Light-regulated translation mediates gated induction of the Arabidopsis clock protein LHY

Abstract

The transcription factor LHY and the related protein CCA1 perform overlapping functions in a regulatory feedback loop that is closely associated with the circadian oscillator of Arabidopsis: Overexpression of LHY abolished function of the circadian clock in constant light, but rhythmic expression of several circadian clock-regulated transcripts was observed under light- dark cycles. These oscillations correlated with high amplitude changes in LHY protein levels, caused by light-induced translation of the LHY transcript. Increases in LHY protein levels were also observed in light-grown wild-type plants, when light signals coincided with the circadian-regulated peak of LHY transcription at dawn. Unexpectedly, translational induction coincided with acute downregulation of LHY transcript levels. We suggest that the simultaneous translational induction and transcriptional repression of LHY expression play a role to narrow the peak of LHY protein synthesis at dawn and increase the robustness and accuracy of circadian oscillations. Strong phase shifting responses to light signals were observed in plants lacking function of LHY, CCA1 or both, suggesting that light-regulated expression of these proteins does not mediate entrainment of the clock to light-dark cycles.

Fig. 1. Diurnal rhythms of gene expression in LHY-overexpressing plants. (A) Temporal patterns of gene expression in 10-day-old wild-type (Ler) and lhy-1^TN104 plants under 12L:12D cycles. CCR2, actin (ACT2) and the overexpressed LHY transcript (LHY-ox) were assayed simultaneously by RNase protection assays. (B) CCR2 mRNA levels from (A) were quantified using a PhosphorImager and normalized to ACT2 mRNA levels. (C) Temporal patterns of CAB::luc expression in wild-type (Ler) and lhy-1 plants under light–dark cycles. Transgenic seedlings expressing a CAB::luc reporter fusion were grown under light–dark cycles of varying total duration (T = 20 h, 6.7L:13.3D; T = 24 h, 8L:16D; T = 28 h, 9.3L:18.7D) for 7 days and imaged every 2 h using a photon-counting camera. Each trace represents luminescence data from a group of 10–15 seedlings. Time scales were normalized to 24 h, to facilitate comparison between the different light–dark cycles. One normalized unit of time corresponds to the total duration of the light–dark cycle (T, in hours) divided by 24. Experiments were carried out at least twice with similar results. White and black boxes at the top of the graphs indicate the intervals of light and darkness, respectively.

Fig. 2. Light regulation of CCA1 and LHY expression in lhy-1^TN104 plants. Expression of the endogenous CCA1 and LHY transcripts was analysed by RNase protection assays. A probe to the 5′-UTR of the LHY gene was used to detect the wild-type LHY transcript, but not the overexpressed transcript, in lhy-1^TN104 plants. (A) Seven-day old etiolated wild-type and lhy-1^TN104 plants were exposed to 2 min of red light, and plants were harvested at different time intervals. Closed symbols, dark controls; open symbols, light-induced plants; circles, endogenous LHY mRNA; squares, CCA1 mRNA. (B) Wild-type (Ler) and lhy-1^TN104 plants were grown under 12L:12D cycles for 10 days then harvested at 4 h intervals. The CCA1 mRNA and the endogenous LHY transcript were analysed as described above. Open symbols, wild-type plants; closed symbols, lhy-1^TN104 plants. White and black boxes at the top of the graphs indicate the intervals of light and darkness, respectively. (C) To emphasize any possible response to light, lhy-1^TN104 mRNA levels from (B) were plotted on the same scale as in (A). (D) Expression of LHY::luc and CCA1::luc reporter constructs in Ler (open symbols) and lhy-1 mutant plants (closed symbols) grown under 12L:12D and then transferred to constant light or darkness. Each trace represents average values from 2–10 individual seedlings. Expression levels in wild-type plants are shown on the primary axis, those in lhy-1 plants on the secondary axis. Experiments were carried out at least twice with similar results.

Fig. 3. Temporal patterns of LHY protein expression in wild-type and lhy-1^TN104 plants under light–dark cycles. Plants were grown under 12L:12D light–dark cycles for 8 days prior to harvesting. Protein extracts were analysed by SDS–PAGE. The upper part of the gel (containing proteins >60 kDa) was blotted onto nylon membrane, and probed using an antibody to LHY. The bottom part of the same gel was stained using Coomassie Blue, and the most abundant band at 55 kDa, corresponding to RBCL, was used as a loading control. (A) Specificity of the LHY antiserum. Plants were harvested either 1 or 10 h after dawn (ZT 1 or ZT 10). Equal amounts of protein were loaded in each lane. Bacterially expressed LHY protein [B] was used as a size marker. (B) Temporal patterns of LHY protein expression in wild-type (Ler) and lhy-1^TN104 plants grown under 12L:12D cycles for 8 days, then harvested over a 49 h time span. The level of LHY protein was determined by western blot analysis. For wild-type samples, 100 µg of total protein were loaded in each lane. For lhy-1 samples, only 30 µg of total protein were used. Exposure times in (B) were optimized to emphasize rhythmic changes in LHY protein levels in the lhy mutant. Similar changes were detected for the experiment in (A) after shorter exposure times. (C) Quantification of protein and mRNA levels, in wild-type (Ler) and lhy-1^TN104 plants grown under 12L:12D. LHY protein levels (closed symbols) were determined by quantification of the western blot shown in (B). Total LHY transcript levels (open symbols) were determined by RNase protection assay of RNA prepared from the same plant samples, using a probe to the LHY coding region. RNA and protein levels are expressed as a percentage of their maximum levels. (D) Arrhythmic expression of the LHY protein in lhy-1^TN104 plants in constant light. Plants were grown under 12L:12D cycles for 8 days then transferred to constant light at time zero. A 50 µg aliquot of total protein was loaded in each lane. White and black boxes at the top of the graphs indicate the intervals of light and darkness, respectively. Solid and hatched white bars in (D) indicate subjective days and nights, respectively. All experiments except those in (D) were carried out at least twice with similar results.

Fig. 4. The half-life of the LHY protein was identical in light or darkness. lhy-1^TN104 plants were grown under 12L:12D cycles for 20 days, then pulse-labelled with [³⁵S]methionine for 3 h starting at ZT 3 (3 h after dawn). Chasing experiments were then carried out either in the light, or after transfer to darkness. (A) LHY protein levels were analysed after immunoprecipitation, followed by SDS–PAGE and autoradiography. Total levels of radioactivity incorporated into protein were quantified by spotting 5 µl of sample onto nylon membrane prior to immunoprecipitation. Time zero corresponds to the beginning of the chase. (B) LHY protein levels were quantified using a PhosphorImager, normalized to the number of counts incorporated into total protein, and expressed as a percentage of their initial value. The experiment in (A) was carried out three times. Data points in (B) correspond to average results from two (30, 90 and 180 min) or three (0, 60 and 120 min) experiments. Error bars represent standard deviations.

Fig. 5. Rate of LHY protein synthesis under light or dark conditions. lhy-1^TN104 plants were grown under 12L:12D cycles for 20 days prior to labelling with [³⁵S]methionine under light or darkness. Two identical experiments were carried out, starting either at dawn (ZT 0) or at dusk (ZT 12). (A and B) LHY protein levels were analysed by immunoprecipitation followed by SDS–PAGE and autoradiography. Total levels of radioactivity incorporated into protein were quantified by spotting 5 µl of sample onto a nylon membrane, prior to immunoprecipitation. Time zero corresponds to the time at which [³⁵S]methionine was added to the plants. (C and D) Quantification of the experiments in (A) and (B). The radioactivity incorporated into LHY and into total protein was determined using a PhosphorImager (A) or by densitometry (B), and normalized to counts incorporated into total protein.

Fig. 6. Light regulation of LHY expression in wild-type (Ler) plants. Seedlings were grown under 12L:12D for 8 days then transferred to constant darkness at the normal dusk (ZT 12). At different times in DD, plants were exposed to white light for 1 h and harvested after 1 h further in darkness. (A) LHY mRNA levels were determined by RNA blot analysis using a probe to the LHY coding region, quantified by PhosphorImager analysis and expressed relative to rRNA levels, determined by ethidium bromide staining of the gel. (B) LHY protein levels were assayed by immunoblot analysis and normalized to RBCL levels as described in Figure 3. Analysis of LHY protein levels was carried out three times with similar results. Hatched and solid black bars indicate subjective days and nights, respectively.

Fig. 7. Effects of 1 h light pulses on expression of the LHY::luc reporter gene. Wild-type and lhy-11 cca1-1 plants were grown under light– dark cycles for 7 days, transferred to constant darkness at ZT 12 and exposed to 1 h pulses of white light (70–80 µmol/m²/s) at the times indicated by the open arrows (A, ZT 18; B, ZT 21; C, ZT 15; and D, ZT 18). Immediate effects on the peak of LHY:luc expression were observed when light pulses were applied 2–3 h before (A and C) and after (B and D) the onset of LHY:luc expression. Dampening levels of luminescence due to exhaustion of the luciferin substrate precluded detection of further oscillations in DD. Traces represent average data from individual seedlings or from groups of 10–15 seedlings, normalized to trough luminescence levels prior to the light pulse. Error bars indicate standard errors, and N indicates the number of plants analysed in each experiment.

Fig. 8. Phase shifting responses to altered light–dark cycles in plants lacking function of LHY, CCA1 or both. Transgenic plants carrying a CCR2::luc reporter construct were grown under 12L:12D for 7 days prior to imaging. From time 24 h, the light–dark cycle was inverted, resulting in a 12 h phase shift. The experiment was carried out twice with similar results. White and black boxes at the top of the graphs indicate the intervals of light and darkness, respectively.