Targeted metabolomics shows plasticity in the evolution of signaling lipids and uncovers old and new endocannabinoids in the plant kingdom

Abstract

The remarkable absence of arachidonic acid (AA) in seed plants prompted us to systematically study the presence of C20 polyunsaturated fatty acids, stearic acid, oleic acid, jasmonic acid (JA), N-acylethanolamines (NAEs) and endocannabinoids (ECs) in 71 plant species representative of major phylogenetic clades. Given the difficulty of extrapolating information about lipid metabolites from genetic data we employed targeted metabolomics using LC-MS/MS and GC-MS to study these signaling lipids in plant evolution. Intriguingly, the distribution of AA among the clades showed an inverse correlation with JA which was less present in algae, bryophytes and monilophytes. Conversely, ECs co-occurred with AA in algae and in the lower plants (bryophytes and monilophytes), thus prior to the evolution of cannabinoid receptors in Animalia. We identified two novel EC-like molecules derived from the eicosatetraenoic acid juniperonic acid, an omega-3 structural isomer of AA, namely juniperoyl ethanolamide and 2-juniperoyl glycerol in gymnosperms, lycophytes and few monilophytes. Principal component analysis of the targeted metabolic profiles suggested that distinct NAEs may occur in different monophyletic taxa. This is the first report on the molecular phylogenetic distribution of apparently ancient lipids in the plant kingdom, indicating biosynthetic plasticity and potential physiological roles of EC-like lipids in plants.

Figure 1. Conventional and alternative biosynthetic pathways for the production of PUFAs in eukaryotes.

In green, the switch between ω-3 and ω-6 pathways characteristic of plants and nematodes through the action of ω-3 desaturases (FAD) is highlighted. Desaturases (D) and elongases (E) are shown together with the position at which the enzymatic reaction takes place. Red arrows highlight a proposed pathway for the biosynthesis of ScA and JuA in gymnosperms. For simplicity, the FA substrates here presented do not differentiate between acyl-lipid, acyl-ACP or acyl-CoA that occur either free or bounded to glycolipids. FAs investigated (bold) as well as the bioactive metabolites (blue squares pointed by blue arrows) that were investigated are shown.

Figure 2. Concentrations (mean values) of AA and JA in pmol/g plant weight found in the 71 plant species analyzed (0.0 = < LOD).

The background colors of the cells are used to highlight concentrations in the column (i.e. concentrations of the respective analyte are coded red for small amounts, yellow for intermediate amounts and green for high amounts). Complete information is available in Supplementary Tables S1 and S11.

Figure 3. Chemical structures of C-20 PUFA metabolites.

Arachidonic acid (1) (AA), arachidonoyl ethanolamide (AEA) (2), 1-arachidonoyl glycerol (1-AG) (3), 2-arachidonoyl glycerol (2-AG) (4), juniperonic acid (JuA) (5), juniperoyl ethanolamide (JEA) (6), 1-juniperoyl glycerol (1-JG) (7) and 2-juniperoyl glycerol (2-JG) (8).

Figure 4. Chromatograms showing the analysis of C20 PUFA metabolites.

First column: AA and JuA; second column: 1/2-AG and 1/2-JG and third column: AEA and JEA. Samples were prepared using HPLC for cleanup (see Sample preparation for identification) and analyzed using LC-MS/MS in the positive and negative modes (see Chromatographic conditions used for the identification of structural isomers (LC-MS/MS method 2)).

Figure 5. Chemotaxonomic distribution of C-20 PUFA metabolites.

AA (pale blue), JuA (orange), 1/2-AG (green), 1/2-JG (pink), AEA (blue) and JEA (red). The intensity of the colors indicates concentration. The concentration ranges used for AA and JuA are 54 to 1E4 (shade 1), 1E4 to 1E5 (shade 2), 1E5 to 1E6 (shade 3) and >1E6 (shade 4); for 1/2-AG and 1/2-JG are 37 to 1E3 (shade 1), 1E3 to 1E4 (shade 2), 1E4 to 1E5 (shade 3) and >1E5 (shade 4); and for AEA and JEA are 0.8 to 1 (shade 1), 1 to 10 (shade 2), 10 to 100 (shade 3) and 100–1000 (shade 4).

Figure 6. 3D-PCA plots depicting 71 plant species distributed in the 9 plant groups investigated.

Nine different colors are used to highlight the plant groups. Three panels (a–c) showing the same analysis in different positions are presented to highlight difference between plant groups and samples. Each panel shows the scores on the left and the loadings on the right sides, respectively. Angiosperms: 1) Viscum album, 2) Arabidopsis thaliana, 3) Theobroma cacao, 4) Cannabis sativa, 5) Rhizophora mangle, 6) Salix glabra, 7) Sorghum bicolor, 8) Hakonechloa macra, 9) Zea mays, 10) Allium sativum, 11) Liriodendron tulipifera, 12) Chloranthus spicatus, 13) Schisandra chinensis, 14) Nymphaea nouchali, 15) Amborella trichopoda; Gymnospers: 16) Welwitschia mirabilis, 17) Pinus peuce, 18) Pinus peuce, 19) Pinus mugo, 20) Pinus cembra, 21) Larix gmelinii, 22) Picea abies, 23) Abies numidica, 24) Abies cephalonica, 25) Abies koreana, 26) Taxus baccata, 27) Cryptomeria japonica, 28) Thujopsis dolabrata, 29) Araucaria araucana, 30) Ginkgo biloba, 31) Cycas revoluta; Monilophytes: 32) Polypodium vulgare, 33) Davallia canariensis, 34) Tectaria zeylanica, 35) Polystichum aculeatum, 36) Onoclea sensibilis, 37) Blechnum spicant, 38) Thelypteris palustri, 39) Gymnocarpium robertianum, 40) Asplenium trichomanes, 41) Phyllitis scolopendrium, 42) Adiantum venustum, 43) Sphaeropteris cooperi, 44) Salvinia natans, 45) Salvinia molesta, 46) Anemia phyllitidis, 47) Lygodium volubile, 48) Osmunda regalis, 49) Angiopteris evecta, 50) Equisetum trachyodon, 51) Psilotum nudum; Lycophytes: 52) Selaginella moellendorffii, 53) Selaginella pallescens, 54) Huperzia phlegmaria; Hornworts: 55) Anthoceros agrestis, 56) Anthoceros punctatus, 57) Phaeoceros laevis; Mosses: 58) Physcomitrella patens, 59) Funaria hygrometrica, 60) Polytrichum juniperinum, 61) Hedwigia ciliata, 62) Hylocomium splendens; Liverworts: 63) Conocephalum conicum, 64) Marchantia polymorpha, 65) Riccia fluitans; Algae: 66) Chara vulgaris, 67) Klebsormidium elegans, 68) Caulerpa prolifera, 69) Halymenia floresii; Lichens: 70) Cetraria islandica, 71) Xanthoria parietina.

Figure 7. Graphical representation of the metabolite distribution within the investigated phylogenetic groups.