CCR has recently been both downregulated and mutated in tobacco (Nicotiana tabacum) (232) and Arabidopsis (233), respectively. The phenotypes obtained for each line were, at first glance, quite similar: both were considerably dwarfed (131, 232) (Figures 7.13C and 7.13D). Such effects, as already noted earlier, are not always a typical consequence of downregulating lignin amounts and/or compositions. Nevertheless, by comprehensively examining the lignin contents and compositions, stem diameters/lengths and anatomy, it was considered that the Arabidopsis irx4 mutant was delayed in overall development (Figure 7.12F), particularly as regards lignification (131). Specifically, the deposition of S-lignin components in the irx4 mutant line initially lagged behind that of wild type. At maturation, the lignin S/G compositions (ratios) were similar though for both lines, with the overall lignin amounts only being 10-15% lower in the mutant (131). On the other hand, the lignin levels reported for the CCR downregulated tobacco stems were circa 50% of wild-type levels, with the xylem cells collapsed (Figure 7.12E), indicative of a much compromised vascular apparatus (77, 232). At the anatomical level, it was of interest that both the Arabidopsis and tobacco stem cross-sections apparently differed substantially in their overall effects on vascular integrity; in neither case were “perfectly viable” plants obtained, given the defects/pleiotropic effects noted.

Detailed analysis of lignin deposition in the Arabidopsis irx4 line had thus established that initially the mutant had a delayed but coherent (normal) program of lignification (131, 132). By contrast, a previous study by other researchers (233) had reported that this plant line contained an “abnormal lignin,” derived from “alternative” phenolics and whose lignin levels were reduced by circa 50%. The “abnormal” nature of the lignin was apparently determined by NMR spectroscopic analyses, although no data were actually provided. These reports have since been revised with more in-depth analyses from our laboratory (131, 132). First, the full extent of the lignification response in the irx4 mutant line was only actually determined by examining the lignin contents and compositions up to maturation (>8 weeks), with only a small reduction (~10—15%) in deposition levels noted. Second, NMR spectroscopic analyses indicated that typical G-S lignins were being formed and not “abnormal” lignins as had been reported. Furthermore, we proposed that the dwarfing phenomenon and reduced lignin levels may be due to CoASH levels being reduced (due to build up of hydroxycinnamoyl CoA derivatives) in the irx4 line, with some sort of, for example, feedback inhibition occurring (131); however, this remains to be fully established in future studies.

Perhaps most importantly, plotting lignin contents versus (thioacidolysis) releasable monomers (representing a subset of cleavable 8- O-4′-linkages) again indicated that both irx4 and wild-type lines had a monomer invariant frequency (Figure 7.14C) of said linkages at all stages of lignin deposition, plant growth, and development. These data are thus again discussed later in terms of further indications of a non-random assembly process.

The original, albeit now incorrect, report by Jones etal. (233) was apparently based on ex­pectations that had been raised from the study of the CCR downregulation in tobacco (173, 174,226). The latter papers described bewildering findings as regards lignin macromolecular assembly and composition. Specifically, it was reported that when CCR was downregulated in tobacco, the plants compensated for reduction in monolignol (lignin precursor) supply by incorporating other “alternative” phenolics into the lignifying cell walls. Initially, the “non-traditional” phenolics reported as incorporated into lignin through CCR downregu­lation included ferulic (11) and sinapic (13) acids, as well as a variety of other phenolics, including acetosyringone (61) (173, 174, 226). It was also reported that feruloyl tyramine (60) was “heavily incorporated” into the lignin as a consequence of CCR downregulation (173, 174, 226), and that this moiety represented a chemical “signature” for CCR downreg­ulation (174). However, no quantification of feruloyl tyramine (60) levels of any sort was carried out.

As regards the reported increases in amounts of hydroxycinnamic acids/benzaldehydes, etc. in the lignins of the highly dwarfed, lignin reduced (~50%), CCR downregulated tobacco lines, relative to the wild type, there was apparently no significant difference in total amounts measured quantitatively (77). For example, the reported levels of such moieties ranged from 0.04 to 0.07% of stem cell wall residues in both wild type and downregulated lines (77). Such minute levels would not constitute compelling evidence for “abnormal” lignin and “aberrant” lignins being formed from “non-traditional monomers.” Indeed, since the lignin contents of CCR downregulated and wild type were ~11 and ~22% of the plant stem dry weight, respectively (77), the amounts of the aldehydes/acids (0.04-0.07%), etc. were minuscule relative to actual lignin levels.

Other studies by Ralph and colleagues (173, 174), which reported that there were ele­vated levels of feruloyl tyramine (60) covalently attached to tobacco lignins, and that these were chemical signatures of CCR downregulation/mutation, could not be independently verified either. Furthermore, the lack of any feruloyl (p-hydroxycinnamoyl) tyramine (60) resonances in the Arabidopsis CCR-irx4 lignin-derived isolates (77, 132) eliminated these as being generic chemical “signatures” for CCR mutation/downregulation as had been pro­posed (174). By contrast, careful isolation (by dissection) ofthe vascular (lignified) apparatus and subsequent detailed NMR spectroscopic analyses of the resulting lignin isolate(s) gave no evidence for the presence of feruloyl tyramine (60) moieties in the lignins from the wild — type lines of tobacco (177). Nor were feruloyl tyramine moieties (60) observed in the lignin preparations by Bernard-Vailhe et al. (234). Additionally, in contrast to reports (173, 174) of feruloyl tyramine (60) moieties being incorporated into lignin, these researchers had never demonstrated that the feruloyl tyramine-like resonances in the lignin-enriched isolate were covalently linked to lignin, or came from the same cell wall types harboring lignins. Nor was evidence provided that the overall amounts of feruloyl tyramine (60) moieties had increased in the isolates from the CCR downregulated lines, relative to the original levels in the wild-type line.