The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B

Abstract

Developmental and physiological responses are regulated by light throughout the entire life cycle of higher plants. To sense changes in the light environment, plants have developed various photoreceptors, including the red/far-red light-absorbing phytochromes and blue light-absorbing cryptochromes. A wide variety of physiological responses, including most light responses, also are modulated by circadian rhythms that are generated by an endogenous oscillator, the circadian clock. To provide information on local time, circadian clocks are synchronized and entrained by environmental time cues, of which light is among the most important. Light-driven entrainment of the Arabidopsis circadian clock has been shown to be mediated by phytochrome A (phyA), phytochrome B (phyB), and cryptochromes 1 and 2, thus affirming the roles of these photoreceptors as input regulators to the plant circadian clock. Here we show that the expression of PHYB::LUC reporter genes containing the promoter and 5' untranslated region of the tobacco NtPHYB1 or Arabidopsis AtPHYB genes fused to the luciferase (LUC) gene exhibit robust circadian oscillations in transgenic plants. We demonstrate that the abundance of PHYB RNA retains this circadian regulation and use a PHYB::Luc fusion protein to show that the rate of PHYB synthesis is also rhythmic. The abundance of bulk PHYB protein, however, exhibits only weak circadian rhythmicity, if any. These data suggest that photoreceptor gene expression patterns may be significant in the daily regulation of plant physiology and indicate an unexpectedly intimate relationship between the components of the input pathway and the putative circadian clock mechanism in higher plants.

Figure 1

Organ specificity of luminescence in transgenic PHYB∷LUC seedlings. (A) NtPHYB∷LUC expression was imaged in 3-week-old, LD-grown tobacco seedlings. (B) AtPHYB∷LUC expression was imaged in 1-week-old, LD-grown Arabidopsis seedlings. (Left) Reflected-light image. (Right) Luminescence image presented in false-color (blue, low intensity; red, high intensity).

Figure 2

Circadian regulation of PHY gene expression in tobacco seedlings. Luminescence of NtPHYB∷LUC (■) and CAB2∷LUC (▵) was imaged in seedlings (A) grown under LDs, or after transfer to (B) constant light or (C) constant darkness. Open box on time axis, light interval; filled box, dark interval.

Figure 3

Circadian regulation of PHYB gene expression in Arabidopsis. Luminescence of AtPHYB∷LUC (■) and CAB2∷LUC (▵) was imaged in seedlings grown under LDs, after transfer to (A) constant light or (B) constant darkness. Open box on time axis, light interval; filled box, dark interval.

Figure 4

Rhythmic accumulation of PHYB RNA. RNA from PHYB (■) and the CAB gene family (▵) were detected by S1 nuclease protection and RNA gel blot hybridization, respectively, in extracts of tobacco seedlings (A) grown under LDs, or after transfer to (B) constant darkness or (C) constant light. Hybridization signals were quantified by PhosphorImager analysis and are normalized to the highest signal. Open box on time axis, light interval; filled box, dark interval.

Figure 5

Circadian regulation of PHYB protein synthesis and accumulation. (A) The abundance of PHYB and histone H2/2b proteins was determined by Western blot analysis, in extracts of tobacco plants grown under LD and transferred to constant light. (B) Luminescence of NtPHYB∷LUC (■) and PHYB protein quantification (□). Western blots were analyzed by scanning densitometry. PHYB protein levels were normalized to histone levels after local background subtraction. (C) Luminescence of NtPHYB∷LUC (■) and NtPHYB∷PHYB∷LUC (□) tobacco plants was measured by imaging plants grown under LDs and transferred to constant darkness at time 12 h. NtPHYB∷LUC levels are divided by three to facilitate phase comparison. (Inset) The detection of the PHYB∷LUC fusion protein (*) specifically in protein extracts of the NtPHYB∷PHYB∷LUC plants, using the PHYB-specific antiserum. The major band at 120 kDa is PHYB. Lanes a and b, NtPHYB∷LUC; lanes c and d, NtPHYB∷PHYB∷LUC; lanes a and c, harvested after 12 h dark (time 24 h); lanes b and d, harvested after 24 h dark (time 36 h). Open box on time axis, light interval; filled box, dark interval.