The systematic analyses ofvarious transgenic/mutant lines has identified important trends in lignin macromolecular assembly. These analyses (71, 72,131,132,215) have also eliminated non-monolignol entities, such as feruloyl tyramine (60), acetosyringone (61), etc. as being involved in core lignification in contrast to previous assertions (173, 174). Additionally, as summarized in Figure 7.14, modulation of the monolignol-forming pathways in the same organisms gave H, G, 5OH-G, and S-enriched lignins whose subset of identifiable, cleavable, interunit linkages were apparently invariant of hydroxylation/methoxylation patterns of aromatic ring substitution. In the case of H-monolignol deposition, however, this was prematurely terminated at a “metabolic checkpoint” (72, 215), the reasons for which need to be established. A similar situation also held for the po/y-p-hydroxycinnamaldehydes (71), with severe adverse effects being noted on plant structure overall and thus on vascular integrity. For the H, G, 5OH-G, and S enriched lignins, this subunit invariance would not be expected a priori as the H, G, 5OH-G, and S monolignols differ in having from 5 (H) to 3 (S) potential sites available for radical-radical coupling. Additionally, the catechol nature of 5-hydroxyconiferyl alcohol (4) represents yet another potentially confounding feature, as discussed earlier.

Yet the data obtained in lignin subunit characterization and frequency suggest a limited substrate degeneracy during the dehydropolymerization step, i. e., whereby the amounts of (cleavable) 8- O-4′ and 8-1′ interunit linkages present are kept directly proportional to lignin content, but apparently invariant of hydroxyl/methoxyl group aromatic ring substitution pattern. Moreover, for the H — and G-enriched lignins, the quantifiable amounts of releasable 8-5′, 5-5′, and 5-O-4′-derived dimers followed similar trends, whereas neither the 8-8′ linked ligballinol (58) and pinoresinol (69) were released in any appreciable amount. The latter is likely consistent with their presumed covalent modification during native lignin formation, perhaps at C-5. Analyses of the S-enriched Arabidopsis line also provided useful insights: While only ~32% of the linkages could be accounted thus far via thioacidolysis, the 8-8′ linked syringaresinol (70) subunits were only present in very small amount as their thioacidolysis derivatives. These data suggest that the S-enriched lignin was not composed primarily of S-units linked through 8-O-4′ and 8-8′ interunits as previously envisaged (i. e., substructures Ic and IIIc, Figure 7.2D), the reasons for which need to be determined. Additionally, the COMT knockout line (in Arabidopsis) resulted in an apparently equivalent reduction of G/S monomeric moieties leading to benzodioxane formation. Such data, for the COMT knockout mutant line in Arabidopsis, provisionally suggest that in the cell wall types normally forming S-units, each of the 5-hydroxyconiferyl alcohol (4) moieties is linked to a coniferyl alcohol (3) moiety; this is again envisaged to place another severe limitation on how native lignin macromolecular configuration is achieved.

Taken together, the data have nevertheless begun to provide useful insight into the trends involved in lignin macromolecular assembly, and give an impetus to explore and determine lignin primary structure. We do not consider, however, that these data are consistent with a “random assembly/combinatorial chemistry” model with 1066 or more isomers, but instead reflect limited substrate degeneracy during native lignin formation, as is observed in many areas of metabolism. It is presumably significant that several of the preceding enzymes in the monolignol-forming pathway (4CL, CCR, and CAD) are also substrate versatile. This, in turn, may have an important bearing on the limited substrate degeneracy observed for lignin macromolecular assembly.