Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley
Naoki Ube, Atsushi Ishihara, Yukinori Yabuta, Shin Taketa, Yasuo Kato, Taiji Nomura
Abstract
SUMMARY Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p ‐coumaroylagmatine ( p CA), is an antifungal compound accumulated at high levels in young barley ( Hordeum vulgare ) seedlings. The enzyme responsible for the oxidative dimerization of p CA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H 2 O 2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat ( Triticum aestivum ) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes ( HvLAC1 and HvLAC2 ) that are highly expressed at the seedling stage and may encode enzymes that oxidize p CA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis‐related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo ( Phyllostachys nigra ) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N . benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo‐configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of p CA during HA biosynthesis.