Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid.

The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.

Rhamnolipids and fengycins, very promising amphiphilic antifungal compounds from bacteria secretomes, act on Sclerotiniaceae fungi through different mechanisms.

Rhamnolipids (RLs) and fengycins (FGs) are amphiphilic lipid compounds from bacteria secretomes proposed to replace synthetic pesticides for crop protection. They both display plant defense triggering properties and direct antimicrobial activities. In particular, they have well reported antifungal effects against phytopathogenic fungi. RLs and FGs are considered to act through a direct interaction with membrane lipids and a destabilization of microorganism plasma membrane, thereby limiting the risk of resistance emergence. The main objective of this work was to gain insights in the antimycelial mode of action of these metabolites to promote them as environment and human health friendly biocontrol solutions. Their biocidal effects were studied on two Sclerotiniaceae fungi responsible for diseases in numerous plant species worldwide. We show here that different strains of Botrytis cinerea and Sclerotinia sclerotiorum have opposite sensitivities to RLs and FGs on plate experiments. Overall, B. cinerea is more sensitive to FGs while S. sclerotiorum is more sensitive to RLs. Electron microscopy observations demonstrated that RLs induce mycelial destructuring by asperities emergence and hyphal fusions whereas FGs promote swelling and formation of vesicle-like structures due to vacuole fusions and autophagy. Permeability studies, phosphatidylserine externalization and reactive oxygen species production assessments showed a programmed cell death triggering by RLs at medium concentrations (until 50 µg mL-1) and necrosis characteristics at higher concentration. Programmed cell death was always observed on hyphae treated with FGs. Quantifications of mycelial ergosterol content indicated that a higher ergosterol rate in S. sclerotiorum correlates with increasing sensitivity to RLs. Oppositely, a lower ergosterol rate in B. cinerea correlates with increasing sensitivity to FGs, which was confirmed by ergosterol biosynthesis inhibition with tebuconazole. This gain of knowledge will help to better understand the mode of action of RLs and FGs to fight specific plant fungal diseases.

Characterization and impact of sunflower plastidial octanoyltransferases (Helianthus annuus L.) on oil composition.

Prosthetic lipoyl groups are essential for the metabolic activity of several multienzyme complexes in most organisms. In plants, octanoyltransferase (LIP2) and lipoyl synthase (LIP1) enzymes in the mitochondria and plastids participate in the de novo synthesis of lipoic acid, and in the attachment of the lipoyl cofactors to their specific targets. In plastids, the lipoylated pyruvate dehydrogenase complex catalyzes the synthesis of the acetyl-CoA that is required for de novo fatty acid synthesis. Since lipoic acid transport across plastid membranes has not been demonstrated, these organelles require specific plastidial LIP1 and LIP2 activities for the in situ synthesis of this cofactor. Previously, one essential LIP1 enzyme and two redundant LIP2 enzymes have been identified within Arabidopsis chloroplasts. In this study, two plastidial sunflower (Helianthus annuus L.) LIP2 genes (HaLIP2p1 and HaLIP2p2) were identified, cloned and characterized. The expression of these genes in different tissues was studied and the tertiary structure of the peptides they encode was modeled by protein docking. These genes were overexpressed in Escherichia coli and their impact on bacterial fatty acid synthesis was studied. Finally, transgenic Arabidopsis plants overexpressing HaLIP2p1 were generated and their seed lipid profiles analyzed. The lipid composition of the transgenic seeds, particularly their TAG species, differed from that of wild-type plants, revealing a relationship between lipoic acid synthesis and the accumulation of storage lipids in Arabidopsis seeds.

Lifespan prolonging mechanisms and insulin upregulation without fat accumulation in long-lived reproductives of a higher termite.

Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it.

Characterization of Helianthus annuus Lipoic Acid Biosynthesis: The Mitochondrial Octanoyltransferase and Lipoyl Synthase Enzyme System.

Lipoic acid (LA, 6,8-dithiooctanoic acid) is a sulfur containing coenzyme essential for the activity of several key enzymes involved in oxidative and single carbon metabolism in most bacteria and eukaryotes. LA is synthetized by the concerted activity of the octanoyltransferase (LIP2, EC 2.3.1.181) and lipoyl synthase (LIP1, EC 2.8.1.8) enzymes. In plants, pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase or glycine decarboxylase are essential complexes that need to be lipoylated. These lipoylated enzymes and complexes are located in the mitochondria, while PDH is also present in plastids where it provides acetyl-CoA for de novo fatty acid biosynthesis. As such, lipoylation of PDH could regulate fatty acid synthesis in both these organelles. In the present work, the sunflower LIP1 and LIP2 genes (HaLIP1m and HaLIP2m) were isolated sequenced, cloned, and characterized, evaluating their putative mitochondrial location. The expression of these genes was studied in different tissues and protein docking was modeled. The genes were also expressed in Escherichia coli and Arabidopsis thaliana, where their impact on fatty acid and glycerolipid composition was assessed. Lipidomic studies in Arabidopsis revealed lipid remodeling in lines overexpressing these enzymes and the involvement of both sunflower proteins in the phenotypes observed is discussed in the light of the results obtained.

Colistin Treatment Affects Lipid Composition of Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii (A. baumannii) causes severe and often fatal healthcare-associated infections due partly to antibiotic resistance. There are no studies on A. baumannii lipidomics of susceptible and resistant strains grown at lethal and sublethal concentrations. Therefore, we analyzed the impact of colistin resistance on glycerolipids' content by using untargeted lipidomics on clinical isolate. Nine lipid sub-classes were annotated, including phosphatidylcholine, rarely detected in the bacterial membrane among 130 different lipid species. The other lipid sub-classes detected are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), lysophosphatidylethanolamine, hemibismonoacylglycerophosphate, cardiolipin, monolysocardiolipin, diacylglycerol, and triacylglycerol. Under lethal and sublethal concentrations of colistin, significant reduction of PE was observed on the resistant and susceptible strain, respectively. Palmitic acid percentage was higher at colistin at low concentration but only for the susceptible strain. When looking at individual lipid species, the most abundant PE and PG species (PE 34:1 and PG 34:1) are significantly upregulated when the susceptible and the resistant strains are cultivated with colistin. This is, to date, the most exhaustive lipidomics data compilation of A. baumannii cultivated in the presence of colistin. This work is highlighting the plasma membrane plasticity used by this gram-negative bacterium to survive colistin treatment.

The Impact of Plasma Membrane Lipid Composition on Flagellum-Mediated Adhesion of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell's plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions.IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

13C-Metabolic Flux Analysis in Developing Flax (Linum usitatissinum L.) Embryos to Understand Storage Lipid Biosynthesis.

Flax (Linum usitatissinum L.) oil is an important source of α-linolenic (C18:3 ω-3). This polyunsaturated fatty acid is well known for its nutritional role in human and animal diets. Understanding storage lipid biosynthesis in developing flax embryos can lead to an increase in seed yield via marker-assisted selection. While a tremendous amount of work has been done on different plant species to highlight their metabolism during embryo development, a comprehensive analysis of metabolic flux in flax is still lacking. In this context, we have utilized in vitro cultured developing embryos of flax and determined net fluxes by performing three complementary parallel labeling experiments with 13C-labeled glucose and glutamine. Metabolic fluxes were estimated by computer-aided modeling of the central metabolic network including 11 cofactors of 118 reactions of the central metabolism and 12 pseudo-fluxes. A focus on lipid storage biosynthesis and the associated pathways was done in comparison with rapeseed, arabidopsis, maize and sunflower embryos. In our hands, glucose was determined to be the main source of carbon in flax embryos, leading to the conversion of phosphoenolpyruvate to pyruvate. The oxidative pentose phosphate pathway (OPPP) was identified as the producer of NADPH for fatty acid biosynthesis. Overall, the use of 13C-metabolic flux analysis provided new insights into the flax embryo metabolic processes involved in storage lipid biosynthesis. The elucidation of the metabolic network of this important crop plant reinforces the relevance of the application of this technique to the analysis of complex plant metabolic systems.

Lipidomic Analysis of Plastidial Octanoyltransferase Mutants of Arabidopsis thaliana.

Plant de novo fatty acid synthesis takes place in the plastid using acetyl-coenzyme A (acetyl-CoA) as the main precursor. This first intermediate is produced from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO2, and NADH. For the proper functioning of this complex, lipoic acid is required to be bound to the dihydrolipoamide S-acetyltransferase E2 subunit of PDH. Octanoyltransferase (LIP2; EC 2.3.1.181) and lipoyl synthase (LIP1; EC 2.8.1.8) are the enzymes involved in the biosynthesis of this essential cofactor. In Arabidopsis plastids, an essential lipoyl synthase (AtLIP1p) and two redundant octanoyltransferases (AtLIP2p1 and AtLIP2p2) have been described. In the present study, the lipidomic characterization of Arabidopsis octanoyltransferase mutants reveals new insight into the lipoylation functions within plastid metabolism. Lipids and fatty acids from mature seeds and seedlings from Atlip2p1 and Atlip2p2 mutants were analysed by gas chromatography (GC) and liquid chromatography-electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS2), the analysis revealed changes in fatty acid profiles that showed similar patterns in both mutant seeds and seedlings and in the lipid species containing those fatty acids. Although both mutants showed similar tendencies, the lack of the AtLIP2p2 isoform produced a more acute variation in its lipids profile. These changes in fatty acid composition and the increase in their content per seed point to the interference of octanoyltransferases in the fatty acid synthesis flux in Arabidopsis thaliana seeds.

Untargeted Analysis of Semipolar Compounds by LC-MS and Targeted Analysis of Fatty Acids by GC-MS/GC-FID: From Plant Cultivation to Extract Preparation.

The way plants are grown and samples are harvested, prepared, and extracted has a profound impact on the output of a metabolomics experiment. In this chapter, we detail the experimental procedures from plant cultivation to extract preparation, in order to avoid difficulties that could result in contamination or undesired changes of the analytes. Two plant organs are mentioned as examples: tomato fruits (Solanum lycopersicum) and flax seeds (Linum usitatissimum). Extractions designed for the untargeted analysis of semipolar compounds by liquid chromatography-mass spectrometry (LC-MS) and targeted analysis of fatty acids by gas chromatography-mass spectrometry (GC-MS) or gas chromatography with flame ionization detector (GC-FID) are described.

Analysis of 13C labeling amino acids by capillary electrophoresis - High resolution mass spectrometry in developing flaxseed.

In context of fluxomic studies, 13C labeling analysis of amino acids are very important for solving the carbon flow calculation, because they are synthesized in various biosynthesis pathways and cellular compartments in plant cells. Traditionally, 13C labeling analysis are performed using low resolution mass spectrometry detector by GC-MS. We compared a method using capillary electrophoresis-high resolution mass spectrometry without derivatization and with better accuracy assessment of labeling measurements comparing to classical GC-MS. Our method allowed us to show that valine, leucine, alanine are not synthesized from the same pyruvate pool during the period of reserves accumulation in flax seeds.

Glycerolipid analysis during desiccation and recovery of the resurrection plant Xerophyta humilis (Bak) Dur and Schinz.

Xerophyta humilis is a poikilochlorophyllous monocot resurrection plant used as a model to study vegetative desiccation tolerance. Dehydration imposes tension and ultimate loss of integrity of membranes in desiccation sensitive species. We investigated the predominant molecular species of glycerolipids present in root and leaf tissues, using multiple reaction monitoring mass spectrometry, and then analysed changes therein during dehydration and subsequent rehydration of whole plants. The presence of fatty acids with long carbon chains and with odd numbers of carbons were detected and confirmed by gas chromatography. Dehydration of both leaves and roots resulted in an increase in species containing polyunsaturated fatty acids and a decrease in disaturated species. Upon rehydration, lipid saturation was reversed, with this being initiated immediately upon watering in roots but only 12-24 hr later in leaves. Relative levels of species with short-chained odd-numbered saturated fatty acids decreased during dehydration and increased during rehydration, whereas the reverse trend was observed for long-chained fatty acids. X. humilis has a unique lipid composition, this report being one of the few to demonstrate the presence of odd-numbered fatty acids in plant phosphoglycerolipids.

Data documenting the comparison between the theoretically expected values of free sugars mass isotopomer composition with standards using GC-MS and LC-HRMS for Metabolic Flux Analysis.

The data presented in this article are related to the research article entitled "13C labeling analysis of sugars by high resolution-mass spectrometry for Metabolic Flux Analysis" (Acket et al., 2017) [1]. This article provides data concerning the comparison between the theoretically expected values of free sugars mass isotopomer composition with standards using our previous methods using low resolution mass spectrometry by GC-MS (Koubaa et al., 2012, 2014) [2,3], and your new method using high resolution-mass spectrometry (LC-HRMS) for Metabolic Flux Analysis [1]. For discussion and a more comprehensive data interpretation and analysis, please refer to Acket et al. (2017) [1].

13C labeling analysis of sugars by high resolution-mass spectrometry for metabolic flux analysis.

Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography-high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].

Cell wall compositional modifications of Miscanthus ecotypes in response to cold acclimation.

Miscanthus, a potential energy crop grass, can be damaged by late frost when shoots emerge too early in the spring and during the first winter after planting. The effects of cold acclimation on cell wall composition were investigated in a frost-sensitive clone of Miscanthus x giganteus compared to frost-tolerant clone, Miscanthus sinensis August Feder, and an intermediate frost-tolerant clone, M. sinensis Goliath. Cellulose and lignin contents were higher in M. x giganteus than in the M. sinensis genotypes. In ambient temperature controls, each clone displayed different glucuronoarabinoxylan (GAX) contents and degree of arabinose substitution on the xylan backbone. During cold acclimation, an increase in (1→3),(1→4)-ß-D-glucan content was observed in all genotypes. Uronic acid level increased in the frost sensitive genotype but decreased in the frost tolerant genotypes in response to cold. In all clones, major changes in cell wall composition were observed with modifications in phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) activities in both non- and cold-acclimated experiments. A large increase in CAD activity under cold stress was displayed in each clone, but it was largest in the frost-tolerant clone, M. sinensis August Feder. The marked increase in PAL activity observed in the frost-tolerant clones under cold acclimation, suggests a reorientation of the products towards the phenylpropanoid pathway or aromatic synthesis. How changes in cell wall physical properties can impact frost tolerance is discussed.