Three out of the several ripe epidermis-specific TFs were selected to study their biological role, one of them belonging to the MYB family (FvMYB29), and two bHLH-like proteins (FvbHLH22 and FvbHLH67). Protein interaction assays revealed that the FvMYB29 protein physically interacts independently with the two FvbHLHs forming a heterocomplex. Furthermore, these two bHLHs might participate together in this FvMYB29-FvbHLHs heterocomplex, as evidenced by an in vivo assay. Genome-wide binding sites of these TFs were identified by DAP-seq, revealing that genes involved in flavonoid biosynthesis and cuticle composition are among the FvMYB29 targets, which were validated by transactivation assays (Luciferase/Renilla system), while the bHLH TFs did not bind to DNA by themselves. Interestingly, transactivation assays combining FvMYB29 and the two FvbHLHs separately or together showed that the latter modulates the transcription of these targets by FvMYB29.
Stable FvMYB29 overexpression led to pleiotropic phenotypes evident in both vegetative and reproductive organs compared to the wild type. These plants exhibited significantly smaller leaves, crown elongation, taller inflorescences with more branching, earlier flowering, and a higher rate of fruit abortion due to a reduction in pollen content and viability. As expected from the enrichment in cuticle formation-related genes among the FvMYB29 targets, the overexpression lines showed a misregulation of genes related to cutin and wax biosynthesis in ripe fruits and leaves, which is consistent with a higher content in epicuticular waxes, in particular alkanes, aldehydes, and esters, on the abaxial surface of their leaves compared to those of the control. All these results support the role of the FvMYB29-FvbHLH TF complex as an important regulator of cuticle structure in F. vesca.
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