Article Figures & Data
Figures
- Figure 1.
Diurnal regulation makes major contributions to cold-responsive transcriptome differences between experiments. PCA was performed on several independent studies investigating gene expression after 1 d of cold treatment (Table I). GCRMA expression estimates (Wu et al., 2004) were used to calculate the cold minus control log2 differences. Probe sets that were detected in at least one experiment were retained. Data were mean centered and plotted using classical PCA (Stacklies et al., 2007). Samples from each experiment are denoted by letters, with lowercase denoting soil-grown plants. Colors indicate the light regime: red, continuous light for control and cold; blue, diurnal for control and continuous light for cold; green, diurnal for control and cold. A, PC 1 versus PC 2. B, PC 3 versus PC 4. Axis labels indicate the proportion of the total variance explained by each PC and the P value (Fisher's exact test) for the significance of the overlap between the top 500 genes contributing to it and those that are diurnally regulated (Blasing et al., 2005; Table II). [See online article for color version of this figure.]
- Figure 2.
Circadian-regulated genes make coordinated phase-specific contributions to the major differences between experiments. Following PCA (Fig. 1) to identify the main differences between independent experiments to identify cold-responsive genes, we extracted the top 500 genes contributing to PC 1 to PC 5. These genes were separated into those that contributed positively (blue) or negatively (red) to the separation. To visualize the time of day these genes have maximum expression, the numbers and the phases of those genes classified as circadian regulated (Edwards et al., 2006) are plotted for each PC. [See online article for color version of this figure.]
- Figure 3.
The oscillations of circadian clock components are dampened in light-dark cycles in the cold. Targeted expression analysis for several circadian clock (black panel labels), circadian output (dark red panel labels), and cold-regulated (blue panel labels) genes was performed using qRT-PCR. Plants were grown under warm diurnal conditions under normal light in long days (16 h) before transfer to 4°C at 8 h after dawn. Whole rosettes were sampled from individual plants every 4 h across the 1st d in warm conditions and for days 1, 2, 7, and 14 in the cold. The y axes show raw expression (Ct; log2 scale) values normalized by subtracting the mean of three control genes. The x axes show time after dawn, with night shown in dark gray. Data are means from three biological replicate plants. sd values are not shown for clarity but averaged 0.3 Ct.
- Figure 4.
The oscillations of circadian clock components are stopped in continuous light in the cold. Targeted expression analysis for several circadian clock (black panel labels), circadian output (dark red panel labels), and cold-regulated (blue panel labels) genes was performed using qRT-PCR. Plants were grown under warm diurnal conditions under low light in long days (16 h) before transfer to continuous light at 20°C or 4°C at 14 h after dawn. Whole rosettes were sampled from individual plants every 4 h until 58 h. The y axes show raw expression (Ct; log2 scale) values normalized by subtracting the mean of three control genes. The x axes show time after subjective dawn, with subjective night shown in light gray. Data are means from three biological replicate plants. sd values are not shown for clarity but averaged 0.5 Ct.
- Figure 5.
Diurnal gating of cold-responsive TFs. qRT-PCR for 1,880 Arabidopsis TFs was used to select strongly cold-responsive TFs (>4-fold change) using pooled samples from two independent experiments. Data are presented for the 60 TFs that were subsequently confirmed to change significantly using within-experiment biological replicates. Prior to the experiments, plants were grown under warm diurnal conditions at either low or normal light in long days (16 h). Plants were then transferred to 4°C (or simulated control transfer) at 2 h (ZT2) or 14 h (ZT14) after dawn. Whole rosettes were sampled from control plants before cold (ZT2 and ZT14), from paired diurnal controls (ZT5 and ZT17), or from plants cold treated for 3 h at ZT2 (Cm) or ZT14 (Ce). The sampling scheme and sample names are illustrated at the bottom. Only the 56 up-regulated and four down-regulated TFs that were significantly (P < 0.05, t test) induced at least 4-fold against both controls in both experiments for either ZT2 and/or ZT14 are shown. Normalized values were compared to generate log2 ratios between samples of interest, and these were used to plot heat maps. The left panel shows cold induction versus the time zero and paired control for each experiment, indicating gating of relative induction. The first column of the right panel shows absolute gating as the difference between the expression attained after morning cold treatment at ZT2 (Cm) versus evening cold treatment at ZT14 (Ce). The second column reveals diurnal regulation by the difference in expression between ZT2 and ZT14. Data are mean log2 ratios from five replicates.
- Figure 6.
Experiment-specific bias in the cold response of circadian-regulated genes that peak at different phases of the day. The overlap between circadian-regulated genes that peak at different phases (Edwards et al., 2006) of the day (ZT, time after subjective dawn) and those responding to cold in independent studies (Table I) were compared. For direct comparability, we selected the 1,000 most induced (blue) and 1,000 most repressed (red) genes in each experiment and made the comparison using Fisher's exact test. Experiments are lettered as in Table I and labeled as in Figure 1; lowercase letters denote soil-grown plants. Colors indicate the light regime: red, continuous light for control and cold; blue, diurnal for control and continuous light for cold; green, diurnal for control and cold. The bars show the log odds ratios, which show whether the genes at a specific phase are more or less likely to be cold responsive than expected by chance. Significance (false discovery rate-corrected P < 0.05) is denoted by solidly colored bars, while nonsignificant log odd ratios are shown in hatched bars. [See online article for color version of this figure.]
- Figure 7.
Simple model to illustrate the time-of-day effects on the identity of cold-responsive genes. In the cold, many genes, particularly of the core oscillator, show low-amplitude cycles in diurnal conditions, while in continuous light (circadian conditions) they stop to cycle. Therefore, even when paired controls are used, there are considerable time-of-day effects on measured gene expression changes. In reality, diurnal gating of gene expression, phase advances, and delays as well as the continued cycles of many genes mean that time-of-day influences will be much greater and more diverse than illustrated. [See online article for color version of this figure.]
Tables
- Table I.
Cold treatment expression profiling data sets used in this study
Experimental details are shown for the 11 data sets used to investigate the main contributions to variation in the identity of cold-responsive genes. Labeling is as in Figure 1. Experiments are denoted by letters, with lowercase indicating soil-grown plants. Bold, italic, and underlined typefaces indicate the light regime: bold, continuous light for control and cold; italic, diurnal for control and continuous light for cold; underlined, diurnal for control and cold. The Light columns show both intensity (μmol m−2 s−1) and duration. The Age column gives the age in days (d) or, where available, the growth stage (Boyes et al., 2001). MS, Murashige and Skoog; NA, not applicable.
Identifier Control Plants Cold Treatment Tissue Reference Medium Temperature Light Age Temperature Time Light Start A MS 24°C 150 18 d 3°C 24 h 60 ZT3 Shoot Kilian et al. (2007) Liquid 16 h 24 h B MS 22°C NA 14 d 0°C 24 h NA ZT12 Seedling Lee et al. (2005) Agar, 3% Suc 16 h 24 h C MS 21°C 100 1.1 4°C 24 h 100 NA Shoot Nottingham Arabidopsis Stock Centre Agar 24 h 4 24 h NASCARRAYS-70 D B5 24°C 100 10 d 4°C 24 h 25 NA Seedling Vogel et al. (2005) Agar 24 h 24 h e Soil 22°C 100 18 d 4°C 24 h 25 NA Shoot Vogel et al. (2005) 24 h 24 h f Soil 20°C 150 3.70 4°C 22 h 90 ZT14 Shoot New 18°C 16 h 16 h g Soil 20°C 150 3.7 4°C 26 h 90 ZT14 Shoot New 18°C 16 h 0 16 h h Soil 20°C 150 3.90 4°C 24 h 90 ZT8 Leaf discs New 18°C 16 h 16 h i Soil 20°C 125 21 d 4°C 24 h 125 ZT2 Shoot Kaplan et al. (2007) 15 h 15 h j Soil 21°C 150 3.70 4°C 22 h 90 ZT4 Shoot New 16°C 16 h 16 h k Soil 21.5°C NA 1.12 4°C 24 h Same ZT4 Shoot Nottingham Arabidopsis Stock Centre 8 h 8 h NASCARRAYS-24 - Table II.
Significant overlap between diurnal-, circadian-, and Suc-regulated genes and those contributing to variance between cold experiments
Following PCA (Fig. 1) to identify the major differences between independent experiments to identify cold-responsive genes, we extracted the top 500 genes contributing to PC 1 to PC 5. These genes were compared with diagnostic sets of diurnal-regulated (Blasing et al., 2005), circadian-regulated (Edwards et al., 2006), and Suc-regulated (Solfanelli et al., 2005) genes, and the significance of the overlap was calculated. Absolute numbers of genes as well as P values from Fisher's exact test are shown. The numbers of genes in parentheses indicate the size of each diagnostic gene set. Venn diagrams showing the overlap between gene lists are shown in Supplemental Figure S2.
PC Diurnal (3,409) Circadian (3,480) Suc (1,890) PC 1 303 4.4 × 10−88 255 1.2 × 10−51 137 1.5 × 10−23 PC 2 342 1.2 × 10−122 240 9.4 × 10−43 263 1.3 × 10−118 PC 3 259 4.8 × 10−56 161 2.4 × 10−09 123 3.1 × 10−17 PC 4 308 3.5 × 10−92 245 1.2 × 10−45 133 9.8 × 10−22 PC 5 234 7.3 × 10−41 152 4.3 × 10−07 120 6.9 × 10−16 - Table III.
The induction of the CBF TFs is diurnally gated
Expression of the CBF TFs was measured using qRT-PCR. Plants were grown under warm diurnal conditions under normal or low light in long days (16 h) before transfer to 4°C (or simulated control transfer) either at 2 h (ZT2) or 14 h (ZT14) after dawn. At both points, samples were harvested after 3 h at 4°C (Cm and Ce) and at 0 h (ZT2 and ZT14) and 3 h (ZT5 and ZT17) in control conditions. The sampling scheme is illustrated in Figure 5. Data are mean log2 ratios (n = 5). In low light, CBF1 was not detected at ZT2 generating infinite ratios (±inf).
CBF Cold Induction Morning Versus Evening Low Light Normal Light Low Light Normal Light Cm–ZT2 Cm–ZT5 Ce–ZT14 Ce–ZT17 Cm–ZT2 Cm–ZT5 Ce–ZT14 Ce–ZT17 Cm–Ce ZT2–ZT14 Cm–Ce ZT2–ZT14 CBF1 +inf 8.3 10.5 9.7 8.5 8.4 5.7 5.2 −2.5 −inf 1.7 −1.1 CBF2 9.7 8.6 8.5 9.1 10.3 8.5 4.9 6.9 2.8 1.5 3.1 −2.3 CBF3 13.0 8.1 5.3 7.4 10.2 7.5 2.9 5.8 3.3 −4.4 4.0 −3.3
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figure 1
- Supplemental Data - Supplemental Figure 2
- Supplemental Data - Supplemental Figure 3
- Supplemental Data - Supplemental Figure 4
- Supplemental Data - Supplemental Figure 5
- Supplemental Data - Supplemental Figure Legends
- Supplemental Data - Supplemental Table I
- Supplemental Data - Supplemental Table II
- Supplemental Data - Supplemental Table III
