Key message The investigation provides initial knowledge on the distribution and evolution of the high and low methyl salicylate-producing trait in the Betula genus. Mislabelled birch species could be identified and removed. Abstract The genus Betula is the largest group of ecologically and economically dominant perennial woody plants in subalpine forests. The taxonomy of Betula is complex due to an extensive history of hybridization and periodic introgression events among the species. Although almost all land plants including birches produce methyl salicylate (MeSA) as a signaling molecule and in response to stress (“low MeSA producer”), some birch species produce high amounts of MeSA in the leaves and bark (“high MeSA producer”). Unfortunately, the evolution of high levels of MeSA production in the genus Betula remains unclear. The salicylic acid-binding protein 2 (SABP2) and salicylic acid methyltransferase (SAMT) genes involved in MeSA biosynthesis were incorporated into this study to examine the interspecific relationship of high and low MeSA-producing birches. Additionally, eight chloroplast and three nuclear regions were included to evaluate their potential application in species determination. The analysis resulted in 25 and 61 nucleotide variations, respectively, which allowed for a visualization of the genetic architecture in the 18 Betula species investigated. The high MeSA-producing B. lenta, B. grossa, and B. alleghaniensis formed the basal clade in the phylogenetic analysis, thus revealing their ancestral status, and the network analysis postulates that the diploid B. lenta is one of the ancestors of the genus Betula. The results indicate that the ability to produce high levels of MeSA that were initially present in the genus has been lost several times during its evolution. Placing species of the subgenus Acuminata alongside the subgenus Betula, together with a fragrance analysis, questions their ability to produce high levels of MeSA.

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.

Key message This review for the first time gathers the current state of knowledge on the role of plant and microbial methyl salicylate (MeSA) signaling processes in forest ecosystems. It aims to establish a basis for the use of high-MeSA-emitting trees as a silvicultural tool aiming to enhance stability and resilience in managed temperate forests affected by climate change. Abstract Methyl salicylate (MeSA) is a volatile plant and microbial signaling compound involved in systemic acquired resistance (SAR) and defense against pests and microbial pathogens, and antagonists. MeSA emitted by plants is also believed to trigger SAR in neighboring plant individuals, thus contributing to the resilience of the entire plant community. In this review, we discuss volatile plant-to-plant communication processes with a special focus on MeSA and provide an overview about the occurrence of MeSA in fungi and other microbes. We summarize present findings on the role of MeSA in plants and particularly in birches (Betula spp.) and discuss the potential use of MeSA and MeSA-emitting plants in agriculture and forestry. MeSA levels in plant tissues are adjusted by methylation of salicylic acid to MeSA and the reverse process of demethylation. Some plant species possess constitutively high MeSA levels and thus are suitable for experiments of admixture of high MeSA plants, e.g., birches of the subgenera Betulenta and Acuminata in plant communities such as mixed forests. Furthermore, knowledge of candidate genes and the molecular pathways underlying high MeSA emission is expected to offer a basis for altering MeSA levels and/or the selection of high MeSA mutants.

Plant SABATH family members catalyze the methylation of many hormones, signaling molecules, and floral scent metabolites, including salicylic acid (SA), jasmonic acid (JA), and indol-3 acetic acid (IAA). Demethylation of resulting methyl esters was executed by members of the MES family. Members of both families are significantly involved in plant developmental processes. Here, using different bioinformatics tools, we studied the evolutionary relationship and characterized the putative functions of the family members in silver birch (Betula pendula). It is a socio-ecologically important tree species and plays a vital role in reforestation. Ten and twelve members of the SABATH (BpSABATH1-10) and MES (BpMES1-12) family were identified in silver birch, respectively at the gene and enzyme levels. The BpSABATH and BpMES genes were distributed on seven of fourteen chromosomes, indicating the occurrence of moderate duplication events important for the expansion of both families. Phylogenetic clustering and the gene ontology database suggest, BpSA-BATH8 is involved in the methylation of indole-3-acetic acid (IAA), while BpSABATH5, BpSABATH6, and BpSABATH7 methylate JA to methyl jasmonate (MeJA). BpSABATH9 was alone in the phylogenetic functional group 1 and prefers SA as a substrate to synthesize methyl salicylate (MeSA). Likewise, BpMES5 and BpMES12 are possibly involved in the demethylation of the methyl ester of IAA, while BpMES6, BpMES7, and BpMES8 are responsible for the demethylation of MeJA. BpMES9 clustered with MES and prefers MeSA as a substrate. The current analysis helped to select candidate genes that could be subjected to further molecular breeding of birch varieties adapted to biotic and abiotic stress conditions.

Climate change alters the seasonal synchronization between plants and respective pests plus pathogens. The geographical infiltration helps to shift their hosts, resulting in novel outbreaks that damage forests and ecology. Traditional management schemes are unable to control such outbreaks, therefore unconventional and competitive governance is needed to manage forest pests and pathogens. RNA interference (RNAi) mediated double-stranded RNA (dsRNA) treatment method can be implemented to protect forest trees. Exogenous dsRNA triggers the RNAi-mediated gene silencing of a vital gene, and suspends protein production, resulting in the death of targeted pathogens and pests. The dsRNA treatment method is successful for many crop insects and fungi, however, studies of dsRNA against forest pests and pathogens are depleting. Pesticides and fungicides based on dsRNA could be used to combat pathogens that caused outbreaks in different parts of the world. Although the dsRNA has proved its potential, the crucial dilemma and risks including species-specific gene selection, and dsRNA delivery methods cannot be overlooked. Here, we summarized the major fungi pathogens and insect pests that have caused outbreaks, their genomic information, and studies on dsRNA fungi-and pesticides. Current challenges and opportunities in dsRNA target decision, delivery using nanoparticles, direct applications, and a new method using mycorrhiza for forest tree protection are discussed. The importance of affordable next-generation sequencing to minimize the impact on non-target species is discussed. We suggest that collaborative research among forest genomics and pathology institutes could develop necessary dsRNA strategies to protect forest tree species.