Norlignan biosynthesis in Asparagus officinalis L.: the norlignan originates from two non-identical phenylpropane units

Abstract

Little is known about the biosynthetic mechanism(s) of norlignans with C₆–C₅–C₆ skeletons in spite of their important contributions to heartwood formation in conifers. To clarify the mechanism(s), we have established cell-suspension cultures of Asparagus officinalis that produce a norlignan, (Z)-hinokiresinol, after fungal elicitor treatment. Feeding experiments with ring- or side chain-¹³C- and/or ²H-labelled phenylpropanoid monomers show that two units of L-phenylalanine, cinnamic acid, 4-coumaric acid, or 4-coumaryl alcohol are efficiently incorporated into the norlignan. ¹³C NMR of (Z)-hinokiresinols isolated after individual administration of [7-¹³C]cinnamic acid, [8-¹³C]cinnamic acid, and [9-¹³C]cinnamic acid conclusively demonstrate that the side chain 7-C, 8-C, and 9-C atoms of cinnamic acid are incorporated into C-1 and C-3, C-2 and C-4, and C-5 of (Z)-hinokiresinol, respectively. Thus, ring- and side-chain-labelled tracer results indicate that all carbon atoms of (Z)-hinokiresinol are found to originate from C₆–C₃ (phenylpropanoid) monomers, and this compound is formed with a loss of one carbon atom at the 9-position of one of the coupling monomers. Furthermore, a competitive tracer experiment with simultaneous administration of 4-[ring-¹³C₆]coumaric acid and 4-[7,9,9-²H₃]coumaryl alcohol indicates that the C₆–C₃ moiety of (Z)-hinokiresinol is derived from 4-coumaryl alcohol, while the C₆–C₂ moiety originates from a 4-coumaroyl compound such as 4-coumaroyl CoA and not directly from 4-coumaryl alcohol.