A negative regulatory factor for the dark repression of Arabidopsis thaliana cab1 gene

A protein factor and its binding site involved in light-responsive gene expression of Arabidopsis thaliana cab1 were investigated. Mobility shift assays were performed to identify a nuclear protein factor and its binding sites on the cab1 promoter. For the binding assay, the Arabidopsis cab1 promoter was cleaved with endonucleases into small fragments (65-200 bp) and end-labeled with Klenow fragments. Nuclei were prepared from the light-grown plants and nuclear proteins were prepared by extracting the purified nuclei with 0.5 M ammonium sulfate. The binding site of the nuclear protein factor was scattered throughout the whole promoter region from the transcription start site to the far upstream region of the promoter. To identify the binding sites that are involved in the light responsiveness, mobility shift assays were performed between the cab1 promoter fragments and the nuclear extracts prepared from the 2 day dark-adapted sample. The mobility shift assay of the 65 bp (-318/ -254) fragment with nuclear extract from the dark-adapted sample showed an additional band, not seen with the light-grown sample. Because the new band was present only in the dark-adapted sample that repressed cab1 expression, it may represent a negative regulatory factor (NRF). The NRF was separable on a heparin-Sepharose column from the other factor present in both the light-grown and dark-adapted samples. The implications of the presence of the NRF have been discussed with respect to gene products of the photosignal transduction Arabidopsis mutants.