The transcription factor EMISSION OF BENZENOIDS II activates the MYB ODORANT1 promoter at a MYB binding site specific for fragrant petunias.

Fragrance production in petunia flowers is highly regulated. Two transcription factors, ODORANT1 (ODO1) and EMISSION OF BENZENOIDS II (EOBII) have recently been identified as regulators of the volatile benzenoid/phenylpropanoid pathway in petals. Unlike the non-fragrant Petunia hybrida cultivar R27, the fragrant cultivar Mitchell highly expresses ODO1. Using stable reporter lines, we identified the 1.2-kbp ODO1 promoter from Mitchell that is sufficient for tissue-specific, developmental and rhythmic expression. This promoter fragment can be activated in non-fragrant R27 petals, indicating that the set of trans-acting factors driving ODO1 expression is conserved in these two petunias. Conversely, the 1.2-kbp ODO1 promoter of R27 is much less active in Mitchell petals. Transient transformation of 5' deletion and chimeric Mitchell and R27 ODO1 promoter reporter constructs in petunia petals identified an enhancer region, which is specific for the fragrant Mitchell cultivar and contains a putative MYB binding site (MBS). Mutations in the MBS of the Mitchell promoter decreased overall promoter activity by 50%, highlighting the importance of the enhancer region. We show that EOBII binds and activates the ODO1 promoter via this MBS, establishing a molecular link between these two regulators of floral fragrance biosynthesis in petunia.