Isolation and functional characterization of two rapeseed genes orthologous to Arabidopsis thaliana Phenylalanine Ammonia-lyase 2

Two rapeseed (Brassica napus L.) phenylalanine ammonia-lyase 2 genes (BnPAL2-1 and BnPAL2-2) were isolated. The full-length cDNAs of BnPAL2-1 and BnPAL2-2 are 2422 bp and 2421 bp, with corresponding genomic sequences of 2735 bp and 2742 bp, respectively. Clues from NCBI BLAST, multi-alignment, the phylogenetic tree, conserved domains, preferential amino acids, and tertiary structures all indicated that they are orthologs of Arabidopsis thaliana PAL2 (AtPAL2). The purified His-tagged BnPAL2-1 and BnPAL2-2 proteins, expressed in Escherichia coli, were both active for PAL catabolism, but BnPAL2-2 was much more active than BnPAL2-1. Southern blot analysis showed that the BnPAL2 subfamily might contain 2-6 members. Transcripts of both BnPAL2-1 and BnPAL2-2 were detectable in all tested organs, but BnPAL2-2 was more organ-specific than BnPAL2-1. Transcription of BnPAL2-1 was most abundant in buds, with sequentially decreased abundance in stems, roots, flowers, seeds of all stages, silique pericarps and leaves, and was weak in hypocotyls and cotyledons. Expression of BnPAL2-2 was most intensive in roots, declining sequentially in buds, stems, flowers, and 30D seeds, and was weak in all other organs. Transcription of both genes in yellow-seeded rapeseed line L2's most reproductive organs, including developing seeds, was lower than that of black-seeded line L1. These results, combined with our previous investigations on BnPAL1 genes, demonstrate that most Brassicaceae species contain orthologs of all 4 AtPAL genes. Both PAL1 and PAL2 were most probably triplicated in Brassiceae ancestor and simultaneous down-regulation of all/most PAL paralogs contributes to the formation of the yellow seed trait in L2.