The phenylalanine ammonia lyase (PAL) gene family shows a gymnosperm-specific lineage

Abstract

Background: Phenylalanine ammonia lyase (PAL) is a key enzyme of the phenylpropanoid pathway that catalyzes the deamination of phenylalanine to trans-cinnamic acid, a precursor for the lignin and flavonoid biosynthetic pathways. To date, PAL genes have been less extensively studied in gymnosperms than in angiosperms. Our interest in PAL genes stems from their potential role in the defense responses of Pinus taeda, especially with respect to lignification and production of low molecular weight phenolic compounds under various biotic and abiotic stimuli. In contrast to all angiosperms for which reference genome sequences are available, P. taeda has previously been characterized as having only a single PAL gene. Our objective was to re-evaluate this finding, assess the evolutionary history of PAL genes across major angiosperm and gymnosperm lineages, and characterize PAL gene expression patterns in Pinus taeda.

Methods: We compiled a large set of PAL genes from the largest transcript dataset available for P. taeda and other conifers. The transcript assemblies for P. taeda were validated through sequencing of PCR products amplified using gene-specific primers based on the putative PAL gene assemblies. Verified PAL gene sequences were aligned and a gene tree was estimated. The resulting gene tree was reconciled with a known species tree and the time points for gene duplication events were inferred relative to the divergence of major plant lineages.

Results: In contrast to angiosperms, gymnosperms have retained a diverse set of PAL genes distributed among three major clades that arose from gene duplication events predating the divergence of these two seed plant lineages. Whereas multiple PAL genes have been identified in sequenced angiosperm genomes, all characterized angiosperm PAL genes form a single clade in the gene PAL tree, suggesting they are derived from a single gene in an ancestral angiosperm genome. The five distinct PAL genes detected and verified in P. taeda were derived from a combination of duplication events predating and postdating the divergence of angiosperms and gymnosperms.

Conclusions: Gymnosperms have a more phylogenetically diverse set of PAL genes than angiosperms. This inference has contrasting implications for the evolution of PAL gene function in gymnosperms and angiosperms.

Figure 1

Alignment between the five PtPAL genes in P. taeda. Arrow indicates position of the conserved MIO region (Ala-Ser-Gly triad).

Figure 2

Consensus tree of the Phenylalanine ammonia lyase gene family. Numbers at nodes are nonparametric Bootstrap values (BS) from Maximum Likelihood (ML) and Posterior Probabilities (PP) from Bayesian Inference (BI), respectively, separated by a slash. Asterisks (*/*) symbol indicates [90-100]/[0.9 - 1.00] support values. The # symbol indicates BS values lesser than 50%. Plus (+) symbol indicate variation in branching patterns between the ML and BI consensus trees.

Figure 3

NOTUNG: reconciled gene tree. A reconciled gene tree with duplication events as obtained from Notung is depicted. Duplication nodes are marked with circles. The branch shading corresponds to the pattern of gymnosperms branching. The blue branch indicates gymnosperm sequences that clustered with angiosperm PAL genes. The green branch indicates a unique gymnosperm branch, while the brown branch indicates gymnosperm sequences clustering with sequences from basal taxa.