A cad-4 cad-5 (cad-c cad-d) double mutant of Arabidopsis (ecotype Wassilewskja) was also recently successfully generated (57) and subsequently comprehensively analyzed (71). Figure 7.13E shows the prostrate phenotype that results from the double mutation relative to that of wild type (71); as anticipated, the dynamic modulus properties were also substan­tially reduced, this being a further indication of a structurally weakened vascular apparatus (71). Several other important features were identified following the comprehensive study of different stages of growth/development and polyphenolic deposition into the cell walls of the double mutant, using both chemical degradation and 13C NMR spectroscopic analyses. Specifically, the double mutant contained very small amounts of monolignols (circa 10% of the polyphenolics present), as well as polymeric p-hydroxycinnamaldehyde moieties. At plant maturity, these constituted together about 11.3% of the cell wall residue, in contrast to lignin in the wild-type line which was almost double this amount (~22.5%). Chemical degradation (thioacidolysis) analyses though established that the total amount of monomer — cleavable 8-0-4′ interunit linkages, i. e., monolignol 1,3, and 5 andstyryl — O-aryl ether (sub­structure XII, Figure 7.11B), etc. derived substructures closely mirrored that for monolignol — derived lignin deposition (Figure 7.14D) at the same stages of growth/development. [This was not previously recognized by other researchers (57) since their analyses of the styryl — O-aryl ether-derived substructures (XII, Figure 7.11B) were lower by almost an order of magnitude, due to a lack of either authentic standards and/or correct response factors.]

That is, the 8-0-4′ interunit linkage frequency was again apparently directly propor­tional to polyphenolic content in a manner somewhat analogous to that of lignification proper in the wild-type line; however, the p-hydroxycinnamaldehyde deposition was ter — minated/aborted prematurely (see arrowhead, Figure 7.14D). Perhaps significantly, this metabolic “checkpoint” — arresting polyphenolic deposition — also appears to be coincident to that of termination of H-lignin deposition in the pC3H line, suggesting a common mech­anism is in place for terminating/aborting both processes. Interestingly, 8-5′ linkages were also very evident in the p-hydroxycinnamaldehyde isolates, but in this case with substruc­ture IX (Figure 7.11B); others [e. g., substructures X and XI (239)] were not detected (71).

Taken together, it is proposed that the double mutant has attempted, in a futile manner, to produce a poly-p-hydroxycinnamaldehyde facsimile (of inferior structural properties) to that of monolignol 1-, 3-, and 5-derived lignins. This can thus be provisionally envisaged to occur through very limited substrate degeneracy during template polymerization, that would normally be operative for constitutive macromolecular lignin assembly. On the other hand, the poor biophysical/structural integrity properties of the phenotype so obtained presumably provide useful insight as to why poly-p-hydroxycinnamaldehydes did not evolve as a substitute for the monolignols 1, 3, and 5 in lignification proper.