Accurate timekeeping is controlled by a cycling activator in Arabidopsis
- University of California, Davis, United States
Figures
Figure 1
Activation of PRR5 by RVE8 induction is stronger in the afternoon.
(A) and (B) Luciferase activity in mock (A) and DEX-treated (B) Col, rve8-1 and rve8-1 RVE8::RVE8:GR plants transgenic for the CCR2::LUC reporter. Plants were entrained in 12:12 light/dark (LD) …
Figure 2 with 1 supplement
Identification of RVE8 targets by RNA-seq. RNA-seq experimental design and data analysis.
(A) Relative timing of RVE8 induction and RVE8 protein abundance during a day. Adapted from Rawat et al. (2011). (B) Scheme of experimental design. (C) and (D) Weighted Venn diagrams of genes …
Figure 2—figure supplement 1
Expression levels as determined by RNA-seq and qRT-PCR are highly correlated.
Expression levels of selected genes defined as RVE8-regulated in the RNA-seq experiment were examined using qRT-PCR. The logarithm of fold change values in the RNA-seq and the qRT-PCR data were …
Figure 3 with 1 supplement
RVE8 activates evening genes directly.
(A)–(F) Transcript levels of evening genes in response to RVE8 induction in the absence or presence of cycloheximide (CHX). 7-day-old rve8-1 and rve8-1 RVE8::RVE8:GR plants were grown in light:dark …
Figure 3—figure supplement 1
Morning-phased genes are indirectly repressed in response to RVE8 induction.
(A)–(C) Levels of transcripts controlled by nonsense-mediated mRNA decay (NMD) in response to cycloheximide (CHX) treatment. N.D.: not detectable. (D)–(G) Transcript levels of morning genes in …
Figure 4 with 1 supplement
RVE8 functions through the EE.
(A) Circadian phase distributions of all EE-containing CCGs and RVE8-induced EE-containing CCGs. The RVE8-induced EE-containing CCGs are enriched for an earlier phase than that of all EE-containing …
Figure 4—figure supplement 1
Characterization of RVE4, RVE6, and RVE8 mutant alleles.
(A)–(C) RVE4, RVE6 and RVE8 transcript levels in Col and the rve4 rve6 rve8 triple mutant. 7-day-old seedlings (about 30 plants each) were grown in 12:12 LD and harvested at ZT 0 and ZT 4. RNA was …
Figure 5 with 1 supplement
Expression of clock genes is altered in rve4 rve6 rve8 triple mutants.
(A), (B), (E), (F), (I), (J), (M) and (N) Expression of evening genes in Col and rve4 rve6 rve8. (C), (D), (G), (H), (K), (L), (O) and (P) Expression of morning genes in Col and rve4 rve6 rve8. (A)–(…
Figure 5—figure supplement 1
Clock gene expression in wild type and the rve4 rve6 rve8 mutant.
(A) PRR5 expression in Col, rve6 rve8 and rve4 rve6 rve8 in LL. Seedlings were grown in LD for 7 days, released to constant light, and then harvested at the indicated times after the last …
Figure 6
RVE8 expression is likely controlled by other clock genes through PRR5, 7 and 9.
(A) RVE8 expression in Col, toc1-4, lux-1 and CCA1-OX in LD. 7-day-old seedlings were collected at the times indicated and qRT-PCR was performed. Data are double-plotted to facilitate visualization. …
Tables
Table 1
Enrichment of EE, G-box-like and ME-like motifs in CCGs regulated by RVE8 compared to their occurrence in all CCGs previously defined as either evening-phased or morning-phased (Hsu and Harmer, 2012)
| (A) Evening-phased genes (CT 8 to CT 14) | ||||||
| Motif | Sequence | CCGs (2709 genes) | RVE8-induced CCGs (278 genes) | p | ||
| Genes with the motif | Coverage (%) | Genes with the motif | Coverage (%) | |||
| Short EE | AAATATCT | 794 | 29.3 | 152 | 54.5 | <2.2 × 10−16*** |
| Long EE | AAAATATCT | 444 | 16.4 | 104 | 37.5 | 2.06 × 10−15*** |
| EE-like | AATATCT | 1360 | 50.2 | 190 | 68.2 | 7.39 × 10−09*** |
| (B) Morning-phased genes (CT 20 to CT 2) | ||||||
| Motif | Sequence | CCGs (1572 genes) | RVE8-repressed CCGs (328 genes) | p | ||
| Genes with the motif | Coverage (%) | Genes with the motif | Coverage (%) | |||
| G-box-like | BACGTRD | 1187 | 75.5 | 266 | 81.0 | 0.0317* |
| ME-like | CCACA | 1429 | 90.9 | 308 | 93.9 | 0.08297 |
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To determine whether the over-represented motifs found in RVE8 targets (Supplementary file 2) are enriched when compared to the morning-phased and evening-phased CCG groups, the number of genes containing the motif in each phase group was compared to that in the up- or down-regulated RVE8 targets. Fisher's exact test was performed to determine if the ratios in both groups are significantly different (*p<0.05; **p<0.01; ***p<0.001).
Additional files
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Supplementary file 1
RNA-seq analysis of RVE8-regulated genes.
(A) Pipeline and summary of RNA-seq data analysis. (B) Pseudo-normalized counts for visualization. Counts for each library were normalized (Robinson et al., 2010) according to the library size and represented as counts per million. (C) Genes identified as significantly induced by RVE8 by RNA-seq. Genes significantly differentially expressed (adjusted p<0.01) between RVE8:GR + DEX and RVE8:GR + mock or between RVE8:GR + DEX and rve8 + DEX, but not between rve8 + DEX and rve8 + mock, are defined as RVE8-induced or RVE8-repressed targets. (D) Genes identified as significantly repressed by RVE8 by RNA-seq. Genes significantly differentially expressed (adjusted p<0.01) between RVE8:GR + DEX and RVE8:GR + mock or between RVE8:GR + DEX and rve8 + DEX, but not between rve8 + DEX and rve8 + mock, are defined as RVE8-induced or RVE8-repressed targets. (E) Genes identified as significantly induced by DEX by RNA-seq. Genes significantly differentially expressed between rve8 + DEX and rve8 + mock are defined as DEX-induced or DEX-repressed genes (adjusted p<0.01). (F) Genes identified as significantly repressed by DEX by RNA-seq. Genes significantly differentially expressed between rve8 + DEX and rve8 + mock are defined as DEX-induced or DEX-repressed genes (adjusted p<0.01). (G) Clock genes identified as RVE8 targets in the RNA-seq experiment. Genes found to be differentially expressed in response to RVE8 induction (adjusted p<0.01). Evening-phased genes are highlighted in yellow. (H) Significantly overrepresented functional classifications among RVE8-induced genes. The enrichment of the functional terms was identified using BioMaps in Virtual Plant 1.2 (http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/) (Katari et al., 2010). Functional classifications were provided by the Munich Information Center for Protein Sequences (MIPS) (Schoof et al., 2005). All genes classified as expressed in the RNA-seq experiment were used for the background. Fisher's exact test (with FDR correction) was performed and the cut-off value for statistical significance was set to 0.01.
- https://doi.org/10.7554/eLife.00473.014
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Supplementary file 2
Promoter motifs overrepresented in RVE8-regulated CCGs (related to Table 1).
Promoters were defined as the 1500 bp region upstream of the translational start site and motifs were identified using the SCOPE motif finder (Carlson et al., 2007). Both strands were considered for calculation of significance. Background frequency was determined using all genes in the genome. (A) Up-regulated CCGs (376 genes). (B) Down-regulated CCGs (525 genes).
- https://doi.org/10.7554/eLife.00473.015
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Supplementary file 3
Primers used in this study.
- https://doi.org/10.7554/eLife.00473.016
