Lipidomic Analysis of Endocannabinoid Signaling: Targeted Metabolite Identification and Quantification

This article reviews several methodologies to quantify endocannabinoids in various biological systems

N-Palmitoylethanolamine depot injection increased its tissue levels and those of other acylethanolamide lipids

Article discussing how the N-palmitoylethanolamine depot injection increased its tissue levels and those of other acylethanolamide lipids

Overexpression of Fatty Acid Amide Hydrolase Induces Early Flowering in Arabidopsis thaliana

N-Acylethanolamines (NAEs) are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE). In animal systems this reaction is part of the “endocannabinoid” signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH), which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440) lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA), enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD) and inductive long day (LD) conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT) gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3), were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways

Metabolism and action of polyunsaturated n-acylethanolamines in Arabidopsis thaliana seedlings

The lipoxygenase (LOX) pathway plays an important role in the oxidative metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). The LOX pathway functions in conjugation with hydrolysis by fatty acid amide hydrolase (FAAH) and to produce oxidized NAEs during seed germination and early seedling development. When Arabidopsis seedlings were grown in low micromolar concentrations of lauroylethanolamide (NAE 12:0), growth retardation and elevated endogenous PU-NAE levels were observed due to the competitive inhibition of LOX by NAE 12:0. The elevated levels of endogenous PU-NAEs were more pronounced in genotypes with reduced NAE hydrolase capacity (faah knockouts), and less evident with overexpression of FAAH. Alterations in PU-NAE metabolism were studied in seedlings of various lox and FAAH mutants. The partitioning of PU-NAEs into oxylipin metabolites was exaggerated in the presence of exogenous linolenoylethanolamide (NAE18:3) and resulted in bleaching of cotyledons. The bleaching phenotype was restricted to a narrow developmental window (3-to-5 days after sowing), and was attributed to a reversible disruption of thylakoid membranes in chloroplasts. Biochemical and genetic evidence suggested that 9-hydro(pero)xy and 13-hydro(pero)xy octadecatrienoylethanolamides (9- and 13-NAE-H(P)OT), but not their corresponding hydro(pero)xy free fatty acids, induced cotyledon bleaching. The LOX-mediated metabolites of NAE18:3 shared some overlapping effects on seedling development with those of linoleoylethanolamide (NAE18:2) such as a reduction in seedling root growth. On the other hand, NAE18:3 oxylipin metabolites also exhibited distinct effects during seedling development such as the inhibition of photomorphogenesis. Biochemical and genetic evidence indicated that a LOXmediated metabolite of NAE18:2, 9-hydro(pero)xy octadecadienoylethanolamide (9-NAEH( P)OD), acted as a potent negative regulator of seedling root development, and this depended on an intact abscisic acid (ABA) signaling pathway. Synergistic inhibition of root elongation between 9-NAE-H(P)OD and ABA was restricted to a narrow developmental window (3-to-5 d after sowing) of seedling development. Genetic evidence with Arabidopsis mutants in ABA synthesis (aba1, aba2), perception (pyr1, pyl2, pyl4, pyl5, pyl8) and transcriptional regulation (abi3-1) suggested that negative regulation of growth by 9-NAE-H(P)OD likely was mediated through an increase in ABA synthesis, and this was confirmed biochemically. Induction of a secondary dormancy program in Arabidopsis seedlings by environmental stresses also requires an intact ABA signaling cascade, and our study has shown that this regulatory seedling program is dependent, in large part, on NAE oxylipin formation. Together, results presented here indicated that LOX-mediated metabolites of NAE18:3 and NAE18:2 in Arabidopsis represent a newlydiscovered group of bioactive metabolites, and their accumulation during the embryo-toseedling transition of plant development may act to synchronize seedling establishment with environmental cues

Expression of a Bacterial Trehalose-6-phosphate Synthase otsA Increases Oil Accumulation in Plant Seeds and Vegetative Tissues

We previously demonstrated that exogenous trehalose 6-phosphate (T6P) treatment stabilized WRINKLED1 (WRI1), a master transcriptional regulator of fatty acid (FA) synthesis and increased total FA content in Brassica napus (B. napus) embryo suspension cell culture. Here, we explore Arabidopsis lines heterologously expressing the Escherichia coli T6P synthase (otsA) or T6P phosphatase (otsB) to refine our understanding regarding the role of T6P in regulating fatty acid synthesis both in seeds and vegetative tissues. Arabidopsis 35S:otsA transgenic seeds showed an increase of 13 in fatty acid content compared to those of wild type (WT), while seeds of 35:otsB transgenic seeds showed a reduction of 12 in fatty acid content compared to WT. Expression of otsB significantly reduced the level of WRI1 and expression of its target genes in developing seeds. Like Arabidopsis seeds constitutively expressing otsA, transient expression of otsA in Nicotiana benthamiana leaves resulted in strongly elevated levels of T6P. This was accompanied by an increase of 29 in de novo fatty acid synthesis rate, a 2.3-fold increase in triacylglycerol (TAG) and a 20 increase in total fatty acid content relative to empty vector (EV) controls. Taken together, these data support the heterologous expression of otsA as an approach to increasing TAG accumulation in plant seeds and vegetative tissues

N-Acylethanolamines: Lipid Metabolites with Functions in Plant Growth and Development

Twenty years ago, N‐acylethanolamines (NAEs) were considered by many lipid chemists to be biological ‘artifacts’ of tissue damage, and were, at best, thought to be minor lipohilic constituents of various organisms. However, that changed dramatically in 1993, when anandamide, an NAE of arachidonic acid (N‐arachidonylethanolamine), was shown to bind to the human cannabinoid receptor (CB1) and activate intracellular signal cascades in mammalian neurons. Now NAEs of various types have been identified in diverse multicellular organisms, in which they display profound biological effects. Although targets of NAEs are still being uncovered, and probably vary among eukaryotic species, there appears to be remarkable conservation of the machinery that metabolizes these bioactive fatty acid conjugates of ethanolamine. This review focuses on the metabolism and functions of NAEs in higher plants, with specific reference to the formation, hydrolysis and oxidation of these potent lipid mediators. The discussion centers mostly on early seedling growth and development, for which NAE metabolism has received the most attention, but also considers other areas of plant development in which NAE metabolism has been implicated. Where appropriate, we indicate cross‐kingdom conservation in NAE metabolic pathways and metabolites, and suggest areas where opportunities for further investigation appear most pressing

Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry

In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol