Understanding the Implications of Anandamide, an Endocannabinoid in an Early Land Plant, Physcomitrella patens

Endocannabinoid signaling is well studied in mammals and known to be involved in numerous pathological and physiological processes. Fatty acid amide hydrolase (FAAH) terminates endocannabinoid signaling in mammals. In Physcomitrella patens, we identified nine orthologs of FAAH (PpFAAH1 to PpFAAH9) with the characteristic catalytic triad and amidase signature sequence. Kinetics of PpFAAH1 showed specificity towards anandamide (AEA) at 37°C and pH 8.0. Further biophysical and bioinformatic analyses revealed that, structurally, PpFAAH1 to PpFAAH4 were closely associated to the plant FAAH whereas PpFAAH6 to PpFAAH9 were more closely associated to the animal FAAH. A substrate entry gate or ‘dynamic paddle’ in FAAH is fully formed in vertebrates but absent or not fully developed in non-vertebrates and plants. In planta analysis revealed that PpFAAH responded differently with saturated and unsaturated N-acylethanolamines (NAEs). In vivo amidohydrolase activity showed specificity associated with developmental stages. Additionally, overexpression of PpFAAH1 indicated the need for NAEs in developmental transition. To understand and identify key molecules related to endocannabinoid signaling in P. patens, we used high-throughput RNA sequencing. We analyzed temporal expression of mRNA and long non-coding RNA (lncRNA) in response not only to exogenous anandamide but also its precursor arachidonic acid and abscisic acid (ABA, a stress hormone). From the 40 RNA-seq libraries generated, we identified 4244 novel lncRNAs. The highest number of differentially expressed genes (DEGs) for both mRNA and lncRNA were detected on short-term exposure (1 h) to AEA. Furthermore, gene ontology enrichment analysis showed that 17 genes related to activation of the G protein-coupled receptor signaling pathway were highly expressed along with a number of genes associated with organelle relocation and localization. We identified key signaling components of AEA that showed significant difference when compared with ABA. This study provides a fundamental understanding of novel endocannabinoid signaling in early land plants and a future direction to elucidate its functional role

An Endocannabinoid Catabolic Enzyme Faah and Its Paralogs in an Early Land Plant Reveal Evolutionary and Functional Relationship With Eukaryotic Orthologs

Endocannabinoids were known to exist only among Animalia but recent report of their occurrence in early land plants prompted us to study its function and metabolism. In mammals, anandamide, as an endocannabinoid ligand, mediates several neurological and physiological processes, which are terminated by fatty acid amide hydrolase (FAAH). We identified nine orthologs of FAAH in the moss Physcomitrella patens (PpFAAH1 to PpFAAH9) with amidase signature and catalytic triad. The optimal amidase activity for PpFAAH1 was at 37 °C and pH 8.0, with higher specificity to anandamide. Further, the phylogeny and predicted structural analyses of the nine paralogs revealed that PpFAAH1 to PpFAAH4 were closely related to plant FAAH while PpFAAH6 to PpFAAH9 were to the rat FAAH, categorized based on the membrane binding cap, membrane access channel and substrate binding pocket. We also identified that a true ‘dynamic paddle’ that is responsible for tighter regulation of FAAH is recent in vertebrates and absent or not fully emerged in plants and non-vertebrates. These data reveal evolutionary and functional relationship among eukaryotic FAAH orthologs and features that contribute to versatility and tighter regulation of FAAH. Future studies will utilize FAAH mutants of moss to elucidate the role of anandamide in early land plants

Tobacco Methyl Salicylate Esterase Mediates Nonhost Resistance

Nonhost resistance is a type of broad-spectrum resistance exhibited by a given plant species to most strains of a pathogen which are generally pathogenic to other plant species. In this study, we have examined the role of tobacco SABP2 (Salicylic acid-Binding Protein 2) in nonhost resistance. SABP2, a methyl salicylate esterase is a critical component of SA-signaling pathway in tobacco plants. The transgenic tobacco SABP2-silenced lines treated with tetraFA, a known inhibitor of esterase activity of SABP2 exhibited enhanced susceptibility to nonhost pathogen, Pseudomonas syringae pv. phaseolicola compared to the control plants. The increased accumulation of SABP2 transcripts upon Psp infection supports the involvement of SABP2 in nonhost resistance. The tetra-FA treated plants also showed delayed expression of pathogenesis related-1 gene upon Psp inoculations. The expression of nonhost marker genes CDM1 and HIN1 was also monitored in tobacco plants infected with host-pathogen P.s. pv. tabaci and P.s. pv. phaseolicola. Overall, results presented in this manuscript suggest that SABP2 has a role in nonhost resistance in tobacco plants

Antioxidant and antimicrobial activities of Sonneratia albain vitro and In vivo : comparative study with Rhizophora mucronata and Bruguiera gymnorrhiza / Md. Imdadul Haq

The present study evaluated the antioxidant and antimicrobial potential of three mangrove species namely, Sonneratial alba, Rhizophora mucronat and Bruguiera gymnorrhiza. The bioactivities of methanol, ethanol and chloroform extracts of in vivo and in vitro explants of S. alba and in vivo explants of R. mucronata and B. gymnorrhiza were evaluated. The effects of growth regulators on seed germination of S. alba, to obtain the in vitro explants, were also studied. In the first part of bioactivity study, total phenolic contents were detected by using the Folin-ciocalteu assay. Ethanol extract of leaves of R. mucronata had the highest result for total phenolic content as 358.6 ± 0.53 mg of GAE/g of dry sample. Both enzymatic and non-enzymatic antioxidant assays were used to examine the potential of antioxidant activities of all tested samples. Ethanol and methanol extracts of barks showed better antioxidant activities compared to the leaves. Chloroform extracts in both leaf and bark samples exhibited poor antioxidant activities for all selected mangrove plants. Disc diffusion method was used to detect the antimicrobial activity of the extracts of all three plants. Two gram positive (Bacillus cereus and Staphylococcus aureus) and two gram negative (Escherichia coli and Pseudomonas aeruginosa) bacterial pathogens were used to evaluate antimicrobial activity. Methanol and ethanol extracts of leaves and barks showed promising antimicrobial activities when tested for all four abovementioned bacterial pathogens. Nevertheless, chloroform extracts exhibited poor or no antimicrobial activities against all tested microbial. Ethanol extract of barks of S. alba was fractionated into water and ethyl acetate fraction, and their antioxidant and antimicrobial activities were assessed. Water fraction exhibited better bioactive potential compared with ethyl acetate fraction. Liquid chromatography tandem mass spectrometry (LCMS/MS) analysis was also carried out with water fraction to obtain the nominal mass of the major active components in the water fraction

Characterization of a Mammalian Endocannabinoid Hydrolyzing Enzyme in Physcomitrella patens

The discovery of a mammalian endocannabinoid, anandamide (N-arachidonylethanolamide; AEA or NAE 20:4) in Physcomitrella patens but not in higher plants prompted our interest in characterizing its metabolism and physiological role in the early land plant. Anandamide acts as an endocannabinoid ligand in the mammalian central and peripheral systems and mediates various physiological responses. Endocannabinoid signaling is terminated by a membrane-bound fatty acid amide hydrolase (FAAH). Based on sequence identity and in silico analyses, we identified nine orthologs of human and Arabidopsis FAAH in P. patens (PpFAAH1 to PpFAAH9). Predicted structural analysis revealed that all the nine PpFAAH contain characteristic amidase signature sequence with a highly conserved catalytic triad and share a number of key features of both plant and animal FAAH. These include a membrane binding cap, membrane access channel, substrate binding pocket and as well as potential for dimerization. Among the nine, gene expression levels for PpFAAH1 and PpFAAH9 were enhanced with exogenous anandamide treatment. Further cloning and heterologous expression, followed by radiolabeled in vitro enzyme assays revealed that PpFAAH1 activity was optimal at 37 °C and pH 8.0. Furthermore, PpFAAH1 showed higher specificity to NAE 20:4 than to other N-acylethanolamines such as NAE 16:0. Highest in planta amide hydrolase activity was noted in microsomes of gametophyte tissues, suggesting the possibility for membrane localization of active FAAH. Interestingly, when FAAH1 was overexpressed, the moss cultures not only showed reduced growth but their transition from protonemal stage to gametophyte was inhibited, which was rescued in part by exogenous AEA. Unlike overexpressors of AtFAAH1, which showed enhanced growth and hypersensitivity to abscisic acid, PpFAAH1 overexpressors showed tolerance to abscisic acid. Together, these data suggest that the occurrence of anandamide and distinct properties of PpFAAH1 in early land plants have physiological implications that are different from that of higher plants

Functional Characterization of Anandamide Hydrolyzing Enzyme in Physcomitrella patens

The discovery of a mammalian endocannabinoid, anandamide (AEA or NAE 20:4) in Physcomitrella patens but not in higher plants prompted our interest in characterizing its metabolism and physiological role in the early land plants. Anandamide acts as an endocannabinoid ligand in the mammalian central and peripheral systems and mediates various physiological responses. Endocannabinoid signaling is terminated by a membrane-bound fatty acid amide hydrolase (FAAH). Using in silico analyses, we identified nine orthologs of human and Arabidopsis FAAH in P. patens (PpFAAH1 to PpFAAH9). Predicted structural analysis revealed that all the nine PpFAAH contain characteristic amidase signature sequence with a highly conserved catalytic triad and share a number of key features of both plant and animal FAAH. These include a membrane binding cap, membrane access channel, substrate binding pocket and as well as potential for dimerization. Among the nine, gene expression for PpFAAH1 and PpFAAH9 was enhanced with exogenous AEA treatment. Cloning and heterologous expression, followed by radiolabeled in vitro enzyme assays revealed that PpFAAH1 activity was optimal at 37 °C and pH 8.0. Furthermore, PpFAAH1 showed higher specificity to NAE 20:4 than to other N- acylethanolamines such as NAE 16:0. Highest in planta amide hydrolase activity was noted in microsomes of gametophytes, suggesting the possibility for membrane localization of active FAAH. Interestingly, when FAAH1 was overexpressed, the moss cultures not only showed reduced growth but their transition from protonema to gametophyte was inhibited, which was rescued by exogenous AEA. Unlike overexpressors of AtFAAH1, which showed enhanced growth and hypersensitivity to abscisic acid, PpFAAH1 overexpressors showed tolerance to abscisic acid. Together, these data suggest that the occurrence of anandamide and distinct properties of PpFAAH1 in early land plants have physiological implications that are different from that of higher plants

Fatty Acid Amide Hydrolase in an Early Land Plant, Physcomitrella patens

Fatty acid amide hydrolase (FAAH) belongs to a diverse class of enzymes in amidase signature family. In mammals, FAAH is targeted to affect neurological functions because it terminates endocannabinoid signaling by degrading anandamide, a 20C N-acylethanolamine (NAE 20:4). In higher plants, FAAH is known to modulate growth, development and stress tolerance by degrading 12-18C NAEs. Since anandamide was reported to exclusively occur in early land plants, we investigated its metabolic pathway in the moss Physcomitrella patens. Based on the highest identity with ratFAAH, we identified nine orthologs in moss, PpFAAH1 to PpFAAH9. Several bioinformatic tools were used to understand the structural basis of how catalytic residues fold for amidohydrolase activity. Based on these in silico analyses of PpFAAH homologs and their gene expression in response to saturated (NAE16:0) and unsaturated NAE (NAE 20:4) treatment, PpFAAH1 was selected for biochemical characterization. Heterologously expressed PpFAAH1 showed highest amidohydrolase activity at 37°C and pH 8.0. Both in vivo and in vitro studies showed that unsaturated NAE substrate is hydrolyzed faster than the saturated NAE (\u3e 10-fold in vivo and 50-fold in vitro). Additionally, transgenic moss lines over expressing FAAH1 showed slower growth and disrupted gametophyte formation when compared to wild type. These data suggest that PpFAAH1-mediated NAE metabolism is likely involved in developmental transition in moss