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Arachidonylethanolamide (AEA) is a bioactive lipid ligand for mammalian cannabinoid receptors (CB). Thus far, AEA was reported to occur only in animals and was shown to regulate a wide range of physiological responses. Our recent fi nding of the occurrence of AEA in moss has led us hypothesize that AEA might mediate stress responses in plants, similar to that in animals. In mammals, AEA is generated from hydrolysis of N-acylphosphatidylethanolamine (NAPE) by a NAPE-specifi c phospholipase D (NAPE-PLD), and degraded by a fatty acid amide hydrolase (FAAH) and this metabolic pathway is highly conserved among eukaryotes. Here, using in silico approach, putative genes encoding for AEA pathway enzymes, were identifi ed in moss. Full-length coding sequences for putative NAPE-PLD and FAAH were isolated from Physcomitrella patens and were cloned and expressed into a heterologous expression vector. Biochemical characterization of AEA pathway enzymes is underway and is expected to lead to generation of AEA metabolite mutants in moss. Such mutants will allow for elucidation of the role of AEA in development of moss and mediating stress responses. Overall, this study will provide novel insights into functional and evolutionary role of lipid-mediated signaling in plants.

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.

N-Acylethanolamines (NAEs) are bioactive acylamides which are involved in diverse biological functions in eukaryotes. Although NAEs are ubiquitous in plants and animals, occurrence of N-arachidonoylethanolamide (anandamide, AEA, NAE20:4) is limited to mammals and early land plants. Metabolism of NAEs and their functional implications in plants are yet to be fully discovered. Unlike seed plants, bryophytes possess unique fatty acid composition that includes abundance of polyunsaturated fatty acids such as arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5). Moss Physcomitrella patens contains ~18.7 and 15.9 % of AA in gametophores and protonemata, respectively. Therefore, it is hypothesized that P. patens may exhibit a unique NAE metabolite profile. To this extent, we performed lipid profiling and discovered long-chain NAEs and their corresponding N-acyl-phosphatidylethanolamine (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; minor amounts of N-20:3-PE, N-20:4-PE and N-20:5-PE were also present with only a traceable quantity of NAE20:4. To understand biological implications of AEA, 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. Additionally, we identified moss ortholog for NAPE-hydrolyzing phospholipase D (NAPE-PLD) enzyme that likely generates AEA. Putative PpNAPE-PLD has been expressed in E. coli for further characterization. Our data demonstrates the occurrence of evolutionarily conserved NAE metabolic pathway in the moss, with an exclusive occurrence of AEA. However, functional and evolutionary implications of this mammalian endocannabinoid in early land plants remain elusive.

N-Acylethanolamines (NAEs) including anandamide (NAE 20:4) are fatty acid ethanolamides generated by the hydrolysis of N-acylphoshotidylethanolamine (NAPE) by phospholipase D (PLD) and degraded by fatty acid amide hydrolase (FAAH). In mammals, ligands such as NAE 20:4 act through cannabinoid receptors and regulate several physiological processes like neuroprotection, pain perception, mental depression, and appetite suppression. In plants, NAE with chain length C12 to C18 are common and affect physiological processes such as cytoskeletal organization, endomembrane trafficking, cell wall and cell shape formation, seedling growth and response to stress. However, our recent identification of NAE 20:4 in moss, Physcomitrella patens prompted us to elucidate its metabolic pathway and physiological implications. We hypothesize that unique NAE metabolites such as anandamide in moss might play a role in rendering moss its ability to tolerate temperature, dehydration, salt and osmotic stress. To address the above hypothesis, three main objectives are being pursued using P patens. 1)Biochemical and molecular characterization of NAE metabolic pathway, 2) Generation and phenotypic characterization of NAE metabolite mutants, and 3) Elucidation of the physiological role of NAEs in abscisic acid-mediated dehydration tolerance. A NAPE-PLD, known to synthesize NAE 20:4 has been identified in mammals and FAAH in several eukaryotes, including plants. Here, identification and cloning of putative NAPE-PLD and FAAH genes that are likely involved in NAE synthesis and degradation, respectively, in P patens is discussed. Our long-term objective is to understand lipid-mediated stress responses in plants.

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Limnological study of Moss Reservoir from May, 1975 through August, 1976 was conducted to evaluate the use of ATP assay in describing planktonic changes within the water column.

Thesis (M.F.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 2031-01-01

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