Arabidopsis FHY3 specifically gates phytochrome signaling to the circadian clock

Abstract

Circadian gating of light signaling limits the timing of maximum responsiveness to light to specific times of day. The fhy3 (for far-red elongated hypocotyl3) mutant of Arabidopsis thaliana is involved in independently gating signaling from a group of photoreceptors to an individual response. fhy3 shows an enhanced response to red light during seedling deetiolation. Analysis of two independent fhy3 alleles links enhanced inhibition of hypocotyl elongation in response to red light with an arrhythmic pattern of hypocotyl elongation. Both alleles also show disrupted rhythmicity of central-clock and clock-output gene expression in constant red light. fhy3 exhibits aberrant phase advances under red light pulses during the subjective day. Release-from-light experiments demonstrate clock disruption in fhy3 during the early part of the subjective day in constant red light, suggesting that FHY3 is important in gating red light signaling for clock resetting. The FHY3 gating function appears crucial in the early part of the day for the maintenance of rhythmicity under these conditions. However, unlike previously described Arabidopsis gating mutants that gate all light signaling, gating of direct red light-induced gene expression in fhy3 is unaffected. FHY3 appears to be a novel gating factor, specifically in gating red light signaling to the clock during daytime.

Figure 1.

Hypocotyl Length in Wild-Type and fhy3 Seedlings in Red and Far-Red Light. Seedlings were germinated in darkness for 1 d before transfer to continuous red or far-red light of the fluence rate indicated for a further 3 d. Values shown are means ± se from 30 seedlings. Col, Columbia.

Figure 2.

Hypocotyl Elongation in Constant Red Light. Wild-type and fhy3 seedlings were germinated in LD(12:12) for 3 d before transfer to Rc. Left, representative traces produced by individual seedlings recording the vertical pixel position of the apical meristem. Position 0 represents the pixel position at the start of recording. Right, percentage of traces found to be rhythmic by rhythm analysis software. Data shown are from between 6 and 17 seedlings.

Figure 3.

Mean CAB2:LUC Bioluminescence in Constant Light. Wild-type and fhy3 seedlings expressing the CAB2:LUC reporter construct were entrained in LD(12:12) for 6 d before transfer to constant monochromatic red, white, or monochromatic blue light. Each data point represents the mean of between 16 and 29 seedlings.

Figure 4.

Normalized Mean CCR2:LUC Bioluminescence in DD and in Constant Red Light. Wild-type and fhy3 seedlings expressing the CCR2:LUC reporter construct were entrained in LD(12:12) for 6 d before transfer to DD (A) or to constant red light (B). Each data point represents the normalized mean of between 17 and 59 seedlings. CPS, counts per second.

Figure 5.

Expression Patterns of CCA1, LHY, and TOC1 in Constant Red Light. Wild-type and fhy3 seedlings were entrained in LD(12:12) for 6 d before transfer to Rc. After 24 h in red light, individual batches of seedlings were harvested at 3-h intervals for a further 24 h. Expression of CCA1 (left), LHY (middle), and TOC1 (right), relative to the wild type at time 0, was measured using quantitative PCR.

Figure 6.

Effect of Red Light on Direct Induction of CAB2:LUC Expression and on Clock Control of CAB2:LUC Expression. (A) Gating of the induction of CAB2:LUC expression by a pulse of red light. Wild-type and fhy3 seedlings expressing the CAB2:LUC reporter construct were entrained in LD(12:12) before transfer to darkness at subjective dawn. CAB2:LUC bioluminescence was recorded at the indicated times immediately before seedlings were given a pulse of red light. After return to darkness, CAB2:LUC bioluminescence was recorded for a further 2 h. The mean induction of CAB2:LUC shown was calculated by subtracting basal luminescence before light treatment. Gray and black shading represent subjective day and subjective night, respectively. Each data point represents the mean of between 11 and 24 seedlings. CPS, counts per second. (B) Release from red light. Wild-type and fhy3 seedlings expressing the CAB2:LUC reporter construct were entrained in LD(12:12) for 6 d and then transferred to Rc at subjective dawn for the duration indicated before transfer to darkness, during which the timing of the first peak of CAB2:LUC bioluminescence was recorded. Each data point represents the mean ± se of between 11 and 28 seedlings.

Figure 7.

Phase-Response Curves for Red and Blue Light Resetting Pulses. Wild-type and fhy3 seedlings expressing the CCR2:LUC reporter construct were entrained in LD(12:12) for 6 d before transfer to DD at subjective dawn. After a further 24 h in darkness, individual batches of seedlings were given a 1-h pulse of red light (A) or blue light (B) at 4-h intervals over the next 24 h before being returned to darkness. Data represent the mean phase shifts (±pooled se) plotted against the circadian time of the pulse. Phase advances are plotted as positive values, and delays are plotted as negative values. The data are double plotted to clarify the shape of the phase-response curve. Each data point represents the mean of between 7 and 28 seedlings.

Figure 8.

Scheme Demonstrating the Possible Sites of Action of FHY3, TIC, and ELF3 in the Gating of Signals from the Phytochrome (PHY) and Cryptochrome (CRY) Photoreceptors. FHY3 acts to gate signals on a branch of the red light pathway mediating light input to the clock. TIC and ELF3 affect all light-signaling pathways tested. TIC and ELF3 together may act to gate both the red and blue light input pathways upstream of a divergence of a branch mediating photomorphogenic responses (light inhibition of hypocotyl elongation and direct induction of CAB2 gene expression) and a branch mediating light input to the clock.