The essential oil of German chamomile (Matricaria chamomilla L.) is rich in sesquiterpenoids, which contribute to its extensive applications in the pharmaceutical, tea, and cosmetics industries. Sesquiterpenoids are synthesized through the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. In this study, we identified and cloned genes encoding for three key enzymes involved in these pathways: 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), 1-deoxy-D-xylulose 5-phosphate synthase (DXS), and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) from German chamomile. Expression analysis revealed that McDXS2a, McDXS2b, McDXR, and McHMGR were highly expressed during flower development. Sampling was performed on the whole flower before the initial flowering stage, and subsequently, disk florets and ray florets were sampled separately for further analysis. Subcellular localization analysis indicated that McDXS1, McDXS2a, McDXS2b, and McDXR localized to the chloroplasts, while McHMGR localized to the cytoplasm. Overexpressing these genes in German chamomile hairy roots significantly increased sesquiterpenoid accumulation, confirming their functional roles. Transcription factors associated with these genes were identified and cloned, providing a data reference for further investigation into their regulatory mechanisms. Additionally, precursor feeding experiments demonstrated that the MEP pathway play an important role in the biosynthesis of sesquiterpenoids in German chamomile. These findings provide a basis for future research on the biosynthetic regulation of sesquiterpenoids.

The essential oil of German chamomile (Matricaria recutita L.) is widely used in food, cosmetics, and the pharmaceutical industry. α-Bisabolol is the main active substance in German chamomile. Farnesyl diphosphate synthase (FPS) and α-bisabolol synthase (BBS) are key enzymes related to the α-bisabolol biosynthesis pathway. However, little is known about the α-bisabolol biosynthesis pathway in German chamomile, especially the transcription factors (TFs) related to the regulation of α-bisabolol synthesis. In this study, we identified MrFPS and MrBBS and investigated their functions by prokaryotic expression and expression in hairy root cells of German chamomile. The results suggest that MrFPS is the key enzyme in the production of sesquiterpenoids, and MrBBS catalyzes the reaction that produces α-bisabolol. Subcellular localization analysis showed that both MrFPS and MrBBS proteins were located in the cytosol. The expression levels of both MrFPS and MrBBS were highest in the extension period of ray florets. Furthermore, we cloned and analyzed the promoters of MrFPS and MrBBS. A large number of cis-acting elements related to light responsiveness, hormone response elements, and cis-regulatory elements that serve as putative binding sites for specific TFs in response to various biotic and abiotic stresses were identified. We identified and studied TFs related to MrFPS and MrBBS, including WRKY, AP2, and MYB. Our findings reveal the biosynthesis and regulation of α-bisabolol in German chamomile and provide novel insights for the production of α-bisabolol using synthetic biology methods.

German chamomile (Matricaria chamomilla L.) is one of the most ancient medicinal species in the world and terpenoids from their flowers have important medicinal value. We cloned three sesquiterpene synthase genes, McGDS1, McGDS2 and McGDS3, and performed sequence alignment and phylogenetic analysis. The encoded proteins possess three conserved structural features: an RRxxxxxxxxW motif, an RxR motif, and a DDxxD motif. McGDS1, McGDS2 and McGDS3 were confirmed to be (E)-farnesene synthase, germacrene D synthase, and germacrene A synthase, respectively. Subcellular localization revealed diffuse GFP reporter-gene signals in the cytoplasm and nucleus. qPCR indicated that McGDS1, McGDS2 and McGDS3, were more highly expressed in young flowers than in old flowers and the expression was highly correlated with amounts of the end-product essential oils ((E)-β-farnesene, germacrene D and β-elemene), with coefficients of 0.76, 0.83 and 0.68, respectively. We also established a transformation system for chamomile hairy roots. The overexpression of McGDS1, McGDS2 and McGDS3 resulted in γ-muurolene accumulation in hairy roots. The activity of three aphid alarm pheromones here forms the molecular basis for the study of the biosynthesis and regulation of volatile terpenes. Transformation of chamomile hairy roots provides a simple system in which to study terpene biosynthesis in chamomile.