The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects

Abstract

APETALA3 is a MADS box gene required for normal development of the petals and stamens in the Arabidopsis flower. Studies in yeast, mammals and plants demonstrate that MADS domain transcription factors bind with high affinity to a consensus sequence called the CArG box. The APETALA3 promoter contains three close matches to the consensus CArG box sequence. To gain insights into the APETALA3 regulatory circuitry, we have analyzed the APETALA3 promoter using AP3::uidA(GUS) fusions. 496 base pairs of APETALA3 promoter sequence 5′ to the transcriptional start directs GUS activity in the same temporal and spatial expression pattern as the APETALA3 RNA and protein in wild-type flowers. A synthetic promoter consisting of three tandem repeats of a 143 base pair sequence directs reporter gene activity exclusively to petals and stamens in the flower. We have analyzed the role of the CArG boxes by site-specific mutagenesis and find that the three CArG boxes mediate discrete regulatory effects. Mutations in CArG1 result in a decrease in reporter expression suggesting that CArG1 is the binding site for a positively acting factor or factors. Mutations in CArG2 result in a decrease in reporter expression in petals, but the expression pattern in stamens is unchanged. By contrast, mutations in CArG3 result in an increase in the level of reporter gene activity during early floral stages suggesting that CArG3 is the binding site for a negatively acting factor.