Metabolomics of Green-Tea Catechins on Vascular-Endothelial-Growth-Factor-Stimulated Human-Endothelial-Cell Survival.

Neovascularization causes serious oculopathy related to upregulation of vascular-endothelial-growth factor (VEGF) causing new capillary growth via endothelial cells. Green-tea-extract (GTE) constituents possess antiangiogenesis properties. We used VEGF to induce human umbilical-vein endothelial cells (HUVECs) and applied GTE, epigallocatechin gallate (EGCG), and mixtures of different compositions of purified catechins (M1 and M2) to evaluate their efficacies of inhibition and their underlying mechanisms using cell-cycle analysis and untargeted metabolomics techniques. GTE, EGCG, M1, and M2 induced HUVEC apoptosis by 22.1 ± 2, 20.0 ± 0.7, 50.7 ± 8.5, and 69.8 ± 4.1%, respectively. GTE exerted a broad, balanced metabolomics spectrum, involving suppression of the biosynthesis of cellular building blocks and oxidative-phosphorylation metabolites as well as promotion of the biosynthesis of membrane lipids and growth factors. M2 mainly induced mechanisms associated with energy and biosynthesis suppression. Therefore, GTE exerted mechanisms involving both promotion and suppression activities, whereas purified catechins induced extensive apoptosis. GTE could be a more promising antineovascularization remedy for ocular treatment.

HOTHEAD-Like HTH1 is Involved in Anther Cutin Biosynthesis and is Required for Pollen Fertility in Rice.

The cuticle covering the outer surface of anthers is essential for male reproductive development in plants. However, the mechanism underlying the synthesis of these lipidic polymers remains unclear. HOTHEAD (HTH) in Arabidopsis thaliana is a presumptive glucose-methanol-choline (GMC) oxidoreductase involved in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids. In this study, we characterized the function of an anther-specific gene HTH1 in rice. HTH1 contains a conserved GMC oxidoreductase-like domain, and the sequence of HTH1 was highly similar to that of HTH in A. thaliana. Quantitative real-time PCR (qRT-PCR) and in situ hybridization analyses showed that HTH1 was highly expressed in epidermal cells of anthers. Rice plants with HTH1 suppression through CRISPR (clustered regularly interspaced short palindromic repeats) and RNA interference (RNAi) displayed defective anther wall and aborted pollen. Disorganized cuticle layers in anthers and shriveled pollen grains were observed in HTH1-RNAi lines. The total amounts of long-chain fatty acids and cutin monomers in anthers of HTH1-RNAi lines were significantly reduced compared with the wild type. Our results suggested that HTH1 is involved in cutin biosynthesis and is required for anther development and pollen fertility in rice.

Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis.

The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis.

Lipid biochemists salute the genome.

The biochemistry of plant metabolic pathways has been studied for many generations; nevertheless, numerous new enzymes and metabolic products have been discovered in the last 5-10 years. More importantly, many intriguing questions remain in all areas of metabolism. In this review, we consider these issues with respect to several pathways of lipid metabolism and the contributions made by the Arabidopsis genome sequence and the tools that it has spawned. These tools have allowed identification of enzymes and transporters required for the mobilization of seed storage lipids, as well as transporters that facilitate movement of lipids from the endoplasmic reticulum to the chloroplast in green leaf cells. Genomic tools were important in recognition of novel components of the cutin and suberin polymers that form water-impermeable barriers in plants. The waxes that also contribute to these barriers are exported from cells of the epidermis by transporters that are now being identified. Biochemical and genetic knowledge from yeast and animals has permitted successful homology-based searches of the Arabidopsis genome for genes encoding enzymes involved in the elongation of fatty acids and the synthesis of sphingolipids. Knowledge of the genome has identified novel enzymes for the biosynthesis of the seed storage lipid, triacylglycerol, and provided a refined understanding of how the pathways of fatty acid and triacylglycerol synthesis are integrated into overall carbon metabolism in developing seeds.

Cytochrome P450 family member CYP704B2 catalyzes the {omega}-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice.

The anther cuticle and microspore exine act as protective barriers for the male gametophyte and pollen grain, but relatively little is known about the mechanisms underlying the biosynthesis of the monomers of which they are composed. We report here the isolation and characterization of a rice (Oryza sativa) male sterile mutant, cyp704B2, which exhibits a swollen sporophytic tapetal layer, aborted pollen grains without detectable exine, and undeveloped anther cuticle. In addition, chemical composition analysis indicated that cutin monomers were hardly detectable in the cyp704B2 anthers. These defects are caused by a mutation in a cytochrome P450 family gene, CYP704B2. The CYP704B2 transcript is specifically detected in the tapetum and the microspore from stage 8 of anther development to stage 10. Heterologous expression of CYP704B2 in yeast demonstrated that CYP704B2 catalyzes the production of omega -hydroxylated fatty acids with 16 and 18 carbon chains. Our results provide insights into the biosynthesis of the two biopolymers sporopollenin and cutin. Specifically, our study indicates that the omega -hydroxylation pathway of fatty acids relying on this ancient CYP704B family, conserved from moss to angiosperms, is essential for the formation of both cuticle and exine during plant male reproductive and spore development.

Competition between 24:5n-3 and ALA for Delta 6 desaturase may limit the accumulation of DHA in HepG2 cell membranes.

The use of Delta 6 desaturase (D6D) twice in the conversion of alpha-linolenic acid (ALA; 18:3n-3) to docosahexaenoic acid (DHA; 22:6n-3) suggests that this enzyme may play a key regulatory role in the synthesis and accumulation of DHA from ALA. We examined this using an in vitro model of fatty acid metabolism to measure the accumulation of the long-chain metabolites of ALA in HepG2 cell phospholipids. The accumulation of ALA, eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and 24:5n-3 in cell phospholipids was linearly related to the concentration of supplemented ALA over the range tested (1.8-72 microM). The accumulation of the post-D6D products of 22:5n-3, 24:6n-3 and DHA, in cell phospholipids was saturated at concentrations of >18 microM ALA. Supplementation of HepG2 cells with preformed DHA revealed that, although the accumulation of DHA in cell phospholipids approached saturation, the level of DHA in cell phospholipids was significantly greater compared with the accumulation of DHA from ALA, indicating that the accumulation of DHA from ALA was not limited by incorporation. The parallel pattern of accumulation of 24:6n-3 and DHA in response to increasing concentrations of ALA suggests that the competition between 24:5n-3 and ALA for D6D may contribute to the limited accumulation of DHA in cell membranes.

Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces.

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.

Effect of UV-B on photosynthesis, membrane lipids and MAAs in marine cyanobacterium, Phormidium corium (Agardh) Gomont.

UV-B radiation (0.8 +/- 0.1 mW cm(-2)) and UV-B radiation supplemented with low intensity PAR (approximately 80 micro mol m(-2) s(-1)) affected photosynthesis at the level of antenna system as well as PS II reaction centre (Fo and Fm declined) in Phormidium corium (Agardh) Gomont. UV-B radiation resulted in decline in sugar content, peroxidation of membrane lipids as well as quantitative and qualitative changes in phosphoglycolipids and neutral lipids. Fatty acid profile did not show any qualitative changes due to the treatment, however, UV-B supplemented with low PAR resulted in slightly higher level of unsaturation. P. corium synthesized MAAs in response to UV-B. Quantity of MAAs increased when UV-B treatment was supplemented with low level PAR.

Oral uridine-5'-monophosphate (UMP) increases brain CDP-choline levels in gerbils.

We examined the biochemical pathways whereby oral uridine-5'-monophosphate (UMP) increases membrane phosphatide synthesis in brains of gerbils. We previously showed that supplementing PC12 cells with uridine caused concentration-related increases in CDP-choline levels, and that this effect was mediated by elevations in intracellular uridine triphosphate (UTP) and cytidine triphosphate (CTP). In the present study, adult gerbils received UMP (1 mmol/kg), a constituent of human breast milk and infant formulas, by gavage, and plasma samples and brains were collected for assay between 5 min and 8 h thereafter. Thirty minutes after gavage, plasma uridine levels were increased from 6.6 +/- 0.58 to 32.7 +/- 1.85 microM (P < 0.001), and brain uridine from 22.6 +/- 2.9 to 89.1 +/- 8.82 pmol/mg tissue (P < 0.001). UMP also significantly increased plasma and brain cytidine levels; however, both basally and following UMP, these levels were much lower than those of uridine. Brain UTP, CTP, and CDP-choline were all elevated 15 min after UMP (from 254 +/- 31.9 to 417 +/- 50.2, [P < 0.05]; 56.8 +/- 1.8 to 71.7 +/- 1.8, [P < 0.001]; and 11.3 +/- 0.5 to 16.4 +/- 1, [P < 0.001] pmol/mg tissue, respectively), returning to basal levels after 20 and 30 min. The smallest UMP dose that significantly increased brain CDP-choline was 0.05 mmol/kg. These results show that oral UMP, a uridine source, enhances the synthesis of CDP-choline, the immediate precursor of PC, in gerbil brain.

Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility.

Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells. Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT. The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant. The functional significance of one isoform, AtGPAT1, is the focus of the present study. Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development. Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion. In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene. Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds. Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content.

Reactive oxygen species production in association with suberization: evidence for an NADPH-dependent oxidase.

In response to wounding, potato tubers generate reactive oxygen species (ROS) in association with suberization. Immediately following wounding, an initial burst of ROS occurs, reaching a maximum within 30 to 60 min. In addition to this initial oxidative burst, at least three other massive bursts occur at 42, 63 and 100 h post-wounding. These latter bursts are associated with wound healing and are probably involved in the oxidative cross-linking of suberin poly(phenolics). The source of ROS is likely to be a plasma membrane NADPH-dependent oxidase immunorelated to the human phagocyte plasma membrane oxidase. The initial oxidative burst does not appear to be dependent on new protein synthesis, but the subsequent bursts are associated with an increase in oxidase protein components. Oxidase activity is enhanced in vitro by hydroxycinnamic acids and conjugates associated with the wound healing response in potato.

Biosynthesis, molecular structure, and domain architecture of potato suberin: a (13)C NMR study using isotopically labeled precursors.

Although suberin in potato wound periderm is known to be a polyester containing long-chain fatty acids and phenolics embedded within the cell wall, many aspects of its molecular structure and polymer-polymer connectivities remain elusive. The present work combines biosynthetic incorporation of site-specifically (13)C-enriched acetates and phenylalanines with one- and two-dimensional solid-state (13)C NMR spectroscopic methods to monitor the developing suberin polymer. Exogenous acetate is found to be incorporated preferentially at the carboxyl end of the aliphatic carbon chains, suggesting addition during the later elongation steps of fatty acid synthesis. Carboxyl-labeled phenylalanine precursors provide evidence for the concurrent development of phenolic esters and of monolignols typical of lignin. Experiments with ring-labeled phenylalanine precursors demonstrate a predominance of sinapyl and guaiacyl structures among suberin's phenolic moieties. Finally, the analysis of spin-exchange (solid-state NOESY) NMR experiments in ring-labeled suberin indicates distances of no more than 0.5 nm between pairs of phenolic and oxymethine carbons, which are attributed to the aromatic-aliphatic polyester and the cell wall polysaccharide matrix, respectively. These results offer direct and detailed molecular information regarding the insoluble intermediates of suberin biosynthesis, indicate probable covalent linkages between moieties of its polyester and polysaccharide domains, and yield a clearer overall picture of this agriculturally important protective material.

Cress and potato soluble epoxide hydrolases: purification, biochemical characterization, and comparison to mammalian enzymes.

Affinity chromatographic methods were developed for the one-step purification to homogeneity of recombinant soluble epoxide hydrolases (sEHs) from cress and potato. The enzymes are monomeric, with masses of 36 and 39 kDa and pI values of 4.5 and 5.0, respectively. In spite of a large difference in sequence, the two plant enzymes have properties of inhibition and substrate selectivity which differ only slightly from mammalian sEHs. Whereas mammalian sEHs are highly selective for trans- versus cis-substituted stilbene oxide and 1,3-diphenylpropene oxide (DPPO), plant sEHs exhibit far greater selectivity for trans- versus cis-stilbene oxide, but little to no selectivity for DPPO isomers. The isolation of a covalently linked plant sEH-substrate complex indicated that the plant and mammalian sEHs have a similar mechanism of action. We hypothesize an in vivo role for plant sEH in cutin biosynthesis, based on relatively high plant sEH activity on epoxystearate to form a cutin precursor, 9,10-dihydroxystearate. Plant sEHs display a high thermal stability relative to mammalian sEHs. This stability and their high enantioselectivity for a single substrate suggest that their potential as biocatalysts for the preparation of enantiopure epoxides should be evaluated.

NMR studies of structure and dynamics in fruit cuticle polyesters.

Cutin and suberin are support polymers involved in waterproofing the leaves and fruits of higher plants, regulating the flow of nutrients among various plant organs, and minimizing the deleterious impact of microbial pathogens. Despite the complexity and intractable nature of these plant biopolyesters, their molecular structure and development are amenable to study by suitable solid-state and solution-state NMR techniques. Interactions of tomato cutin with water were examined by solid-state 2H and 13C NMR, showing that water films enhance rapid segmental motions of the acyl chains and are associated with a fivefold increase in surface elasticity upon cutin hydration. The suberization of wounded potato tissues was studied by solid-state 13C NMR, revealing the likely phenylpropanoid structures that permit dense cross-linking of the suberin structure and their proximity to the cell-wall polysaccharides. Finally, two new approaches were developed to elucidate the molecular structures of these biopolymers: partial depolymerization followed by spectroscopic analysis of the soluble oligomers; and swelling of the intact materials followed by magic-angle spinning (MAS) NMR analysis.

Induced phenylpropanoid metabolism during suberization and lignification: a comparative analysis.

Induction of the biosynthesis of phenylpropanoids was monitored at the enzyme level through measurement of the temporal change in the activity of two marker enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase, (PAL, E.C. 4.1.3.5) and 4-coumaryl-CoA ligase (4-CL, E.C. 6.2.1.12) and two marker enzymes for hydroxycinnamyl alcohol biosynthesis, cinnamoyl-CoA:NADP+ oxidoreductase (CCR, E.C. 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) in both suberizing potato (Solanum tuberosum) tubers and lignifying loblolly pine (Pinus taeda) cell cultures. While measurable activities of PAL, 4-CL and CAD increased upon initiation of suberization in potato tubers, that of CCR did not. By contrast, all four enzymes were induced upon initiation of lignification in pine cell cultures. The lack of CCR induction in potato by wound treatment is consistent with the channelling of hydroxycinnamoyl-CoA derivatives away from monolignol formation and toward other hydroxycinnamoyl derivatives such as those that accumulate during suberization.

Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis.

The chemical tagging of a cytochrome P-450-dependent lauric acid omega-hydroxylase from clofibrate-treated Vicia sativa seedlings with [1-14C]11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1. We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana. The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1. The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1. Both E/Z and Z/E configurations of 9, 12-octadecadienoic acids were omega-hydroxylated. Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1). The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized. Similar to animal omega-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment. Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug. The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.

The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C.

Our analysis of epidermal lipids revealed that (glucosyl)ceramide profiles in various human skin equivalents are different from those of native tissue. The main difference is the reduced content in skin equivalents of ceramides 4-7 and especially the very low content of the most polar ceramides 6 and 7, which contain hydroxylated sphingoid base and/or fatty acid. To facilitate hydroxylation, the culture medium was supplemented with vitamins C and E. Although in vitamin E-supplemented medium lipogenesis was not affected, in vitamin C-supplemented medium the content of glucosylceramides and of ceramides 6 and 7 was markedly increased, both in the presence and absence of serum and irrespective the substrate used (inert or natural, populated or not with fibroblasts). The improvement of the lipid profile was accompanied by a marked improvement of the barrier formation as judged from extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/stratum corneum interface, and the formation of multiple broad lipid lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle x-ray diffraction. Some differences between native and reconstructed epidermis, however, were noticed. Although the long-range lipid lamellar phase was present in both the native and the reconstructed epidermis, the short lamellar phase was present only in native tissue. It remains to be established whether these differences can be ascribed to small differences in relative amounts of individual ceramides, to differences in fatty acid profiles, or to differences in cholesterol sulfate, pH, or calcium gradients. The results indicate the key role vitamin C plays in the formation of stratum corneum barrier lipids.

Hydroxycinnamic acid-derived polymers constitute the polyaromatic domain of suberin.

Suberin is an abundant, complex, intractable, plant cell wall polymeric network that forms both protective and wound-healing layers. Its function is, therefore, critical to the survival of all vascular plants. Its chemical structure and biosynthesis are poorly defined, although it is known to consist of both aromatic and aliphatic domains. While the composition of the aliphatic component has been fairly well characterized, that of the phenolic component has not. Using a combination of specific carbon-13 labeling techniques, and in situ solid state 13C NMR spectroscopic analysis, we now provide the first direct evidence for the nature of the phenolic domain of suberin and report here that it is almost exclusively comprised of a covalently linked, hydroxycinnamic acid-derived polymeric matrix.

Cutinase gene disruption in Fusarium solani f sp pisi decreases its virulence on pea.

Fusarium solani f sp pisi (Nectria haematococca) isolate 77-2-3 with one cutinase gene produced 10 to 20% of the cutinase produced by isolate T-8 that has multiple cutinase genes, whereas cutinase gene-disrupted mutant 77-102 of isolate 77-2-3 did not produce cutinase. On the surface of pea stem segments, lesion formation was most frequent and most severe with T-8, less frequent and less severe with 77-2-3, and much less frequent and much milder with the gene-disrupted mutant. Microscopic examination of the lesions caused by the mutant strongly suggest that it penetrated the host mostly via the stomata. In seedling assays, 77-2-3 caused severe lesions on every seedling and stunted growth, whereas the mutant showed very mild lesions on one-third of the seedlings with no stunting. Thus, cutinase gene disruption resulted in a significant decrease in the pathogenicity of F. s. pisi on pea.