pC3H is also considered to have an important regulatory role in carbon allocation (77) to the monolignol/lignin-forming pathway, at least as far as the G and S segments are involved. Two different mutant/transgenic lines (Arabidopsis and alfalfa) altered in pC3H activity have thus far been obtained using standard biotechnological manipulations (72, 213, 214). These manipulations resulted in one of the most dramatic examples of disparity seen in morphology between two different species, i. e., as a result of the same genetic target (pC3H) (72,215).

In this regard, the chemically generated (ethane methyl sulfonate, EMS) Arabidopsis ref8 mutant, isolated following screening of ~ 100 000 plants, contains a random mutation in the pC3H gene (213). The line was severely dwarfed (Figure 7.13A), reaching only ~3% of the height of wild type at maturity (215). It was also sterile and its proto-/metaxylem and xylary fiber elements had collapsed in the stem cross-sections examined (Figure 7.12B). This latter observation presumably reflects the very low levels of lignin, reduced by ~64% relative to wild type, and which is primarily of H character (H:G:S ratios of ~85:8:7) (215). This observation contrasts with previous assertions that the ref8 lignin was exclusively H-derived (175,213).

Although pC3H has a rate-limiting role in lignin biosynthesis and can thus be expected to produce vascular defects, the multiple and extreme morphological characteristics of ref8 suggested that additional pleiotropic effects were manifested in this line (215). Because of tissue availability limitations, the ref8 line was not readily amenable to more detailed analy­ses and thus these questions were not explored further. On the other hand, the alfalfa pC3H-I line was generated using a standard antisense transgenic method (214) with presumably less “unintended” effects resulting than that of chemical mutagenesis. In stark contrast to the Arabidopsis ref8, the alfalfa pC3H-I has a phenotype nearly like that of wild type (Figure 7.13B), with vessel anatomy also being very similar to wild type (data not shown) (72).

The remarkably different outcomes of modulation of pC3H between the Arabidopsis ref8 mutant and alfalfa pC3H-I lines (215) reflect further the weaknesses of current methods in the genetic manipulation of plants, and of our very limited understanding of said ma­nipulations. Indeed, it is for reasons such as the unknown (pleiotropic) effects observed herein that some segments of humanity currently distrust the use of genetically modified organisms. From a more scientific perspective, these limitations also highlight the need for comprehensive, careful, systematic data collection in order to more accurately assess,

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Figure 7.13 (continued) of Botany, vol. 94, Patten, A. M., Jourdes, M., Brown, E. E., Laborie, M.-P., Davin, L. B. & Lewis, N. G., Reaction tissue formation and stem tensile modulus properties in wild type and p — coumarate-3-hydroxylase downregulated lines of alfalfa, Medicago sativa (Fabaceae), pp. 912-925, Copy­right 2007, with permission from the Botanical Society of America. (D) Plant Journal, vol. 13, Piquemal, J., Lapierre, C., Myton, K., O’Connell, A., Schuch, W., Grima-Pettenati, J. & Boudet, A.-M., Downregulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants, pp. 71-83, Copyright 1998, with permission from Blackwell;and (F) The Plant Cell, vol. 19, Mitsuda, N., Iwase, A., Yamamoto, H., Yoshida, M., Seki, M., Shinozaki, K. & Ohme-Takagi, M., NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Ara­bidopsis, pp. 270-280, Copyright 2007, with permission from the American Society of Plant Biologists.] (Reproduced in color as Plate 22.) analyze, and understand the effects of such manipulations. Clearly, we need to get such manipulations “right,” for example, before contemplating dedicating large swaths of lands to lignocellulosic-modified organisms.

The alfalfa pC3H line also had lignin contents significantly reduced (>68% of wild type) (72, 215), which were again mainly of H-character (H:G:S ratios of ~77:12:11) as to be predicted (31,34,35,77). Significantly, biomechanical testing of stem vascular tissue integrity showed essentially no differences in tensile dynamic modulus properties, at least under the test conditions employed (72), i. e., where both the pC3H-I and wild-type lines apparently displayed more or less equivalent structural integrity of the vascular apparatus. More detailed anatomical analyses ofthe stem tissue inboththe pC3H-I and wild-type lines also established that the vasculature contained lignin-deficient, cellulose-enriched, gelatinous layers that are characteristic of reaction (tension) wood (see Figure 7.12C). Moreover, this occurred both earlier in growth/development and to a greater extent in the pC3H-I line (72), suggesting that vascular/structural properties are (at least in part) maintained by partially “switching” metabolic outcome to that of reaction tissue formation. Although the latter tissue is (both constitutively and inducibly) employed to control branching and realignment of leaning stem(s) back to the vertical, there are many applications, such as in forestry and in pulp/paper manufacture, where its formation is not desirable; gelatinous fibers, for example, result in poor paper strength quality due to decreased bonding properties (216).

Analyses of the lignins in both pC3H-I and wild-type lines were most instructive: the 13C NMR spectroscopic analysis of solubilized lignin derivatives established the presence of a H-enriched lignin (215) in pC3H-I as predicted (31, 34, 35, 77), with substructures I-VIII (Figure 7.2D) being in evidence. More importantly, the thioacidolysis degradation products (monomers and dimers) released, which corresponded to cleavage products from subsets of 8-0-4′, 8-5′, 5-0-4′, 3-3′ (5-5′), and 8-1′ (substructures I/II, IV, VI, VII, and VIII in Figure 7.2D) lignin interunit linkages, were determined quantitatively at vari­ous stages of stem growth/development/lignification until maturation (215) (Figures 7.14A, F-I). The frequency of the interunit linkages indicated that they were directly proportional to increasing lignin content, at least for the (monomeric/dimeric) cleavable 8-0-4′, 8-1′, 8-5′, 5-0-4′, and 3-3′ (5-5′) moieties being released. Significantly, the 8-8′-linked dimer (lig — ballinol 58) was not released as such (Figure 7.14J), even though its “substructure IIIa” was readily detectable by NMR spectroscopic analyses. This observation is also consistent with the previous “conundrum” of pinoresinol-like substructures IIIb not being released either from native lignins, whether by acidolysis (193) or thioacidolysis (217). These substructure­like moieties are, nevertheless, also readily detectable by NMR spectroscopic analyses. In direct contrast, synthetic lignin DHPs readily release such entities (218) upon cleavage. This, in turn, is presumably indicative of a very different mode of lignin assembly/organization in vivo than that occurring randomly in vitro.

Moreover, when compared to the lignin levels and interunit linkage frequencies in the wild-type line, the same correlations and trends exist for all levels of lignin deposition, with the amounts of individual products released and quantified apparently being invariant of methoxyl group composition. Significantly, the H-monolignol (1) did not seamlessly replace the G/S components (3/5), and thus presumably was unable to extend deposition into the domains normally designated for G/S monolignols in the lignifying cell wall(s). This, in turn, indicated that the H-monolignols (1) presumably could not substitute fully for either coniferyl (3) or sinapyl (5) alcohols, this representing yet another constraint on

Figure 7.14 Correlations of current best estimates of amounts of (A-E) total monomeric derivatives re­leasable by thioacidolysis as a function of estimated AcBr lignin contents);(F) 8-1′ dimeric derivatives after thioacidolysis follow by desulphurization as a function of estimated AcBr lignin contents;8-5′ (G), 5-5′ (H) and 5-0-4′ (I) dimeric derivatives after thioacidolysis followed by desulphurization as a function of total releasable monomeric derivatives by thioacidolysis;(J) 8-8′ dimeric derivatives after thioacidolysis followed by desulphurization as a function of total S unit releasable monomeric derivatives by thioacidol­ysis.

the notion of a seamless random coupling/combinatorial chemistry being in effect (175). On the other hand, the overall amounts of H-moieties appeared to increase slightly over that of wild-type levels, but where designated patterns of a subset of interunit linkage frequencies (for the products released) were being maintained (relative to wild-type lignin) until the overall deposition process was prematurely terminated — for reasons still to be determined. Such reasons for terminating this process could include the plant perceiving that the biophysical properties of the H-lignin being deposited in the cell wall were, for example, limiting and/or defective in some way, e. g., through feedback inhibition. Nevertheless, the partial replacement of G/S moieties by H-monomers can presumably be explained via limited substrate degeneracy during lignin template polymerization.

Reduction in lignin contents have also been reported with HCT silencing in Nicotiana benthamiana and Arabidopsis (219, 220). For example, RNA silencing was achieved in Ara — bidopsis (ecotype Columbia) by transformation with a hairpin repeat of a portion of the HCT gene whereas in N. benthamiana, a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system was used (219). In Arabidopsis, this resulted in stunted plants with apparently reduced lignin levels based on collapsed xylem cells (220). The yields of thioacidolysis monomers 54-56 of stems from 2-month-old plants were, however, very low, i. e., ~75.7 ^mol g-1 dry sample for the wild type and only ~1.0 ^mol g-1 dry sample for the silenced line (HCT-). Such values for the wild type (control) are uncharacteristically low and presumably indicate that the lines were of an unknown maturation state, and/or were defective in some manner, and/or the analyses had yielded unreliable data; in our studies and others, the thioacidolysis yields are reproducibly ~350 ^mol g-1 dry sample (cell wall residue, CWR) for wild-type lines at plant maturity (see Figures 7.14A-E). Thus, these data again underscore the need to determine at the minimum lignin contents and compositions at several stages until maturation has definitively been reached, rather than the single point analyses that have become typical of most studies in this field. On the other hand, the H:G:S ratios of 1:83:16 in wild type were now 83:12:2 in the HCT- line in agreement with HCT involvement in G/S lignin biosynthesis. In N. benthamiana, plants ranging from no visual growth phenotype to severely stunted phenotypes were also obtained, yet with estimated lignin levels decreased only by ~ 15% of wild type in the HCT-silenced plants as measured by the Klason method, i. e., the results for both species were apparently quite different in terms of effects on lignification. Although the reasons are not yet known for these differences, Arabidopsis and tobacco differ in having only one HCT homologue in the former, whereas in the latter both HCT/HQT enzymes are present. Yet, HQT silencing was also studied in tomato (Lycopersicum esculentum) where it was shown to only affect chlorogenic acid (26) levels but not lignin content/composition (111).