Ultradian and circadian rhythms of phototaxis in chlamydomonas reinhardii

Abstract

We investigated phototactic and circadian rhythmicity in Chlamydomonas reinhardii under photoautotrophic (HSM) and photoorganotrophic (HSA) conditions to explore the influence of metabolic context on rhythmic behavior. Cultures were grown under controlled conditions (LD 12:12, 28 °C). Phototaxis was monitored using an automated multi-channel system that continuously recorded light transmission changes associated with photoaccumulation. Cell density and chlorophyll a were also quantified. Organisms incubated in continuous dim light showed a robust free-running circadian rhythm (∼24 h) with phase-dependent responses to prior light-dark entrainment, with the maximum rate of phototaxis occurring during the subjective day. The phototaxis rhythm could also be initiated by a decrease in light intensity, and showed temperature compensation for organisms in photoorganotrophic and phototoautotrophic growth media respectively over a temperature range 17°- 28 °C. Time series of phototactic activity were analysed using the GaMoSEC framework-a multiscale wavelet-based approach for identifying periodicities and quantifying rhythmic phase and power. The rhythm persisted longer under photoautotrophic conditions and was temperature-compensated (Q10 of 1.01 for HSM- and 1.07 for HSA-grown organisms). Wavelet analyses also revealed faster oscillations (60-80 min) superimposed on the circadian component, particularly under photoorganotrophic growth. These findings demonstrate that phototactic rhythmicity in C. reinhardii is influenced by metabolic conditions and light history and that GaMoSEC analysis effectively captures multiscale temporal organization in biological time series.

Keywords: Chlamydomonas; Circadian; GaMoSec wavelet analyses; Oscillations; Phototaxis; Rhythms; Ultradian.

Fig. 1

Representation of the experimental setup. (a) The apparatus used for measuring phototaxis: the complete system. (b) photograph of the setup: The phototaxis 4 x 3 array of Petri dishes containing the samples of Chlamydomonas reinhardii in position on the reciprocal shaker under the overhead strip lights illuminating the test samples, (c)A single channel, and (d) table indicating the state of the apparatus used for the sequence of operation during and between gradient measurements.

Fig. 2

Change in cell number during photoautotrophic growth of Chlamydomonas reinhardii and on subsequent exposure to continuous dim light. Dynamics of (○) cell number; (●) fluorescence spectra of whole cells; (□) extracted chlorophyll a expressed in micrograms/ml and obtained by spectrophotometric determination, and (■) in vivo fluorescence (a.u./micrograms chlorophyll a/ml). ZT, Zeitgeber time; CT, circadian time; ss, factorial increase in cell number; light period (13,000 lux); dark period; continuous dim light (3,000 lux).

Fig. 3

Phototaxis of Chlamydomonas reinhardii measured at different circadian times. Organisms were grown under photoorganotrophic conditions (LD: 12,12; 13,000 lux) using HSM medium before measurements of phototaxis were made under dim continuous illumination (3,000 lux). Photoaccumulation of organisms gradually occluded the test light leading to a decreased photodiode signal. CT0, transition from dark to subjective dawn; CT 6, middle of subjective day; CT12 transition from subjective day to subjective night; CT18 middle of subjective night; temperature, 28 °C throughout.

Fig. 4

Circadian rhythm of rates of phototaxis in Chlamydomonas reinhardii following growth under light-dark cycles (L, D; 12,12; 13,000 lux) synchronized by the LD cycles. Organisms were growing either under photoautotrophic, HSA (a,b,e) or photoorganotrophic conditions, HSA (c, d, f) On transfer to the phototaxis set-up, they were exposed to continuous (a–d) 3000 or 13000 lux from the overhead lights. During measurement of phototaxis (5 min once an hour), these overhead lights would turn off. Note that (a) and (c) were transferred at the beginning of the subjective day, while (b) and (d) at the beginning of subjective night. White/grey bars indicate subjective day L, D: 12, 12: light, dark; 12 h, 12 h ▱ , ▯. Temperature was 28 °C throughout. Waiting time of 30s. Gradient was calculated from the decrease in light transmitted over time. The y-axes refer to appropriately scaled measurement of rates of phototaxis. T values indicate periods of rhythms.

Fig. 5

Circadian rhythm of phototaxis in Chlamydomonas reinhardii following growth in continuous light (LL, 13,000 lux) under photoautotrophic conditions using HSM medium (a–c), or photoorganotrophic conditions using HSA medium (d–f). Phototaxis measurements were initiated at time 0 at which point, light intensity was decreased to 3,000 lux. Cultures were in early- (a, d), mid- (b, e) exponential or stationary (c, f) phase of growth; temperature, 28 °C throughout.

Fig. 6

Effect of temperature on the free-running rhythm of phototaxis in Chlamydomonas reinhardii following growth under photoautotrophic conditions using HSM Medium (LL, 13,000 lux; 28 °C). Phototaxis measurement was initiated at time 0 when light intensity was decreased to 3,000 lux and the temperature was either maintained at 28 °C (a), or decreased to 21 °C (b), or 17 °C (c). T values indicate periods of rhythms. Prior to the single drop in light intensity, cultures were grown for at least 10 cell divisions in continuous light of 13,000 lux.

Fig. 7

Analysis using GaMoSEC of phototaxis in Chlamydomonas reinhardii following growth in continuous light (LL, 13,000 lux) under photoorganotrophicanic conditions using HSA medium (a) Representative time series (same as in Fig. 5e) and the first three steps of GaMoSEC, namely the Gaussian continuous wavelet transform cwt, (b), the real part of the complex Morlet cwt (c) and Synchrosqueezing. A strong daily rhythm is observable at the 24th scale (y axis) with the 3 methods. Peak activity estimated with the Complex Morlet cwt (panel c, ‘‘x’’) is observed daily between 13.3 and 15.5 h showing a strong daily rhythm with peak activity estimated with the between ZT 13.3 and 15.5 h; also indicated in green bars in (a), with a period of 22.8 h as estimated with Synchrosqueezing (yellow horizontal line in panel (d). Fluctuations at shortest time scales are evident as explored in Fig. 7.

Fig. 8

Higher-frequency fluctuations of phototaxis in Chlamydomonas reinhardii. (a) Zoom-in on the representation of the example time series presented in Fig. 5e, and analysed Fig. 7 (inset). Here the Gaussian cwt is not shown (see Supplementaty Fig. 3). (b) The real part of the Morlet shows at the 1 h scale a characteristic pattern of blue (valleys) and red (peaks) in the oscillation. (c) Syncrosqueezing shows that the frequency varies over time. (d) The dotted line shows the ridge marking the estimated period of the fluctuation, with the grey background highlighting the observed range. (e) Empirical wavelet decomposition confirming the fluctuation with a period of close to 1 h.