When ferns go BAHD: characterization of acyltransferases responsible for shaping the phenolic landscape of the fern Neoblechnum brasiliense

Main conclusion 47 BAHD acyltransferase sequences were retrieved in a transcriptome of the rosmarinic acid-accumulating fern Neoblechnum brasiliense . One sequence encoded a hydroxycinnamoyltransferase accepting (hydroxy)phenyllactates leading to the formation of rosmarinic acid. Abstract The fern Neoblechnum brasiliense (Blechnaceae) is known for its accumulation of rosmarinic acid and related phenolic compounds. Previous studies failed to identify the enzyme catalyzing the transfer of a hydroxycinnamoyl moiety onto a phenyllactic acid—the key step in rosmarinic acid biosynthesis. A long-read transcriptome of young and mature fronds of Neoblechnum brasiliense was established, yielding 47 transcribed BAHD acyltransferase sequences. The encoded enzymes were tested for catalytic activity using p -coumaroyl-CoA and 70 putative acceptor substrates. NbHCT10/11/12/13 primarily accepted shikimic and quinic acids and grouped into BAHD clade V, alongside other enzymes utilizing the same acceptors. NbHCT19/20 acted as ω-hydroxypalmitic acid hydroxycinnamoyltransferases, clustering in clade VI alongside similar enzymes. NbHCT21/22 were also members of clade VI but displayed different substrate acceptance profiles. NbHCT22 acylates tryptophan, while NbHCT21 utilizes phenyllactic, 4-hydroxyphenyllactic and 3,4-dihydroxyphenyllactic acid as acyl acceptors, qualifying it as an enzyme able to form rosmarinic acid. The biochemical characterization of NbHCT21 showed a high affinity for p -coumaroyl- and caffeoyl-CoA (K m 9.2 and 4.4 µM, respectively), but rather low affinity for 4-hydroxyphenyllactic acid (K m 3.2 mM). The phylogenetic placement of NbHCT21 indicates a convergent evolution of enzymes capable of synthesizing rosmarinic acid. Experiments on NbCYP98 as the meta -hydroxylating enzyme revealed that p -coumaroyltryptamine was preferentially hydroxylated along three other p -coumaroyl amides. However, not the p -coumaroyl but the tryptamine moiety was hydroxylated, presumably forming p -coumaroyl- N -serotonin. Our transcriptome analysis provides novel results regarding the phenolic metabolism in the fern Neoblechnum brasiliense , which has an elaborated spectrum of phenolic compounds, and a basis for future research.