Functional analysis of (E)-β-farnesene synthases involved in accumulation of (E)-β-farnesene in German chamomile (Matricaria chamomilla L.)

German chamomile (Matricaria chamomilla L.) is a traditional medicinal aromatic plant, and the sesquiterpenoids in its flowers have important medicinal value. The (E)-β-farnesene (EβF) is one of the active sesquiterpenoid components and is also a major component of aphid alarm pheromones. In this study, two EβF synthase (βFS) genes (McβFS1 and McβFS2), were cloned from German chamomile. Subcellular localization analysis showed that both McβFS1 and McβFS2 were localized in the cytoplasm and nucleus. Tissue-specific expression analysis revealed that McβFS1 and McβFS2 were expressed in all flower stages, with the highest levels observed during the tubular flower extension stage. Prokaryotic expression and enzyme activity results showed that McβFS1 and McβFS2 possess catalytic activity. Overexpression of McβFS1 and McβFS2 in the hairy roots of German chamomile led to the accumulation of EβF, demonstrating enzyme activity in vivo. The promoters of McβFS1 and McβFS2 were cloned and analyzed. After treating German chamomile with methyl jasmonate (MeJA) and methyl salicylate (MeSA), the transcription levels of McβFS1 and McβFS2 were found to be regulated by both hormones. In addition, feeding experiments showed that aphid infestation upregulated the expression levels of McβFS1 and McβFS2. Our study provides valuable insights into the biosynthesis of EβF, laying a foundation for further research into its metabolic pathways.