A systematic survey in Arabidopsis thaliana of transcription factors that modulate circadian parameters

Abstract

Background: Plant circadian systems regulate various biological processes in harmony with daily environmental changes. In Arabidopsis thaliana, the underlying clock mechanism is comprised of multiple integrated transcriptional feedbacks, which collectively lead to global patterns of rhythmic gene expression. The transcriptional networks are essential within the clock itself and in its output pathway.

Results: Here, to expand understanding of transcriptional networks within and associated to the clock, we performed both an in silico analysis of transcript rhythmicity of transcription factor genes, and a pilot assessment of functional phenomics on the MYB, bHLH, and bZIP families. In our in silico analysis, we defined which members of these families express a circadian waveform of transcript abundance. Up to 20% of these families were over-represented as clock-controlled genes. To detect members that contribute to proper oscillator function, we systematically measured rhythmic growth via an imaging system in hundreds of misexpression lines targeting members of the transcription-factor families. Three transcription factors were found that conferred aberrant circadian rhythms when misexpressed: MYB3R2, bHLH69, and bHLH92.

Conclusion: Transcript abundance of many transcription factors in Arabidopsis oscillates in a circadian manner. Further, a developed pipeline assessed phenotypic contribution of a panel of transcriptional regulators in the circadian system.

Figure 1

Oscillation peaks of various transcription factors. Transcripts from 42 transcription factors oscillate with certain peaks in circadian phase of one subjective diurnal day. The peak expression of these genes is illustrated (ZT h). MYB, bHLH, and bZIP transcription factors are colored in pink, yellow, and blue, respectively. Each trace illustrating the cycling patterns of expression is shown on the right side of this figure.

Figure 2

Representative leaf movement data for lines expressing a clock phenotype. Leaf movement rhythms were assayed under constant light for approx. 1 week (n = 14–28). (A, C, E) Representative traces of rhythmic leaf movement of wild-type (blue circles) and ox-lines (other colored symbols) are shown. (B, D) The phase angles normalized to a 24-h cycle (CT phase) are plotted with relative amplitude errors (RAE), which indicate the robustness of the rhythm (the lower the RAE the more robust the rhythm). The center of the circle represents a high RAE (= 1). (A, B) MYB3R2-ox, (C, D) bHLH69, and (E) bHLH92.

Figure 3

Confirmation of transcriptional clock phenotypes under constant light. Seedlings harboring CCA1:LUC or CCR2:LUC reporter genes were monitored under constant light for 4–5 days. Representative traces of rhythmic expression of ox-plants (pink squares) and wild-type (blue circles) are shown. (A, B) MYB3R2-ox, (C, D) bHLH69-ox. (A, C) CCA1:LUC, (B, D) CCR2:LUC.

Figure 4

Confirmation of transcriptional clock phenotypes in constant dark. Seedlings harboring CCA1:LUC or CCR2:LUC reporter genes were monitored in constant darkness for 4–5 days. Representative traces of rhythmic expression of oxplants (pink squares) and wild-type (blue circles) are shown. (A, B) MYB3R2-ox, (C, D) bHLH69-ox. (A, C) CCA1:LUC, (B, D) CCR2:LUC.

Figure 5

Clock-gene expression profiles in selected transcription factor misexpression lines. Seedlings from ox- and wild-type plants were harvested every 4 hours. Total RNA was the substrate for RT-PCR against the coding regions of the core-clock genes CCA1, LHY, GI, or TOC1, and as a control, TUBULIN (TUB). Results are presented as proportional to the average value after normalization with respect to TUB. Expression profiles in the control, MYB3R2-ox and bHLH69-ox were represented as blue, orange, and pink lines, respectively. (A) LHY, (B) TOC1, (C) CCA1, and (D) GI.

Figure 6

Transcript accumulation of transcription factors that have clockcontrolling properties. Replicate seedlings from wild-type plants maintained under constant light were harvested every 4 hours. Total RNA was the substrate for RT-PCR of the coding regions of the transcription factors MYB3R2, and bHLH69 and bHLH92. Results are presented as proportional to the average value after normalization with respect to TUB. (A) MYB3R2, (B) bHLH69, and (C) bHLH92.