Agmatine coumaroyltransferase gene on the short arm of chromosome 2H is involved in hordatine biosynthesis in barley

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound that accumulates substantially in young barley (Hordeum vulgare) seedlings. The first committed step of the HA biosynthetic pathway is the formation of pCA via condensation of p-coumaroyl-CoA and agmatine, which is catalyzed by agmatine coumaroyltransferase (ACT). Although two ACT-encoding genes (HvACT-2HL1/2) on the long arm of barley chromosome 2H (2HL) have been identified, our previous study suggested the presence of another ACT locus on the short arm of chromosome 2H (2HS). In this study, an analysis of dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected pCA in wheat lines carrying the distal region of 2HS. This chromosomal region, which includes genes encoding the laccase catalyzing the last committed step of the HA biosynthetic pathway, was revealed to also contain a putative ACT gene (HvACT-2HS1), with the encoded amino acid sequence similar to that of HvACT-2HL1 (46% sequence identity). Changes in HvACT-2HS1 transcript levels were in accordance with those in the pCA-forming enzymatic activity and the pCA level in barley seedlings. Additionally, recombinant HvACT-2HS1 heterologously expressed in Escherichia coli had pCA-forming enzymatic activity, with high specificity for agmatine as the acyl acceptor. Moreover, a phylogenetic analysis indicated that HvACT-2HS1 is not a paralog of HvACT-2HL1/2. These results suggest that HvACT-2HS1 and HvACT-2HL1/2, which originated from different ancestral genes, jointly mediate the formation of pCA for HA biosynthesis in barley.