Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis.

Abstract

Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanold synthesis. The role of PAL In pathway regulation was investigated by measurement of product accumulation as a function of enzyme activity in a coilection of near-isogenic transgenic tobacco plants exhibiting a range of PAL levels from wild type to 0.2% of wild type. In leaf tissue, PAL level is the dominant factor regulating accumulation ofthe major product chlorogenic acid and overall flux into the pathway. In stems, PAL at wild-type levels contributes, together with downstream steps, in the regulation of lguin deposition and becomes the dominant, rate-determining step at levels 3-to 4-fold below wild type. The metabolic impact of elevated PAL levels was investigated in transgenic leaf callus that overexpressed PAL. Accumulation of the flavonoid rutin, the major product in wild-type callus, was not increased, but several other products accumulated to similarly high levels. These data indicate that PAL is a key step in the regulation of overall flux into the pathway and, hence, accumulation of major phenylpropanoid products, with the regulatory architecture of the pathway poised so that downstream steps control partitioning into different branch pathways.

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

“…Although PAL catalyzes the first step in phenylpropanoid biosynthesis and plays a major role in regulating carbon flow into the pathway (Bate et al, 1994), additional flux control points occur at various downstream branches (Howles et al, 1996). Superimposed on regulation of the individual pathway enzymes is the allocation of chorismate to Phe biosynthesis and partitioning of Phe to PAL; chorismate and Phe are branch point intermediates that are substrates for multiple enzymes.…”

Transgene-Mediated and Elicitor-Induced Perturbation of Metabolic Channeling at the Entry Point into the Phenylpropanoid Pathway

“…PAL -overexpressing tobacco lines, resulting from constitutive expression of a bean PAL2 gene, overproduce phenylpropanoid compounds (Howles et al, 1996), whereas underexpressing lines, resulting from epigenetic gene silencing (Elkind et al, 1990;Bate et al, 1994), have reduced levels of phenylpropanoid compounds. We initiated callus and then cell suspension cultures from PAL -overexpressing transgenic tobacco; constitutive PAL activities in the cell suspension cultures were at least threefold higher than in comparable cultures derived from wild-type plants.…”

“…These results indicate that externally added unlabeled transcinnamic acid does not dilute isotope incorporation from 3 H-Phe into phenylpropanoid compounds in tobacco cell cultures, whereas externally added unlabeled 4-coumaric acid does, suggesting a specific metabolic compartmentalization of trans -cinnamic acid. This does not directly prove channeling or necessarily indicate the presence of more than one endogenous pool of trans -cinnamic acid in the absence of an externally fed compound.PAL -overexpressing tobacco lines, resulting from constitutive expression of a bean PAL2 gene, overproduce phenylpropanoid compounds (Howles et al, 1996), whereas underexpressing lines, resulting from epigenetic gene silencing (Elkind et al, 1990;Bate et al, 1994), have reduced levels of phenylpropanoid compounds. We initiated callus and then cell suspension cultures from PAL -overexpressing transgenic tobacco; constitutive PAL activities in the cell suspension cultures were at least threefold higher than in comparable cultures derived from wild-type plants.…”

“…All plants were grown under greenhouse conditions (18ЊC at night and 27ЊC by day) and harvested just before flowering. Stem samples were taken from internodes seven to 11, counting from the top, frozen in liquid N 2 , and ground in a tissue grinder.Callus cultures were initiated from leaf discs of wild-type and transformed tobacco plants, as described previously (Bate et al, 1994). Liquid cultures were initiated and maintained in a modified Schenk and Hildebrandt medium, as described previously (Dixon et al, 1981), and subcultured every 7 to 10 days.…”

Beta-aminobutyric acid-induced resistance in grapevine against downy mildew: involvement of pterostilbene

“…These authors hypothesised that flux into the phenylpropanoid pathway is controlled, at least in part, via feedback regulation of PAL sensed through the production of cinnamic acid. Furthermore, when reduced below a threshold of 20% to 25% of wild-type activity, PAL becomes a rate-limiting step for lignin biosynthesis in tobacco (Bate et al 1994;Howles et al 1996). Reduced C4H activity is also correlated with reduced levels of lignin and phenolics (Sewalt et al 1997).…”