Jasmonic acid/ethylene signaling coordinates hydroxycinnamic acid amides biosynthesis through ORA59 transcription factor.

Hydroxycinnamic acid amides (HCAAs) are a class of antimicrobial metabolites involved in plant defense against necrotrophic pathogens, including Alternaria brassicicola and Botrytis cinerea. The agmatine coumaryl transferase (AtACT) is the key enzyme that catalyzes the last reaction in the biosynthesis of HCAAs, including p-coumaroylagmatine (CouAgm) and feruloylagmatine in Arabidopsis thaliana. However, the regulatory mechanism of AtACT gene expression is currently unknown. Yeast one-hybrid screening using the AtACT promoter as bait isolated the key positive regulator ORA59 that is involved in jasmonic acid/ethylene (JA/ET)-mediated plant defense responses. AtACT gene expression and HCAAs biosynthesis were synergistically induced by a combination of JA and ET. In the AtACT promoter, two GCC-boxes function equivalently for trans-activation by ORA59 in Arabidopsis protoplasts, and mutation of either GCC-box abolished AtACT mRNA accumulation in transgenic plants. Site-directed mutation analysis demonstrated that the specific Leu residue at position 228 of the ORA59 EDLL motif mainly contributed to its transcriptional activity on AtACT gene expression. Importantly, MEDIATOR25 (MED25) and ORA59 homodimer are also required for ORA59-dependent activation of the AtACT gene. These results suggest that ORA59 and two functionally equivalent GCC-boxes form the regulatory module together with MED25 that enables AtACT gene expression and HCAAs biosynthesis to respond to simultaneous activation of the JA/ET signaling pathways.