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Copyright � 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.097808

Rhythmic Conidiation in Constant Light in Vivid Mutantsof Neurospora crassa

Kevin Schneider,* Sabrina Perrino,* Kim Oelhafen,* Sanshu Li,† Artiom Zatsepin,†

Patricia Lakin-Thomas† and Stuart Brody*,1

*Division of Biological Sciences, University of California, San Diego, California 92093-0116 and †Department ofBiology, York University, Toronto, Ontario M3J 1P3, Canada

Manuscript received December 3, 2008Accepted for publication January 2, 2009

ABSTRACT

In Neurospora crassa, a circadian rhythm of conidiation (asexual spore formation) can be seen on thesurface of agar media. This rhythm has a period of 22 hr in constant darkness (D/D). Under constantillumination (L/L), no rhythm is visible and cultures show constant conidiation. However, here we reportthat strains with a mutation in the vivid (vvd) gene, previously shown to code for the photoreceptor involvedin photo-adaptation, exhibit conidiation rhythms in L/L as well as in D/D. The period of the rhythm of vvdstrains ranges between 6 and 21 hr in L/L, depending upon the intensity of the light, the carbon source, andthe 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 aftereffects; i.e., the short period of a vvd strain in L/L gradually lengthens over 2–3 days in D/D. The rhythm inL/L requires the white collar (wc-1) gene, but not the frequency ( frq) gene. FRQ protein shows no rhythm inL/L in a vvd strain. The conidiation rhythm in L/L in vvd is therefore driven by a FRQ-less oscillator (FLO).

CIRCADIAN rhythms are biological rhythms witha period of about a day (Sweeney 1976). These

rhythms and their regulation play important roles innearly all organisms and control numerous biologicalprocesses ranging from sexual and asexual reproduc-tion to complex behavioral changes. A typical circadianclock possesses several characteristics, such as having aperiod close to that of the geophysical day, is endog-enous and self-sustaining, and is entrained by envi-ronmental cues such as light and temperature (Edmunds

1988; Pittendrigh 1993).Neurospora crassa, a model organism for the study of

circadian rhythms (Lakin-Thomas et al. 1990; Dunlap

and Loros 2006; Liu and Bell-Pedersen 2006), ex-presses its rhythm by alternating areas of asexual sporeformation (conidiation) with areas of thinner hyphalgrowth without spore formation when growing on solidagar medium. Areas of spore formation are known as‘‘bands’’ and thinner areas without spore formation areknown as ‘‘interbands.’’ The period and phase of aculture can be easily determined by the position of thebands on the agar surface and can be shifted andentrained by pulses or cycles of light or temperature(Sargent and Briggs 1967; Francis and Sargent 1979).

The frequency ( frq) and white-collar (wc-1, wc-2) genesplay important roles in the expression of the circadianrhythm. Point mutations in the frq gene can increase ordecrease the period of the conidiation rhythm (Feldman

and Hoyle 1973; Aronson et al. 1994). Null mutationsat the frq locus, either a point mutation (Loros andFeldman 1986) or a complete deletion of the codingsequence (Aronson et al. 1994), show a residual rhythmthat lacks temperature compensation. Wc-1 and wc-2mutants were originally isolated as ‘‘blind’’ mutants anddo not display conidiation banding under ordinarylaboratory conditions (Degli-Innocenti and Russo

1984; Crosthwaite et al. 1997). It is generally heldthat the conidiation cycle in Neurospora is partially theproduct of a negative molecular feedback loop, the frq/wc oscillator (FWC), involving the rhythmic levels andactivities of products of the frq, wc-1, and wc-2 genes.There are also positive feedback mechanisms involvingthe influence of frq expression on wc-1 and wc-2 activityand an important role played by post-translationalmodifications and protein degradation. Details may befound in recent reviews (Lakin-Thomas and Brody

2004; Liu 2005; Brunner and Schafmeier 2006;Dunlap 2006; Liu and Bell-Pedersen 2006).

Oscillators other than the FWC oscillator, sometimesreferred to as frq-less oscillators, or FLOs (Iwasaki andDunlap 2000), have been proposed on the basis of theobservation of residual rhythmicity in the frq null strainsfrq9 (Loros and Feldman 1986) and frq10 (Aronson et al.

1Corresponding author: Division of Biological Sciences (0116), Universityof California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0116.E-mail: [email protected]

Genetics 181: 917–931 (March 2009)

1994) in wc mutants (Dragovic et al. 2002) and in the csn-2 mutants defective in FRQ degradation (He et al. 2005).Strains carrying null mutations of frq or wc-1 showoscillator-like behavior when entrained to cycles oftemperature pulses (Merrow et al. 1999; Lakin-Thomas

2006a). Additionally, rhythms have been found in the frq10

strain in the levels of nitrate reductase (Christensen et al.2004), the levels of the neutral lipid diacylglycerol(Ramsdale and Lakin-Thomas 2000) and in the mRNAlevels of some clock-controlled genes (ccgs) (Correa et al.2003). The existence of a FLO or FLOs is furthersuggested by the finding that ‘‘clock null’’ mutants, suchas deletions of frq, wc-1, or wc-2, that may lack conidiationrhythmicity under standard growth conditions will exhibitrhythmicity when grown on media enriched with farnesolor geraniol (Granshaw et al. 2003; Lombardi et al. 2007)or when combined with certain mutations affecting lipidbiosynthesis, such as cel or chol-1 (Lakin-Thomas andBrody 2000). As these rhythms are seen in strains carryingdeletions of particular FWC genes, with frq10 and Dwc-2expressing very low levels of WC-1 and FRQ, respectively(Lee et al. 2000; Collett et al. 2002), the observation ofconidiation rhythms in these strains suggests the presenceof an oscillator (or oscillators) that is not dependent onFWC whose rhythmic output can be expressed as con-idiation bands upon addition of farnesol or geraniol or byalteration in lipid synthesis. Discussion of the nature ofthe FLO or FLOs can be found in several recent pub-lications (Bell-Pedersen et al. 2005; de Paula et al. 2006;Lakin-Thomas 2006b).

Photoresponses of the Neurospora circadian systemhave been known for some time (Sargent and Briggs

1967). In addition to the effects on the circadian rhythm,light has effects on development, on conidiation, andon other processes such as carotenogenesis. Thesestudies have been summarized recently in several re-views (Liu et al. 2003; Dunlap and Loros 2005;Corrochano 2007). All known Neurospora light re-sponses are sensitive to blue light, not red (Froehlich

et al. 2005), and several blue-light receptors are knownin Neurospora. The key blue-light photoreceptor is theWC-1 protein, which also functions in the FWC feedbackloop. The Vivid (VVD) protein has recently been shownto be a photoreceptor as well (Schwerdtfeger andLinden 2003). Both WC-1 and VVD contain a conservedlight-oxygen-voltage (LOV) domain. If this domain isremoved from WC-1, many of the light-regulatedresponses of WC-1 are lost (Cheng et al. 2003). If theLOV domain of WC-1 is replaced with that from VVD,the LOV domain of VVD partially restores the functionof the WC-1 protein (Cheng et al. 2003). The crystalstructure of the VVD protein has been determined andthe mechanism of light-induced conformational changeand dimerization have been reported (Zoltowski et al.2007; Zoltowski and Crane 2008). The vvd transcriptis not ordinarily found in cultures grown in the dark, butthe vvd gene is transcribed and the VVD protein is

produced following exposure to light (Heintzen et al.2001). These observations support the involvement ofVVD in light-induced cellular processes.

Two null mutants at the vvd locus are known: theP4246 allele (vvdP) is due to a translocation event in themiddle of the gene leading to a frameshift while the SS-692 allele (vvdSS) is due to a 10-bp insertion resulting ina frameshift and early termination at amino acid 146(Heintzen et al. 2001). These loss-of-function mutantsthat lack the VVD protein appear to be deficient in theirgeneral adaptation to light, thus affecting several blue-light-regulated genes. In a vvd mutant, rapid light-induced transcripts, such as con-10, con-6, wc-1, frq, andvvd itself, are induced to a higher level and down-regulate more slowly than in wild type (Heintzen et al.2001; Shrode et al. 2001). The most obvious phenotypicdifference found in the vvd mutants, and the origin ofthe name, is the presence in mycelia of bright orangecolor when grown in constant light, which is caused byan increased accumulation of carotenoids. The pro-duction of carotenoids is carried out by the products ofthe al-1, -2, and -3 genes, which are activated when theWC-1 protein is transiently phosphorylated in responseto an increase in light intensity. In the vvd mutant, theWC-1 protein is constitutively, rather than transiently,phosphorylated (Heintzen et al. 2001) and thereforethe al-1 transcript is not downregulated normally(Schwerdtfeger and Linden 2003), resulting in over-production of carotenoids.

Mutations in vvd have very little effect on the cir-cadian rhythm of conidiation in constant darkness (DD)(Heintzen et al. 2001; Shrode et al. 2001), consistentwith the observation that VVD protein is not expressedin DD. The vvd mutation delays the phase of the firstband in DD and this effect is more prominent at lowertemperatures; VVD may normally act to compensate fortemperature effects on the rate of conidiation (Hunt

et al. 2007). Vvd mutants show an exaggerated phase-resetting response to light pulses, consistent with thefunction of wild-type VVD in downregulating rapidresponses to light (Heintzen et al. 2001). In wild-typeNeurospora, conidiation rhythms are not observed inconstant light (LL), and several groups have reportedthe same for vvd mutants (Shrode et al. 2001; Elvin

et al. 2005). In this study, we have found conditions thatallow expression of the conidiation rhythm in the vvdmutant in LL, and we have characterized the propertiesof that rhythm. Moreover, we have found that rhythmic-ity in LL is not dependent on the expression of frq.

MATERIALS AND METHODS

Strains: The bd or csp-1; bd strains were used as the vvd1

control strains. The bd mutation has been characterized as amutation that permits conidiation even in the presence ofhigh CO2 concentrations in closed cultures (Perkins et al.

918 K. Schneider et al.

2001) and has recently been found to be a mutation in the ras-1gene (Belden et al. 2007). The csp-1 mutation shows improperseparation of conidia from hyphae (Perkins et al. 2001),preventing self-contamination of cultures during handling.The csp-1 gene has been found to code for a light-inducibletranscription factor (Lambreghts et al. 2009).

Two alleles at the vvd locus were used: P4246 (abbreviatedas vvdP) and SS-692 (abbreviated as vvdSS). The bd vvdP strainwas obtained from D. Bell-Pedersen. The vvdSS strain wasobtained from the Fungal Genetics Stock Center (FGSC)(McCluskey 2003). Progeny from crosses were identified asvvd by the bright orange color in LL or by using the closelylinked pan-2 mutation (see below). The csp-1 mutation wasintroduced into the vvd strains by crossing to csp-1; bd.

The triple mutant bd; vvdP pan-2 was constructed by crossingbd; vvdP to bd; pan-2. Of 78 progeny, 38 were judged to be vvdand 4 were found to be pan-2. One isolate was selected forfurther characterization.

The strain al-1; bd; vvdP pan-2 was constructed by crossing bd;vvdP pan-2 to bd; al-1. Of 32 total progeny, 14 were identified asal-1 on the basis of their pale color, and 5 of the 14 were foundto require pantothenate. The five al-1; bd; pan-2 strains wereassumed to be vvdP as well.

The strain bd; vvdP pan-2; frq10 was constructed by crossing bd;vvdP pan-2 to bd; frq10. The bd; frq10 strain was obtained from J.Dunlap. Of 42 isolates, 22 were hygromycin sensitive and 20were hygromycin resistant, indicating the presence of the frqknockout. Of the 20 resistant strains, 12 were pan-21 and 8were pan-2. Two pan-2 isolates were further characterized.

The wc knockout strains were constructed by crossing bd;vvdP pan-2 to bd strains carrying either the wc-1 or wc-2knockouts. The wc-2KO strain was received from J. Dunlap.The his-3; bd; wc-1KO strain was obtained from the FGSC.Progeny were characterized for pan-2 and for hygromycinresistance to identify the wc knockouts. Two isolates from eachcross were selected for further study and were backcrossed toverify the genotypes.

The bd; vvdP pan-2; frq7 strain was constructed by crossing bd;vvdP pan-2 to a bd; frq7 strain. A total of 58 progeny werecharacterized and 28 pan-2 isolates were screened for period inDD. Several long-period (�30 hr) strains that were also pan-2were identified and two isolates (#3 and #22) were furthercharacterized.

Crosses were carried out using standard genetics proce-dures for Neurospora. Details of methods can be found in thesupplemental methods of Lombardi et al. (2007). Strains notdescribed above were obtained from the FGSC or have beendescribed previously.

Growth conditions and media: The general growth con-ditions were as previously described (Granshaw et al. 2003).Cultures were grown either in 150-mm disposable petri dishes(plates) or in 27-cm-long (1.1-cm internal diameter) glassgrowth tubes (race tubes). All growth media contained 2%agar, 2% Vogel’s 503 minimal medium (Vogel 1956), acarbon source, and a trace of food coloring. The carbonsources were as follows: maltose medium (0.5%), glucosemedium (0.1% glucose plus 0.17% arginine), acetate medium[1.2% acetate plus 0.05% casamino acids (Feldman andHoyle 1973)], sugarless medium (0.5% arginine plus 1 mg/mlbiotin), and maltose/arginine medium (0.5% maltose plus 0.1%arginine). Auxotrophic strains were supplemented with 0.01mg/ml pantothenate or 0.5 mg/ml histidine, as appropriate.After sterilization for 20 min, the media were dispensed intoplates (25 ml/plate) or race tubes (9 ml/tube) and allowed tocool for 1 day before inoculation. Cultures were inoculatedwith conidia taken from slant cultures. After inoculation,cultures were incubated either immediately under various LLconditions or overnight in 5.4 mmol/m2/sec light before

transfer to DD. Illumination was provided by cool-whitefluorescent bulbs (Phillips Alto 17 w, F17T8/TL735). Lightintensity for most experiments was measured with a Westonlight meter or Fisher Scientific light meter in lux andconverted to micromoles/square meter/second using theconversion factors found in Thimijan and Heins (1983). Forthe experiments in Figure 8, light intensity was measureddirectly using a LiCor PAR quantum sensor.

Period and growth rate calculations: The growth fronts ofcultures were marked once a day under red safelight (forcultures in DD) or under lighting conditions similar to growthconditions (for cultures in LL). After growth had finished, thecenters or peaks of bands were marked on the cultures andthe growth marks and band marks were transferred to paper.The paper record was scanned and the growth rates andperiods were calculated using software developed by FredHajjar and Mike Ferry that calculates every period and growthrate between adjacent band marks and growth marks. Valuesfrom at least five replicate cultures were averaged for eachcondition. For the experiments of Figure 8 and Table 2, plateswere imaged by placing them directly on a scanner. Periodsand growth rates were calculated by fitting a least-squaresregression line to the data from an entire culture andaveraging the values from several cultures (Lakin-Thomas

1998).Dark-pulse phase-shift experiments: Cultures of the csp-1;

bd; vvdP strain growing under 8.1 mmol/m2/sec light at 22�were subjected to 1-hr dark pulses during the fourth day afterinoculation, thus providing at least three bands prior to thepulse and three bands after. Phase shifts were determined bycomparing the timing of four consecutive band centers afterthe pulse to band centers over the same time interval inunpulsed control cultures. Band times were averaged acrossa set of 10 plates. The phase shift for each band center wascalculated and the four or more phase shift values were thenaveraged.

To calculate the phase of the culture at the time of the pulse,we used a phase reference different from the standardmethod. In standard light-pulse experiments for culturesgrowing in DD, circadian time (CT) CT 12 is defined as lightsoff and CT 0 or 24 is lights on. In Neurospora growing in a12 hr light and 12 hr dark (12:12 L:D) cycle, a band forms8–10 hr after lights off, or at about CT 21. In cultures free-running in DD, the band center can therefore be defined asCT 21. Because the vvd cultures made bands in LL, we used thetiming of the band that formed after lights on as our phasereference. Under the conditions employed in our dark-pulseexperiments, cultures with a period of 16 hr formed a band at�5.5 hr after lights on. When period is normalized to a 24-hrcircadian day, the center of the band that formed after lightson occurred at CT 8.

The phase at which a dark pulse was given was calculatedfrom the timing of the preceding band center. Phases andphase shifts were normalized so that a 16-hr period equals 24circadian hours. For example, a pulse occurring 10 hr after theband center in a culture with a 16-hr period would be plottedas a pulse at CT 23¼ CT 8 1 10*(24/16). A phase shift of 3 hrwould be plotted as 4.5 hr ¼ 3*(24/16).

Light:dark entrainment: Cultures were grown under cool-white fluorescent lights for artificial light cycles of 12:12 L:D.Cultures were exposed to natural lighting cycles of variousdurations by placing the plates in front of north-facingwindows at different times of the year. Light intensity atmidday was measured as 2.7 mmol/m2/sec. Lights on wasdefined as 30 min after sunrise, and lights off was defined as30 min after sunset.

Imaging and densitometry: For the images in Figure 1,plates were illuminated from above and pictures were taken

Vivid Mutants of Neurospora 919

with a digital camera from directly above. For the images inFigure 8, plates were placed directly on a scanner. The densityprofiles in Figure 2 were obtained by using the Plot Profilefunction of ImageJ software [National Institutes of Health(NIH)] to obtain pixel densities, which were then analyzedwith either the MatLab moving average smoothing functionor R-loess. The dimensions of the profiles were �1400 pixelsand were smoothed using a smoothing window of 41, or �3%of the profile.

Western blotting for expression of FRQ and WC-1 protein:The csp-1; bd; vvdSS strain and the control strain csp-1; bd weregrown on maltose/arginine medium in petri plates on top ofcellophane to aid in harvesting of cultures for protein ex-traction. The cellophane (350 POO) was a generous gift ofInnovia Films (Smyrna, GA). Plates were initially grown at 30�in LL at 40 mmol/m2/sec for various lengths of time and thentransferred to 22� in either DD or LL at 13.5 mmol/m2/sec.Plates were transferred at 3-hr intervals and all plates wereharvested after 72 hr total growth. Samples were collectedstarting 24 hr after transfer to DD or LL. Cultures in DD wereharvested under red safelight and cultures in LL wereharvested in the light. To harvest samples for protein extrac-tion, the entire growth front of a culture was scraped off of thecellophane using a plastic spatula that removed a 1-cm-widestrip and the sample was immediately frozen in liquidnitrogen.

Protein was extracted by grinding the frozen samples to apowder in liquid nitrogen using a mortar and pestle. Thefrozen powder was transferred to extraction buffer on ice,immediately heated to 100� for 5 min, and then cooled on ice.The extraction buffer contained 50 mm Tris pH 6.8, 2% SDS,10% glycerol, 50 mm EDTA–Na2, 0.1 m DTT, and 1 mm PMSF.Samples were centrifuged at room temperature at 12,000 3 gfor 5 min. The supernatant was collected and Bradford proteinassay was carried out. Bromophenol blue (0.1%) was addedand the samples were boiled again for 5 min before loading ongels. Equal amounts of protein (100 mg) were loaded on 7.5%SDS–PAGE. After electrophoresis, the proteins were trans-ferred to PVD membrane (Immobilon-P, Millipore) using asemidry electroblotter (Owl Panther) and the three-buffersystem (tris/methanol/glycine) recommended by Millipore.The blot was dried after transfer. Rapid immunodetection forchemiluminescence was carried out according to the methodsrecommended by Millipore for Immobilon membrane. Theblot was probed sequentially with 1:40 FRQ or 1:10 WC-1primary monoclonal antibodies and 1:1500 goat anti-mouseIgG–horseradish peroxidase conjugate (Bio-Rad Laborato-ries). The primary antibodies were generously supplied byM. Merrow (Gorl et al. 2001) and M. Brunner. The blot wasdeveloped by chemiluminescence (ECL, Amersham), imagedon film or with a CCD camera, and quantitated with ImageJ(NIH). FRQ and WC-1 were normalized against total proteinby staining the membrane with Ponceau S (Sigma) beforeimmunodetection.

RESULTS

Effects of growth conditions on rhythmicity of vvdmutants in LL: In initial experiments, rhythmicity of thebd; vvdP strain was studied. The bd mutation permitsconidiation under high CO2 concentrations in closedculture vessels and is commonly used in circadianrhythm experiments to allow the conidiation rhythmto be easily visualized.The bd; vvdP strain was grown onmaltose-containing medium in large petri dishes in LL

(5.4 mmol/m2/sec) and produced rhythmic bandingwith a period of 11 hr. Under these conditions the bdstrain was arrhythmic. The csp-1 mutation was thenintroduced into the bd; vvdP strain. The csp-1 mutationprevents conidiospores from detaching from the hy-phae and facilitates the handling of cultures by prevent-ing self-contamination (see below under ‘‘csp-1’’). Thebd; vvdP strain was crossed to csp-1; bd and 10 isolates ofgenotype csp-1; bd; vvdP and 10 of csp1; bd; vvdP wereobtained. A different allele of vvd, the vvdss strain, wasalso crossed to csp-1; bd and six csp-1; bd; vvdSS and twobd; vvdss isolates were obtained. All vvd strains showedsimilar 21- to 22-hr periods in DD and shorter-periodrhythmicity in LL. Table 1A presents period and growthrate data for a representative isolate of each of the fourgenotypes grown in DD and in LL at 2.7 mmol/m2/sec.A representative photo of a culture of csp-1; bd; vvdP isshown in Figure 1A, and the corresponding densitytrace is shown in Figure 2A.

It was consistently found that the growth rates of thevvd strains were significantly slower in LL than in DD(Table 1 and Table 3), in contrast to the vvd1 strains inwhich there was little difference in growth rate betweenDD and LL. The VVD gene product normally down-regulates light-induced gene expression (Heintzen

et al. 2001; Shrode et al. 2001; Schwerdtfeger andLinden 2003), as shown by the vivid orange color of themutants due to overproduction of light-induced carot-enoid pigments. The light-induced growth inhibition ofthe vvd mutants may be due to overexpression of somelight-induced gene product(s) that have toxic effects ongrowth at high levels.

Several other culture conditions were tested for ef-fects on the expression of rhythms in LL. The two bd; vvdstrains were cultured on growth tubes instead of petriplates and showed the same 21- to 22-hr period in DD ason plates, but in LL the rhythms were unclear and theperiod could not be measured accurately (Table 1B). Noimprovement in the clarity of banding was found after3 days of entrainment to a 12:12 L:D cycle followed byfree run in LL.

The effects of growth media were investigated byreplacing the maltose medium with glucose as theprincipal carbon source. In both petri plates and growthtubes, the vvd strains showed a 21- to 22-hr period in DDbut rhythms in LL were unclear. A medium withoutadded sugar (Dragovic et al. 2002) and an acetate/casamino acids medium (Feldman and Hoyle 1973)were also tested. Both of these media produced clearrhythms in both vvd strains in DD in growth tubes andpetri plates, but in LL only the plate cultures showedclear rhythms. A photo of a representative culture onacetate/casamino acids is shown in Figure 1C and thecorresponding density trace is in Figure 2C.

The acetate/casamino acids medium produced clearbands in petri plates and shortened the period: in LL(2.7 mmol/m2/sec), the period of the csp-1; bd; vvdP strain


was 8 hr and the period of the csp-1; bd; vvdSS strain was 12hr. Increasing the light intensity slightly decreased theperiods on this growth medium. (Effects of LL on theperiod of cultures on maltose medium are describedbelow in Effects of light intensity and temperature) For the csp-1; bd; vvdP strain, the period at 8.1 mmol/m2/sec LL was10.5 6 1.5 hr (n ¼ 30), and at 27.0 mmol/m2/sec it was9.8 6 2.0 hr (n¼ 23). Growth rates in both cases were 0.6mm/hr. The pan-2 mutation is closely linked to vvd andwas introduced into the vvd strains to facilitate theselection of multiple-mutant strains in which the vvdphenotype might be difficult to detect (see below under‘‘pan-2 and al-1’’). For the bd; vvdP pan-2 strain, the periodsat 8.1 and 27.0 mmol/m2/sec were 7.6 6 0.7 hr (n ¼ 23)and 6.9 6 0.7 hr (n¼ 26), respectively. Growth rates were0.7 mm/hr.

In summary, we did not find conditions that sup-ported sustained rhythms of the vvd strains in LL ingrowth tubes. Our results therefore repeat previousfindings that vvd strains are not rhythmic for .1 dayin growth tubes in LL (Shrode et al. 2001; Elvin et al.2005). However, we have found that clear, sustainedrhythms are expressed in LL on maltose medium andacetate/casamino acids medium in petri plates, andthese conditions were used to investigate the propertiesof this rhythm.

Genetic interactions with other mutations: csp-1: Thecsp-1 mutation was introduced into the vvd strains toreduce contamination of cultures by self-inoculation.We unexpectedly found that the csp-1 mutation length-ened the period of vvd strains by 6–8 hr in LL but notin DD (Table 1A). To determine whether the length-ened period cosegregated with the csp-1 phenotype, wecrossed bd; vvdss to csp-1; bd and isolated 14 csp-1; bd; vvdprogeny and 14 csp1; bd; vvd progeny. Progeny weretested for period at 26� in LL of 27 mmol/m2/sec. All 14csp-1 strains had periods longer than the csp1 strains: theaverage period of csp-1 progeny was 16 hr (range 13–21hr), and the average period of csp1 progeny was 9.9 hr(range 8.7–11.6 hr). The period-lengthening effect inLL therefore appears to segregate with csp-1. The csp-1gene has been identified as a light-induced transcrip-tion factor (Lambreghts et al. 2009) and this mayexplain why there is a difference between csp1 and csp-1in LL but not in DD.

pan-2 and al-1: To introduce the vvd mutation intostrains such as albino (al) where the intense color of vvdmight not be easy to see, a closely linked (,5 MU)marker pan-2 was utilized. The triple mutant bd; vvdP

pan-2 was constructed and its properties are presentedin Table 1C. The introduction of the pan-2 mutation hadno significant effect on the period or growth rate of

TABLE 1

Periods and growth rates in DD and LL

DD LL (2.7 mmol/m2/sec)

Genotype Period (hr)a Growth rate (mm/hr) Period (hr)a Growth rate (mm/hr)

A. vvd strains in petri dishesbd 22.4 6 3.2 (41) 1.0 NR 1.0csp-1; bd 21.5 6 2.7 (54) 1.1 NR 0.9bd; vvdP 22.2 6 3.1 (49) 1.2 10.9 6 1.9 (44) 0.8csp-1; bd; vvdP 21.5 6 2.6 (50) 1.1 17.4 6 2.5 (27) 0.6bd; vvdSS 20.7 6 2.3 (42) 1.1 13.5 6 2.3 (23) 0.9csp-1; bd; vvdSS 21.5 6 2.4 (51) 1.0 20.9 6 2.7 (25) 0.5

B. vvd strains in growth tubesbd 21.8 6 2.6(122) 1.3 NR 1.4csp-1; bd 21.0 6 1.8 (71) 1.3 NR 1.3bd; vvdP 21.7 6 2.8 (29) 1.4 Unclear 0.8csp-1; bd; vvdP 21.0 6 2.3 (57) 1.3 Unclear 0.6bd; vvdSS 21.4 6 2.4 (26) 1.4 Unclear 1.2csp-1; bd; vvdSS 21.7 6 2.4 (28) 1.4 Unclear 0.6

C. Multiple mutant strains in petri dishesbd; frq10 NR 1.0 NR 0.8bd; frq7 31.6 6 4.5 (17) 1.2 NR 1.0csp-1; bd; frq7 31.8 6 3.9 (15) 1.2 NR 1.0bd; vvdP pan-2 22.0 6 2.8 (48) 1.1 11.2 6 1.5 (31) 0.9bd; vvdP pan-2; frq10 NR 1.2 10.3 6 2.0 (36) 1.0bd; vvdP pan-2; frq7 #3 31.3 6 3.7 (24) 1.1 11.7 6 2.0 (17) 0.7bd; vvdP pan-2; frq7 #22 29.1 6 2.2 (11) 1.3 10.9 6 1.3 (19) 0.9bd; vvdP pan-2; wc-1 NR 1.1 NR 1.1

Maltose medium, 22�; NR, not rhythmic.a Mean 6 SD. Numbers in parentheses are the total number of individual periods averaged.


bd; vvdP strains in either DD or LL. Several additionalisolates of bd; vvdP pan-2 were found to have similarproperties.

It was of interest to determine whether the over-production of carotenoids in vvd strains was a factor inthe short-period rhythmicity seen in LL. To investigatethis, the albino-1 (al-1) mutation was introduced into abd; vvdP pan-2 strain. Five isolates of al-1; bd; vvdP pan-2were constructed by using pan-2 as a marker for vvd. Allfive isolates still exhibited periods of 11 hr in LL. Thegrowth rates of the al-1; bd; vvdP pan-2 strains were slowerin LL than in DD (data not shown), as seen for the othervvd strains (Table 1). This indicates that it is not theoverproduction of carotenoids that is responsible forthe rhythmicity and slow growth of vvd strains in LL.

frq10, wc-1 and wc-2: To determine whether any com-ponents of the FWC are required for rhythmicity in LL,knockout mutants of the three core genes of FWC wereintroduced into vvd strains. The introduction of the frq10

mutation led to the loss of the rhythmicity of the vvdstrain in DD, but not in LL (Table 1C, Figure 1B, andFigure 2B). The conidiation bands in LL were not asrobust in frq10 as in frq1 (compare A and B in Figure 1).This deletion of the frq gene did not significantly affectthe period of the vvd strain in LL (Table 1C). Twoisolates of the frq10 strain gave similar results.

The introduction of the wc-1 knockout into the vvdP

strain led to loss of rhythmicity in both DD and LL(Table 1C). This suggests that the LL response of vvdrequires the blue-light photoreceptor encoded by thewc-1 gene. This is in agreement with preliminary studiesemploying filters with various transmission spectra thatshowed blue-green light to be the effective wavelengthfor period shortening in vvd in LL (data not shown).The introduction of the wc-2 knockout did not producea clear-cut result. Cultures did not produce bands in DD,and LL cultures showed signs of weak rhythmicity (datanot shown).

frq7: To determine whether mutations at the frq locusthat affect the period in DD would affect the rhythm ofvvd in LL, the long-period frq7 mutation was introducedinto the bd; vvdP pan-2 strain. Two isolates (#3 and #22)were characterized, and the period and growth rate dataare given in Table 1C. A density trace of a typical plateculture (isolate #22) is shown in Figure 2G. There wasno significant effect of the frq7 mutation on the periodin the vvd background in LL. This is consistent with thelack of effect of the frq10 knockout on vvd in LL.

Effects of light intensity and temperature: The re-lationship between light intensity and period is shown inFigure 3. The period of the vvd strains decreased as lightintensity increased, and the period was proportional tothe log of the light intensity. The csp1 strain had a slope 3-to 4-fold greater than the csp-1 strain. This indicates that300- to 1000-fold higher light intensity was required toachieve the same period-shortening effect in csp-1 as inthe csp1 strain. This accounts for the difference in periodlength seen for the csp-1 vs. csp1 strains under low lightconditions (Table 1A) and is consistent with the findingthat csp-1 codes for a light-inducible transcription factor(Lambreghts et al. 2009).

Figure 4 shows the effect of temperature on the periodof the csp-1; bd; vvdP strain at several light intensities. InDD, the period is temperature compensated, with a Q10

of 1.15, similar to the Q10 for the csp-1; bd strain(Mattern et al. 1982; Lakin-Thomas 1998). At highlight intensity (27 mmol/m2/sec), temperature com-pensation is poor, with a Q10 of 2.2. At intermediate lightintensities, the Q10’s are intermediate values, indicatingthat temperature compensation is lost gradually as thelight intensity increases. Similar results were found forthe bd; vvdP pan-2 strain and for both strains on acetate/casamino acids medium (data not shown).

After effects of LL-to-DD and DD-to-LL transfers:The difference in period between vvd cultures grown inLL vs. DD raised the question of how rapidly the period

Figure 1.—Representative cultures of vvd strains. Plates wereinoculated at the lower edge. All cultures were grown at 22�on maltose medium unless otherwise noted. (A) csp-1; bd;vvdP (LL, 2.7 mmol/m2/sec). (B) bd; vvdP pan-2; frq10 (LL,2.7 mmol/m2/sec). (C) bd; vvdP pan-2 on acetate medium (LL,13.5 mmol/m2/sec). (D) bd; vvdP pan-2 (LL, 2.7 mmol/m2/sec, toDD at 95 hr). (E) bd; vvdP pan-2; frq10 (LL, 2.7 mmol/m2/sec, toDDat95hr).(F)bd;vvdP pan-2(12:12L:Dcycle,8.1mmol/m2/sec).


would adjust to a change in lighting conditions. Forthese experiments, growth conditions were chosen tomaximize the difference in period between DD and LL.For the experiments reported below, the bd; vvdP pan-2strain was grown on petri plates with acetate/casaminoacid medium in bright light (13.5 mmol/m2/sec) at 22�.These experiments were also repeated using the malt-ose medium, and the same after effects reported belowwere seen. Because the difference in period between LLand DD is not as large on maltose, the effect was not asstriking (data not shown).

For the experiments of Figure 5, cultures were grownin LL and transferred to DD or grown in DD andtransferred to LL. The periods were calculated betweenpairs of adjacent bands and the periods were plotted

against the time corresponding to the center of thesecond band in each pair. Figure 5A shows that the vvd1

control strain, which is arrhythmic in LL, rapidly re-sumed the normal 22-hr banding rhythm in DD after atransfer from LL to DD and produced its first band �8hr after the transfer. It also rapidly (within 1 day) be-came arrhythmic when transferred from DD to LL.

In contrast, the vvd strain required almost 3 days toadjust from the 8-hr period in LL to the steady-state DDperiod of 22 hr (Figure 5B). These data are replotted inFigure 5C as frequency (bands/day) rather than period,because frequency is a rate measurement and can moreeasily be related to rates of biochemical processes andhalf-lives of molecules. An approximately linear de-crease in frequency is seen. This may represent a decline

Figure 2.—Densitometry tracesof representative cultures of vvdstrains. All cultures grown at 22�on maltose medium unless other-wise noted. (A–F) As for Figure1. (G) bd; vvdP pan-2; frq7 (LL,2.7 mmol/m2/sec); (H) Overlayof D (solid line) and E (dashedline).


in the level of a molecular species produced in the lightthat decays in DD at a constant rate or with a constanthalf-life, or some other phenomenon. The mechanismof such after effects is unknown. This experiment wasrepeated at 28� and the period after effects persisted for2 days instead of 3, suggesting that the system returns tothe DD steady state faster at higher temperature. Inother experiments, cultures were transferred to LL atvarious phases of the conidiation rhythm, and no effectsof transfer phase on period were found (data notshown). Figure 5C also includes the csp-1; bd; vvdP strain,and it can be seen that similar after effects were seen,

except that the longer period (shorter frequency) of thecsp-1 strain in LL is rapidly matched by the csp1 strainafter transfer to DD.

A representative plate culture from the experiment inFigure 5B is shown in Figure 1D, with the correspondingdensity trace in Figure 2D. What is not obvious from thegray-scale photo is the clear difference in the color of

Figure 3.—Effect of light intensity on the period of vvdstrains. Cultures were grown on maltose medium in platesat 22�. Diamonds, csp-1; bd; vvdP; triangles, bd; vvdP pan-2. Stan-dard deviations of all points were �15%.

Figure 4.—Effect of temperature on the period of a vvdstrain. The csp-1; bd; vvdP strain was grown on maltose mediumat various temperatures and light intensities. Circles, DD;squares, 0.54 mmol/m2/sec; diamonds, 2.7 mmol/m2/sec; tri-angles, 27 mmol/m2/sec. Standard deviations of all pointswere �15%.

Figure 5.—Effects on the period of vvd of transfer from DDto LL and from LL to DD. Cultures were grown at 22� on ac-etate medium. Vertical shaded bars indicate time of transferfrom DD to LL (13.5 mmol/m2/sec) or from LL to DD. The x-axis is time in hours after inoculation of cultures. The periodwas calculated between adjacent bands, and the value wasplotted on the x-axis at the time corresponding to the centerof the second band. Standard deviations of all points were�15%. (A) csp-1; bd strain. (B) bd; vvdP pan-2 strain. (C) Trans-fer from LL to DD, replotted as frequency (bands/day). Thebd; vvdP pan-2 strain is compared with the csp-1; bd; vvdP strain.


the colony, which displays bright orange bands duringthe incubation in LL and much lighter coloring in DD.Figure 1D does demonstrate that the bands formed inDD were considerably wider and more robust than thoseseen in LL.

The transfer from DD to LL of the vvd strain (Figure5B) produced a result different from the LL-to-DDtransfer. The vvd strain adjusted its period from thesteady-state 22 hr to the LL period of 8 hr in ,1 day.Incidentally, this demonstrates that the period aftereffects seen after an LL-to-DD transfer are not likely tobe caused by changes in growth rate. Although growthrate did change by �30% in vvd strains after switchingfrom LL to DD and vice versa, the period changes werenot reciprocal and did not correlate with growth rate.

A set of LL-to-DD transfer experiments similar to that inFigure 5 was also carried out with the bd; vvdP pan-2; frq10

strain. It was found that the 11-hr rhythm in LL persistedfor several days after transfer to DD with little lengtheningof the period. A representative culture is shown in Figure1E and the corresponding density trace is shown in Figure2E. In Figure 2H, this trace is superimposed on the densitytrace of the frq1 control strain (Figure 2D) to demonstratethe period difference in DD. This is an unexpectedfinding of another condition that supports rhythmicityof a frq-null strain in DD.

Phase resetting by dark pulses: In organisms that arenormally rhythmic in LL, dark-pulse phase responsecurves (PRCs) have been reported ( Johnson 1999).Because rhythmic conidiation in LL has not previouslybeen reported for Neurospora, the effect of dark pulseson the phase of the rhythm had not been determined.The occurrence of a reproducible rhythm in LL in thevvd strains provided the opportunity to determine thedark-pulse PRC for the Neurospora rhythm. The csp-1;bd; vvdP strain was chosen to minimize self-inoculationduring handling of the cultures. Cultures growingunder 8.1 mmol/m2/sec at 22� were subjected to 1-hrdark pulses during the fourth day after inoculation, thusproviding at least three bands prior to the pulse andthree bands after. The results are plotted as a phase-

response curve (Figure 6) in which the data are doubleplotted (plotted twice along the x-axis to display thecontinuity of the data). The x-axis is normalized to theperiod of the culture so that 24 circadian hours equalone period. In contrast to the PRC for light in Neuros-pora and all other organisms, in which the peak ofadvances follows the peak of delays, the dark-pulse PRCshows the opposite pattern, with peak delays followingpeak advances. This mirror-image pattern has been seenfor dark-pulse PRCs in some other organisms ( Johnson

1999).When the data from the dark-pulse PRC are replotted

as a phase-transition curve (PTC) in Figure 7, it can beseen that 14 of the 17 data points fall within a range ofnew phases between CT 12.5 and CT 23.5. This shadedarea has been named the ‘‘reset zone’’ (Shaw andBrody 2000). A clustering of points within a narrowrange of new phases indicates a strong type 0 resetting. Aweak type 1 resetting response would produce a range ofnew phases spanning the entire 24 hr and following thediagonal from the bottom left to the top right in Figure7. The data points outside the reset zone may indicatethat this resetting pattern is transitional between type 1and type 0 and that a stronger resetting signal (such as alonger dark pulse) would have produced a pure type 0pattern. This dark-pulse PTC differs from a typical lightPTC in that the reset zone has its midpoint at new phaseCT 18, while the light-pulse reset zone has its midpointat new phase CT 9 (Shaw and Brody 2000). Similarresults have been seen before for temperature pulses(reviewed in Shaw and Brody 2000) where the mid-point of the reset zone is CT 7 for temperature pulses upand CT 22 for temperature pulses down.

The effects of dark-pulse length on the phase re-setting were also determined. Several different phaseswere chosen and dark pulses of 15 min, 30 min, and 1 hrwere used. No consistent effects were seen with 15-minpulses. The 30-min pulses consistently produced smallerphase shifts than the 1-hr pulses for both advances anddelays, as expected if the strength of the resetting signaldepends on the length of the dark pulse.

Figure 6.—Phase-response curve for 1-hr darkpulses. The csp-1; bd; vvdP strain was grown in LLat 8.1 mmol/m2/sec on maltose medium at 22�.One data set of 17 points from CT 0 to CT 24is double plotted to show the continuity of thedata.


Entrainment to light/dark cycles: To determine howthe vvd mutation affected entrainment of cultures tolight/dark (L:D) cycles, cultures were grown in artificialcycles of 12:12 L:D on maltose medium at 22� under4.0 mmol/m2/sec during the light phase. Under theseconditions, the bd; vvdP pan-2 strain showed an �12-hrrhythm (Table 2) with a band in the dark phase and asecond band in the light phase, which we describe as‘‘double banding.’’ A representative plate culture isshown in Figure 1F with the corresponding densitytrace in Figure 2F. This double banding may be causedby the reduction in period in LL: the short period of11.2 hr in this strain in LL (Table 1) would allow theformation of a second band during the 12-hr light pulsein a 12:12 light:dark cycle. The phase angle for the darkband (number of hours between lights off and thecenter of the subsequent band) was 3.0 6 2.0 hr. Thephase angle for the light band (number of hoursbetween lights on and the subsequent band center)was 1.8 6 2.0 hr. The bd strain produced a few doublebands in some cultures, and the reported period of15.9 hr (Table 2) is the mean of long (�24) and short(�12 hr) cycles. The phase angle of the dark band in thebd cultures was 8.3 6 2.0 hr. The csp-1 strains csp-1; bd andcsp-1; bd; vvdP showed no double banding and producedlonger periods closer to 24 hr (Table 2).

Entrainment studies were also carried out with shortL:D cycles (Table 2). The bd; vvdP pan-2 strain entrainedto cycles of 4:4, 6:6, and 8:8 with periods of 8.3, 11.7, and15.0 hr, respectively. The bd strain in these same L:Dcycles produced periods of 10.7, 11.2, and 14.1 hr,respectively. The csp-1; bd; vvdP strain entrained to 6:6and 8:8 with periods of 12.5 and 15.0 hr, but the csp-1; bdstrain failed to entrain to any of the short L:D cycles.

Natural light/dark regimes of various ratios were alsoused. The natural lighting regime was employed to

determine whether the gradual ramp up and rampdown of light intensity at dawn and dusk, and thechanges in wavelength proportion compared to fluo-rescent lights, would affect the entrainment of therhythm. Under conditions of L:D 9:15, 11:13, 12:12,and 15:9, with an intensity of 2.7 mmol/m2/sec duringthe light phase, the bd; vvdP pan-2 strain displayeddouble banding and periods of�12 hr for all L:D cyclestested (data not shown).

Expression of FRQ protein in vvd cultures: Thepersistence of rhythmicity in vvd cultures in LL raisedthe question of whether FRQ protein continued to berhythmically expressed in these cultures. The csp-1; bd;vvdSS strain and the control strain csp-1; bd were grownon maltose/arginine medium on top of cellophane inpetri plates at 22� in DD or in LL at 13.5 mmol/m2/sec,and samples were harvested every 3 hr. In Figure 8A,representative photos of unharvested plates demon-strate that the rhythmicity in both strains was similar inDD but that the vvd1 strain was arrhythmic in LL whilethe vvdSS strain continued to form bands rhythmically.The periods and growth rates under these conditionsare shown in Table 3.

Figure 8, B and C, demonstrates that FRQ protein levelswere rhythmic as expected in the vvd1 and vvdSS cultures inDD. Representative blots are shown in Figure 8G, and theexpected rhythm of FRQ phosphorylation can also beseen in these cultures. In Figure 8, B and C, we have alsoplotted the times at which band centers or peaks wereformed on control unharvested plates. The band peakswere consistently formed when FRQ protein was at itslowest level. This is the first report of the relationshipbetween the banding phase of cultures grown on agar and

Figure 7.—Phase-transition curve for 1-hr dark pulses. Thedata from Figure 6 have been replotted as new phase insteadof phase shift. The shaded area is the reset zone.

TABLE 2

Periods in L:D cycles

Genotype L:D cyclea Period (hr)b

bd 4:4 10.7 6 1.6 (15)6:6 11.2 6 1.4 (19)8:8 14.1 6 2.2 (24)

12:12 15.9 6 2.7 (70)csp-1; bd 4:4 Variable (34)

6:6 Variable (20)8:8 20.6 6 4.7 (14)

12:12 23.2 6 1.9 (53)bd; vvdP pan-2 4:4 8.3 6 0.8 (40)

6:6 11.7 6 1.5 (44)8:8 15.0 6 1.5 (26)

12:12 13.0 6 4.0 (90)csp-1; bd; vvdP 4:4 10.0 6 2.4 (31)

6:6 12.5 6 6.9 (28)8:8 15.0 6 1.7 (22)

12:12 19.4 6 6.0 (60)

Maltose media, 22�; variable, periods highly variable.a Hours of light:hours of dark.b Mean 6 SD. Numbers in parentheses are the total number

of individual periods averaged.


the level of FRQ protein assayed under identical con-ditions. All previous reports of FRQ protein levels (such asGarceau et al. 1997; Lee et al. 2000) have used a liquidculture system in which banding is not expressed.

In vvd1 in LL, the level of FRQ was highly variable andnonrhythmic (Figure 8D), as previously reported (Elvin

et al. 2005). FRQ levels in LL in the vvdSS culture (Figure8E) were also nonrhythmic and were less variable than in

Figure 8.—Expression of FRQ protein. Cultures of csp-1; bd (labeled ‘‘vvd1’’) and csp-1; bd; vvdSS (labeled ‘‘vvdSS’’) were grown oncellophane on petri plates at 22� and harvested every 3 hr for protein extraction. (A) Representative unharvested plates. Plateswere inoculated at the lower edge. Semicircular lines on the vvd1 plates mark the growth fronts at 24-hr intervals. (B–E) Timecourses of FRQ expression relative to total protein. The x-axis is the hours after transfer from 30� in LL at 40 mmol/m2/sec to 22�in either DD or LL at 13.5 mmol/m2/sec. Error bars are SEM for three (B–D) or four (E) independent experiments. Arrows in B,C, and E indicate the average times (6SEM) at which the peak of a band was formed on the unharvested plates; n¼ 4 and 2 (B), 10and 6 (C), and 17 and 17 (E). (F) Data from B to E are replotted after correcting for relative levels of expression. (G) Repre-sentative Western blots. Numbers below the lanes are harvesting times in hours after transfer.


vvd1 (compare error bars in Figure 8E to those in Figure8D). No phosphorylation rhythm could be seen in eitherstrain in LL (Figure 8G). Selected samples from all foursets were run together on one gel to estimate the relativelevels of FRQ in the four conditions, and the data arereplotted in Figure 8F. FRQ levels are very similar in bothvvd1and vvdSS in DD, as expected, and are also similar inLL and higher than in DD. This is also expected, as FRQexpression is induced by light (Crosthwaite et al. 1995).We conclude that rhythmic FRQ levels and/or phosphor-ylation are not required for conidiation rhythms in LL.This is consistent with the persistence of rhythms in LLin frq null strains (Table 1 and Figure 1B). Preliminaryresults (data not shown) indicate that levels of WC-1protein are also rhythmic in DD but arrhythmic in LL inboth strains.

DISCUSSION

The results presented in this article have revealed thefunctioning of an oscillator that drives conidiationunder a new condition, LL, in which robust rhythmicityhas not previously been observed in Neurospora. Thishas allowed us to characterize this oscillator in LL, andwe have found that it responds to dark pulses with astrong type 0 PRC and that some of its properties, suchas period and temperature compensation, can be grad-ually altered by changing the light intensity.

We assayed six growth conditions—three media (malt-ose, acetate, or glucose) in two culture vessels (petri platesor growth tubes)—and found undamped rhythmicity inthe vvd mutant strain growing in LL on petri plates withmaltose or acetate media. Shrode et al. (2001) and Elvin

et al. (2005) found no rhythmicity in vvd mutant strains inLL. Elvin et al. (2005) found rapidly damped conidiationrhythms in LL for vvd1 but not for vvd. These authors usedrace tube cultures, and we have also failed to see robustrhythmicity in race tubes. Our results using petri platecultures, however, demonstrate robust, undamped rhyth-micity in LL for vvd mutants but not for vvd1. We concludetherefore that when the VVD gene product is present, itrepresses or masks rhythmic conidiation in LL, resultingin constant conidiation. Under appropriate culture con-ditions (in plates on maltose or acetate media), the vvdmutation reveals an oscillator in LL. We do not have an

explanation for the difference between plate and tubecultures. We can only speculate that it may have somethingto do with the more aerobic conditions on plates in whichgas exchange is more rapid than on tubes closed withplugs.

We have shown that levels of FRQ are high andarrhythmic under conditions that allow conidiationrhythms to be expressed in vvd (Figure 8). We have alsoshown that conidiation rhythms continue in LL in afrq10 null mutant (Figures 1B and 2B) and that rhythmscontinue for several days after frq10 cultures are re-turned to DD (Figures 1E and 2E). FRQ-less rhythmicityhas been reported many times: rhythms in conidiation(Loros and Feldman 1986; Merrow et al. 1999; Lakin-Thomasand Brody 2000; Granshaw et al. 2003; Lakin-Thomas 2006a; Lombardi et al. 2007) and rhythms atthe molecular level (Ramsdale and Lakin-Thomas

2000; Christensen et al. 2004; de Paula et al. 2006)are found in frq null mutants. The combination of thevvd mutation and constant light is therefore another ofmany conditions that allow rhythmicity to be assayedin the absence of FRQ function. The nature of theoscillator or oscillators that drive(s) FLO (Iwasaki andDunlap 2000) is currently unknown and is the subjectof debate (Dunlap and Loros 2004; de Paula et al.2006; Lakin-Thomas 2006b). There may be one ormore independent FLOs in addition to a FWC oscilla-tor; a single FLO that is coupled to, and closely interactswith, a FWC oscillator; or a single oscillator that interactswith FWC as part of the entire circadian system.

Because conidiation in the vvd1 strains is not rhyth-mic at the light levels that we used, but is rhythmic in DDor very low LL intensity, it appears that the vvd mutantsare less sensitive to the rhythm-damping effects of light.This presents us with an apparent paradox: the VVDgene product has been shown to attenuate the effects oflight, and vvd mutants should therefore be more, notless, sensitive to damping in LL. Previously publisheddata (Heintzen et al. 2001; Shrode et al. 2001;Schwerdtfeger and Linden 2003) demonstrate thatVVD is a light-induced factor that downregulates therapid wc-dependent responses to light. In the absence ofVVD function in vvd null mutants, rapid light responsespersist longer than normal (such as al-1 gene expres-sion) and/or are exaggerated (such as phase resetting

TABLE 3

Periods and growth rates of unharvested cultures for FRQ protein extraction

DD LL (13.5 mmol/m2/sec)

Genotype Period (hr)a Growth rate (mm/hr) Period (hr)a Growth rate (mm/hr)

csp-1; bd 22.0 6 0.3 (9) 1.2 6 0.02 NR 1.2 6 0.04csp-1; bd; vvdSS 24.4 6 0.3 (9) 1.3 6 0.03 21.7 6 0.7 (16) 0.5 6 0.01

Maltose–arginine medium in petri plates with cellophane, 22�; NR, not rhythmic.a Mean 6 SEM. Numbers in parentheses are the total number of individual plate cultures averaged.


of the conidiation rhythm). Schwerdtfeger and Lin-

den (2003) found that VVD is required for a secondwave of light-induced gene expression if light intensity isincreased after .15 min exposure to lower intensity.They concluded that VVD has two functions: the first isto downregulate the expression of rapidly light-inducedgenes, and the second is to activate expression of asubset of light-induced genes in response to an increasein light intensity after the initial induction. Thisestablishes a function for VVD as an activator of somelight-induced genes, and it may be the absence of thisfunction that allows the vvd mutant strains in ourexperiments to express a rhythm of conidiation in LL.Hunt et al. (2007) have proposed a role for VVD in theconidiation output pathway, and this may explain theactivation of continuous conidiation, rather than rhyth-mic conidiation, in LL in vvd1. The loss of VVD proteinin the vvd mutants would remove this activationfunction of VVD and allow expression of the conidiationrhythm in LL. Our results with the wc-1 deletion mutant(Table 1C) indicate that WC-1 is a necessary lightreceptor for both of these light-dependent functionsof VVD.

Elvin et al. (2005) assayed conidiation rhythms invvd1 and a vvd mutant after release from LD cycles intoLL and found that vvd1 rhythms damped out in one totwo cycles but vvd rhythms damped faster. In experi-ments in which the cultures were exposed to various daylengths and released into DD, the vvd strain demon-strated a stronger resetting response. Elvin et al. (2005)propose a separate daytime oscillator controlling rhyth-mic conidiation in LL, which is dependent on VVD, andthey propose that ‘‘as yet undiscovered processes maycontribute to the extended overt rhythmicity we seein DD.’’ Our results clearly demonstrate that rhythmicconidiation in LL is not dependent on VVD. Elvin et al.(2005, p. 2597) carried out their experiments in racetubes, and we also failed to see sustained rhythms in LLin race tubes. The stronger resetting response of thedamped rhythm in vvd during the first day in LL in racetubes can be explained as simply a consequence of thestronger responses to light in vvd mutants that havebeen previously reported.

What can we deduce about the nature of the oscillatorthat drives rhythmic conidiation in LL? The phase-resetting behavior (Figures 6 and 7) indicates theexistence of strong type 0 resetting, which is found inoscillators with at least two state variables (Lakin-Thomas 1995), and may indicate that this oscillator isa limit cycle. This is the first report of what may be limitcycle behavior in a FLO. Some of the properties of therhythm of the vvd strains in LL differ from those of thewell-characterized conidiation rhythm seen in vvd1

strains in DD. At high light intensity, the vvd rhythmhas poor temperature compensation. The period ismore strongly influenced by the growth medium than inDD and can be as short as 8 hr, shorter than the short-

period frq1 mutant (�14–16 hr). The period in LL isinfluenced by the csp-1 mutation, but is not influencedby loss of frq and does not require FRQ protein cycling.The FWC feedback loop does not operate correctly inLL, as shown by the constant high levels of FRQ proteinand lack of a FRQ phosphorylation rhythm in LL(Figure 8). One explanation for the appearance ofrhythmicity in vvd in LL is that it is driven by anoscillator, a FLO, that is independent of the FWCoscillator. It is unlikely that the loss of one gene, vvd,would lead to the creation of an entirely new oscillatorin LL. It can be argued that the mechanism for thisoscillator that drives conidiation rhythms in LL is alwayspresent in both DD and LL. It may be strongly coupledto the FWC in DD or may run at a low amplitude. Thecombination of high light intensity and the vvd muta-tion may lead to amplification of the amplitude of thesecond oscillator (FLO) so that it overrides signals fromthe FWC oscillator.

One problem with the two-oscillator model is that ourdata do not demonstrate any obvious demarcationbetween a DD circadian oscillator and an LL FLO invvd: properties of period and temperature compensa-tion change gradually as the light intensity increases,and the rhythm in LL responds to dark pulses asexpected of a circadian system. To explain the gradualchange in period with light intensity, we would need topropose that two oscillators could blend their propertiesgradually as conditions change and one oscillatorgradually dominates the other. An alternative, andsimpler, explanation is that within the intact circadiansystem of Neurospora there is a single oscillator thatdrives conidiation under all conditions. This oscillatorwould lack the canonical circadian properties of a stable24-hr period and temperature compensation. The FWCfeedback loop would be an important part of the entirecircadian system, conferring robust temperature com-pensation and period control when coupled to theconidiation oscillator.

If the conidiation oscillator is assumed to be a limitcycle (Peterson 1980; Lakin-Thomas 1995; Winfree

2001; Johnson et al. 2003), then the effects of vvd onrhythmicity in LL can be explained by assuming thatthe parameters of the oscillator are affected by inputfrom light. In the light, the oscillator would continue tocycle but with altered levels of state variables. Theresults of Elvin et al. (2005) demonstrate that the vvdmutant has a stronger phase-shifting response to light.Their results can be explained by proposing that theoscillator may cycle in a different region of state spacein LL and will display a larger phase shift when itreturns to the DD limit cycle. Gooch (1984) carriedout experiments in which Neurospora cultures wereentrained to LD cycles, held in LL for various lengthsof time, and then released into DD; the resultingphases provided evidence for continued cycling of thecircadian oscillator in LL. Using our culture conditions


allows the cycling of the oscillator in LL to be visualizedas a conidiation rhythm.

The effect of light on the free-running period of thevvd strains (Figure 3) recalls the effects first noted byAschoff and later referred to by Pittendrigh as ‘‘Aschoff’srule’’ (for review see Johnson et al. 2003): The free-running circadian period of day-active animals tends toshorten in constant light, while the period of nocturnalanimals tends to lengthen in constant light, althoughthe correlation is not exact ( Johnson 1999), and theeffects on period increase with increasing light intensity.If an oscillator is modeled as a limit cycle, then the effectof light on the period can be explained as an effect onthe level of a state variable and/or an effect on the valueof a parameter of that oscillator ( Johnson et al. 2003).Our data also show a gradual change in temperaturecompensation as light intensity increases. This is alsoconsistent with an effect of light on a parameter of anoscillator. In several organisms, single mutations inclock-associated genes change both free-running periodand temperature compensation of the circadian rhythmsimultaneously, such as frq mutations in Neurosporaand per mutations in Drosophila (reviewed in Lakin-Thomas et al. 1991). In the case of vvd, light may beacting (through transcriptional effects on light-inducedgenes) to alter the levels of a parameter that affects bothperiod and temperature compensation of the oscillatorsimultaneously.

The slow return of the period length to the DD valuewhen vvd cultures are switched from LL to DD (Figure5B) may be caused by the slow decay of a light-inducedfactor that affects the oscillator parameters and wouldcorrespond to a slow return of the oscillator to the DDlimit cycle. Remarkably, the short 12-hr period of thefrq10 strain in LL does not immediately disappear orslowly revert to 21 hr when these cultures are shifted toLL (Figures 1E and 2E). This may indicate that thesteady-state period of the frq10 strain is close to 12 hrand that the influence of FWC in a frq1 strain is tomaintain the period in the circadian range (�24 hr).

S.B. thanks the following for their contributions to this work: MichelleShuff, Michael Ferry, and Karthika Balasubramanian. The data in Figure8 were collected by S.L. and A.Z. in the laboratory of P.L.-T., and all otherdata were collected in the laboratory of S.B. P.L.-T. gratefully acknowl-edges the contributions of Sarah Farooq and Pardeep Heir for datacollection and Duane Lakin-Thomas for figure preparation. Antibodiesfor Figure 8 Western blots were generously supplied by Martha Merrowand Michael Brunner. This work was supported in part by NationalScience Foundation grant MCB 0212190 to S.B. and by Natural Sciencesand Engineering Research Council of Canada Discovery grant 250133-07 to P.L.-T.

LITERATURE CITED

Aronson, B. D., K. A. Johnson and J. C. Dunlap, 1994 Circadianclock locus Frequency: protein encoded by a single open readingframe defines period length and temperature compensation.Proc. Natl. Acad. Sci. USA 91: 7683–7687.

Belden, W. J., L. F. Larrondo, A. C. Froehlich, M. Shi, C. H. Chen

et al., 2007 The band mutation in Neurospora crassa is a domi-nant allele of ras-1 implicating RAS signaling in circadian output.Genes Dev. 21: 1494–1505.

Bell-Pedersen, D., V. M. Cassone, D. J. Earnest, S. S. Golden, P. E.Hardin et al., 2005 Circadian rhythms from multiple oscilla-tors: lessons from diverse organisms. Nat. Rev. Genet. 6: 544–556.

Brunner, M., and T. Schafmeier, 2006 Transcriptional and post-transcriptional regulation of the circadian clock of cyanobacteriaand Neurospora. Genes Dev. 20: 1061–1074.

Cheng, P., Q. Y. He, Y. H. Yang, L. X. Wang and Y. Liu,2003 Functional conservation of light, oxygen, or voltage do-mains in light sensing. Proc. Natl. Acad. Sci. USA 100: 5938–5943.

Christensen, M. K., G. Falkeid, J. J. Loros, J. C. Dunlap, C. Lillo

et al., 2004 A nitrate induced frq-less oscillator in Neurospora cras-sa. J. Biol. Rhythms 19: 280–286.

Collett, M. A., N. Garceau, J. C. Dunlap and J. J. Loros,2002 Light and clock expression of the Neurospora clock genefrequency is differentially driven by but dependent on WHITECOLLAR-2. Genetics 160: 149–158.

Correa, A., A. Z. Lewis, A. V. Greene, I. J. March, R. H. Gomer et al.,2003 Multiple oscillators regulate circadian gene expression inNeurospora. Proc. Natl. Acad. Sci. USA 100: 13597–13602.

Corrochano, L. M., 2007 Fungal photoreceptors: sensory mole-cules for fungal development and behaviour. Photochem. Photo-biol. Sci. 6: 725–736.

Crosthwaite, S. K., J. J. Loros and J. C. Dunlap, 1995 Light-induced resetting of a circadian clock is mediated by a rapid in-crease in frequency transcript. Cell 81: 1003–1012.

Crosthwaite, S. K., J. C. Dunlap and J. J. Loros, 1997 Neurosporawc-1 and wc-2: transcription, photoresponses, and the origins ofcircadian rhythmicity. Science 276: 763–769.

Degli-Innocenti, F., and F. E. A. Russo, 1984 Isolation of new whitecollar mutants of Neurospora crassa and studies of their behaviorin the blue light-induced formation of protoperithecia. J. Bacter-iol. 159: 757–761.

de Paula, R. M., Z. A. Lewis, A. V. Greene, K. S. Seo, L. W. Morgan

et al., 2006 Two circadian timing circuits in Neurospora crassacells share components and regulate distinct rhythmic processes.J. Biol. Rhythms 21: 159–168.

Dragovic, Z., Y. Tan, M. Gorl, T. Roenneberg and M. Merrow,2002 Light reception and circadian behavior in ‘‘blind’’ and‘‘clock-less’’ mutants of Neurospora crassa. EMBO J. 21: 3643–3651.

Dunlap, J. C., 2006 Proteins in the Neurospora circadian clock-works. J. Biol. Chem. 281: 28489–28493.

Dunlap, J. C., and J. J. Loros, 2004 The Neurospora circadian system.J. Biol. Rhythms 19: 414–424.

Dunlap, J. C., and J. J. Loros, 2005 Neurospora photoreceptors, pp.371–389 in Handbook of Photosensory Receptors, edited by W. R.Briggs and J. L. Spudich. Wiley-VCH, Weinheim, Germany.

Dunlap, J. C., and J. J. Loros, 2006 How fungi keep time: circadiansystems in Neurospora and other fungi. Curr. Opin. Microbiol. 9:579–587.

Edmunds, L. N., 1988 Cellular and Molecular Bases of Biological Clocks.Springer-Verlag, New York.

Elvin, M., J. J. Loros, J. C. Dunlap and C. Heintzen, 2005 ThePAS/LOV protein VIVID supports a rapidly dampened daytimeoscillator that facilitates entrainment of the Neurospora circa-dian clock. Genes Dev. 19: 2593–2605.

Feldman, J. F., and M. N. Hoyle, 1973 Isolation of circadian clockmutants of Neurospora crassa. Genetics 75: 606–613.

Francis, C. D., and M. L. Sargent, 1979 Effects of temperature per-turbations on circadian conidiation in Neurospora. Plant Physiol.64: 1000–1004.

Froehlich, A. C., B. Noh, R. D. Vierstra, J. Loros and J. C. Dunlap,2005 Genetic and molecular analysis of phytochromes from thefilamentous fungusNeurosporacrassa.Eukaryot.Cell4:2140–2152.

Garceau, N. Y., Y. Liu, J. J. Loros and J. C. Dunlap, 1997 Alter-native initiation of translation and time-specific phosphorylationyield multiple forms of the essential clock protein FREQUENCY.Cell 89: 469–476.

Gooch, V. D., 1984 Effects of light and temperature steps on circa-dian rhythms of Neurospora and Gonyaulax, pp. 232–237 in Tempo-ral Order, edited by L. Rensing and N. I. Jaeger. Springer-Verlag,New York.


Gorl, M., M. Merrow, B. Huttner, J. Johnson, T. Roenneberg

et al., 2001 A PEST-like element in FREQUENCY determinesthe length of the circadian period in Neurospora crassa. EMBOJ. 20: 7074–7084.

Granshaw, T., M. Tsukamoto and S. Brody, 2003 Circadianrhythms in Neurospora crassa: farnesol or geraniol allow expres-sion of rhythmicity in the otherwise arrhythmic strains frq10, wc-1, and wc-2. J. Biol. Rhythms 18: 287–296.

He, Q., P. Cheng, Q. He and Y. Liu, 2005 The COP9 signalosomeregulated the Neurospora circadian clock by controlling the stabil-ity of the SCFFWD-1 complex. Genes Dev. 19: 1518–1531.

Heintzen, C., J. J. Loros and J. C. Dunlap, 2001 The PAS proteinVIVID defines a clock-associated feedback loop that represseslight input, modulates gating, and regulates clock resetting. Cell104: 453–464.

Hunt, S. M., M. Elvin, S. K. Crosthwaite and C. Heintzen,2007 The PAS/LOV protein VIVID controls temperature com-pensation of circadian clock phase and development in Neuros-pora crassa. Genes Dev. 21: 1964–1974.

Iwasaki, H., and J. C. Dunlap, 2000 Microbial circadian oscillatorysystems in Neurospora and Synechococcus: models for cellularclocks. Curr. Opin. Microbiol. 3: 189–196.

Johnson, C. H., 1999 Forty years of PRCs: What have we learned?Chronobiol. Int. 16: 711–743.

Johnson, C. H., J. A. Elliot and R. G. Foster, 2003 Entrainment ofcircadian programs. Chronobiol. Int. 20: 741–774.

Lakin-Thomas, P. L., 1995 A beginner’s guide to limit cycles, theiruses and abuses. Biol. Rhythm Res. 26: 216–232.

Lakin-Thomas, P. L., 1998 Choline depletion, frq mutations, andtemperature compensation of the circadian rhythm in Neurosporacrassa. J. Biol. Rhythms 13: 268–277.

Lakin-Thomas, P. L., 2006a Circadian clock genes frequency and whitecollar-1 are not essential for entrainment to temperature cycles inNeurospora crassa. Proc. Natl. Acad. Sci. USA 103: 4469–4474.

Lakin-Thomas, P. L., 2006b New models for circadian systems in mi-croorganisms. FEMS Microbiol. Lett. 259: 1–6.

Lakin-Thomas, P. L., and S. Brody, 2000 Circadian rhythms in Neu-rospora crassa: lipid deficiencies restore robust rhythmicity to nullfrequency and white-collar mutants. Proc. Natl. Acad. Sci. USA 97:256–261.

Lakin-Thomas, P. L., and S. Brody, 2004 Circadian rhythms in mi-croorganisms: new complexities. Annu. Rev. Microbiol. 58: 489–519.

Lakin-Thomas, P. L., G. G. Cote and S. Brody, 1990 Circadianrhythms in Neurospora crassa: biochemistry and genetics. Crit.Rev. Microbiol. 17: 365–416.

Lakin-Thomas, P. L., S. Brody and G. G. Cote, 1991 Amplitudemodel for the effects of mutations and temperature on periodand phase resetting of the Neurospora circadian oscillator. J. Biol.Rhythms 6: 281–297.

Lambreghts, R., M. Shi, W. J. Belden, D. deCaprio, D. Park et al.,2009 A high-density single nucleotide polymorphism map forNeurospora crassa. Genetics 181: 767–781.

Lee, K., J. J. Loros and J. C. Dunlap, 2000 Interconnected feedbackloops in the Neurospora circadian system. Science 289: 107–110.

Liu, Y., 2005 Analysis of posttranslational regulations in the Neuros-pora circadian clock. Methods Enzymol. 393: 379–393.

Liu, Y., and D. Bell-Pedersen, 2006 Circadian rhythms in Neurosporacrassa and other filamentous fungi. Eukaryot. Cell 5: 1184–1193.

Liu, Y., Q. He and P. Cheng, 2003 Photoreception in Neurospora: atale of two White Collar proteins. Cell. Mol. Life Sci. 60: 2131–2138.

Lombardi, L., K. Schneider, M. Tsukamoto and S. Brody,2007 Circadian rhythms in Neurospora crassa: Clock mutant ef-fects in the absence of a frq-based oscillator. Genetics 175:1175–1183.

Loros, J. J., and J. F. Feldman, 1986 Loss of temperature compen-sation of circadian period length in the frq-9 mutant of Neurosporacrassa. J. Biol. Rhythms 1: 187–198.

Mattern, D. L., L. R. Forman and S. Brody, 1982 Circadianrhythms in Neurospora crassa: a mutation affecting temperaturecompensation. Proc. Natl. Acad. Sci. USA. 79: 825–829.

McCluskey, K., 2003 The Fungal Genetics Stock Center: frommolds to molecules. Adv. Appl. Microbiol. 52: 245–262.

Merrow, M., M. Brunner and T. Roenneberg, 1999 Assignment ofcircadian function for the Neurospora clock gene frequency. Nature399: 584–586.

Perkins, D. D., A. Radford and M. S. Sachs, 2001 The NeurosporaCompendium: Chromosomal Loci. Academic Press, San Diego.

Peterson, E. L., 1980 A limit cycle interpretation of a mosquito cir-cadian oscillator. J. Theor. Biol. 84: 281–310.

Pittendrigh, C. S., 1993 Temporal organization: reflections of aDarwinian clockwatcher. Annu. Rev. Physiol. 55: 17–54.

Ramsdale, M., and P. L. Lakin-Thomas, 2000 sn-1,2-diacylglycerollevels in the fungus Neurospora crassa display circadian rhythmic-ity. J. Biol. Chem. 275: 27541–27550.

Sargent, M. L., and W. R. Briggs, 1967 The effects of light on acircadian rhythm of conidiation in Neurospora. Plant Physiol. 42:1504–1510.

Schwerdtfeger, C., and H. Linden, 2003 VIVID is a flavoproteinand serves as a fungal blue light photoreceptor for photoadapta-tion. EMBO J. 22: 4846–4855.

Shaw, J., and S. Brody, 2000 Circadian rhythms in Neurospora: anew measurement, the reset zone. J. Biol. Rhythms 15: 225–240.

Shrode, L. B., Z. A. Lewis, L. D. White, D. Bell-Pedersen and D. J.Ebbole, 2001 vvd is required for light adaptation of conidia-tion-specific genes of Neurospora crassa, but not circadian conidia-tion. Fungal Genet. Biol. 32: 169–181.

Sweeney, B. M., 1976 Circadian rhythms, definition and generalcharacterization, pp. 77–83 in The Molecular Basis of CircadianRhythms, edited by J. W. Hastings and H.-G. Schweiger.Dahlem Konferenzen, Berlin.

Thimijan, R. W., and R. D. Heins, 1983 Photometric, radiometric,and quantum light units of measure: a review of proceduresfor interconversion. HortScience 18: 818–822.

Vogel, H. J., 1956 A convenient growth medium for Neurospora.Microbiol. Genet. Bull. 13: 42.

Winfree, A. T., 2001 The Geometry of Biological Time. Springer, NewYork.

Zoltowski, B. D., and B. R. Crane, 2008 Light activation of theLOV protein vivid generates a rapidly exchanging dimer. Bio-chemistry Mosc. 47: 7012–7019.

Zoltowski, B. D., C. Schwerdtfeger, J. Widom, J. J. Loros, A. M.Bilwes et al., 2007 Conformational switching in the fungal lightsensor vivid. Science 316: 1054–1057.

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