A Region of the Arabidopsis Lhcb1*3 Promoter That Binds to CA-1 Activity Is Essential for High Expression and Phytochrome Regulation

We have previously characterized a protein from Arabidopsis thaliana, called CA-1, that bound to a specific region of the Lhcb1*3 promoter. This binding activity was of interest because the sequence to which it bound is included in a portion of the promoter that is sufficient for phytochrome regulation and because the activity was absent in photomorphogenic mutant det1 seedlings (L. Sun, R.A. Doxsee, E. Harel, E.M. Tobin [1993] Plant Cell 5: 109-121). We have now directly tested whether the nucleotide sequence to which CA-1 binds is required for regulation of the transcription of this gene by phytochrome. A mutation that abolished CA-1 binding in vitro was introduced into a 1.15-kb segment of the Lhcb1*3 promoter, and both the wild-type and mutant promoter fragments were fused to a uidA reporter gene and used to stably transform A. thaliana. Ten different homozygous lines were examined for phytochrome responsiveness for each of the two constructs by assaying beta-glucuronidase activity. The wild-type construct showed normal phytochrome responsiveness. The mutant construct showed no phytochrome response, and the overall level of beta-glucuronidase activity in etiolated seedlings was decreased by about 2 orders of magnitude. We did not detect a response to a B photoreceptor other than phytochrome itself for either the wild-type or mutant construct. We conclude that information essential for both a high level of expression and phytochrome responsiveness is contained in a 27-bp region to which the CA-1 activity binds.