The maize bm3 mutant (61), now known to affect COMT activity (66), is a plant line with reduced stem stalk strength and prone to stalk lodging (falling and collapsing) relative to wild type, thereby currently precluding its commercial utilization (250) [see discussion in Anterola and Lewis (77)]. Not having a rate-limiting function in the pathway in terms of carbon allocation, modulation of COMT activity does not affect overall lignin contents. Instead, the monolignol, 5-hydroxyconiferyl alcohol (4), is formed rather than sinapyl alcohol (5); the former is then used in place of sinapyl alcohol (5), but whereby a polymeric structure is generated whose properties are such that it results in a weakening of overall plant vascular integrity due to the incorporation of (4) into the lignifying matrix. This is again considered to reflect a tightly programmed lignification response, albeit where the outcome is affected by a limited substrate degeneracy during template polymerization. This, however, results in diminished cell wall properties and helps explain why 5-hydroxyconiferyl alcohol (4) never evolved to be an effective lignin precursor.

As indicated earlier, the role of COMT in the second methylation step of syringyl lignin biosynthesis was first demonstrated by Atanassova et al. (136) in tobacco, and later fur­ther confirmed by others in poplar (182), aspen (251), and alfalfa (252). Downregulation/ mutation of COMT though had no apparent adverse effect on overall lignin amounts as indicated using Klason lignin estimations (77), with the striking exceptions reported from the Dixon laboratory (186, 212). These latter researchers had again used the unreliable thioglycolic acid lignin (186) and the neutral detergent fiber (NDF)/Klason lignin methods (212), the results of which appeared to indicate that lignin contents had been (significantly) reduced (by circa 60%). While this interpretation can now be viewed as incorrect, this was the same methodology used for the PAL (207, 212) and C4H (207) studies; hence, the over­all findings from these studies should also be reexamined with more robust and reliable technologies.

Additionally, in preliminary studies, COMT downregulation had no apparent significant adverse effect on the amounts of thioacidolysis-releasable G-monomers in the lignins from either tobacco or poplar, given that the total amounts, relative to lignin contents, were very similar to the wild-type lines (77). On the other hand, little to no S-units could be detected. To account for this, we can provisionally propose that in the COMT downregulated lines, the 5-hydroxyconiferyl alcohol (4) moieties were undergoing homo-coupling, whereas in the wild-type lines, sinapyl alcohol (5) was also doing the same. However, instead of 8- O-4′ bond formation, etc. occurring as in the wild-type line (see Figure 7.15A), the presence of the flanking 5-hydroxyl group (from 4) has resulted in an apparently near-quantitative replacement with the benzodioxane substructure (Figure 7.15B), rather than the “simple” 8-O-4′ interunit linkage present in the wild-type line. That is, the main difference be­tween the 8-O-4′ linkage (Figure 7.15A) and the benzodioxane substructure formation (Figure 7.15B) is only in “trapping” of the quinone methide intermediate during template polymerization. [By contrast, if the incoming monolignol radical was a p-coumaryl (1), coniferyl (3), or sinapyl (5) alcohol moiety, the “trapping” would be performed by an exter­nal water or possibly carbohydrate molecule.] Nevertheless, formation of the benzodioxane substructure can thus still be considered as an 8-O-4′ interunit linkage, even if the overall substructure generated differs.

Furthermore, with the Arabidopsis COMT-downregulated line, there provisionally appears to be a near equivalent reduction in amounts of G-releasable monomers to that of the original S levels (see Figure 7.15C which plots G and S 8- O-4′ interunit cleavage, leading to monomer release, at different stages of Arabidopsis growth and development; Jourdes et al., manuscript in preparation). In this case, the near equivalent reduction of both G and 5-OHG (previously S) moieties at different stages of growth and development suggests that G-S hetero-coupling was mainly occurring in the wild-type line, i. e., via hetero-coupling of coniferyl (3) and sinapyl (5) alcohol-derived monomers, thereby affording cleavable 8-O-4′ interunit linkages. This is now apparently replaced by an equivalent level of hetero ­coupling between coniferyl (3) and 5-hydroxyconiferyl (4) alcohol-derived radicals to afford the mixed benzodioxane substructure (Figure 7.15B). This near 1:1 reduction in releasable G:S moieties thus places another considerable constraint on how lignin macromolecular configuration is actually being achieved. It could, for example, possibly indicate that G and S monomers are being alternately laid down during macromolecular lignin assembly;

such observations once again underscore the urgent need for technology development to determine lignin primary structure(s).

In alfalfa, however, COMT downregulation resulted in reduction of both G-S and S-S amounts, this in turn presumably reflecting differing cell-type lignification processes. In this regard, although the incorporation level of 5-hydroxyconiferyl alcohol (4) moieties into the

lignin was not quantitatively estimated using chemical lignin degradative analysis, attempts to do this indirectly were carried out, i. e., by attempting to estimate the level of benzodioxane substructures using NMR spectroscopic analyses of the extracted lignin-enriched isolates by measuring volume integrals in the 2D HMQC spectra (253). Reassessment of this data herein also results in the provisional conclusion that overall levels of 8— O—4′ interunit linkages were actually conserved in both wild-type and COMT-deficient alfalfa lines. That is, the wild-type line apparently had circa 81% of 8— 0—4 linkages, whereas the COMT-deficient alfalfa had ~38% of benzodioxane and 44% of 8—0—4′ linkages. Together, these represent ~82% of substructures with 8— O—4′ linkages, and thus represent a high degree of conservation of the overall 8— O—4′ linkage type in both wild type and COMT lines.

These data are thus also in apparent harmony with a proposed limited template degeneracy in assembly of the lignin macromolecule — but where both the chemistry on the proposed template is altered in the mutation (to involve catechol, rather than phenolic, chemistry). It is worthwhile reflecting that the alfalfa data has been known since 2003 (253), and yet has been interpreted by others as evidence for random coupling/combinatorial chemistry leading to 1066 isomers, etc. (175). On the other hand, while the COMT mutation results — in a presumably ordered way — in formation of benzodioxane substructures (Figure 7.15B), the biophysical effects, nevertheless, resulted in weakening of the overall vasculature.