Nepetalactones profiling and putative iridoid synthase expression analysis indicate trichome specific localization and developmental regulation of iridoids biosynthesis in leaves of Nepeta rtanjensis

Abstract

The majority of species belonging to genus Nepeta (fam. Lamiaceae) produce and accumulate nepetalactones as the main secondary metabolites. Nepetalactones are iridoid monoterpenoids recognized for their wide spectrum of high-value bioactive properties, namely antimicrobial, cytostatic, anti-inflammatory, phytotoxic, etc. However, biosynthetic pathway of these compounds in plants remains to a great extent unexplored. Most probably they derive through general iridoid biosynthetic pathway leading to nepetalactol, a common intermediate of all plant-produced iridoids, that is formed in a reaction catalyzed by iridoid synthase (IS, EC 1.3.1.99). Present investigation entails homology based degenerative primer approach which led to the identification of two promising iridoid synthase candidates (NrIS1 and NrIS2) in leaves of Nepeta rtanjensis Diklić & Milojević, rich in trans,cis-nepetalactone (trans,cis-NL) and dehydronepetalactone (DNL). Comparative UHPLC/qqqMS profiling of nepetalactones in detached trichomes and abraded leaves, complemented with Raman spectroscopy and dichloromethane dipping experiment highlighted trichomes as the main site of their accumulation. This was further supported by the significantly high trichome/abraded leaves expression ratio of NrIS1 (~31) and NrIS2 (~25), which strongly indicated the involvement of these genes in nepetalactone biosynthesis. High correlation between NrIS1 expression profile and trans,cis-NL and DNL contents in five developmental stages of N. rtanjensis leaves confirmed developmental regulation of nepetalactone biosynthesis. Concentrations of trans,cis-NL and DNL were 22-fold and 80-fold lower in the oldest leaves investigated than in the youngest leaves, respectively. In addition, the expression pattern of the NrIS1 followed the same trend exhibiting 7.4-fold higher expression in the oldest leaves compared to the youngest ones. The present study on spatial and developmental regulation of the nepetalactones production and accumulation in N. rtanjensis leaves, as well as the identification of two IS candidate genes is a proof-of-the-concept research that serves as a cornerstone for future studies on elucidation of the nepetalactones biosynthesis.