DEVELOPMENTAL
BIOLOGY
The Development
72, 175-181
(1979)
of Sporulation ADAM
Competence
in Physarum polycephalum
S. WILKINS AND GAIL REYNOLDS
Department of Microbiology and Genetics, Massey University, Palmerston North, New Zealand Received October 23, 1978; accepted in revised form February 27, 1979 Plasmodial cells of the slime mold Physarum polycephalum become “competent” for sporulation following a prolonged period of dark starvation in the presence of nicotinamide. Sporulation can then be induced by illumination. Plasmodia are found to release into the medium during starvation one or more cellular products that promote sporulation. These products exert their effect specifically during the dark starvation period, rather than during the final phase of fruiting body construction. The sporulation control factor(s) (SCF) is nondialyzable and can stimulate the development of sporulation competence in the absence of nicotinamide. INTRODUCTION
The potential of slime molds as model systems for the study of mechanisms of eucaryotic differentiation has long been recognized. Investigation of developmental controls in these organisms has, however, been primarily concentrated on one group, the Acrasiales, where sporulation involves the transformation of multicellular amoebal aggregates into fruiting bodies (Garrod and Ashworth, 1973). Relatively less is known about sporulation in the other class of slime molds, the Myxomycetes. In these organisms, fruiting body formation takes place in single macroscopic, syncytial cells called plasmodia (Guttes et al., 1961). In the best-known member of this group, Physarum polycephalum, sporulation takes place following a prolonged starvation period in the dark and subsequent illumination with visible light; in these circumstances, a typical cell yields dozens of fruiting bodies. During the sustained dark starvation phase, each plasmodial cell becomes a highly branched network of connected strands. Following illumination, the intracellular material accumulates in nodules, which first elongate into small pillars and then assume the lobular shape of mature sporangia. The final stages of development include melanization
and the cleavage of the cytoplasm to monoand oligonucleate spores. Meiosis takes place during the last stages of sporulation (Laane and Haugli, 1976). Starvation and illumination trigger necessary but presumably different biochemical events in myxomycete sporulation. While the second phase of the developmental program, the induction of sporangium formation by light, has been extensively investigated (Daniel, 1966; Wormington and Weaver, 1976), comparatively little is known about the role of starvation in potentiating the cells for sporulation, a state termed one of “competence” (Daniel and Rusch, 1962a). Because the photoreception system, including the pigment active in induction, appears much the same in both starved and growing cells (Wormington and Weaver, 1976; Daniel and Jarlfors, 1972), the development of competence probably does not involve a change in this system but rather a novel priming of the cell to initiate sporulation in response to light-generated signals. In plasmodia starved at 21.5”C, competence development is reported to require a minimum 96-hr starvation period and the presence of exogenous nicotinamide, or its precursors (Daniel and Rusch, 1962a,b). The initial effect of nicotinamide addition
175 eon-1606/79/090175-07$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
DEVELOPMENTAL BIOLOGY VOLUME72,1979
during starvation is probably an expansion of the NAD+ or NADP+ pool (see review by Gorman and Wilkins (1979)), but the precise function of this pool expansion in sporulation is uncertain. Evidence is presented here that the development of competence involves an interaction between the cells and one or more substances that they release into the medium. The factor or factors are nondialyzable and produce their effect specifically during the period of dark starvation. The respective roles of nicotinamide metabolism and these cellular products in establishing competence are discussed. MATERIALS
AND METHODS
Organism and culture media. Physarum polycephalum strain CL-2, a derivative of the haploid apogamic strain CL, was donated by Dr. Tom Laffler. The particular advantages of CL for the analysis of cellular phenomena in Physarum have been dis. cussed elsewhere (Dee, 1975). A diploid CL was used in this work to facilitate comparison with earlier work on the heterothallic strain M~c (Daniel and Rusch, 1962a,b; Sauer et al., 1969), and was constructed by heat shock of CL during plasmodial mitosis (Brewer and Rusch, 1968). Although meiosis in CL displays aberrant features (Laane et al., 1976), diploidization of this strain increases the number of viable spores (Laffler and Dove, 1977). Liquid shake cultures of microplasmodia are grown in the standard semidefined medium (Daniel and Baldwin, 1964). Sporulation medium (SM) is a simple salt medium, adapted from Daniel and Rusch (1962a), and consists of the following (milligrams/ liter): citric acid. H20, 489; FeC12.4Hz0,60; HCl, 40; MnCb . 4H20, 84; ZnSOl - 7Hz0, 33.6; CaC12- 2Hz0,600; MgS04.7H20,600; CuClz . 2H20, 23; KH~POI, 0.4; CaC03, 1; nicotinamide, 0.1. Where noted, SM lacking nicotinamide @M-N) was employed. Sporulation protocol. The method is based on that of Sauer et al. (1969). Fifty-
milliliter cultures of microplasmodia are grown to late log phase, from 2.5-5.0 ml inocula, in l-liter flasks with rotary shaking at 22°C. Microplasmodia are harvested by centrifuging at 500g for 1 min, and are resuspended in an equal volume of SM. Aliquots of the suspension (containing typically 7.5 mg/ml protein) are placed on individual Whatman No. 50 filter papers, each supported by a stainless-steel mesh, inside a 50-mm-diameter petri plate. The microplasmodia then undergo coalescence to form surface plasmodia. Platings of a given volume of suspension produce uniform-size plasmodia, and, in the experiments described here, plasmodial sizes are given in terms of the volume of microplasmodial suspension plated. After 1 to 3 hr to permit coalescence, 3.3 ml SM is introduced beneath each filter (0 hr of starvation). Plates are then wrapped in aluminum foil and incubated at 22°C. Following starvation, plasmodia are illuminated at 22°C by placing them 30 cm below 40-W cool white fluorescent lights for 4 hr. Plasmodia are then returned to the dark for further incubation and subsequent scoring. Where noted a further period of continuous illumination was given to facilitate scoring. (Reconstruction experiments show little effect of secondary illumination on final sporulation frequencies). In experiments involving transfers during starvation, filters were aseptically transferred under conditions of reduced (noninducing) illumination, first to a wash plate with SM and briefly swirled, and then to fresh test medium. Preparation of preconditioned medium (PCM). Four mililiter aliquots of microplasmodial suspension are placed on filters in standard @O-mm-diameter) petri plates. After 2 to 4 hr drying to produce coalescence, 12 ml SM is added, and the plates incubated in the dark for 60 to 70 hr. The preconditioned medium (PCM) is then collected and stored at 4°C. Dialysis of PCM is carried out against three changes of SM
177
BRIEF NOTES
(or SM-N, as noted) at 4°C for approximately 70 hr. Dialyzed PCM (D-PCM) is stored at 4’C without further treatment.
TABLE
RESULTS
In strain Cl-2, competence for sporulation can develop well before 96 hr of starvation at 22°C. We have found that the development of sporulation capacity in this strain is a function of both the duration of starvation and of the ratio of plasmodial mass to the volume of medium. In general, larger plasmodia develop sporulation capability sooner and with higher frequency than smaller cells, for a given medium volume, while an increase in the volume of medium decreases the frequency of sporulation. In Table lA, the effects of cell size and the length of the starvation interval, for a constant medium volume, are illustrated. The advantage of large cells relative to small cells is most pronounced when illimination is given early in starvation (2630 hr). (The delay in morphogenesis seen under these induction conditions is discussed below.) For later times of illumination (51-55 hr), the effect of larger cell mass is still observed, but it is less marked and exceptions are sometimes seen (e.g., the O.lml cells in this experiment). The opposite effect of increasing the medium volume, while maintaining fixed cell sizes, is shown in Table 1B. A fourfold increase in the volume of the medium dramatically reduces the sporulation frequency. Similar relationships between cell size and starvation intervals and the sporulation frequency are seen when starvation is imposed by exhaustion of a limiting amount of nutrient on solid medium (data not shown). The effect of cell size is thus not limited to one particular starvation regimen. The importance of the plasmodial size/ medium volume ratio in governing the development of sporulation capacity is indicative of some interaction between the cells and the medium. One hypothesis to explain
1
EFFECT OF MASS, STARVATION INTERVAL, AND MEDIUM VOLUME ON SPORULATION FREQUENCY”
A.
Time of illumination during starvation (hr)
Sample volume6 (d)
26-30
0.60 0.40 0.20 0.15 0.10
51-55
0.60 0.40 0.20 0.15 0.10
Time of illumination during starvation (hr) B.
54-58
%z: (ml) 0.25 0.25 0.15 0.15
Number sporulated 25 hr o/15 o/15 o/15 o/15 o/15
39 hr 7/15 (3) l/15 l/15 o/15 o/15
12/15 (10) 11/15 (9)
WI5 (6) 2/15 (2) lo/15 (61 Medium volume (ml) 3.3 14.0 3.3 14.0
Number sporulated 24 hr 13/15 (13) o/15 15/15(15) l/15
“A. Two sets of plasmodia were prepared as described under Materials and Methods, over a fixed medium volume (3.3 ml), and illuminated at the indicated times. The time of scoring for each set is given from the end of the illumination period, and plasmodia were counted as having sporulated if manifesting any stage of sporulation. The numbers in parentheses give the number that had completed fruiting body development. B. Procedure as in A, except that the medium volume was varied as shown, and standard petri plates were used for the larger medium volumes. *Volume of microplasmodial suspension used to form test cells.
the results is that plasmodial cells release a substance essential for sporulation into the medium; larger cells might release more of the substance(s) and thus develop sporulation capability sooner while smaller cells would make less and develop this capacity later or with reduced frequency. If this hypothesis is correct, then daily removal of medium during starvation should inhibit sporulation, while repeated transfer to preconditioned medium (PCM) should promote sporulation. These predictions have been confirmed, as shown in Table 2 for one experiment.
178
DEVELOPMENTAL BIOLOGY TABLE
2
EFFECT OF PCM ON SPORULATION CAPACITY” Treatment
during starvation -
Number sporulated 19.5 hr
1. Not transferred 2. Transferred to fresh SM at ‘72 hr 3. Transferred sequentially to fresh SM at 22, 45.5, and 72 hr 4. Transferred sequentially to PCM-medium at 22 and 45.5 hr, and to SM at 72 hr 5. Transferred sequentially to D-PCM-medium at 22 and 45.5 hr, and to SM at 72 hr
30.5 hr
4/6 (3) 5/6 (4) o/10
l/IO
l/10 (1)
8/10 (1)
4/10 (4)
7/10 (4)
n Procedures as described under Materials and Methods, except that PCM was collected at 88 hr of starvation. Cells were prepared from 0.4-m] aliquots of microplasmodial suspension. PCM-medium consists of 1 part PCM:2 parts SM. D-PCM-medium consists of 1 part dialyzed PCM:2 parts SM. All plates illuminated from 72 to 76 hr, then returned to dark incubation for 19.5 hr; further incubation was under illumination. Scoring procedure as described for Table 1, from end of initial illumination period.
Successive transfers to fresh medium during the course of starvation significantly reduces sporulation (line 3) while transfer to medium consisting of 0.33 vol PCM permits retention of sporulation capacity (line 4). To determine whether this effect of PCM is due to a small molecule, PCM that had been extensively dialyzed was also tested. Dialyzed PCM retains high activity (line 5), although dialysis efficiently removes small molecular weight material (a seven- to ninefold reduction in uv-absorbing material in this experiment). Since the biological activity is expressed against a background of fresh medium, we conclude that: (1) fresh medium is not in itself inhibitory to sporulation, and, (2) there are one or more factors, of MW approximately 10,000 or greater, in conditioned medium that actively promote sporulation. Significantly, this positive influence of conditioned medium on sporulation takes
VOLUME 72,1979
place primarily during the dark starvation period, the time of competence development. In the transfer experiments, all plasmodia exposed to PCM during dark starvation are transferred to fresh medium prior to illumination, with a wash step included to reduce carry-over of medium. Likewise, transfer of control competent cells to fresh medium prior to illumination does not inhibit sporulation (line 2). Thus, conditioned medium does not promote sporulation simply through provision of accumulated metabolites during fruiting body construction, but acts during the preillumination dark starvation period to prime the cells for sporulation. Furthermore, the response to PCM is not uniformly distributed throughout the dark starvation period but is restricted to later segments of this interval. If plasmodia are placed over PCM during specific 24-hr periods within a 72-hr starvation span, the stimulatory effect is not produced by exposure during the first 24 hr, but only by treatment during the second and third days (Table 3). In this experiment, a few plasmodia treated with PCM during the final TABLE
3
TIME OF ACTION OF PCM FACTORY Treatment during starvation
SM on Days 1, 2, and 3 PCM on Day 1; SM on Days 2 and 3 PCM on Day 2; SM on Days 1 and 3 PCM on Day 3; SM on Days 1 and 2 PCM on Days 2 and 3; SM on Day 1 PCM on Days 1,2, and 3
Number sporulated (total) 23.5 hr
39.5 hr
Number sporulated in dark
l/14
l/14
-
l/14
l/14
-
7/15
7/15
-
3/13
4/13
2/13
9/15
11/15
4/15
6/15
8/15
2/15
’ Cells prepared from 0.15~ml aliquots. dium consists of 1 part PCM:P parts SM. transferred as indicated, and ilhrminated hr; then incubated in the dark, and scored 39.5 hr from end of illumination period.
PCM-meCells were from 72-76 at 23.5 and
179
BRIEF NOTES
24 hr were found to have sporulated in the dark (last column). While the occurrence of preillumination sporulations is a variable feature in these experiments (as is the relative extents of priming by PCM on the final 2 days of starvation), it is correlated with exposure to PCM late in starvation. To date, two requirements for competence development have been described: the sporulation control factor(s) in conditioned medium, discussed above and designated here as SCF, and the previously reported requirement for nicotinamide (Daniel, 196213). In strain CL-2, however, the nicotinamide-dependent pathway is dispensable. In this strain, nicotinamide is stimulatory for sporulation, rather than strictly essential (Table 4): in the absence of nicotinamide, sporulation frequencies are TABLE
4
EFFECT OF NICOTINAMIDE ON SPORULATION FREQUENCY= Time of illuminFr r
Volume
Nicotinamide
(ml)
50-54
0.60 0.60 0.20 0.20 0.10 0.10
+ + + -
Number sporulated 15.5 hr
23 hr
39 hr
9/10 O/IO 3/10 O/IO o/10 o/10
9/10 (9) l/10 4/10 (3) o/10 o/10 o/10
7/IO (5) 6/10 (5) 3/10 o/10 l/10
” Plasmodia were harvested, then resuspended in SM-N, recentrifuged, and suspended again in SM-N. Other procedures were as described under Materials and Methods, and the scoring procedure was as in Table 1.
reduced and morphogenesis is delayed. These quantitative effects are similar to those produced, in the presence of nicotinamide, by employing relatively small cells or short starvation periods (see Table 1). In addition, cells can develop competence efficiently in the complete absence of nicotinamide, simply by exposure to SCF. As shown in Table 5, treatment with dialyzed PCM, in the absence of nicotinamide, can yield sporulation frequencies greater than those produced by incubation with nicotinamide. Evidently, interaction with SCF can stimulate competence development independently of exogenous nicotinamide. DISCUSSION
During a period of dark starvation, plasmodial cells of Physarum polycephatum strain CL-2 release into the medium and then interact with one or more factors (SCF) that actively promote the development of sporulation competence. At least part of the activity is in molecules 3 10,000 MW. Experiments to further characterize the factor(s) are in progress. The development of competence appears to involve at least two events during the period of dark starvation: the production of SCF by the cells and the development of responsiveness to the factor(s) during the later stages of starvation. The initial incapacity of starving plasmodia to respond to SCF presumably reflects either a failure of uptake or the absence of some cellular component required for SCF action. The initial
TABLE
5
SUBSTITUTION OF D-PCM Starvation
FOR NICOTINAMIDE” Number sporulated
medium
Expt No. 2
Expt No. 1
1. SM 2. SM-N 3. D-PCM-medium
24.5 hr
44.5 hr
24.5 hr
37.5 hr
1/12 (1)
m2 (2) I/3 (1) 5/I2 (4)
l/20 (1) O/6
‘J/20 (1)
o/13 o/12
3/17 (3)
O/16 10/17 (3)
n The cells were prepared as described for Table 3, and starved over the indicated media. PCM was dialyzed against SM-N. D-PCM-medium consists of 1 part D-PCM:2 parts SM-N. All plasmodia were starved for a period of 48 hr, then transferred to SM-N, and illuminated. Plates were subsequently incubated for 24.5 hr in the dark, scored, and placed under continuous illumination until the second scoring.
180
DEVELOPMENTALBIOLOGY
refractory period in starvation probably explains the observation (Sauer, 1973; Wilkins, unpublished experiments) that placing previously unstarved plasmodia over PCM does not make the cells immediately inducible for sporulation. The nature of the relationship between nicotinamide metabolism and the production or action of SCF remains to be fully determined. However, the observation that SCF can efficiently prime cells for sporulation in the absence of nicotinamide is compatible with either of two hypotheses of competence development. The first explanation is that different and independent pathways for developing competence may exist. In this case, supplying either nicotinamide or SCF to plasmodia undergoing starvation would serve to develop competence, but by different respective routes. The alternative possibility is that the function of nicotinamide is to boost SCF production by the cells. Under this hypothesis, experimental addition of SCF to the medium eliminates the nicotinamide requirement by furnishing the end product. The quantitative effects on sporulation produced by nicotinamide deprivation (Table 4) are quite consistent with this second hypothesis. In the course of these experiments on competence development, several observations pertinent to the induction process itself were made. The specific starvation conditions (cell size, duration of starvation, presence or absence of nicotinamide) determine not only the extent of competence development in a set of cells (as inferred from the final sporulation frequency) but also influence the timing of the onset of morphogenesis. When large cells are starved for only a short interval, sporulation ensues after illumination, but exhibits a characteristic delay (Table 1A). For longer starvation periods, larger cells are frequently observed to complete morphogenesis sooner than smaller ones. (One example of this can be seen in Table 4.) If one
VOLUME 72, 1979
assumes that SCF accumulation is proportional to both the length of starvation and to cell mass, delayed morphogenesis is correlated with lower SCF concentrations at the time of illumination, and conversely, rapid morphogenesis with higher SCF concentrations. (Interestingly, one major consequence of nicotinamide deprivation during starvation is a delay in morphogenesis.) On the other hand, exposure to exogenously added SCF during prolonged dark incubation can result in some frequency of induction without illumination (Table 3). Taken together, these findings are suggestive of a possible quantitative relationship between SCF concentration and the rate or likelihood of the induction reaction. This relationship and its implications for the induction process will be discussed more fully elsewhere (Wilkins and Reynolds, manuscript in preparation). We thank Drs. D. F. Bacon and E. Terzaghi for criticisms of the manuscript. REFERENCES BREWER, H. D., and RUSCH, H. P. (1968). Effect of elevated temperature shocks in mitosis and on the initiation of DNA replication in Physarum polycephalum. Exp. Cell. Res. 49, 79-86.
DANIEL, J. W. (1966). Light-induced synchronous sporulation of a myxomycete. In “Cell Synchrony” (I. Cameron and G. M. Padilla, eds.), pp. 117-152. Academic Press, New York. DANIEL, J., and BALDWIN, H. (1964). Methods of culture for plasmodial myxomycetes. In “Methods in Cell Physiology” (D. M. Prescott., ed.), Vol. 1, pp. 9-41. Academic Press, New York. DANIEL, J. W., and JARLFORS,U. (1972). Light-induced changes in the ultrastructure of a plasmodial myxomycete. Tissue Cell 4,405-426. DANIEL, J. W., and RUSCH,H. P. (1962a). Method for inducing sporulation of pure culture of the myxomycete Physarum polycephalum. J. Bacterial. 83, 234-240.
DANIEL, J. W., and RUSCH, H. P. (1962b). Niacin requirement for sporulation in Physarumpolycephalum. J. Bacterial. 83, 1244-1250. DEE, J. (1975). Slime molds in biological research. Sci. Progr.
(Oxford)
62, 523-542.
GARROD,D., and ASHWORTH, J. M. (1973). Development of the cellular slime mould Dictyostelium discoideum. In “Microbial Differentiation” (J. M. Ash-
BRIEF NOTES
worth and J. E. Smith, eds.), pp. 407-435. Cambridge Univ. Press, Cambridge. GORMAN, J., and WILKINS, A. S. (1979) Developmental phases in the life cycle of Physarum and related myxomycetes. In “Growth and Differentiation in Physarum polycephalum” (W. F. Dove and H. P. Rusch, eds.), in press. GUITES, E., GUTTES, S., and RUSCH, H. P. (1961). Morphological observations on growth and differentiation of Physarum polycephalum growth in pure culture. Develop. Biol. 3.588-614. LAANE, M. M., and HAUGLI, F. B. (1976). Nuclear behaviour during meiosis in the myxomycete Physarum polycephalum. Norw. J. Bot. 23, 7-21. LAANE, M. M., HAUGLI, F. B., and MELLUM, T. R. (1976). Nuclear behaviour during sporulation and
181
germination in the Colonia strain of Physarumpolycephalum. Norw. J. Bot. 23, 177-189. LAFFLER, T., and DOVE, W. F. (1977). The viability of Physarum polycephalum spores and ploidy of plasmodial nuclei. J. Bacterial. 131.473-476. SAUER, H. W. (1973). Differentiation in Physarum polycephalum. In “Microbial Differentiation” (J. M. Ashworth and J. E. Smith, eds.), pp. 375-405. Cambridge Univ. Press, Cambridge. SAUER, H. W., BABCOCK,K. L., and RUSCH, H. P. (1969). Sporulation in Physarum polycephalum. Exp. Cell. Res. 57, 319-327. WORMINGTON,W. M., and WEAVER, R. F. (1976). Photoreceptor pigment that induces differentiation in the slime mold Physarum polycephalum. Proc. Nut. Acad. Sci. USA 73,3896-3899.
BIOLOGY
The Development
72, 175-181
(1979)
of Sporulation ADAM
Competence
in Physarum polycephalum
S. WILKINS AND GAIL REYNOLDS
Department of Microbiology and Genetics, Massey University, Palmerston North, New Zealand Received October 23, 1978; accepted in revised form February 27, 1979 Plasmodial cells of the slime mold Physarum polycephalum become “competent” for sporulation following a prolonged period of dark starvation in the presence of nicotinamide. Sporulation can then be induced by illumination. Plasmodia are found to release into the medium during starvation one or more cellular products that promote sporulation. These products exert their effect specifically during the dark starvation period, rather than during the final phase of fruiting body construction. The sporulation control factor(s) (SCF) is nondialyzable and can stimulate the development of sporulation competence in the absence of nicotinamide. INTRODUCTION
The potential of slime molds as model systems for the study of mechanisms of eucaryotic differentiation has long been recognized. Investigation of developmental controls in these organisms has, however, been primarily concentrated on one group, the Acrasiales, where sporulation involves the transformation of multicellular amoebal aggregates into fruiting bodies (Garrod and Ashworth, 1973). Relatively less is known about sporulation in the other class of slime molds, the Myxomycetes. In these organisms, fruiting body formation takes place in single macroscopic, syncytial cells called plasmodia (Guttes et al., 1961). In the best-known member of this group, Physarum polycephalum, sporulation takes place following a prolonged starvation period in the dark and subsequent illumination with visible light; in these circumstances, a typical cell yields dozens of fruiting bodies. During the sustained dark starvation phase, each plasmodial cell becomes a highly branched network of connected strands. Following illumination, the intracellular material accumulates in nodules, which first elongate into small pillars and then assume the lobular shape of mature sporangia. The final stages of development include melanization
and the cleavage of the cytoplasm to monoand oligonucleate spores. Meiosis takes place during the last stages of sporulation (Laane and Haugli, 1976). Starvation and illumination trigger necessary but presumably different biochemical events in myxomycete sporulation. While the second phase of the developmental program, the induction of sporangium formation by light, has been extensively investigated (Daniel, 1966; Wormington and Weaver, 1976), comparatively little is known about the role of starvation in potentiating the cells for sporulation, a state termed one of “competence” (Daniel and Rusch, 1962a). Because the photoreception system, including the pigment active in induction, appears much the same in both starved and growing cells (Wormington and Weaver, 1976; Daniel and Jarlfors, 1972), the development of competence probably does not involve a change in this system but rather a novel priming of the cell to initiate sporulation in response to light-generated signals. In plasmodia starved at 21.5”C, competence development is reported to require a minimum 96-hr starvation period and the presence of exogenous nicotinamide, or its precursors (Daniel and Rusch, 1962a,b). The initial effect of nicotinamide addition
175 eon-1606/79/090175-07$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
DEVELOPMENTAL BIOLOGY VOLUME72,1979
during starvation is probably an expansion of the NAD+ or NADP+ pool (see review by Gorman and Wilkins (1979)), but the precise function of this pool expansion in sporulation is uncertain. Evidence is presented here that the development of competence involves an interaction between the cells and one or more substances that they release into the medium. The factor or factors are nondialyzable and produce their effect specifically during the period of dark starvation. The respective roles of nicotinamide metabolism and these cellular products in establishing competence are discussed. MATERIALS
AND METHODS
Organism and culture media. Physarum polycephalum strain CL-2, a derivative of the haploid apogamic strain CL, was donated by Dr. Tom Laffler. The particular advantages of CL for the analysis of cellular phenomena in Physarum have been dis. cussed elsewhere (Dee, 1975). A diploid CL was used in this work to facilitate comparison with earlier work on the heterothallic strain M~c (Daniel and Rusch, 1962a,b; Sauer et al., 1969), and was constructed by heat shock of CL during plasmodial mitosis (Brewer and Rusch, 1968). Although meiosis in CL displays aberrant features (Laane et al., 1976), diploidization of this strain increases the number of viable spores (Laffler and Dove, 1977). Liquid shake cultures of microplasmodia are grown in the standard semidefined medium (Daniel and Baldwin, 1964). Sporulation medium (SM) is a simple salt medium, adapted from Daniel and Rusch (1962a), and consists of the following (milligrams/ liter): citric acid. H20, 489; FeC12.4Hz0,60; HCl, 40; MnCb . 4H20, 84; ZnSOl - 7Hz0, 33.6; CaC12- 2Hz0,600; MgS04.7H20,600; CuClz . 2H20, 23; KH~POI, 0.4; CaC03, 1; nicotinamide, 0.1. Where noted, SM lacking nicotinamide @M-N) was employed. Sporulation protocol. The method is based on that of Sauer et al. (1969). Fifty-
milliliter cultures of microplasmodia are grown to late log phase, from 2.5-5.0 ml inocula, in l-liter flasks with rotary shaking at 22°C. Microplasmodia are harvested by centrifuging at 500g for 1 min, and are resuspended in an equal volume of SM. Aliquots of the suspension (containing typically 7.5 mg/ml protein) are placed on individual Whatman No. 50 filter papers, each supported by a stainless-steel mesh, inside a 50-mm-diameter petri plate. The microplasmodia then undergo coalescence to form surface plasmodia. Platings of a given volume of suspension produce uniform-size plasmodia, and, in the experiments described here, plasmodial sizes are given in terms of the volume of microplasmodial suspension plated. After 1 to 3 hr to permit coalescence, 3.3 ml SM is introduced beneath each filter (0 hr of starvation). Plates are then wrapped in aluminum foil and incubated at 22°C. Following starvation, plasmodia are illuminated at 22°C by placing them 30 cm below 40-W cool white fluorescent lights for 4 hr. Plasmodia are then returned to the dark for further incubation and subsequent scoring. Where noted a further period of continuous illumination was given to facilitate scoring. (Reconstruction experiments show little effect of secondary illumination on final sporulation frequencies). In experiments involving transfers during starvation, filters were aseptically transferred under conditions of reduced (noninducing) illumination, first to a wash plate with SM and briefly swirled, and then to fresh test medium. Preparation of preconditioned medium (PCM). Four mililiter aliquots of microplasmodial suspension are placed on filters in standard @O-mm-diameter) petri plates. After 2 to 4 hr drying to produce coalescence, 12 ml SM is added, and the plates incubated in the dark for 60 to 70 hr. The preconditioned medium (PCM) is then collected and stored at 4°C. Dialysis of PCM is carried out against three changes of SM
177
BRIEF NOTES
(or SM-N, as noted) at 4°C for approximately 70 hr. Dialyzed PCM (D-PCM) is stored at 4’C without further treatment.
TABLE
RESULTS
In strain Cl-2, competence for sporulation can develop well before 96 hr of starvation at 22°C. We have found that the development of sporulation capacity in this strain is a function of both the duration of starvation and of the ratio of plasmodial mass to the volume of medium. In general, larger plasmodia develop sporulation capability sooner and with higher frequency than smaller cells, for a given medium volume, while an increase in the volume of medium decreases the frequency of sporulation. In Table lA, the effects of cell size and the length of the starvation interval, for a constant medium volume, are illustrated. The advantage of large cells relative to small cells is most pronounced when illimination is given early in starvation (2630 hr). (The delay in morphogenesis seen under these induction conditions is discussed below.) For later times of illumination (51-55 hr), the effect of larger cell mass is still observed, but it is less marked and exceptions are sometimes seen (e.g., the O.lml cells in this experiment). The opposite effect of increasing the medium volume, while maintaining fixed cell sizes, is shown in Table 1B. A fourfold increase in the volume of the medium dramatically reduces the sporulation frequency. Similar relationships between cell size and starvation intervals and the sporulation frequency are seen when starvation is imposed by exhaustion of a limiting amount of nutrient on solid medium (data not shown). The effect of cell size is thus not limited to one particular starvation regimen. The importance of the plasmodial size/ medium volume ratio in governing the development of sporulation capacity is indicative of some interaction between the cells and the medium. One hypothesis to explain
1
EFFECT OF MASS, STARVATION INTERVAL, AND MEDIUM VOLUME ON SPORULATION FREQUENCY”
A.
Time of illumination during starvation (hr)
Sample volume6 (d)
26-30
0.60 0.40 0.20 0.15 0.10
51-55
0.60 0.40 0.20 0.15 0.10
Time of illumination during starvation (hr) B.
54-58
%z: (ml) 0.25 0.25 0.15 0.15
Number sporulated 25 hr o/15 o/15 o/15 o/15 o/15
39 hr 7/15 (3) l/15 l/15 o/15 o/15
12/15 (10) 11/15 (9)
WI5 (6) 2/15 (2) lo/15 (61 Medium volume (ml) 3.3 14.0 3.3 14.0
Number sporulated 24 hr 13/15 (13) o/15 15/15(15) l/15
“A. Two sets of plasmodia were prepared as described under Materials and Methods, over a fixed medium volume (3.3 ml), and illuminated at the indicated times. The time of scoring for each set is given from the end of the illumination period, and plasmodia were counted as having sporulated if manifesting any stage of sporulation. The numbers in parentheses give the number that had completed fruiting body development. B. Procedure as in A, except that the medium volume was varied as shown, and standard petri plates were used for the larger medium volumes. *Volume of microplasmodial suspension used to form test cells.
the results is that plasmodial cells release a substance essential for sporulation into the medium; larger cells might release more of the substance(s) and thus develop sporulation capability sooner while smaller cells would make less and develop this capacity later or with reduced frequency. If this hypothesis is correct, then daily removal of medium during starvation should inhibit sporulation, while repeated transfer to preconditioned medium (PCM) should promote sporulation. These predictions have been confirmed, as shown in Table 2 for one experiment.
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DEVELOPMENTAL BIOLOGY TABLE
2
EFFECT OF PCM ON SPORULATION CAPACITY” Treatment
during starvation -
Number sporulated 19.5 hr
1. Not transferred 2. Transferred to fresh SM at ‘72 hr 3. Transferred sequentially to fresh SM at 22, 45.5, and 72 hr 4. Transferred sequentially to PCM-medium at 22 and 45.5 hr, and to SM at 72 hr 5. Transferred sequentially to D-PCM-medium at 22 and 45.5 hr, and to SM at 72 hr
30.5 hr
4/6 (3) 5/6 (4) o/10
l/IO
l/10 (1)
8/10 (1)
4/10 (4)
7/10 (4)
n Procedures as described under Materials and Methods, except that PCM was collected at 88 hr of starvation. Cells were prepared from 0.4-m] aliquots of microplasmodial suspension. PCM-medium consists of 1 part PCM:2 parts SM. D-PCM-medium consists of 1 part dialyzed PCM:2 parts SM. All plates illuminated from 72 to 76 hr, then returned to dark incubation for 19.5 hr; further incubation was under illumination. Scoring procedure as described for Table 1, from end of initial illumination period.
Successive transfers to fresh medium during the course of starvation significantly reduces sporulation (line 3) while transfer to medium consisting of 0.33 vol PCM permits retention of sporulation capacity (line 4). To determine whether this effect of PCM is due to a small molecule, PCM that had been extensively dialyzed was also tested. Dialyzed PCM retains high activity (line 5), although dialysis efficiently removes small molecular weight material (a seven- to ninefold reduction in uv-absorbing material in this experiment). Since the biological activity is expressed against a background of fresh medium, we conclude that: (1) fresh medium is not in itself inhibitory to sporulation, and, (2) there are one or more factors, of MW approximately 10,000 or greater, in conditioned medium that actively promote sporulation. Significantly, this positive influence of conditioned medium on sporulation takes
VOLUME 72,1979
place primarily during the dark starvation period, the time of competence development. In the transfer experiments, all plasmodia exposed to PCM during dark starvation are transferred to fresh medium prior to illumination, with a wash step included to reduce carry-over of medium. Likewise, transfer of control competent cells to fresh medium prior to illumination does not inhibit sporulation (line 2). Thus, conditioned medium does not promote sporulation simply through provision of accumulated metabolites during fruiting body construction, but acts during the preillumination dark starvation period to prime the cells for sporulation. Furthermore, the response to PCM is not uniformly distributed throughout the dark starvation period but is restricted to later segments of this interval. If plasmodia are placed over PCM during specific 24-hr periods within a 72-hr starvation span, the stimulatory effect is not produced by exposure during the first 24 hr, but only by treatment during the second and third days (Table 3). In this experiment, a few plasmodia treated with PCM during the final TABLE
3
TIME OF ACTION OF PCM FACTORY Treatment during starvation
SM on Days 1, 2, and 3 PCM on Day 1; SM on Days 2 and 3 PCM on Day 2; SM on Days 1 and 3 PCM on Day 3; SM on Days 1 and 2 PCM on Days 2 and 3; SM on Day 1 PCM on Days 1,2, and 3
Number sporulated (total) 23.5 hr
39.5 hr
Number sporulated in dark
l/14
l/14
-
l/14
l/14
-
7/15
7/15
-
3/13
4/13
2/13
9/15
11/15
4/15
6/15
8/15
2/15
’ Cells prepared from 0.15~ml aliquots. dium consists of 1 part PCM:P parts SM. transferred as indicated, and ilhrminated hr; then incubated in the dark, and scored 39.5 hr from end of illumination period.
PCM-meCells were from 72-76 at 23.5 and
179
BRIEF NOTES
24 hr were found to have sporulated in the dark (last column). While the occurrence of preillumination sporulations is a variable feature in these experiments (as is the relative extents of priming by PCM on the final 2 days of starvation), it is correlated with exposure to PCM late in starvation. To date, two requirements for competence development have been described: the sporulation control factor(s) in conditioned medium, discussed above and designated here as SCF, and the previously reported requirement for nicotinamide (Daniel, 196213). In strain CL-2, however, the nicotinamide-dependent pathway is dispensable. In this strain, nicotinamide is stimulatory for sporulation, rather than strictly essential (Table 4): in the absence of nicotinamide, sporulation frequencies are TABLE
4
EFFECT OF NICOTINAMIDE ON SPORULATION FREQUENCY= Time of illuminFr r
Volume
Nicotinamide
(ml)
50-54
0.60 0.60 0.20 0.20 0.10 0.10
+ + + -
Number sporulated 15.5 hr
23 hr
39 hr
9/10 O/IO 3/10 O/IO o/10 o/10
9/10 (9) l/10 4/10 (3) o/10 o/10 o/10
7/IO (5) 6/10 (5) 3/10 o/10 l/10
” Plasmodia were harvested, then resuspended in SM-N, recentrifuged, and suspended again in SM-N. Other procedures were as described under Materials and Methods, and the scoring procedure was as in Table 1.
reduced and morphogenesis is delayed. These quantitative effects are similar to those produced, in the presence of nicotinamide, by employing relatively small cells or short starvation periods (see Table 1). In addition, cells can develop competence efficiently in the complete absence of nicotinamide, simply by exposure to SCF. As shown in Table 5, treatment with dialyzed PCM, in the absence of nicotinamide, can yield sporulation frequencies greater than those produced by incubation with nicotinamide. Evidently, interaction with SCF can stimulate competence development independently of exogenous nicotinamide. DISCUSSION
During a period of dark starvation, plasmodial cells of Physarum polycephatum strain CL-2 release into the medium and then interact with one or more factors (SCF) that actively promote the development of sporulation competence. At least part of the activity is in molecules 3 10,000 MW. Experiments to further characterize the factor(s) are in progress. The development of competence appears to involve at least two events during the period of dark starvation: the production of SCF by the cells and the development of responsiveness to the factor(s) during the later stages of starvation. The initial incapacity of starving plasmodia to respond to SCF presumably reflects either a failure of uptake or the absence of some cellular component required for SCF action. The initial
TABLE
5
SUBSTITUTION OF D-PCM Starvation
FOR NICOTINAMIDE” Number sporulated
medium
Expt No. 2
Expt No. 1
1. SM 2. SM-N 3. D-PCM-medium
24.5 hr
44.5 hr
24.5 hr
37.5 hr
1/12 (1)
m2 (2) I/3 (1) 5/I2 (4)
l/20 (1) O/6
‘J/20 (1)
o/13 o/12
3/17 (3)
O/16 10/17 (3)
n The cells were prepared as described for Table 3, and starved over the indicated media. PCM was dialyzed against SM-N. D-PCM-medium consists of 1 part D-PCM:2 parts SM-N. All plasmodia were starved for a period of 48 hr, then transferred to SM-N, and illuminated. Plates were subsequently incubated for 24.5 hr in the dark, scored, and placed under continuous illumination until the second scoring.
180
DEVELOPMENTALBIOLOGY
refractory period in starvation probably explains the observation (Sauer, 1973; Wilkins, unpublished experiments) that placing previously unstarved plasmodia over PCM does not make the cells immediately inducible for sporulation. The nature of the relationship between nicotinamide metabolism and the production or action of SCF remains to be fully determined. However, the observation that SCF can efficiently prime cells for sporulation in the absence of nicotinamide is compatible with either of two hypotheses of competence development. The first explanation is that different and independent pathways for developing competence may exist. In this case, supplying either nicotinamide or SCF to plasmodia undergoing starvation would serve to develop competence, but by different respective routes. The alternative possibility is that the function of nicotinamide is to boost SCF production by the cells. Under this hypothesis, experimental addition of SCF to the medium eliminates the nicotinamide requirement by furnishing the end product. The quantitative effects on sporulation produced by nicotinamide deprivation (Table 4) are quite consistent with this second hypothesis. In the course of these experiments on competence development, several observations pertinent to the induction process itself were made. The specific starvation conditions (cell size, duration of starvation, presence or absence of nicotinamide) determine not only the extent of competence development in a set of cells (as inferred from the final sporulation frequency) but also influence the timing of the onset of morphogenesis. When large cells are starved for only a short interval, sporulation ensues after illumination, but exhibits a characteristic delay (Table 1A). For longer starvation periods, larger cells are frequently observed to complete morphogenesis sooner than smaller ones. (One example of this can be seen in Table 4.) If one
VOLUME 72, 1979
assumes that SCF accumulation is proportional to both the length of starvation and to cell mass, delayed morphogenesis is correlated with lower SCF concentrations at the time of illumination, and conversely, rapid morphogenesis with higher SCF concentrations. (Interestingly, one major consequence of nicotinamide deprivation during starvation is a delay in morphogenesis.) On the other hand, exposure to exogenously added SCF during prolonged dark incubation can result in some frequency of induction without illumination (Table 3). Taken together, these findings are suggestive of a possible quantitative relationship between SCF concentration and the rate or likelihood of the induction reaction. This relationship and its implications for the induction process will be discussed more fully elsewhere (Wilkins and Reynolds, manuscript in preparation). We thank Drs. D. F. Bacon and E. Terzaghi for criticisms of the manuscript. REFERENCES BREWER, H. D., and RUSCH, H. P. (1968). Effect of elevated temperature shocks in mitosis and on the initiation of DNA replication in Physarum polycephalum. Exp. Cell. Res. 49, 79-86.
DANIEL, J. W. (1966). Light-induced synchronous sporulation of a myxomycete. In “Cell Synchrony” (I. Cameron and G. M. Padilla, eds.), pp. 117-152. Academic Press, New York. DANIEL, J., and BALDWIN, H. (1964). Methods of culture for plasmodial myxomycetes. In “Methods in Cell Physiology” (D. M. Prescott., ed.), Vol. 1, pp. 9-41. Academic Press, New York. DANIEL, J. W., and JARLFORS,U. (1972). Light-induced changes in the ultrastructure of a plasmodial myxomycete. Tissue Cell 4,405-426. DANIEL, J. W., and RUSCH,H. P. (1962a). Method for inducing sporulation of pure culture of the myxomycete Physarum polycephalum. J. Bacterial. 83, 234-240.
DANIEL, J. W., and RUSCH, H. P. (1962b). Niacin requirement for sporulation in Physarumpolycephalum. J. Bacterial. 83, 1244-1250. DEE, J. (1975). Slime molds in biological research. Sci. Progr.
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62, 523-542.
GARROD,D., and ASHWORTH, J. M. (1973). Development of the cellular slime mould Dictyostelium discoideum. In “Microbial Differentiation” (J. M. Ash-
BRIEF NOTES
worth and J. E. Smith, eds.), pp. 407-435. Cambridge Univ. Press, Cambridge. GORMAN, J., and WILKINS, A. S. (1979) Developmental phases in the life cycle of Physarum and related myxomycetes. In “Growth and Differentiation in Physarum polycephalum” (W. F. Dove and H. P. Rusch, eds.), in press. GUITES, E., GUTTES, S., and RUSCH, H. P. (1961). Morphological observations on growth and differentiation of Physarum polycephalum growth in pure culture. Develop. Biol. 3.588-614. LAANE, M. M., and HAUGLI, F. B. (1976). Nuclear behaviour during meiosis in the myxomycete Physarum polycephalum. Norw. J. Bot. 23, 7-21. LAANE, M. M., HAUGLI, F. B., and MELLUM, T. R. (1976). Nuclear behaviour during sporulation and
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germination in the Colonia strain of Physarumpolycephalum. Norw. J. Bot. 23, 177-189. LAFFLER, T., and DOVE, W. F. (1977). The viability of Physarum polycephalum spores and ploidy of plasmodial nuclei. J. Bacterial. 131.473-476. SAUER, H. W. (1973). Differentiation in Physarum polycephalum. In “Microbial Differentiation” (J. M. Ashworth and J. E. Smith, eds.), pp. 375-405. Cambridge Univ. Press, Cambridge. SAUER, H. W., BABCOCK,K. L., and RUSCH, H. P. (1969). Sporulation in Physarum polycephalum. Exp. Cell. Res. 57, 319-327. WORMINGTON,W. M., and WEAVER, R. F. (1976). Photoreceptor pigment that induces differentiation in the slime mold Physarum polycephalum. Proc. Nut. Acad. Sci. USA 73,3896-3899.
