No description available.

No description available.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. We identified FAAH in Physcomitrella patens and expressed heterologously in E. coli using Gateway cloning system. Radiolabeled NAE 16:0 and 20:4 were used as substrates to test amide hydrolase activity in vitro. In order to understand the role of PpFAAH in vivo, knock out (KO) and overexpressors (OE) were generated by homologous recombination. PpFAAH KO construct was generated by inserting 5‟- and 3‟-flanking regions into pMP1159 plasmid. Full length PpFAAH with stop codon was cloned into pTHUBlGATE vector in order to make OE construct. KO and OE constructs were then transformed into protoplasts of P. patens by using PEG-mediated transformation to generate mutant lines. To identify potential interacting proteins of PpFAAH, it was cloned into pDEST15 plasmid with N-terminus GST tag. Interaction between GST-tagged PpFAAH and proteins from 14-day old protonema will be visualized by SDS-PAGE and then subjected to LC-MS/MS analysis for identification. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in P. patens.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. We identified FAAH in Physcomitrella patens and expressed heterologously in E. coli using Gateway cloning system. Radiolabeled NAE 16:0 and 20:4 were used as substrates to test amide hydrolase activity in vitro. In order to understand the role of PpFAAH in vivo, knock out (KO) and overexpressors (OE) were generated by homologous recombination. PpFAAH KO construct was generated by inserting 5'- and 3'-flanking regions into pMP1159 plasmid. Full length PpFAAH with stop codon was cloned into pTHUBlGATE vector in order to make OE construct. KO and OE constructs were then transformed into protoplasts of P. patens by using PEG-mediated transformation to generate mutant lines. To identify potential interacting proteins of PpFAAH, it was cloned into pDEST15 plasmid with Nterminus GST tag. Interaction between GST-tagged PpFAAH and proteins from 14-day old protonema will be visualized by SDS-PAGE and then subjected to LC-MS/MS analysis for identification. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in P. patens.

In plants, saturated and unsaturated N-Acylethanolamines (NAEs) with acyl chains 12C to 18C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, its metabolism and functional implications in plants however, remains incomplete. Recently, anandamide (NAE 20:4), an essential fatty acid neurotransmitter in mammalian system, was identified in moss Physcomitrella patens, in addition to other types of NAEs. Bryophytes display high tolerance to abiotic stress and thus presence of anandamide in moss, but not in higher plants, suggests that NAE 20:4 might have contributed to their survival in harsh environmental conditions. Therefore, we hypothesize the anandamide metabolic pathway might play a role in mediating stress responses in P. patens. To this extent, using previously identified NAE-metabolic genes in mouse and/or Arabidopsis as templates, we identified moss orthologs for enzymes that likely participate in anandamide metabolism. We identified members of metallo-hydrolase superfamily and a/Β-hydrolase4, and five putative fatty acid amide hydrolases, which may hydrolyze N-acylphosphatidylethanolamine and NAE, respectively. Electronic fluorescent pictograph analyses of these orthologs in moss revealed differential developmental stage-specific expression patterns in gametophyte and sporophyte stages. We are currently examining expression pattern for these putative NAE-metabolic pathway genes, along with anandamide levels, in different tissues and developmental stages of moss subjected to water stress in the presence of anandamide. These transcript and metabolite levels in moss subjected to stress are expected to offer better understanding of the role of anandamide in mediating stress responses and further allow us to identify candidate genes that might participate in NAE metabolism. Our studies are aimed at functional validation of candidate genes and generating moss transgenic lines with altered NAE metabolite profile. Our long-term goal is to conduct comprehensive analyses of NAE metabolite mutants to determine their role in growth and development, and mediating stress responses in plants.

N-acylethanolamines (NAEs) are fatty acid ethanolamides that mediate stress responses in plants and animals. NAEs such as NAE 20:4 (anandamide) have only been reported in mammals and they regulate processes like neuroprotection and pain perception. Interestingly, we discovered the unique occurrence of anandamide in moss, Physcomitrella patens, a stress tolerant early land plant. Since NAEs including anandamide are degraded by fatty acid amide hydrolase (FAAH), it is hypothesized that a functional homolog of FAAH occurs in P. patens. I specifically propose to biochemically characterize FAAH enzyme that degrades anandamide. For this, Arabidopsis FAAH (AtFAAH) homolog was identified in moss database using BLASTP. The predicted protein structure of putative moss FAAH (PpFAAH) closely resembled to that of AtFAAH with conserved amidase signature sequence and catalytic triad residues: Lys205, Ser281, Ser305. Transcript levels of PpFAAH increased five-fold when moss was grown on excess NAE containing media. PpFAAH cDNA was PCR amplified and cloned into pET23a expression vector and transformed into RIL E. coli cells and confirmed by colony PCR. Heterologously expressed protein will be purified by Ni+2 affinity column chromatography and confirmed by western blot using anti-His-tag antibody. For biochemical characterization, enzyme will be presented with 14C NAE 20:4 substrate and rate of product free fatty acid formed will be quantified by extracting lipids from reaction mixture and separating by thin layer chromatography followed by radiometric scanning. E. coli cells expressing AtFAAH enzyme will be used as control. A complete characterization of the PpFAAH enzyme will be carried out to determine the kinetics, optimal temperature and pH conditions. Characterization of the enzyme that hydrolyzes anandamide in moss is expected to lead us to develop NAE metabolite mutants that will subsequently allow us to study the physiological role of anandamide in early land plants.

In higher plants, a class of bioactive fatty acid ethanolamides or N-acylethanolamines (NAEs) mediate growth, development, cellular organization and response to stress, in an abscisic acid (ABA)-dependent or independent manner. Unlike in higher plants, Physcomitrella patens, a bryophyte contains anandamide or NAE 20:4, a mammalian endocannabinoid ligand that mediates a multitude of physiological functions including development and stress. Unique lipids in mosses are considered vital for their resilience to environmental stresses; such lipids might enable them to recognize stress at the cellular level, and respond with membrane reorganization and altered growth. Since the identification of anandamide in moss, we have shown that, like abscisic acid (ABA), it inhibits gametophyte growth in a dose-dependent manner and reduced chlorophyll content. It is hypothesized that moss gametophores undergo morphological and cellular changes during anandamide-mediated growth inhibition. To test this, gametophyte growth and morphological changes in phyllodes, under different concentrations of NAE 20:4, were digitally captured using Canon EOS 70D, and analyzed using ImageJ software. NAE 20:4 but not its free fatty acid, arachidonic acid, not only inhibited growth of both shoots and rhizoids in a dose-dependent manner but also showed remarkable cellular changes. Phyllodes and protonemal cells of NAE 20:4 treated plants were further examined under stereo and compound light microscopes. Long- and short-term treatment with anandamide resulted in reduced chloroplast number, cytoplasmic shrinkage and plasmolysis in phyllodes and protonemal cells. A 100 micromolar NAE 20:4 treatment resulted in complete loss of green pigmentation in phyllodes. Effects of anandamide on cytoskeletal organization will be studied using Physcomitrella plants expressing GFP-talin and tubulin, via confocal microscopy. Together, these data will provide insights into anandamide-mediated cellular responses during growth inhibition.

No description available.

No description available.

N-acylethanolamines (NAEs) are a group of fatty acid ethanolamides and their metabolic pathway is highly conserved in eukaryotes. However, metabolites such as NAE 20:4 (anandamide) are known to occur in mammalian systems but not in higher plants. Anandamide is an endocannabinoid receptor ligand and mediates stress responses and regulates various physiological processes such as neuroprotection, pain perception and appetite suppression in animals. Interestingly anandamide occurrence was recently reported in a highly stress tolerant early land plant, Physcomitrella patens but its physiological role remains to be elucidated. Since NAEs including anandamide are degraded by fatty acid amide hydrolase (FAAH), it is hypothesized that a functional homolog of FAAH occurs in P. patens. To test this hypothesis, arabidopsis FAAH homolog was used to search moss database using BLASTP. Eight putative FAAH candidates (PpFAAH1-8), with an amidase signature sequence and conserved catalytic sites, were identified. Among these, PpFAAH1 and PpFAAH2 responded to exogenous NAE, and their 3D predicted protein structure closely resembled to that of AtFAAH1. The 1.8Kb coding region of putative PpFAAH1 was chosen for further characterization and was PCR amplified, cloned into TrcHis2 expression vector and transformed into E. coli TOP10 cells. Upon confirmation of the positive clones and induction of proteins, expressed proteins will be purified by Ni+2 affinity column chromatography, confirmed by western blot and analyzed for its substrate specificity using radiolabelled anandamide. Lipids extracted from reaction mixture will be separated by thin layer chromatography and detected by radiometric scanning. Characterization of the enzyme that hydrolyzes anandamide in moss is expected to lead us to develop NAE metabolite mutants that will subsequently allow us to study the physiological role of anandamide in early land plants.