Phytol Metabolism Research Articles

An Arabidopsis mutant line lacking the mitochondrial calcium transport regulator MICU shows an altered metabolite profile

ABSTRACT Plant metabolism is constantly changing and requires input signals for efficient regulation. The mitochondrial calcium uniporter (MCU) couples organellar and cytoplasmic calcium oscillations leading to oxidative metabolism regulation in a vast array of species. In Arabidopsis thaliana, genetic deletion of AtMICU leads to altered mitochondrial calcium handling and ultrastructure. Here we aimed to further assess the consequences upon genetic deletion of AtMICU. Our results confirm that AtMICU safeguards intracellular calcium transport associated with carbohydrate, amino acid, and phytol metabolism modifications. The implications of such alterations are discussed.

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.

Phytol and its metabolites phytanic and pristanic acids for risk of cancer: current evidence and future directions.

This review summarizes the current evidence on the potential role of phytol, a microbial metabolite of chlorophyl A, and its metabolites, phytanic and pristanic acids, in carcinogenesis. Primary food sources in Western diets are the nut skin for phytol and lipids in dairy, beef and fish for its metabolites. Phytol and its metabolites gained interest as dietary compounds for cancer prevention because, as natural ligands of peroxisome proliferator-activated receptor-α and -γ and retinoid X receptor, phytol and its metabolites have provided some evidence in cell culture studies and limited evidence in animal models of anti-carcinogenic, anti-inflammatory and anti-metabolic-syndrome properties at physiological concentrations. However, there may be a narrow range of efficacy, because phytol and its metabolites at supra-physiological concentrations can cause in vitro cytotoxicity in non-cancer cells and can cause morbidity and mortality in animal models. In human studies, evidence for a role of phytol and its metabolites in cancer prevention is currently limited and inconclusive. In short, phytol and its metabolites are potential dietary compounds for cancer prevention, assuming the challenges in preventing cytotoxicity in non-cancer cells and animal models and understanding phytol metabolism can be mitigated.

Metabolic quirks and the colourful history of the Euglena gracilis secondary plastid.

Euglena spp. are phototrophic flagellates with considerable ecological presence and impact. Euglena gracilis harbours secondary green plastids, but an incompletely characterised proteome precludes accurate understanding of both plastid function and evolutionary history. Using subcellular fractionation, an improved sequence database and MS we determined the composition, evolutionary relationships and hence predicted functions of the E.gracilis plastid proteome. We confidently identified 1345 distinct plastid protein groups and found that at least 100 proteins represent horizontal acquisitions from organisms other than green algae or prokaryotes. Metabolic reconstruction confirmed previously studied/predicted enzymes/pathways and provided evidence for multiple unusual features, including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism, and dual sets of the SUF pathway for FeS cluster assembly, one of which was acquired by lateral gene transfer from Chlamydiae. Plastid paralogues of trafficking-associated proteins potentially mediating fusion of transport vesicles with the outermost plastid membrane were identified, together with derlin-related proteins, potential translocases across the middle membrane, and an extremely simplified TIC complex. The Euglena plastid, as the product of many genomes, combines novel and conserved features of metabolism and transport.

Lipid profiles reveal different responses to brown planthopper infestation for pest susceptible and resistant rice plants.

Brown planthopper (BPH) is the most destructive insect pest for rice, causing major reductions in rice yield and large economic losses. More than 31 BPH-resistance genes have been located, and several of them have been isolated. Nevertheless, the metabolic mechanism related to BPH-resistance genes remain uncharacterized. To elucidate the resistance mechanism of the BPH-resistance gene Bph6 at the metabolic level, a Bph6-transgenic line R6 (BPH-resistant) and the wild-type Nipponbare (BPH-susceptible) were used to investigate their lipid profiles under control and BPH treatments. In conjunction with multivariate statistical analysis and quantitative real-time PCR, BPH-induced lipid changes in leaf blade and leaf sheath were investigated by GC-MS-based lipidomics. Forty-five lipids were identified in leaf sheath extracts. Leaf sheath lipidomics analysis results show that BPH infestation induces significant differences in the lipid profiles of Nipponbare and R6. The levels of hexadecanoic acid, methyl ester, linoleic acid, methyl ester, linolenic acid, methyl ester, glycidyl palmitate, eicosanoic acid, methyl ester, docosanoic acid, methyl ester, beta-monolinolein, campesterol, beta-sitosterol, cycloartenol, phytol and phytyl acetate had undergone enormous changes after BPH feeding. These results illustrate that BPH feeding enhances sterol biosynthetic pathway in Nipponbare plants, and strengthens wax biosynthesis and phytol metabolism in R6 plants. The results of quantitative real-time PCR of 5 relevant genes were consistent with the changes in metabolic level. Forty-five lipids were identified in the leaf blade extracts. BPH infestation induces distinct changes in the lipid profiles of the leaf blade samples of Nipponbare and R6. Although the lipid changes in Nipponbare are more drastic, the changes within the two varieties are similar. Lipid profiles in leaf sheath brought out significant differences than in leaf blade within Nipponbare and R6. We propose that Bph6 mainly affects the levels of lipids in leaf sheath, and mediates resistance by deploying metabolic re-programming during BPH feeding. The results indicate that wax biosynthesis, sterol biosynthetic pathway and phytol metabolism play vital roles in rice response to BPH infestation. This finding demonstrated that the combination of lipidomics and quantitative real-time PCR is an effective approach to elucidating the interactions between brown planthopper and rice mediated by resistance genes.

Sex-dependent impact of Scp-2/Scp-x gene ablation on hepatic phytol metabolism

While prior studies focusing on male mice suggest a role for sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x; DKO) on hepatic phytol metabolism, its role in females is unresolved. This issue was addressed using female and male wild-type (WT) and DKO mice fed a phytoestrogen-free diet without or with 0.5% phytol. GC/MS showed that hepatic: i) phytol was absent and its branched-chain fatty acid (BCFA) metabolites were barely detectable in WT control-fed mice; ii) accumulation of phytol as well as its peroxisomal metabolite BCFAs (phytanic acid » pristanic and 2,3-pristenic acids) was increased by dietary phytol in WT females, but only slightly in WT males; iii) accumulation of phytol and BCFA was further increased by DKO in phytol-fed females, but much more markedly in males. Livers of phytol-fed WT female mice as well as phytol-fed DKO female and male mice also accumulated increased proportion of saturated straight-chain fatty acids (LCFA) at the expense of unsaturated LCFA. Liver phytol accumulation was not due to increased SCP-2 binding/transport of phytol since SCP-2 bound phytanic acid, but not its precursor phytol. Thus, the loss of Scp-2/Scp-x contributed to a sex-dependent hepatic accumulation of dietary phytol and BCFA.

Impact of Fabp1/Scp-2/Scp-x gene ablation (TKO) on hepatic phytol metabolism in mice

Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.

Transcriptional regulation of tocopherol biosynthesis in tomato

Tocopherols, compounds with vitamin E (VTE) activity, are potent lipid-soluble antioxidants synthesized only by photosynthetic organisms. Their biosynthesis requires the condensation of phytyl-diphosphate and homogentisate, derived from the methylerythritol phosphate (MEP) and shikimate pathways (SK), respectively. These metabolic pathways are central in plant chloroplast metabolism and are involved in the biosynthesis of important molecules such as chlorophyll, carotenoids, aromatic amino-acids and prenylquinones. In the last decade, few studies have provided insights into the regulation of VTE biosynthesis and its accumulation. However, the pathway regulatory mechanism/s at mRNA level remains unclear. We have recently identified a collection of tomato genes involved in tocopherol biosynthesis. In this work, by a dedicated qPCR array platform, the transcript levels of 47 genes, including paralogs, were determined in leaves and across fruit development. Expression data were analyzed for correlation with tocopherol profiles by coregulation network and neural clustering approaches. The results showed that tocopherol biosynthesis is controlled both temporally and spatially however total tocopherol content remains constant. These analyses exposed 18 key genes from MEP, SK, phytol recycling and VTE-core pathways highly associated with VTE content in leaves and fruits. Moreover, genomic analyses of promoter regions suggested that the expression of the tocopherol-core pathway genes is trancriptionally coregulated with specific genes of the upstream pathways. Whilst the transcriptional profiles of the precursor pathway genes would suggest an increase in VTE content across fruit development, the data indicate that in the M82 cultivar phytyl diphosphate supply limits tocopherol biosynthesis in later fruit stages. This is in part due to the decreasing transcript levels of geranylgeranyl reductase (GGDR) which restricts the isoprenoid precursor availability. As a proof of concept, by analyzing a collection of Andean landrace tomato genotypes, the role of the pinpointed genes in determining fruit tocopherol content was confirmed. The results uncovered a finely tuned regulation able to shift the precursor pathways controlling substrate influx for VTE biosynthesis and overcoming endogenous competition for intermediates. The whole set of data allowed to propose that 1-deoxy-D-xylulose-5-phosphate synthase and GGDR encoding genes, which determine phytyl-diphosphate availability, together with enzyme encoding genes involved in chlorophyll-derived phytol metabolism appear as the most plausible targets to be engineered aiming to improve tomato fruit nutritional value.

Metabolism of phytol to phytanic acid in the mouse, and the role of PPARα in its regulation

Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha). To investigate the metabolism of phytol and the role of PPARalpha in its regulation, wild-type and PPARalpha knockout (PPARalpha-/-) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARalpha agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARalpha-/- mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARalpha agonist resulted in a PPARalpha-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARalpha and is specific for the breakdown of (E)-phytol.

Polyunsaturated Fats Attenuate the Dietary Phytol–Induced Increase in Hepatic Fatty Acid Oxidation in Mice

The effects of dietary phytol and the type of dietary fat on hepatic fatty acid oxidation were examined in male ICR mice. Mice were fed diets containing 0 or 5 g/kg phytol and 100 g/kg palm, safflower, or fish oil for 21 d. Among the groups fed phytol-free diets, the activities and mRNA abundance of various enzymes involved in fatty acid oxidation were greater in mice fed fish oil than in those fed palm or safflower oil. Dietary phytol profoundly increased the activities and mRNA abundance of hepatic fatty acid oxidation enzymes in mice fed palm oil. However, safflower and fish oils, especially the latter, greatly attenuated the phytol-dependent increase in hepatic fatty acid oxidation. The hepatic concentration of phytanic acid, a metabolite of phytol that is the ligand and activator of retinoid X receptors and peroxisome proliferator-activated receptors, was higher in mice fed fish oil than safflower or palm oil, and in those administered safflower oil than palm oil. The hepatic mRNA abundance of sterol carrier protein-2, a lipid-binding protein involved in phytol metabolism, was inversely correlated with the hepatic concentration of phytanic acid. We demonstrated that polyunsaturated fats attenuate the enhancing effect of dietary phytol on hepatic fatty acid oxidation. Dietary fat-dependent changes in the hepatic phytanic acid concentration cannot account for this phenomenon.

Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis.

Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR) α, PPARγ or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5′ enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis.

Aerobic bacterial metabolism of phytol in seawater: effect of particle association on an abiotic intermediate step and its biogeochemical consequences

The biodegradation of phytol by a bacterial community isolated from the oxic zone of a Recent temperate marine sediment was studied in the presence, and in the absence, of a solid support. Analyses of isoprenoid wax esters produced by condensation of bacterial metabolites with themselves, or with phytol, showed that bacterial metabolism of phytol was strongly modified in the presence of the solid support. Aerobic metabolism of phytol involves the transient production of (E)-phytenal, which in turn can be quickly abiotically converted to 6,10,14-trimethylpentadecan-2-one or (E)-phytenic acid. Sorption on mineral particles appears to hinder the addition of water to the activated double bond of (E)-phytenal, which constitutes the first step of its transformation to the C18 isoprenoid ketone. Consequently, it is concluded that in surface temperate sediments phytol must be metabolised mainly via (E)-phytenic acid and that results obtained from in vitro experiments with free cell cultures may not be comparable to processes occuring in marine sedimentary environments.

The aerobic bacterial metabolism of phytol in seawater: Temperature dependence of an abiotic intermediate step and its consequences

A non-negligible production of 6,10,14-trimethylpentadecan-2-one has been often observed during aerobic phytol incubations with bacteria. In this work involving a marine Acinetobacter, a combination of biotic and abiotic processes is proposed in order to explain this phenomenon. In seawater, phytenal produced by bacterial oxidation of phytol can be converted abiotically to this isoprenoid ketone after addition of a water molecule and subsequent retro-aldol reaction. This abiotic process is very sensitive to temperature. It was demonstrated in this study that at 30°C an important part of phytol is metabolized through 6,10,14-trimethylpentadecan-2-one, whereas this pathway practically disappeares at 13°C. Consequently, in studies concerning phytol biodegradation, it will be important to take into account the effect of temperature.

Stereochemical studies of acyclic isoprenoid compounds. 3. The stereochemistry of naturally occurring (marine) 2,6,10,14-tetramethylpentadecane.

High resolution gas chromatographic analysis of natural 2,6,10,14-tetramethylpentadecane from shark lipids shows it to comprise only the 6(R),10(S)-meso-isomer. This was confirmed by gas chromatographic analysis of esters of the acidic products of dichromate oxidation. These data support the hypothesis that the alkane derives from zooplanktonic metabolism of phytol (trans-3,7(R),11 (R),15-tetramethylhexadec-2-en-1-ol).

Fate of 14 C-phytol administered orally to sheep.

The metabolism of phytol was investigated in 12 sheep, each given a single oral dose of 14C-phytol. In the unsaponifiable material of abomasum liquor or liver tissue. 14C-phytol radioactivity was recovered unchanged as the administered compound or as an unidentified metabolite of phytol. Most of the radioactivity in the saponifiable material of tissues, after esterification. occurred in the methyl ester of phytanic acid. A portion of the phytol occurring in the rumen lumen was converted to phytanic acid.