RESUMEN
Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of ß-carotene and, consequently, ß-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing ß-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from ß-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and ß-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from ß-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.
Asunto(s)
Arabidopsis/metabolismo , Carotenoides/metabolismo , Proteínas de Plantas/metabolismo , Transcriptoma , Xenobióticos/metabolismo , Arabidopsis/genética , Radicales Libres/metabolismo , Perfilación de la Expresión Génica , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Xantófilas/metabolismoRESUMEN
KEY MESSAGE: Analysis of carotenoid-accumulating roots revealed that oxidative carotenoid degradation yields glyoxal and methylglyoxal. Our data suggest that these compounds are detoxified via the glyoxalase system and re-enter primary metabolic pathways. Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation. We recently identified redox enzymes previously known to be involved in the detoxification of fatty acid-derived reactive carbonyl species which were able to convert apocarotenoids into corresponding alcohols and carboxylic acids. However, their subsequent metabolization pathways remain unresolved. Interestingly, we found that carotenoid-accumulating roots have increased levels of glutathione, suggesting apocarotenoid glutathionylation to occur. In vitro and in planta investigations did not, however, support the occurrence of non-enzymatic or enzymatic glutathionylation of ß-apocarotenoids. An alternative breakdown pathway is the continued oxidative degradation of primary apocarotenoids or their derivatives into the shortest possible oxidation products, namely glyoxal and methylglyoxal, which also accumulated in carotenoid-accumulating roots. In fact, combined transcriptome and metabolome analysis suggest that the high levels of glutathione are most probably required for detoxifying apocarotenoid-derived glyoxal and methylglyoxal via the glyoxalase pathway, yielding glycolate and D-lactate, respectively. Further transcriptome analysis suggested subsequent reactions involving activities associated with photorespiration and the peroxisome-specific glycolate/glyoxylate transporter. Finally, detoxified primary apocarotenoid degradation products might be converted into pyruvate which is possibly re-used for the synthesis of carotenoid biosynthesis precursors. Our findings allow to envision carbon recycling during carotenoid biosynthesis, degradation and re-synthesis which consumes energy, but partially maintains initially fixed carbon via re-introducing reactive carotenoid degradation products into primary metabolic pathways.
Asunto(s)
Carbono , Piruvaldehído , Carotenoides/metabolismo , Glutatión/metabolismo , Redes y Vías MetabólicasRESUMEN
Geranylgeranyl diphosphate (GGPP), a prenyl diphosphate synthesized by GGPP synthase (GGPS), represents a metabolic hub for the synthesis of key isoprenoids, such as chlorophylls, tocopherols, phylloquinone, gibberellins, and carotenoids. Protein-protein interactions and the amphipathic nature of GGPP suggest metabolite channeling and/or competition for GGPP among enzymes that function in independent branches of the isoprenoid pathway. To investigate substrate conversion efficiency between the plastid-localized GGPS isoform GGPS11 and phytoene synthase (PSY), the first enzyme of the carotenoid pathway, we used recombinant enzymes and determined their in vitro properties. Efficient phytoene biosynthesis via PSY strictly depended on simultaneous GGPP supply via GGPS11. In contrast, PSY could not access freely diffusible GGPP or time-displaced GGPP supply via GGPS11, presumably due to liposomal sequestration. To optimize phytoene biosynthesis, we applied a synthetic biology approach and constructed a chimeric GGPS11-PSY metabolon (PYGG). PYGG converted GGPP to phytoene almost quantitatively in vitro and did not show the GGPP leakage typical of the individual enzymes. PYGG expression in Arabidopsis resulted in orange-colored cotyledons, which are not observed if PSY or GGPS11 are overexpressed individually. This suggests insufficient GGPP substrate availability for chlorophyll biosynthesis achieved through GGPP flux redirection to carotenogenesis. Similarly, carotenoid levels in PYGG-expressing callus exceeded that in PSY- or GGPS11-overexpression lines. The PYGG chimeric protein may assist in provitamin A biofortification of edible plant parts. Moreover, other GGPS fusions may be used to redirect metabolic flux into the synthesis of other isoprenoids of nutritional and industrial interest.
Asunto(s)
Arabidopsis/genética , Carotenoides/biosíntesis , Fosfatos de Poliisoprenilo/metabolismo , Arabidopsis/metabolismo , Unión Competitiva , Biofortificación , Carotenoides/química , Carotenoides/metabolismo , Ingeniería Genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Biología SintéticaRESUMEN
The Arabidopsis carotenoid cleavage dioxygenase 4 (AtCCD4) is a negative regulator of the carotenoid content of seeds and has recently been suggested as a candidate for the generation of retrograde signals that are thought to derive from the cleavage of poly-cis-configured carotene desaturation intermediates. In this work, we investigated the activity of AtCCD4 in vitro and used dynamic modeling to determine its substrate preference. Our results document strict regional specificity for cleavage at the C9-C10 double bond in carotenoids and apocarotenoids, with preference for carotenoid substrates and an obstructing effect on hydroxyl functions, and demonstrate the specificity for all-trans-configured carotenes and xanthophylls. AtCCD4 cleaved substrates with at least one ionone ring and did not convert acyclic carotene desaturation intermediates, independent of their isomeric states. These results do not support a direct involvement of AtCCD4 in generating the supposed regulatory metabolites. In contrast, the strigolactone biosynthetic enzyme AtCCD7 converted 9-cis-configured acyclic carotenes, such as 9-cis-ζ-carotene, 9'-cis-neurosporene, and 9-cis-lycopene, yielding 9-cis-configured products and indicating that AtCCD7, rather than AtCCD4, is the candidate for forming acyclic retrograde signals.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , Dioxigenasas/fisiología , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/metabolismo , Carotenoides/metabolismo , Cromatografía Líquida de Alta Presión , Dioxigenasas/metabolismo , Espectrometría de Masas , Especificidad por Sustrato , Xantófilas/metabolismoRESUMEN
Metabolite accumulation in plant tissues represents the transient net result of their constant biosynthesis and degradation. For carotenoids, degradation might occur enzymatically by carotenoid cleavage producing plant hormones and volatiles or by nonenzymatic oxidation, both depending on environmental and developmental conditions. Carotenoid biosynthesis is therefore constantly regulated at various levels to attain sufficient carotenoid accumulation, mainly for photosynthesis and photoprotection. Due to the plenitude of carotenoids and their degradation products, it is not feasible to investigate overall carotenoid biosynthetic activity and its regulation by the quantification of all carotenoids including their derivatives. This is an issue encountered in investigations on many other highly branched pathways. We therefore present protocols to determine carotenoid biosynthesis flux in a given plant tissue by HPLC quantification of phytoene, the first pathway-specific intermediate and precursor of all carotenoids synthesized by phytoene synthase (PSY). For this purpose, enzymatic metabolization of phytoene in the tissue under investigation is prevented by treatment with the bleaching herbicide norflurazon, blocking the carotenogenic pathway downstream of PSY. As phytoene is more resistant to oxidation than desaturated carotenoids, the rate of phytoene biosynthesis serves as a good measure for total carotenogenic flux in a given tissue. The method is described for Arabidopsis for two photosynthetically active sample types, namely, seedlings and leaves, as well as for seed-derived callus as nongreen tissue. It should be realizable using only a relatively simple experimental setup and is applicable to other plant tissues as well as to different plant species. Additionally, similar experimental setups could be a useful tool to investigate total flux and turnover rates in other biosynthetic pathways.
Asunto(s)
Carotenoides/metabolismo , Redes y Vías Metabólicas , Plantas/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Carotenoides/química , Carotenoides/aislamiento & purificación , Extracción Líquido-Líquido , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Plantas/química , Plantones/química , Plantones/metabolismo , Semillas/química , Semillas/metabolismoRESUMEN
Cauliflower Orange (Or) mutant is characterized by high level of ß-carotene in its curd. Or mutation affects the OR protein that was shown to be involved in the posttranslational control of phytoene synthase (PSY), a major rate-limiting enzyme of carotenoid biosynthesis, and in maintaining PSY proteostasis with the plastid Clp protease system. A transposon integration into the cauliflower wild-type Or gene (BoOR-wt) results in the formation of three differently spliced transcripts. One of them is characterized by insertion (BoOR-Ins), while the other two have exon-skipping deletions (BoOR-Del and BoOR-LD). We investigated the properties of individual BoOR variants and examined their effects on carotenoid accumulation. Using the yeast split-ubiquitin system, we showed that all variants were able to form OR dimers except BoOR-LD. The deletion in BoOR-LD eliminated the first of two adjacent transmembrane domains and was predicted to result in a misplacement of the C-terminal zinc finger domain to the opposite side of membrane, thus preventing OR dimerization. As interaction with PSY is mediated by the N-terminus of BoOR, which remains unaffected after splicing, all BoOR variants including BoOR-LD maintained interactions with PSY. Expression of individual BoOR mutant variants in Arabidopsis revealed that their protein stability varied greatly. While expression of BoOR-Del and BoOR-Ins resulted in increased BoOR protein levels as BoOR-wt, minimal amounts of BoOR-LD protein accumulated. Carotenoid accumulation showed correlated changes in calli of Arabidopsis expressing these variants. Furthermore, we found that OR also functions in E. coli to increase the proportion of native, enzymatically active PSY from plants upon co-expression, but not of bacterial phytoene synthase CrtB. Taken together, these results suggest that OR dimerization is required for OR stability in planta and that the simultaneous presence of PSY interaction-domains in both OR and PSY proteins is required for the holdase function of OR. The more pronounced effect of simultaneous expression of all BoOR variants in cauliflower Or mutant compared with individual overexpression on carotenoid accumulation suggests an enhanced activity with possible formation of various BoOR heterodimers.
RESUMEN
Synthetic biology applies engineering concepts to build cellular systems that perceive and process information. This is achieved by assembling genetic modules according to engineering design principles. Recent advance in the field has contributed optogenetic switches for controlling diverse biological functions in response to light. Here, the concept is introduced to apply synthetic biology switches and design principles for the synthesis of multi-input-processing materials. This is exemplified by the synthesis of a materials system that counts light pulses. Guided by a quantitative mathematical model, functional synthetic biology-derived modules are combined into a polymer framework resulting in a biohybrid materials system that releases distinct output molecules specific to the number of input light pulses detected. Further demonstration of modular extension yields a light pulse-counting materials system to sequentially release different enzymes catalyzing a multistep biochemical reaction. The resulting smart materials systems can provide novel solutions as integrated sensors and actuators with broad perspectives in fundamental and applied research.
Asunto(s)
Biología Sintética , Ingeniería Genética , PolímerosRESUMEN
Cyanobacteria and plants synthesize carotenoids via a poly-cis pathway starting with phytoene, a membrane-bound C40 hydrocarbon. Phytoene desaturase (PDS) introduces two double bonds and concomitantly isomerizes two neighboring double bonds from trans to cis. PDS assembles into homo-tetramers that interact monotopically with membranes. A long hydrophobic tunnel is proposed to function in the sequential binding of phytoene and the electron acceptor plastoquinone. The herbicidal inhibitor norflurazon binds at a plastoquinone site thereby blocking reoxidation of FADred. Comparison with the sequence-dissimilar bacterial carotene desaturase CRTI reveals substantial similarities in the overall protein fold, defining both as members of the GR2 family of flavoproteins. However, the PDS active center architecture is unprecedented: no functional groups are found in the immediate flavin vicinity that might participate in dehydrogenation and isomerization. This suggests that the isoalloxazine moiety is sufficient for catalysis. Despite mechanistic differences, an ancient evolutionary relation of PDS and CRTI is apparent.
Asunto(s)
Herbicidas/farmacología , Oxidorreductasas/química , Proteínas de Plantas/química , Piridazinas/farmacología , Carotenoides/química , Carotenoides/metabolismo , Herbicidas/química , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Oryza/enzimología , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Plastoquinona/química , Plastoquinona/metabolismo , Unión Proteica , Piridazinas/químicaRESUMEN
Provitamin A biofortification, the provision of provitamin A carotenoids through agriculture, is regarded as an effective and sustainable intervention to defeat vitamin A deficiency, representing a global health problem. This food-based intervention has been questioned in conjunction with negative outcomes for smokers and asbestos-exposed populations of the CARET and ATBC trials in which very high doses of ß-carotene were supplemented. The current notion that ß-carotene cleavage products (apocarotenoids) represented the harmful agents is the basis of the here-presented research. We quantitatively analyzed numerous plant food items and concluded that neither the amounts of apocarotenoids nor ß-carotene provided by plant tissues, be they conventional or provitamin A-biofortified, pose an increased risk. We also investigated ß-carotene degradation pathways over time. This reveals a substantial nonenzymatic proportion of carotene decay and corroborates the quantitative relevance of highly oxidized ß-carotene polymers that form in all plant tissues investigated.
Asunto(s)
Productos Agrícolas/química , Alimentos Fortificados/análisis , Provitaminas/química , Vitamina A/química , beta Caroteno/química , Biofortificación , Suplementos Dietéticos , Inocuidad de los AlimentosRESUMEN
Phytoene desaturase (PDS) is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides. PDS catalyzes the introduction of two double bonds into 15-cis-phytoene, yielding 9,15,9'-tri-cis-ζ-carotene via the intermediate 9,15-di-cis-phytofluene. We present the necessary data to scrutinize functional implications inferred from the recently resolved crystal structure of Oryza sativa PDS in a complex with norflurazon. Using dynamic mathematical modeling of reaction time courses, we support the relevance of homotetrameric assembly of the enzyme observed in crystallo by providing evidence for substrate channeling of the intermediate phytofluene between individual subunits at membrane surfaces. Kinetic investigations are compatible with an ordered ping-pong bi-bi kinetic mechanism in which the carotene and the quinone electron acceptor successively occupy the same catalytic site. The mutagenesis of a conserved arginine that forms a hydrogen bond with norflurazon, the latter competing with plastoquinone, corroborates the possibility of engineering herbicide resistance, however, at the expense of diminished catalytic activity. This mutagenesis also supports a "flavin only" mechanism of carotene desaturation not requiring charged residues in the active site. Evidence for the role of the central 15-cis double bond of phytoene in determining regio-specificity of carotene desaturation is presented.
Asunto(s)
Oryza/enzimología , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Biocatálisis/efectos de los fármacos , Carotenoides/química , Carotenoides/metabolismo , Cromatografía Liquida , Simulación por Computador , Pruebas de Enzimas , Cinética , Espectrometría de Masas , Modelos Moleculares , Mutación/genética , Oxidorreductasas/antagonistas & inhibidores , Multimerización de Proteína , Piridazinas/farmacología , Estereoisomerismo , Especificidad por Sustrato , Factores de TiempoRESUMEN
Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FAD(red), while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.