Upper level and cross hierarchical regulation of predominantly expressed phenolic genes in maize

There is strong interest in deciphering the gene regulatory networks (GRNs) that govern plant specialized metabolism to assist in plant breeding. Here, we investigated the GRN governing phenolic biosynthesis pathways from which ∼ 8000 secondary metabolites are derived in plants. Previously it was established that 19 predominantly expressed phenolic (PEP) genes in maize are sufficient to explain >70 % of the metabolic flux through the core phenylpropanoid, monolignol, and flavonoid branches of this pathway. A yeast-1-hybrid (Y1H) gene centric screening approach was employed to discover upper level (tier 2, 3, and 4) regulators of maize PEP genes. These regulators were further examined by co-expression analyses, and a subset of protein-DNA interactions (PDIs) validated in vivo by ChIP-qPCR and luciferase reporter assays in maize protoplasts. This study reveals a comprehensive GRN composed of 429 PDIs that exhibits hubs with high connectivity and cross hierarchical regulation of PEP genes in different branches of the pathway. The core GRN includes TFs that are conserved in other plant species and that are implicated in phenolic gene regulation including ZmMYB40/53/100, ZmMADS9, and ZmWD40.1/PAC1. The GRN also includes conserved TFs (e.g., ZmC3H9, ZmHB20/79, ZmNAC103/123, ZmMYB19/26, ZmMYBR87, ZmDOF3, ZmbZIP67, ZmTCP30, and ZmbHLH128) which indicate that maize PEP genes are developmentally regulated but also fall under the control of biotic and abiotic stress signals. Together, the maize PEP GRN provides a complex regulatory mechanism that has evolved to coordinately regulate many phenolic genes in response to multiple internal and external signals and can guide efforts aimed at manipulating phenolic levels in plants towards targeted breeding improvement.