Phase-specific circadian clock regulatory elements in Arabidopsis

We have defined a minimal Arabidopsis CATALASE 3 (CAT3) promoter sufficient to drive evening-specific circadian transcription of a LUCIFERASE reporter gene. Deletion analysis and site-directed mutagenesis reveal a circadian response element, the evening element (EE: AAAATATCT), that is necessary for evening-specific transcription. The EE differs only by a single base pair from the CIRCADIAN CLOCK ASSOCIATED 1-binding site (CBS: AAAAAATCT), which is important for morning-specific transcription. We tested the hypothesis that the EE and the CBS specify circadian phase by site-directed mutagenesis to convert the CAT3 EE into a CBS. Changing the CAT3 EE to a CBS changes the phase of peak transcription from the evening to the morning in continuous dark and in light-dark cycles, consistent with the specification of phase by the single base pair that distinguishes these elements. However, rhythmicity of the CBS-containing CAT3 promoter is dramatically compromised in continuous light. Thus, we conclude that additional information normally provided in the context of a morning-specific promoter is necessary for full circadian activity of the CBS.