The Potassium Content of Gonyaulax Polyedra Andphase Changes Short

Plant Physiol. (1974) 53, 337-342

The Potassium Content of Gonyaulax polyedra and Phase Changes in the Circadian Rhythm of Stimulated Bioluminescence by Short Exposures to Ethanol and Valinomycin1

Received for publication August 7, 1973 and in revised form October 29, 1973 BEATRICE M. SWEENEY Department of Biological Sciences, University of California, Santa Barbara, California 93106

ABSTRACT cillation of the physical properties or permeability of membranes may mediate the observed rhythms (6, 9, 11). Support A circadian rhythm in the intracellular level of K+ in for such a hypothesis cites the phase changes brought about in Gonyaulax polyedra is reported. When axenic cultures of Phaseolus by short exposures to substances which can interact Gonyaulax in continuous light (60-75 foot candles) are ex- with membranes such as alcohol (3), D20 (4), or valinomycin posed for 4 hours to 0.1 or 0.2 % ethanol, the subsequent free- (6), or by wilting for short times (5). Exposure of Phaseolus running rhythm in stimulated bioluminescence is phase-shifted, plants to ethanol, methanol (23), and D2O (4) increases the the amount and direction of the shift being dependent on the length of the free-running period of the leaf movement rhythm. time in the circadian cycle when cells are treated. The phase- Similar effects of ethanol and D20 on the period of the tidal response curve for ethanol closely resembles that for light in rhythm in activity in the isopod Excirolana have also been re- similarly maintained cells. When valinomycin (0.1 or 0.2 ,ug ported (9, 10). The rhythmic firing of the optic nerve in the ml-') is present in addition to ethanol, the phase of the bio- excised eye of Aplysia can be phase-shifted by short exposures luminescence rhythm is returned to that of an untreated cell to higher than normal concentrations of K+ (11). The rhythmic suspension. Valinomycin thus negates the effect of ethanol on opening and closing of the leaflets of Albizzia are mediated by phase. The intracellular K+ level immediately after treatment of movement of K+ (30). All these effects could be understood as a cell suspension for 4 hours with ethanol (0.1% ) is about half temporary cyclic alterations in membrane properties, although that of untreated cells. If valinomycin (0.1 ug ml-') is also no direct measurements of such changes have been reported. present during the 4-hour treatment, the intracellular K+ is only Because the unicellular marine dinoflagellate Gonyaulax slightly lower than in untreated cells. Increasing the external polyedra is known to show four circadian rhythms, in stimu- concentration of K+ or Na+ for 4 hours has no effect on the lated bioluminescence, in bioluminescent glow, in photosyn- rhythm of stimulated bioluminescence. These results are thetic capacity, and in cell division (33), and because these interpreted as support for the hypothesis that the mechanism rhythms persist for many cycles in continuous light and con- by which circadian oscillations are generated involves changes stant temperature, it is a particularly favorable test object for in membrane properties. examining the role of membrane changes in circadian rhythmicity. Since valinomycin at low concentrations is known to form a complex with K+, enhancing its transport through biological membranes (14, 27), its effects on rhythms are especially interesting. This paper reports experiments in which cell suspensions of Gonyaulax in continuous light were exposed for short times to ethanol and valinomycin, and the rhythm in A number of models for the generation of circadian oscilla- stimulated bioluminescence was subsequently examined for tions have been proposed, but none have proven very satisfac- phase shifts. Determinations of intracellular potassium levels tory. The most explicit recent hypothesis is the chronon theory at different times in the circadian cycle are also reported. (8), which postulates that oscillations arise from the repeated sequential transcription of a portion of the cellular DNA, pre- sumably composed of genes controlling the various processes MATERIALS AND METHODS which show rhythmicity. This model suffers from the fact that it predicts a severe disruption of rhythmicity by inhibitors of Cultures of a strain of Gonyaulax polyedra Stein isolated at transcription; Scripps Institution of Oceanography in 1960 were used in these although effects of such substances on rhythms experiments. In all cases where cells were exposed to ethanol, can be observed (12, 21, 22), they are not always present (31, axenic cultures of this strain purified by Dr. R. R. L. Guillard 36, 37). For example, actinomycin D inhibits the glow rhythm and designated "Gonyaulax 60P" were employed, because, if in Gonyaulax but not the photosynthetic rhythm (21). Such an bacteria were present, they used ethanol as a carbon source, effect could be due to uncoupling the glow from the clock or multiplied excessively, and killed the Gonyaulax. The medium inhibiting the glow. Furthermore, it has not been possible to for cultures with bacteria was that employed previously (35), demonstrate cyclic synthesis and degradation of the enzymes in which sea water was diluted to 75% salinity and enriched concerned in the various rhythmic processes such as luciferase with nitrate, phosphate, iron, EDTA, and soil extract. For in Gonyaulax (25). Recently, it has been suggested that an os- axenic cultures, "f' medium (15) with half-strength nutrients and the addition of 2% v/v soil extract was used. Cultures IThis research was supported in part by Grant GB 8418 from were grown to stationary phase (cell density of 5,000-10,000 the National Science Foundation. cells ml') in alternating light (300 ft-c) and darkness, each of 337 338 SWEENEY Plant Physiol. Vol. 53, 1974

12 hr duration at 22 C. All light sources were cool white fluo- RESULTS rescent lamps. For experiments, cells were transferred to continuous light If the circadian rhythms that are observed in Gonyaulax have a common origin in cyclic differences in membrane prop- (60-75 ft-c, 22 C) at the end of a light period. In one experi- a ex- ment, a large sample of "red tide," composed of more than erties, then rhythm in intracellular ion levels might be 99.5% Gonyaulax polyedra, was collected at night from Santa pected. A preliminary examination of the potassium concentra- Barbara Harbor, divided into liter aliquots, and transferred to tion in Gonyaulax cells showed changes over the course of the continuous light at 0700 the next morning. Liter samples were environmental light-dark cycle, cells containing about twice as much K+ at the end of the light period as at its (Ta- harvested at intervals over 2 days for the determination of in- beginning tracellular potassium. ble I). Since such a change could be the result of ion accumu- lation in photosynthesis during the light period, measurements In experiments where the effects of valinomycin were to be of the ion content in cells in continuous light were made. An observed, 100-ml samples were withdrawn from a culture of Gonyaulax in continuous light, and valinomycin dissolved in intensity was chosen where the circadian rhythm of photosynthesis is not expressed, so that the rate of photosynthesis was 95% ethanol was added. The concentration of ethanol in the constant over time (32). These measurements also showed a cell suspension was 0.1 or 0.2% v/v. Four hours later, cells in intracellular at end of were sedimented by centrifugation at 30g for 30 sec in an In- maximum potassium the the biologi- ternational clinical centrifuge, the medium containing valino- cal day phase, at 12 hr circadian time (Table II). Two cycles in potassium content were observed in the "red mycin was decanted, and the cells were resuspended in medium tide" sample in continuous light (Table III). Thus, differences in in without additive. Two-milliliter aliquots were then pipetted K+ persist into sterile shell vials and replaced in continuous light for the continuous light and represent another circadian rhythm in measurement of stimulated luminescence. Ethanol at same Gonyaulax. While a rhythm in intracellular K+ is consistent the with the hypothesis that the overt rhythms in Gonyaulax have concentration as that present in the valinomycin was added to a another sample of the culture and removed in the same manner common origin in cyclic membrane permeability, it does not as valinomycin. A third sample of the cell suspension without constitute proof of such an hypothesis. the addition of either ethanol or valinomycin was centrifuged and resuspended as a further control. Experiments were re- Table I. Intracellular Cation Concentration of Gonyaulax polyedra peated at least twice. at Different Times in a Light-Dark Cycle Experiments were carried out at different circadian times The light intensity was 300 ft-c and the temp was 22 C. The after the cell suspensions were transferred to continuous light light-dark cycle was 12:12 hr. Ion concentrations are mm. at 1200 circadian time. The time of the 4-hr exposure is given according to the convention of a circadian time scale of 24 hr, Circadian Time Cells Sampled in which 0 hr is taken as biological dawn (25). In one experi- Ion Medium ment, the effects of continuous exposure to ethanol and valino- O hr 6 hr 12 hr 18 hr 24 hr mycin were also examined. The bioluminescence of aliquots was measured by stimulat- K+ 9 15 24 31 18 16 ing a fresh sample of the cell suspension in a photomultiplier Na+ 315 54 photometer previously described (35). Mechanical stimulation Mg2+ 51 27 30 was provided by a small motor-driven stirrer like that employed by Hamman and Seliger (16) and was continued for 1 min. The Table II. Intracellular Potassium Ion Concentration of Gonyaulax total light emitted from each of two or three samples was re- polyedra at Different Times in Continuous Light corded at 3-hr intervals over three or four cycles of the rhythm in stimulated bioluminescence. Valinomycin (mol wt 1111.3) A culture was divided and half (No. 2) was rephased by 1800, so that all sample preparation could be carried out during a 12-hr was obtained from Cal Biochemical Co. time space. The concentration of K+ in the medium was 12 mm. Experiments in which the sodium or potassium concentration The light intensity was 75 ft-c, and the temperature was 22 C. in the cell suspension was increased by 100 mm by the addition of NaCl or and the medium after 4 were car- KCl, replaced hr, Circadian Time Cells Sampled ried out as described for ethanol and valinomycin. Medium For the determination of the potassium content of Gonyau- O hr 6 hr 12 hr 18 hr 24 hr lax, cells were harvested by filtration on a No. 1 filter, 2.5 cm in diameter, which had been prewashed three times in No. Amoles/mg protein double-distilled water. Cells were washed from the ifiter with 1 5.7 7.8 8.5 double-distilled water and were extracted by freezing and thaw- 2 8.9 6.9 7.1 ing and grinding in a glass homogenizer. Cell debris was removed by centrifugation, and the K+ and Na+ in the supernatant were determined with an Eppendorf flame photometer. Table III. Intracellular Potassium Ion Concentration of a The extent of contamination by the ions from the medium was Gonyaulax "Red Tide" at Different Times after estimated by adding "C-inulin (0.5 ptc) to the cell suspension Transfer to Continuous Light at Circadian Time 0 just before harvesting, and determining the radioactivity of The concentration of K+ in the sea water was 10 mm. The tem- both the original cell suspension and the supernatant after ex- perature was 22 C, and the light intensity was 75 ft-c. traction, in a Packard scintillation counter in 5 ml of POPOP in toluene. The protein content of the extract was determined Time Cells Sampled after by the Lowry procedure (24), after hydrolyzing the extract and Transfer to Continuous 8 11 14 17 26 31 36 39 the bovine serum albumin used as a standard in 0.5 N NaOH Light (hr) overnight at room temperature. In some experiments, the packed cell volume was estimated by centrifugation in cali- Concn K+ (mM) 23 23 21 21 17 19 20 14 brated tubes. The standard error for the determinations of ,smoles/mg protein 4.4 5.0 4.3 3.9 3.2 4.9 5.2 3.7 ,umoles K+ per mg protein by this procedure was + 3.5%. Plant Physiol. Vol. 53, 1974 GONYAULAX RHYTHM: K+, ETHANOL, VALINOMYCIN 3a9 Gonyaulax does not contain a large central vacuole, and cells examined by electron microscopy (34) are seen to consist VALINOMYCIN O.IlLg/ml 12-16c.t; largely of membrane-bound organelles including the large nu- cleus, chloroplasts, mitochondria, trichocysts, and vesicles of unknown nature. The total intracellular potassium is only slightly higher than that in the medium, but the concentration within small vaculoes could be much higher, and could change by a greater fraction over a cycle than the total potassium. Thus nothing can be said at present concerning the accumula- tion of K+ in Gonyaulax. Although measurements of Na+ and 1.0 F Mg`+ in extracts were made, the contamination of the extracts -J with the medium, which contain high concentrations of these w ions, prevented accurate determinations of intracellular levels. 0 Increasing the potassium or the sodium content of the me- so dium by 100 mm for 4 hr had no effect on the phase of the x rhythm in stimulated bioluminescence in Gonyaulax, irrespec- oex - I- tive of the time in the cycle when the high ion pulse occurred. z L) z . x However, Gonyaulax can tolerate rather large changes in the atD salinity of the medium, and did not lose motility on the addi- 0.5- / / tion of either ion, so it is likely that this cell is able to regulate v / x the internal ion concentration irrespective of the external me- x dium. If a change in the permeability to potassium is a component of the process by which oscillations are generated, then artifi- cially altering the intracellular potassium would be expected to alter the phase of the observable rhythms. Valinomycin is known to change the permeability of many biological membranes to potassium (27). Therefore, cells were exposed for short times to valinomycin and the rhythm of stimulated bio- o i luminescence was measured over several cycles in continuous 75 80 85 90^l-ft light. Since valinomycin is relatively insoluble in water, stock HOURS IN CONTINUOUS LIGI4T solutions were made up in ethanol and ethanol controls were FIG. 1. Effect of exposure of Gonyaulax polyedra strain 60P to included in all experiments. Phase shifts were observed in cells ethanol and valinomycin for 4 hr at 12 to 16 hr circadian time treated with valinomycin, as compared with ethanol controls, on the phase of the rhythm in stimulated bioluminescence in con- and the amount and direction of phase shift depended on the tinuous light (60 ft-c.) at 22 C. Data points are average of total circadian time of exposure, confirming the findings of Binning light emitted by two or three 2-ml samples of cell suspension. Luminescence of cells exposed to 0.1 Ag ml-' valinomycin and and Moser (6) in Phaseolus. However, considerable phase shifts 0.1% ethanol (-X-); luminescence of cells exposed to 0.1% occurred in the ethanol controls, as compared with the lumi- ethanol (-A-); luminescence of cells resuspended in new medium nescence rhythm in cells which had merely been centrifuged without other treatment (-O-). The time of maximum lumi- and resuspended in fresh medium. Changing the medium had nescence is shown by arrow. no effect on phase, confirming previous experience (18). Phase shifts with ethanol also depended on the time when the cells jAg mP was assayed for phase changes. Valinomycin at 0.25 were treated. For example, exposing a Gonyaulax cell suspen- ,ug ml-' or more damaged cells so extensively that a rhythm in sion to 0.1% ethanol for 4 hr between 12 and 16 hr circadian bioluminescence could not be measured. The effects on phase time resulted in a 3-hr phase delay in the bioluminescent were concentration-dependent in the concentration range 0.05 rhythm (Fig. 1). The presence of valinomycin (0.1 jug ml-') in to 0.2 Pg ml' (Table IV). addition to ethanol (0.1%) returned the phase to that of the The intracellular K+ of cells extracted at the end of a 4-hr control, in which the medium was replaced but no additions exposure to ethanol from 12 to 16 hr circadian time was about were made. Thus, as compared with the ethanol control, va- half that of the untreated control cells, while the K+ content of linomycin-treated cells were phase-advanced by 3 hr. Given cells exposed to valinomycin in ethanol was only slightly lower later in the circadian cycle, ethanol alone advanced rather than than that of the untreated control (Table V). Thus valinomycin delayed phase (Fig. 2), and again valinomycin reversed this ef- partially reverses the effect of ethanol on intracellular K+. It is fect, bringing the phase back to that of the untreated control. interesting to note that the greatest phase delay after ethanol Neither valinomycin nor ethanol had any effect on the phase treatment occurs at about 12 hr circadian time, at the peak of of the rhythm in bioluminescence between 5 and 9 hr circadian the rhythm in intracellular K+ in untreated cells. time, the biological day phase. The time-dependent phase Gonyaulax cell suspensions continuously exposed to valino- changes caused by 4-hr exposures to ethanol are summarized mycin (0.2 jug ml') in 0.2% ethanol showed no rhythm in bio- in Figure 3A, a phase-response curve for ethanol. A similar luminescence over 3 days in continuous light. The level of phase-response curve for the effect of pulses of valinomycin luminescence was high and the cells were motile during this as compared to those of ethanol alone is given in Figure 3B. A time. This observation requires repetition, however. comparison of these curves shows that they are mirror images of each other and documents the opinion that valinomycin re- DISCUSSION verses the phase changes expected from the ethanol in which it is dissolved. The phase of all circadian rhythms that have been examined The effects of 0.2% ethanol were indistinguishable from in this respect is altered by short exposures to light. The amount those of 0.1% ethanol and were reversed by 0.2 ,ug ml-' valino- and direction of these phase changes is dependent on when in mycin. A range of valinomycin concentrations from 0.02 to 10 the circadian cycle the cells are exposed to light and may be 340 SWEENEY Plant Physiol. Vol. 53, 1974 firing of the optic nerve is phase-shifted, and the amount and VALINOMYCIN direction of the change in phase is dependent on the time of O.1 Lg/ml 20-24c.t. the high K+ pulse. It is also interesting in this connection that a circadian rhythm in intracellular K+ can be observed in Go- nyaulax. Since the results of experiments with valinomycin in Phaseolus and increased K+ in Aplysia have been plotted on -I IJ coordinates of time and phase shift different from each other LuJ and from those used in this paper, the data from these experiments have been replotted (Fig. 4), to allow a comparison between them, and with the data from Gonyaulax. Phase changes x with increased potassium levels in Aplysia follow a pattern z quite similar to the phase response curve for valinomycin

n 75 80 85 90 HOURS IN CONTINUOUS LIGHT crt D 0 exposure polyedra strain 60P to FIG. 2. Effect of of Gonyaulax I ethanol and valinomycin for 4 hr at 20 to 24 hr circadian time on the phase of the rhythm in stimulated bioluminescence in continuous light (60 ft-c.) at 22 C. Data points are averages of total (I) light emitted by two or three 2-ml samples of cell suspension. Luminescence of cells exposed to 0.1 ,ug mP valinomycin and 0.1% ethanol (--- x---); luminescence of cells exposed to 0.1% ethanol 0- (-A-); luminescence of cells resuspended in new medium without other treatment (---0---). The time of maximum luminescence is shown by arrow. Wi -3 represented by a phase response curve (28). Many attempts have -u a04 been made to duplicate this phase-response curve by treating organisms with various biologically active substances for short times, with the hope of identifying components of the biochemical processes by which oscillations are generated. Until recently, these have been remarkably unsuccessful, Most meta- z bolic inhibitiors are ineffective in this respect (2, 18), although some appear to render biological timekeeping more erratic than usual. Specific inhibitors of RNA and protein synthesis like- wise fail to duplicate the effects of light on phase (21, 22). Of Uf) a long list of substances assayed for phase-shifting in the Gony- 0 aulax glow rhythm (18), only arsenite appeared to delay phase I somewhat. More recently, however, some success in experiments of this type has been achieved. A high concentration of CLL ethanol (25%) given to shoots of Phaseolus via the transpira- U.) Lli tion stream was reported to delay phase by 3 hr if given for 4 Cl) to 5 hr some time after the middle of the biological night aL- phase (3). Temporary deuteration of Phaseolus is followed by transient cycles in leaf movement, but stable phase delays are also obtained (4). In the Euglena phototactic rhythm also, exposure for 24 hr or more to D20 is followed by phase delays w c) (1). Unfortunately, a complete phase-response curve for ethanol or deuterium pulses cannot be constructed for either 12 18 24 Phaseolus or Euglena from the published data. Phaseolus plants CIRCADIAN TIME - HOURS allowed to wilt for 6 hr responded with phase changes in the leaf movement rhythm, the nature of the response depending FIG. 3. Phase-response curves for the effects on the phase of on when wilting occurred (5). the circadian rhythm in stimulated bioluminescence in Gonyaulax In Phaseolus, valinomycin appears to be one of the most polyedra strain 60P brought about by 4-hr exposures to 0.1% successful substances in imitating the phase changes caused by ethanol (A) and to 0.1 ,ug ml-" valinomycin plus 0.1% ethanol calcu- light (6). The effectiveness of this substance, both in Phaseolus lated with reference to ethanol only (B). Note that phase advances are considered positive in sign and are plotted upward from zero suggests K+ in the gen- and in Gonyaulax, the implication of on the ordinate. The abscissa is circadian time, using the con- eration of rhythmicity. This is supported by experiments with vention that dawn equals 0 hr. The time of the dark period of the a circadian rhythm in a very different system, the isolated previous entraining light-dark cycle is shown as a shaded bar on Aplysia eye (11). WVhen the level of K+ in the medium sur- the abscissa. Cells in all experiments were in 60 to 75 ft-c. con- rounding this tissue is temporarily increased, the rhythm in the tinuous light at 22 C. Plant Physiol. Vol. 53, 1974 GONYAULAX RHYTHM: K+, ETHANOL, VALINOMYCIN 341 pulses in the leaf movement rhythm in Phaseolus. Although the change from delay to advance is more sudden in the Ap- lysia data, the maximum phase advance occurs in both systems at 24 hr circadian time. The phase-response curve for ethanol 0 in Gonyaulax (Fig. 3A) is also similar in general form, and shows a maximum phase advance at 24 hr circadian time. This zi similarity suggests that all three effects share a common mechanism, possibly a transitory change in intracellular K+. In Gonyaulax, valinomycin clearly negates the effect of eth- D anol alone, both in phase shifting the rhythm of stimulated 0 bioluminescence and in changing the total intracellular K+. T Both ethanol and valinomycin would be expected to exert ef- IL fects on the structure and permeability of biological mem- cr~ branes. Ethanol is a lipid solvent and has been shown to sta- Ui bilize red blood cell membranes against hemolysis (20, 26). Valinomycin is known to act as an ionophore for K+ and has recently been found to substitute for the K+ requirement of a membrane-bound K+ and Na+-dependent ATPase active in ion I transport (19). The biochemical basis for the opposite effect of 0. ethanol and valinomycin in Gonyaulax awaits further experi- mentation on isolated membrane systems. Spin-label and other such studies are in progress in this laboratory. In Gonyaulax, the phase-response curve for the effect of short exposures to dilute ethanol mimics that to light reason- ably closely. Phase changes are of about the same magnitude as those produced by bright light pulses in cells in continuous 0 6 12 18 24 6 12 light (7). The effects of both light and ethanol pulses change CIRCADIAN TIME-HOURS from a phase delay to a phase advance in the early part of the FIG. 4. Phase-response curves for the effect of 0.1 ,ug ml[' va- biological night phase (7). Exposure to light or to ethanol in the linomycin in 0.1% ethanol on the phase of the leaf movement middle of the biological day phase has no effect the rhythm in Phaseolus coccineus calculated with reference to the eth- on phase anol control, from Buinning and Moser (6), (-O-); and for the of the subsequent rhythm in stimulated bioluminescence. Light effects of high potassium ion (100 mM) pulses on the phase of the has been found to alter the electrical properties of Acetabularia rhythm in firing rate of the optic nerve of isolated eyes of Aplysia (13, 17, 29). Thus it is possible that the phase-shifting effects California, from Eskin (11) (-X--). The data have been replotted of light may be membrane-mediated. so that circadian time 0 is dawn in both cases and phase advances are plotted upward. The time of the dark period of the previous Table IV. Phase Shifts after Treatment of Gonyaulax polyedra entraining light-dark cycle is shown as a shaded bar on the abscissa. Strain 60P by Valinomycin The organism was treated for 4 hr with different concentrations The phase and the period of a circadian rhythm are proper- of valinomycin from 8 to 12, or from 12 to 16 hr circadian time. ties of the underlying oscillator, and are the only measurable Phase shifts are calculated relative to the ethanol control. features which can be unequivocally assigned to this oscillator at present, as distinct from the physiological processes which Phase Shift this oscillator controls. Substances which affect phase or period Valinomycin Concn are thus indicators of the nature of this oscillator. The findings at 8-12 c.t.1 at 12-16 c.t. that the circadian rhythm in stimulated bioluminescence in Gonyaulax is phase-shifted by ethanol, that this phase shift is pg mU' hr reversed by the presence of valinomycin in low concentrations, 0.02 0 and that the intracellular K+ in untreated cells shows a cir- 0.05 1.9 cadian rhythm, strengthen the hypothesis that the mechanism 0.1 3 3 by which circadian rhythms are generated involves an oscilla- 0.2 3.8 tion in the physical properties of biological membranes, or in- 0.25 toxic tracellular ion concentration. 1 Circadian time. Acknowledgments-The author wishes to express her appreciation to J. Nel- son and A. Swerdloff for their excellent technical assistance, to R. R. L. Guillard for the axenic culture of Gonyaulax, strain 60P, and to P. Laris for the use of his Table V. 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