Functional study of an O-methyltransferase in eupatilin biosynthesis in Artemisia argyi by an efficient hairy roots transformation system.

Artemisia argyi is a well-known medicinal plant, and the reference genome has been worked out. However, the absence of an effective genetic transformation system obstructs research into the genetic functions associated with the biosynthesis of its active ingredients. Here, we first established an efficient hairy roots (HRs) transgenic system for A. argyi. MSU440 was the most suitable R. rhizogenes strain for HRs transformation in A. argyi. After 20 days of suspension culture, the biomass of HRs could grow almost 16-fold. The external application of MeJA could considerably boost the levels of flavonoids and phenolic acids in the HRs of A. argyi. RUBY-expressing vector was transformed into the HRs of A. argyi and the transgenic red roots could be obtained with a 24.3 % positive rate. Eupatilin is a polyoxymethyl flavonoid modified by O-methyltransferase, which is the main active component of A. argyi. Furthermore, we cloned an O-methyltransferase gene from A. argyi, AYFOMT2. Enzymatic assays confirmed that AYFOMT2 could catalyze methylation at the 4'-OH of ring B in jaceosidin to produce eupatilin. The AYFOMT2 gene was transformed into the HRs of A. argyi through the established transformation system, which remarkably increased the content of eupatilin. Overall, we established an efficient system for the induction of HRs and genetic transformation technology. Genetic transformation will be a valuable tool for the functional study of A. argyi. A key O-methyltransferase enzyme was cloned, which provided genetic resources for the subsequent biosynthesis of eupatilin.