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Sources of endogenous glucose during endotrophic differentiation of zoosporangia in Allomyces arbuscula
[ 547 ] Trans. Br. mycol. Soc. 81 (3) 547-552 (1983)
Primed in Great Britain
SOURCES OF ENDOGENOUS GLUCOSE DURING ENDOTROPHIC DIFFERENTIATION OF ZOOSPORANGIA IN ALLOMYCES ARBUSCULA By M . OJHA AND G. TURIAN Laboratory of General Microbiology, University of Geneva, 3 rue de Candolle, CH-l 21 1 Geneva 4 The induction of zoosporangial differentiation in dilute salt solution results in accumulation and utilization of free glucose from the suspending medium. Actively growing plants synthesize glycogen and trehalose. None of them is degraded intracellularly during growth. Much of the intracellular trehalose synthesized is secreted into the medium during differentiation. Although part of the extracellular trehalose might be degraded into glucose, the bulk of the glucose required for differentiation comes from the degradation of cell wall through the action of fJ-glucanases (jJ-1,3 and fJ-1,6-glucanases). Glucano-lactone, an inhibitor of fJ-glucanases, blocks the differentiation if added early (0-1 h). In Allomyces, as in many other aquatic fungi, the initiation of reproductive differentiation can be induced by transfer of an actively growing culture to distilled water or dilute salt solution (Kobr & Turian, 1967). Apparently this does not require exogenous glucose for its commitment, although exogenously added glucose can be taken up and metabolized (Youatt, 1980) but without preventing commitment to differentiation (Kobr & Turian, 1967). Since induction of differentiation in aquatic fungi can take place in the absence of any external substrate, the catabolism of existing cellular components must provide the metabolites necessary for such so-called endotrophic differentiation. Enzymic degradation of cell-wall polysaccharides has been shown to be one of the most salient features of reproductive differentiation in the basidiomycete Schizophyllum commune Fr. (Wessels, 1965). It was therefore of interest to search for the endogenous source of glucose, the main carbohydrate metabolic substrate required for zoosporangial differentiation of the hypogynous Allomyces arbuscula, induced to differentiate in dilute salt solution. MA TERIALS AND METHODS
Strains and cultural conditions Allomyces arbuscula Butl. obtained from the collection of Professor C. Stumm (Haaren, Netherlands) was used throughout this study. Culture maintenance and the production of inoculum have been described earlier (Ojha & Turian, 1981). An inoculum of 104 spores/rnl (final concentration) was used to inoculate 2 I of GCY medium (T urian,
1963) in a 3 I Erlenmeyer flask. Cells were kept in a homogeneous suspension with constant aeration by forced sterile humidified air. The culture was grown at 32°C, filtered through nylon and washed with sterile DS (dilute salt solution, Machlis, 1953). The washed plants thus obtained were suspended either in 1 I sterile DS or GCY medium and incubated at 32° with forced aeration of sterile humidified air. At intervals, samples were taken, filtered, the filtrate saved for the analysis of glucose and the cells washed with cold distilled water and frozen. Determination of intra- and extracellular glucose For the determination of intracellular glucose extracts were prepared as follows : immediately after filtration the cell pellet was suspended in 1 ml 500 mM-TCA and extracted for 60 min at room temperature. After centrifugation the pellet was re-extracted and the two supernatants were pooled. TCA was removed by three extractions with water-saturated ether and the latter removed by bubbling with nitrogen. Culture fluid from the DS medium taken at intervals was either used directly or lyophilized. Glucose was estimated by the glucose-peroxidase method (Bergmeyer & Bernt, 1974)· Determination of trehalose The method is essentially as described by Lillie & Pringle (1980). Trehalose was measured in the cellular extract prepared as described above using anthrone reagent. Our preliminary test did not reveal the presence of other carbohydrates in the extract in appreciable quantities. Glucose was used 18-2
Differentiation in Allomyces arbuscula as standard and the values of trehalose were expressed as glucose equivalents. Extracellular lyophilized medium was used to determine the anthrone-positive material. The amount of trehalose was determined by the difference between total anthrone-positive material and glucose, assayed by the glucose oxidase and peroxidase method.
16
14
.,
12
e;, Determination of glycogen The method followed was essentially as described ~ 10 by Rickenberg et al. (1975) and Nazly, Carter & C Knowles (1980). Cell pads collected at intervals OIl E during growth and differentiation were suspended 8 in 2 ml 30 % KOH; the clumps were broken to "r5 obtain a homogeneous suspension. The suspension :lCJ 6 was digested by heating in a boiling water bath for 00 OIl 30 min. After cooling 0'2 ml8 % (wIv ) Na 2SO. and :t 5"3 ml absolute ethanol were added. The suspension 4 was kept for at least 1 h in the cold, centrifuged at 1700 g for 10 min, the precipitate dissolved in 1 ml H 2 0 , reprecipitated with 2'4 ml absolute eth2 anol and re-washed twice with distilled water and ethanol. The washed pellet thus obtained was dissolved in 1 ml 1 N-H 2SO. and centrifuged, and the supernatant was hydrolysed in a water bath at 2 3 100° for 3 h, neutralized to pH 7'0 with 6 M-KOH Tim e (h) and the glucose liberated was estimated by the glucose-peroxidase method (Bergmeyer & Bernt, Fig. 1. Intra- and extracellular glucose during zoosporangial differentiation. Intracellular glucose in growing 1974)· .~
/
•
•
(. - - . ) and differentiating (e - - e ) plants. Extracellular glucose (0- -0) in the culture filtrate.
Intracellular and extracellular p-glucanase The cells collected at intervals during growth and differentiation were homogenized with acid-washed quartz sand in a chilled mortar and suspended in sorbitol-tris-EDTA buffer (sorbitol o·5 M,tris-H Cl 0 '01 M (pH 7), EDTA 0'001 M). The homogenate was subsequently dialysed overnight in tris-HClEDTA buffer. The dialysed extract was used to measure p-glucanase activity. In certain experiments the cells were homogenized in 0'05 M-Naacetate buffer pH 5'1 and glucanase activity was measured directly. The culture filtrate from the growth and induction medium (DS) was collected at intervals, lyophilized, dialysed against 0'05 M-phosphate buffer (pH 5'5) for 24 h with two changes. p-glucanase activity was measured according to Cortat (1971) using both laminarin (P-l,3-glucan) and pullulan (P-1,6-glucan). The reducing sugar produced was measured with the glucose oxidase-peroxidase method. Protein was determined according to Lowry et al. ( 1951).
RESULTS
Intracellular and extracellular glucose The level of free glucose in the cellular extracts of plants suspended either in dilute salt solution or rich medium was measured at intervals for 3-4 h. The apices of plants suspended in dilute salt solution (DS) differentiated more or less synchronously into zoosporangia whilst those in the rich medium continued to grow. The intracellular glucose in plants suspended either in DS or rich medium remained at a low level; however, glucose accumulated progressively in the filtrate of the differentiating culture (Fig. 1). This accumulation began immediately after suspension of the plants in the DS solution and a concentration of approximately 32 pM was attained by 1'5 h when young zoosporangia appeared. Subsequently, the level declined to 20 pM and remained at this level until the end of differentiation. The intracellular free glucose remained at 2 pM in both growing and differentiating plants.
M. Ojha and G. Turian 80
500
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(0)
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400
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300
60
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o
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3
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70
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.c:.. 7 .---. 4
20
Tim e (11)
Fig.
549
Cellular glycogen levels during growth Ce--e) and zoosporangial differentiation C . - - . )·
2.
Accumulation of glycogen and trehalose
The amount of glycogen in the plants placed in OS solution varied little during the course of development. A minor increase was observed during the first 60 min of transfer. The plants placed in the growth medium began to accumulate immediately, consequently the rate and amount of glycogen was higher than those in the OS medium (Fig . 2) . In the absence of degradation of glycogen we looked for the synthesis or loss of another storage carbohydrate, trehalose. Measurements of cellular trehalose showed a considerable loss in differentiating plants ' and accumulation in growing plants (Fig. 3a ). In the absence of any increase in the intracellular glucose during differentiation it was anticipated that the loss of cellular trehalose was due to secretion in the medium and not degradation. The measurements of trehalose in the culture fluid of the differentiating cultures showed that indeed this was the case (Fig. 3b). The loss of trehalose in the plants corresponded to its accumulation in the culture fluid. Activities of trehalase and fl-glucanases in the culture filtrates
Since trehalose was massively secreted in the culture medium during differentiation it might serve as a source of glucose through the action of trehalase. Therefore we measured the activity of this enzyme in the culture filtrate . Although some activity was found it was not sufficient to account for the amount of glucose found in the culture filtrate. The possibility that it carne from the degradation of cell-wall glucan through the action
10
-.
•
.
.~/.-
•
0 0
3
2 T ime ( h)
120
(b)
0
100
/\
o
80
60
40
0
~
0
-..--.--.--.
20
0 0
2
3
Time (h )
c. - -.)
Fig. 3.(a) Intracellular trehalose during growth and differentiation Ce - - e ). (b) Trehalose in the plants (e - - e) and culture filtrate (0- - 0) during differentiation.
of glucanases was analysed by measuring the activity of fl-glucanase during development, and the result is shown in Fig. 4. The evolut ion of glucanase activity corresponded well with the appearance of glucose in the medium.
Differentiation in Allomyces arbuscula
550
Table
Time of observation after transfer
15
I
e 0.
•
OIl
" "
on 0
:: Oil OIl
::t
5
/
lactone (mM) 1
o
0-05
0 -1
+2'5*
mM gluc ose
0 '5
0
0
0
0
0
0
0
0-5
0
0
0
0
0
0
0 0 100 7 0 100 90
0
0
30
6
0 0
70
50
0
1 2
3
•
"'.-.
•
• o
%hyphalapices differentiated withglucano-
(h)
/\
10
c
-;:;
E
1 . Effect of glu cano-lactone on zoosporangial differentiation in Allomyces arbuscula
:2
3
Time (11)
Fig. 4. ,8-glucanase in the culture filtrate of differentiating culture. ,8-1,3-C.--.), /1-1,6-C.--. ) glucanase.
Inhibition of fl-glucanase and differentiation Glucano-lactone, an inhibitor of fl-glucanase (Fleet & Phaff, 1974), inhibited zoosporangial differentiationat low concentrations (Table 1).A concentration of 0 '5 mM completely inhibited the differentiation, apparently without affecting any other morphological feature. At 1 mM glucano-lactone provoked cytolysis besides inhibiting zoosporangial differentiation. Exogenous supplement of glucose (2' 5 mM) even at 1 msr-glucano-lactone overcame the inhibitory effect and zoosporangia developed synchronously. Addition of glucano-lactone (0'5 mM) at any time between 0 and 1 h of transfer to DS solution prevented zoosporangial differentiation (Figs 5-7). Subsequently, addition of the inhibitor had no effect and the plants differentiated normally (Fig. 8). DISCUSSION
In many microbial systems, accumulation of reserve carbohydrates takes place at the onset of nutrient depletion (Nazly et al., 1980; Lillie & Pringle, 1980). Nutrient depletion, particularly of C, Nand/or P, also initiates lifting of catabolite
0 30 100
*Added at the time of transfer.
repressionand inducedreproductive differentiation. However, reproductive differentiation requires energy and substrate for the synthesis of new wall constituents. Therefore, against a background of external depletion, the substrate for new synthesis must come from the breakdown of internal constituents. As a matter of fact, the degradation of reserve polymers is often associated with differentiation (Fonzi, Shanley & Opheim, 1979; Zonneveld, 1972; Wessels, 1965; Wolf, 1980). In Saccharomyces cereoisiae Hansen glycogen hydrolysis and accumulation of intracellular glucose is specific to sporulation-committed cells (Fonzi et al., 1979). Likewise, glycogen degradation, accumulation and degradation oftrehalose and increase in intracellular glucose are stage-specific in Dictyostelium development (Rosness & Wright, 1974; Wilson & Rutherford, 1978). By contrast there are examples in other eukaryotic microbes where glucose does not come from the degradation of these intracellular carbohydrate reserves, instead it comes from the degradation of wall polymers , In Aspergillus nidulans (Eidam) Winter, degradation of a-1,3-glucan is associated with conidiation and development of cleistothecia (Zonneveld, 1972). Mutants lacking a-1,3-glucan and melanin are also defective in cleistothecial development (Polachek & Rosenberger, 1977; Zonneveld, 1972). Our results with A. arbuscula show that carbohydrate reserves like glycogen and trehalose are synthesized in the growth medium. Whilst glycogen continues to be synthesized in the induction medium, trehalose synthesis slows down and the cellular trehalose is secreted in the medium. The intracellular trehalase activity during differentiation was found to be very low. This and the absence ofincrease in intracellular glucose indicated that decrease in cellular trehalose was due to secretion in the medium. Indeed, the measurement of trehalose in the culture filtrate showed a progressive accumulation in the early period of differentiation. However, this does not seem to
M. Ojha and G. Turian
551
Figs S-8. Effect of glucano-lactone on zoosporangial differentiation. The plants suspended in DS solution were supplemented with glucano-lactone (o'S mM) at specified intervals and the culture incubated at 32°C on a reciprocal shaker. The zoosporangial differentiation was checked after 3 h of incubation. The control culture was without glucano-lactone (Fig. S); glucano-lactone was added at 0 h (Fig. 6),30 min (Fig. 7), 1'S h (Fig. 8).
provide all the glucose necessary for differentiation, since in the presence of glucano-lactone, a specific inhibitor of {J-1, 3- and 1,6-glucanases, the plants suspended in DS medium do not differentiate. Youatt (1980) reported that apparently trehalose is not metabolized by growing cultures of A.
macrogynus (Emerson) Emerson & Wilson. Therefore, the role of trehalose synthesis and excretion during differentiation remains obscure. The main internal provider of glucose for zoosporangial differentiation in Allomyces appears therefore to be {J-glucan as shown by (a) the
552
Differentiation in Allomyces arbuscula
stimulation of fJ-glucanase during the initiation phase of differentiation; (b) the negative consequence on differentiation of the inhibition of fJ-glucanase by the competitor of glucose , glucano-Iactone; (c) the simultaneous overcoming of both these inhibitions by the provision of external glucose. The requirement of glucose for zoosporangial differentiation is limited to the early period of induction (1'5 h after transfer) and coincides with the peak of glucose accumulation and fJ-1,3glucanase activity. This period is also sensitive to glucano-lactone inhibition of zoosporangial differentiation. These results are in agreement with the conclusion of Youatt (1980), who found that active metabolism of glucose during zoosporangial differentiation takes place before septation occurs to delimit the zoosporangia and that glucose is not metabolized actively by differentiated cultures. a-1,3-g1ucans are the usual storage compounds as recently shown at wall changes during microcycle conidiation of A. niger Van Tieghem (Smith et al., 1980). It is therefore interesting to see that fJglucans, as first shown by Wessels (1965), can also be deployed in the Allomyces as carbon source if necessary, even though their main function remains more of a structural nature. We are grateful to Mrs Arlette Cattaneo for her technical help and to Mrs Barbara Duchoud for manuscript preparation and typing. REFERENCES
BERGMEYER, H. U. & BERNT, E. (1974). Determination with glucose oxidase and peroxidase. In Methods of Enzymatic Analysis (ed. H. U . Bergmeyer), pp . 1205-1212. London, New York : Verlag Chemie, Academic Press. CORTAT, M . (1971). Localisation intracellulaire et fonction des P-1,3-glucanases lors du bourgeonnement chez Saccharomyces cerevisiae. These Ecole Polytechnique Federale, Zurich. FLEET, G. H . & I'HAFF, H . J. (1974). Glucanases in Schizosaccharomyces. Isolation and properties of the cell wall associated P-1,3-glucanases. Journal of Biological Chemistry 249, 1717-1728. FONZI, W. A., SHANLEY, M. & OPHEIM, D. J. (1979). Relationship of glycolytic intermediates, glycolytic enzymes and ammonia to glycogen metabolism during sporulation in the yeast Saccharomyces cerevisiae. Journal of Ba cteriology 137, 285-294. KOBR, M. J. & TURIAN, G. (1967). Metabolic changes during sexual differentiation in Allomy ces. Archiv fur Mikrobiologie 57, 271-279.
LILLIE, S. H . & PRINGLE, J. R. (1980). Reserve carbohydrate metabolism in Sa ccharomy ces cerevisiae. J ournal of Ba cteriology 143, 1384-1394. LOWRY, O. H ., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. MACHLIS, L. (1953). Growth and nutrition in water molds in the subgenus Euallomyces. I. Growth factor requirements. AmericanJournal of Botany 4°,189-195 . NAlLY, N ., CARTER, I. S. & KNOWLES, C. J. (1980). Adenine nucleotide pools during starvation of Benekia natriegens. Journal of General Microbiology 116, 295-3 03 . 0JHA, M . & TURIAN, G. (1981). DNA synthesis during zoosporangial differentiation in Allomyces arbuscula. Journal of General Microbiology 122, 263-269. POLACHEK, I. & ROSENBERGER, R. F. (1977). Aspergillus nidulans mutant lacking a-(1,3)-glucan, melanin, and cleistothecia. Journal of Bacteriology 132, 650-656. RICKENBERG, H . V., RAMSDORF, H. J., CAMPBELL, A., NORTH, M . J., KWASNIAK, J. & ASHWORTH, J. M. (1975). Inhibition of development in Dictyostelium discoideum by sugars . Journal of Bacteriology 124, 212-219· ROSNESS, P. A. & WRIGHT, B. E. (1974). In vivo changes of cellulose , trehalose and glycogen during differentiation of Dictyosteliumdiscoideum.ArchivesofBiochemistry and Biophysics 164, 60-72. SMITH, J. E ., ANDERSON, J. G ., KRISTIANSEN, B., AL-RAWI, A. & YAYHA, A. G. (1980). Microcyc1ic conidiation. In The Fungal Spor e: Morphogenetic Controls (ed, G . Turian & H. R. Hohl), pp. 625--{)50. London, New York: Academic Press. T URIAN, G. (1963). Synthese differentielle d'acide ribonucleique et differenciation sexuelle chez I' A 110myces. Developmental Biology 6, 61-72. WESSELS, J. G . H . (1965). Biochemical processes in S chizophyllum commune. Wentia 13, 1-113. WILSON, J. B. & RUTHERFORD, C. L. (1978). ATP, trehalose, glucose and ammonium ion localization in the two cell types of Dictyostelium discoideum. Journal of Cellular Phy siology 94, 37-45 . WOLF, D. H . (1980). Proteinases and sporulation in yeast . In The Fungal Spore : Morphogenetic Controls (ed. G . Turian & H . R . Hohl), pp. 355-374. London, New York : Academic Press. YOUATT, J. (1980). Changes in carbohydrates of Allomyces macrogynus during selective development of either zoosporangia or resistant sporangia. Australian fournal of Biological S ciences 33, 505-511. ZONNEVELD,B. J. M. (1972). Morphogenesis in Aspergillus nidulans. The significance of a-1 ,3-glucans of the cell wall and a-1 ,3-glucanase for c1eistothecia development. Biochimica B iophysica A cta 273,174-187.
(R eceived for publication
20
January 1983)
Primed in Great Britain
SOURCES OF ENDOGENOUS GLUCOSE DURING ENDOTROPHIC DIFFERENTIATION OF ZOOSPORANGIA IN ALLOMYCES ARBUSCULA By M . OJHA AND G. TURIAN Laboratory of General Microbiology, University of Geneva, 3 rue de Candolle, CH-l 21 1 Geneva 4 The induction of zoosporangial differentiation in dilute salt solution results in accumulation and utilization of free glucose from the suspending medium. Actively growing plants synthesize glycogen and trehalose. None of them is degraded intracellularly during growth. Much of the intracellular trehalose synthesized is secreted into the medium during differentiation. Although part of the extracellular trehalose might be degraded into glucose, the bulk of the glucose required for differentiation comes from the degradation of cell wall through the action of fJ-glucanases (jJ-1,3 and fJ-1,6-glucanases). Glucano-lactone, an inhibitor of fJ-glucanases, blocks the differentiation if added early (0-1 h). In Allomyces, as in many other aquatic fungi, the initiation of reproductive differentiation can be induced by transfer of an actively growing culture to distilled water or dilute salt solution (Kobr & Turian, 1967). Apparently this does not require exogenous glucose for its commitment, although exogenously added glucose can be taken up and metabolized (Youatt, 1980) but without preventing commitment to differentiation (Kobr & Turian, 1967). Since induction of differentiation in aquatic fungi can take place in the absence of any external substrate, the catabolism of existing cellular components must provide the metabolites necessary for such so-called endotrophic differentiation. Enzymic degradation of cell-wall polysaccharides has been shown to be one of the most salient features of reproductive differentiation in the basidiomycete Schizophyllum commune Fr. (Wessels, 1965). It was therefore of interest to search for the endogenous source of glucose, the main carbohydrate metabolic substrate required for zoosporangial differentiation of the hypogynous Allomyces arbuscula, induced to differentiate in dilute salt solution. MA TERIALS AND METHODS
Strains and cultural conditions Allomyces arbuscula Butl. obtained from the collection of Professor C. Stumm (Haaren, Netherlands) was used throughout this study. Culture maintenance and the production of inoculum have been described earlier (Ojha & Turian, 1981). An inoculum of 104 spores/rnl (final concentration) was used to inoculate 2 I of GCY medium (T urian,
1963) in a 3 I Erlenmeyer flask. Cells were kept in a homogeneous suspension with constant aeration by forced sterile humidified air. The culture was grown at 32°C, filtered through nylon and washed with sterile DS (dilute salt solution, Machlis, 1953). The washed plants thus obtained were suspended either in 1 I sterile DS or GCY medium and incubated at 32° with forced aeration of sterile humidified air. At intervals, samples were taken, filtered, the filtrate saved for the analysis of glucose and the cells washed with cold distilled water and frozen. Determination of intra- and extracellular glucose For the determination of intracellular glucose extracts were prepared as follows : immediately after filtration the cell pellet was suspended in 1 ml 500 mM-TCA and extracted for 60 min at room temperature. After centrifugation the pellet was re-extracted and the two supernatants were pooled. TCA was removed by three extractions with water-saturated ether and the latter removed by bubbling with nitrogen. Culture fluid from the DS medium taken at intervals was either used directly or lyophilized. Glucose was estimated by the glucose-peroxidase method (Bergmeyer & Bernt, 1974)· Determination of trehalose The method is essentially as described by Lillie & Pringle (1980). Trehalose was measured in the cellular extract prepared as described above using anthrone reagent. Our preliminary test did not reveal the presence of other carbohydrates in the extract in appreciable quantities. Glucose was used 18-2
Differentiation in Allomyces arbuscula as standard and the values of trehalose were expressed as glucose equivalents. Extracellular lyophilized medium was used to determine the anthrone-positive material. The amount of trehalose was determined by the difference between total anthrone-positive material and glucose, assayed by the glucose oxidase and peroxidase method.
16
14
.,
12
e;, Determination of glycogen The method followed was essentially as described ~ 10 by Rickenberg et al. (1975) and Nazly, Carter & C Knowles (1980). Cell pads collected at intervals OIl E during growth and differentiation were suspended 8 in 2 ml 30 % KOH; the clumps were broken to "r5 obtain a homogeneous suspension. The suspension :lCJ 6 was digested by heating in a boiling water bath for 00 OIl 30 min. After cooling 0'2 ml8 % (wIv ) Na 2SO. and :t 5"3 ml absolute ethanol were added. The suspension 4 was kept for at least 1 h in the cold, centrifuged at 1700 g for 10 min, the precipitate dissolved in 1 ml H 2 0 , reprecipitated with 2'4 ml absolute eth2 anol and re-washed twice with distilled water and ethanol. The washed pellet thus obtained was dissolved in 1 ml 1 N-H 2SO. and centrifuged, and the supernatant was hydrolysed in a water bath at 2 3 100° for 3 h, neutralized to pH 7'0 with 6 M-KOH Tim e (h) and the glucose liberated was estimated by the glucose-peroxidase method (Bergmeyer & Bernt, Fig. 1. Intra- and extracellular glucose during zoosporangial differentiation. Intracellular glucose in growing 1974)· .~
/
•
•
(. - - . ) and differentiating (e - - e ) plants. Extracellular glucose (0- -0) in the culture filtrate.
Intracellular and extracellular p-glucanase The cells collected at intervals during growth and differentiation were homogenized with acid-washed quartz sand in a chilled mortar and suspended in sorbitol-tris-EDTA buffer (sorbitol o·5 M,tris-H Cl 0 '01 M (pH 7), EDTA 0'001 M). The homogenate was subsequently dialysed overnight in tris-HClEDTA buffer. The dialysed extract was used to measure p-glucanase activity. In certain experiments the cells were homogenized in 0'05 M-Naacetate buffer pH 5'1 and glucanase activity was measured directly. The culture filtrate from the growth and induction medium (DS) was collected at intervals, lyophilized, dialysed against 0'05 M-phosphate buffer (pH 5'5) for 24 h with two changes. p-glucanase activity was measured according to Cortat (1971) using both laminarin (P-l,3-glucan) and pullulan (P-1,6-glucan). The reducing sugar produced was measured with the glucose oxidase-peroxidase method. Protein was determined according to Lowry et al. ( 1951).
RESULTS
Intracellular and extracellular glucose The level of free glucose in the cellular extracts of plants suspended either in dilute salt solution or rich medium was measured at intervals for 3-4 h. The apices of plants suspended in dilute salt solution (DS) differentiated more or less synchronously into zoosporangia whilst those in the rich medium continued to grow. The intracellular glucose in plants suspended either in DS or rich medium remained at a low level; however, glucose accumulated progressively in the filtrate of the differentiating culture (Fig. 1). This accumulation began immediately after suspension of the plants in the DS solution and a concentration of approximately 32 pM was attained by 1'5 h when young zoosporangia appeared. Subsequently, the level declined to 20 pM and remained at this level until the end of differentiation. The intracellular free glucose remained at 2 pM in both growing and differentiating plants.
M. Ojha and G. Turian 80
500
rc
.;:; '5
(0)
. -: -:
400
/
li or,
E
300
60
•
•
50
40 - -• ..---•
\
30 01--_---'_ _--1..._ _- ' - _ - - - - '
o
2
3
•
•
70
•
.c:.. 7 .---. 4
20
Tim e (11)
Fig.
549
Cellular glycogen levels during growth Ce--e) and zoosporangial differentiation C . - - . )·
2.
Accumulation of glycogen and trehalose
The amount of glycogen in the plants placed in OS solution varied little during the course of development. A minor increase was observed during the first 60 min of transfer. The plants placed in the growth medium began to accumulate immediately, consequently the rate and amount of glycogen was higher than those in the OS medium (Fig . 2) . In the absence of degradation of glycogen we looked for the synthesis or loss of another storage carbohydrate, trehalose. Measurements of cellular trehalose showed a considerable loss in differentiating plants ' and accumulation in growing plants (Fig. 3a ). In the absence of any increase in the intracellular glucose during differentiation it was anticipated that the loss of cellular trehalose was due to secretion in the medium and not degradation. The measurements of trehalose in the culture fluid of the differentiating cultures showed that indeed this was the case (Fig. 3b). The loss of trehalose in the plants corresponded to its accumulation in the culture fluid. Activities of trehalase and fl-glucanases in the culture filtrates
Since trehalose was massively secreted in the culture medium during differentiation it might serve as a source of glucose through the action of trehalase. Therefore we measured the activity of this enzyme in the culture filtrate . Although some activity was found it was not sufficient to account for the amount of glucose found in the culture filtrate. The possibility that it carne from the degradation of cell-wall glucan through the action
10
-.
•
.
.~/.-
•
0 0
3
2 T ime ( h)
120
(b)
0
100
/\
o
80
60
40
0
~
0
-..--.--.--.
20
0 0
2
3
Time (h )
c. - -.)
Fig. 3.(a) Intracellular trehalose during growth and differentiation Ce - - e ). (b) Trehalose in the plants (e - - e) and culture filtrate (0- - 0) during differentiation.
of glucanases was analysed by measuring the activity of fl-glucanase during development, and the result is shown in Fig. 4. The evolut ion of glucanase activity corresponded well with the appearance of glucose in the medium.
Differentiation in Allomyces arbuscula
550
Table
Time of observation after transfer
15
I
e 0.
•
OIl
" "
on 0
:: Oil OIl
::t
5
/
lactone (mM) 1
o
0-05
0 -1
+2'5*
mM gluc ose
0 '5
0
0
0
0
0
0
0
0-5
0
0
0
0
0
0
0 0 100 7 0 100 90
0
0
30
6
0 0
70
50
0
1 2
3
•
"'.-.
•
• o
%hyphalapices differentiated withglucano-
(h)
/\
10
c
-;:;
E
1 . Effect of glu cano-lactone on zoosporangial differentiation in Allomyces arbuscula
:2
3
Time (11)
Fig. 4. ,8-glucanase in the culture filtrate of differentiating culture. ,8-1,3-C.--.), /1-1,6-C.--. ) glucanase.
Inhibition of fl-glucanase and differentiation Glucano-lactone, an inhibitor of fl-glucanase (Fleet & Phaff, 1974), inhibited zoosporangial differentiationat low concentrations (Table 1).A concentration of 0 '5 mM completely inhibited the differentiation, apparently without affecting any other morphological feature. At 1 mM glucano-lactone provoked cytolysis besides inhibiting zoosporangial differentiation. Exogenous supplement of glucose (2' 5 mM) even at 1 msr-glucano-lactone overcame the inhibitory effect and zoosporangia developed synchronously. Addition of glucano-lactone (0'5 mM) at any time between 0 and 1 h of transfer to DS solution prevented zoosporangial differentiation (Figs 5-7). Subsequently, addition of the inhibitor had no effect and the plants differentiated normally (Fig. 8). DISCUSSION
In many microbial systems, accumulation of reserve carbohydrates takes place at the onset of nutrient depletion (Nazly et al., 1980; Lillie & Pringle, 1980). Nutrient depletion, particularly of C, Nand/or P, also initiates lifting of catabolite
0 30 100
*Added at the time of transfer.
repressionand inducedreproductive differentiation. However, reproductive differentiation requires energy and substrate for the synthesis of new wall constituents. Therefore, against a background of external depletion, the substrate for new synthesis must come from the breakdown of internal constituents. As a matter of fact, the degradation of reserve polymers is often associated with differentiation (Fonzi, Shanley & Opheim, 1979; Zonneveld, 1972; Wessels, 1965; Wolf, 1980). In Saccharomyces cereoisiae Hansen glycogen hydrolysis and accumulation of intracellular glucose is specific to sporulation-committed cells (Fonzi et al., 1979). Likewise, glycogen degradation, accumulation and degradation oftrehalose and increase in intracellular glucose are stage-specific in Dictyostelium development (Rosness & Wright, 1974; Wilson & Rutherford, 1978). By contrast there are examples in other eukaryotic microbes where glucose does not come from the degradation of these intracellular carbohydrate reserves, instead it comes from the degradation of wall polymers , In Aspergillus nidulans (Eidam) Winter, degradation of a-1,3-glucan is associated with conidiation and development of cleistothecia (Zonneveld, 1972). Mutants lacking a-1,3-glucan and melanin are also defective in cleistothecial development (Polachek & Rosenberger, 1977; Zonneveld, 1972). Our results with A. arbuscula show that carbohydrate reserves like glycogen and trehalose are synthesized in the growth medium. Whilst glycogen continues to be synthesized in the induction medium, trehalose synthesis slows down and the cellular trehalose is secreted in the medium. The intracellular trehalase activity during differentiation was found to be very low. This and the absence ofincrease in intracellular glucose indicated that decrease in cellular trehalose was due to secretion in the medium. Indeed, the measurement of trehalose in the culture filtrate showed a progressive accumulation in the early period of differentiation. However, this does not seem to
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Figs S-8. Effect of glucano-lactone on zoosporangial differentiation. The plants suspended in DS solution were supplemented with glucano-lactone (o'S mM) at specified intervals and the culture incubated at 32°C on a reciprocal shaker. The zoosporangial differentiation was checked after 3 h of incubation. The control culture was without glucano-lactone (Fig. S); glucano-lactone was added at 0 h (Fig. 6),30 min (Fig. 7), 1'S h (Fig. 8).
provide all the glucose necessary for differentiation, since in the presence of glucano-lactone, a specific inhibitor of {J-1, 3- and 1,6-glucanases, the plants suspended in DS medium do not differentiate. Youatt (1980) reported that apparently trehalose is not metabolized by growing cultures of A.
macrogynus (Emerson) Emerson & Wilson. Therefore, the role of trehalose synthesis and excretion during differentiation remains obscure. The main internal provider of glucose for zoosporangial differentiation in Allomyces appears therefore to be {J-glucan as shown by (a) the
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stimulation of fJ-glucanase during the initiation phase of differentiation; (b) the negative consequence on differentiation of the inhibition of fJ-glucanase by the competitor of glucose , glucano-Iactone; (c) the simultaneous overcoming of both these inhibitions by the provision of external glucose. The requirement of glucose for zoosporangial differentiation is limited to the early period of induction (1'5 h after transfer) and coincides with the peak of glucose accumulation and fJ-1,3glucanase activity. This period is also sensitive to glucano-lactone inhibition of zoosporangial differentiation. These results are in agreement with the conclusion of Youatt (1980), who found that active metabolism of glucose during zoosporangial differentiation takes place before septation occurs to delimit the zoosporangia and that glucose is not metabolized actively by differentiated cultures. a-1,3-g1ucans are the usual storage compounds as recently shown at wall changes during microcycle conidiation of A. niger Van Tieghem (Smith et al., 1980). It is therefore interesting to see that fJglucans, as first shown by Wessels (1965), can also be deployed in the Allomyces as carbon source if necessary, even though their main function remains more of a structural nature. We are grateful to Mrs Arlette Cattaneo for her technical help and to Mrs Barbara Duchoud for manuscript preparation and typing. REFERENCES
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(R eceived for publication
20
January 1983)