The Formation and Sequestration of Nonendogenous Ketocarotenoids in Transgenic Nicotiana glauca.

Ketolated and hydroxylated carotenoids are high-value compounds with industrial, food, and feed applications. Chemical synthesis is currently the production method of choice for these compounds, with no amenable plant sources readily available. In this study, the 4,4' ß-oxygenase (crtW) and 3,3' ß-hydroxylase (crtZ) genes from Brevundimonas sp. SD-212 were expressed under constitutive transcriptional control in Nicotiana glauca, which has an emerging potential as a biofuel and biorefining feedstock. The transgenic lines produced significant levels of nonendogenous carotenoids in all tissues. In leaf and flower, the carotenoids (∼0.5% dry weight) included 0.3% and 0.48%, respectively, of nonendogenous ketolated and hydroxylated carotenoids. These were 4-ketolutein, echinenone (and its 3-hydroxy derivatives), canthaxanthin, phoenicoxanthin, 4-ketozeaxanthin, and astaxanthin. Stable, homozygous genotypes expressing both transgenes inherited the chemotype. Subcellular fractionation of vegetative tissues and microscopic analysis revealed the presence of ketocarotenoids in thylakoid membranes, not predominantly in the photosynthetic complexes but in plastoglobules. Despite ketocarotenoid production and changes in cellular ultrastructure, intermediary metabolite levels were not dramatically affected. The study illustrates the utility of Brevundimonas sp. SD-212 CRTZ and CRTW to produce ketocarotenoids in a plant species that is being evaluated as a biorefining feedstock, the adaptation of the plastid to sequester nonendogenous carotenoids, and the robustness of plant metabolism to these changes.

Effect of sleep deprivation on the human metabolome.

Sleep restriction and circadian clock disruption are associated with metabolic disorders such as obesity, insulin resistance, and diabetes. The metabolic pathways involved in human sleep, however, have yet to be investigated with the use of a metabolomics approach. Here we have used untargeted and targeted liquid chromatography (LC)/MS metabolomics to examine the effect of acute sleep deprivation on plasma metabolite rhythms. Twelve healthy young male subjects remained in controlled laboratory conditions with respect to environmental light, sleep, meals, and posture during a 24-h wake/sleep cycle, followed by 24 h of wakefulness. Two-hourly plasma samples collected over the 48 h period were analyzed by LC/MS. Principal component analysis revealed a clear time of day variation with a significant cosine fit during the wake/sleep cycle and during 24 h of wakefulness in untargeted and targeted analysis. Of 171 metabolites quantified, daily rhythms were observed in the majority (n = 109), with 78 of these maintaining their rhythmicity during 24 h of wakefulness, most with reduced amplitude (n = 66). During sleep deprivation, 27 metabolites (tryptophan, serotonin, taurine, 8 acylcarnitines, 13 glycerophospholipids, and 3 sphingolipids) exhibited significantly increased levels compared with during sleep. The increased levels of serotonin, tryptophan, and taurine may explain the antidepressive effect of acute sleep deprivation and deserve further study. This report, to our knowledge the first of metabolic profiling during sleep and sleep deprivation and characterization of 24 h rhythms under these conditions, offers a novel view of human sleep/wake regulation.

Functional characterization of long-chain prenyl diphosphate synthases from tomato.

The electron transfer molecules plastoquinone and ubiquinone are formed by the condensation of aromatic head groups with long-chain prenyl diphosphates. In the present paper we report the cloning and characterization of two genes from tomato (Solanum lycopersicum) responsible for the production of solanesyl and decaprenyl diphosphates. SlSPS (S. lycopersicum solanesyl diphosphate synthase) is targeted to the plastid and both solanesol and plastoquinone are associated with thylakoid membranes. A second gene [SlDPS (S. lycopersicum solanesyl decaprenyl diphosphate synthase)], encodes a long-chain prenyl diphosphate synthase with a different subcellular localization from SlSPS and can utilize geranyl, farnesyl or geranylgeranyl diphosphates in the synthesis of C45 and C50 prenyl diphosphates. When expressed in Escherichia coli, SlSPS and SlDPS extend the prenyl chain length of the endogenous ubiquinone to nine and ten isoprene units respectively. In planta, constitutive overexpression of SlSPS elevated the plastoquinone content of immature tobacco leaves. Virus-induced gene silencing showed that SlSPS is necessary for normal chloroplast structure and function. Plants silenced for SlSPS were photobleached and accumulated phytoene, whereas silencing SlDPS did not affect leaf appearance, but impacted on primary metabolism. The two genes were not able to complement silencing of each other. These findings indicate a requirement for two long-chain prenyl diphosphate synthases in the tomato.

Proteome changes in tomato lines transformed with phytoene synthase-1 in the sense and antisense orientations.

The commercial cultivation of genetically engineered (GE) crops in Europe has met with considerable consumer resistance, which has led to vigorous safety assessments including the measurement of substantial equivalence between the GE and parent lines. This necessitates the identification and quantification of significant changes to the metabolome and proteome in the GE crop. In this study, the quantitative proteomic analysis of tomato fruit from lines that have been transformed with the carotenogenic gene phytoene synthase-1 (Psy-1), in the sense and antisense orientations, in comparison with a non-transformed, parental line is described. Multidimensional protein identification technology (MudPIT), with tandem mass spectrometry, has been used to identify proteins, while quantification has been carried out with isobaric tags for relative and absolute quantification (iTRAQ). Fruit from the GE plants showed significant alterations to their proteomes compared with the parental line, especially those from the Psy-1 sense transformants. These results demonstrate that MudPIT and iTRAQ are suitable techniques for the verification of substantial equivalence of the proteome in GE crops.