A similar situation to that for PAL downregulation/mutation holds for C4H. Although this enzyme is considered to be one of the important rate-limiting processes in phenylpropanoid metabolism (such as for differential carbon allocation) (34, 35), it is also a common step leading to various metabolites, including monolignols, lignins, flavonoids, and so forth. Downregulation of C4H has though only been studied in a very preliminary way thus far

Wild type B3 Tobacco Cinnamoyl CoA reductase
Wild type AOPP — treated Mungbean Phenylalanine ammonia lyase
Normal > development	Delayed development
CCRt-irx4
Wild type cad-4 cad-5 Arabidopsis Cinnamyl alcohol dehydrogenase
Wild type pC3H-l Alfalfa p-Coumarate-3-hydroxylase
Larger vessels
Normal vessels
Wild type CS-1 Tobacco l	Wild type 1074 Tobacco s p

(77, 207, 210-212) as regards overall effects on lignification and the vasculature. As noted above for PAL, the methodologies employed in two of these studies (207,212) were again very questionable (i. e., estimations of Klason lignin of neutral detergent fiber), and ultimately found to be unreliable as analytical protocols. Nevertheless, in all three studies the lignin levels provisionally appeared to be reduced — at least by >25% (207, 210, 211). Various alterations were also noted in S/G ratios of the lignified tissues, the reasons for which are yet unknown. As for PAL modulation, no evidence was obtained indicating deployment of non-monolignol moieties to “compensate” for reductions in lignin levels. There were also no experiments conducted to quantitatively ascertain the effects on the vascular integrity through biomechanical analyses. In hindsight, such studies need to be expanded significantly in order to ascertain, at the very least, the true effects of reductions in lignin levels on plant growth/development, on overall vascular structure/anatomy, and on lignin macromolecular configuration, etc.

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Figure 7.12 Examples of effects on vascular anatomy of inhibiting/mutating/downregulating various en­zymes and/or genes in the phenylpropanoid pathway. (A) AOPP-treatment of mungbean (204), results in collapsed xylem. (B) Similar effects occurred in a pC3H mutated Arabidopsis line (ref8). (C) On the other hand, formation of gelatinous fibers such as in wild-type alfalfa (Medicago sativa) apparently help mechanically offset deleterious effects of pC3H downregulation without forming "abnormal" lignins (72).

(D) Collapsed xylem observed in tobacco (Nicotiana tabacum) lines 4CL downregulated (223), as was

(E) xylem in a tobacco line CCR downregulated (232). (F) Detailed analysis of the Arabidopsis irregular xylem4 mutant (CCR1-irx4) identified pleiotropic effects, including delayed xylem formation (131). (G) An Arabidopsis CAD double mutant, AtCAD4/5, (cad-4, cad-5) with unusual red coloration in the xylem attributed to presence of p-hydroxycinnamyl aldehydes (71). (H) Qualitatively larger vessels observed in a tobacco line downregulated for a lignin-specific peroxidase (257). All images are of transverse sections and taken using brightfield microscopy, unless otherwise specified. Images shown in (A) are SEM analyses, while images (B and F) are images of semi-thin resin embedded sections stained with either toluidine blue O or Stevenel’s Blue. Image (C) was taken of cryosections stained with zinc chloro-iodide. Image (D) was taken of hand-cut sections stained with phloroglucinol-HCl. Image (E) was taken using UV light microscopy. Image (G) was recorded using unstained hand-cut sections, while image (H) was taken of phloroglucinol-HCl stained sections. [Reprinted from (A) The European Journal of Cell Biology, vol. 29, Amrhein, N., Frank, G., Lemm, G. & Luhmann, H.-B., Inhibition of lignin formation by L-a-aminooxy-P — phenylpropionic acid, an inhibitor of phenylalanine ammonia-lyase, pp. 139-144, Copyright 1983, with permission from Elsevier. (D) Plant Science, vol. 128, Kajita, S., Mashino, Y., Nishikubo, N., Katayama, Y. & Omori, S., Immunological characterization of transgenic tobacco plants with a chimeric gene for 4-coumarate:CoA ligase that have altered lignin in their xylem tissue, pp. 109-118, Copyright 1997, with permission from Elsevier. (E) 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. (F) Phytochemistry, vol. 66, Patten, A. M., Cardenas, C. L., Cochrane, F. C., Laskar, D. D., Bedgar, D. L., Davin, L. B. & Lewis, N. G., Reassessment of effects on lignification and vascular devel­opment in the irx4 Arabidopsis mutant, pp. 2092-2107, Copyright 2005, with permission from Elsevier. (G) Phytochemistry, vol. 68, Jourdes, M., Cardenas, C. L., Laskar, D. D., Moinuddin, S. G.A., Davin, L. B. & Lewis, N. G., Plant cell walls are enfeebled when attempting to preserve native lignin configuration with poly-p-hydroxycinnamaldehydes: Evolutionary implications, pp. 1932-1956, Copyright 2007, with per­mission from Elsevier;and (H) Phytochemistry, vol. 64, Blee, K. A., Choi, J. W., O’Connell, A. P., Schuch, W., Lewis, N. G. & Bolwell, G. P., A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification, pp. 163-176, Copyright 2003, with permission from Elsevier.] (Reproduced in color as Plate 21.)

Figure 7.13 Gross phenotypical changes/effects of either mutating or downregulating various phenyl — propanoid genes, as well as vascular related transcription factors. (A) p C3H mutant with various pleiotropic effects, resulted in a severely dwarfed line (ref8) (213, 215) (shown 23 days post seed-sown). (B) pC3H downregulation in alfalfa (Medicago sativa) resulted in a phenotype (pC3H-I) very similar to wild type without visible pleiotropic effects (shown 4 weeks post-cut-back). (C) Dwarfed Arabidopsis CCR irx4 mutant with pleiotropic effects (shown 6 weeks post seed-sown) (131). (D) CCR downregulation in to­bacco, with stunted morphology relative to wild type (232). (E) CAD double mutation in Arabidopsis (AtCAD4/5, cad-4, cad-5) resulted in a limp to prostrate stem phenotype (shown after 4 weeks growth) (71). (F) Double T-DNA tagged Arabidopsis nst1 nst3 line (199). [Reprinted from (B) The American Journal