REVEILLE8 and PSEUDO-REPONSE REGULATOR5 form a negative feedback loop within the Arabidopsis circadian clock

Abstract

Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE-binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE-containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.

Figure 1. Circadian expression patterns of the CCA1, LHY, and RVE genes.

Expression of the RVE family of genes was assayed in a previously published microarray experiment . Seedlings were entrained in LD 12:12 for 7 days before being transferred to constant white light. Plants were harvested at the indicated times and subjected to microarray analysis . Relative expression levels of the different Myb-like genes are shown; all are graphed using the same scale. Underlined names indicate that peptides from the corresponding gene product were identified by mass spectrometry after affinity purification of EE-binding proteins.

Figure 2. RVE8 binds specifically to the EE both in vivo and in vitro.

(A, B) Four copies of wild-type or mutant EE were multimerized and used to drive expression of the HIS3 or lacZ genes in S. cerevisiae. The CCA1, RVE1, and RVE8 cDNAs were fused to the GAL4 activation domain, transformed into yeast containing the above bait vectors, and plated on media lacking histidine (A, left panel). They were also assayed for β-galactosidase activity on a filter (A, right panel) and in a liquid assay (B). (C) Recombinant RVE1 or RVE8 was incubated with a multimerized, radiolabeled, EE probe sequence. A 2, 5, 10, 20, or 50-fold molar excess of unlabeled EE or a 50-fold molar excess of unlabeled mutant EE (indicated by the letter M) was added to each reaction as competitor. Protein/DNA complexes were separated on a non-denaturing polyacrylamide gel and the radiolabeled DNA visualized using a phosphorimager. The arrowhead indicates the position of protein/DNA complexes. (D) The fraction of probe shifted in each lane was quantified and normalized to the fraction shifted in the lane with no added competitor. (E) An EMSA assay was performed with protein extracted from wild-type or plants overexpressing HA-tagged RVE8. 1 µg of anti-HA antibody was added to some reactions, as indicated. The arrow indicates the mobility of the shifted DNA/protein complex using extracts made from wild type while the arrowhead indicates the mobility of the complex using extracts made from plants overexpressing RVE8. Data are representative of three independent experiments.

Figure 3. RVE8 affects seedling growth and flowering time.

(A, B) Plants were grown in either long-day (16 hr white light/8 hr dark; LD 8:16) or short day conditions (8 hr white light/16 hr dark; SD 8:16). Plants were classified as bolting when a 1 cm bolt was observed; n = 18–25. (C–E) Seedlings were grown in darkness (DD) or constant white light (C) or in SD (D) at the indicated fluence rates for 6 days and hypocotyl lengths were measured using ImageJ. In constant light, hypocotyl lengths of rve8-1 and RVE8-OX were significantly different from Col at all except for the highest fluence rate tested (p<0.05) whereas in short days, hypocotyl lengths of rve8-1 and RVE8-OX were significantly different from Col at all fluences rates tested (p<0.0001). (E) Wild type, rve8-1, and independent rve8-1 lines transformed with a RVE8 gene driven by the native promoter were grown in constant white light at 2 mol m⁻² s⁻¹. n = 20–30 for all experiments; means ± SEM are shown. Data are representative of at least two independent experiments; ** indicates p<0.005; Student's two-tail heteroscedastic t test.

Figure 4. RVE8 protein accumulates in the subjective afternoon.

Col, RVE8::RVE8-HA and 35S::RVE8-HA seedlings were entrained in 12 hr light:12 hr dark for 6 days before being released to constant white light (55 mol m⁻² s⁻¹) (A) Plants were harvested at the indicated times and extracts were subjected to immunoblot analysis using either an anti-HA antibody (upper panel) or an anti-UGPase antibody (lower panel). (B) Data shown in panel (A) was quantified using ImageQuant software. Data are representative of two independent experiments.

Figure 5. Perturbation of RVE8 expression changes free-running period.

Seedlings were entrained in 12 hr light:12 hr dark for 6 days and then transferred to either constant red (44 mol m⁻² s⁻¹) (A–B), blue (19 mol m⁻² s⁻¹) (C–D), or red + blue (36 mol m⁻² s⁻¹ red and18 mol m⁻² s⁻¹ blue) (E–F) light; CCR2::LUC activity rhythms were then monitored. (A, C, E) Average luciferase activity of Col, rve8-1, and RVE8-OX plants expressing CCR2::LUC; each point is the average of 20–25 seedlings and error bars represent ± SEM. (B, D, F) RAE, a measure of rhythmic robustness (with smaller values indicating stronger rhythms) is plotted relative to free-running period. (A, B) In red light, the free-running periods of rve8-1 and RVE8-OX were significantly different from Col (Col = 24.75±0.15 hr; rve8-1 = 25.73±0.07 hr; RVE8-OX = 23.44±0.07 hr; p<0.0001 for both comparisons). (C, D) In blue light, rve8-1 had a significantly longer period than Col (Col = 24.93±0.11 hr; rve8-1 = 25.70±0.09 hr; p<0.005); RVE8-OX had a shorter period (RVE8-OX = 24.60±0.09 hr) than wild type but this was not statistically significant (p = 0.08). (E, F) In red + blue, rve8-1 had a significantly longer period and RVE8-OX had a significantly shorter period than Col (Col = 24.84±0.07 hr; rve8-1 = 25.82±0.06 hr; RVE8-OX = 24.06±0.09 hr; p<.0001 for both; Student's two-tail heteroscedastic t test used for all comparisons). These data are representative of at least two independent experiments.

Figure 6. Expression of CCA1, LHY, and TOC1 in rve8 and RVE8-OX.

Seedlings were entrained 12 hr light:12 hr dark for 6 days before being transferred to continuous white light and harvested at the indicated times. Expression of CCA1 (A, D), LHY (B, E), and TOC1 (C, F) was determined using qRT-PCR in (A–C) Col, rve8-1, RVE8-OX, and in (D–F) Col, rve8-1, and rve8-1 plants transformed with the RVE8 genomic region. Values are expressed relative to PP2a. Error bars represent ± SE. These data are representative of at least two independent experiments.

Figure 7. RVE8 clock function is both light- and temperature-dependent.

Seedlings were entrained in 12 hr light:12 hr dark for 6 days at 22°C before being analyzed for CCR2::LUC activity in different environmental conditions. (A–B) Plants were transferred to constant red (A) or blue (B) light of the indicated fluence rates; average free-running period at each fluence rate, ± SEM is indicated. (C) Plants were maintained in the same light/dark regimen as during entrainment and then subjected to one long night before resumption of light/dark cycles. Average luciferase activity, ± SEM, is depicted. White bars represent times lights were on and grey bars times lights were off during imaging. (D) After entrainment, plants were transferred to continuous darkness. The average free-running periods were: Col = 25.87±0.14 hr; rve8-1 = 25.42±0.22 hr; RVE8-OX = 24.94±0.12 hr. The average period of RVE8-OX, but not rve8-1 plants, was significantly different from Col (p = 0.000002 and p = 0.10, respectively). (E) Seedlings were released to constant red light at the indicated temperatures; free-running period ± SEM is shown. (* indicates p<0.05; ** indicates p<0.001; Student's two-tail heteroscedastic t test used for all comparisons). These data are representative of at least two independent experiments. Note that in many cases the error bars are smaller than the symbols in the graphs.

Figure 8. RVE8 and PRR5 form a negative feedback loop.

(A, B) RVE8 binds to promoter regions containing EE motifs. Col, rve8-1 + RVE8::RVE8-HA and rve8-1 + 35S::RVE8-HA seedlings were entrained in white light/dark cycles for six days before release into continuous red light (35 mol m⁻² sec⁻¹). Plants were harvested at ZT48 (A) or ZT 32 (B); chromatin immunoprecipitations (IPs) were carried out using anti-HA and anti-GST antibodies. qRT-PCR was performed using primers that amplify the EE-containing regions of the PRR5 and TOC1 promoters and the CBS-containing region of the PRR7 promoter; primers that amplify a portion of the UBQ10 locus were used as a background control. The ratios of the amount of DNA isolated in the anti-HA IPs vs. the control anti-GST IPs are presented. (C–E) Expression of clock-associated genes in Col, rve8-1 and RVE8-OX. Plants were entrained as described for panels A–B and samples were harvested at the indicated times. Expression of PRR5 (C), TOC1 (D), and PRR7 (E) was determined using qRT-PCR. Values are expressed relative to PP2a. Similar results were obtained in two independent experiments. (F–H) Regulatory interactions between RVE8 and PRR5. (F) Relative abundance of RVE8 protein and PRR5 mRNA (re-plotted from Figure 4B and Figure 8C). (G) RVE8 transcript levels are elevated in prr5 prr7 prr9 mutants; data are derived from previously-published microarray data , . (H) RVE8 transcript levels are reduced in plants expressing PRR5 under the control of the strong viral 35S promoter. Data are derived from microarray data available at NASC (submitted by Hitoshi Sakakibara; NASCArrays experiment reference number NASCARRAYS-420). ** indicates p<0.01 and * indicates p<0.05; Student's two-tail heteroscedastic t test. Error bars represent ± SE.