Positive and negative factors confer phase-specific circadian regulation of transcription in Arabidopsis

Abstract

The circadian clock exerts a major influence on transcriptional regulation in plants and other organisms. We have previously identified a motif called the evening element (EE) that is overrepresented in the promoters of evening-phased genes. Here, we demonstrate that multimerized EEs are necessary and sufficient to confer evening-phased circadian regulation. Although flanking sequences are not required for EE function, they can modulate EE activity. One flanking sequence, taken from the PSEUDORESPONSE REGULATOR 9 promoter, itself confers dawn-phased rhythms and has allowed us to define a new clock promoter motif (the morning element [ME]). Scanning mutagenesis reveals that both activators and repressors of gene expression act through the ME and EE. Although our experiments confirm that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are likely to act as repressors via the EE, they also show that they have an unexpected positive effect on EE-mediated gene expression as well. We have identified a clock-regulated activity in plant extracts that binds specifically to the EE and has a phase consistent with it being an activator of expression through the EE. This activity is reduced in CCA1/LHY null plants, suggesting it may itself be part of a circadian feedback loop and perhaps explaining the reduction in EE activity in these double mutant plants.

Figure 1.

The EE Is Necessary and Sufficient for Evening-Phased Rhythms. (A) Reporter constructs were made with four copies of either wild-type or mutant EE sequences (shown) placed upstream of the NOS minimal promoter and modified firefly luciferase. (B) generic_EE and generic_EEmt T1 transformants were grown on selective medium in 12-h-light/12-h-dark cycles for 8 d and then transferred to constant white light. Luminescence levels were monitored every 2.5 h for 5 d. Period, phase, and relative amplitude error (RAE; a measure of the robustness of the rhythm) were calculated according to the method of Plautz et al. (1997). The average luminescence (±se of the mean) levels for the numbers of T1 plants noted in (A) are plotted. Data on additional T1 plants are presented in Table 1. generic_EE plants are plotted on the primary and generic_EEmt plants are plotted on the secondary y axis. (C) CCR2_EE and CCR2_EEmt T1 plants were imaged and analyzed as described in (B). (D) Circular plot of the phase and RAE for all generic_EE (n = 56) and CCR2_EE (n = 69) T1 plants that returned a rhythm. Phase is indicated in CT (phase/period × 24 h) and RAE is graphed radially such that plants with RAE = 1 (no significant rhythm detected; error in the amplitude equals the amplitude value itself) would be graphed at the center of the circle and plants with RAE = 0 (very robust rhythms with an infinitely well determined rhythmic component) would be graphed on the periphery of the outermost circle.

Figure 2.

The PRR9 Promoter Contains MEs and EEs. (A) Four copies of either the wild-type EE-containing region of the PRR9 promoter or various mutants were placed upstream of luciferase, and T1 plants were assayed as described in Figure 1. The EE is indicated with red underlined text. The experimentally defined ME (this work) is underscored with a black line. (B) Luciferase data for PRR9_EE and PRR9_EEmt plants. Data from each plant were normalized to its median expression level. Averages (± se) of all plants that returned an RAE (i.e., for which a rhythm was detected) in this experiment are depicted, with PRR9_EE plants plotted on the primary y axis and PRR9_EEmt plants plotted on the secondary y axis. (C) Average luminescence for T1 plants transformed with the mutant constructs indicated in (A). No RAE cutoff was applied, but only plants with detectable luciferase activity were considered. EE_scan4 plants are plotted on the secondary y axis, and all other lines are plotted on the primary y axis. The number of plants averaged for the data depicted in (B) and (C) is indicated in (A); data for additional T1 plants is summarized in Table 1. (D) and (E) Phase and RAE are plotted for all PRR9 and ME constructs that conferred substantially rhythmic luciferase activity (defined as >50% of the visible T1 plants transformed with a given construct showing statistically significant rhythms in luciferase activity).

Figure 3.

Multimerized CBS Confer Evening-Phased Rhythms. (A) One nucleotide of each EE in the CCR2- and PRR9-derived multimers was mutated from T to A, creating CCR2_CBS and PRR9_CBS multimers driving luciferase expression. (B) T1 plants were assayed as described in Figure 1, except that plants were transferred to constant red light for luciferase assays. Data from each plant were normalized to its median expression level. Averages (± se) of all plants that returned an RAE (i.e., for which a rhythm was detected) are depicted. The number of plants averaged for each trace is indicated in (A). Data for additional T1 plants are summarized in Table 1. (C) Phase and RAE are plotted for all CCR2_CBS and PRR9_CBS plants for which a rhythmic component of luciferase activity was determined.

Figure 4.

CCA1 Acts Both Positively and Negatively via the EE. (A) to (D) Plants that constitutively overexpress CCA1 (Wang and Tobin, 1998) were crossed to plants that express luciferase under the control of the generic_EE (A), CCR2_EE (B), PRR9_EE (C), or PRR9_EEmt (D) multimers. F1 progeny and the parental EE:luc+ lines were assayed as described in Figure 1. The average of between 9 and 18 plants, ± se, is depicted. (E) and (F) Plants from two different CCR2_EE:luc+ lines (Col) were introgressed twice into the cca1-1 lhy-12 mutant background (Ler). F2 populations segregating for the transgene and both mutations were assayed; all plants with wild-type rhythms were compared with all plants with no significant rhythms detected. Twenty-six plants with wild-type rhythms and 24 plants with no detectable rhythms (total population = 130) were compared for (E); for (F), 23 plants with wild-type rhythms and 37 plants with no detectable rhythms (total population = 155) were compared. Averages ± se are shown.

Figure 5.

Both Positive and Negative Factors Mediate Cycling through the EE. (A) Sequence of EE mutants used in DNA binding experiments or to create luciferase reporter vectors. (B) Extracts from bacteria expressing GST-CCA1 or GST were incubated with radiolabeled double-stranded DNA containing the CCR2_EE sequence. A 5-, 15-, 50-, 150-, or 500-fold molar excess of unlabeled competitor DNA was added to each reaction as indicated. DNA and protein/DNA complexes were separated by nondenaturing gel electrophoresis and visualized using a phosphor imager. The arrowhead indicates unbound probe. (C) Binding curves for each competitor is indicated, based on data from (B) and from Supplemental Figure 1 online. (D) Average luciferase expression (± se) of visible plants is shown for T1 plants transformed with constructs with luciferase expression regulated by the mutant EE sequences indicated in (A). Experiments were performed as described in Figure 1. Plants transformed with the EE_scan2 and EE_scan6 constructs have similar expression patterns to the EE_scan1 plants and have been omitted for clarity. The number of plants averaged is indicated in (A). Data on additional T1 plants are in Table 1.

Figure 6.

A Clock-Regulated Activity in Plant Extracts Binds the EE. (A) Extracts made from Arabidopsis seedlings (Col) harvested at ZT 6 were incubated with radiolabeled double-stranded DNA containing the CCR2_EE sequence. A 5-, 15-, or 50-fold molar excess of unlabeled competitor DNA was added to each reaction as indicated. Protein/DNA complexes were separated by nondenaturing gel electrophoresis and visualized using a phosphor imager. Specificity of binding is shown by the ability of fragments with wild-type EE sequences, but not fragments in which these sequences are altered, to compete for binding to proteins in the extracts. (B) Arabidopsis seedlings (Col) were harvested at the indicated times of the subjective day and night, and extracts were made. Binding assays were performed in duplicate. A 50-fold molar excess of the indicated competitor DNAs was used. (C) Extracts were made from Col, Ler, and cca1-1 lhy-12 (Ler) plants (all harvested at ZT 8) and binding assays performed as described in (A). The arrowheads indicate unbound probe.

Figure 7.

A Secondary Loop Acts within the Plant Central Clock. CCA1 and LHY bind to the EE in many clock-regulated promoters, acting to repress transcription. They also, directly or indirectly, increase expression and/or activity of an EE binding protein (EEBP) that binds to the EE and acts as a transcriptional activator. TOC1 protein, directly or indirectly, promotes expression of CCA1 and LHY. Steps thought to occur directly are indicated by black arrows; steps that may be direct or indirect are indicated by gray arrows.