A developmental cycle masks output from the circadian oscillator under conditions of choline deficiency in Neurospora

Abstract

In Neurospora, metabolic oscillators coexist with the circadian transcriptional/translational feedback loop governed by the FRQ (Frequency) and WC (White Collar) proteins. One of these, a choline deficiency oscillator (CDO) observed in chol-1 mutants grown under choline starvation, drives an uncompensated long-period developmental cycle ( approximately 60-120 h). To assess possible contributions of this metabolic oscillator to the circadian system, molecular and physiological rhythms were followed in liquid culture under choline starvation, but these only confirmed that an oscillator with a normal circadian period length can run under choline starvation. This finding suggested that long-period developmental cycles elicited by nutritional stress could be masking output from the circadian system, although a caveat was that the CDO sometimes requires several days to become consolidated. To circumvent this and observe both oscillators simultaneously, we used an assay using a codon-optimized luciferase to follow the circadian oscillator. Under conditions where the long-period, uncompensated, CDO-driven developmental rhythm was expressed for weeks in growth tubes, the luciferase rhythm in the same cultures continued in a typical compensated manner with a circadian period length dependent on the allelic state of frq. Periodograms revealed no influence of the CDO on the circadian oscillator. Instead, the CDO appears as a cryptic metabolic oscillator that can, under appropriate conditions, assume control of growth and development, thereby masking output from the circadian system. frq-driven luciferase as a reporter of the circadian oscillator may in this way provide a means for assessing prospective role(s) of metabolic and/or ancillary oscillators within cellular circadian systems.

Fig. 1.

FRQ oscillation with a circadian period (≈22 h) under conditions of choline deficiency. (A) Race tube assay of a chol-1;csp-1 strain under choline-supplemented (100 μM) and choline-starved conditions. The chol-1;csp-1 strain showed a normal circadian period length of ≈22 h with choline supplementation but an elongated period length (≈60 h) under choline starvation. The mycelial growth front was marked by a black line every day on race tubes. (B) Western blot analysis of FRQ expression in a 2-day time course from liquid culture. Oscillations of FRQ were observed in chol-1 strains in both choline-replete and choline-starved conditions.

Fig. 2.

Normal circadian rhythms in liquid cultures not supplemented with choline. (A) Diagram of the conidia transfer assay (see Materials and Methods). If the clock is not reset by conidia transfer from liquid culture to race tube media, phases should be similar in all samples (light to dark reset). See Results for details. (B) Phase in WT conidia transfer assay. Phases of all WT cultures are similar regardless of the time of conidial transfer, indicating that liquid-to-solid medium transfer does not reset clock (error bars = 1 SD; n = 6). (C) Predicted phase differences in strains with longer (filled circles, period = 66 h), equal (open circles, period = 22 h), or shorter (solid inverted triangles, period = 11 h) period lengths in liquid culture compared with solid culture (see Results for details). (D) Conidia transfer assay of chol-1. Phase is reported from chol-1 cultured in liquid with no choline and inoculated onto race tubes supplemented with 100 μM choline (error bars = 1 SD; n = 6). Lack of significant phase differences among cultures transferred at different times suggests that the period under choline deficiency conditions is similar to that of fully choline-supplemented cultures.

Fig. 3.

frq allele-dependent luciferase rhythms with normal period lengths in a race tube assay under choline starvation conditions. (A) Characteristically elongated conidiation rhythms in chol-1;csp-1,frqP-luc and chol-1;csp-1,frqP-luc;frq⁷ strains. (B) Race tube assay containing 12.5 μM luciferin. Black lines were marked as reference points on the tube before strain growth. The 3 h of saturating light given after 292 h of growth (white bar) did not change the conidiation banding pattern but served to synchronize the FWOs in the culture. (C) Bioluminescence data collected from each of the whole race tubes and detrended to remove background reveals rhythms of ≈22-h period in WT strains and ≈29-h period in frq⁷ strains. The rhythms were sustained for >10 days under choline starvation whereas ≈70-h conidiation rhythms were still observed on the race tubes (B). Time values are marked on individual peaks of the detrended luciferase rhythms that show similar period and phase. (D) Periodogram analysis of the luciferase data using a window of 14–100 h shows a strong 22.5-h component in WT strain and a 29.3-h component in frq⁷ with their multiples but no appreciable contribution of rhythms with periods near 70 h.

Fig. 4.

Temperature-compensated luciferase rhythms under conditions of choline depletion. (A) Race tube assays of chol-1 strains at 20°C and 28°C show the period of CDO-driven rhythmicity to be strongly temperature-dependent. (B) Luciferase rhythm at 20°C and 28°C. (C) FWO-driven luciferase rhythms are temperature-compensated in chol-1;csp-1,frqP-luc frq⁺ and undercompensated in chol-1;csp-1,frqP-luc;frq⁷, whereas the conidiation rhythm of CDO is not. Note the difference in scale (error bars = 1 SD; n = 3–17).