRev. 57, 367-382 (1993).
KRYg'TOFOVA S., VARF2XA L., BEns^ V.: The 45Ca 2+ uptake by Trichoderma viride mycelium. Correlation with growth and conidiation. Gen.Physiol.Biophys. 14, 323-337 (1995).
MUTHUKUMAR G., NICKERSON A.W.: Ca(lI)-calmodulin regulation of fungal dimorphism in Ceratocystis ulmL ZBacteriol. 154, 219-230 (1983).
MU'I~UKUMAR G., JENSEN E.C., NICKERSON A.W., ECKLES M.K., NICKER.SON K.W.: Photomorphogenesis in Penicillium isariae- forme: exogenous calcium substitutes for light. Photochem.PhotobioL 53, 287-291 (1991).
O'DAY D.H.: Calcium as an intracellular messenger in eucaryotic microbes, pp. 3-13 in D.H. O'Day (Ed.): Calcium as an Intracellular Messenger in Eucaryotic Microbes. American Society for Microbiology, Washington (DC) 1990.
Prrr D., POOLE P.C.: Calcium induces conidiation in Penicillium notatum in submerged cultures. Trans.Brit.Mycol.Soc. 76, 219- 230 (1984).
Prrr D., BARNES J.C.: Calcium homeostasis, signalling and protein phosphorylation during calcium-induced conidiation in Peni- cillium notatum. J.Gen.Microbiol. 139, 3053-3063 (1993).
REISSlG J.L., KINNEY S.G.: Calcium as branching signal in Neurospora crassa. J.BacterioL 154, 219-230 (1983).
ROBSON G.D., WIEBE M.G., Tinsel A.PJ.: Involvement Ca2+ in the regulation of hyphal extension and branching in Fusarium graminearum A 3/5. F_xp.Mycol. 15, 263-272 (1991a).
RoB.sOS G.D., WiEBE M.G., TRlIqCl A.PJ.: Low calcium concentrations induce increased branching in Fusarium graminearum.
Mycol.Res. 95, 561-565 (1991b).
ROSEN B.: Calcium transport in microorganisms, pp. 187-216 in E. Carafoli (Ed.): Membrane Transport of Calcium. Academic Press, London 1982.
RUDOLPH H.IC, ANTEm A., FtNK G.IL, BUCKLEY C.M., DORMAN T.E., LEVrrRE J., DAValX)W L.S., MAO J.I., Morn D.T.: The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca ?-+ ATPase family. Cell 58, 133-145 (1989).
YOUATr J.: Calcium and microorganisms. Crit.Rev.Microbiol. 19, 83-97 (1993).
