Plant age-dependent dynamics of annatto pigment (bixin) biosynthesis in Bixa orellana.

This article investigates how the conserved microRNA156 (miR156)–SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) regulatory module controls age-dependent biosynthesis of bixin, an economically important apocarotenoid, in the leaves of the achiote tree (Bixa orellana L.). Overexpression of miR156 in transgenic annatto plants prolonged the juvenile phase, altered leaf development, reduced bixin content and secretory structures (bixin channels), and increased abscisic acid (ABA) levels, suggesting a shift in carbon flux from bixin to ABA biosynthesis. Molecular analyses revealed that miR156 overexpression downregulated key carotenoid and bixin biosynthetic genes (e.g., BoCCD4-4, BoSABATHs) and upregulated others (e.g., BoCCD1, lycopene β-cyclases), while proteomic data showed a broad reduction in secondary metabolite-related enzymes. These findings indicate that miR156-targeted BoSPL transcription factors are likely essential for proper leaf maturation and activation of secondary metabolic pathways, providing insights that may inform breeding strategies to optimize bixin production in B. orellana.