1-deoxy-2-pentulose and mevalonolactone

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (

Topics: Arabidopsis; Carbon Isotopes; Chromatography, Gas; Dolichols; Erythritol; Isotope Labeling; Metabolic Networks and Pathways; Mevalonic Acid; Models, Theoretical; Osmotic Pressure; Phytosterols; Sorbitol; Spectrometry, Mass, Electrospray Ionization; Sugar Phosphates; Terpenes; Xylulose

Isopentenyl/dimethylallyl diphosphate isomerase (IPI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal C(5) units of isoprenoids. In plants, IPP and DMAPP are synthesized via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways, respectively. However, the role of IPI in each pathway and in plant development is unknown due to a lack of genetic studies using IPI-defective mutants. Here, we show that the atipi1atipi2 double mutant, which is defective in two Arabidopsis IPI isozymes, exhibits dwarfism and male sterility under long-day conditions and decreased pigmentation under continuous light, whereas the atipi1 and atipi2 single mutants are phenotypically normal. We also show that the sterol and ubiquinone levels in the double mutant are <50% of those in wild-type plants, and that the male-sterile phenotype is chemically complemented by squalene, a sterol precursor. In vivo isotope labeling experiments using the atipi1atipi2 double mutant revealed a decrease in the incorporation of MVA (in its lactone form) into sterols, with no decrease in the incorporation of MEP pathway intermediates into tocopherol. These results demonstrate a critical role for IPI in isoprenoid biosynthesis via the MVA pathway, and they imply that IPI is essential for the maintenance of appropriate levels of IPP and DMAPP in different subcellular compartments in plants.

Topics: Arabidopsis; Carbon Isotopes; Carbon-Carbon Double Bond Isomerases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genetic Complementation Test; Hemiterpenes; Mevalonic Acid; Mutation; Organ Specificity; Phenotype; Plant Infertility; Protein Transport; Subcellular Fractions; Terpenes; Xylulose

The biosynthesis of the monoterpene (S)-linalool and the sesquiterpene trans-(S)-nerolidol in fruits of Fragaria x ananassa Duch. cv. Eros and Florence and of the monoterpene (-)-alpha-pinene in Fragaria vesca was investigated by in vivo feeding experiments with [5,5-2H2]mevalonic acid lactone (d2-MVL) and [5,5-2H2]-1-deoxy-d-xylulose (d2-DOX). The feeding experiments indicate that (S)-linalool and trans-(S)-nerolidol in Fragaria x ananassa Duch. and (-)-alpha-pinene in F. vesca are exclusively synthesized via the cytosolic mevalonic acid pathway without any contribution from the plastidial 1-deoxy-D-xylulose/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) route. Inhibition experiments revealed that even the presence of mevastatin, an export of plastid-derived isopentyl diphosphate/dimethylallyl diphosphate, cannot be induced. However, the enantioselective analysis shows that in Fragaria x ananassa Duch. cv. Eros and Florence both linalool enantiomers are present and that only (S)-linalool is labeled after administration of d2-MVL. Therefore, the origin of (R)-linalool in these fruits remains unknown. Contrarily, in Fragaria x ananassa Duch. foliage (R)-linalool is the dominant enantiomer. Feeding experiments revealed an incorporation of d2-MVL and d2-DOX at equal rates exclusively into (S)-linalool. Only in F. vesca foliage, where (R)-linalool is present at high enantiomeric purity (ee > 90%), is a de novo biosynthesis of the (R)-enantiomer via the DOXP/MEP pathway detectable. These results demonstrate a complex intraplant variation of (R)- and (S)-linalool biosynthesis via the cytosolic and plastidial route.

Topics: Acyclic Monoterpenes; Deuterium; Fragaria; Fruit; Gas Chromatography-Mass Spectrometry; Mevalonic Acid; Monoterpenes; Plant Leaves; Sesquiterpenes; Stereoisomerism; Xylulose

The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.

Topics: Hemiterpenes; Mevalonic Acid; Organophosphorus Compounds; Pentanols; Pinus; Pinus ponderosa; Terpenes; Xylulose