N-acylethanolamines (NAEs) are present in wide range of organisms and belong to family of functionally diverse signaling lipids. They consist of a fatty acid with varying chain lengths and ethanolamine linked via an amide bond. The level of NAEs is modulated by their hydrolysis to ethanolamine and fatty acid by an enzyme fatty acid amide hydrolase (FAAH). FAAH is an integral membrane protein that belongs to “amidase signature” superfamily of proteins, which is characterized by highly conserved region rich in serine, glycine and alanine. FAAH directly or indirectly plays a role in modulation of various physiological processes by regulating NAE levels. Although the role of NAEs and its key modulator FAAH has been studied in other plants, their role in tomato model is limited and unknown. More recently, SlFAAH1, an ortholog of AtFAAH1, was identified in tomato and cloned into bacterial expression system. However, putative SlFAAH1 function and distinct features are yet to be determined. It is hypothesized that the putative SlFAAH1 catalyzes the hydrolysis of NAEs and modulates the level of NAEs during the seedling development in tomato. To this extent, a putative SlFAAH1 (previously identified and cloned in pET-23a vector) will be biochemically characterized and also effect of NAEs on seedling development will be studied. Thus far, SlFAAH1 cloned in pET-23a vector was expressed in RIL cell line (prokaryotic expression system) followed by conformation of positive transformant by colony PCR. Currently, protein expression and confirmation of SlFAAH in the positive transformant is being done. The expressed protein will be characterized for its hydrolytic activity using radiolabelled substrate. The effect of exogenous NAEs during seedling development will be studied with regards to expression level of SlFAAH1 by qPCR and composition of NAE during the seedling development to determine the role of NAE during seedling development. Thus, this study is expected to not only characterize a protein in tomato but also determine its role in mediating NAE metabolism and seedling development. Longterm studies will identify the significance of highly conserved NAE pathway in eukaryotes.

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.

N-acylethanolamines(NAEs) are fatty acid amides derived from a minor membrane lipid constituent Nacylphosphatidylethanolamine, structurally consisting the linkage of fatty acid tothe ethanolamines. NAE is hydrolysed by fatty acid amide hydrolase (FAAH) into free fatty acid and ethanolamine in both plants and animals. In plants, FAAH gene has been thus far characterized in Arabidopsis, where it was shown to act as a modulator of endogenous NAE levels, seedling growth and their ability to respond to biotic and biotic stress. Based on the evidence that NAEs occur in tomato (Solanum lycopersicum) seeds, we hypothesized that there is a functional FAAH that hydrolyzes NAEs in tomato. To test this, we performed in silico analysis using AtFAAH sequence as a template and identified six orthologs in tomato. These six S. lycopersicum FAAH homologs have the characteristic amidase signature sequence and conserved catalytic residues. Protein structures of putative SlFAAH1 and 2 were predicted using molecular visualization system (PyMOL). They showed similar domain structure with minor differences in spatial arrangement when compared with that of AtFAAH. Among the six homologs only putative SlFAAH1 and SlFAAH2 expression levels were associated with seedling development. Therefore the study was focused on cloning and characterization of SlFAAH1 and 2. Thus far, full-length coding sequence of putative SlFAAH1 was cloned into a heterologous expression system and its expression was confirmed by Western blot. Biochemical characterization of its hydrolytic activity against radiolabelled NAE substrates is underway. Furthermore, expression of SlFAAH1 and SlFAAH2 will be quantified and correlated with the NAE levels and hydrolytic activity at different developmental stages. This study is expected to reveal how NAE metabolite levels are modulated in tomato plant during its development.

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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.

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