Identification of the Structure and Origin of a Thioacidolysis Marker Compound for Ferulic Acid Incorporation into Angiosperm Lignins (and a pseudo-Marker Compound for Cinnamoyl-CoA Reductase Deficiency)

S1. Comment: Tyramine Ferulates in Tobacco Lignins Tobacco lignins were found to have a substantial component derived from tyramine ferulate. The levels of this component appeared elevated in certain CCRand CAD-deficient transgenics (Ralph et al., 1998). Although we were careful to note that tyramine ferulate is a wellknown wound-response product in tobacco, we remained convinced by the correlation data from detailed NMR experiments that the component was well integrated into the polymeric fraction characterized as lignin. It has, however, been claimed that tyramine ferulates were not part of the polymer fraction at all and were only low molecular-mass impurities (Anterola and Lewis, 2002). The argument, based on noting that the tyramine peaks in 1D 13C-NMR spectra were much sharper than those from the bulk lignin polymer, is flawed. As is well known, terminal units that are especially rotationally mobile have lower NMR relaxation rates and hence sharper peaks than the bulk polymer (Ralph et al., 1999). Thus the terminal free-phenolic tyramine units attached to ferulates incorporated into the backbone of these isolated polymers, as well as free-phenolic p-coumarate units found acylating the γ-methylol of sidechain units in grass lignins (Ralph et al., 1994), and p-hydroxybenzoates similarly found acylating lignin units in palms, poplars and willows (Smith, 1955; Nakano et al., 1961; Landucci et al., 1992; Sun et al., 1999; Meyermans et al., 2000; Li and Lundquist, 2001; Lu and Ralph, 2003), all display such sharp peaks. A simple argument, beyond noting the fact that the lignins were molecular-mass-fractionated, as stated in the original CCR paper (Ralph et al., 1998), would have mollified the criticism—if the tyramine ferulate units were from low molecular mass components, the ferulate moieties would also display observably sharp peaks. However, finding the ferulate moieties, and proving that they were attached to the tyramines, required more sophisticated NMR experiments. The simple reason is that the ferulate moiety cross-couples integrally into the polymer, combinatorially coupling at its 4–O-, 5-, and 8-positions like the monolignols themselves, as has been reviewed (Ralph et al., 2004). Ferulates are consequently involved in such a variety of structures that they become difficult to detect in simple 1D spectra. Whether tyramine ferulates are truly part of lignin can be debated, but the contention that they have been “unequivocally disproven” (Anterola and Lewis, 2002; Patten et al., 2005) as being incorporated into the polymer by combinatorial radical coupling reactions is not supported by the facts.