title: High AN1 variability and interaction with basic helix-loop-helix co-factors related to anthocyanin biosynthesis in potato leaves.
abstract: AN1 is a regulatory gene that promotes anthocyanin biosynthesis in potato tubers and encodes a R2R3 MYB transcription factor. However, no clear evidence implicates AN1 in anthocyanin production in leaves, where these pigments might enhance environmental stress tolerance. In our study we found that AN1 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 AN1 expression and that StJAF13 acts as putative AN1 co-regulator for anthocyanin gene expression in leaves of the red leaf variety 'Magenta Love,' while a concomitant expression of StbHLH1 may contribute to anthocyanin accumulation in leaves of 'Double Fun.' Yeast two-hybrid experiments confirmed that AN1 interacts with StbHLH1 and StJAF13 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 AN1 with StJAF13 or AN1 with StbHLH1 showed deeper purple pigmentation with respect to AN1 alone. This further confirmed AN1/StJAF13 and AN1/StbHLH1 interactions. Our findings demonstrate that the classical loci identified for potato leaf anthocyanin accumulation correspond to AN1 and may represent an important step to expand our knowledge on the molecular mechanisms underlying anthocyanin biosynthesis in different plant tissues.
authors.affiliation: Department of Agricultural Sciences, University of Naples Federico II, Via Università 100, 80055, Portici, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: D'Amelia
authors.forename: Riccardo
authors.initials: R
authors.lastname: Aversano
authors.forename: Giorgia
authors.initials: G
authors.lastname: Batelli
authors.forename: Immacolata
authors.initials: I
authors.lastname: Caruso
authors.forename: Mar
authors.initials: M
authors.lastname: Castellano Moreno
authors.forename: Ana Beatriz
authors.initials: AB
authors.lastname: Castro-Sanz
authors.forename: Pasquale
authors.initials: P
authors.lastname: Chiaiese
authors.forename: Carlo
authors.initials: C
authors.lastname: Fasano
authors.forename: Francesca
authors.initials: F
authors.lastname: Palomba
authors.forename: Domenico
authors.initials: D
authors.lastname: Carputo
mesh_terms: D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000378:metabolism*; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D035264:Flowers / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010802:Phylogeny; D010858:Pigmentation / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D036226:Seedlings / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D000872:Anthocyanins; D051792:Basic Helix-Loop-Helix Proteins; D010940:Plant Proteins
keywords: MYB transcription factor; Nicotiana tabacum; Solanum tuberosum; allelic genotyping; bimolecular fluorescence complementation; flavonoids; regulatory complex; transient and stable transformation; yeast two hybrid assay