A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus delfoides X P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAL clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parenta1 and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by Hindlll restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific Hindlll restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues.The enzyme PAL (EC 4.3.1.5) plays a key role in linking primary metabolism to phenylpropanoid metabolism by catalyzing the deamination of L-Phe to produce trans-cinnamic acid. This reaction is considered a key step in phenylpropanoid metabolism (Jones, 1984;Hahlbrock and Scheel, 1989) because it provides an entry point for the biosynthesis of a large number of natural products derived from the phenylpropane skeleton. Consistent with the diverse roles played by these phenylpropanoid-derived compounds, PAL enzyme levels are under both developmental and environmental control (Hahlbrock and Scheel, 1989). The accumulation of PAL mRNA and the activity of PAL promoters varies during the

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Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanoid metabolism and plays a central role in the biosynthesis of phenylpropanoid compounds. We have previously cloned two PAL genes, PALI and PAL2, from a Populus trichocarpa x P. deltoides F1 hybrid. Here, we describe the properties of PALI and PAL2 promoters and their expression patterns in transgenic tobacco and poplar. The promoters were 75% identical in the regions sequenced, and each contained two copies of AC-rich putative cis-acting elements that matched a consensus plant myb transcription factor binding site sequence. In transgenic tobacco, PALI-GUS and PAL2-GUS fusions directed similar patterns of expression in developing primary xylem of leaves, stems, and other organs, and in secondary xylem of stems. Contrary to previously documented patterns of PAL1/2 expression in poplar, no expression of either fusion was detected in epidermal or subepidermal cell layers of young tobacco leaves or stems. In poplar, the PAL2-GUS fusion directed the highest levels of expression in roots and young leaves and stems. In young leaves and stems, high GUS activity was detected in epidermal or subepidermal cells as well as in primary xylem and phloem fibers. GUS activity was low in woody stems, and was weak or absent in developing secondary xylem. The patterns of PAL2-GUS expression in poplar are very similar to those of PAL1/2 mRNA accumulation in poplar. However, the distinct patterns of expression directed by the PAL2 promoter in poplar and tobacco show that PAL2-GUS expression in tobacco does not accurately reflect all aspects of PAL2 expression in poplar.

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