Amino acid catabolism in plants

Amino acids are among the most versatile compounds in plants performing diverse functions within proteins as well as individually. The publications constituting this habilitation thesis focus on different aspects of amino acid catabolism. Not all of the biochemical pathways catalyzing degradation of the twenty proteinogenic amino acids in plants are known, and annotation of amino acid catabolic enzymes in databases and pathway maps is still insufficient for comprehensive evaluation of proteomics or transcriptomics datasets. Thus, a manually assembled pathway map covering 136 reactions involved in amino acid related pathways is presented and used for reevaluation of already published as well as interpretation of new omics datasets. This approach clearly defines gaps in the present knowledge and at the same time postulates candidate proteins for unknown reactions as a starting point for further investigation. In addition, it provides insight into the different metabolic functions of amino acid catabolism and their significance during abiotic and biotic stress response. A major topic of the work presented here is cysteine catabolism. Cysteine in addition to the amino group also contains a thiol moiety and therefore represents an intersection of carbon, nitrogen and sulfur metabolism. The degradation of cysteine via different enzymatic routes releases reduced sulfur in the form of sulfide or persulfide, which act as signaling molecules and can be further metabolized either by reincorporation into cysteine or by oxidation. This thesis describes a mitochondrial cysteine catabolic pathway that oxidizes the thiol group catalyzed by the sulfur dioxygenase ETHE1. In plants, this pathway is essential during early embryo development and also required for energy metabolism during low light availability. Mutations in the ETHE1 gene in humans cause the metabolic disease ethylmalonic encephalopathy, which is characterized by rapidly progressive encephalopathy, chronic diarrhea, and peripheral microangio¬pathy caused by accumulation of toxic sulfide levels. These symptoms can be relieved by combined treatment with a bactericide that represses sulfide production by intestinal anaerobes and N-acetylcysteine as a precursor for glutathione, which can act as a buffer for reduced sulfur

News about amino acid metabolism in plant–microbe interactions

Plants constantly come into contact with a diverse mix of pathogenic and beneficial microbes. The ability to distinguish between them and to respond appropriately is essential for plant health. Here we review recent progress in understanding the role of amino acid sensing, signaling, transport, and metabolism during plant–microbe interactions. Biochemical pathways converting individual amino acids into active compounds have recently been elucidated, and comprehensive large-scale approaches have brought amino acid sensors and transporters into focus. These findings show that plant central amino acid metabolism is closely interwoven with stress signaling and defense responses at various levels. The individual biochemical mechanisms and the interconnections between the different processes are just beginning to emerge and might serve as a foundation for new plant protection strategies

Die Proteinausstattung eines einzelnen pflanzlichen Mitochondriums

The structure and function of mitochondria have been characterized with increasing precision. How the protein inventory defines the characteristics of the organelle remains insufficiently understood, however. Recently we devised a quantitative proteomic approach to estimate the copy numbers of proteins in a single plant mitochondrion, as physical operational unit in the cell. We illustrate how such a simple thought experiment can give fascinating insights into how a mitochondrion works

Die Proteinausstattung eines einzelnen pflanzlichen Mitochondriums

The structure and function of mitochondria have been characterized with increasing precision. How the protein inventory defines the characteristics of the organelle remains insufficiently understood, however. Recently we devised a quantitative proteomic approach to estimate the copy numbers of proteins in a single plant mitochondrion, as physical operational unit in the cell. We illustrate how such a simple thought experiment can give fascinating insights into how a mitochondrion works

Composition and Stability of the Oxidative Phosphorylation System in the Halophile Plant Cakile maritima

Mitochondria play a central role in the energy metabolism of plants. At the same time, they provide energy for plant stress responses. We here report a first view on the mitochondrial Oxidative Phosphorylation (OXPHOS) system of the halophile (salt tolerant) plant Cakile maritima. Mitochondria were purified from suspension cultures of C. maritima and for comparison of Arabidopsis thaliana, a closely related glycophyte (salt sensitive) plant. Mitochondria were treated with digitonin and solubilized protein complexes were analyzed by 2D Blue native/SDS polyacrylamide gel electrophoresis. The OXPHOS systems of the two compared plants exhibit some distinct differences. C. maritima mitochondria include a very abundant respiratory supercomplex composed of monomeric complex I and dimeric complex III. At the same time the complexes II and IV are of reduced abundance. The stability of the OXPHOS complexes was investigated by combined salt and temperature treatments of isolated mitochondria. ATP synthase (complex V) is of increased stability in C. maritima. Also, the I + III2 supercomplex is present in high abundance during stress treatments. These results give insights into the mitochondrial contribution to the plant salt stress response

Isotope-Guided Metabolomics Reveals Divergent Incorporation of Valine into Different Flavor Precursor Classes in Chives

Plants of the genus Allium such as chives, onions or garlic produce S-alk(en)yl cysteine sulfoxides as flavor precursors. Two major representatives are S-propenyl cysteine sulfoxide (isoalliin) and S-propyl cysteine sulfoxide (propiin), which only differ by a double bond in the C3 side chain. The propenyl group of isoalliin is derived from the amino acid valine, but the source of the propyl group of propiin remains unclear. Here, we present an untargeted metabolomics approach in seedlings of chives (Allium schoenoprasum) to track mass features containing sulfur and/or 13C from labeling experiments with valine-13C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13C5, but to a much lesser extent than isoalliin and related propenyl compounds. Our findings provide new insights into the biosynthetic pathways of flavor precursors in Allium species and open new avenues for future untargeted labeling experiments

CoQ deficiency causes disruption of mitochondrial sulfide oxidation, a new pathomechanism associated with this syndrome

Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain, but it also has several other functions in the cellular metabolism. One of them is to function as an electron carrier in the reaction catalyzed by sulfide:quinone oxidoreductase (SQR), which catalyzes the first reaction in the hydrogen sulfide oxidation pathway. Therefore, SQR may be affected by CoQ deficiency. Using human skin fibroblasts and two mouse models with primary CoQ deficiency, we demonstrate that severe CoQ deficiency causes a reduction in SQR levels and activity, which leads to an alteration of mitochondrial sulfide metabolism. In cerebrum of Coq9R239X mice, the deficit in SQR induces an increase in thiosulfate sulfurtransferase and sulfite oxidase, as well as modifications in the levels of thiols. As a result, biosynthetic pathways of glutamate, serotonin, and catecholamines were altered in the cerebrum, and the blood pressure was reduced. Therefore, this study reveals the reduction in SQR activity as one of the pathomechanisms associated with CoQ deficiency syndrome

Proteome adaptations in Ethe1-deficient mice indicate a role in lipid catabolism and cytoskeleton organization via post-translational protein modifications

Synopsis Hydrogen sulfide is a physiologically relevant signalling molecule. However, circulating levels of this highly biologically active substance have to be maintained within tightly controlled limits in order to avoid toxic side effects. In patients suffering from EE (ethylmalonic encephalopathy), a block in sulfide oxidation at the level of the SDO (sulfur dioxygenase) ETHE1 leads to severe dysfunctions in microcirculation and cellular energy metabolism. We used an Ethe1-deficient mouse model to investigate the effect of increased sulfide and persulfide concentrations on liver, kidney, muscle and brain proteomes. Major disturbances in post-translational protein modifications indicate that the mitochondrial sulfide oxidation pathway could have a crucial function during sulfide signalling most probably via the regulation of cysteine S-modifications. Our results confirm the involvement of sulfide in redox regulation and cytoskeleton dynamics. In addition, they suggest that sulfide signalling specifically regulates mitochondrial catabolism of FAs (fatty acids) and BCAAs (branched-chain amino acids). These findings are particularly relevant in the context of EE since they may explain major symptoms of the disease

Quantitative multilevel analysis of central metabolism in developing oilseeds of oilseed rape during in vitro culture

Seeds provide the basis for many food, feed, and fuel products. Continued increases in seed yield, composition, and quality require an improved understanding of how the developing seed converts carbon and nitrogen supplies into storage. Current knowledge of this process is often based on the premise that transcriptional regulation directly translates via enzyme concentration into flux. In an attempt to highlight metabolic control, we explore genotypic differences in carbon partitioning for in vitro cultured developing embryos of oilseed rape (Brassica napus). We determined biomass composition as well as 79 net fluxes, the levels of 77 metabolites, and 26 enzyme activities with specific focus on central metabolism in nine selected germplasm accessions. Overall, we observed a tradeoff between the biomass component fractions of lipid and starch. With increasing lipid content over the spectrum of genotypes, plastidic fatty acid synthesis and glycolytic flux increased concomitantly, while glycolytic intermediates decreased. The lipid/starch tradeoff was not reflected at the proteome level, pointing to the significance of (posttranslational) metabolic control. Enzyme activity/flux and metabolite/flux correlations suggest that plastidic pyruvate kinase exerts flux control and that the lipid/starch tradeoff is most likely mediated by allosteric feedback regulation of phosphofructokinase and ADP-glucose pyrophosphorylase. Quantitative data were also used to calculate in vivo mass action ratios, reaction equilibria, and metabolite turnover times. Compounds like cyclic 39,59-AMP and sucrose-6-phosphate were identified to potentially be involved in so far unknown mechanisms of metabolic control. This study provides a rich source of quantitative data for those studying central metabolism

CoQ deficiency causes disruption of mitochondrial sulfide oxidation, a new pathomechanism associated with this syndrome

Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain, but it also has several other functions in the cellular metabolism. One of them is to function as an electron carrier in the reaction catalyzed by sulfide:quinone oxidoreductase (SQR), which catalyzes the first reaction in the hydrogen sulfide oxidation pathway. Therefore, SQR may be affected by CoQ deficiency. Using human skin fibroblasts and two mouse models with primary CoQ deficiency, we demonstrate that severe CoQ deficiency causes a reduction in SQR levels and activity, which leads to an alteration of mitochondrial sulfide metabolism. In cerebrum of Coq9R239X mice, the deficit in SQR induces an increase in thiosulfate sulfurtransferase and sulfite oxidase, as well as modifications in the levels of thiols. As a result, biosynthetic pathways of glutamate, serotonin, and catecholamines were altered in the cerebrum, and the blood pressure was reduced. Therefore, this study reveals the reduction in SQR activity as one of the pathomechanisms associated with CoQ deficiency syndrome.Ministerio de Economía y CompetitividadERDF/SAF2013-47761-RERDF/SAF2014-55523-RERDF/RD12/0042/0011ERDF/SAF2015-65786-RJunta de AndalucíaNIH/P01HD08064