molecules Article Identification and Quantification of Coumarins by UHPLC-MS in Arabidopsis thaliana Natural Populations Izabela Perkowska 1 , Joanna Siwinska 1, Alexandre Olry 2 ,Jérémy Grosjean 2 , Alain Hehn 2 , Frédéric Bourgaud 3 , Ewa Lojkowska 1 and Anna Ihnatowicz 1,* 1 Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland; [email protected] (I.P.); [email protected] (J.S.); [email protected] (E.L.) 2 Université de Lorraine-INRAE, LAE, 54000 Nancy, France; [email protected] (A.O.); [email protected] (J.G.); [email protected] (A.H.) 3 Plant Advanced Technologies, 54500 Vandœuvre-lès-Nancy, France; [email protected] * Correspondence: [email protected]; Tel.: +48-58-5236330 Abstract: Coumarins are phytochemicals occurring in the plant kingdom, which biosynthesis is induced under various stress factors. They belong to the wide class of specialized metabolites well known for their beneficial properties. Due to their high and wide biological activities, coumarins are important not only for the survival of plants in changing environmental conditions, but are of great importance in the pharmaceutical industry and are an active source for drug development. The identification of coumarins from natural sources has been reported for different plant species including a model plant Arabidopsis thaliana. In our previous work, we demonstrated a presence of naturally occurring intraspecies variation in the concentrations of scopoletin and its glycoside, Citation: Perkowska, I.; Siwinska, J.; scopolin, the major coumarins accumulating in Arabidopsis roots. Here, we expanded this work Olry, A.; Grosjean, J.; Hehn, A.; by examining a larger group of 28 Arabidopsis natural populations (called accessions) and by Bourgaud, F.; Lojkowska, E.; Ihnatowicz, A. Identification and extracting and analysing coumarins from two different types of tissues–roots and leaves. In the Quantification of Coumarins by current work, by quantifying the coumarin content in plant extracts with ultra-high-performance UHPLC-MS in Arabidopsis thaliana liquid chromatography coupled with a mass spectrometry analysis (UHPLC-MS), we detected a Natural Populations. Molecules 2021, significant natural variation in the content of simple coumarins like scopoletin, umbelliferone and 26, 1804. https://doi.org/10.3390/ esculetin together with their glycosides: scopolin, skimmin and esculin, respectively. Increasing molecules26061804 our knowledge of coumarin accumulation in Arabidopsis natural populations, might be beneficial for the future discovery of physiological mechanisms of action of various alleles involved in their Academic Editor: Maria João Matos biosynthesis. A better understanding of biosynthetic pathways of biologically active compounds is the prerequisite step in undertaking a metabolic engineering research. Received: 27 February 2021 Accepted: 18 March 2021 Keywords: analytical methods; model plant; natural genetic variation; natural products; sim- Published: 23 March 2021 ple coumarins Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Coumarins are secondary metabolites widely distributed throughout the plant king- dom. They are synthetized via the phenylpropanoid biosynthesis pathway. We can distin- guish several simple coumarins like coumarin, scopoletin (7-hydroxy-6-methoxycoumarin), Copyright: © 2021 by the authors. esculetin (6,7-dihydroxycoumarin), umbelliferone (7-hydroxycoumarin), fraxetin (7,8- Licensee MDPI, Basel, Switzerland. dihydroxy-6-methoxycoumarin), sideretin (5,7,8-trihydroxy-6-methoxycoumarin) and their This article is an open access article respective glycosylated forms–scopolin, esculetin, skimmin, fraxin and sideretin-glycoside, distributed under the terms and respectively [1]. Figure1 presents the semi-developed formula of simple coumarins and conditions of the Creative Commons their glycosides derivatives identified in this research. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Molecules 2021, 26, 1804. https://doi.org/10.3390/molecules26061804 https://www.mdpi.com/journal/molecules MoleculesMolecules 2021, 202126, x, 26FOR, 1804 PEER REVIEW 2 of 152 of 15 FigureFigure 1. Chemical 1. Chemical structures structures of ofsimple simple coumarins coumarins and and their glycosidesglycosides analysed analysed in in this this work work (www.chem-space.com (www.chem-space.com (accessed(accessed on 20 on January 20 January 2021 2021)).)). Coumarins have been recognized for many years as an important class of pharma- Coumarins have been recognized for many years as an important class of pharmaco- cologically active compounds. They have anticoagulant, anticancer, antiviral and anti- logicallyinflammatory active propertiescompounds [2].. They In addition have anticoagulant, to the listed medicinal anticancer, benefits, antiviral it was and shown anti-in- flammatoryrecently in properties numerous studies [2]. In that addition coumarins to the play listed an important medicinal role benefits in iron (Fe), it was homeosta- shown re- centlysis, oxidativein numerous stress studies response, that plant-microbe coumarins interactions play an important and that they role can in actiron as (Fe) signalling homeosta- sis,molecules oxidative in stress plants response [3–8]. In, the plant last- fewmicrobe years, interactions an increasing and number that they of reports can act concern as signal- lingthe molecules analysis of in root plants extracts [3–8 and]. In root the exudateslast few thatyears, are an rich increasing in phenolic number compounds, of reports such con- cernas the simple analysis coumarins, of root which extracts mediate and multiple root exudates interactions that inare the rich rhizosphere. in phenolic Coumarins compounds, suchwere as shownsimple to coumarins have a strong, which impact mediat on thee multiple plant interactions interactions with in microorganisms the rhizosphere. and Cou- play a crucial role in nutrient acquisition [6,9–18]. Moreover, the root-secreted scopoletin marins were shown to have a strong impact on the plant interactions with microorganisms was proved to exert a selective antimicrobial action in the rhizosphere [8]. These numerous andreports play a examining crucial role the biochemicalin nutrient andacquisition physiological [6,9–18 functions]. Moreover, of coumarins, the root make-secreted this sco- poletinclass ofwas specialized proved metabolitesto exert a selective extremely antimicrobial interesting from action a scientific in the and rhizosphere commercial point[8]. These numerousof view. Thereports vast examining majority of the these biochemical studies were and performed physiological using a functions reference accession,of coumarins, makeCol-0, this of class the model of specialized plant Arabidopsis metabolites thaliana extremely(hereinafter intere Arabidopsis),sting from a and scientific its mutants and com- mercialdefective point at variousof view. steps The ofvast coumarin majority biosynthesis. of these studies were performed using a reference accessionHere,, Col we-0, conducted of the model the qualitative plant Arabidopsis and quantitative thaliana assessment (hereinafter of coumarinArabidopsis), content and its mutantsin leaf anddefective root tissue at various of a set steps of Arabidopsis of coumarin natural biosynthesis. populations (accessions). Numerous studiesHere, on we primary conducted and specializedthe qualitative metabolites and quantitative profiling were assessment conducted of using coumarin the Ara- content bidopsis model system [19–28]. Previously, due to the importance of coumarins for human in leaf and root tissue of a set of Arabidopsis natural populations (accessions). Numerous health, most research on their metabolic profiling were carried out on plants of economic studiesimportance, on primary such as and e.g., specialized sweet potato metabolites (Ipomoea batatas profilingL.), rue were (Ruta graveolensconductedL.) using or lettuce the Ara- bidopsis(Lactuca model sativa L.)system [29–34 [19]. One–28]. of Previously, the first metabolic due to profiling the importance of root exudates of coumarins using Ara- for hu- manbidopsis health, natural most populationsresearch on (Col-0, their metabolic C24, Cvi-0, profiling Ler) was madewere bycarried Micallef out et on al. plants [35] who of eco- nomicattempted importance, to correlate such them as withe.g., thesweet compositions potato (Ipomoea of rhizobacterial batatas L.), communities. rue (Ruta However, graveolens L.) or thelettuce authors (Lactuca of this sativa work L.) did [29 not– undertake34]. One of the the qualitative first metabolic and quantitative profiling evaluation of root exudates of usingthe isolatedArabidopsis compounds. natural Consequently, populations they (Col could-0, C24, only Cvi conclude-0, Ler) that was there made are differences by Micallef et al. between[35] who Arabidopsis attempted accessions to correlate in terms them of with the quality the compositions and quantity of releasedrhizobacterial substances, commu- which may have an impact on the composition of the rhizobacterial communities. nities. However, the authors of this work did not undertake the qualitative and quantita- So far, only a few more studies focusing on the accumulation of coumarins in Ara- tivebidopsis