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)-f3-farnesene (E(3F) is one of the active sesquiterpenoid components and is also a major component of aphid alarm pheromones. In this study, two E(3F synthase ((3FS) genes (Mcf3FS1 and Mcf3FS2), were cloned from German chamomile. Subcellular localization analysis showed that both Mc(3FS1 and Mc(3FS2 were localized in the cytoplasm and nucleus. Tissue-specific expression analysis revealed that Mcf3FS1 and Mcf3FS2 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 Mc(3FS1 and Mc(3FS2 possess catalytic activity. Overexpression of Mc(3FS1 and Mc(3FS2 in the hairy roots of German chamomile led to the accumulation of E(3F, demonstrating enzyme activity in vivo. The promoters of Mcf3FS1 and Mcf3FS2 were cloned and analyzed. After treating German chamomile with methyl jasmonate (MeJA) and methyl salicylate (MeSA), the transcription levels of Mcf3FS1 and Mcf3FS2 were found to be regulated by both hormones. In addition, feeding experiments showed that aphid infestation upregulated the expression levels of Mcf3FS1 and Mcf3FS2. Our study provides valuable insights into the biosynthesis of E(3F, laying a foundation for further research into its metabolic pathways.