N-acylethanolamines (NAEs) are a family of signaling lipids derived from a minor membrane lipid constituent N-acylphosphatidylethanolamine (NAPE). In Arabidopsis, NAE mediates physiological functions such as seedling growth, flowering, and response to stress via abscisic acid (ABA) –dependent and –independent signaling pathways. The function of NAEs is terminated by a highly conserved fatty acid amide hydrolase (FAAH). Studies in model plant Arabidopsis showed the significant role of NAEs that makes it relevant to elucidate the conserved metabolic pathway of NAEs in crop species such as tomato. It is hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. To test this hypothesis, AtFAAH was used as a template to identify putative FAAH sequences in tomato, using BLASTX. Six SlFAAH sequences with the conserved amidase signature sequence and the catalytic triad, formed by Lys205, Ser281, and Ser305 in AtFAAH, were identified. Phylogenetic analysis of putative SlFAAH homologs and other FAAH family proteins (Arabidopsis, rice and moss), using CLUSTALW, revealed the two sequences that are closely related to the functionally characterized AtFAAH1. Using molecular visualization system (PyMOL), protein structures of putative SlFAAH1and 2 were predicted and compared with AtFAAH; both sequences showed similar domain structure to AtFAAH, with minor differences in spatial arrangement. For further biochemical characterization, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. Furthermore, transcript levels for SlFAAH1 and SlFAAH2 will be quantified and correlated with the NAE levels in various tissues to predict their role in tissue-specific NAE hydrolysis. Together, these molecular and biochemical characterization studies in tomato are expected to further validate the conserved nature of NAE metabolic pathway in 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 diverse family of signaling lipids that occur in eukaryotes and their presence is specific to developmental stage and tissue type. In plants, NAEs with an acyl chain ranging from C12 to C18 are common with NAE 18:2 generally being the most abundant type, particularly in desiccated seeds. In Arabidopsis, NAEs negatively regulate growth and mediate stress responses via abscisic acid-dependent and -independent signaling pathway. The function of NAEs is terminated by a highly conserved fatty acid amide hydrolase (FAAH). Because of the significant role NAEs were shown to play in model plant Arabidopsis it is pertinent to elucidate this conserved metabolic pathway in crop species such as tomato. It is hypothesized that NAE pathway occurs in tomato and that there is a functional FAAH that hydrolyzes NAEs.To test this hypothesis, NAE content and composition will be determined in various tissues and developmental stages of tomato by selective lipidomic analysis. Furthermore, a functional homolog of AtFAAH has been identified in tomato and will be biochemically characterized.Thus far, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. Temporal expression of SlFAAH1 and SlFAAH2 in different tissues will also be analyzed by quantitative PCR to correlate with the NAE levels. The molecular and biochemical characterization of FAAH in addition to determining the composition of NAEs in tomato will further validate the conserved nature of NAE metabolic pathway in plants.

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