Catalyzing the final step in monolignol 1-5 biosynthesis, the effects of downregulation and mutating bona fide CAD genes have been extensively studied and reported upon [see Anterola and Lewis (77), for a comprehensive discussion and analysis]. The common phe­notype resulting from CAD mutation/downregulation is that of a red-brown coloration in the xylem region (57, 71, 172, 235-237) and Figure 7.12G shows this effect in a stem cross-section from a double CAD mutant (cad-4 cad-5) in Arabidopsis (57, 71). As men­tioned above, this coloration has been known for nearly eight decades (58) in the brown midrib mutants, with the bm1 associated with expression of the CAD gene (67). This red — brown coloration was also reported as being due to formation of an abnormal “wine-red” lignin in tobacco (236, 237), with this being proposed to have good potential for furniture staining, dyes, and so forth. Moreover, the results from such studies and other analyses of presumably lignin-enriched isolates from CAD downregulation/mutation of tobacco were rationalized by several investigators as further evidence for random coupling/combinatorial biochemistry, and thus of lignin’s composition not being particularly important (173,226).

Our own data and the interpretations thereof, provide very different findings and insights. More importantly, they give an evolutionary perspective as to why lignins are essentially monolignol (1, 3, and 5) derived (and, partially from monolignol esters 30-32 in grasses as well). Specifically, the recent findings now help explain why lignins are not formed from, for example, p-hydroxycinnamaldehydes 19, 21, and 23.

7.6.2.2.1 "RED LIGNIN": A MISNOMER

Comprehensive analyses and reassessment of reports of “red lignin” in tobacco, (235-237) resulting from CAD downregulation, established that there was no “red-lignin” as such (177). Instead, it was simply a pigment [mainly sinapyl aldehyde (23) derived] in near trace amounts in tobacco that could readily be removed under conditions generally used for floral pigment removal (i. e., 0.5% HCl in MeOH) (177). Similar treatment of Arabidopsis “red xylem” also resulted in facile removal of this coloration, with concomitant release of sinapyl aldehyde (23) (71). No evidence was obtained, though, this red pigment was an integral part of the polymeric lignin. Interestingly, the red coloration could also be reconstituted on either preextracted tobacco xylem tissue cross-sections and/or polyamine TLC plates, by dipping either into a dilute solution of sinapyl aldehyde (23) (177). Furthermore, Bernard-Vailhe et al. (234), using modified Bjorkman procedures to isolate lignin derivatives from both wild type and CAD downregulated tobacco xylem, had also noted that the red coloration was completely removed from the lignin-derived preparations. These data also contrast with a contribution by Boerjan and coworkers (238) who reported that the “red xylem” coloration in tobacco could not be removed by treatment with sulfuric acid, methanol, butanol/HCl, acetyl bromide, or triethylene glycol. Such an observation, therefore, needs to be independently confirmed, as it appears incongruous with the findings using both tobacco and Arabidopsis.