Synthesis of Seed-Specific Bidirectional Promoters for Metabolic Engineering of Anthocyanin-Rich Maize

Tissue-specific promoters play an important role in plant molecular farming. Here, we describe a strategy to modify the tissue specificity of a maize embryo-specific bidirectional promoter PZₘBD₁. Six types of cis-elements, i.e. RY repeats (R), GCN4 (G), the prolamin box (P), Skn-1 (S), and the ACGT and AACA (A) motifs, were collected and fused to PZₘBD₁ to generate eight chimeric putative bidirectional promoters. Qualitative and quantitative analysis of reporter genes driven by the promoters showed that two promoters exhibited high seed-specific bidirectional activity in maize transient and stable transformed systems. The stronger one was chosen and fused to the intergenic region of two gene clusters consisting of four anthocyanin biosynthesis-related genes (ZmBz1, ZmBz2, ZmC1 and ZmR2) and seven reporter genes, resulting in the first embryo and endosperm anthocyanin-rich purple maize. Anthocyanin analysis showed that the total anthocyanin content reaches 2,910 mg kg–¹ DW in transgenic maize and cyanidin is the major anthocyanin in transgenic maize, as in natural varieties. The expression profile analysis of endogenous genes showed that the anthocyanin biosynthesis pathway was activated by two transgenic transcription factor genes ZmC1 and ZmR2. Our results indicate that both the modification strategy and these functionally characterized tissue-specific bidirectional promoters generated could be used for genetic research and development of plant biotechnology products. The anthocyanin-rich purple maize could provide economic natural colorants for the food and beverage industry, and valuable germplasm for developing anthocyanin-rich fresh corn.