The Loss of the Circadian Rhythm in Photosynthesis in an Old Strain of Gonyaulax polyedra

Cultures of Gonyaulax polyedra Stein maintained in the laboratory for 15 to 20 years, including an axenic strain isolated in 1960, have gradually lost the ability to survive in darkness. G. polyedra (70A), isolated in 1970 and maintained in a 12:12 light:dark cycle, now tolerates continuous darkness for a much shorter time than a strain isolated in 1981. I have compared the properties of strain 70A with those of this newer strain (81N), to investigate changes in Gonyaulax with length of time in culture, which may account for poor survival in darkness. When grown in continuous light (13, 12, or 4.5 watts per square meter), strains 70A and 81N have similar growth rates, yields, cell diameters, protein contents, C/N ratios, respiration rates, pigment complements, and photosynthetic rates. When entrained by a light:dark cycle (12L:12D), 70A showed no photosynthesis rhythm, although such a rhythm was formerly present. However, the circadian rhythms in bioluminescence and cell division were normal in both strains. Thus, the circadian clock is apparently still intact in 70A as in 81N. The rate of photosynthesis in strain 70A was constant at a low level, the consequent smaller accumulation of photosynthetic products probably accounting for the limited survival in darkness. The defect in strain 70A may be the loss of a component either directly affecting P_max or necessary for transduction from the circadian clock to photosynthesis.     

A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyedra

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.     

A Circadian Rhythm in the Activity of Superoxide Dismutase in the Photosynthetic Alga Gonyaulax polyedra

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.     

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg²⁺ concentration is 3 mm. Heavy metal ions (Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO₃⁻ was also the same (16 to 19 mm).     

Requirement of Indoleamines and a V-type Proton ATPase for the Expression of the Circadian Glow Rhythm in Gonyaulax polyedra

In the dinoflagellate Gonyaulax polyedra, bioluminescence is known to be controlled by proton transfer from an acidic vacuole system to the scintillons. We demonstrate that bafilomycin A 1, a specific blocker of V-type proton ATPases, inhibits at low concentrations (down to 2 × 10 –8 M) bioluminescence and, in particular, the circadian glow maximum. For many hours bafilomycin A 1 does not interfere with the capacity of the bioluminescent system. Therefore, we conclude on the participation of a V-type ATPase in proton accumulation in the acidic vacuoles. Inhibition of tryptophan hydroxylase by p-chlorophenylalanine, p-fluorophenylalanine, or 5-fluorotryptophan also suppresses the circadian glow maximum. After inhibition of the enzyme by p-chlorophenylalanine, the glow peak can be restored, without any additional unspecific effects on bioluminescence, by supplementation with 5-hydroxytryptophan. Therefore, the availability of indoleamines is required for the expression of the glow maximum. Since 5-methoxytryptamine is the only physiologically occurring indoleamine with substantial effects on bioluminescence at low concentrations (below 10 –7 M), and since this substance accumulates in the second half of the night to stimulatory concentrations, this indolic metabolite may represent the physiologically active substance involved in the expression of the glow maximum.     

Requirement of Indoleamines and a V-type Proton ATPase for the Expression of the Circadian Glow Rhythm in Gonyaulax polyedra

In the dinoflagellate Gonyaulax polyedra , bioluminescence is known to be controlled by proton transfer from an acidic vacuole system to the scintillons. We demonstrate that bafilomycin A 1 , a specific blocker of V-type proton ATPases, inhibits at low concentrations (down to 2 × 10 -8 M) bioluminescence and, in particular, the circadian glow maximum. For many hours bafilomycin A 1 does not interfere with the capacity of the bioluminescent system. Therefore, we conclude on the participation of a V-type ATPase in proton accumulation in the acidic vacuoles. Inhibition of tryptophan hydroxylase by p-chlorophenylalanine, p-fluorophenylalanine, or 5-fluorotryptophan also suppresses the circadian glow maximum. After inhibition of the enzyme by p-chlorophenylalanine, the glow peak can be restored, without any additional unspecific effects on bioluminescence, by supplementation with 5-hydroxytryptophan. Therefore, the availability of indoleamines is required for the expression of the glow maximum. Since 5-methoxytryptamine is the only physiologically occurring indoleamine with substantial effects on bioluminescence at low concentrations (below 10 -7 M), and since this substance accumulates in the second half of the night to stimulatory concentrations, this indolic metabolite may represent the physiologically active substance involved in the expression of the glow maximum.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

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

A circadian rhythm in the intracellular level of K⁺ in Gonyaulax polyedra is reported. When axenic cultures of Gonyaulax in continuous light (60-75 fot candles) are exposed for 4 hours to 0.1 or 0.2% ethanol, the subsequent free-running rhythm in stimulated bioluminescence is phase-shifted, the amount and direction of the shift being dependent on the time in the circadian cycle when cells are treated. The phase-response curve for ethanol closely resembles that for light in similarly maintained cells. When valinomycin (0.1 or 0.2 μg ml⁻¹) is present in addition to ethanol, the phase of the bioluminescence rhythm is returned to that of an untreated cell suspension. Valinomycin thus negates the effect of ethanol on phase. The intracellular K⁺ level immediately after treatment of a cell suspension for 4 hours with ethanol (0.1%) is about half that of untreated cells. If valinomycin (0.1 μg ml⁻¹) is also present during the 4-hour treatment, the intracellular K⁺ is only slightly lower than in untreated cells. Increasing the external concentration of K⁺ or Na⁺ for 4 hours has no effect on the rhythm of stimulated bioluminescence. These results are interpreted as support for the hypothesis that the mechanism by which circadian oscillations are generated involves changes in membrane properties.     

Circadian rhythms of cell cycle processes in the marine dinoflagellate Gonyaulax polyedra

The circadian expression of several growth properties was examined in the dinoflagellate Gonyaulax polyedra under constant light and light-dark conditions. The cell concentration, mean cell volume and rate of DNA synthesis varied in a circadian rhythm, with the primary maximum of cytokinesis and DNA synthesis at about dawn. High rates of cell mortality also occurred during phases related to events of cytokinesis, and may be important in the expression of the other rhythms and in "red tide" generation. Flow-cytofluorimetric analysis indicated that cells of a population contain either a relatively high or a low amount of DNA, but the proportion of cells in each of these classes and the absolute amount of DNA in each cell varied rhythmically depending on the circadian time. This DNA-distribution pattern was unlike the usual G1-S-G2+M pattern typical of eukaryotic cell populations. Isotopically labelled thymidine, used as a marker of DNA synthesis, was continuously incorporated; but the incorporation rate fluctuated in a regular pattern that repeated each circadian period.     

The Action Spectrum for Shifting the Phase of the Rhythm of Luminescence in Gonyaulax polyedra

The action spectrum for changing the phase of the rhythm of luminescence in the marine dinoflagellate Gonyaulax polyedra has been determined. Maxima in effectiveness were found at 475 and 650 mµ. The significance of these findings is discussed.     

Peridinin-Chlorophyll a-Protein Is not Implicated in the Photosynthesis Rhythm of the Dinoflagellate Gonyaulax despite Circadian Regulation of its Translation

The levels of peridinin-chlorophyll a -protein (PCP) mRNA, apoprotein and protein bound with peridinin (holoprotein) were measured as a function of circadian time in the dinoflagellate Gonyaulax polyedra to test involvement of this protein in the circadian oxygen evolution rhythm. This involvement was suggested by previous work showing that synthesis of PCP was rhythmic in vivo and in phase with the three-fold rhythm of oxygen evolution. However, Gonyaulax contains six PCP isoforms, only one of which was previously examined. In this report, we extend our analysis to two additional isoforms to encompass roughly 90% of the total cellular PCP. We confirm that synthesis of two additional PCP isoforms is rhythmic in vivo and show that this regulation appears to occur at a translational level as found for two other regulated proteins in this organism. However, PCP is unlikely to be implicated in the oxygen evolution rhythm since both PCP protein levels and the amount of chromophore (OD⁴⁸⁰) bound to protein (OD²⁸⁰) are constant over a circadian period.     

Peridinin-Chlorophyll a-Protein Is not Implicated in the Photosynthesis Rhythm of the Dinoflagellate Gonyaulax despite Circadian Regulation of its Translation

The levels of peridinin-chlorophyll a -protein (PCP) mRNA, apoprotein and protein bound with peridinin (holoprotein) were measured as a function of circadian time in the dinoflagellate Gonyaulax polyedra to test involvement of this protein in the circadian oxygen evolution rhythm. This involvement was suggested by previous work showing that synthesis of PCP was rhythmic in vivo and in phase with the three-fold rhythm of oxygen evolution. However, Gonyaulax contains six PCP isoforms, only one of which was previously examined. In this report, we extend our analysis to two additional isoforms to encompass roughly 90% of the total cellular PCP. We confirm that synthesis of two additional PCP isoforms is rhythmic in vivo and show that this regulation appears to occur at a translational level as found for two other regulated proteins in this organism. However, PCP is unlikely to be implicated in the oxygen evolution rhythm since both PCP protein levels and the amount of chromophore (OD⁴⁸⁰) bound to protein (OD²⁸⁰) are constant over a circadian period.     

Circadian changes in protein-synthesis rate and protein phosphorylation in cell-free extracts of Gonyaulax polyedra

The polysomal pattern of the dinoflagellate Gonyaulax polyedra, cultured under constant conditions, demonstrates a circadian rhythm. The relative amount of polysomes increases during the phase corresponding to the previous night period (=subjective night phase) when the rate of protein synthesis reaches its maximum (Cornelius et al., 1985, Planta 160, 365–370). Cell-free extracts were isolated at different circadian phase. The rate of protein synthesis in the extracts changed rhythmically in the same manner as the rate of protein synthesis in vivo. Substances in the postribosomal supernatants influenced the protein-synthesis rate of the cell-free system, depending on the phase when they were isolated: night factors stimulated protein synthesis in day extracts whereas day factors inhibited protein synthesis in night extracts. These effects were abolished by heating the postribosomal supernatant. In-vitro phosphorylation in parallel probes showed changes in the pattern of phosphorylated proteins. Phosphorylation of one of the proteins (95 kDa) was decreased after addition of night factor(s) and increased after addition of day factor(s). Cyclic-AMP enhanced the rates of protein synthesis and phosphorylation in the day extracts.Abbreviations cAMP cyclic-AMP - CT circadian time - D (N) subjective day (night)phase     

Biochemical Correlates of the Circadian Rhythm in Photosynthesis in Phaseolus vulgaris

A circadian rhythm in photosynthesis occurs in Phaseolus vulgaris after transfer from a natural or artificial light:dark cycle to constant light. The rhythm in photosynthesis persists even when intercellular CO₂ partial pressure is held constant, demonstrating that the rhythm in photosynthesis is not entirely due to stomatal control over the diffusion of CO₂. Experiments were conducted to attempt to elucidate biochemical correlates with the circadian rhythm in photosynthesis. Plants were entrained to a 12-hour-day:12-hour-night light regimen and then monitored or sampled during a subsequent period of constant light. We observed circadian oscillations in ribulose-1,5-bisphosphate (RuBP) levels, and to a lesser extent in phosphoglyceric acid (PGA) levels, that closely paralleled oscillations in photosynthesis. However, the enzyme activity and activation state of the enzyme responsible for the conversion of RuBP to PGA, ribulose-1,5-bisphosphate carboxylase/oxygenase, showed no discernible circadian oscillation. Hence, we examined the possibility of circadian effects on RuBP regeneration. Neither ribulose-5-phosphate kinase activity nor the level of ATP fluctuated in constant light. Oscillations in triose-phosphate levels were out of phase with those observed for RuBP and PGA.     

Circadian oscillation of nitrate reductase activity in Gonyaulax polyedra is due to changes in cellular protein levels.

A circadian rhythm in the activity of nitrate reductase (NR; EC 1.6.6.1) isolated from the marine dinoflagellate Gonyaulax polyedra is shown to be attributable to the daily synthesis and destruction of the protein. The enzyme was purified in three steps: gel filtration on S-300 Sephacryl, an Affigel-Blue column, and a diethylaminoethyl ion-exchange column. Undenatured protein shows a molecular mass of about 310 kD; based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of six possibly identical subunits. The amino acid composition of the G. polyedra NR is very similar to that reported for the NR of barley leaves, Chlorella vulgaris, and Ankistrodesmus braunii. The experiments reported indicate that the cellular expression of NR is under circadian control. In extracts of cells grown under either constant dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at midday phase, as does photosynthesis. Staining with affinity-purified polyclonal antibodies, raised in rabbits against purified NR, shows that the amount of protein changes by a factor of about 10, with the maximum occurring in midday phase.     

A circadian rhythm of population behavior in Gonyaulax polyedra

Like other flagellates, Gonyaulax polyedra exhibits diurnal vertical migration and pattern formation. Shape and size of the aggregations depend on container type, light intensity, and cell density. In Petri dishes, cells form oval "swarms"; within these, cells move downward in the highly dense center and rise up at the periphery. We have investigated the daily rhythm of this swarming activity in Petri dishes illuminated from the side, using time-lapse video recordings. At night, a "lawn" of cells forms at the bottom of the dish toward the light source (independent of light intensity). Before dawn, cells rise toward the surface and aggregate in swarms. The daily vertical migration occurs independent of light direction and intensity. The diurnal swarms, however, form every day at the same location within the dish, at a distance from the light that depends on light intensity, indicating a self-selection of light intensity. In constant light and temperature and with negligible vertical nutrient differences, all aspects of the rhythm continue to oscillate for up to 3 weeks, when the rhythm of the population becomes desynchronized. Under cycles of bright white-dim red light (WR), cell entrain to WR 10:10 but free run in WR 8:8 and shorter cycles, showing relative coordination (von Holst, 1939) to the driving light cycle. They also entrain to the 24-hr multiple of WR 6:6. Under nonentrained conditions, swarming activity is still influenced by light changes, and in spite of the apparent free run, the phasing of the averaged activity varies systematically with different T-cycle frequencies.     

Action Spectra for Two Effects of Light on Luminescence in Gonyaulax polyedra

The luminescence of the marine dinoflagellate Gonyaulax polyedra shows an endogenous diurnal rhythm. The effect of light during the phase of low luminescence capacity may be observed as an enhancement of luminescence during the subsequent bright phase. During the bright phase, however, illumination diminishes the capacity for luminescence. The action spectra for these two effects of light have been determined, and the major pigments of Gonyaulax have been examined. A consideration of the action spectrum and the pigment complement of Gonyaulax suggests that photosynthesis during the day is responsible, directly or indirectly, for the enhancement of luminescence during the following night. Photoinhibition of luminescence is in part attributable to light absorbed by the photosynthetic pigments. However, activity observed in the far red region of the spectrum beyond the absorption maximum of chlorophyll a suggests that an additional pigment, present in small amounts, may also act as sensitizer for photoinhibition.