SYNERGISM BETWEEN CACGTG (G-BOX) AND CACCTG CIS-ELEMENTS IS REQUIRED FOR ACTIVATION OF THE BEAN SEED STORAGE PROTEIN BETA-PHASEOLIN GENE

Expression of bean seed storage protein phaseolin is under strict deve lopmental control. Four distinct nuclear proteins recognize in vitro t he proximal beta-phaseolin promoter (-295/+45) which confers spatial a nd temporal regulation of the native gene. Functional significance of these protein-binding sites was evaluated by substitution mutation of the motifs in the promoter, which was fused to GUS reporter gene, and subsequent transient gene expression assay using protoplasts from deve loping bean cotyledons. DNA-binding protein CAN binds three CANNTG mot ifs, CACGTG (-248/-243), CACCTG (-163/-158), and CATATG (-100/-95). Su bstitution mutation of the CACGTG motif, which is commonly known as G- box, reduced the -295 promoter activity by 75%, indicating that the G- box is a major positive cis-element. Mutation analyses also demonstrat ed that the CACCTG and CATATG motifs act as positive and negative cis- elements, respectively. Substitution mutation of all three CANNTG moti fs essentially eliminated the -295 promoter activity. A construct cont aining the G-box and CACCTG motif resulted in a transcriptional level that is much greater than the sum of the transcriptional levels from t he individual cis-elements, demonstrating that the G-box and CACCTG ac t synergistically. Substitution mutations of two AT-rich sequences, to which a nuclear protein AG-1 binds, showed that these sites function as major negative (-376/-367, -356/-347) or positive (-191/-182) cis-e lements, and that the effect of the two AG-1 binding sites was counter active in the -391 promoter. These results indicate that the three CAN NTG motifs and two AG-1-binding sites play critical roles in transcrip tion of the beta-phaseolin gene in cotyledons.