Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically.

AGAMOUS (AG) is an Arabidopsis MADS box gene required for the normal development of the internal two whorls of the flower. AG RNA accumulates in distinct patterns early and late in flower development, and several genes have been identified as regulators of AG gene expression based on altered AG RNA accumulation in mutants. To understand AG regulatory circuits, we are now identifying cis regulatory domains by characterizing AG::beta-glucuronidase (GUS) gene fusions. These studies show that a normal AG::GUS staining pattern is conferred by a 9.8-kb region encompassing 6 kb of upstream sequences and 3.8 kb of intragenic sequences. Constructs lacking the 3.8-kb intragenic sequences confer a GUS staining pattern that deviates both spatially and temporally from normal AG expression. The GUS staining patterns in the mutants for the three negative regulators of AG, apetala2, leunig, and curly leaf, showed the predicted change of expression for the construct containing the intragenic sequences, but no significant change was observed for the constructs lacking this intragenic region. These results suggest that intragenic sequences are essential for AG regulation and that these intragenic sequences contain the ultimate target sites for at least some of the known regulatory molecules.