Mutually Regulated AP2/ERF Gene Clusters Modulate Biosynthesis of Specialized Metabolites in Plants

Abstract

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) gene clusters regulate the biosynthesis of diverse specialized metabolites, including steroidal glycoalkaloids in tomato (<i>Solanum lycopersicum</i>) and potato (<i>Solanum tuberosum</i>), nicotine in tobacco (<i>Nicotiana tabacum</i>), and pharmaceutically valuable terpenoid indole alkaloids in Madagascar periwinkle (<i>Catharanthus roseus</i>). However, the regulatory relationships between individual AP2/ERF genes within the cluster remain unexplored. We uncovered intracluster regulation of the <i>C. roseus</i> AP2/ERF regulatory circuit, which consists of <i>ORCA3</i>, <i>ORCA4</i>, and <i>ORCA5</i> ORCA3 and ORCA5 activate <i>ORCA4</i> by directly binding to a GC-rich motif in the <i>ORCA4</i> promoter. ORCA5 regulates its own expression through a positive autoregulatory loop and indirectly activates <i>ORCA3</i> In determining the functional conservation of AP2/ERF clusters in other plant species, we found that GC-rich motifs are present in the promoters of analogous AP2/ERF clusters in tobacco, tomato, and potato. Intracluster regulation is evident within the tobacco <i>NICOTINE2</i> (<i>NIC2</i>) <i>ERF</i> cluster. Moreover, overexpression of <i>ORCA5</i> in tobacco and of <i>NIC2 ERF189</i> in <i>C. roseus</i> hairy roots activates nicotine and terpenoid indole alkaloid pathway genes, respectively, suggesting that the AP2/ERFs are functionally equivalent and are likely to be interchangeable. Elucidation of the intracluster and mutual regulation of transcription factor gene clusters advances our understanding of the underlying molecular mechanism governing regulatory gene clusters in plants.