Binding of MYB96 to consensus sequences in the ABI4 promoter. A, R2R3-MYB-binding consensus sequences on the ABI4 promoter. Core binding sequences are marked with arrowheads. Underbars represent amplified genomic regions. The numbers below the underbars indicate amplification product size (bp). B, ChIP assays. Total protein extracts from pMYB96:MYB96-MYC transgenic seeds were immunoprecipitated with an anti-MYC antibody. Fragmented genomic DNA was eluted from the protein-DNA complexes and subjected to quantitative PCR analysis. Biological triplicates were averaged, and statistically significant differences of the measurements were determined using Student’s t test (*P < 0.05). Error bars indicate se. In each experiment, the measurement values in pBA002 were set to 1 after normalization against eIF4a for quantitative PCR analysis. C, Transient expression analysis using Arabidopsis protoplasts. The core promoter sequence elements of ABI4, A-1 (CAAGTAACTTATGA) and A-2 (GCTATAGTTACCCA), were constructed in the reporter plasmid. The recombinant reporter and effector constructs were coexpressed transiently in Arabidopsis protoplasts, and GUS activities were determined fluorimetrically. Luciferase gene expression was used to normalize the GUS activities. The normalized values in control protoplasts were set to 1 and represented as relative activation. Three independent measurements were averaged. Statistically significant differences were determined by Student’s t test (*P < 0.05). Error bars indicate se. CaMV, Cauliflower mosaic virus; Nos-T, nopaline synthase terminator.