Planta

Planta (1982)155:431436

9 Springer-Verlag 1982

Light-induced phase shifting of the circadian clock in Neurospora crassa requires ammonium salts at high pH Hideaki Nakashima and Yoko Fujimura National Institute for Basic Biology, Myodaijicho, Okazaki, Japan 444

Abstract. Effects of external ionic conditions on

light-induced phase shifting of the circadian rhythm of conidiation in Neurospora crassa were examined in simple buffer solutions for discerning effects of individual ions. Mycelia were cultured in liquid media of different pHs and then transferred to 10 mM piperazine-N,N'-bis (2-ethanesulfonic acid) (Pipes) buffer of various pHs and irradiated with white light. The phase of the rhythm of dark controls was not changed by transfer from medium to buffer. When mycelia were cultured in media of pH above 6.7, light did not advance the phase of the clock in Pipes buffer alone. However, light-induced phase advance was restored when an ammonium salt was added to buffer of pH higher than 7.6. An amination-defective mutant, bdam, showed the same response to ammonium nitrate as the wild-type strain, bd. Ammonium must be present before light irradiation for restoration of phase shifting. Free-amino-acid pools in the cells were changed by treatment with Pipes buffer: aspartic acid, glutamic acid, ammonia, glutamine and ornithine levels decreased, while lysine and histidine increased. Addition of ammonium nitrate to Pipes buffer resulted in further changes in amino-acid pools: lysine, histidine, arginine, alanine and ornithine decreased, and glutamine levels increased. Irradiation did not result in significant changes in amino acid pools. Key words: Ammonium - Circadian clock - Light and biological clock - Neurospora - Rhythm, endogenous. Introduction

Light is one of the most important factors regulating circadian rhythm, and phase response curves Abbreviation:

acid)

Pipes = piperazine-N,N'-bis(2-ethanesulfonic

for light are used as indicators of the state of the circadian clock (Pittendrigh 1967). In Neurospora, the photoreceptor for the clock has been suggested to be a flavine-cytochrome b system (Munoz et al. 1974; Munoz and Butler 1975; Schmidt and Butler 1976) or nitrate reductase (Roldan and Butler 1980). However, we have little information about reactions connecting the photoreceptor and the circadian clock. Recently, Nakashima and Feldman (1980) compared phase response curves for light at different temperatures and suggested that a temperature-sensitive process couples photoreception to phase shifting. Furthermore, Nakashima (1982) showed that in Neurospora an inhibitor of plasmamembrane ATPhase, diethylstilbestrol, completely suppresses light-induced phase shifting. In membrane models of the circadian clock, light has usually been assumed to change membrane permeability, altering ionic conditions in the cells, and thereby shifting the phase of the clock (Burgoyne 1978; Njus et al. 1974; Sweeney 1974). In view of these models and the possible involvement of the plasma-membrane ATPase, it seemed possible that external ionic conditions might affect light-induced phase shifting in Neurospora. Using isolated eyes of Aplysia, Eskin (1977) suppressed light-induced phase shifting by changing the ionic conditions of the culture medium. Such findings may contribute to an understanding of the mechanism of phase shifting of the circadian clock. In a particular liquid medium, Neurospora cultures have a normal clock function but do not exhibit growth nor differentiation (Nakashima 1981). Preliminary experiments had shown that, at least at some phases, the phase of the clock is quite stable for at least several hours after mycelia are transferred to 10 mM Pipes buffer. We have studied circadian clock function of cultures in simple buffer solutions so that effects of individual ions can be easily discerned. We have examined c o m 0032-0935/82/0155/0431/$01.20

432

H. Nakashima and Y. Fujimura: Light, NH~ salts, pH and circadian clock in Neurospora

pounds whose presence in the culture medium is necessary for light-induced phase shifting.

Results

Ammonium dependence of light-induced phase shifting. It was reported previously that the clock in Materials and methods Liquid culture. The bd (band) strain of Neurospora crassa was a gift of Dr. J.F. Feldman (University of California, Santa Cruz, USA). The am mutant (allele 32213), an amination-defectire mutant, was obtained from the Fungal Genetics Stock Center, Arcata, Cal., USA. The double mutant, bd am, was isolated by crossing bda with am A. All experiments were done at 26 ~ C. After the concentration of conidia was determined by absorbance at 480 nm, 13.105 eonidia were added to disposable Petri dishes (100 mm diameter, 15 mm high) with 25 ml of liquid medium containing Fries' salts (Fries 1948), 0.3% glucose and 0.5% arginine (Sargent and Kaltenborn 1972). In the case of bdam, glutamic acid (0.1 mgm1-1) and alanine (0,1 mgm1-1) were also added. The pH of this medium was 5.8 and was not adjusted. After 33 h in continuous light, discs were cut from the hyphal mats with a cork borer 11 mm in diameter. Six discs were transferred into 125-ml Erlenmeyer flasks with 25 ml of liquid medium of the appropriate pH containing Fries' salts, 0.03% glucose and 0.05% arginine. The pH of the medium was adjusted by 1N HC1 and 1N NaOH. The flasks were shaken on a reciprocal shaker (about 100 cycles/rain) in continuous darkness; the light-to-dark transition initiates "free-run" of the clock (Perlman et al. 1981). All experimental manipulations after the transition from light to dark were done under a red safe light from 30 W white fluorescent lamps with red acrylic plate filters (Acrylite; Mitsubishi, Tokyo, Japan). After 48 h in the dark, the discs were transferred to 25 ml of 10 mM Pipes-NaOH or Mes(2-N-morpholino) ehtanesulfonic acid)-NaOH buffer of the appropriate pH, incubated while shaking, and after 1 h irradiated with 2.6 W m - Z o f white light from 15-W fluorescent lamps for 5 min. Irradiation at this phase of the cycle results in a phase advance of about 10 h. This phase-shifting by light was maximal (Nakashima 1981). Treatment with simple buffer in the dark does not cause phase shifts at this time. Two h after irradiation, each plug was transferred to a race tube that held 8 ml of solid agar medium containing Fries' salts, 0.15% glucose, 0.25% arginine and 1.5% agar, and was cultured in continuous darkness. In the case of the bd am strain, glutamic acid (0.1 mg ml -~) and alanine (0.J mg m l - ~) were added. The growth fronts in the race tubes were marked every 24 h. The period length and phase, or position of the first band after the initial growth front mark (at the 64th hour), were calculated for each race-tube culture as described by Dharmananda and Feldman (1979).

mycelial discs is differentially sensitive to light and chemicals, including plasma membrane ATPase inhibitors and ethanol, depending on the pH at which the mycelia are cultured (Nakashima 1982). Mycelial discs were cultured in liquid media of different pHs. After 48 h, they were transferred into 10 mM Pipes buffer o f p H 7.6 and then irradiated. As shown in Fig. 1, light did not shift the phase when the discs were cultured at initial pHs higher than 6.7. Culture in media of initial pHs lower than 6.7 resulted in phase shifting by light of about 10 h, which is the same as when the discs were irradiated in culture medium. However, when the discs were transferred into Pipes buffer containing 24 mM ammonium nitrate, light caused normal phase shifts in discs cultured in media of all pHs examined. The phase of the dark controls was the same throughout the series; there was no phase

Irradiated

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Dark

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Determination of free amino acids'. Six discs were washed once with distilled water, placed in 5 ml of 80% ethanol and heated for 3 rain in a boiling water bath. The supernatant was removed by centrifugation, and this procedure was repeated twice, first with 80% ethanol and then with 50% ethanol. The combined extracts were dried using a rotary evaporator at a temperature below 40 ~ C. Amino acids were solubilized in 0.8 ml of 0.02 N HC1 and were analysed using a high-performance amino-acid analyser (Hitachi, model 835). Identification and measurements of individual amino acids were made by comparison with a standard amino-acid mixture (Amino acid calibration mixture, Takara Kohsan Co., Tokyo, Japan). The positions of glutamine and ornithine were determined by co-chromatography with standards. The data shown are the average of four flasks.

4

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Initial

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Fig. 1. Effects of medium pH on light-induced phase shifting in Neurospora. Mycelial discs of the bd strain were cultured in media of different pHs (abscissa) for 48 h after a light-dark transition. They were transferred to 10 m M Pipes (pH 7.6) with or without 24 mM ammonium nitrate, and 1 h later irradiated with 2.6 W m -2 of white light for 5 min. Then, the discs were individually transferred to race tubes and the phases of the conidial bands were determined as described in the text. Solid symbols (e, m), control; open symbols (o, n), irradiated; o, o, Pipes without NH~NO3; N, D, Pipes with NH4NO 3. The phase of discs irradiated in the culture medium is shown on the ordinate: A, dark; A, irradiated with white light. Error bars are -t- SD

H. Nakashima and Y. Fujimura: Light, NH,~ salts, pH and circadian clock in Neurospora

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Fig. 2. Effect of pH on light-induced phase advance of mycelial discs of Neurospora cultured at different pHs. Mycerial discs of the bd strain cultured in medium of pH 5.7 (e) or 6.7 (A) were transferred into buffers of different pHs according to the schedule described in Fig. 1. Error bars are _+SD

shifting by transfer to Pipes buffer with or without ammonium nitrate. Discs cultured in pH 5.7 or 6.7 media were transferred into Mes or Pipes buffers of different pHs and light-induced phase shifting was examined (Fig. 2). Discs which were cultured at pH 5.7 were phase-shifted by light in buffer alone at all, pHs; phase shifts were larger in buffer of acidic pH than in neutral pH. On the other hand, discs cultured in pH 6.7 medium showed very little light-induced phase shifting in buffer of any pH, although small phase shifts occurred in the acidic pH range. The phase of dark controls cultured at either pH was not changed by transfer into buffer of any pH (data not shown). To examine the dependence of light-induced phase-shifting on ammonium nitrate in buffer, mycelial discs of the bd strain and of bd am were cultured in pH 6.7 medium and transferred into Pipes buffer of different pHs containing different concentrations of ammonium nitrate (Fig. 3). Maximum restoration of light sensitivity in Pipes buffer of pH 7.6 and 8.4 occurred in the presence of 12 mM ammonium nitrate, but there was little phase-shifting by light in pH 6.7 buffer even in the presence of 48 mM ammonium nitrate. The recovery of light-induced phase shifting by ammonium nitrate is dependent on the pH of the buffer. Phase shifting in the amination-defective mutant showed the same restoration by ammonium nitrate as in the bd strain (Fig. 3). In the preceding experiments, discs were irradiated by light i h after transfer to Pipes buffer with

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48

Fig. 3. Effect of pH on restoration of light-induced phase advance in Neurospora by different concentrations of ammonium nitrate. Mycelial discs of the bd and the bd am strain were cultured in pH 6.7 medium and transferred to 10 mM Pipes (o, pH 6.7; v, pH 7.6; or e, pH 8.4 for bd; or l , pH 8.2 for bdam) with different concentrations of ammonium nitrate. Phase-advance of discs irradiated in the culture medium is als0 shown on the ordinate (,)

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Fig, 4. Time dependence of restoration of light-induced phase advance in Neurospora by addition of ammonium nitrate. Mycelial discs of the bd strain were cultured in pH 6.7 medium and transferred to 10 mM Pipes (pH 7.6). Ammonium nitrate (24 raM) was added at various times (abscissa). The discs were irradiated with light 1 h after transfer to Pipes. Data are shown as the advance in phase between the light-irradiated series and dark controls without addition of ammonium nitrate. Error bars are_+ SD

or without ammonium nitrate. As shown in Fig. 4, ammonium nitrate was ineffective in restoring light sensitivity to discs cultured in pH 6.7 medium when it was added to Pipes buffer after irradiation; its presence was necessary before irradiation. Several other monovalent ionic compounds were examined for their ability to restore light sen-

H. Nakashima and Y. Fujimura: Light, NHr salts, pH and circadian clock in Neurospora

434

sitivity to cultures from pH 6.7 medium. As shown in Fig. 5, ammonium compounds were the most effective; nitrate and other cationic and anionic monovalent ions were not as effective as ammonium compounds. In addition, the divalent compounds CaC12 and MgSO4, present in Fries salts, were examined at the same concentrations as in the usual medium. Neither restored light sensitivity in Pipes buffer (data not shown).

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Fig. 5. Effect of different salts on restoration of light-induced phase advance of Neurospora in Pipes. Mycelial discs of the bd strain were cultured in pH 6.7 medium. They were transferred to 10 mM Pipes (pH 7.6) with different concentrations of salts: v, NHr o, KC1; t~, KNO3; o, N a C I ; . , NaNO3; and irradiated, Phase advance of discs irradiated in the culture medium is also shown on the ordinate (A). Error bars a r e • SD

Change of amino acid composition. Free-aminoacid pools in discs cultured in medium of pH 5.7 and pH 6.7 were examined before and after 3 h treatment with Pipes buffer (Table 1). When the discs were transferred into Pipes buffer, pool sizes of several amino acids changed markedly: aspartic acid, glutamic acid, glutamine and ornithine levels decreased, and lysine and histidine increased. The largest change was the decrease in ammonium content. Differences in amino-acid pools between discs cultured in media o f p H 5.7 and pH 6.7 were clear for alanine, arginine, ornithine and glutamine. The first-named three amino acids were pres-

Table 1, Changes of free amino acid content in mycelial discs of Neurospora treated with 10 mM Pipes (pH 7.6). The discs of the bd strain were harvested before or after treatment with buffer. Amounts of UK-1 through ornithine were calculated using a calibration coefficient which was the average of calibration coefficients for other known amino acids. Results are the mean • SD. Values are nmol/6 plugs Amino acids

pH 5.7 cultures Before treatment

Aspartic acid Serine Glutamic acid Glycine Alan• Valine Isoleucine Leucine Tyrosine Lysine NH 4 Histidine Arginine Phenylalanine UK~I UK-2 Gluta~ne UK-4 UK-5 UK-6 UK-7 Ornithine a UK=unknown

pH 6.7 cultures After treatment

Before treatment

40 _ 2 25 • 3 322-+ 21 6• 1 135 -+ 5 2• 1 6• 1 3+1 2• 1 5_ 1 2,415 • 304 7 _+1 283 • 12 -

20,,1,,4 25 • 8 132 • 23 8• 3 38 _ 7 6• 1 4_+ 1 7-+2 4_ 1 11 • 2 63 ,,1,,14 10 • 2 134 ,,1,,16 3 -+ 1

39 _-t_-2 22 • 1 348 • 22 5 _+1 23 • 2 1 _+1 3 ,,1,,1 4• 2• 1 2• 1 3,621 • 98 4• 1 197 • 17 2• 1

5• 20• 93• 18-+I 1• 7• 1-+1 88,,1,,4

3• 10-+1 62• 7• 5-+1 1• 31•

6• 16-+1 140• 9-+1 2-+1 7-+1 56,,1,,2

After treatment 17 • 1 27 • 1 130,,1,, 6 11 _+1 21 ,,1,,1 6• 1 4• 1 6• 3• 1 20 • 2 118 • 22 13 • 1 189 ,,1,,22 3• 1 3• 10• 72• 5• 3• 7• 4• 28•

H. Nakashima and Y. Fujimura: Light, N H 4 salts, pH and circadian clock in Neurospora

435

Table 2. Free-amino acid content in mycelial discs of Neurospora treated with 10 m M Pipes (pH 7.6) with or without 24 m M NH4NO 3. The discs of the bd strain were cultured in pH 6.7 medium. Culture schedules were as described in Materials and methods. Discs were harvested 3 h after transfer to buffer. Values are nmol/6 plugs Amino acid

Aspartic acid Serine Glutamic acid Glycine Alanine Valine Isoleucine Leucine Tyrosine Lysine NH 4 Histidine Arginine Phenylalanine UK-I UK-2 Glutamine UK-4 UK-5 UK-6 UK-7 Ornithine

Without N H 4 N O 3

With N H a N O 3

- light

+ light

- light

+ light

14 __ 1 25 _+4 143 +- 13 11 _+1 31 _+4 5 __ 1 5 _+1 7 +- 1 2+ 1 15_+2 203 _+ 14 11 +- 1 160 +_ t 0 2 +- 1

16 +- 1 35 + 2 164 +- 5 12 + 1 39 + 3 6 +- 1 5 _+1 7 +- 1 5 +- 1 18_+1 189 +- 30 14 +- 1 173 +_9 2 +- 1

18 _+5 26 +- 18 154 +- 11 9 _+8 20 __ 5 4 __ 1 3 __+1 6+ 1 4 _+i 6_+1 1,770__+ 184 7 _+3 109 _+11 3 +- 1

16 _+2 20 _+5 146 __20 5 _+1 19 _+4 3 _+1 3 _+ 1 6_+ 1 4_+ 1 7_+1 1,858 _+351 7_ 1 108 +_6 -

2+1 10___1 70_+ 7 5+1

2_+1 11_+1 93 __.5 7_+1

3_+1 12_+1 109 _+11 8_+1

3_+1 14_+3 116 ___21 10_+2

1+_1

1+_0

3_+1 3+_1 37 +- 4

5+1 3+_1 42 +- 2

ent in greater amounts in discs cultured at pH 5.7 than in those from pH 6.7. On the other hand, there was more free glutamine in discs from pH 6.7 than 5.7. In discs from pH 6.7, the addition of ammonium nitrate to Pipes resulted in changes in the sizes of several amino acid pools; amounts of alanine, lysine, histidine, arginine and ornithine decreased, and glutamine increased (Table 2). The ammonium content of the discs treated with Pipes buffer containing ammonium nitrate was about nine times higher than that of discs treated with Pipes buffer alone. Irradiation of discs cultured in pH 6.7 medium did not result in any large changes in aminoacid content (Table 2) whether the discs were incubated in Pipes alone or in P i p e s + a m m o n i u m nitrate. Discussion

When mycelial discs were cultured in medium of initial pH higher than 6.7 for 48 h, the effectiveness of light in phase-shifting the clock in Pipes buffer of pH higher than 7.6 was dependent on ammonium. This dependence was not found when the mycelial discs were cultured in the medium of initial pH lower than 6.3 (Fig. 1). The ammonium

1+_1

1+1

5+1 -26 _+ 13

6+1 -18 _+8

content in discs cultured at pH 6.7 was higher than in those cultured at pH 5.7 but both discs lost almost all of their ammonium pools when they were transferred into Pipes buffer (Table 1). This indicates that external ammonium is essential for lightinduced phase shifting only for discs cultured in media of pH higher than 6.7. The role of ammonium in the process of phase shifting by light in discs cultured at pH 6.7 is unknown. One possibility is that ammonium is simply a component of the general ionic environment in the cells, or regulates biochemical reactions occurring in the cells. For example, plasma-membrane ATPase in Neurospora was reported to be stimulated by ammonium ion in vitro (Bowman and Slayman 1977). Addition of ammonium to Pipes resulted in changes in amino-acid pools in discs cultured in a medium of pH 6.7 (Table 2). Similar differences were found between discs cultured in media of pH 5.7 and pH 6.7 (Table 1). Also, the ammonium content of the latter discs was much higher than that of the former discs. Therefore, ammonium may be one of the factors that regulate amino-acid pools in Neurospora cells. This indicates the possibility that ammonium is needed for synthesis of amino acids or other nitrogenous compounds which are necessary for phase shifting by

436

H. Nakashima and Y. Fujimura: Light, NH 4 salts, pH and circadian clock in Neurospora

light. However, an amination-defective mutant also showed dependence on ammonium of lightinduced phase shifting when it was cultured in the medium of pH 6.7 and incubated in Pipes buffer of pH 8.2. Furthermore, irradiation of the discs cultured at pH 6.7 had little effect on amino-acid pools irrespective of the presence of ammonium nitrate in the medium. These facts indicate that amination itself is not the primary step for lightinduced phase shifting. Ammonium could, however, act as a regulator of specific biochemical reaction(s) involved in phase shifting. Phase shifting of discs cultured in pH 5.7 medium does not show any dependence on external ammonium, even though free ammonium in the cells decreased drastically with treatment with Pipes buffer. Sensitivity to inhibitors of plasma membrane ATPase, ethanol, and light also depends on the pH of the medium in which the discs are cultured after the light-to-dark transition (Nakashima 1982). When discs are cultured in pH 6.7 medium, light does not shift the phase in the presence of these inhibitors or ethanol. On the other hand light can fully shift the phase in discs cultured in pH 5.7 medium even in the presence of higher concentrations of these inhibitors. As we reported here, analogous effects of external conditions were found in the dependence on ammonium of light-induced phase shifting. We wish to express our thanks to Professor Y. Oota for his kind guidance throughout this study and to Dr. J. Perlman for her correction of the manuscript.

References Bowman, B.J., Slayman, C.W. (1977) Characterization of plasma membrane adenosine triphosphatase of Neurospora crassa. J. Biol. Chem. 252, 3357-3363 Burgoyne, R.D. (1978) A model for the molecular basis of circadian rhythms involving monovalent ion-mediated translational control. FEBS Lett. 94, 17-19

Dharmananda, S., Feldman, J.F. (1979) Spatial distribution of circadian clock phase in aging cultures of Neurospora crassa. Plant Physiol. 63, 1049-1051 Eskin, A. (1977) Neurophysiological mechanisms involved in photoentrainment of the circadian rhythm from the Aplysia eye. J. Neurophysiol. 8, 273-299 Fries, N. (1948) The nutrition of fungi from the aspect of growth factor requirements. Trans. Br. Mycol. Soc. 30, 118-134

Munoz, V., Brody, S., Butler, W.L. (1974) Photoreceptor pigment for blue light responses in Neurospora crassa. Biochem. Biophys. Res. Commun. 58, 32~327 Munoz, V., Butler, W.L. (1975) Photoreceptor pigment for blue light in Neurospora crassa. Plant Physiol. 55, 421-426 Nakashima, H. (1981) A liquid culture method for the biochemical analysis of the circadian clock of Neurospora crassa. Plant Cell Physiol. 22, 231-238 Nakashima, H. (1982) Effects of membrane ATPase inhibitors on light-induced phase shifting of the circadian clock in Neurospora crassa. Plant Physiol. 69, 619 625 Nakashima, H., Feldman, J.F. (1980) Temperature-sensitivity of light-induced phase shifting of the circadian clock of Neurospora. Photochem. Photobiol. 32, 247-251 Njus, D., Sulzman, F.M., Hastings, J.W. (1974) Membrane model for the circadian clock. Nature (London) 248, 116-120

Perlman, J., Nakashima, H., Feldman, J.F. (1981) Assay and characteristics of circadian rhythmicity in liquid cultures of Neurospora crassa. Plant Physiol. 67, 404-407 Pittendrigh, C.S. (1967) Circadian systems. I. The driving oscillator and its assay in Drosophila pseudoobscura. Proc. Natl. Acad. Sci. USA 58, 1762-1767 Roldan, J.M., Butler, W.L. (1980) Photoactivation of nitrate reductase from Neurospora crassa. Photochem. Photobiol. 32, 375-381 Sargent, M.L., Kaltenborn, S.H. (1972) Effects of medium composition and carbon dioxide on circadian conidiation in Neurospora. Plant Physiol. 50, 171-175 Schmidt, W., Butler, W.L. (1976) Light-induced absorbance changes in cell-free extracts of Neurospora crassa. Photochem. Photobiol. 24, 77 80 Sweeney, B.M. (1974) A physiological model for circadian rhythms derived from the Acetabularia rhythm paradoxes. Int. J. Chronobiol. 2, 25-33

Received 28 February; accepted 2 June, 1982