The MBW complex regulates floral volatiles in petunia: transcriptional repressor EOBV is a MYB component of the complex

Anthocyanin biosynthesis in Petunia is regulated by the bHLH AN1 and the WDR protein AN11, which together with interchangeable MYBs form the MYB–bHLH–WDR (MBW) complex. As they share the same biosynthetic pathway, anthocyanins are interlinked with phenylalanine-derived volatiles. However, involvement of the MBW complex in regulating these floral volatiles has not been demonstrated. TRV-based suppression of either AN1 or AN11 led to an increase in volatile emission, indicating that they are involved in negative regulation of this trait. Yeast two-hybrid and in-planta pairwise and three-way protein-interaction assays revealed that EMISSION OF BENZENOIDS V (EOBV) is a component of the MBW complex. Headspace and internal pool analyses of flowers from eobv-knockout lines, generated using a viral-based CRISPR/Cas9 system, revealed that EOBV fine-tunes volatile production: phenylpropenes and vanillin levels increased while those of benzenoids and phenylpropanoid-related compounds decreased. Accordingly, transcript levels of C4H2, directing carbon flux to phenylpropenes and vanillin production, and ADT3 were significantly elevated in eobv-knockout flowers, along with decreases in PAAS and BSMT. Moreover, in-planta promoter activity assays in petunia flowers further demonstrated that C4H2 is negatively transcriptionally regulated by EOBV. In addition to its involvement in volatile production, EOBV is heat-responsive and affects flower development under high-temperature conditions by mitigating the reduction in flower size. EOBV’s participation in the MBW complex that regulates volatiles and anthocyanins reveals an intriguing molecular link between these showy traits and flower development.