Recently, the occurrence of a mammalian endocannabinoid ligand N-arachidonoylethanolamide (anandamide, AEA, NAE20:4), was reported in early land plants. Unlike seed plants, bryophytes such as Physcomitrella patens possess unique fatty acid composition that includes long-chain fatty acids such as arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5). We performed targeted lipid profiling to discovere long-chain N-acylethanolamines (NAEs) and their corresponding N-acylphosphatidylethanolamine (NAPE) precursors in Physcomitrella and Selaginella. In protonemal tissues, N-arachidonyl-PE and N-20:5-PE contributed to about 49 % and 30 %, respectively. Matured gametophytes on the other hand showed a 12 % increase in N-20:4-PE and 20 % decline in N-20:5-PE, relative to NAPE content in protonemata. In all haploid developmental stages analyzed, NAE20:4 levels contributed to ~ 23 % of the total NAE while NAE 20:5 remained as a minor component (5 %). Interestingly, in Selaginella moellendorffi, an early vascular plant, N-18:2-PE species was most abundant and 20C-NAEs were present in trace amounts. To understand biological implications of anadamide, we examined the effects of exogenously applied AEA and its corresponding fatty acid (AA) on moss protonemata growth. Both AEA and AA inhibit growth of gametophytes and protonemata in a dose dependent manner, while AEA exclusively affected actin-mediated tip growth. Additionally, we identified moss ortholog for NAPE-hydrolyzing phospholipase D (NAPE-PLD) enzyme that likely generates AEA and a fatty acid amide hydrolase (FAAH) that catabolizes AEA. Both putative PpNAPEPLD and PpFAAH are expressed in E. coli for further characterization. Our data demonstrates the occurrence of evolutionarily conserved NAE metabolic pathway in the moss, with unique composition. Functional and evolutionary implications of this mammalian endocannabinoid in early land plants, however, remains elusive.

The endogenous arachidonate-based lipids that activate cannabinoid receptors have been well characterized in mammals. In plants only 12-18 carbon fatty acid ethanolamides have been identifi ed so far and have been shown to modulate a number of physiological processes including seed and seedling development. However, since moss plants contain arachidonic acid, we hypothesized the occurrence of arachidonate-based metabolites in their tissues. Using selective lipidomics approach, we identifi ed the presence of anandamide or arachidonylethanolamide (a 20C polyunsaturated fatty acid ethanolamide) and its precursors, in Physcomitrella patens that were previously not reported in plants. Comprehensive lipid profi les for protonema and gametophyte tissues of moss also revealed the occurrence of other saturated and unsaturated fatty acid ethanolamides and a distinct phospholipid and galactolipid composition. Further studies showed that anandamide, like abscisic acid, inhibits the growth of gametophytes more severely than saturated fatty acid ethanolamides. Our current studies are focused on understanding the physiological and developmental role of polyunsaturated fatty acid ethanolamides in nonseed plants. In conclusion, discovery of anandamide in moss provided us with an exciting possibility to identify fatty acid ethanolamide metabolic pathway in early land plants and elucidate receptor-mediated endocannabinoid signaling responses in plants that is akin to mammals.