The fruit of the pomegranate (Punica granatum L.) is an important nutraceutical food rich in polyphenolic compounds, including hydrolysable tannins, anthocyanins and flavonols. Their composition varies according to cultivar, tissue and fruit development stage and is probably regulated by a combination of MYB and bHLH type
transcription factors (TFs). In this study, metabolomics analysis during fruit developmental stages in the main pomegranate cultivars, Wonderful and Valenciana with contrasting colour of their ripe fruits, showed that flavonols were mostly present in flowers while catechins were highest in unripe fruits and anthocyanins in late fruit
maturation stages. A novel MYB TF, PgMYB5-like, was identified, which differs from previously isolated pomegranate TFs by unique C-terminal protein motifs and lack of the amino-acid residues conserved among anthocyanins promoting MYBs. In both pomegranate cultivars the expression of PgMYB5-like was high at flowering stage, while it decreased during fruit ripening. A previously identified bHLH-type TF, PgbHLH, also showed high transcript levels at flowering stage in both cultivars, while it showed a decrease in expression during fruit ripening in cv. Valenciana, but not in cv. Wonderful. Functional analysis of both TFs was performed by agroinfiltration into Nicotiana benthamiana leaves. Plants infiltrated with the PgMYB5-like+PgbHLH combined construct showed a specific and significant accumulation of intermediates of the flavonoid pathway, especially dihydroflavonols, while anthocyanins were not produced. Thus, we propose a role for PgMYB5-like and PgbHLH in the first steps of flavonoid production in flowers and in unripe fruits. The expression patterns of these two TFs may be key in determining the differential flavonoid composition in both flowers and fruits of the pomegranate varieties Wonderful and Valenciana.