Regulation of the floral organ identity gene APETALA3 in Arabidopsis thaliana

APETALA3 (AP3) is a MADS box gene required for normal development of the petals and stamens in the Arabidopsis flower. AP3 transcriptional regulation occurs in two stages; an initial establishment phase, which is mediated by LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO), and a later maintenance phase, which is mediated by an autoregulatory circuit. To gain insights into the AP3 regulatory circuitry we have analyzed the AP3 promoter using fusions to the uidA reporter gene (AP3::uidA(GUS) fusions). Either 496 base pairs of AP3 promoter sequence 5′ to the transcriptional start, or a synthetic promoter consisting of three tandem repeats of a 143 base pair sequence (from −83 to −225) directs GUS activity in the same temporal and spatial expression pattern as the AP3 RNA and protein in wild-type flowers. This 143 base pair sequence contains three CArG boxes. We have analyzed the role of the CArG boxes by site-specific mutagenesis and find that while CArG1 positively mediates both establishment and maintenance, CArG3 may mediate repression, since mutating CArG3 increases GUS activity. In addition, our data suggests that UFO acts through CArG3, as well as a second sequence between −908 and −650. LFY acts through the sequence between −83 and −225 and our results support the hypothesis that LFY acts through CArG1. To determine if LFY and UFO exhibit synergistic effects on AP3 expression, we assayed the effects of simultaneous ectopic expression of LFY and UFO on AP3 expression. We found that although LFY and UFO do exhibit synergistic effects on the floral phenotype, both in terms of floral organ identity and organ number, they do not exhibit synergistic effects on AP3 expression.