Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure

Abstract

Our understanding of the complex, transcriptional feedback loops in the circadian clock mechanism has depended upon quantitative, timeseries data from disparate sources. We measure clock gene RNA profiles in Arabidopsis thaliana seedlings, grown with or without exogenous sucrose, or in soil-grown plants and in wild-type and mutant backgrounds. The RNA profiles were strikingly robust across the experimental conditions, so current mathematical models are likely to be broadly applicable in leaf tissue. In addition to providing reference data, unexpected behaviours included co-expression of PRR9 and ELF4, and regulation of PRR5 by GI. Absolute RNA quantification revealed low levels of PRR9 transcripts (peak approx. 50 copies cell(-1)) compared with other clock genes, and threefold higher levels of LHY RNA (more than 1500 copies cell(-1)) than of its close relative CCA1. The data are disseminated from BioDare, an online repository for focused timeseries data, which is expected to benefit mechanistic modelling. One data subset successfully constrained clock gene expression in a complex model, using publicly available software on parallel computers, without expert tuning or programming. We outline the empirical and mathematical justification for data aggregation in understanding highly interconnected, dynamic networks such as the clock, and the observed design constraints on the resources required to make this approach widely accessible.

Keywords: biological clocks; circadian rhythms; data management; gene regulatory networks; model optimization; plant biology.

Figure 1.

The clock gene network and experimental protocols. (a) The clock gene network summarized in the activity-flow language of SBGN v. 1.0 [8], with the principal connections in the P2012 model [9]. The repressilator is denoted by green lines; morning loop components are filled yellow; LHY/CCA1, red; evening loop components, blue. Light inputs are shown in electronic supplementary material, figure S1 and all modelled connections of P2011 [10] in electronic supplementary material, figure S2. (b) Peak-normalized RNA profiles of genes depicted in (a), in plants of the Col-0 accession under a 12 h light : 12 h dark cycle (LD 12 : 12; experiment 2b of panel (c)). (c) Graphical representation of the growth conditions. Experiments 1, 4, 5, 6 and 7 used seedlings grown in LD 12 : 12 for the number of days indicated; experiments 2 and 3 used plants grown on soil in LD 12 : 12 for the number of days indicated. Sucrose concentrations, growth temperatures and genotypes tested are shown for each experiment. Open box, light interval; black box, dark interval; light grey box, predicted darkness in constant light; dark grey box, predicted light in constant darkness; red box, red light. Sampling time in ZT (h), relative to lights-on of the first day of sampling or the last dawn before experimental treatment (ZT0). Ros, rosette; sd, seedling.

Figure 2.

Clock gene expression in wild-type plants under LD cycles. Transcript levels in Col-0 and Ws-2 WT under LD 12 : 12 were measured by qRT-PCR, in experiment 2 (TiMet ros) including eight external RNA standards to allow absolute quantification in Col-0 and Ws-2 (a,c,e) and in experiment 1 (ROBuST) normalized to the ACTIN7 control in Col-4 and Ws-2 (b,d,f). Data represent transcripts of (a,b) LHY and CCA1, (c,d) PRR9, and (e,f) TOC1 and GI. Error bars show SD, for two to three biological replicates. Electronic supplementary material, figure S3 shows the data on logarithmic plots.

Figure 3.

Waveforms of clock gene expression across experiments at different plant age and in the absence and presence of exogenous sucrose. This plot compares transcript abundance of CCA1, TOC1 and GI in 12 h photoperiods in three WTs grown in different experimental conditions in different laboratories. The data are taken from the following experiments (figure 1): WS ROBuST (1, seedlings), Col4 ROBuST (1, seedlings), Col0 suc Ed (6, seedlings provided with 3% exogenous sucrose), Col0 suc McW (5, seedlings provided with 3% sucrose), Col0 TiMet ros (2B, 21 day-old rosettes), WS TiMet ros (2, 21 day-old rosettes), WS TiMet sd1 (3, 10 day-old seedlings), WS TiMet sd2 (4, 13-day-old seedlings). All plants were entrained in LD 12 : 12 (figure 1). Values for each transcript are normalized to the peak. The results are the mean of duplicate or triplicate samples, double-plotted; error bars are not shown for clarity.

Figure 4.

Range of transcript abundance for clock genes in clock mutants. The bars show the highest and lowest mean values for the absolute abundance of transcripts for clock genes in a given genotype. The genotypes are, from left to right, Col-0 wild-type, gi-201, prr9 prr7 double mutant, toc1, WS WT, lhy cca1 double mutant (from experiments 2 and 2B of figure 1c, 21-day-old rosettes) and WS (designated WS_2) and elf3 from experiment 3 (13-day-old seedlings), (a) LHY, (b) CCA1, (c) PRR9, (d) PRR7, (e), PRR5, (f), TOC1, (g) LUX, (h) GI, (i) ELF3, (j) ELF4. The underlying data are as in figures 5 and 6.

Figure 5.

Clock gene expression in wild-type plants and clock mutants in LD, and after transition to constant light (LL) or darkness (DD). Col-0 and Ws-2 WT, the lhy-21 cca1-11 and prr7-3 prr9-1 double mutants, and the toc1-101 and gi-201 single mutants were grown in a 12 h photoperiod for 20 days, harvested through a LD cycle and then transferred to LL (a–j) or DD (k–t; TiMet ros, dataset 2 of figure 1c). Transcript levels for clock genes were measured by qRT-PCR, including eight external RNA standards to allow absolute quantification. (a,k) LHY, (b,l) CCA1, (c,m) PRR9, (d,n) PRR7, (e,o), PRR5, (f,p), TOC1, (g,q) LUX, (h,r) GI, (i,s) ELF3, (j,t) ELF4. The results are the mean of duplicate samples, error bars show SD. Open box, light interval; black box, dark interval; light grey box, predicted darkness in LL; dark grey box, predicted light in DD.

Figure 5.

Clock gene expression in wild-type plants and clock mutants in LD, and after transition to constant light (LL) or darkness (DD). Col-0 and Ws-2 WT, the lhy-21 cca1-11 and prr7-3 prr9-1 double mutants, and the toc1-101 and gi-201 single mutants were grown in a 12 h photoperiod for 20 days, harvested through a LD cycle and then transferred to LL (a–j) or DD (k–t; TiMet ros, dataset 2 of figure 1c). Transcript levels for clock genes were measured by qRT-PCR, including eight external RNA standards to allow absolute quantification. (a,k) LHY, (b,l) CCA1, (c,m) PRR9, (d,n) PRR7, (e,o), PRR5, (f,p), TOC1, (g,q) LUX, (h,r) GI, (i,s) ELF3, (j,t) ELF4. The results are the mean of duplicate samples, error bars show SD. Open box, light interval; black box, dark interval; light grey box, predicted darkness in LL; dark grey box, predicted light in DD.

Figure 6.

Clock gene expression in wild-type plants and elf3 mutants in LD. Ws-2 WT (solid lines) and elf3–4 mutant plants (dashed lines) were grown in a 12 h photoperiod for 12 days and harvested through one LD cycle (TiMet sd, dataset 3 of figure 1c). Transcript levels for clock genes were measured by qRT-PCR, including eight external RNA standards to allow absolute quantification. (a) LHY, (b) CCA1, (c) PRR9, (d) PRR7, (e), PRR5, (f), TOC1, (g) LUX, (h) GI, (i) ELF3, (j) ELF4. The results are the mean of duplicate samples. Error bars show SD.

Figure 7.

Phase plane diagrams reveal pairwise gene interactions. (a–c) Normalized RNA profiles of figure 3 are represented as phase plane diagrams, plotting (a) GI and TOC1, and TOC1 and CCA1 on (b) linear and (c) logarithmic scales. Larger markers indicate ZT0 datapoint, arrows indicate the direction of time. (d–f) RNA profiles of figure 5 are represented as phase plane diagrams on logarithmic scales, plotting data for ELF4 and PRR9 (d) in wild-type Col plants under LD and LL (0–22 h in figure 5, dashed line; 24–70 h, solid line), and (e) in Col plants under LD and lhy cca1 double mutants under LD and LL (solid blue line), with (f) a rescaled view of a subset of the data from the lhy cca1 double mutants. Larger markers indicate 0 (ZT0) and 12 h (ZT12) datapoints in the cycle labelled LD. These timepoints are equivalent to 24 (ZT0) and 36 h (ZT12) in the cycle labelled LL. Arrows indicate the direction of time. (d) Red dashed line marks falling ELF4 levels during the night-time trough of PRR9 in LD. (f) Red dashed line marks correlated PRR9 and ELF4 levels; arrowheads mark an earlier peak on each cycle in PRR9. Timepoints 48 (ZT24) to 70 h (ZT46) under LL are plotted in brown to emphasize the similar profiles on successive days.

Figure 8.

Computational infrastructure for systems chronobiology. Customized wizards in the Pedro XML editor capture detailed metadata (right panel, showing CCA1 : LUC in sample wizard). Rather than filling 3705 metadata fields for this experiment, as a naive spreadsheet would require, Pedro captures the information with only 156 entries. After uploading the metadata and numerical data to BioDare, results can be displayed in the web browser (centre panel) with powerful secondary processing functions. The left-hand sidebar in this screen has shortcuts to common tasks and recent activity. A naive text search for ‘CCA1’ returned 394 experiments (exp'ts), whereas BioDare's ‘aggregate’ function retrieved six specific results by searching the structured metadata, with secondary filters. The search shown (right panel) aggregated qPCR assays of CCA1 in wild-type plants (see main text) including datasets 1, 3, 4 and 6 of figure 1c. The export button above the graph downloads the data shown to a spreadsheet-compatible file.

Figure 9.

Model re-optimization. Comparison of measured transcript levels from figure 5 (experimental data, symbols), with simulation of models P2011.1.2 (old model, dotted line) and P2011.2.1 (new model, solid line), which resulted from fitting to these data using SBSI. 0–24 h, LD; 24–72 h, LL. (a) LHY and CCA1 transcripts are combined in the model, so the average of LHY and CCA1 data is plotted. The peak of LHY/CCA1 under LL was delayed in the P2011.1.2 model (52.4 h) relative to the peak in the data (50 h), which was closely matched by the P2011.2.1 model (50.5 h). (b) GI transcript, (c) TOC1 transcript and (d) PRR7 transcript in Col-0 WT. (e) PRR7 transcript in the toc1 mutant shows a greater phase-advance in LL than either model. Chi-square cost value for match to TiMet ros Col-0 data in LD-LL was 20.2 for P2011.1.2, 7.6 for P2011.2.1. Chi-square cost for match to TiMet ros toc1 data in LD-LL was 39.7 for P2011.1.2, 13.1 for P2011.2.1.