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.     

Circadian rhythmicity of nitrate reductase activity in barley leaves

Nitrate reductase (EC 1.6.6.1) activity showed circadian rhythmicity in the first leaf of 8–11 days old barley ( Hordeum vulgare L. cv. Herta) plants. Circadian rhythms were found using both the in vitro and in vivo method for testing the enzyme activity. When the light intensity was reduced from 65 to 20 W m⁻², the amplitude was smaller and the oscillations were damped sooner. In continuous darkness nitrate reductase activity decreased in a two step process. Three different light qualities were tested which all gave the same results.     

Properties and cellular localization of a luciferin binding protein in the bioluminescence reaction of Gonyaulax polyedra

A luciferin binding protein LBP involved in the bioluminescence reaction of Gonyaulax polyedra was purified and used for antibody production. Luciferin bound to LBP is fluorescent and can be used as a marker in living cells, allowing the localization of LBP in cortical organelles to be visualized. In cell sections, the same peripheral localization was observed using anti-LBP and immunofluorescence microscopy. The amount of LBP is ten-fold greater from cells from in night phase compared to those from in day phase, as determined both by immunoblots of cell extracts, and in vivo fluorescence. These changes correlate with the circadian changes in bioluminescence of living cells.     

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.     

Circadian-clock control of protein synthesis and degradation in Gonyaulax polyedra

In growing cultures of the dinoflagellate, Gonyaulax polyedra, total protein synthesis showed a circadian rhythm with a maximum during the phase of the cycle which corresponded to the previous darktime. The maximum coincided with the maximal phase shift of the glow rhythm caused by lower concentrations of the antibiotic anisomycin (Taylor, W., et al., 1982). J. Comp. Physiol. 148 B, 11–25. The dose reponses of inhibition of protein synthesis correlated well with the phase shifting by anisomycin. The amplitude and level of the total-protein synthesis rhythm increased with the growth rate, indicating that the majority of proteins controlled by the circadian clock were cell cycle-dependent. The degradation rate showed the same circadian rhythm as the synthesis rate. Slight variations in uptake and pool size of amino acids were not responsible for the rhythm in the protein-synthesis rate.     

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.     

Effects of aldehydes and alcohols on protein synthesis in the dinoflagellate, Gonyaulax polyedra: relation to phase-shifting potency in the circadian oscillation

In Gonyaulax polyedra, protein synthesis was measured in vivo and in an in vitro translating system. Aldehydes applied in concentrations by which the circadian oscillator is phase-shifted inhibited protein synthesis considerably, both in vivo and in vitro. The efficiency of the drugs decreased with increasing chain length. Alcohols, though given in much higher concentrations, showed only weak effects. Therefore, the phase-shifting potency of aldehydes can be explained by a general mechanism influencing the circadian oscillator, namely through inhibition of protein synthesis, whereas this is obviously not the case in alcohols.     

Role of a luciferin-binding protein in the circadian bioluminescent reaction of Gonyaulax polyedra

A luciferin-binding protein (LBP), which binds and protects from autoxidation the substrate of the circadian bioluminescent reaction of Gonyaulax polyedra, has been purified to near homogeneity. The purified protein is a dimer with two identical 72-kDa subunits, and an isoelectric point of 6.7. LBP is a major component of the cells, comprising about 1% of the total protein during the night phase, but drops to only about 0.1% during the day. The luciferin is protected from autoxidation by binding to LBP, and one luciferin is bound per dimer at alkaline pH (Ka approximately 5 x 10(7) M-1). The protein undergoes a conformational change with release of luciferin at pH values below 7, concurrent with an activation of Gonyaulax luciferase. LBP thus has a dual role in the circadian bioluminescent system.     

Phase of the circadian clock is accurately transferred from mother to daughter cells in the dinoflagellate Gonyaulax polyedra.

In the first cycle following transfer from a 12 h light-12 h dark cycle (LD12:12) to constant darkness (DD), the standard deviation in circadian phase among individual clocks in populations of Gonyaulax polyedra is approximately 60 min. When a culture is transferred to constant light conditions (LL) from an LD 12:12 cycle, the standard deviation increases in the first 2-3 d, but then remains unchanged, suggesting a lack of observable desynchronization in LL after the transient period. The synchrony in a cell population is preserved even after several cell divisions. The results indicate that variations in period among cells are small, that the period of an individual clock does not fluctuate randomly from day to day, and that the circadian phase of a mother cell is faithfully passed to the clocks of the daughter cells.     

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.     

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.     

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

Regulation of Chlorella nitrate reductase: control of enzyme activity and immunoreactive protein levels by ammonia

Nitrate reductase catalyzes the initial step in the conversion of nitrate to organic nitrogen and is thought to be repressed by ammonia and induced by nitrate. Induction by nitrate and repression by ammonia were studied by following changes in NADH:nitrate reductase and the associated partial activities NADH:cytochrome c reductase and methylviologenr:nitrate reductase. Immunoreactive protein was assessed by enzyme-linked immunosorbent assay and immunoblotting. Molybdenum cofactor levels were investigated using the nit-1 complementation assay as well as fluorescence of the oxidized cofactor. The results indicate that the NADH:cytochrome c reductase activity is "induced" faster than the nitrate-reducing activity and suggest that incorporation of the molybdo-pterin cofactor may be rate limiting in the expression of activity. Molybdenum cofactor levels are significantly elevated in nitrate-treated cells. Under "repressing" conditions all activities decreased at approximately the same rate. A more rapid conversion of the enzyme to a reversibly inactive form also occurred under these conditions. Changes in immunoreactive protein levels correlated most closely with NADH:cytochrome c reductase activity but appeared to increase faster during induction and decrease slightly slower during repression than the enzyme activities. Removal of exogenous ammonia results in the appearance of nitrate reducing activity, as well as immunoreactive protein (derepression). Studies using protein and RNA synthesis inhibitors indicated that de novo synthesis is required for nitrate reductase induction and were in agreement with the results of the immunoreactive studies.     

Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.