AUTHOR=Aničić Neda , Matekalo Dragana , Skorić Marijana , Gašić Uroš , Nestorović Živković Jasmina , Dmitrović Slavica , Božunović Jelena , Milutinović Milica , Petrović Luka , Dimitrijević Milena , Anđelković Boban , Mišić Danijela 

TITLE=Functional iridoid synthases from iridoid producing and non-producing Nepeta species (subfam. Nepetoidae, fam. Lamiaceae)

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2024

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1211453

DOI=10.3389/fpls.2023.1211453

ISSN=1664-462X

ABSTRACT=<p>Iridoids, a class of atypical monoterpenes, exhibit exceptional diversity within the <italic>Nepeta</italic> genus (subfam. Nepetoidae, fam. Lamiaceae).The majority of these plants produce iridoids of the unique stereochemistry, with nepetalactones (NLs) predominating; however, a few <italic>Nepeta</italic> species lack these compounds. By comparatively analyzing metabolomics, transcriptomics, gene co-expression, and phylogenetic data of the iridoid-producing <italic>N. rtanjensis</italic> Diklić &amp; Milojević and iridoid-lacking <italic>N. nervosa</italic> Royle &amp; Bentham, we presumed that one of the factors responsible for the absence of these compounds in <italic>N. nervosa</italic> is iridoid synthase (ISY). Two orthologues of ISY were mined from leaves transcriptome of <italic>N. rtanjensis</italic> (<italic>Nr</italic>PRISE1 and <italic>Nr</italic>PRISE2), while in <italic>N. nervosa</italic> only one (<italic>Nn</italic>PRISE) was identified, and it was phylogenetically closer to the representatives of the Family 1 isoforms, designated as P5βRs. Organ-specific and MeJA-elicited profiling of iridoid content and co-expression analysis of IBG candidates, highlighted <italic>NrPRISE2</italic> and <italic>NnPRISE</italic> as promising candidates for ISY orthologues, and their function was confirmed using <italic>in vitro</italic> assays with recombinant proteins, after heterologous expression of recombinant proteins in <italic>E. coli</italic> and their His-tag affinity purification. <italic>Nr</italic>PRISE2 demonstrated ISY activity both <italic>in vitro</italic> and likely <italic>in planta</italic>, which was supported by the 3D modeling and molecular docking analysis, thus reclassification of <italic>Nr</italic>PRISE2 to <italic>Nr</italic>ISY is accordingly recommended. <italic>Nn</italic>PRISE also displays <italic>in vitro</italic> ISY-like activity, while its role under <italic>in vivo</italic> conditions was not here unambiguously confirmed. Most probably under <italic>in vivo</italic> conditions the <italic>Nn</italic>PRISE lacks substrates to act upon, as a result of the loss of function of some of the upstream enzymes of the iridoid pathway. Our ongoing work is conducted towards re-establishing the biosynthesis of iridoids in <italic>N. nervosa</italic>.</p>