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Validation of biomarker assays is crucial for effective drug development and clinical applications. Interlaboratory reproducibility is vital for reliable comparison and combination of data from different centers. This review summarizes interlaboratory studies of quantitative LC-MS-based biomarker assays using reference standards for calibration curves. The following points are discussed: trends in reports, reference and internal standards, evaluation of analytical validation parameters, study sample analysis and normalization of biomarker assay data. Full evaluation of these parameters in interlaboratory studies is limited, necessitating further research. Some reports suggest methods to address variations in biomarker assay data among laboratories, facilitating organized studies and data combination. Method validation across laboratories is crucial for reducing interlaboratory differences and reflecting target biomarker responses.
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Cromatografía Líquida con Espectrometría de Masas , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Estándares de ReferenciaRESUMEN
Nicotinamide adenine dinucleotides (NAD+ and NADP+) are electron mediators involved in various metabolic pathways. NADP(H) are produced by NAD kinase (NADK) through the phosphorylation of NAD(H). The Arabidopsis NADK3 (AtNADK3) is reported to preferentially phosphorylate NADH to NADPH and is localized in the peroxisome. To elucidate the biological function of AtNADK3 in Arabidopsis, we compared metabolites of nadk1, nadk2 and nadk3 Arabidopsis T-DNA inserted mutants. Metabolome analysis revealed that glycine and serine, which are intermediate metabolites of photorespiration, both increased in the nadk3 mutants. Plants grown for 6 weeks under short-day conditions showed increased NAD(H), indicating a decrease in the phosphorylation ratio in the NAD(P)(H) equilibrium. Furthermore, high CO2 (0.15%) treatment induced a decrease in glycine and serine in nadk3 mutants. The nadk3 showed a significant decrease in post-illumination CO2 burst, suggesting that the photorespiratory flux was disrupted in the nadk3 mutant. In addition, an increase in CO2 compensation points and a decrease in CO2 assimilation rate were observed in the nadk3 mutants. These results indicate that the lack of AtNADK3 causes a disruption in the intracellular metabolism, such as in amino acid synthesis and photorespiration.
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Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Glicina/metabolismo , NAD/metabolismo , NADP/metabolismo , Serina/metabolismoRESUMEN
Background: Although the fit-for-purpose approach has been proposed for biomarker assay validation, practical data should be compiled to facilitate the predetermination of acceptance criteria. Methods: Immunoaffinity LC-MS was used to analyze glucagon-like peptide-1 as a model biomarker in six laboratories. Calibration curve, carryover, parallelism, precision, relative accuracy and processed sample stability were evaluated, and their robustness among laboratories was assessed. The rat glucagon-like peptide-1 concentrations in four blinded samples were also compared. Results: The obtained results and determined concentrations in the blinded samples at all laboratories were similar, with a few exceptions, and robust, despite the difference in optimization techniques among laboratories. Conclusion: The results provide insights into the predefinition of the acceptance criteria of immunoaffinity LC-MS-based biomarker assays.
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Laboratorios , Espectrometría de Masas en Tándem , Ratas , Animales , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Péptido 1 Similar al Glucagón , BiomarcadoresRESUMEN
Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.
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Terapia Biológica , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , CalibraciónRESUMEN
2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and <1 s for the separation. We compared three derivatization methods and found that a method using (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine enables the separation. In addition, we were able to detect dl-2-hydroxyglutaric acid in standard solution at lower concentrations than that previously reported for the serum. These results show the potential of the method to be used in clinical analysis.
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Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5-250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.
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Terapia Biológica/métodos , Cromatografía Liquida/métodos , ADN sin Sentido/metabolismo , Espectrometría de Masas/métodos , Oligonucleótidos/metabolismo , Calibración , HumanosRESUMEN
Hydroxyoctadecadienoic acids (HODEs) are produced by oxidation and reduction of linoleates. There are several regio- and stereo-isomers of HODE, and their concentrations in vivo are higher than those of other lipids. Although conformational isomers may have different biological activities, comparative analysis of intracellular function of HODE isomers has not yet been performed. We evaluated the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ), a therapeutic target for diabetes, and analyzed PPARγ agonist activity of HODE isomers. The lowest scores for docking poses of 12 types of HODE isomers (9-, 10-, 12-, and 13-HODEs) were almost similar in docking simulation of HODEs into PPARγ ligand-binding domain (LBD). Direct binding of HODE isomers to PPARγ LBD was determined by water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiments. In contrast, there were differences in PPARγ agonist activities among 9- and 13-HODE stereo-isomers and 12- and 13-HODE enantio-isomers in a dual-luciferase reporter assay. Interestingly, the activity of 9-HODEs was less than that of other regio-isomers, and 9-(E,E)-HODE tended to decrease PPARγ-target gene expression during the maturation of 3T3-L1 cells. In addition, 10- and 12-(Z,E)-HODEs, which we previously proposed as biomarkers for early-stage diabetes, exerted PPARγ agonist activity. These results indicate that all HODE isomers have PPARγ-binding affinity; however, they have different PPARγ agonist activity. Our findings may help to understand the biological function of lipid peroxidation products.
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Ácidos Linoleicos/farmacología , PPAR gamma/agonistas , Células 3T3-L1 , Animales , Ácidos Linoleicos/química , Peroxidación de Lípido , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , PPAR gamma/química , PPAR gamma/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
NAD is a well-known co-enzyme that mediates hundreds of redox reactions and is the basis of various processes regulating cell responses to different environmental and developmental cues. The regulatory mechanism that determines the amount of cellular NAD and the rate of NAD metabolism remains unclear. We created Arabidopsis thaliana plants overexpressing the NAD synthase (NADS) gene that participates in the final step of NAD biosynthesis. NADS overexpression enhanced the activity of NAD biosynthesis but not the amounts of NAD+, NADH, NADP+ or NADPH. However, the amounts of some intermediates were elevated, suggesting that NAD metabolism increased. The NAD redox state was greatly facilitated by an imbalance between NAD generation and degradation in response to bolting. Metabolite profiling and transcriptional analysis revealed that the drastic modulation of NAD redox homeostasis increased tricarboxylic acid flux, causing the ectopic generation of reactive oxygen species. Vascular bundles suffered from oxidative stress, leading to a malfunction in amino acid and organic acid transportation that caused early wilting of the flower stalk and shortened plant longevity, probably due to malnutrition. We concluded that the mechanism regulating the balance between NAD synthesis and degradation is important in the systemic plant response to developmental cues during the growth-phase transition.
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Arabidopsis/metabolismo , Arabidopsis/fisiología , Longevidad , NAD/metabolismo , Desarrollo de la Planta , Amida Sintasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Vías Biosintéticas , Coenzimas/metabolismo , Regulación de la Expresión Génica de las Plantas , Homeostasis , Peróxido de Hidrógeno/metabolismo , Metabolómica , Modelos Biológicos , Oxidación-Reducción , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , ReproducciónRESUMEN
The existence of glucose conjugates of fumonisin B2 (FB2) and fumonisin B3 (FB3) in corn powder was confirmed for the first time. These "bound-fumonisins" (FB2 and FB3 bound to glucose) were identified as N-(1-deoxy-D-fructos-1-yl) fumonisin B2 (NDfrc-FB2) and N-(1-deoxy-D-fructos-1-yl) fumonisin B3 (NDfrc-FB3) respectively, based on the accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS) analysis. Treatment on NDfrc-FB2 and NDfrc-FB3 with the o-phthalaldehyde (OPA) reagent also supported that D-glucose binding to FB2 and FB3 molecules occurred to their primary amine residues.
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Fumonisinas/aislamiento & purificación , Zea mays/microbiología , Cromatografía Liquida , Contaminación de Alimentos/análisis , Microbiología de Alimentos , Espectrometría de MasasRESUMEN
Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition.
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Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Germinación , NAD/metabolismo , Polen/crecimiento & desarrollo , Polen/metabolismo , Vías Biosintéticas , Humedad , NAD/biosíntesis , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Supervivencia TisularRESUMEN
2-Hydroxy fatty acids (2-HFAs) are predominantly present in sphingolipids and have important physicochemical and physiological functions in eukaryotic cells. Recent studies from our group demonstrated that sphingolipid fatty acid 2-hydroxylase (FAH) is required for the function of Arabidopsis (Arabidopsis thaliana) Bax inhibitor-1 (AtBI-1), which is an endoplasmic reticulum membrane-localized cell death suppressor. However, little is known about the function of two Arabidopsis FAH homologs (AtFAH1 and AtFAH2), and it remains unclear whether 2-HFAs participate in cell death regulation. In this study, we found that both AtFAH1 and AtFAH2 had FAH activity, and the interaction with Arabidopsis cytochrome b5 was needed for the sufficient activity. 2-HFA analysis of AtFAH1 knockdown lines and atfah2 mutant showed that AtFAH1 mainly 2-hydroxylated very-long-chain fatty acid (VLCFA), whereas AtFAH2 selectively 2-hydroxylated palmitic acid in Arabidopsis. In addition, 2-HFAs were related to resistance to oxidative stress, and AtFAH1 or 2-hydroxy VLCFA showed particularly strong responses to oxidative stress. Furthermore, AtFAH1 interacted with AtBI-1 via cytochrome b5 more preferentially than AtFAH2. Our results suggest that AtFAH1 and AtFAH2 are functionally different FAHs, and that AtFAH1 or 2-hydroxy VLCFA is a key factor in AtBI-1-mediated cell death suppression.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Esfingolípidos/metabolismo , Estrés Fisiológico , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sistema Enzimático del Citocromo P-450/genética , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Ésteres/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Peróxido de Hidrógeno/farmacología , Hidroxilación/efectos de los fármacos , Oxigenasas de Función Mixta/genética , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Células Vegetales/efectos de los fármacos , Células Vegetales/metabolismo , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genéticaRESUMEN
When ammonium is the sole nitrogen (N) source, plant growth is suppressed compared with the situation where nitrate is the N source. This is commonly referred to as ammonium toxicity. It is widely known that a combination of nitrate and ammonium as N source alleviates this ammonium toxicity (nitrate-dependent alleviation of ammonium toxicity), but the underlying mechanisms are still not completely understood. In plants, ammonium toxicity is often accompanied by a depletion of organic acids and inorganic cations, and by an accumulation of ammonium. All these factors have been considered as possible causes for ammonium toxicity. Thus, we hypothesized that nitrate could alleviate ammonium toxicity by lessening these symptoms. We analyzed growth, inorganic N and cation content and various primary metabolites in shoots of Arabidopsis thaliana seedlings grown on media containing various concentrations of nitrate and/or ammonium. Nitrate-dependent alleviation of ammonium toxicity was not accompanied by less depletion of organic acids and inorganic cations, and showed no reduction in ammonium accumulation. On the other hand, shoot growth was significantly correlated with the nitrate concentration in the shoots. This suggests that nitrate-dependent alleviation of ammonium toxicity is related to physiological processes that are closely linked to nitrate signaling, uptake and reduction. Based on transcript analyses of various genes related to nitrate signaling, uptake and reduction, possible underlying mechanisms for the nitrate-dependent alleviation are discussed.
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Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Ácidos Carboxílicos/metabolismo , Nitratos/farmacología , Compuestos de Amonio Cuaternario/metabolismo , Compuestos de Amonio Cuaternario/toxicidad , Aminoácidos/biosíntesis , Arabidopsis/genética , Biomasa , Tampones (Química) , Cationes , Ciclo del Ácido Cítrico/efectos de los fármacos , Medios de Cultivo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Glucólisis/efectos de los fármacos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Nitrógeno/farmacología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/crecimiento & desarrollo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Extractos de TejidosRESUMEN
Nonphotochemical quenching (NPQ) regulates energy conversion in photosystem II and protects plants from photoinhibition. Here we analyze NPQ capacity in a number of rice cultivars. NPQ was strongly induced under medium and high light intensities in rice leaves. Japonica cultivars generally showed higher NPQ capacities than Indica cultivars when we measured a rice core collection. We mapped NPQ regulator and identified a locus (qNPQ1-2) that seems to be responsible for the difference in NPQ capacity between Indica and Japonica. One of the two rice PsbS homologues (OsPsbS1) was found within the qNPQ1-2 region. PsbS protein was not accumulated in the leaf blade of the mutant harboring transferred DNA insertion in OsPsbS1. NPQ capacity increased as OsPsbS1 expression increased in a series of transgenic lines ectopically expressing OsPsbS1 in an Indica cultivar. Indica cultivars lack a 2.7-kb region at the point 0.4 kb upstream of the OsPsbS1 gene, suggesting evolutionary discrimination of this gene.
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Transferencia de Energía , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Complejo de Proteína del Fotosistema II/genética , Sitios Genéticos , Complejos de Proteína Captadores de Luz , Oryza/metabolismo , Hojas de la Planta , Proteínas de Plantas/genética , Especificidad de la EspecieRESUMEN
Nicotinamide adenine dinucleotide (NAD) and its derivative nicotinamide adenine dinucleotide phosphate (NADP) are indispensable co-factors in broad-spectrum metabolic events for the maintenance of cellular homeostasis in all living organisms. In this study, the cellular expression levels of NAD biosynthesis genes in Arabidopsis were investigated. A very high expression of nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) was observed in the differentiated stomatal guard cells of the leaf surface. Transcriptional analysis confirmed that several genes in the biosynthesis pathway were also highly expressed in stomatal guard cells. In fact, NAD and NADP metabolisms were investigated during stomatal movement. Importantly, the generation of phytohormone ABA-induced reactive oxygen species, which acts as a signal for stomatal closure, was accompanied by markedly decreased levels of NAD. The ABA-induced oxidative stress caused stomatal cell death in the nmnat mutant. Furthermore, stomata partially lost their ability to close leading to drought susceptibility. The stomata were less responsive to opening cues as well. These results indicate that NAD biosynthesis is involved in protecting guard cells from ABA-induced local oxidative stress via the regulation of NMNAT activity. In this study, it is demonstrated that NMNAT is essential for the maintenance of NAD homeostasis enabling sustainable stomatal movement.
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Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , NAD/biosíntesis , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Estomas de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/enzimología , Arabidopsis/genética , Deshidratación , NAD/metabolismo , Estrés Oxidativo/fisiología , Reguladores del Crecimiento de las Plantas/farmacología , Estomas de Plantas/efectos de los fármacos , Transducción de Señal/fisiología , Estrés Fisiológico/fisiologíaRESUMEN
Oxygen uptake rates are increased when concentrated ammonium instead of nitrate is used as sole N source. Several explanations for this increased respiration have been suggested, but the underlying mechanisms are still unclear. To investigate possible factors responsible for this respiratory increase, we measured the O2 uptake rate, activity and transcript level of respiratory components, and concentration of adenylates using Arabidopsis thaliana shoots grown in media containing various N sources. The O2 uptake rate was correlated with concentrations of ammonium and ATP in shoots, but not related to the ammonium assimilation. The capacity of the ATP-coupling cytochrome pathway (CP) and its related genes were up-regulated when concentrated ammonium was sole N source, whereas the ATP-uncoupling alternative oxidase did not influence the extent of the respiratory increase. Our results suggest that the ammonium-dependent increase of the O2 uptake rate can be explained by the up-regulation of the CP, which may be related to the ATP consumption by the plasma-membrane H+ -ATPase.
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Arabidopsis/metabolismo , Grupo Citocromo c/metabolismo , Consumo de Oxígeno , Compuestos de Amonio Cuaternario/metabolismo , Adenosina Trifosfato/metabolismo , Arabidopsis/genética , Respiración de la Célula , Grupo Citocromo c/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Mitocondriales , Mutación , Nitratos/metabolismo , Nitrógeno/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas , ARN de Planta/genéticaRESUMEN
Expression of alternative oxidase (AOX) and cyanide (CN)-resistant respiration are often highly enhanced in plants exposed to low-nitrogen (N) stress. Here, we examined the effects of AOX deficiency on plant growth, gene expression of respiratory components and metabolic profiles under low-N stress, using an aox1a knockout transgenic line (aox1a) of Arabidopsis thaliana. We exposed wild-type (WT) and aox1a plants to low-N stress for 7 d and analyzed their shoots and roots. In WT plants, the AOX1a mRNA levels and AOX capacity increased in proportion to low-N stress. Expression of the genes of the components for non-phosphorylating pathways and antioxidant enzymes was enhanced, but differences between WT and aox1a plants were small. Metabolome analyses revealed that AOX deficiency altered the levels of certain metabolites, such as sugars and sugar phosphates, in the shoots under low-N stress. However, the carbon (C)/N ratios and carbohydrate levels in aox1a plants were similar to those in the WT under low-N stress. Our results indicated that the N-limited stress induced AOX expression in A. thaliana plants, but the induced AOX may not play essential roles under stress due to low-N alone, and the C/N balance under low-N stress may be tightly regulated by systems other than AOX.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Nitrógeno/metabolismo , Oxidorreductasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Carbono/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Metaboloma , Proteínas Mitocondriales , Oxidorreductasas/genética , Proteínas de Plantas , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , ARN de Planta/genéticaRESUMEN
The chloroplastic NAD kinase (NADK2) is reported to stimulate carbon and nitrogen assimilation in Arabidopsis (Arabidopsis thaliana), which is vulnerable to high light. Since rice (Oryza sativa) is a monocotyledonous plant that can adapt to high light, we studied the effects of NADK2 expression in rice by developing transgenic rice plants that constitutively expressed the Arabidopsis chloroplastic NADK gene (NK2 lines). NK2 lines showed enhanced activity of NADK and accumulation of the NADP(H) pool, while intermediates of NAD derivatives were unchanged. Comprehensive analysis of the primary metabolites in leaves using capillary electrophoresis mass spectrometry revealed elevated levels of amino acids and several sugar phosphates including ribose-1,5-bisphosphate, but no significant change in the levels of the other metabolites. Studies of chlorophyll fluorescence and gas change analyses demonstrated greater electron transport and CO2 assimilation rates in NK2 lines, compared to those in the control. Analysis of oxidative stress response indicated enhanced tolerance to oxidative stress in these transformants. The results suggest that NADP content plays a critical role in determining the photosynthetic electron transport rate in rice and that its enhancement leads to stimulation of photosynthesis metabolism and tolerance of oxidative damages.
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Arabidopsis/genética , Metaboloma , Oryza/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fotoquímica , Arabidopsis/enzimología , Electroforesis Capilar , Espectrometría de Masas , Plantas Modificadas GenéticamenteRESUMEN
Bax inhibitor-1 (BI-1) is a cell death suppression factor widely conserved in higher plants and animals. Overexpression of Arabidopsis BI-1 (AtBI-1) in plants confers tolerance to various cell death-inducible stresses. However, apart from the cell death-suppressing activity, little is known about the physiological roles of BI-1-overexpressing plants. In this study, we evaluated the effects of AtBI-1 overexpression on the rice metabolome in response to oxidative stress. AtBI-1-overexpressing rice cells in suspension displayed enhanced tolerance to menadione-induced oxidative stress compared with vector control cells, whereas AtBI-1 overexpression did not influence the increase of intracellular H(2)O(2) concentration or inhibition of oxidative stress-sensitive aconitase activity. Capillary electrophoresis-mass spectrometry (CE-MS)-based metabolome analysis revealed dynamic metabolic changes in oxidatively stressed rice cells, e.g. depletion of the central metabolic pathway, imbalance of the redox state and energy charge, and accumulation of amino acids. Furthermore, comparative metabolome analysis demonstrated that AtBI-1 overexpression did not affect primary metabolism in rice cells under normal growth conditions but significantly altered metabolite composition within several distinct pathways under cell death-inducible oxidative stress. The AtBI-1-mediated metabolic alteration included recovery of the redox state and energy charge, which are known as important factors for metabolic defense against oxidative stress. These observations suggest that although AtBI-1 does not affect rice metabolism directly, its cell death suppression activity leads to enhanced capacity to acclimate oxidative stress.
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Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Metaboloma/genética , Oryza/genética , Oryza/metabolismo , Estrés Oxidativo/genética , Adaptación Fisiológica/genética , Aminoácidos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Muerte Celular/genética , Resistencia a Medicamentos/genética , Electroforesis Capilar , Metabolismo Energético/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Espectrometría de Masas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oryza/citología , Oxidación-Reducción , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Vitamina K 3/toxicidad , Vitaminas/toxicidadRESUMEN
The paper derives a simple way to calculate the linear relationships between all separable groups of rate constants for de-excitation of Chl a excitation energy. This is done by comparison of the inverse values of chlorophyll fluorescence intensities and is based on the matrix model of Kitajima and Butler and on the lake model of energy exchange among PSII centers. Compared with the outputs of earlier, similar calculations, the results presented here add some linear comparisons of the relative sizes of rate constants without the need for F(0)' measurement. This enables us to regenerate the same alternative formula to calculate q(L) as presented previously, in a different and simple form. The same former equation to calculate F(0)' value from F(m), F(m)' and F(0) values is also regenerated in our calculation system in a simple form. We also apply relaxation analysis to separate the rate constant for non-photochemical quenching (k(NPQ)) into the rate constant for a fast-relaxing non-photochemical quenching (k(fast)) and the rate constant for slow-relaxing non-photochemical quenching (k(slow)). Changes in the sizes of rate constants were measured in Arabidopsis thaliana and in rice.
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Clorofila/química , Fluorescencia , Modelos Químicos , Arabidopsis/química , Clorofila A , Oryza/química , FotosíntesisRESUMEN
Nicotinamide nucleotides (NAD and NADP) are important cofactors in many metabolic processes in living organisms. In this study, we analyzed transgenic Arabidopsis (Arabidopsis thaliana) plants that overexpress NAD kinase2 (NADK2), an enzyme that catalyzes the synthesis of NADP from NAD in chloroplasts, to investigate the impacts of altering NADP level on plant metabolism. Metabolite profiling revealed that NADP(H) concentrations were proportional to NADK activity in NADK2 overexpressors and in the nadk2 mutant. Several metabolites associated with the Calvin cycle were also higher in the overexpressors, accompanied by an increase in overall Rubisco activity. Furthermore, enhanced NADP(H) production due to NADK2 overexpression increased nitrogen assimilation. Glutamine and glutamate concentrations, as well as some other amino acids, were higher in the overexpressors. These results indicate that overexpression of NADK2 either directly or indirectly stimulates carbon and nitrogen assimilation in Arabidopsis under restricted conditions. Importantly, since neither up-regulation nor down-regulation of NADK2 activity affected the sum amount of NAD and NADP or the redox state, the absolute level of NADP and/or the NADP/NAD ratio likely plays a key role in regulating plant metabolism.