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Abstract Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, perhaps best known for inactivating N -acylethanolamine lipid mediators. However, FAAH enzymes hydrolyze a wide range of acylamide substrates. Analysis of FAAHs from multiple angiosperm species revealed two conserved phylogenetic groups that differed in key conserved residues in the substrate binding pocket. While the foundation group of plant FAAHs, designated FAAH1, has been studied at the structural and functional level in Arabidopsis thaliana , nothing is known about FAAH2 members. Here, we combined computational and biochemical approaches to compare the structural and enzymatic properties of two FAAH isoforms in the legume Medicago truncatula designated MtFAAH1 and MtFAAH2a. Differences in structural and physicochemical properties of the substrate binding pockets, predicted from homology modeling, molecular docking, and molecular dynamic simulation experiments, suggested that these two FAAH isoforms would exhibit differences in their amidohydrolase activity profiles. Indeed, kinetic studies of purified, recombinant MtFAAHs indicated a reciprocal preference for acylamide substrates with MtFAAH1 more efficiently utilizing long-chain acylamides, and MtFAAH2a more efficiently hydrolyzing short-chain and aromatic acylamides. This first report of the enzymatic behavior of two phylogenetically distinct plant FAAHs will provide a foundation for further investigations regarding FAAH isoforms in legumes and other plant species. 

Abstract Polyunsaturated N-acylethanolamines (NAEs) can be hydrolyzed by fatty acid amide hydrolase (FAAH) or oxidized by lipoxygenase (LOX). In Arabidopsis (Arabidopsis thaliana), the 9-LOX product of linoleoylethanolamide, namely, 9-hydroxy linoleoylethanolamide (9-NAE-HOD), is reported to negatively regulate seedling development during secondary dormancy. In upland cotton (Gossypium hirsutum L.), six putative FAAH genes (from two diverged groups) and six potential 9-LOX genes are present; however, their involvement in 9-NAE-HOD metabolism and its regulation of seedling development remain unexplored. Here, we report that in cotton plants, two specific FAAH isoforms (GhFAAH Ib and GhFAAH IIb) are needed for hydrolysis of certain endogenous NAEs. Virus-induced gene silencing (VIGS) of either or both FAAHs led to reduced seedling growth and this coincided with reduced amidohydrolase activities and elevated quantities of endogenous 9-NAE-HOD. Transcripts of GhLOX21 were consistently elevated in FAAH-silenced tissues, and co-silencing of GhLOX21 and GhFAAH (Ib and/or IIb) led to reversal of seedling growth to normal levels (comparable with no silencing). This was concomitant with reductions in the levels of 9-NAE-HOD, but not of 13-NAE-HOD. Pharmacological experiments corroborated the genetic and biochemical evidence, demonstrating that direct application of 9-NAE-HOD, but not 13-NAE-HOD or their corresponding free fatty acid oxylipins, inhibited the growth of cotton seedlings. Additionally, VIGS of GhLOX21 in cotton lines overexpressing AtFAAH exhibited enhanced growth and no detectable 9-NAE-HOD. Altogether, we conclude that the growth of cotton seedlings involves fine-tuning of 9-NAE-HOD levels via FAAH-mediated hydrolysis and LOX-mediated production, expanding the mechanistic understanding of plant growth modulation by NAE oxylipins to a perennial crop species. 

The combination of 13C-isotopic labeling and mass spectrometry imaging (MSI) offers an approach to analyze metabolic flux in situ. However, combining isotopic labeling and MSI presents technical challenges ranging from sample preparation, label incorporation, data collection, and analysis. Isotopic labeling and MSI individually create large, complex data sets, and this is compounded when both methods are combined. Therefore, analyzing isotopically labeled MSI data requires streamlined procedures to support biologically meaningful interpretations. Using currently available software and techniques, here we describe a workflow to analyze 13C-labeled isotopologues of the membrane lipid and storage oil lipid intermediate―phosphatidylcholine (PC). Our results with embryos of the oilseed crops, Camelina sativa and Thlaspi arvense (pennycress), demonstrated greater 13C-isotopic labeling in the cotyledons of developing embryos compared with the embryonic axis. Greater isotopic enrichment in PC molecular species with more saturated and longer chain fatty acids suggest different flux patterns related to fatty acid desaturation and elongation pathways. The ability to evaluate MSI data of isotopically labeled plant embryos will facilitate the potential to investigate spatial aspects of metabolic flux in situ. 

Abstract Fatty acid amide hydrolase (FAAH) is a conserved amidase that is known to modulate the levels of endogenousN‐acylethanolamines (NAEs) in both plants and animals. The activity of FAAH is enhancedin vitroby synthetic phenoxyacylethanolamides resulting in greater hydrolysis of NAEs. Previously, 3‐n‐pentadecylphenolethanolamide (PDP‐EA) was shown to exert positive effects on the development of Arabidopsis seedlings by enhancing Arabidopsis FAAH (AtFAAH) activity. However, there is little information regarding FAAH activity and the impact of PDP‐EA in the development of seedlings of other plant species. Here, we examined the effects of PDP‐EA on growth of upland cotton (Gossypium hirsutumL. cv Coker 312) seedlings including two lines of transgenic seedlings overexpressingAtFAAH. Independent transgenic events showed accelerated true‐leaf emergence compared with non‐transgenic controls. Exogenous applications of PDP‐EA led to increases in overall seedling growth in AtFAAH transgenic lines. These enhanced‐growth phenotypes coincided with elevated FAAH activities toward NAEs and NAE oxylipins. Conversely, the endogenous contents of NAEs and NAE‐oxylipin species, especially linoleoylethanolamide and 9‐hydroxy linoleoylethanolamide, were lower in PDP‐EA treated seedlings than in controls. Further, transcripts for endogenous cottonFAAHgenes were increased following PDP‐EA exposure. Collectively, our data corroborate that the enhancement of FAAH enzyme activity by PDP‐EA stimulates NAE‐hydrolysis and that this results in enhanced growth in seedlings of a perennial crop species, extending the role of NAE metabolism in seedling development beyond the model annual plant species,Arabidopsis thaliana. 

Abstract In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.