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

• Published in: Plant physiology and biochemistry 2021-10, Vol.167, p.31-41
• Main Authors: Liu, Licheng, Yin, Mengjiao, Lin, Guyin, Wang, Qian, Zhou, Peina, Dai, Shilin, Sang, Mengru, Liu, Chanchan, Wu, Qinan
• Format: Article
• Language: English
• Subjects: BASIC BIOLOGICAL SCIENCES; Functional characterization; Monoterpene biosynthesis; Nepeta tenuifolia Briq; qRT-PCR
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/j.plaphy.2021.07.026

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. •Comparative transcriptome analysis between shoots and roots provides differential unigenes in monoterpene biosynthesis in N. tenuifolia.•Six p-menthane monoterpene biosynthetic genes on (+)-menthone pathway were identified and cloned, and function of limonene 3-hydroxylase in N. tenuifolia was reported firstly.•Networks between secondary metabolites and gene expression report crucial activity of pulegone reductase during development for accumulation of (+)-menthone.