The circadian conidiation rhythm inNeurospora crassa

The filamentous fungusNeurospora crassais one of the best organisms for analysing the molecular basis of the circadian rhythm observed in asexual spore formation, conidiation. Many clock mutants in which the circadian conidiation rhythm has different characteristics compared to those in the wild-type strain have been isolated since the early 1970s. With the cloning of one of these clock genes,frq, the molecular basis of the circadian clock inNeurosporahas become gradually clearer. Physiological and pharmacological studies have also contributed to our understanding of the physiological basis of the circadian clock inNeurospora. These studies strongly indicate that the circadian clock is based on or is closely related to a network of metabolic processes for cellular activities. Based on these studies, it may be possible to isolate new types of clock mutants which should contribute to a better understanding of the molecular basis of the circadian clock inNeurospora.     

Rhythmic Conidiation in Constant Light in Vivid Mutants of Neurospora crassa

In Neurospora crassa, a circadian rhythm of conidiation (asexual spore formation) can be seen on the surface of agar media. This rhythm has a period of 22 hr in constant darkness (D/D). Under constant illumination (L/L), no rhythm is visible and cultures show constant conidiation. However, here we report that strains with a mutation in the vivid (vvd) gene, previously shown to code for the photoreceptor involved in photo-adaptation, exhibit conidiation rhythms in L/L as well as in D/D. The period of the rhythm of vvd strains ranges between 6 and 21 hr in L/L, depending upon the intensity of the light, the carbon source, and the presence of other mutations. Temperature compensation of the period also depends on light intensity. Dark pulses given in L/L shift the phase of the rhythm. Shifts from L/L to D/D show unexpected after effects; i.e., the short period of a vvd strain in L/L gradually lengthens over 2–3 days in D/D. The rhythm in L/L requires the white collar (wc-1) gene, but not the frequency (frq) gene. FRQ protein shows no rhythm in L/L in a vvd strain. The conidiation rhythm in L/L in vvd is therefore driven by a FRQ-less oscillator (FLO).     

ENTRAINMENT ELICITS PERIOD AFTEREFFECTS IN NEUROSPORA CRASSA

Circadian clocks continue to oscillate in constant conditions with their own period (τ) and entrain to a cyclic environment by adjusting their intrinsic period to that of the zeitgeber. When circadian clocks are released from entrained to constant conditions, the τ of their initial free-run often depends on the nature of the prior zeitgeber. These postentrainment effects on period (τ-aftereffects) have predominantly been reported for animals but, so far, not fungi. The authors therefore investigated τ aftereffects in the classic circadian model system Neurospora crassa. The standard laboratory strain frq⁺, the short-period mutant frq¹, and the long-period mutant frq⁷ were entrained to 11 different photoperiods in a 24-h day (2–22?h) and to zeitgebers with six different T (16–26?h), and then released to constant darkness. τ-Aftereffects in response to different photoperiods correlated weakly with prior photoperiod in frq⁺ and were unsystematic in both period mutant strains. Strength and direction of the τ-aftereffect in zeitgeber cycles with different T depended on their length and on the strain, showing a negative correlation with zeitgeber length in frq⁺ and positive correlations in frq¹ and frq⁷. It has been proposed that τ-aftereffects are based on interactions of oscillators within a cellular network. The present findings in Neurospora, which grows as a syncytium, suggest that τ-aftereffects also exist in circadian systems based on multioscillatory networks organized at the molecular level. (Author correspondence: roenneberg@lmu.de)     

A Direct Comparison between Circadian and Noncircadian Rhythms in Neurospora crassa

A method has been devised for observing both circadian and noncircadian rhythms in a single wild type strain of Neurospora crassa. This method allows a direct comparison of the properties of the two types of rhythm. The circadian rhythm of conidiation always entrains to a light-dark cycle, damps out in constant light, and has a temperature-compensated period length. The noncircadian rhythm of hyphal branching, expressed by the same strain under different environmental conditions, does not entrain to a light-dark cycle, persists in constant light, and its period length is temperature-dependent. These results suggest that the two rhythms have different underlying mechanisms and demonstrate that the differences in the rhythms previously observed in different strains (patch, band, and clock) are not due to genetic differences between these strains but rather are inherent properties of the rhythms themselves.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

Circadian Nature of a Rhythm Expressed by an Invertaseless Strain of Neurospora crassa

A new strain of Neurospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth tube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is about 24 hours (22.7 hours at 25 degrees ); B) the period is relatively temperature-independent (Q(10) is between 0.95 and 1.21 for temperature range of 18 to 35 degrees ); C) the rhythm persists in continuous darkness at constant temperature for a minimum of 14 days without damping out; and D) the phase of the rhythm can be shifted by a single brief exposure to light. The sensitivity of this strain to light has been demonstrated further by the entrainment of the rhythm to a period of 24.0 hours using a suitable light-dark regime, and by the inhibition by light of the appearance of a rhythm; i.e., continuous conidiation occurs when the strain is subjected to continuous light. The new strain is compared to 2 other strains of Neurospora which also express a rhythm, patch and clock.     

Circadian periodicity in cockroaches altered by high frequency light-dark cycles

Summary The circadian period of the freerunning activity rhythm in the cockroach is systematically altered by high frequency light-dark cycles (HF-LD) according to the ratio of light to dark within each cycle. With a standard 10 min cycle time, brief (e.g., 0.5 min) exposure to light each cycle causes the free-running period to shorten significantly in comparison to its steady-state value in constant darkness. As the ratio of light to dark in HF-LD is increased, the period of the rhythm is progressively lengthened. These findings are discussed in terms of the general proposition that light, applied throughout the circadian cycle, predictably modifies periodicity according to the asymmetrical shape of the circadian phase response curve.Abbreviations LD light-dark cycles in which cycle length is in hours - HF-LD light-dark cycles in which cycle length is in min; period of the activity rhythm; change in period of the activity rhythm - PRC phase response curve - LL constant light     

Complementation Analysis of Linked Circadian Clock Mutants of NEUROSPORA CRASSA

A fourth mutant of Neurospora crassa, designated frq-4, has been isolated in which the period length of the circadian conidiation rhythm is shortened to 19.3 ± 0.3 hours. This mutant is tightly linked to the three previously isolated frq mutants, and all four map to the right arm of linkage group VII about 10 map units from the centromere. Complementation tests suggest, but do not prove, that all four mutations are allelic, since each of the four mutants is co-dominant with the frq⁺ allele—i.e., heterokaryons have period lengths intermediate between the mutant and wild-type—and since heterokaryons between pairs of mutants also have period lengths intermediate between those of the two mutants.     

The frq Locus in NEUROSPORA CRASSA: A Key Element in Circadian Clock Organization

Four new circadian clock mutants of Neurospora crassa have been isolated that alter the period length of the circadian conidiation rhythm. Three of these are at the frq locus on linkage group VIIR, where four other clock mutants are located. In contrast to wild type, which has a period length of 21.6 hr, frq-6 has a period length of 19 hr, while frq-7 and frq-8 have period lengths of 29 hr and represent the largest effects of any single gene mutants on circadian periodicity. Thus, seven mutants have now been isolated that map to the frq locus, with period lengths ranging from 16.5 to 29 hr, and each mutant alters clock periodicity by an integral multiple of 2.5 hr. In addition, all frq mutants show incomplete dominance in heterokaryons. The large percentage of clock mutants that map to this locus, coupled with their unique properties, suggests that the frq locus plays an important role in clock organization.—The fourth mutant, designated chrono (chr), has a period length of 23.5 hr, shows incomplete dominance and is unlinked to either of the previously identified clock loci, frq or prd (formerly called frq-5). Double mutants between various combinations of clock mutants show additive effects and indicate no significant gene interaction among mutants at these three loci.     

Mutation of the gene for the second-largest subunit of RNA polymerase I prolongs the period length of the circadian conidiation rhythm in Neurospora crassa

The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation rhythm. Below 22° C, the un-18 mutation did not affect the period length, but at temperatures between 22° C and 32° C, the period length of the un-18 mutant was ∼2 h longer than that of the wild-type strain. The un-18 ⁺ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis, lengthens the period of the circadian conidiation rhythm in Neurospora. Received: 17 October 1997 / Accepted: 26 April 1998     

CIRCADIAN GROWTH RHYTHM IN JUVENILE SPOROPHYTES OF LAMINARIALES (PHAEOPHYTA)1

A circadian rhythm in growth was detected by computer-aided image analysis in 3–4-cm-long, juvenile sporophytes of the kelp species Pterygophora California Rupr. and in seven Laminaria spp. In P. californica, the free-running rhythm occurred in continuous white fluorescent light, had a period of 26 h at 10°or 15°C, and persisted for at least 2 weeks in white or blue light. The rhythm became insignificant in continuous green or red light after 3 cycles. Synchronization by white light-dark regimes, e.g. by 16 h light per day, resulted in an entrained period of 24 h and in a shift of the circadian growth minimum into the middle of the light phase. A morning growth peak represented the decreasing portion of the circadian growth curve, and an evening peak the increasing portion. The circadian growth peak was not visible during the dark phase, because growth rate decreased immediately after the onset of darkness. At night, some growth still occurred at 16 or 12 h light per day, whereas growth stopped completely at 8 h light per day, as in continuous darkness. During 11 days of darkness, the thallus area became reduced by 3.5%, but growth rate recovered in subsequent light–dark cycles, and the circadian growth rhythm reappeared in subsequent continuous light.     

On the Role of Energy Metabolism in Neurospora Circadian Clock Function

Neurospora crassa (bdA) mycelia were kept in liquid culture. Without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. Maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at CT 0-6 hr. Pulses of metabolic inhibitors such as vanadate (Na₃Vo₄), molybdate (Na₂MoO₄: 2 H₂O), N-ethylmaleimide (NEM), azide (NaN₃), cyanide (NaCN) and oligomycin phase shift the circadian conidiation rhythm of Neurospora crassa. Maximal advance phase shifts are observed at about CT 6 with all inhibitors.

Pulses of N,N'dicyclohexylcarbodiimide (DCCD) and light phase shift the conidiation rhythm following a phase response curve different from those of the other agents (maximal advance at about CT 18-24). The phase shifts with DCCD and light are significantly larger in the wild type compared to the mitochrondrial mutant poky. Such differences are not found in PRCs of the protein synthesis inhibitor cycloheximide.

[³¹P] NMR spectra of wild type Neurospora crassa and the clock mutants frq 1 and frq 7 which differ in their circadian period lengths did not reveal differences in the concentrations of adenine nucleotides, pyridine nucleotides or sugar phosphates. Starvation causes drastic changes of the levels of adenine nucleotides, phosphate and mobile polyphosphate without effecting phase or period length of the circadian rhythm.     

Mutation of the gene for the second-largest subunit of RNA polymerase I prolongs the period length of the circadian conidiation rhythm in Neurospora crassa

The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation rhythm. Below 22°?C, the un-18 mutation did not affect the period length, but at temperatures between 22°?C and 32°?C, the period length of the un-18 mutant was ～2 h longer than that of the wild-type strain. The un-18 ⁺ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis, lengthens the period of the circadian conidiation rhythm in Neurospora.     

PRESENCE OF CIRCADIAN RHYTHMS IN THE LOCOMOTOR ACTIVITY OF A CAVE-DWELLING MILLIPEDE GLYPHIULUS CAVERNICOLUS SULU (CAMBALIDAE,SPIROSTREPTIDA)

The locomotor activity of the millipede Glyphiulus cavernicolus (Spirostreptida), which occupies the deeper recesses of a cave, was monitored in light-dark (LD) cycles (12h light and 12h darkness), constant darkness (DD), and constant light (LL) conditions. These millipedes live inside the cave and are apparently never exposed to any periodic factors of the environment such as light-dark, temperature, and humidity cycles. The activity of a considerable fraction of these millipedes was found to show circadian rhythm, which entrained to a 12:12 LD cycle with maximum activity during the dark phase of the LD cycle. Under constant darkness (DD), 56.5% of the millipedes (n = 23) showed circadian rhythms, with average free-running period of 25.7h ± 3.3h (mean ± SD, range 22.3h to 35.0h). The remaining 43.5% of the millipedes, however, did not show any clear-cut rhythm. Under DD conditions following an exposure to LD cycles, 66.7% (n = 9) showed faint circadian rhythm, with average free-running period of 24.0h ± 0.8h (mean ± SD, range 22.9h to 25.2h). Under constant light (LL) conditions, only 2 millipedes of 11 showed free-running rhythms, with average period length of 33.3h ± 1.3h. The results suggest that these cave-dwelling millipedes still possess the capacity to measure time and respond to light and dark situations. (Chronobiology International, 17(6), 757–765, 2000)     

Locomotor activity rhythm in two wrasses,Halichoeres tenuispinnis andPteragogus flagellifera,under various light conditions

The locomotor activity rhythms were examined by using an actograph with infra-red photo-electric switches for two species of wrasses, (Halichoeres tenuispinnis andPteragogus flagellifera) under various light conditions. InH. tenuispinnis, the locomotor activity of almost all fish under light-dark cycle regimen (LD12:12; 06:00–18:00 light, 18:00–06:00 dark) commenced somewhat earlier than the beginning of light period and continued till somewhat earlier than the beginning of the dark period. This species clearly showed free-running activity rhythms under both constant illumination (LL) and constant darkness (DD). Therefore,H. tenuispinnis appeared to have a circadian rhythm. The length of the circadian period ranged from 23 hr. 30 min. to 23 hr. 44 min. under LL, and was from 23 hr. 39 min. to 24 hr. 18 min. under DD. On the other hand, the locomotor activity ofP. flagellifera occurred mostly in the light period under LD 12:12. The activity of this species continued through LL, but was greatly suppressed in DD, so that none of the fish had any activity rhythm in both constant conditions. It was known from field observations thatH. tenuispinnis burrowed and lay in sandy bottoms, whileP. flagellifera hid and rested in bases of seagrasses and shallow crevices of rocks during the night. In the present two wrasses, it seemed that the above-mentioned difference of noctural behavior was closely related to the intensity of the endogenous factor in the activity rhythm.     

Photoperiod modifies daily maps of light and dark sensitivity for N-acetyltransferase activity in pineal glands of 3-week oldGallus domesticus

Summary N-acetyltransferase (NAT) activity in pineal glands exhibits a circadian rhythm with peak activity occurring in the dark-time. We previously showed that inGallus domesticus chicks pretreated with LD12:12, NAT activity was increased by dark exposure (peak dark sensitivity occurred during the expected dark-time) or decreased by light at night (peak light sensitivity occurred early in the night during the time of dark sensitivity). In this study we mapped dark sensitivity vs time (for NAT activity increase in response to 2 h dark pulses), and light sensitivity vs time (for NAT activity decrease in response to 10 min or 30 min light pulses) over a cycle for 3-week old chicks,Gallus domesticus, pretreated with long (LD16:8) or short photoperiod (LD8:16). Sensitivity to light was increased in the second 8 h after L/D by LD8:16. Sensitivity to dark was increased in the first 8 h after L/D by LD16:8.Abbreviations LD16:8 a light-dark cycle consisting of 16 h of light alternating with 8 h of dark - LD8:16 a light-dark cycle consisting of 8 h of light alternating with 16 h of dark - DD constant dark - LL constant light - L/D lights-off - D/L lights-on - NAT pineal serotonin N-acetyltransferase - NAT activity is given in nmoles/pineal gland/h - chick used here to denote a young bird of either sex of the speciesGallus domesticus from hatching to three weeks of age     

Light- and temperature-entrained circadian regulation of activity and mRNA accumulation of 1-aminocyclopropane-1-carboxylic acid oxidase in Stellaria longipes

Stem and leaf tissues of Stellaria longipes Goldie (prairie ecotype) exhibit circadian rhythmicity in the activity and mRNA abundance for 1-aminocyclopropane-1-carboxylic acid oxidase (EC 1.4.3). The steady-state mRNA levels and enzymatic activity levels fluctuated with a period of approximately 24 h and reached their maxima by the middle of the light phase and minima by the middle of the dark phase. The oscillations showed damping under constant light, constant dark and constant temperature conditions, indicating that the rhythm is entrained by an external signal. The results indicate that light/dark cycles have greater entraining effects than temperature cycles. A 15-min red light pulse, but not a blue light pulse, could reset rhythm in continuous darkness, suggesting the possible role of a red-light signal transduction pathway in the circadian regulation of 1-aminocyclopropane-1-carboxylic acid oxidase.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DD continuous dark - LD light-dark - LL continuous light - ZT Zeitgeber time (start of light period for circadian entrainment) This study was supported by operating grants to C.C.C., and D.M.R. from the Natural Sciences and Engineering Research Council of Canada.The authors gratefully acknowledge the award of a Bettina Bahlsen memorial Graduate Scholarship by University of Calgary to A.K. We are grateful to Dr. M.M. Moloney for allowing the use of his laboratory facilities.     

Circadian rhythms in Kalanchoë: effects of irradiance and temperature on gas exchange and carbon metabolism

Gas exchange in K. blossfeldiana shows a circadian rhythm in net CO₂ uptake and transpiration when measured under low and medium irradiances. The period length varies between 21.4 h at 60 W m^-2 and 24.0 h at 10 W m^-2. In bright light (80 W m^-2) or darkness there are no rhythms. High leaf temperatures result in a fast dampening of the CO₂-uptake rhythm at moderate irradiances, but low leaf temperatures can not overcome the dampening in bright light. The rhythm in CO₂ uptake is accompanied by a less pronounced and more rapidly damped rhythm in transpiration and by oscillations in malate levels with the amplitude being highly reduced. The oscillations in starch content, usually observed to oscillate inversely to the acidification in light-dark cycles, disappear after the first cycle in continuous light. The balance between starch and malate levels depends in continuous light on the irradiance applied. Leaves show high malate and low starch content at low irradiance and high starch and low malate in bright light. During the first 12 h in continuous light replacing the usual dark period, malate synthesis decreases with the increasing irradiance. Up to 50 W m^-2 starch content decreases; at higher irradiances it increases above the values usually measured at the end of the light period of the 12:12 h light-dark cycle.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - PEP phosphoenolpyruvate