Transcription of a CAT3::LUC transgene is regulated by the circadian clock. Plants were grown under 12-h light/12-h dark photoperiod at 22°C for 7 d. Plants were moved to a Packard TopCount luminometer and further entrained for 3 d in the LD cycle before being released into LL. The LD regime is indicated by the bars beneath the traces, with day (light) indicated by white bars, night (dark) indicated by black bars, and subjective night (dark of the entraining cycle) indicated by hatched bars. A, Traces present average values (± se, n = 12) from individual seedlings expressing CAT3::LUC (squares) or TOC1::LUC (triangles). B, Traces present average values (± se, n = 12) from individual seedlings expressing CAB2::LUC (circles) and CAT1::LUC (diamonds). C, Phase plot in which phases of individual seedlings are plotted against the strength of the rhythm. Phase is expressed in CT (phase/period × 24 h) around the circumference of a 24-h clock face. Strength of the rhythm is expressed as relative amplitude error (RAE), where a perfect sine wave is defined as 0 and a value of 1 defines the weakest rhythm considered to be statistically significant. The strength of the rhythm is plotted along the radius with the strongest rhythms (RAE = 0) at the outer edge of the circle and weakest rhythms (RAE = 1) at the center. CAT3::LUC, squares; CAB2::LUC, circles; TOC1::LUC, triangles. [−221/−103]₂ CAT3::LUC seedlings are depicted because of their highly reproducible and accurate representation of endogenous CAT3 circadian-regulated transcription. Similar results have been obtained with all other rhythmic CAT3::LUC fusions tested.

CAT3::LUC activity continues to oscillate in DD. Plants grown as described in the Figure 1 legend and released into DD conditions instead of LL. Traces present average values (± se, n = 12), normalized as described in the legend to Figure 2, from CAB2::LUC (black triangles), CAT3::LUC (red squares), and TOC1::LUC (blue circles) seedlings. The LD regime is indicated by the bars beneath the traces, with subjective day indicated by white bars and subjective night indicated by gray bars. As discussed in the Figure 1 legend, [−221/−103]₂ CAT3::LUC seedlings are depicted.

Deletion and site-directed mutagenesis show that the EE is necessary for circadian-regulated transcription of CAT3::LUC. Plants were grown as described in the Figure 1 legend and released into LL (A) or DD (B). The LD regime is indicated by the bars beneath the traces, with subjective day indicated by white bars and subjective night indicated by gray bars. A, Traces present average values (± se, n = 12), normalized as described in the legend to Figure 2, from −281/+1 CAT3::LUC (black squares), −281/+1 delEE CAT3::LUC (blue circles), and −281/+1 mutEE CAT3::LUC (red triangles) seedlings assayed in LL. B, Traces present average values (± se, n = 12), normalized as described in the legend to Figure 2, from −281/+1 CAT3::LUC (black squares), −281/+1 delEE CAT3::LUC (blue circles), and −281/+1 mutEE CAT3::LUC (red triangles) seedlings assayed in DD. C, Average proportion (%) of seedlings per each independent transgenic line exhibiting circadian rhythmicity in LL and DD.

The CBS and EE are phase-specific motifs. Plants were grown under a 12/12 LD photoperiod at 22°C for 7 d. Plants were grown as described in the Figure 1 legend and released into LL (A) or into DD (B) or retained in LD cycles (C). Traces present average values (± se, n = 12), normalized as described in the legend to Figure 2, from −199/+1 EE CAT3::LUC (triangles) and −199/+1 CBS CAT3::LUC (squares) seedlings. The LD regime is indicated by the bars beneath the traces, with subjective day indicated by white bars and subjective night indicated by hatched bars. C, The entraining LD cycle is indicated with white and black bars, respectively. D, Average proportion (%) of seedlings per each independent transgenic line exhibiting circadian rhythmicity in LL, DD, and LD cycles. E, Phase plots of all rhythmic seedlings, assayed in DD, from five transgenic lines of −199/+1 EE CAT3::LUC (triangles) and for eight transgenic lines of −199/+1 CBS CAT3::LUC (squares).

CAT3::LUC expression can be entrained by light or temperature cycles. A, Plants were grown at 22°C either under a 12/12 LD photoperiod (LD HH; triangles) or under a 12-h/12-h dark-light (DL HH; circles) photoperiod for 7 d before release into LL at T = 0. Traces present average values (± se, n = 12) from individual independent transgenic lines. Data are normalized to the average luciferase activity of the individual seedling and are presented as relative bioluminescence. B, Plants were grown in LL either under a 12-h hot (22°C)/12-h cold (18°C) thermoperiod (LL HC; circles) or under a 12-h cold (18°C)/12-h hot (22°C) thermoperiod (LL CH; triangles) for 7 d before release into constant temperature (22°C) and LL at T = 0. Traces present average values (± se, n = 12) from individual independent transgenic lines. C and D, Phase plots as described in the legend to Figure 1C for multiple seedlings from A and C, respectively. [−221/−103]₂ CAT3::LUC seedlings are depicted, but similar results have been obtained with all other CAT3::LUC fusions tested, except those constructs that have lost rhythmicity.

Deletion analysis of the CAT3::LUC promoter reveals an EE that is necessary for evening-specific circadian transcription. A, Summary of the CAT3::LUC promoter resection indicating the proportion of independent transgenic lines expressing evening-specific circadian LUC activity in LL. B, Plants were grown as described in the Figure 1 legend and released into LL. The LD regime is indicated by the bars beneath the traces, with subjective day indicated by white bars and subjective night indicated by gray bars. Traces present average values (± se, n = 12), normalized as described in the legend to Figure 2, from −1,130/+1 CAT3::LUC (blue triangles), −199/+1 CAT3::LUC (red squares), and −174/+1 CAT3::LUC (black circles) seedlings. C, Phase plots of 12 seedlings from single transgenic lines carrying either the −1,130/+1 CAT3::LUC (blue triangles) or the −199/+1 CAT3::LUC (red squares) constructs. D, Nucleotide sequence of the 25-bp CAT3 promoter region between −199 and −174, which is required for rhythmicity and contains the EE, AAAATATCT (highlighted), and the lhc motif, CAN_2–4ATC (underlined; Piechulla et al., 1998).

Gain of function experiments show that a 118-bp region from the CAT3 promoter is sufficient to confer evening-specific circadian LUC activity. Plants were grown as described in the Figure 1 legend and released into LL. A, Traces present average values (± se, n = 12), normalized as described in the legend to Figure 2, from (−221/−118)₂ CAT3::LUC (red circles), −281/+1 CAT3::LUC (black squares), and (−203/−163)₁ CAT3::LUC (blue triangles) seedlings. The LD regime is indicated by the bars beneath the traces, with subjective day indicated by white bars and subjective night indicated by gray bars. B, Cartoon comparing CAT3 promoter fragments used in gain-of-function experiments. C, Phase plots of 12 seedlings from one transgenic line for each of the two rhythmic constructs shown in A.