OVEREXPRESSION OF L-PHENYLALANINE AMMONIA-LYASE IN TRANSGENIC TOBACCOPLANTS REVEALS CONTROL POINTS FOR FLUX INTO PHENYLPROPANOID BIOSYNTHESIS

Transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the en zyme L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) were grown from s eeds of a primary transformant containing the bean PAL2 gene, which ha d shown homology-dependent silencing of the endogenous tobacco PAL gen es. Analysis of endogenous and transgene-encoded PAL transcripts and p rotein in the primary transformant (T-0) and first-generation (T-1) ov erexpressor plants indicated that the transgene-encoded PAL is the cau se of the greater than wild-type levels of PAL activity (up to 5- and 2-fold greater in leaf and stem tissue, respectively) in the T-1 plant s. Leaves of PAL-overexpressing plants contained increased levels of t he hydroxycinnamic acid ester chlorogenic acid but not of the flavonoi d rutin, indicating that PAL is the key control point for flux into ch lorogenic acid. In addition, levels of the glucoside of 4-coumaric aci d increased in the overexpressing plants, suggesting that the 4-coumar ate:coenzyme A ligase or coumarate hydroxylase reactions might have be come limiting. These results help to define the regulatory architectur e of the phenylpropanoid pathway and indicate the possibility of engin eering-selective changes in this complex metabolic pathway by overexpr ession of a single early pathway gene.