HighAN1variability and interaction with basic helix-loop-helix co-factors related to anthocyanin biosynthesis in potato leaves

Summary AN 1 is a regulatory gene that promotes anthocyanin biosynthesis in potato tubers and encodes a R2R3 MYB transcription factor. However, no clear evidence implicates AN 1 in anthocyanin production in leaves, where these pigments might enhance environmental stress tolerance. In our study we found that AN 1 displays intraspecific sequence variability in both coding/non‐coding regions and in the promoter, and that its expression is associated with high anthocyanin content in leaves of commercial potatoes. Expression analysis provided evidence that leaf pigmentation is associated to AN 1 expression and that St JAF 13 acts as putative AN 1 co‐regulator for anthocyanin gene expression in leaves of the red leaf variety ‘Magenta Love,’ while a concomitant expression of Stb HLH 1 may contribute to anthocyanin accumulation in leaves of ‘Double Fun.’ Yeast two‐hybrid experiments confirmed that AN 1 interacts with Stb HLH 1 and St JAF 13 and the latter interaction was verified and localized in the cell nucleus by bimolecular fluorescence complementation assays. In addition, transgenic tobacco ( Nicotiana tabacum ) overexpressing a combination of either AN 1 with St JAF 13 or AN 1 with Stb HLH 1 showed deeper purple pigmentation with respect to AN 1 alone. This further confirmed AN 1/St JAF 13 and AN 1/Stb HLH 1 interactions. Our findings demonstrate that the classical loci identified for potato leaf anthocyanin accumulation correspond to AN 1 and may represent an important step to expand our knowledge on the molecular mechanisms underlying anthocyanin biosynthesis in different plant tissues.