Modulation of 4CL activity can also, depending upon the gene(s), be targeted to affect monolignol, flavonoid, suberin, and related metabolism. Standard biotechnological ma­nipulations of overall 4CL activity have been described thus far using tobacco (221-223), Arabidopsis (185), and aspen (Populus tremuloides) (224). Very preliminary descriptions of each ofthe corresponding phenotypes so obtained were also reported to be dramatically dif­ferent, even though lignin levels were apparently diminished to a similar extent by circa 50% or so, but only when 4CL activity levels were greatly reduced (down to ~7% of wild-type levels) (77). The reports on the transformants ranged from having no visible phenotype in Arabidopsis, to dwarfing in tobacco, and to a claim of spectacularly enhanced growth in aspen.

In the case of 4CL downregulation in tobacco, the data obtained were the most compre­hensive and generally appeared the most reliable (77, 221-223), albeit where several of the transformants were dwarfed at the flowering stage. In terms of the 13C NMR spectra of the lignin-enriched preparations isolated from the wild type and 4CL downregulated lines, both were very similar except for a few slightly enhanced resonances which were subsequently interpreted by Anterola and Lewis (77) as being mis-assigned by Kajita etal. (222). We con­sidered these to correspond instead to p-coumaroyl (59)/feruloyl (60) tyramine moieties, which if correct, were then in accordance with our previous work on this molecular species in tobacco (225). There was no evidence that the overall amounts of these well-known tyramine-derived moieties in the Solanaceae had either increased, or were covalently linked to the lignin in the lignin-enriched isolates. Interestingly, and as expected, the 4CL downreg — ulated line displayed a significant reduction in vascular integrity as evidenced by collapsed xylem in the tobacco stem cross-sections (223) (see Figure 7.12D). Again, though, there were no comprehensive studies on tobacco growth/development and structural integrity of the vasculature of these transformants carried out to gain a more full understanding of the overall effects engendered by this manipulation.

For 4CL downregulation of Arabidopsis (185), the phenotype was apparently similar to wild type, although no data were provided describing either growth and/or developmental processes. Additionally, the results obtained were not explicable based on known lignin chemistry, even though ostensibly there were reductions in lignin contents. For example, the approach taken by these researchers to estimate lignin amounts used the unreliable thio — glycolic acid method. Nitrobenzene oxidation (discussed earlier) was also carried out, this being a degradative method which generally accounts at best for circa 15-25% of the lignin present in plant tissue (Figure 7.10E). Recalculation and comparison of the thioglycolic acid and nitrobenzene oxidation data by Anterola and Lewis (77) determined, however, that the latter data accounted for >115% of the thioglycolic acid lignin perhaps indicat­ing that either the true lignin contents were grossly underestimated, or the alkaline NBO analyses were incorrect. This is an important matter as it again underscores the serious discrepancies in which many lignin “analyses” are often being carried out in this field. For example, currently many researchers typically do not either compare or contrast results be­tween the different methods used for consistency, accuracy and/or quantification reliability. Accordingly, meaningful trends are potentially being missed in terms of overall effects of manipulation of lignin contents and compositions. Hence, taken together, the effects of Arabidopsis 4CL downregulation provided little insight yet other than perhaps (and quite unexceptionally) possibly reducing lignin levels. Again, these studies simply represent very preliminary analyses and now need to be extended to probe both effects on lignification proper and on vascular apparatus assembly.

4CL downregulation in aspen resulted in quite unusual assertions based on the data actu­ally obtained (224). In that study, it was reported that the downregulated lines grew ~50% taller and that cellulose synthesis was markedly increased. Our reanalysis (77) gave a very different interpretation. In terms of accelerated growth, the lignin contents of the controls were 21.62%, whereas the “growth acceleration” reportedly occurred in the various trans­genic lines having between 20.6 and 11.8% lignin, respectively (224). That is, a very small reduction in lignin content from 21.62 to 20.6% was considered sufficient to result in acceler­ated growth. This effect has not been observed in any other study of lignin downregulation/ mutation, and requires scientific explanation, if correct. Moreover, to date, there have been no further reports of effects on poplar growth by 4CL downregulation. These investigators, or others, should thus clarify at the earliest opportunity whether growth enhancements are still observed for both greenhouse and field-grown 4CL downregulated lines, and what ef­fects on lignification, cell wall architecture, and physiology in general result. One possibility to be considered is that this plant line is capable of overproducing reaction (tension wood) tissue as a mechanism of partially “compensating” for reduced lignin levels, i. e., as noted for pC3H downregulation in alfalfa (72).

If so, this could further explain the reportedly enhanced cellulose contents, which had also only “increased” proportionally in large part because of reductions in lignin levels. Furthermore, although the full NMR spectroscopic data was not provided for that study (224), it was stated that the lignin in the 4CL downregulated line (reduced by ~45%) was apparently the same as that of wild type, as were also the G/S ratios by thioacidolysis. This would again provisionally be expected from first principles (77): namely that there were simply reductions in lignin content when 4CL activity was greatly suppressed without any “compensation” by other non-monolignol phenolic entities into the lignin core structure as suggested by others (173-175, 226). Finally, these preliminary studies again emphasize the need to conduct more extensive analyses of the lignins and to determine the effects on the vascular apparatus integrity in 4CL downregulated lines.