Temporal changes of global gene expression during an extended night. The plots show the changes of transcript levels of the 200 most strongly induced and the 200 most strongly repressed genes. Line shading is used to distinguish genes that respond early and later in the treatment.

K-means clustering of the responses of sugar-responsive genes during the diurnal cycle and an extended night. The 200 genes that are induced and the 200 genes that are repressed most strongly 3 h after adding 15 mm Suc to C-depleted seedlings were downloaded from Osuna et al. (2007) and corresponding sets of genes for the responses 3 h after adding 100 mm Glc to C-starved seedlings and after illuminating 5-week-old plants for 4 h in 350 or 50 ppm [CO₂] were downloaded from Bläsing et al. (2005). The genes were combined to provide a test set of 484 C-induced genes and 383 C-repressed genes. The responses of these genes in the diurnal cycle (Bläsing et al., 2005) and the extended night were extracted from Supplemental Table S4. All transcript levels were normalized on the level at the end of the night in the same experiment and K-means clustered. Clusters for C-repressed (clusters 1–7) and C-induced (clusters 8–14) genes are shown. The numbers of genes in each cluster are indicated.

Ontology-based overview of the global responses of transcript levels during a diurnal cycle and an extended night. Transcript levels were expressed as a log₂ ratio compared to the value at the end of the night in the same experiment. The response was averaged across all data points available for that time point. This procedure was repeated for each time point during the diurnal cycle and each time point during the extended night treatment. The left-hand image shows the average change of the transcript level (on a log₂ scale) for all of the genes in a given BIN or subBIN. An increasingly deep blue or deep red color indicates an increasingly large average increase or decrease, respectively, of all the transcripts in a given BIN or subBIN. The right-hand image shows the P value (calculated using Wilcoxon's test) that the changes of transcript levels of genes in a given BIN or subBIN are significantly different from the response of all the other genes represented on the array. An increasingly deep blue or red color indicates an increasingly significant P value that transcript levels for genes in a given category increase or decrease compared to the level at the end of the night. The major BINS and subBINs are indicated on the left-hand side of the diagram. Selected BINS or subBINS that showed highly significant changes are indicated on the right-hand side of the diagram together with the P value. A, Schematic illustration of selected biologically relevant responses. B, Responses for selected functional categories related to metabolism and cellular growth. C, Responses for selected functional categories related to metabolism and cellular growth.

Changes of transcripts for clock genes. The display is as described in the legend of Figure 7. A, Central clock genes. B to D, Genes that entrain the clock.

Temporal responses of light-regulated genes during a diurnal cycle and an extended night. Test sets of approximately 200 light-induced and 200 light-repressed genes were abstracted from a treatment in AtGenExpress in which dark-grown seedlings were exposed to weak white light for 4 h. To investigate how these genes respond during a more prolonged exposure to darkness, the responses of this test set were reanalyzed in a dataset combining the diurnal cycle (Bläsing et al., 2005) and an extended night treatment. The genes in the test sets are listed and datasets are provided in Supplemental Table S4. Clusters 1 to 7 show light-induced genes and clusters 8 to 14 show light-repressed genes. The pie chart inserts show the proportion of the genes in a given cluster that are present in the sets of C-induced (blue) or C-repressed (red) genes (white, no overlap). Lists of genes in the various clusters are provided in Supplemental Table S4.

Changes of metabolites measured in an extended night. Arabidopsis Col-0 was grown for 5 weeks at moderate light intensity (130 μmol m⁻² s⁻¹) in a 14-h light/10-h dark cycle at 20°C in well-fertilized soil. After 5 weeks, plants were subjected to an extended night by not illuminating them at the start of the light period and whole rosettes harvested at the end of the night and at different times into the treatment. A, Changes of metabolites summarized as a heat map; all data are normalized on the values at the end of the night, with blue and red indicating an increase and decrease, respectively (see legend for scale). Gray indicates missing values. Some of the data are recalculated from Gibon et al. (2006). B to J, Starch (B), Suc (C), Glc-6-P (D), Fru-6-P (E), ATP (F), total amino acids (G), Asn (H), Phe (I), protein (J). The results in B to I are the mean and sd of five independent samples.

PCA of an extended night treatment in Col-0 (x) combined with datasets for a diurnal cycle in wild-type Col-0 (d) and pgm (p). A cluster analysis of the same datasets is shown in Supplemental Figure S2. White symbols and black symbols represent day and (extended) night time points, respectively. The star, circles (○), rectangles (□), and triangles (▵) represent time points −2, 4, 8, and 12 h into the day or night. Further, all extended night treatments are also marked with a closed triangle (▴). Treatments with ambient and compensation point [CO₂] are represent by diamonds. White diamonds (⋄) represent the light treatment at ambient [CO₂], black diamonds (♦) the dark treatment at compensation point [CO₂], and the half-black diamond represents the light treatment at compensation point [CO₂].

Temporal responses during a diurnal cycle and an extended night of a set of 158 genes, which respond rapidly to Suc addition. A, Clustering of rapidly responding genes. Osuna et al. (2007) short listed 158 genes as showing marked changes (>1.41, log₂ scale) of their transcript levels 30 min after adding 15 mm Suc to C-depleted seedlings. The responses of these genes in the diurnal cycle (Bläsing et al., 2005) and the extended night in wild-type Col-0 and a diurnal cycle in pgm (Bläsing et al., 2005) were extracted from Supplemental Table S4. The genes were manually sorted into different response groups. The panels show groups of C-repressed (groups 1–5) and C-induced (groups 6–10) genes. The plots show absolute transcript levels. B, Heat map showing correlations between rapidly responding genes and other more slowly responding C-responsive genes. The genes in the rapid- and slow-responding groups were preordered based on internal clustering. Positive correlations are shown as pink and negative as mauve, with a cutoff filter of R² > 0.7. The inset shows a frequency diagram of pair-pair correlations between members of a rapidly responding gene set and the other genes.

Antagonistic interaction between C and the clock. The images show, from left to right, the response of shoots after illumination of dark-grown seedlings with weak white light for 4 h (AtGenExpress), the response after illumination of rosettes for 4 h at 50 ppm [CO₂] compared to 4-h darkness (two independent inputs for light responsiveness), the response after illumination of rosettes for 4 h at 350 ppm compared to 50 ppm [CO₂], and the response 30 min or 3 h after adding Suc to C-starved seedlings (two independent inputs for the C responsiveness), the response in a free-running cycle over 48 h (input for the clock) and the measured changes during a diurnal cycle and extended night in Col-0, and a diurnal cycle in the starchless pgm mutant. The original data and identities of the genes are given in Supplemental Table S4. Transcript levels in the wild-type diurnal cycle, the pgm diurnal cycle are normalized on the levels at the end of the night in wild-type plants. Treatments with light and 50 ppm or 350 ppm [CO₂] are normalized on the respective dark or 50 ppm [CO₂]. Transcript levels in the extended night treatment are normalized on the levels at the start of the extended night treatment (i.e. the end of the normal night in these experiments). Transcript levels in a free-running cycle are normalized on an estimated level at the end of the 24-h period. Transcript levels after illuminating etiolated seedlings are normalized on the levels in nonilluminated controls. Transcript levels in seedlings in full nutrition and 30 min and 3 h after adding 15 mm Suc to C-starved seedlings are normalized on the levels in the C-starved seedlings. The plots show genes that are regulated by C and the clock (Table III) and peak 6 h (A) and 10 h (B) into the subjective day in a free-running cycle.

Changes of transcripts for selected genes that are antagonistically regulated by the clock output pathways and C. The left-hand display shows, from left to right, the input s for light, C, and the clock (see legend of Fig. 7 for details), and the observed responses in a diurnal cycle and extended night treatment in Col-0, and a diurnal cycle in pgm. Transcript levels were normalized as in Figure 8. The right-hand images show the response predicted by our linear model (for details, see the legend to Table IV; also “Materials and Methods” and “Results”). A, Genes involved in Tre-6-P signaling: TPS5, TPS8, TPS9, TPS10, and TPS11. B, Genes involved in the regulation of protein synthesis and degradation: EF1B α-subunit 1 (At5g12110), nucleolin organizer (NUC-L1, At1g48920), and autophagy protein 8e (ATG8e, At2g41570). C, Genes involved in starch degradation (AMY3, PHS1, ISA3, DPE2, PHS2, DPE2, SEX4, GWD, PWD, MEX1, BMY9, LDA1). D, bZIP1 (Atg5g49450), which is implicated in AKIN10 signaling (Baena-Gonzalez et al., 2007).

Modeled contribution of C. A, Comparison of the weighting factors for sugar extracted from the models and the level of Suc in the tissue at the corresponding time point. The Suc levels are normalized on the reference (wild type, end of the night) and expressed as a log ratio of Suc. B, Estimated relative contribution of the clock, light, and C to the global transcript level. For each treatment and time point, the weighting factors generated by the model were calculated by scaling the variance of the corresponding input dataset to 1 to estimate their relative contribution. The variances of the input datasets before scaling were 0.01, 0.04, 0.11, 0.08, 0.08, 0.05, and 0.04 for CC at the 2-, 4-, 6-, 8-, 12-, 16-, and 20-h time points, 0.16 for light, and 0.41 for sugar.