Metabolite Profiling in Arabidopsisthaliana with Moderately Impaired Photorespiration Reveals Novel Metabolic Links and Compensatory Mechanisms of Photorespiration.

Photorespiration is an integral component of plant primary metabolism. Accordingly, it has been often observed that impairing the photorespiratory flux negatively impacts other cellular processes. In this study, the metabolic acclimation of the Arabidopsisthaliana wild type was compared with the hydroxypyruvate reductase 1 (HPR1; hpr1) mutant, displaying only a moderately reduced photorespiratory flux. Plants were analyzed during development and under varying photoperiods with a combination of non-targeted and targeted metabolome analysis, as well as 13C- and 14C-labeling approaches. The results showed that HPR1 deficiency is more critical for photorespiration during the vegetative compared to the regenerative growth phase. A shorter photoperiod seems to slowdown the photorespiratory metabolite conversion mostly at the glycerate kinase and glycine decarboxylase steps compared to long days. It is demonstrated that even a moderate impairment of photorespiration severely reduces the leaf-carbohydrate status and impacts on sulfur metabolism. Isotope labeling approaches revealed an increased CO2 release from hpr1 leaves, most likely occurring from enhanced non-enzymatic 3-hydroxypyruvate decarboxylation and a higher flux from serine towards ethanolamine through serine decarboxylase. Collectively, the study provides evidence that the moderate hpr1 mutant is an excellent tool to unravel the underlying mechanisms governing the regulation of metabolic linkages of photorespiration with plant primary metabolism.

First Steps Towards an Understanding of a Mode ofCarcinogenic Action for Vanadium Pentoxide.

Inhalation of vanadium pentoxide clearly increases the incidence of alveolar/bronchiolar neoplasms in male and female B6C3F1 mice at all concentrations tested (1, 2 or 4 mg/m(3)), whereas responses in F344/N rats was, at most, ambiguous. While vanadium pentoxide is mutagenic in vitro and possibly in vivo in mice, this does not explain the species or site specificity of the neoplastic response. A nose-only inhalation study was conducted in female B6C3F1 mice (0, 0.25, 1 and 4 mg/m(3), 6 h/day for 16 days) to explore histopathological, biochemical (α-tocopherol, glutathione and F2-isoprostane) and genetic (comet assays and 9 specific DNA-oxo-adducts) changes in the lungs. No treatment related histopathology was observed at 0.25 mg/m(3). At 1 and 4 mg/m(3), exposure-dependent increases were observed in lung weight, alveolar histiocytosis, sub-acute alveolitis and/or granulocytic infiltration and a generally time-dependent increased cell proliferation rate of histiocytes. Glutathione was slightly increased, whereas there were no consistent changes in α-tocopherol or 8-isoprostane F2α. There was no evidence for DNA strand breakage in lung or BAL cells, but there was an increase in 8-oxodGuo DNA lesions that could have been due to vanadium pentoxide induction of the lesions or inhibition of repair of spontaneous lesions. Thus, earlier reports of histopathological changes in the lungs after inhalation of vanadium pentoxide were confirmed, but no evidence has yet emerged for a genotoxic mode of action. Evidence is weak for oxidative stress playing any role in lung carcinogenesis at the lowest effective concentrations of vanadium pentoxide.

A cytosolic pathway for the conversion of hydroxypyruvate to glycerate during photorespiration in Arabidopsis.

Deletion of any of the core enzymes of the photorespiratory cycle, one of the major pathways of plant primary metabolism, results in severe air-sensitivity of the respective mutants. The peroxisomal enzyme hydroxypyruvate reductase (HPR1) represents the only exception to this rule. This indicates the presence of extraperoxisomal reactions of photorespiratory hydroxypyruvate metabolism. We have identified a second hydroxypyruvate reductase, HPR2, and present genetic and biochemical evidence that the enzyme provides a cytosolic bypass to the photorespiratory core cycle in Arabidopsis thaliana. Deletion of HPR2 results in elevated levels of hydroxypyruvate and other metabolites in leaves. Photosynthetic gas exchange is slightly altered, especially under long-day conditions. Otherwise, the mutant closely resembles wild-type plants. The combined deletion of both HPR1 and HPR2, however, results in distinct air-sensitivity and a dramatic reduction in photosynthetic performance. These results suggest that photorespiratory metabolism is not confined to chloroplasts, peroxisomes, and mitochondria but also extends to the cytosol. The extent to which cytosolic reactions contribute to the operation of the photorespiratory cycle in varying natural environments is not yet known, but it might be dynamically regulated by the availability of NADH in the context of peroxisomal redox homeostasis.

Integration of metabolomic and proteomic phenotypes: analysis of data covariance dissects starch and RFO metabolism from low and high temperature compensation response in Arabidopsis thaliana.

Statistical mining and integration of complex molecular data including metabolites, proteins, and transcripts is one of the critical goals of systems biology (Ideker, T., Galitski, T., and Hood, L. (2001) A new approach to decoding life: systems biology. Annu. Rev. Genomics Hum. Genet. 2, 343-372). A number of studies have demonstrated the parallel analysis of metabolites and large scale transcript expression. Protein analysis has been ignored in these studies, although a clear correlation between transcript and protein levels is shown only in rare cases, necessitating that actual protein levels have to be determined for protein function analysis. Here, we present an approach to investigate the combined covariance structure of metabolite and protein dynamics in a systemic response to abiotic temperature stress in Arabidopsis thaliana wild-type and a corresponding starch-deficient mutant (phosphoglucomutase-deficient). Independent component analysis revealed phenotype classification resolving genotype-dependent response effects to temperature treatment and genotype-independent general temperature compensation mechanisms. An observation is the stress-induced increase of raffinose-family-oligosaccharide levels in the absence of transitory starch storage/mobilization in temperature-treated phosphoglucomutase plants indicating that sucrose synthesis and storage in these mutant plants is sufficient to bypass the typical starch storage/mobilization pathways under abiotic stress. Eventually, sample pattern recognition and correlation network topology analysis allowed for the detection of specific metabolite-protein co-regulation and assignment of a circadian output regulated RNA-binding protein to these processes. The whole concept of high-dimensional profiling data integration from many replicates, subsequent multivariate statistics for dimensionality reduction, and covariance structure analysis is proposed to be a major strategy for revealing central responses of the biological system under study.

Deletion of glycine decarboxylase in Arabidopsis is lethal under nonphotorespiratory conditions.

The mitochondrial multienzyme glycine decarboxylase (GDC) catalyzes the tetrahydrofolate-dependent catabolism of glycine to 5,10-methylene-tetrahydrofolate and the side products NADH, CO(2), and NH(3). This reaction forms part of the photorespiratory cycle and contributes to one-carbon metabolism. While the important role of GDC for these two metabolic pathways is well established, the existence of bypassing reactions has also been suggested. Therefore, it is not clear to what extent GDC is obligatory for these processes. Here, we report on features of individual and combined T-DNA insertion mutants for one of the GDC subunits, P protein, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana). The individual knockout of either of these two genes does not significantly alter metabolism and photosynthetic performance indicating functional redundancy. In contrast, the double mutant does not develop beyond the cotyledon stage in air enriched with 0.9% CO(2). Rosette leaves do not appear and the seedlings do not survive for longer than about 3 to 4 weeks under these nonphotorespiratory conditions. This feature distinguishes the GDC-lacking double mutant from all other known photorespiratory mutants and provides evidence for the nonreplaceable function of GDC in vital metabolic processes other than photorespiration.

Integrative profiling of metabolites and proteins: improving pattern recognition and biomarker selection for systems level approaches.

Methods such as mRNA expression profiling have provided a vast amount of genomic and transcriptomic information about plants and other organisms. However, there is explicit indication that considerable metabolic control is executed on the metabolite and on the protein level including protein modifications, thereby constituting the phenotypic plasticity. Consequently, the analysis of the molecular phenotype demands the step toward mass spectrometry (MS)-based postgenomic techniques such as metabolomics and proteomics. This chapter describes a straightforward protocol for simultaneously extracting metabolites and proteins from the same biological sample in preparation for MS analysis. Furthermore, protocols for profiling polar metabolites using gas chromatography time-of-flight MS and for shotgun proteomics using liquid chromatography-MS are discussed. A practical course is laid out that outlines all the basic steps, from harvesting to data analysis. These steps enable the correlative study of metabolite and protein dynamics with minimal technical variation. Biological variability of independent samples is exploited for variance analysis and pattern recognition.

A gentle guide to the analysis of metabolomic data.

Modern molecular biology crucially relies on computational tools to handle and interpret the large amounts of data that are generated by high-throughput measurements. To this end, much effort is dedicated to devise novel sophisticated methods that allow one to integrate, evaluate, and analyze biological data. However, prior to an application of specifically designed methods, simple and well-known statistical approaches often provide a more appropriate starting point for further analysis. This chapter seeks to describe several well-established approaches to data analysis, including various clustering techniques, discriminant function analysis, principal component analysis, multidimensional scaling, and classification trees. The chapter is accompanied by a webpage, describing the application of all algorithms in a ready-to-use format.

Metabolomics: from pattern recognition to biological interpretation.

Metabolomics is a technology that aims to identify and quantify the metabolome -- the dynamic set of all small molecules present in an organism or a biological sample. In this sense, the technique is distinct from metabolic profiling, which looks for target compounds and their biochemical transformation. The combination of both approaches is an emerging technique for the characterization of biological samples and for drug treatment. Metabolomics has proven to be very rapid and superior to any other post-genomics technology for pattern-recognition analyses of biological samples. Changing steady state concentrations and fluctuations of metabolites that occur within milliseconds are a result of biochemical processes such as signalling cascades: metabolomic techniques are instrumental in measuring these changes rapidly and sensitively. Metabolite data can be complemented by protein, transcript and external (environmental) data, thereby leading to the identification of multiple physiological biomarkers embedded in correlative molecular networks that are not approachable with targeted studies.

D-GLYCERATE 3-KINASE, the last unknown enzyme in the photorespiratory cycle in Arabidopsis, belongs to a novel kinase family.

D-GLYCERATE 3-KINASE (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C2 cycle, an indispensable ancillary metabolic pathway to the photosynthetic C3 cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C2 cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO2, providing direct evidence of the obligatory nature of the ultimate step of the C2 cycle. The newly identified GLYK is both structurally and phylogenetically distinct from known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated.