Amino acids have various prominent functions in plants. Besides their usage during protein biosynthesis, they also represent building blocks for several other biosynthesis pathways and play pivotal roles during signaling processes as well as in plant stress response. In general, pool sizes of the 20 amino acids differ strongly and change dynamically depending on the developmental and physiological state of the plant cell. Besides amino acid biosynthesis, which has already been investigated in great detail, the catabolism of amino acids is of central importance for adjusting their pool sizes but so far has drawn much less attention. The degradation of amino acids can also contribute substantially to the energy state of plant cells under certain physiological conditions, e.g. carbon starvation. In this review, we discuss the biological role of amino acid catabolism and summarize current knowledge on amino acid degradation pathways and their regulation in the context of plant cell physiology.

Hydrogen sulfide was known as a toxic pollutant long before its physiological functions became apparent. The main effects of sulfide poisoning are the loss of central respiratory drive due to lesions in the brain stem, and inhibition of cytochrome oxidase, leading to impaired aerobic energy metabolism [1]. Sulfide-rich environments occur naturally in the sediments and grass marshes of the intertidal zone, in deep-sea hydrothermal vents, and in the hypolimneon of eutrophic lakes [2]. Moreover, mammalian as well as invertebrate tissues, such as the body wall of the lugworm Arenicola marina, enzymatically produce sulfide [3,4].In mammals, H 2 S acts as a gaseous transmitter and regulates several physiological processes [5]. Altered sulfide metabolism is associated with a number of disorders, such as Alzheimer's disease, Down's syndrome and ulcerative colitis [6][7][8]. Within a narrow concentration range, the effects of sulfide change from physiological to highly toxic, and therefore regulatory mechanisms are necessary to control endogenous sulfide levels within the physiological range.Sulfide is catabolized mainly by oxidation to nontoxic sulfur compounds. Mammals rapidly oxidize sublethal concentrations of sulfide to sulfate and excrete it in the urine [1,9]. The main sites of sulfide oxidation are liver and colon tissues, and the enzyme activity is present in the mitochondria [10,11]. Thiosulfate is produced as an obligate intermediate by a putative sulfide oxidase, which has not yet been identified [12].A mitochondrial pathway also catalyzes sulfide oxidation in many sulfide-adapted invertebrates including polychaetes, crustaceans and bivalves [2]. The lugworm A. marina is often used to study strategies of sulfide tolerance, because it is highly abundant in sandy to muddy intertidal flats, where high micromolar concentrations of sulfide regularly occur [2]. Hydrogen sulfide is a potent toxin of aerobic respiration, but also has physiological functions as a signalling molecule and as a substrate for ATP production. A mitochondrial pathway catalyzing sulfide oxidation to thiosulfate in three consecutive reactions has been identified in rat liver as well as in the body-wall tissue of the lugworm, Arenicola marina. A membranebound sulfide : quinone oxidoreductase converts sulfide to persulfides and transfers the electrons to the ubiquinone pool. Subsequently, a putative sulfur dioxygenase in the mitochondrial matrix oxidizes one persulfide molecule to sulfite, consuming molecular oxygen. The final reaction is catalyzed by a sulfur transferase, which adds a second persulfide from the sulfide : quinone oxidoreductase to sulfite, resulting in the final product thiosulfate. This role in sulfide oxidation is an additional physiological function of the mitochondrial sulfur transferase, rhodanese.Abbreviations GSH, glutathione; GSSH, glutathione persulfide; SQR, sulfide : quinone oxidoreductase.

Ethylmalonic encephalopathy is an autosomal recessive, invariably fatal disorder characterized by early-onset encephalopathy, microangiopathy, chronic diarrhea, defective cytochrome c oxidase (COX) in muscle and brain, high concentrations of C4 and C5 acylcarnitines in blood and high excretion of ethylmalonic acid in urine. ETHE1, a gene encoding a beta-lactamase-like, iron-coordinating metalloprotein, is mutated in ethylmalonic encephalopathy. In bacteria, ETHE1-like sequences are in the same operon of, or fused with, orthologs of TST, the gene encoding rhodanese, a sulfurtransferase. In eukaryotes, both ETHE1 and rhodanese are located within the mitochondrial matrix. We created a Ethe1(-/-) mouse that showed the cardinal features of ethylmalonic encephalopathy. We found that thiosulfate was excreted in massive amounts in urine of both Ethe1(-/-) mice and humans with ethylmalonic encephalopathy. High thiosulfate and sulfide concentrations were present in Ethe1(-/-) mouse tissues. Sulfide is a powerful inhibitor of COX and short-chain fatty acid oxidation, with vasoactive and vasotoxic effects that explain the microangiopathy in ethylmalonic encephalopathy patients. Sulfide is detoxified by a mitochondrial pathway that includes a sulfur dioxygenase. Sulfur dioxygenase activity was absent in Ethe1(-/-) mice, whereas it was markedly increased by ETHE1 overexpression in HeLa cells and Escherichia coli. Therefore, ETHE1 is a mitochondrial sulfur dioxygenase involved in catabolism of sulfide that accumulates to toxic levels in ethylmalonic encephalopathy.

SUMMARYMitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein-protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot-gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re-annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource (https://complexomemap.de/at_mito_lea ves) to deposit the data and to allow straightforward access and custom data analyses.

During abiotic stress low abundant amino acids are not synthesized but they accumulate due to increased protein turnover under conditions inducing carbohydrate starvation (dehydration, salt stress, darkness) and are degraded. Metabolic adaptation is crucial for abiotic stress resistance in plants, and accumulation of specific amino acids as well as secondary metabolites derived from amino acid metabolism has been implicated in increased tolerance to adverse environmental conditions. The role of proline, which is synthesized during Arabidopsis stress response to act as a compatible osmolyte, has been well established. However, conclusions drawn about potential functions of other amino acids such as leucine, valine, and isoleucine are not entirely consistent. This study reevaluates published datasets with a special emphasis on changes in the free amino acid pool and transcriptional regulation of the associated anabolic and catabolic pathways. In order to gain a comprehensive overview about the general direction of amino acid metabolism under abiotic stress conditions a complete map of all currently known enzymatic steps involved in amino acid synthesis and degradation was assembled including also the initial steps leading to the synthesis of secondary metabolites. Microarray datasets and amino acid profiles of Arabidopsis plants exposed to dehydration, high salinity, extended darkness, cold, and heat were systematically analyzed to identify trends in fluxes of amino acid metabolism. Some high abundant amino acids such as proline, arginine, asparagine, glutamine, and GABA are synthesized during abiotic stress to act as compatible osmolytes, precursors for secondary metabolites, or storage forms of organic nitrogen. In contrast, most of the low abundant amino acids are not synthesized but they accumulate due to increased protein turnover under conditions inducing carbohydrate starvation (dehydration, salt stress, extended darkness) and are degraded.

Plant responses to abiotic stress include various modifications in amino acid metabolism. By using a hydroponic culture system, we systematically investigate modification in amino acid profiles and the proteome of Arabidopsis thaliana leaves during initial recovery from low water potential or high salinity. Both treatments elicited oxidative stress leading to a biphasic stress response during recovery. Degradation of highly abundant proteins such as subunits of photosystems and ribosomes contributed to an accumulation of free amino acids. Catabolic pathways for several low abundant amino acids were induced indicating their usage as an alternative respiratory substrate to compensate for the decreased photosynthesis. Our results demonstrate that rapid detoxification of potentially detrimental amino acids such as Lys is a priority during the initial stress recovery period. The content of Pro, which acts as a compatible osmolyte during stress, was adjusted by balancing its synthesis and catabolism both of which were induced both during and after stress treatments. The production of amino acid derived secondary metabolites was up-regulated specifically during the recovery period, and our dataset also indicates increased synthesis rates of the precursor amino acids. Overall, our results support a tight relationship between amino acid metabolism and stress responses.

Summary Mitochondria host vital cellular functions, including oxidative phosphorylation and co‐factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity‐based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage‐Dependent Anion Channel 1 to sub‐stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, gamma-glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine-synthase complex.