Comparative transcriptomics of anthocyanin accumulation in the pericarp of pigmented purple corn

KEY MESSAGE: RNA-seq, bulked segregant analysis, anthocyanin quantification, and WGCNA identified Pl1, Lc1, P1, and Wrky33 as important regulatory factors for enhancing anthocyanin content in maize pericarp. Anthocyanins are plant pigments that can be used as natural food colorants. We developed Midwestern, purple corn lines with enhanced anthocyanin content exclusively in the pericarp tissue layer: two lines near-isogenic to elite inbreds B73 and Mo17 (B73 Color Converted and Mo17 Color Converted, respectively) and two recombinant inbred lines (RILs) with diverse anthocyanin profiles (Amazonas and Maize Morado). In Experiment 1, a time-series, RNA-sequencing (RNA-seq) analysis of whole pericarp tissue was conducted on three pigmented genotypes (B73 Color Converted, Amazonas, and Maize Morado). Ultra-High-Pressure Liquid Chromatography (UHPLC) identified a dramatic increase in anthocyanin accumulation between 15 and 20 days after pollination (DAP) in pigmented genotypes. Bulk segregant analysis discovered Leaf color1 (Lc1) and Purple plant1 (Pl1) as the major contributors to pericarp pigmentation in B73 Color Converted. Additional loci Bronze2 (Bz2) and Pericarp color1 (P1) were also donated by the purple parent. In Experiment 2, RNA-seq was performed on 18 DAP kernels of four pigmented maize lines (B73 Color Converted, Mo17 Color Converted, Amazonas, and Maize Morado), comparing pigmented and unpigmented pericarp fractions from bulked individual kernels. Upregulation of Lc1, Pl1, and P1 suggests a distinct MBW protein complex in pigmented pericarp. Correlational analyses of 18 DAP pigmented pericarp fractions revealed enriched expression of anthocyanin transporters, Bz2 and Multidrug resistance-associated protein3 (Mrpa3), and a candidate transcription factor, WRKY-transcription factor 33 (Wrky33). These candidate genes can be used in breeding programs as a source of natural food and beverage colorants and improve our understanding of the mechanisms underlying maize pericarp pigmentation.