Imaging plant metabolism in situ.

Mass spectrometry imaging (MSI) has emerged as an invaluable analytical technique for investigating the spatial distribution of molecules within biological systems. In the realm of plant science, MSI is increasingly employed to explore metabolic processes across a wide array of plant tissues, including those in leaves, fruits, stems, roots, and seeds, spanning various plant systems such as model species, staple and energy crops, and medicinal plants. By generating spatial maps of metabolites, MSI has elucidated the distribution patterns of diverse metabolites and phytochemicals, encompassing lipids, carbohydrates, amino acids, organic acids, phenolics, terpenes, alkaloids, vitamins, pigments, and others, thereby providing insights into their metabolic pathways and functional roles. In this review, we present recent MSI studies that demonstrate the advances made in visualizing the plant spatial metabolome. Moreover, we emphasize the technical progress that enhances the identification and interpretation of spatial metabolite maps. Within a mere decade since the inception of plant MSI studies, this robust technology is poised to continue as a vital tool for tackling complex challenges in plant metabolism.

Analysis of Pectin-derived Monosaccharides from Arabidopsis Using GC-MS.

Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes. Mutants with altered pectin composition have been reported to exhibit altered stress or pathogen resistance. Understanding the link between mutant phenotypes and their pectin composition requires robust analytical methods to detect changes in the relative monosaccharide composition. Here, we describe a quick and efficient gas chromatography-mass spectrometry (GC-MS)-based method that allows the differential analysis of pectin monosaccharide composition in plants under different conditions or between mutant plants and their respective wild types. Pectin is extracted from seed mucilage or from the alcohol-insoluble residue prepared from leaves or other organs and is subsequently hydrolysed with trifluoracetic acid. The resulting acidic and neutral monosaccharides are then derivatised and measured simultaneously by GC-MS. Key features Comparative analysis of monosaccharide content in Arabidopsis-derived pectin between different genotypes or different treatments. Procedures for two sources of pectin are shown: seed coat mucilage and alcohol-insoluble residue. Allows quick analyses of neutral and acidic monosaccharides simultaneously. Graphical overview.

Quantification of Botrytis cinerea Growth in Arabidopsis thaliana.

Yield losses attributed to plant pathogens pose a serious threat to plant productivity and food security. Botrytis cinerea is one of the most devastating plant pathogens, infecting a wide array of plant species; it has also been established as a model organism to study plant-pathogen interactions. In this context, development of different assays to follow the relative success of B. cinerea infections is required. Here, we describe two methods to quantify B. cinerea development in Arabidopsis thaliana genotypes through measurements of lesion development and quantification of fungal genomic DNA in infected tissues. This provides two independent techniques that are useful in assessing the susceptibility or tolerance of different Arabidopsis genotypes to B. cinerea. Key features Protocol for the propagation of the necrotrophic plant pathogen fungus Botrytis cinerea and spore production. Two methods of Arabidopsis thaliana infection with the pathogen using droplet and spray inoculation. Two readouts, either by measuring lesion size or by the quantification of fungal DNA using quantitative PCR. The two methods are applicable across plant species susceptible the B. cinerea. Graphical overview A simplified overview of the droplet and spray infection methods used for the determination of B. cinerea growth in different Arabidopsis genotypes.

Two legume fatty acid amide hydrolase isoforms with distinct preferences for microbial- and plant-derived acylamides.

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.

Lipid Droplets: Packing Hydrophobic Molecules Within the Aqueous Cytoplasm.

Lipid droplets, also known as oil bodies or lipid bodies, are plant organelles that compartmentalize neutral lipids as a hydrophobic matrix covered by proteins embedded in a phospholipid monolayer. Some of these proteins have been known for decades, such as oleosins, caleosins, and steroleosins, whereas a host of others have been discovered more recently with various levels of abundance on lipid droplets, depending on the tissue and developmental stage. In addition to a growing inventory of lipid droplet proteins, the subcellular machinery that contributes to the biogenesis and degradation of lipid droplets is being identified and attention is turning to more mechanistic questions regarding lipid droplet dynamics. While lipid droplets are mostly regarded as storage deposits for carbon and energy in lipid-rich plant tissues such as seeds, these organelles are present in essentially all plant cells, where they display additional functions in signaling, membrane remodeling, and the compartmentalization of a variety of hydrophobic components. Remarkable metabolic engineering efforts have demonstrated the plasticity of vegetative tissues such as leaves to synthesize and package large amounts of storage lipids, which enable future applications in bioenergy and the engineering of high-value lipophilic compounds. Here, we review the growing body of knowledge about lipid droplets in plant cells, describe the evolutionary similarity and divergence in their associated subcellular machinery, and point to gaps that deserve future attention.

Fatty acid amide hydrolase and 9-lipoxygenase modulate cotton seedling growth by ethanolamide oxylipin levels.

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.

A lipidomics platform to analyze the fatty acid compositions of non-polar and polar lipid molecular species from plant tissues: Examples from developing seeds and seedlings of pennycress (Thlaspi arvense).

The lipidome comprises the total content of molecular species of each lipid class, and is measured using the analytical techniques of lipidomics. Many liquid chromatography-mass spectrometry (LC-MS) methods have previously been described to characterize the lipidome. However, many lipidomic approaches may not fully uncover the subtleties of lipid molecular species, such as the full fatty acid (FA) composition of certain lipid classes. Here, we describe a stepwise targeted lipidomics approach to characterize the polar and non-polar lipid classes using complementary LC-MS methods. Our "polar" method measures 260 molecular species across 12 polar lipid classes, and is performed using hydrophilic interaction chromatography (HILIC) on a NH2 column to separate lipid classes by their headgroup. Our "non-polar" method measures 254 molecular species across three non-polar lipid classes, separating molecular species on their FA characteristics by reverse phase (RP) chromatography on a C30 column. Five different extraction methods were compared, with an MTBE-based extraction chosen for the final lipidomics workflow. A state-of-the-art strategy to determine and relatively quantify the FA composition of triacylglycerols is also described. This lipidomics workflow was applied to developing, mature, and germinated pennycress seeds/seedlings and found unexpected changes among several lipid molecular species. During development, diacylglycerols predominantly contained long chain length FAs, which contrasted with the very long chain FAs of triacylglycerols in mature seeds. Potential metabolic explanations are discussed. The lack of very long chain fatty acids in diacylglycerols of germinating seeds may indicate very long chain FAs, such as erucic acid, are preferentially channeled into beta-oxidation for energy production.

Effective Mechanisms for Improving Seed Oil Production in Pennycress (Thlaspi arvense L.) Highlighted by Integration of Comparative Metabolomics and Transcriptomics.

Pennycress is a potentially lucrative biofuel crop due to its high content of long-chain unsaturated fatty acids, and because it uses non-conventional pathways to achieve efficient oil production. However, metabolic engineering is required to improve pennycress oilseed content and make it an economically viable source of aviation fuel. Research is warranted to determine if further upregulation of these non-conventional pathways could improve oil production within the species even more, which would indicate these processes serve as promising metabolic engineering targets and could provide the improvement necessary for economic feasibility of this crop. To test this hypothesis, we performed a comparative biomass, metabolomic, and transcriptomic analyses between a high oil accession (HO) and low oil accession (LO) of pennycress to assess potential factors required to optimize oil content. An evident reduction in glycolysis intermediates, improved oxidative pentose phosphate pathway activity, malate accumulation in the tricarboxylic acid cycle, and an anaplerotic pathway upregulation were noted in the HO genotype. Additionally, higher levels of threonine aldolase transcripts imply a pyruvate bypass mechanism for acetyl-CoA production. Nucleotide sugar and ascorbate accumulation also were evident in HO, suggesting differential fate of associated carbon between the two genotypes. An altered transcriptome related to lipid droplet (LD) biosynthesis and stability suggests a contribution to a more tightly-packed LD arrangement in HO cotyledons. In addition to the importance of central carbon metabolism augmentation, alternative routes of carbon entry into fatty acid synthesis and modification, as well as transcriptionally modified changes in LD regulation, are key aspects of metabolism and storage associated with economically favorable phenotypes of the species.

N-acylethanolamide metabolizing enzymes are upregulated in human neural progenitor-derived neurons exposed to sub-lethal oxidative stress.

N-acyl amides (NAAs) are a class of lipids that consist of an acyl group N-linked to an amino acid, neurotransmitter, taurine or ethanolamide group (N-acylethanolamines or NAEs) and include some endocannabinoids (eCB) such as anandamide. These lipids are synthesized in a wide variety of organisms and in multiple cell types, including neurons. NAEs are involved in numerous cellular and physiological processes and their concentrations are elevated in response to ischemia and physical trauma to play a role in neuroprotection. The neuroprotective properties of eCB NAEs make the protein targets of these compounds attractive targets for clinical intervention for a variety of conditions. The most promising of these targets include cannabinoid receptor type 1 (CB1), cannabinoid receptor type 2 (CB2), fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA), and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD). Further characterization of these targets in a more contemporary model system of neurodegeneration and neuroprotection will allow us to fully describe their role and mechanism of action in neuroprotection against oxidative stress leading to better utilization in the clinical setting. Human stem cell-derived or human neural progenitor cell-derived cells, such as ReN cells, have become more utilized for the study of human neuronal development and neurodegenerative diseases. ReN cells can be easily differentiated thereby circumventing the need for using transformed cell lines and primary neurons as cell model systems. In this study, we determined whether ReN cells, a superior cell model system for studying neurodevelopment, differentiation, and neuroprotection, express proteins involved in canonical eCB NAE signaling and whether oxidative stress can induce their expression. We determined that sublethal oxidative stress upregulates the expression of all eCB proteins tested. In addition, we determined that oxidative stress increases the nuclear localization of FAAH, and to a lesser extent, NAAA and NAPE-PLD. This study is a first step toward determining how oxidative stress affects CB1, CB2, FAAH, NAAA, and NAPE-PLD expression and their potential defense against oxidative stress. As such, our data is important for further determining the role of eCB metabolizing proteins and eCB receptors against oxidative stress.

Enhanced seedling growth by 3-n-pentadecylphenolethanolamide is mediated by fatty acid amide hydrolases in upland cotton (Gossypium hirsutum L.).

Fatty acid amide hydrolase (FAAH) is a conserved amidase that is known to modulate the levels of endogenous N-acylethanolamines (NAEs) in both plants and animals. The activity of FAAH is enhanced in vitro by 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 hirsutum L. cv Coker 312) seedlings including two lines of transgenic seedlings overexpressing AtFAAH. 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 cotton FAAH genes 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.

Finding new friends and revisiting old ones - how plant lipid droplets connect with other subcellular structures.

The number of described contact sites between different subcellular compartments and structures in eukaryotic cells has increased dramatically in recent years and, as such, has substantially reinforced the well-known premise that these kinds of connections are essential for overall cellular organization and the proper functioning of cellular metabolic and signaling pathways. Here, we discuss contact sites involving plant lipid droplets (LDs), including LD-endoplasmic reticulum (ER) connections that mediate the biogenesis of new LDs at the ER, LD-peroxisome connections, that facilitate the degradation of LD-stored triacylglycerols (TAGs), and the more recently discovered LD-plasma membrane connections, which involve at least three novel proteins, but have a yet unknown physiological function(s).

Overexpression of phospholipid: diacylglycerol acyltransferase in Brassica napus results in changes in lipid metabolism and oil accumulation.

The regulation of lipid metabolism in oil seeds is still not fully understood and increasing our knowledge in this regard is of great economic, as well as intellectual, importance. Oilseed rape (Brassica napus) is a major global oil crop where increases in triacylglycerol (TAG) accumulation have been achieved by overexpression of relevant biosynthetic enzymes. In this study, we expressed Arabidopsis phospholipid: diacylglycerol acyltransferase (PDAT1), one of the two major TAG-forming plant enzymes in B. napus DH12075 to evaluate its effect on lipid metabolism in developing seeds and to estimate its flux control coefficient. Despite several-fold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG, towards less unsaturation. Mass spectrometry imaging of seed sections confirmed the shift in lipid compositions and indicated that PDAT overexpression altered the distinct heterogeneous distributions of phosphatidylcholine (PC) molecular species. Similar, but less pronounced, changes in TAG molecular species distributions were observed. Our data indicate that PDAT exerts a small, negative, flux control on TAG biosynthesis and could have under-appreciated effects in fine-tuning of B. napus seed lipid composition in a tissue-specific manner. This has important implications for efforts to increase oil accumulation in similar crops.

Transgenic manipulation of triacylglycerol biosynthetic enzymes in B. napus alters lipid-associated gene expression and lipid metabolism.

Oilseed rape (Brassica napus) is an important crop that is cultivated for the oil (mainly triacylglycerol; TAG) it produces in its seeds. TAG synthesis is controlled mainly by key enzymes in the Kennedy pathway, such as glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) but can also be produced from phosphoglycerides such as phosphatidylcholine (PC) by the activity of the enzyme phospholipid: diacylglycerol acyltransferase (PDAT). To evaluate the potential for these enzymes to alter oil yields or composition, we analysed transgenic B. napus lines which overexpressed GPAT, LPAT or PDAT using heterologous transgenes from Arabidopsis and Nasturtium and examined lipid profiles and changes in gene expression in these lines compared to WT. Distinct changes in PC and TAG abundance and spatial distribution in embryonic tissues were observed in some of the transgenic lines, together with altered expression of genes involved generally in acyl-lipid metabolism. Overall our results show that up-regulation of these key enzymes differentially affects lipid composition and distribution as well as lipid-associated gene expression, providing important information which could be used to improve crop properties by metabolic engineering.

Better together: Protein partnerships for lineage-specific oil accumulation.

Plant-derived oils are a major agricultural product that exist in both ubiquitous forms such as common vegetable oils and in specialized forms such as castor oil and coconut oil. These specialized oils are the result of lineage-specific metabolic pathways that create oils rich in unusual fatty acids. Considerable progress has been made toward understanding the enzymes that mediate fatty acid biosynthesis, triacylglycerol assembly, and oil storage. However, efforts to translate this knowledge into renewable bioproducts via engineered oil-producing plants and algae have had limited success. Here, we review recent evidence that protein-protein interactions in each of the three major phases of oil formation appear to have profound effects on specialized oil accumulation. We suggest that furthering our knowledge of the noncatalytic attributes of enzymes and other proteins involved in oil formation will be a critical step toward creating renewable bioproducts derived from high performing, engineered oilseeds.

A glossary of plant cell structures: Current insights and future questions.

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.

LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.).

Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ∼90% and ∼60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 single mutants reduced to less than 5%. MALDI-MS spatial imaging analyses of phosphatidylcholine (PC) and triacylglycerol (TAG) molecular species in wild-type pennycress embryo sections from mature seeds revealed that erucic acid is highly enriched in cotyledons which serve as storage organs, suggestive of a role in providing energy for the germinating seedling. In contrast, PUFA-containing TAGs are enriched in the embryonic axis, which may be utilized for cellular membrane expansion during seed germination and seedling emergence. Under standard growth chamber conditions, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 double mutant plants exhibited delayed growth and overall reduced heights and seed yields, suggesting that reducing PUFAs below a threshold in pennycress had negative physiological effects. Taken together, our results suggest that combinatorial knockout of ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.

Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION 7 Localizes to Lipid Droplets via Its Senescence Domain.

Lipid droplets (LDs) are neutral-lipid-containing organelles found in all kingdoms of life and are coated with proteins that carry out a vast array of functions. Compared to mammals and yeast, relatively few LD proteins have been identified in plants, particularly those associated with LDs in vegetative (non-seed) cell types. Thus, to better understand the cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. Here, we performed a proteomics analysis of LDs isolated from drought-stressed Arabidopsis leaves and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and the cytosol when ectopically expressed in plant cells, and the protein's C-terminal senescence domain (SD) was both necessary and sufficient for LD targeting. Phylogenetic analysis revealed that ERD7 belongs to a six-member family in Arabidopsis that, along with homologs in other plant species, is separated into two distinct subfamilies. Notably, the SDs of proteins from each subfamily conferred targeting to either LDs or mitochondria. Further, the SD from the ERD7 homolog in humans, spartin, localized to LDs in plant cells, similar to its localization in mammals; although, in mammalian cells, spartin also conditionally localizes to other subcellular compartments, including mitochondria. Disruption of ERD7 gene expression in Arabidopsis revealed no obvious changes in LD numbers or morphology under normal growth conditions, although this does not preclude a role for ERD7 in stress-induced LD dynamics. Consistent with this possibility, a yeast two-hybrid screen using ERD7 as bait identified numerous proteins involved in stress responses, including some that have been identified in other LD proteomes. Collectively, these observations provide new insight to ERD7 and the SD-containing family of proteins in plants and suggest that ERD7 may be involved in functional aspects of plant stress response that also include localization to the LD surface.

Isolation of Lipid Droplets for Protein and Lipid Analysis.

Cytosolic lipid droplets (LDs) are organelles which emulsify a variety of hydrophobic molecules in the aqueous cytoplasm of essentially all plant cells. Most familiar are the LDs from oilseeds or oleaginous fruits that primarily store triacylglycerols and serve a storage function. However, similar hydrophobic particles are found in cells of plant tissues that package terpenoids, sterol esters, wax esters, or other types of nonpolar lipids. The various hydrophobic lipids inside LDs are coated with a phospholipid monolayer, mostly derived from membrane phospholipids during their ontogeny. Various proteins have been identified to be associated with LDs, and these may be cell-type, tissue-type, or even species specific. While major LD proteins like oleosins have been known for decades, more recently a growing list of LD proteins has been identified, primarily by proteomics analyses of isolated LDs and confirmation of their localization by confocal microscopy. LDs, unlike other organelles, have a density less than that of water, and consequently can be isolated and enriched in cellular fractions by flotation centrifugation for composition studies. However, due to its deep coverage, modern proteomics approaches are also prone to identify contaminants, making control experiments necessary. Here, procedures for the isolation of LDs, and analysis of LD components are provided as well as methods to validate the LD localization of proteins.

In Situ Localization of Plant Lipid Metabolites by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI).

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a major analytical platform for the determination and localization of lipid metabolites directly from tissue sections. Unlike analysis of lipid extracts, where lipid localizations are lost due to homogenization and/ or solvent extraction, MALDI-MSI analysis is capable of revealing spatial localization of metabolites while simultaneously collecting high chemical resolution mass spectra. Important considerations for obtaining high quality MALDI-MS images include tissue preservation, section preparation, MS data collection and data processing. Errors in any of these steps can lead to poor quality metabolite images and increases the chance for metabolite misidentification and/ or incorrect localization. Here, we present detailed methods and recommendations for specimen preparation, MALDI-MS instrument parameters, software analysis platforms for data processing, and practical considerations for each of these steps to ensure acquisition of high-quality chemical and spatial resolution data for reconstructing MALDI-MS images of plant tissues.