Identification and analysis of key genes involved in methyl salicylate biosynthesis in different birch species

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.

Methyl salicylate as a signaling compound that contributes to forest ecosystem stability

“…In bark tissues, B. alleghaniensis and B. lenta showed higher relative expression of SAMT than low-MeSAproducing species. The higher expression of SAMT in bark tissues of constitutively high-MeSA-producing birches Ashburner et al (2013) and Singewar et al (2020b) MeSA is present in the young bark of all species listed supports its significance for MeSA synthesis (Singewar et al 2020a). In contrast, high SABP2 expression in the bark of low-MeSA-producing species B. utilis and B. pendula, appears consistent with a high rate of reversion of MeSA to SA, resulting in lower MeSA levels.…”

“…The enzymes were functionally characterized through gene expression studies and biochemical analyses in various plant species such as Vitis vinifera, A. thaliana, Clarkia breweri, N. tabacum, and Populus trichocarpa (Chen et al 2003;Kumar and Klessig 2003;Ross et al 1999;Zhao et al 2008Zhao et al , 2009Zhao et al , 2016. However, very little is known about SAMT and SABP2 at gene and enzyme levels in birches (Singewar et al 2020a).…”

“…In a recent study, the SAMT and SABP2 genes were cloned from four high (B. lenta, B. alleghaniensis, B. grossa, and B. medwediewii) and four low (B. utilis, B, pendula, B. nana, and B. alnoides) MeSA-producing birches (Singewar et al 2020a). In bark tissues, B. alleghaniensis and B. lenta showed higher relative expression of SAMT than low-MeSAproducing species.…”

Transcriptome analysis of North American sweet birch (Betula lenta) revealed a higher expression of genes involved in the biosynthesis of secondary metabolites than European silver birch (B. pendula)

“…org/), providing an opportunity to explore other therapeutically important and largely ignored species of the genus. Although the cost of sequencing technology has reduced drastically which is a good sign for more scientific research, a recent survey on available genome sequences of the species within the genus Betula in the NCBI database clearly shows the scarcity of knowledge within the subgenus Aspera (Singewar 2020;Zoladeski and Hayes 2013). We performed the transcriptome analysis of B. lenta in comparison to B. pendula to reduce the information gap.…”

“…Birch sap can be combined with corn and fermented to make beer (Suryawanshi 2000 ). Previously it was the only source of wintergreen oil extraction since it has the aroma of wintergreen emanatingfrom crushed bark and leaves (Ashburner and McAllister 2013 ; Singewar et al 2020a , b ). Extensive harvesting of sweet birch caused to become it endangered until the 1950–1970s (Leak 1965 ).…”