Wine-red lignin revisited, dirigent proteins, and native lignin macromolecular configuration

The wine-red coloration of xylem in antisense cinnamyl alcohol dehydrogenase (CAD) down-regulated tobacco (Nicotiana tabacum) has long been reported as due to wine-red colored lignin. To investigate validity of the wine-red lignin report, both wild type (WT) and anti-sense CAD down-regulated tobacco stem xylem tissues were examined, with the red pigment found to be facilely removed by treatment with an 0.5 % HCl in MeOH solution at 4 °C. The red pigment in the xylem tissues was rapidly reconstituted in both pre-treated antisense CAD and WT xylem tissues by incubation with sinapyl aldehyde in MeOH solution, and then facilely removed by subsequent 0.5 % HCl in MeOH treatment. Only sinapyl aldehyde was demonstrated as contributing to the red pigmentation, this being consistent with its anionic quinone methide canonical form. No evidence was obtained for a wine-red lignin. Additionally, lignin-derived isolates from both antisense CAD and WT lines essentially differed only in presence of small amounts (ca. 1.41 %) of 8-O-4' linked sinapyl aldehyde end groups and a slightly smaller molecular weight distribution (MWD) of the lignin from the antisense CAD line. No evidence was obtained that poly-hydroxycinnamyl aldehydes contributed to bulk lignification; the slightly lower MWD may result from hydroxycinnamyl aldehydes functioning as terminators of polymerization during lignification. More importantly, these findings are placed in context of what is now known about 1) lignin macromolecular assembly proper in planta, 2) dirigent protein functions, 3D structures, and active sites as entry points to distinct plant phenol metabolic classes, including lignins, 3) AlphaFold2 modeling of Dir-e subfamily proteins involved in lignification, and 4) what urgently remains to be done in the study of lignification.