Storage duration of human blood samples for fatty acid concentration analyses - How long is too long?

Polyunsaturated fatty acids such as DHA have known anti-inflammatory properties. The therapeutic implication highlights the importance of accurate serum measurements. Sample preservation is challenging when performed parallel to the clinical obligations. Impact of time between sample collection and processing regarding concentration alterations of fatty acids in human blood remains to be elucidated. Therefore, more information is required with respect to the stability and storage options in the context of potential degradation and concentration changes. This study investigates the stability of DHA in serum samples over time, given the challenges of timely sample analysis in clinical settings. Blood samples from three patients were collected and stored at +4 °C. Concentrations were analysed between 6 h and 7 days post-collection. Our data indicate that DHA concentrations remained unchanged during the observational period. Our results suggest that storage duration up to 7 days before sample processing does not affect accuracy of the results. DHA measurements is crucial for ongoing and future research in cardiovascular and inflammatory diseases. Our results reveal that DHA stability remains consistent over one week. This information is important for further clinical studies investigating PUFA concentrations, providing researches the option to postpone processing of samples if required along the clinical obligations.

Degradation of FATTY ACID EXPORT PROTEIN1 by RHOMBOID-LIKE PROTEASE11 contributes to cold tolerance in Arabidopsis.

Plants need to acclimate to different stresses to optimize growth under unfavorable conditions. In Arabidopsis (Arabidopsis thaliana), the abundance of the chloroplast envelope protein FATTY ACID EXPORT PROTEIN1 (FAX1) decreases after the onset of low temperatures. However, how FAX1 degradation occurs and whether altered FAX1 abundance contributes to cold tolerance in plants remains unclear. The rapid cold-induced increase in RHOMBOID-LIKE PROTEASE11 (RBL11) transcript levels, the physical interaction of RBL11 with FAX1, the specific FAX1 degradation after RBL11 expression, and the absence of cold-induced FAX1 degradation in rbl11 loss-of-function mutants suggest that this enzyme is responsible for FAX1 degradation. Proteomic analyses showed that rbl11 mutants have higher levels of FAX1 and other proteins involved in membrane lipid homeostasis, suggesting that RBL11 is a key element in the remodeling of membrane properties during cold conditions. Consequently, in the cold, rbl11 mutants show a shift in lipid biosynthesis towards the eukaryotic pathway, which coincides with impaired cold tolerance. To test whether cold sensitivity is due to increased FAX1 levels, we analyzed FAX1 overexpressors. The rbl11 mutants and FAX1 overexpressor lines show superimposable phenotypic defects upon exposure to cold temperatures. Our re-sults show that the cold-induced degradation of FAX1 by RBL11 is critical for Arabidop-sis to survive cold and freezing periods.

Tocopherol and phylloquinone biosynthesis in chloroplasts requires the phytol kinase VITAMIN E PATHWAY GENE5 (VTE5) and the farnesol kinase (FOLK).

Chlorophyll degradation causes the release of phytol, which is converted into phytyl diphosphate (phytyl-PP) by phytol kinase (VITAMIN E PATHWAY GENE5 [VTE5]) and phytyl phosphate (phytyl-P) kinase (VTE6). The kinase pathway is important for tocopherol synthesis, as the Arabidopsis (Arabidopsis thaliana) vte5 mutant contains reduced levels of tocopherol. Arabidopsis harbors one paralog of VTE5, farnesol kinase (FOLK) involved in farnesol phosphorylation. Here, we demonstrate that VTE5 and FOLK harbor kinase activities for phytol, geranylgeraniol, and farnesol with different specificities. While the tocopherol content of the folk mutant is unchanged, vte5-2 folk plants completely lack tocopherol. Tocopherol deficiency in vte5-2 plants can be complemented by overexpression of FOLK, indicating that FOLK is an authentic gene of tocopherol synthesis. The vte5-2 folk plants contain only ∼40% of wild-type amounts of phylloquinone, demonstrating that VTE5 and FOLK both contribute in part to phylloquinone synthesis. Tocotrienol and menaquinone-4 were produced in vte5-2 folk plants after supplementation with homogentisate or 1,4-dihydroxy-2-naphthoic acid, respectively, indicating that their synthesis is independent of the VTE5/FOLK pathway. These results show that phytyl moieties for tocopherol synthesis are completely but, for phylloquinone production, only partially derived from geranylgeranyl-chlorophyll and phytol phosphorylation by VTE5 and FOLK.

Analysis of circulating ceramides and hexosylceramides in patients with coronary artery disease and type II diabetes mellitus.

BACKGROUND: Cardiovascular disease (CVD) remains the leading cause of death worldwide. The main driving force behind this association is coronary artery disease (CAD), the manifestation of atherosclerosis in the coronary circulation. Cornerstones in the development of CAD are pathologies in lipid metabolism. In recent years, ongoing research has identified ceramides, a subclass of sphingolipids to be mediators of CVD. The aim of this study is to investigate the influence of type II diabetes mellitus (DM) on circulating ceramides and hexosylceramides (HexCers) in CAD patients. METHODS: 24 patients aged 40-90 years with CAD confirmed by angiography were included into a pilot study. Patients with DM were identified by analysis of discharge letters or other medical documents available at the study center. During coronary angiography, arterial blood samples were collected and quantification of sphingolipids in patient serum was performed by mass spectrometry. RESULTS: Statistical analysis showed nine significantly different HexCers in CAD patients with DM compared to patients without DM. Among the nine significantly regulated HexCers, we identified seven d18:1 HexCers. This group contributes to the fourth most abundant subgroup of total ceramides and HexCers in this dataset. HexCer-d18:1-23:1(2-OH) showed the strongest downregulation in the patient group with DM. CONCLUSION: This study suggests that levels of circulating HexCers are downregulated in patients with CAD and concomitant DM compared to patients without DM. Further research is needed to investigate the underlying mechanisms and the suitability of HexCers as possible mediators and/or prognostic markers in CAD.

Analysis of isoprenyl-phosphates by liquid chromatography-mass spectrometry.

Isoprenoids in plants are synthesized following the plastidial methylerythritol-4-phosphate (MEP) pathway or the mevalonate pathway localized to the cytosol and peroxisomes. Isoprenyl-diphosphates (isoprenyl-PP) are important intermediates for the synthesis of chlorophyll, carotenoids, sterols, and other isoprenoids in plants. The quantification of isoprenyl-PP is challenging due to the amphipathic structure, the low abundance, and the susceptibility to hydrolysis during extraction and storage. Different methods for the measurement of isoprenyl-phosphates have been developed. Isoprenyl-phosphates can be measured after radioactive labeling or after derivatization. Liquid chromatography-mass spectrometry (LC-MS) methods provide enhanced sensitivity, but still require the extraction from large amounts of sample material. In the protocol presented here, the monophosphates and diphosphates of farnesol, geranylgeraniol and phytol are isolated from plant material with an isopropanol-containing buffer and quantified by LC-MS using citronellyl-P and citronellyl-PP as internal standards. With a low limit of detection for phytyl-P, geranylgeranyl-P, phytyl-PP, and geranylgeranyl-PP, isoprenyl-phosphates can be accurately measured in Arabidopsis leaves or seeds starting with only 20mg of fresh weight.

Lack of a protective effect of the Tmem106b "protective SNP" in the Grn knockout mouse model for frontotemporal lobar degeneration.

Genetic variants in TMEM106B are a common risk factor for frontotemporal lobar degeneration and the most important modifier of disease risk in patients with progranulin (GRN) mutations (FTLD-GRN). TMEM106B is encoding a lysosomal transmembrane protein of unknown molecular function. How it mediates its disease-modifying function remains enigmatic. Several TMEM106B single nucleotide polymorphisms (SNPs) are significantly associated with disease risk in FTLD-GRN carriers, of which all except one are within intronic sequences of TMEM106B. Of note, the non-coding SNPs are in high linkage disequilibrium with the coding SNP rs3173615 located in exon six of TMEM106B, resulting in a threonine to serine change at amino acid 185 in the minor allele, which is protective in FTLD-GRN carriers. To investigate the functional consequences of this variant in vivo, we generated and characterized a knockin mouse model harboring the Tmem106bT186S variant. We analyzed the effect of this protective variant on FTLD pathology by crossing Tmem106bT186S mice with Grn-/- knockout mice, a model for GRN-mediated FTLD. We did not observe the amelioration of any of the investigated Grn-/- knockout phenotypes, including transcriptomic changes, lipid alterations, or microgliosis in Tmem106bT186S/T186S × Grn-/- mice, indicating that the Tmem106bT186S variant is not protective in the Grn-/- knockout mouse model. These data suggest that effects of the associated SNPs not directly linked to the amino acid exchange in TMEM106B are critical for the modifying effect.

2-Hydroxy-phytanoyl-CoA lyase (AtHPCL) is involved in phytol metabolism in Arabidopsis.

During chlorophyll degradation, large amounts of the isoprenoid alcohol phytol are released. The pathway of phytol catabolism has been studied in humans, because chlorophyll is part of the human diet, but little is known for plants. In humans, phytanoyl-CoA derived from phytol is degraded via α-oxidation by phytanoyl-CoA hydroxylase (PAHX) and 2-hydroxy-phytanoyl-CoA lyase (HPCL). Arabidopsis contains two sequences homologous to the human proteins AtPAHX and AtHPCL. Insertional mutants of Arabidopsis (pahx, hpcl) were grown under N deprivation to stimulate chlorophyll breakdown or supplemented with phytol to increase the endogenous amount of phytol. During N deprivation, chlorophyll, phytol, phytenal, upstream metabolites of phytol breakdown, and tocopherol and fatty acid phytyl esters, alternative phytol-derived lipids, accumulated in pahx and hpcl mutants, in line with the scenario that the mutations interfere with phytol degradation. AtHPCL was localized to the peroxisomes. Expression analysis of the AtHPCL sequence in the yeast Δpxp1 or Δmpo1 mutants followed by supplementation with 2-hydroxy-palmitic acid and enzyme assays of peroxisomal proteins from Col-0 and hpcl plants with 2-hydroxy-stearoyl-CoA revealed that AtHPCL harbors 2-hydroxy-acyl-CoA lyase activity. The α-dioxygenases αDOX1 and αDOX2 are involved in α-oxidation of fatty acids and could be involved in an alternative pathway of phytol degradation. However, phytol-related lipids in the αdox1, αdox2, or αdox1 αdox2 mutants were not altered compared with Col-0, indicating that αDOX1 and αDOX2 are not involved in phytol degradation. These results demonstrate that phytol degradation in Arabidopsis involves α-oxidation by AtPAHX and AtHPCL, but that it is independent of αDOX1/αDOX2.

Activation of neutral sphingomyelinase 2 through hyperglycemia contributes to endothelial apoptosis via vesicle-bound intercellular transfer of ceramides.

BACKGROUND: Pro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development. Local cellular ceramide accumulation mediates endothelial apoptosis, especially in type 2 diabetes mellitus, which is a major cardiovascular risk factor. In recent years, large extracellular vesicles (lEVs) have been identified as an important means of intercellular communication and as regulators of cardiovascular health and disease. A potential role for lEVs as vehicles for ceramide transfer and inductors of diabetes-associated endothelial apoptosis has never been investigated. METHODS AND RESULTS: A mass-spectrometric analysis of human coronary artery endothelial cells (HCAECs) and their lEVs revealed C16 ceramide (d18:1-16:0) to be the most abundant ceramide in lEVs and to be significantly increased in lEVs after hyperglycemic injury to HCAECs. The increased packaging of ceramide into lEVs after hyperglycemic injury was shown to be dependent on neutral sphingomyelinase 2 (nSMase2), which was upregulated in glucose-treated HCAECs. lEVs from hyperglycemic HCAECs induced apoptosis in the recipient HCAECs compared to native lEVs from untreated HCAECs. Similarly, lEVs from hyperglycemic mice after streptozotocin injection induced higher rates of apoptosis in murine endothelial cells compared to lEVs from normoglycemic mice. To generate lEVs with high levels of C16 ceramide, ceramide was applied exogenously and shown to be effectively packaged into the lEVs, which then induced apoptosis in lEV-recipient HCAECs via activation of caspase 3. Intercellular transfer of ceramide through lEVs was confirmed by use of a fluorescently labeled ceramide analogue. Treatment of HCAECs with a pharmacological inhibitor of nSMases (GW4869) or siRNA-mediated downregulation of nSMase2 abrogated the glucose-mediated effect on apoptosis in lEV-recipient cells. In contrast, for small EVs (sEVs), hyperglycemic injury or GW4869 treatment had no effect on apoptosis induction in sEV-recipient cells. CONCLUSION: lEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides.

Urine metabolites for the identification of Onchocerca volvulus infections in patients from Cameroon.

BACKGROUND: The tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,ß-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged. METHODS: We performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome. RESULTS: The mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls. CONCLUSIONS: On an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques.

24(R, S)-Saringosterol - From artefact to a biological medical agent.

Enhancing the cholesterol turnover in the brain via activation of liver x receptors can restore memory in a mouse model for Alzheimer's disease. The edible Asian brown alga Sargassum fusiforme (Hijiki) contains high amounts of oxysterols such as (3ß, 24ξ)-stigmasta-5, 28-dien-3, 24-diol (24[R, S]-saringosterol) that are a potent liver x receptor agonists. We aimed to find native European seaweed species with contents of 24(R, S)-saringosterol that are comparable to those found in Sargassum fusiforme. Additionally, we hypothesize that seasonal variations modify the amount of 24(R, S)-saringosterol in seaweeds. Sterols and oxysterols were extracted with chloroform/methanol from various seaweed species harvested in the Eastern Scheldt in different seasons between October 2016 and September 2017. Identification and quantification of the lipids was performed by gas chromatography- mass spectrometry and gas chromatography- flame ionization detection. We confirmed that brown algae Undaria pinnatifida harvested in February and Sargassum muticum harvested in October contained the highest amounts of 24(R, S)-saringosterol (32.4 ± 15.25 µg/g, mean ± S.D. and 32.95 ± 2.91 µg/g, respectively) and its precursor fucosterol (1.48 ± 0.11 mg/g), higher than Sargassum fusiforme (20.94 ± 3.00 µg/g, mean ± S.D.), while Ascophyllum nodosum and Fucus vesiculosus and Fucus serratus contained amounts of 24(R, S)-saringosterol (22.09 ± 3.45 µg/g, 18.04 ± 0.52 µg/g and 19.47 ± 9.01 µg/g, mean ± S.D., respectively) comparable to Sargassum fusiforme. In other algae only minor amounts of these sterols were observed. The green algae Ulva lactuca contained only 0.29 mg/g fucosterol and 10.3 µg/g 24 (R, S)-saringosterol, while all investigated red algae did not contain any 24(R, S)-saringosterol or fucosterol. In the Eastern Scheldt algae harvested in September/October delivered the highest yield for 24(R, S)-saringosterol, with the exception of Undaria pinnatifida that showed the highest levels in February. We showed that exposure of lipid extracts of Ulva lactuca to sunlight at room temperature or in the presence of oxygen to UV-C light lead to the quantitative conversion of fucosterol into 24(R, S)-saringosterol. Exposing pure fucosterol to UV-light did not convert any fucosterol into 24(R, S)-saringosterol underscoring the requirement of seaweed constituents in the conversion of fucosterol into 24(R, S)-saringosterol. In conclusion, we showed that brown seaweeds harvested from the Eastern Scheldt contain amounts of 24(R, S)-saringosterol comparable to Sargassum fusiforme, varying per season and showing the highest amounts in spring. In accordance with these observations the amount of 24(R, S)-saringosterol in the brown seaweeds can be modulated by light.

Direct Infusion Mass Spectrometry for Complex Lipid Analysis.

Direct infusion or "shotgun" mass spectrometry provides a fast strategy to measure different classes of lipids, combining rapid analysis and short idle time. In contrast to liquid chromatography-mass spectrometry (LC-MS), the lipids are infused into the mass spectrometer without prior separation by liquid chromatography. Ions are separated in the quadrupole of a tandem mass spectrometer, and after collision-induced dissociation fragments are quantified relative to internal standards in the third quadrupole or in the time-of-flight mass analyzer of a triple quadrupole or quadrupole time of flight (Q-TOF) mass spectrometer. Abundant lipids, that is, galactolipids and phospholipids in leaves, are measured in crude lipid extracts, while less abundant lipids can be measured after enrichment by solid-phase extraction. Here we describe protocols for the quantification of the major plant glycerolipids (galactolipids, phospholipids, diacylglycerol, and triacylglycerol) using nanospray direct infusion mass spectrometry. This provides a strategy for comprehensive, highly sensitive, high-throughput lipidomic analyses.

Phytol derived from chlorophyll hydrolysis in plants is metabolized via phytenal.

Phytol is the isoprenoid alcohol bound in ester linkage to chlorophyll, the most abundant photosynthetic pigment in plants. During leaf senescence, large amounts of phytol are released by chlorophyll degradation. However, the pathway of phytol catabolism in plants is unknown. We hypothesized that phytol degradation in plants might involve its oxidation into the long-chain aldehyde phytenal. Using GC-MS for aldehyde quantification after derivatization with methylhydroxylamine, phytenal was identified in leaves, whereas other long-chain aldehydes (phytanal and pristanal) were barely detectable. We found that phytenal accumulates during chlorotic stresses, for example, salt stress, dark-induced senescence, and nitrogen deprivation. The increase in the phytenal content is mediated at least in part independently of enzyme activities, and it is independent of light. Characterization of phytenal accumulation in the pao1 mutant affected in chlorophyll degradation revealed that phytenal is an authentic phytol metabolite derived from chlorophyll breakdown. The increase in phytenal was even stronger in mutants affected in the production of other phytol metabolites including vte5-2 (tocopherol deficient) and pes1 pes2 (fatty acid phytyl ester deficient). Therefore, phytenal accumulation is controlled by competing, alternative pathways of phosphorylation (leading to tocopherol production) or esterification (fatty acid phytyl ester production). As a consequence, the content of phytenal is maintained at low levels, presumably to minimize its toxic effects caused by its highly reactive aldehyde group that can form covalent bonds with and inactivate the amino groups of proteins.

Fatty acid isoprenoid alcohol ester synthesis in fruits of the African Oil Palm (Elaeis guineensis).

The African Oil Palm (Elaeis guineensis; family Arecaceae) represents the most important oil crop for food and feed production and for biotechnological applications. Two types of oil can be extracted from palm fruits, the mesocarp oil which is rich in palmitic acid and in carotenoids (provitamin A) and tocochromanols (vitamin E), and the kernel oil with high amounts of lauric and myristic acid. We identified fatty acid phytyl esters (FAPEs) in the mesocarp and kernel tissues of mature fruits, mostly esterified with oleic acid and very long chain fatty acids. In addition, fatty acid geranylgeranyl esters (FAGGEs) accumulated in mesocarp and kernels to even larger amounts. In contrast, FAPEs and FAGGEs amounts and fatty acid composition in leaves were very similar. Analysis of wild accessions of African Oil Palm from Cameroon revealed a considerable variation in the amounts and composition of FAPEs and FAGGEs in mesocarp and kernel tissues. Exogenous supplementation of phytol or geranylgeraniol to mesocarp slices resulted in the incorporation of these alcohols into FAPEs and FAGGEs, respectively, indicating that they are synthesized via enzymatic reactions. Three candidate genes of the esterase/lipase/thioesterase (ELT) family were identified in the Oil Palm genome. The genes are differentially expressed in mesocarp tissue with EgELT1 showing the highest expression. Geranylgeraniol from FAGGE might be recycled and used as a substrate for the synthesis of carotenoids and tocotrienols during fruit development. Thus, FAPEs and FAGGEs in the mesocarp and kernel of Oil Palm provide an additional metabolic source for fatty acids and phytol or geranylgeraniol, respectively.

Inhibition of acetyl-CoA carboxylase by spirotetramat causes growth arrest and lipid depletion in nematodes.

Plant-parasitic nematodes pose a significant threat to agriculture causing annual yield losses worth more than 100 billion US$. Nematode control often involves the use of nematicides, but many of them including non-selective fumigants have been phased out, particularly due to ecotoxicological concerns. Thus new control strategies are urgently needed. Spirotetramat (SPT) is used as phloem-mobile systemic insecticide targeting acetyl-CoA carboxylase (ACC) of pest insects and mites upon foliar application. However, in nematodes the mode of action of SPT and its effect on their development have not been studied so far. Our studies revealed that SPT known to be activated in planta to SPT-enol acts as a developmental inhibitor of the free-living nematode Caenorhabditis elegans and the plant-parasitic nematode Heterodera schachtii. Exposure to SPT-enol leads to larval arrest and disruption of the life cycle. Furthermore, SPT-enol inhibits nematode ACC activity, affects storage lipids and fatty acid composition. Silencing of H. schachtii ACC by RNAi induced similar phenotypes and thus mimics the effects of SPT-enol, supporting the conclusion that SPT-enol acts on nematodes by inhibiting ACC. Our studies demonstrated that the inhibition of de novo lipid biosynthesis by interfering with nematode ACC is a new nematicidal mode of action addressed by SPT, a well-known systemic insecticide for sucking pest control.

Triacylglycerol and phytyl ester synthesis in Synechocystis sp. PCC6803.

Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacterium Synechocystis sp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis in Synechocystis The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the gene slr2103 responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.

Surface wax esters contribute to drought tolerance in Arabidopsis.

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.

Phytol metabolism in plants.

Phytol, the prenyl side chain of chlorophyll, is derived from geranylgeraniol by reduction of three double bonds. Recent results demonstrated that the conversion of geranylgeraniol to phytol is linked to chlorophyll synthesis, which is catalyzed by protein complexes associated with the thylakoid membranes. One of these complexes contains light harvesting chlorophyll binding like proteins (LIL3), enzymes of chlorophyll synthesis (protoporphyrinogen oxidoreductase, POR; chlorophyll synthase, CHLG) and geranylgeranyl reductase (GGR). Phytol is not only employed for the synthesis of chlorophyll, but also for tocopherol (vitamin E), phylloquinol (vitamin K) and fatty acid phytyl ester production. Previously, it was believed that phytol is derived from reduction of geranylgeranyl-diphosphate originating from the 4-methylerythritol-5-phosphate (MEP) pathway. The identification and characterization of two kinases, VTE5 and VTE6, involved in phytol and phytyl-phosphate phosphorylation, respectively, indicated that most phytol employed for tocopherol synthesis is derived from reduction of geranylgeranylated chlorophyll to (phytol-) chlorophyll. After hydrolysis from chlorophyll, free phytol is phosphorylated by the two kinases, and phytyl-diphosphate employed for the synthesis of tocopherol and phylloquinol. The reason why some chloroplast lipids, i.e. chlorophyll, tocopherol and phylloquinol, are derived from phytol, while others, i.e. carotenoids and tocotrienols (in some plant species) are synthesized from geranylgeraniol, remains unclear.

Species-specific differences in nonlysosomal glucosylceramidase GBA2 function underlie locomotor dysfunction arising from loss-of-function mutations.

The nonlysosomal glucosylceramidase ß2 (GBA2) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Mutations in the human GBA2 gene have been associated with hereditary spastic paraplegia (HSP), autosomal-recessive cerebellar ataxia (ARCA), and the Marinesco-Sjögren-like syndrome. However, the underlying molecular mechanisms are ill-defined. Here, using biochemistry, immunohistochemistry, structural modeling, and mouse genetics, we demonstrate that all but one of the spastic gait locus #46 (SPG46)-connected mutations cause a loss of GBA2 activity. We demonstrate that GBA2 proteins form oligomeric complexes and that protein-protein interactions are perturbed by some of these mutations. To study the pathogenesis of GBA2-related HSP and ARCA in vivo, we investigated GBA2-KO mice as a mammalian model system. However, these mice exhibited a high phenotypic variance and did not fully resemble the human phenotype, suggesting that mouse and human GBA2 differ in function. Whereas some GBA2-KO mice displayed a strong locomotor defect, others displayed only mild alterations of the gait pattern and no signs of cerebellar defects. On a cellular level, inhibition of GBA2 activity in isolated cerebellar neurons dramatically affected F-actin dynamics and reduced neurite outgrowth, which has been associated with the development of neurological disorders. Our results shed light on the molecular mechanism underlying the pathogenesis of GBA2-related HSP and ARCA and reveal species-specific differences in GBA2 function in vivo.

A pair of nonspecific phospholipases C, NPC2 and NPC6, are involved in gametophyte development and glycerolipid metabolism in Arabidopsis.

Phospholipases play crucial roles in plant membrane lipid homeostasis. Nonspecific phospholipase C (NPCs) establish a unique class of phospholipases found only in plants and certain bacteria. Here, we show that two previously uncharacterized NPC isoforms, NPC2 and NPC6, are required for male and female gametophyte development in Arabidopsis. Double mutant plants of npc2-1 npc6-2 could not be retrieved because npc2-1 npc6-2 ovule and pollen development is affected. Genetic complementation, reciprocal crossing and microscope observation of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants suggest that NPC2 and NPC6 are redundant and are required for normal gametophyte development. Both NPC2 and NPC6 proteins are localized to the plastids. Promoter-GUS assays in transgenic Arabidopsis revealed that NPC2 and NPC6 are preferentially expressed in floral organs rather than in leaves. In vitro enzyme assays showed that NPC2 and NPC6 hydrolyze phosphatidylcholine and phosphatidylethanolamine, but not phosphatidate, being consistent with the reported substrate selectivity of NPCs. The amounts of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol were increased in buds but not in flowers of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- plants, presumably due to reduced phospholipid hydrolysis activity in developing flowers. Our results demonstrate that NPC2 and NPC6 play crucial roles in gametogenesis during flower development.

Inactivation of ceramide synthase 2 catalytic activity in mice affects transcription of genes involved in lipid metabolism and cell division.

The replacement of two consecutive histidine residues by alanine residues in the catalytic center of ceramide synthase 2 in a new transgenic mouse mutant (CerS2 H/A) leads to inactivation of catalytic activity and reduces protein level to 60% of the WT level. We show here by qRT-PCR and transcriptome analyses that several transcripts of genes involved in lipid metabolism and cell division are differentially regulated in livers of CerS2 H/A mice. Thus, very long chain ceramides produced by CerS2 are required for transcriptional regulation of target genes. The hepatocellular carcinomata previously described in old CerS2 KO mice were already present in 8-week-old CerS2 H/A animals and thus are caused by the loss of CerS2 catalytic activity already during early life.