R2R3-MYB EVER links emission of volatiles with epicuticular wax biosynthesis in petunia petal epidermis

The epidermal cells of petunia flowers are the main site of volatile emission. However, data on the mechanisms underlying the release of volatiles into the environment are lacking. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by VIGS and CRISPR, and metabolomics we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)--a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. Using a three-step viral-based CRISPR/Cas9 editing system, ever knockout lines were generated and together with transient suppression assays, revealed EVERs involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. LC/GC-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. Thus, we reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a new regulatory layer in the scent-emission machinery in petunia flowers.