Circadian period lengths of lipid synthesis mutants (cel,chol-1) of Neurospora show defective temperature,but intact pH-compensation

The influence of extracellular pH on the circadian sporulation rhythm of Neurospora crassa has been investigated for the mutants chol-1 and cel. Both mutants have a defect in the lipid synthesis pathway and require either choline or palmitate, respectively, as supplements for normal growth. The chol-1 and cel mutants also show an impaired temperature-compensation when growing on minimal medium. We investigated the possible correlation between loss of temperature- and pH-compensation in cel and chol-1 similar to the correlation found earlier for the frq⁷ mutant. Our results show that the cel and the chol-1 mutants, although defective in temperature-compensation have an intact pH-compensation of their circadian rhythms. At present, the products of the frq-locus are the only components of the clock that affect the sporulation rhythm of Neurospora both through pH- and temperature-compensation.     

Temperaure Compensation and Memberane Composition in Neurospora Crassa

The link between temperature compensation of the circadian rhythm and temperature-induced adjustment of membrane composition in Neurospora crassa is briefly reviewed. In common with most organisms, Neurospora responds to changes in growth temperature by adjusting its lipid composition, primarily by increasing the degree of unsaturation of its fatty acids at low temperature. This may result in maintenance of either membrane fluidity or phase transition behavior over a range of temperatures. In Neurospora, there are three mutations (frq, eel, and chol-1) that affect temperature compensation of the circadian rhythm; cel and chol-1 are defective in lipid synthesis, and frq interacts with the other two in double-mutant strains. This suggests that lipid metabolism may play a role in temperature compensation of the rhythm, and that the FRQ gene product may also be involved in membrane function, either in regulating lipid composition or as a sensor responding to changes in lipid composition. (Chronobiology International, 14(5), 445–454, 1997)     

pH HOMEOSTASIS OF THE CIRCADIAN SPORULATION RHYTHM IN CLOCK MUTANTS OF NEUROSPORA CRASSA

The influence of environmental (extracellular) pH on the sporulation rhythm in Neurospora crassa was investigated for wild-type (frq⁺) and the mutants chr, frq¹, frq⁷, and frq⁸. In all mutants, including wild type, the growth rate was found to be influenced strongly by extracellular pH in the range 4–9. On the other hand, for the same pH range, the period length of the sporulation rhythm is little influenced in wild type, chr, and frq¹. A loss of pH homeostasis of the period, however, was observed in the mutants frq⁷ and frq⁸, which also are known to have lost temperature compensation. Concerning the influence of extracellular pH on growth rates, a clear correspondence between growth rates and the concentration of available H₂PO₄^? ion has been found, indicating that the uptake of H₂PO₄^? may be a limiting factor for growth under our experimental conditions. The loss of pH compensation in the frq⁷ and frq⁸ mutants may be related to less easily degradable FRQ^7,8 proteins when compared with wild-type FRQ. Results from recent model considerations and experimental results predict that, with increasing extra-and intracellular pH, the FRQ⁷ protein degradation increases and should lead to shorter period lengths. (Chronobiology International, 17(6), 733–750, 2000)     

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq ⁺ and clock mutantsfrq ⁺,frq ³,frq ⁷,frq ⁹ andfrq ¹¹. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq ⁺,frq ³ andfrq ⁷ strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq ¹¹ strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq ¹ strain in all light-dark cycles examined and in thefrq ⁺ andfrq ³ strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq ⁷ strain was dependent on the light to dark transition when the light period was shorter than 10 hr.     

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.     

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.     

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)     

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.     

Circadian rhythms in Neurospora crassa: Dynamics of the clock component frequency visualized using a fluorescent reporter

The frequency (frq) gene of Neurospora crassa has long been considered essential to the function of this organism’s circadian rhythm. Increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. To address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins such as FRQ. However, due to the difficulty of studying the expression of circadian rhythm genes in aerobic N. crassa cultures, little is known about the dynamics of this gene under physiologically realistic conditions. To address these issues we report a fluorescent fusion to the frq gene using a codon optimized version of the mCherry gene. To trace the expression and accumulation of FRQ–mCherryNC (FRQ–mCh) during the circadian rhythm, growing vegetative hyphae were scanned every hour under confocal microscopy (100×). Fluorescence of FRQ–mCh was detected only at the growing edge of the colony, and located in the cytoplasm and nuclei of vegetative hyphae for a distance of approximately 150–200 μm from the apices of leading hyphae. When driven by the frq promoter, apparently there was also a second FRQ entrance into the nucleus during the circadian cycle; however the second entrance had a lower accumulation level than the first entrance. Thus this fluorescent fusion protein has proven useful in tracking the spatial dynamics of the frq protein and has indicated that the dynamics of the FRQ protein’s nuclear trafficking may be more complex than previously realized.     

Genetics of Circadian Clocks

The isolation of circadian clock mutants in Neurospora crassa and Drosophila melanogaster have identified numerous genes whose function is necessary for the normal operation of the circadian clock. In Neurospora many of these mutants map to a single locus called frq, whose properties suggest that its gene product is intimately involved in clock function. In Drosophila mutations at the per locus also suggest a significant role for the product of this gene in the insect clock mechanism. The per gene has been cloned and its gene product identified as a proteoglycan, most likely a membrane protein involved in affecting the ionic or electrical properties of cells in which it is located. Future progress in elucidating the mechanisms of circadian clocks are likely to come from continued analysis of clock mutants, both at the genetic and molecular levels.     

Utilization of betaine aldehyde by choline (chol-) mutants of Neurospora crassa

Summary The addition of betaine aldehyde to solid and liquid minimal media supported the growth of two choline-requiring mutants of Neurospora crassa (chol-1 and chol-2). The results were interpreted as evidence for the occurrence of the enzyme choline dehydrogenase in Neurospora crassa.     

Attenuation of the Petal Movement Rhythm in Kalanchoë with Light Pulses

The circadian petal movement rhythm of Kalanchoë flowers has been studied. The amplitude of the rhythm can be drastically reduced by an appropriate stimulus of a light pulse. It has also been shown that it is possible to stop the rhythm permanently by administering a single light pulse to the flowers. This is interpreted to indicate that the light pulse has sent the circadian rhythm into a stable state of singularity. The conditions which attenuate the rhythm have been investigated both theoretically (on the basis of a previously published model for circadian rhythms) and experimentally. 120 min red light of 230 μW · cm^?2, starting briefly before the second petal closure about 30 h after transfer to constant safe light conditions is optimal in inducing rhythm-damping. Damping requires the same duration when the light is given at the corresponding phase during the third or fourth cycle of the rhythm. However, in the first cycle 240 min red light of 230 μW · cm^?2 is required to get optimal damping of the rhythm. Conditions to achieve damping for other irradiances are investigated. Individual recordings are presented which show the behaviour of the rhythm when perturbed by light stimuli close to its singularity.     

Circadian rhythm mutants of the prokaryoticSynechococcus RF-1

Synechococcus RF-1 established circadian rhythms in nitrogen fixation and leucine uptake when growing in a diurnal light/dark regimen. The rhythms persisted in subsequent uniform light/light conditions. In order to analyze the circadian rhythm at the genetic level, mutants were induced by N-methyl-N-nitro-N-nitrosoguanidine and then isolated by procedures with the circadian nitrogen-fixing rhythm as a selecton marker. Characterization of the mutants with respect to the circadian rhythm indicated that some mutants were abnormal only in the nitrogen-fixing rhythm, while some simultaneously lost the ability to establish the nitrogen-fixing and leucine-uptake rhythms. The physiological properties of the circadian rhythm were compared. The genetic potential of the mutants that were abnormal in both rhythms is emphasized.     

Temperature Compensation of Circadian Period Length in Clock Mutants of Neurospora crassa

Temperature compensation of circadian period length in 12 clock mutants of Neurospora crassa has been examined at temperatures between 16 and 34°C. In the wild-type strain, below 30°C (the “breakpoint” temperature), the clock is well-compensated (Q₁₀ = 1), while above 30°C, the clock is less well-compensated (Q₁₀ = 1.3). For mutants at the frq locus, mutations that shorten the circadian period length (frq-1, frq-2, frq-4, and frq-6) do not alter this temperature compensation response. In long period frq mutants (frq-3, frq-7, frq-8), however, the breakpoint temperature is lowered, and the longer the period length of the mutants the lower the breakpoint temperature. Long period mutants at other loci exhibit other types of alterations in temperature compensation—e.g. chr is well-compensated even above 30°C, while prd-3 has a Q₁₀ significantly less than 1 below 30°C. Prd-4, a short period mutant, has several breakpoint temperatures. Among four double mutants examined, the only unusual interaction between the individual mutations occurred with chr prd, which had an unusually low Q₁₀ value of 0.86 below 27°C. There was no correlation between circadian period length and growth rate. These strains should be useful tools to test models for the temperature compensation mechanism.