Alternatively Spliced BnaPAP2.A7 Isoforms Play Opposing Roles in Anthocyanin Biosynthesis of Brassica napus L.

Abstract

<i>Brassica napus</i> L. (rapeseed, oilseed rape, and canola) and varieties of its two diploid parents, <i>B. oleracea</i> and <i>B. rapa</i>, display a large amount of variation in anthocyanin pigmentation of the leaf, stem, and fruit. Here, we demonstrate that <i>BnaPAP2.A7</i>, an ortholog of the <i>B. oleracea</i> anthocyanin activator <i>BoMYB2</i> that confers purple traits, positively regulates anthocyanin biosynthesis in leaves of <i>B. napus</i>. Sequencing of <i>BnaPAP2.A7</i> and transgenic analysis suggests that activation of this gene in purple rapeseed may result from a single nucleotide and/or 2bp insertion in its promoter region. <i>BnaPAP2.A7</i> gives rise to three splice variants, designated <i>BnaPAP2.A7-744, BnaPAP2.A7-910</i>, and <i>BnaPAP2.A7-395</i> according to the length of the transcripts. While <i>BnaPAP2.A7-744</i> encodes a full-length R2R3-MYB, both <i>BnaPAP2.A7-910</i> and <i>BnaPAP2.A7-395</i> encode truncated proteins that lack both a partial R3 repeat and the complete C terminal domain, and so <i>in vitro</i> are unable to interact with the <i>Arabidopsis</i> bHLH protein AtTT8. Although expression of either <i>BnaPAP2.A7-910</i> or <i>BnaPAP2.A7-395</i> in green rapeseed does not result in purple leaves, both genes do modify genome-wide gene expression, with a strong repression of anthocyanin-related genes. We have demonstrated that <i>BnaPAP.A7</i> regulates anthocyanin accumulation in leaves of <i>B. napus</i> and propose a potential mechanism for modulation of anthocyanin biosynthesis by alternative splicing.