In the study of effects of downregulating/mutating CAD in loblolly pine and tobacco, there have been various reports of “abnormal” lignins being produced. These have been de­scribed as affording lignin-enriched isolates harboring significant levels of incorporated hydroxycinnamaldehydes 19, 21, and 23 and hydroxybenzaldehydes 45-47, albeit without quantification to indicate what “significant” meant (173, 239).

7.62.23 CAD MUTANT LOBLOLLY PINE, 2-METHOXYBENZALDEHYDE AND DIHYDROMONOLIGNOL/LIGNIN DERIVATIVES One of the unusual findings apparently made initially as regards a CAD mutant loblolly pine isolate was that it biosynthesized 2-methoxybenzaldehyde (62, Figure 7.11A) (172); this was a quite unexpected finding given that such a metabolic response would not have been anticipated, from first principles, to result from CAD downregulation. This finding was then retracted (173), being a consequence of an error in the NMR spectroscopic assignments. Additionally, various dihydroconiferyl alcohol (63, Figure 7.11A) containing substructures were reported as apparently present in the “abnormal” lignin (discussed below), linked to a “conventional lignin monomer,” thereby indicating that at least 50% of the monomers were still monolignol-derived (172). Such moieties with a reduced side-chain had, however, been previously described in Pinus taeda (loblolly pine) cell suspension culture (171). Indeed, the allylic double bond reductases responsible for this reduction step, and the encoding genes have since been fully reportedby our laboratory from both P. taeda (240,241) and Arabidopsis (242); the X-ray structure of the ternary complex has also been obtained and the proposed biochemical catalytic mechanism described (242). Moreover, the CAD loblolly pine mutant has been described as presumably harboring a number of mutations (74), with the plant line apparently stunted. There are no reports of effects on the vascular integrity of this loblolly pine CAD mutant, which is presumed to be weakened, assuming that lignification proper has been modulated/reduced.

7.622.4 CAD DOWNREGULATED TOBACCO: NMR SPECTROSCOPIC

ANALYSIS AND PRELIMINARY EFFECTS ON VASCULAR INTEGRITY CAD downregulated tobacco lines were preliminarily studied by several researchers (235­237,243), who concluded that overall gross lignin contents were unaffected by CAD down — regulation. As discussed in Anterola and Lewis (77), there were significant differences though in the presumed overall polymeric architecture/properties of the lignified tissue, as evidenced byincreased susceptibility to alkaline treatment (77,243). Additionally, various CAD down­regulated lignin-enriched isolates were subjected to detailed NMR spectroscopic analyses (173,177,234). In our own analyses, the extent of the aldehyde-enhanced resonances present in the CAD downregulated tobacco [generated by Halpin etal. (235)] was very modest (177). Quantification, as determined by NMR spectroscopy (estimated from the normalized :H NMR spectra with respect to the aromatic region of lignin isolates), gave small amounts of p-hydroxybenzaldehydes 45-47, p-hydroxycinnamaldehydes 19/21/23 and 8-0-4′-linked

p-hydroxycinnamaldehyde (substructure XIIa-с, Figure 7.11B) end groups in the lignin isolates: ~0.68, 1.5, and 1.41% of total lignin, respectively (177).

In terms of overall effects on the vascular integrity, preliminary studies have also yielded quite predictable results, in the sense of weakening of the vascular apparatus (68-70, 244). In particular, the antisense CAD line had reduced longitudinal tensile stiffness and lower shear and Young’s moduli, respectively, compared to the controls (68). Moreover, these lines apparently responded to periodic flexural stem bending by increasing the thickness of the xylem cylinder (70), although none of the anatomical data (e. g., cross sections and cell types affected) were described to give insight into the changes occurring. Incongruously, as described earlier, Hepworth et al. (69) reported that the downregulated tobacco lines had lignin contents of 40-50%, again further reflecting the ongoing problems experienced in this field for reliable measurement of lignin contents/composition: generally, for tobacco, lignin contents are in the range of ~20-25% at maturation. Nevertheless, once again the effect of manipulation of a gene involved in the monolignol/lignin pathway had deleteriously altered structural properties of the lignocellulosic matrix.