Abstract
Species of the perennial woody plant genus Betula dominate subalpine forests and play a significant role in preserving biological diversity. In addition to their conventional benefits, birches synthesize a wide range of secondary metabolites having pharmacological significance. Methyl salicylate (MeSA) is one of these naturally occurring compounds constitutively produced by different birch species. MeSA is therapeutically important in human medicine for muscle injuries and joint pain. However, MeSA is now mainly produced synthetically due to a lack of information relating to MeSA biosynthesis and regulation. In this study, we performed a comprehensive bioinformatics analysis of two candidate genes mediating MeSA biosynthesis, SALICYLIC ACID METHYLTRANSFERASE (SAMT) and SALICYLIC ACID-BINDING PROTEIN 2 (SABP2), of high (B. lenta, B. alleghaniensis, B. medwediewii, and B. grossa) and low (B. pendula, B. utilis, B. alnoides, and B. nana) MeSA-producing birch species. Phylogenetic analyses of SAMT and SABP2 genes and homologous genes from other plant species confirmed their evolutionary relationships. Multiple sequence alignments of the amino acid revealed the occurrence of important residues for substrate specificity in SAMT and SABP2. The analysis of cis elements in different birches indicated a functional multiplicity of SAMT and SABP2 and provided insights into the regulation of both genes. We successfully developed six prominent single nucleotide substitution markers that were validated with 38 additional birch individuals to differentiate high and low MeSA-producing birch species. Relative tissue-specific expression analysis of SAMT in leaf and bark tissue of two high and two low MeSA-synthesizing birches revealed a high expression in the bark of both high MeSA-synthesizing birches. In contrast, SABP2 expression in tissues revealed indifferent levels of expression between species belonging to the two groups. The comparative expression and bioinformatics analyses provided vital information that could be used to apply plant genetic engineering technology in the mass production of organic MeSA.