Comparison of Pulegone and Estragole Chemotypes Provides New Insight Into Volatile Oil Biosynthesis of Agastache rugosa.

The aerial parts of Agastache rugosa are rich in essential oils containing monoterpenoids, phenylpropanoids, and aromatic compounds. These are used as herbs, perfume plants, and ornamental plants. Based on the difference in the constituents of the essential oil, A. rugosa is divided into pulegone and estragole chemotypes, but the mechanism of key metabolite biosynthesis in these two A. rugosa chemotypes remains unclear. In this study, we compared the morphological differences, metabolite constituents, and transcriptomic data between the two chemotypes of A. rugosa. Monoterpenoid was the main compound in the pulegone chemotype, and phenylpropanoid was the main compound in the estragole chemotype; however, limonene was detected in both chemotypes. Furthermore, 46 genes related to pulegone and estragole biosynthesis were identified. Limonene synthase, limonene-3-hydroxylase, and isopiperitenol dehydrogenase were upregulated in the pulegone chemotype, while phenylalanine ammonia-lyase, 4-coumarate: CoA ligase, CYP73A, coumaroyl-aldehyde dehydrogenase, and eugenol synthase were downregulated in the pulegone chemotype. We identified chavicol methyl transferase and limonene-3-hydroxylase in A. rugosa. This work not only provides the difference in morphology and metabolites in pulegone and estragole chemotypes, but also offers a molecular mechanism of volatile oil biosynthesis, which could be a basis for specialized metabolites in specialized chemotypes.

Integrating RNA-seq with functional expression to analyze the regulation and characterization of genes involved in monoterpenoid biosynthesis in Nepeta tenuifolia Briq.

Nepeta tenuifolia Briq. (Lamiaceae) is a medicinal plant historically used in the East Asia region to treat cold and fever, and it is currently used as a clinically effective treatment for respiratory diseases. We previously found that monoterpenoids are the dominant volatile secondary metabolites in N. tenuifolia and their biosynthesis occurs in peltate glandular trichomes. To gain an insight into the molecular mechanisms underlying monoterpenoid biosynthesis in N. tenuifolia, we conducted transcriptome sequencing and examined the expression differences in monoterpene molecular pathway-related genes in different tissues and growth stages by qRT-RCR. In total, six p-menthane monoterpene biosynthetic genes in the (+)-menthone pathway were identified and cloned successfully based on transcriptome data. Moreover, the major constituents, including (+)-limonene, (-)-pulegone and (+)-menthone showed greater accumulation in the spikes than in other organs, such as the expression levels of related key enzyme genes. Additionally, the relative expression of pulegone reductase was the highest at 84 days, showing an inverse trend from (-)-pulegone relative content and leading to (+)-menthone accumulation in peltate glandular trichomes. Finished cloning of the gene for limonene 3-hydroxylase in N. tenuifolia (NtL3OH), heterologous expression in yeast, and in vitro assays were performed for functional characterization. Our study provides an important resource for further research of secondary metabolism of monoterpenes in peltate glandular trichomes of N. tenuifolia and other homologous species.