7.6.1.1 PAL

This step catalyzes the entry point into phenylpropanoid metabolism, and thus can lead to a diverse range ofphenylpropanoid-derived products (i. e., lignins, lignans, hydroxycinnamic acids, suberins, flavonoids, proanthocyanidins, etc.), depending upon the species, tissue, and/or cell type in question. Reduction of overall PAL enzymatic activity (through inhibition, mutation, downregulation, etc.) would thus be expected to have negative consequences on either all aspects of phenylpropanoid metabolism or on specific elements, depending upon which PAL gene(s) is (are) affected. This was facilely demonstrated first using the PAL inhibitor, L-AOPP (57, Figure 7.11), as early as in 1977 and in subsequent studies extending through 1985 (200-205). This resulted, depending upon the plant species investigated, in reductions in formation/accumulation of anthocyanins, isoflavones, hydroxycinnamic acids, and lignins, respectively. As an example, Figure 7.12A depicts the effects of AOPP (57) inhibition on lignification in mungbeans (2004); the cross-section of the AOPP (57)-treated plant line clearly has collapsed xylem due to presumed reductions in lignin contents and thus a weakened vasculature — as would be anticipated from first principles.

Furthermore, although the reliability of many of the analytical techniques used in later studies by other researchers was clearly questionable, the subsequent standard biotechnolog­ical manipulation of PAL activity in tobacco (through antisense and sense co-suppression, etc.) apparently resulted in poorly formed (weakened) xylem tissue (206,207); additionally, the plant lines were also severely stunted, had curled leaves, localized lesions, less pollen, reduced viability and deformed flowers. These are presumably not desirable traits, and in­deed appear to fall under the rubric of pleiotropic “unintended” effects: these, in turn, reflect our severely limited current understanding of overall plant metabolism, growth, and development. Moreover, while the lignin levels were reportedly lower in these studies as in­dicated above, the analytical procedures used were very questionable (i. e., thioglycolic acid lignin determination and Klason lignin analyses of “neutral detergent fibers”) as discussed in Anterola and Lewis (77). Another very preliminary study also reported that generation of double PAL mutants (e. g., pall pal2) in Arabidopsis resulted in lignin levels reduced to circa 30-35% of wild-type levels (208). This study, however, lacked any systematic analysis of the effects on lignification over different phases of growth/development until maturation. It did though provisionally indicate that lignin levels were reduced.

Perhaps, most importantly, none of the above studies explored any (quantitative) mea­surement of effects on structural integrity of the vasculature; nor was there either any new or additional insight gained on lignin macromolecular configuration/assembly, and/or effects of modulation of same. As predicted from first principles by ourselves there was clearly no “instant response,” as proposed by Ralph (197) and Ralph et al. (175), to severe enzymatic shifts that would suggest that lignin’s structure was not important; nor were “perfectly vi­able” plants produced, based on the defects that had been noted as early as 1983. That is, the genetically modified tobacco PAL plant lines produced were only able to survive with a number of serious defects; additionally, these transformants were not stable, and reverted to wild type in subsequent generations (209). Accordingly, such preliminary (phenomeno­logical) studies on PAL modulation now need to be expanded in depth, in order to gain a more definitive understanding of the actual effects on, for example, lignin macromolecular configuration/assembly, vascular integrity and overall physiology.