UBC Theses and Dissertations
Phenylalanine ammonia-lyase (EC 188.8.131.52) from Pinus banksiana: partial cDNA cloning and effect of exogenously supplied trans-cinnamic acid on elicitor-inducible expression Lam, Monica Lee
Phenylalanine ammonia-lyase (PAL; EC 184.108.40.206), the "entrypoint enzyme" of the phenylpropanoid pathway, catalyzes the conversion of L-phenylalanine to trans-cinnamic acid and is an important bridge between primary and secondary metabolism in plants. In many angiosperms, PAL exists as multiple isoforms, encoded by a gene family. PAL has been characterized from two gymnosperms, Pinus banksiana and Pinus taeda, and one form of the enzyme is present. The regulation of constitutive and inducible PAL expression is not well understood. From studies employing angiosperms, transcriptional control appears to be important, and modulation of PAL activity by trans-cinnamic acid has also been proposed. The objectives of this study were to investigate the genomic organization of PAL and the role of transcriptional and metabolic regulation of PAL in a gymnosperm. PAL activity is transiently increased in P. banksiana cell suspension cultures treated with an ectomycorrhizal fungal elicitor preparation. Preliminary results indicated that increases in PAL transcripts precede and are closely correlated with the PAL induction. Thus this system was convenient for constructing a cDNA library enriched in PAL sequences, and for studying the regulation of inducible PAL expression. Six partial PAL cDNA clones (0.2 to 1.8kb) were isolated from a library prepared from elicitor-treated P. banksiana cell cultures. In overlapping regions, the cDNA sequences are highly similar, but not identical, suggesting that multiple genes may encode PAL in P. banksiana. Extensive homology to the P. taeda PAL cDNA sequence was also observed. The chemical environment of elicitor-treated cells was manipulated by exogenously supplying frans-cinnamic acid. Hybridization of a P. banksiana partial PAL cDNA with total RNA slot blots was used to monitor PAL transcript levels. Supplying frans-cinnamic acid delayed the increase in PAL transcript levels and completely inhibited the induction of PAL activity normally associated with elicitor treatment. This suggests trans-cinnamic acid can downregulate PAL expression, although further study is required to determine the specificity of its action. This work represents the first study of the metabolic regulation of PAL in a gymnosperm species.
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