Metabolomic profiling of shade response and in silico analysis of PAL homologs imply the potential presence of bifunctional ammonia lyases in conifers.

Norway spruce and Scots pine show enhanced lignin synthesis under shade, along with differential expression of defense-related genes that render disease resilience. In general, phenylalanine (Phe) is the precursor for lignin synthesis in plants, and tyrosine (Tyr) forms an additional lignin precursor specifically in grasses. Phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) from the lignin biosynthesis pathway use either Phe or Tyr as precursors for lignin production, respectively. Grasses possess a bifunctional phenylalanine/tyrosine ammonia-lyase (PTAL) that potentially can use both Phe and Tyr for lignin biosynthesis. Metabolomic profiles of seedlings revealed higher levels of Phe and Tyr under shade in Scots pine, while Norway spruce showed differential regulation of only Tyr under shade. Sequence analysis and phylogeny of PAL homologs in the two conifers, coupled with correlation of up-regulation of precursors for lignin synthesis (Phe/Tyr) and enhanced lignin synthesis along with differential expression of PAL homologs under shade, suggest the potential presence of a bifunctional ammonia-lyases (BAL) in conifers. This finding is novel and comparable to PTALs in grasses. Exome sequence analysis revealed a latitudinal variation in allele frequencies of SNPs from coding regions of putative PAL and BAL in Norway spruce, which may impact enzyme activity affecting lignin synthesis. Metabolomic analysis additionally identified metabolites involved in plant immunity, defense and stress response.