Photomorphogenesis in Physarum polycephalum - NCBI

JOURNAL

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BACrERIOLOGY, Sept. 1994,

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5541-5543

Vol. 176, No. 17

0021-9193/94/$04.00+0 Copyright C) 1994, American Society for Microbiology

Time-Resolved Detection of Three Intracellular Signals Controlling Photomorphogenesis in Physarum polycephalum CHRISTINE STAROSTZIK AND WOLFGANG MARWAN* Max-Planck-Institut fir Biochemie, 82152 Martinsried, Germany Received 7 February 1994/Accepted 20 June 1994

Incompetent plasmodia of Physarum polycephalum exposed to a light pulse sporulated after reaching the competent stage. Fusion of irradiated plasmodia with dark-incubated plasmodia and analysis of sporulation indicated the presence of a morphogenetic signal. It is concluded that a logic AND gate integrates the photoreceptor signal and the competence signal and controls the formation of the morphogenetic signal. Starved plasmodia of the acellular slime mold Physarum polycephalum (Myxomycetales) can sporulate when exposed to light. About 8 h after induction, morphogenesis starts by cleavage of the plasmodial strands into nodular structures which culminate and finally form sporangia within the next 5 to 6 h (5, 7). The photoreceptors that trigger sporulation have not been identified so far, and the signal transduction mechanism is completely unknown as well. Here we report on physiological experiments which revealed three intracellular signals in the postinduction period. The light signal is stored until plasmodia become competent. Microplasmodia of the albino strain LU897 x LU898 (1) were grown axenically in complete darkness for 4 days (3), washed, allowed to fuse on starvation agar (4), and incubated at 23°C in the dark. The plasmodia developed a veined network, but sporulation did not occur without light induction (Fig. 1A). (Macroplasmodia are referred to as plasmodia throughout.) Plasmodia exposed to a 2-hour pulse of white fluorescent light (10 W/m2) at any time before or during the third day of starvation sporulated on the fourth day (Fig. 1B through F). When irradiated at a later time, plasmodia sporulated without delay, i.e., morphogenesis was completed within 1 day (Fig. 1G through I). By definition, plasmodia are regarded as competent if they sporulate within 16 to 24 h after light induction (2). The experiment shows that light is received by incompetent plasmodia and generates a photoreceptor signal which remains active for several days until morphogenesis finally occurs. It seems that Fig. 1B through E reflect the time course for plasmodia to develop competence for sporulation. At a nonsaturating light exposure the number of sporulated plasmodia did not continually increase with extended starvation, i.e., photoinduction was limiting while the degree of competence was saturated (data not shown). When submersed microplasmodia were irradiated, harvested, and applied to starvation agar, sporulation occurred in complete darkness 4 days later, indicating the presence of a functional photoreceptor. Sporulation was an all-or-none response, i.e., if induced, the entire coenocytic plasmodial mass was transformed into sporangia. The probability of sporulation depended quantitatively on photon exposure. Young, 2-day-starved plasmodia required about a sixfold-higher exposure to obtain the half-maximal response than plasmodia starved for 6 days (Fig. 2). This different photon effectiveness could have two causes. The photoreceptor concentration in the plasmodium might in*

with the length of the starvation period and/or the activated photoreceptor species might not be completely stable over a period of several days. Evidence for a morphogenetic signal and its time-dependent formation. Plasmodia spontaneously fuse as the plasmodial surfaces come into physical contact. By fusing a light-induced, competent plasmodium with a noninduced one, Hildebrandt (6) obtained sporulation of the entire fusion product, independent of whether the noninduced plasmodium was competent. From these experiments it was concluded that a morphogen that spreads throughout the entire plasmodium exists. We have shown that a stable photoreceptor signal can be generated by light even in incompetent plasmodia. If our photoreceptor signal were identical to Hildebrandt's morphogen, fusion of a competent and an incompetent plasmodium should cause sporulation independent of which of the two plasmodia was irradiated. To test this hypothesis, plasmodia were induced by a saturating light pulse. At various delay times after exposure of a competent plasmodium to a light pulse, the agar plates carrying the plasmodial mass were cut into halves with a scalpel. Each part was combined with one half of a noninduced plasmodium, and the main veins were put together to facilitate fusion (manipulations were done under a dim green safelight). After 24 h in the dark, sporulation was evaluated (Fig. 3A). Light-induced plasmodia were used exclusively during the postinduction stage, i.e., before any visible morphological changes had occurred and while vigorous protoplasmic streaming was still visible. Most of the plasmodia either sporulated completely or formed no sporangia at all. Cultures in which one half of the plasmodial mass sporulated and the other half did not were excluded from the analysis on the assumption that fusion of these plasmodia either had not occurred or was incomplete. The frequency of sporulated plasmodia drastically depended on whether the noninduced plasmodium was competent or incompetent. Fusion products of light-induced competent (6day-old) plasmodia and noninduced competent plasmodia (6L x 6D) sporulated with a frequency of nearly 100% independent of the time elapsed between the inductive light pulse and fusion (Fig. 3B). When light-induced competent plasmodia were fused to noninduced incompetent (1-day-old) plasmodia (6L x 1D), the probability that the fusion product would sporulate continually increased with the time elapsed between light induction and fusion (Fig. 3B). The 6L plasmodia sporulated at nearly 100% when fusion to 1D plasmodia was omitted. In a control experiment, 5.5 h after light induction, 6L plasmodia were fused to 1D plasmodia. Independent of crease

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DURATION OF LIGHT EXPOSURE (h) FIG. 2. Increase in light sensitivity of plasmodia upon starvation. Plasmodia starved for 2 days (incompetent [0]) or 6 days (competent [0]) were irradiated with white light for the time indicated on the abscissa and returned to the dark, and the percentage of plasmodia that finally sporulated was evaluated on the seventh day. For each datum point, 11 to 12 plates were evaluated.

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linear connection. Therefore, a logic AND gate which allows the formation of the morphogenetic signal only when the two other signals are present at the same time has to be postulated (Fig. 4). The signal in this context might be a conformational state of a protein molecule, the change in concentration of a

A TIME OF STARVATION (d) FIG. 1. Sporulation of light-induced plasmodia during progressive starvation. Plasmodia were incubated on starvation agar in complete darkness, and the percentage of plasmodia which sporulated was estimated each day under a dim green safelight. Except for the dark control (A), the plasmodia were irradiated with a 2-h pulse of white light at the time indicated by the arrow (B through I). For each experiment, 12 to 20 plates were evaluated. Each plate carried a single, coenocytic plasmodium.

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whether the young (1D) plasmodia were 24, 27, or 30 h old, in each of the three experiments 25% of the fused plasmodia sporulated (n = 20 for each datum point). We conclude that there is no significant change in the incompetent plasmodia with respect to sporulation capability during the course of the experiment. Therefore, the curves in Fig. 3B must reflect time-dependent processes within the light-induced plasmodia that occurred prior to fusion. No sporulation was obtained by fusing a light-exposed incompetent plasmodium to a nonexposed competent one (1L x 6D) (Fig. 3B). A branched signaling pathway controls sporulation. At least two external factors are required to make a plasmodium sporulate, light and starvation. We propose a minimal model for the signal transduction mechanism that controls sporulation in P. polycephalum. Exposure to light generates a photoreceptor signal; a competence signal is formed during starvation. Only if both signals are present at the same time is a morphogenetic signal which then causes sporulation produced. Generation of a photoreceptor signal and generation of a competence signal do not depend on each other, excluding any

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, 40 X 20 0 DELAY TINE at (h) FIG. 3. Fusion of noninduced with light-induced plasmodia at various times after irradiation. (A) Schematic presentation of the fusion experiment. (B) The percent sporulated fused plasmodia was plotted as a function of the time (t) elapsed after start of the inductive light pulse (2 h) and fusion (see panel A). Light-induced plasmodia were either competent (6 days old [6L]) or incompetent (1 day old [1L]). Noninduced (i.e., dark-incubated) plasmodia were either competent (6D) or incompetent (1D). The end of the inductive light pulse is indicated by a dashed line.

NOTES

VOL. 176, 1994 LIGHT

MORPHOGENETIC-

SPORULATION

STARVATION

FIG. 4. Minimal model for the signal transduction chain controlling sporulation in P. polycephalum.

regulatory factor, or the activation or inhibition of a signaling cascade. The plasmodial fusion experiments can be easily interpreted within the framework of this model. A plasmodium formed by fusion of a light-induced competent part and a noninduced incompetent one (6L x 1D) could sporulate although the incompetent part contained neither photoreceptor signal nor competence signal. Sporulation of the 1D part hence is triggered by a morphogenetic signal formed in the 6L part prior to the fusion event. The probability that the fusion product would sporulate increased with time elapsing after irradiation until fusion (Fig. 3B) (6L x 1D), reflecting the time-dependent formation of the morphogenetic signal. Since the 6L plasmodia alone sporulated with 100% efficiency, sporulation of the fused plasmodium must be suppressed by the incompetent 1D part in the early phase of the curve. According to the model the competence signal is quenched by fusion because the formation of the morphogenetic signal would not have been inhibited otherwise. The present experiments do not allow us to distinguish whether competence signal is passively quenched by dilution or actively erased by a quenching factor. Fusion of a 6-day-old light-induced plasmodium with a 6-day-old dark-incubated one (6L x 6D) yielded maximal sporulation even if fusion occurred at an early time after the inductive light pulse, i.e., when little or no morphogenetic

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signal had been formed. Since the competence signal is present in both parts, switching the photoreceptor signal on causes the formation of a morphogenetic signal throughout the entire fused plasmodium and sporulation occurs. Although an incompetent plasmodium contains a photoreceptor signal when induced by light (Fig. 1), sporulation does not occur when it is fused with a noninduced competent plasmodium (1L x 6D). It seems that the competence signal in the fused plasmodium is too weak to allow the formation of sufficient morphogenetic signal. The alternative possibility that the photoreceptor signal and the competence signal are not easily interchangeable between the two parts of the plasmodium (e.g., they are membrane bound) is made unlikely by the 6L x 1D (early-phase) and the 6L X 6D experiments, which suggest that exchange indeed occurs. We thank A. Hildebrandt and S. Renzel for a culture of P. polycephalum, Kathrin Ueberholz for excellent technical assistance, and J. Shiozawa for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Ma 1516/1-1). REFERENCES 1. Anderson, R. W. 1977. A plasmodial color mutation in the myxomycete Physarum polycephalum. Genet. Res. 30:301-306. 2. Chapman, A., and J. G. Coote. 1982. Sporulation competence in Physarum polycephalum CL and the requirement for DNA replication and mitosis. J. Gen. Microbiol. 128:1489-1501. 3. Daniel, J. W., and H. H. Baldwin. 1964. Methods for culture of plasmodial myxomycetes. Methods Cell Physiol. 1:9-41. 4. Daniel, J. W., and H. P. Rusch. 1962. Method for inducing sporulation of pure cultures of the myxomycete Physarum polycephalum. J. Bacteriol. 83:234-240. 5. Gorman, J. A., and A. S. Wilkins. 1980. Developmental phases in the life cycle of physarum and related myxomycetes, p. 157-179. In W. F. Dove and H. P. Rush (ed.), Growth and differentiation in Physarum polycephalum. Princeton University Press, Princeton, N.J. 6. Hildebrandt, A. 1986. A morphogen for the sporulation of Physarum polycephalum detected by cell fusion experiments. Exp. Cell Res. 167:453-457. 7. Sauer, H. W. 1982. Developmental biology of Physarum, p. 1-229. Cambridge University Press, Cambridge.