Role of ripe fruit epidermis-specific FvMYB29-FvbHLH transcription factor complexes in strawberry

Abstract

Transcriptome changes during strawberry fruit ripening have been previously reported. However, we have identified genes with tissue- and stage-specific patterns in the receptacles of Fragaria vesca coupling LCM with RNA-seq analysis. In the study, we have focused on the Gene Regulatory Network at the epidermis in ripe fruits, since it is the external cell layer in direct contact with the environment and it plays an important role in defense, and, in contrast to receptacles of the commercial species, it is the only part of the fruit that accumulates anthocyanins. Consistently, a GO functional analysis of this GRN showed enrichment in genes involved in flavonoid and wax biosynthesis. 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 with the two FvbHLHs. 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 was validated by transactivation assays. However, transactivation assays with different combinations of FvMYB29 and the two FvbHLH showed that the latter modulates the activation of transcription of the targets. Consistently with the role of FvMYB29 in the cuticle formation, stable FvMYB29-overexpressing lines showed a misregulation of genes related to cutin and wax biosynthesis in ripe fruits and leaves. Furthermore, FvMYB29-overexpressing fruits presented cuticular nanoridges. On the other hand, young leaves of FvMYB29-overexpression lines showed denser epicuticular waxes in the abaxial surface and an alteration in wax composition but compared to the control. All these results support the role of the FvMYB29-FvbHLH TF complex as an important regulator of cuticle structure in F. vesca.