Module-specific regulation of the b-phaseolin promoter during embryogenesis

The phas promoter displays stringent spatial regulation, being very highly expressed during embryogenesis and completely silent during all phases of vegetative development in bean, Phaseolus vulgaris. This pattern is maintained in transgenic tobacco and, as shown here, Arabidopsis. Dimethyl sulphate in vivo footprinting analyses revealed that over 20 cis-elements within the proximal 295 bp of the phas promoter are protected by factor binding in seed tissues whereas none are bound in leaves. The hypothesis that this complex profile represents a summation of several module (cotyledon, hypocotyl, and radicle)-specific factor–DNA interactions has been explored by the incorporation of site-directed substitution mutations into 10 locations within the 295phas promoter. Only 2.6% of 295phas promoter activity remained after mutation of the G-box; the CCAAAT box, the E-box and the RY elements were also found to mediate high levels of expression in embryos. Whereas the CACA element has dual positive and negative regulatory roles, the vicilin box was identified as a strong negative regulatory element. The proximal ( 70 to 64) RY motif was found to bestow expression in the hypocotyl while all the RY elements contribute to expression in cotyledons but not to vascular tissue expression during embryogenesis. RY elements at positions 277 to 271, 260 to 254, and 237 to 231 were found to orchestrate radicle-specific repression. The G-box appears to be the functional abscisic acid responsive element and the E-site may be a coupling element. The results substantiate the concept that autarkical cis-element functions generate modular patterning during embryogenesis. They also reflect the existence of both redundancy and hierarchy in cis–element interactions. Importantly, the virtually identical expression patterns observed for the two distantly related plants studied argue strongly for the generality of function for the observed factor–element interactions.