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.

Proteome reorganization and amino acid metabolism during germination and seedling establishment in Lupinus albus.

During germination plants rely entirely on their seed storage compounds to provide energy and precursors for the synthesis of macromolecular structures until the seedling has emerged from the soil and photosynthesis can be established. Lupin seeds use proteins as their major storage compounds, accounting for up to 40% of the seed dry weight. Lupins are therefore a valuable complement to soy as a source of plant protein for human and animal nutrition. The aim of this study was to elucidate how storage protein metabolism is coordinated with other metabolic processes to meet the requirements of the growing seedling. In a quantitative approach, we analyzed seedling growth, as well as alterations in biomass composition, the proteome, and metabolite profiles during germination and seedling establishment in Lupinus albus. The reallocation of nitrogen resources from seed storage proteins to functional seed proteins was mapped based on a manually curated functional protein annotation database. Although classified as a protein crop, Lupinus albus does not use amino acids as a primary substrate for energy metabolism during germination. However, fatty acid and amino acid metabolism may be integrated at the level of malate synthase to combine stored carbon from lipids and proteins into gluconeogenesis.

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 13 C from labeling experiments with valine-13 C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13 C5 , 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.

Estimating the number of protein molecules in a plant cell: protein and amino acid homeostasis during drought.

During drought stress, cellular proteostasis on the one hand and amino acid homeostasis on the other hand are severely challenged, because the decrease in photosynthesis induces massive proteolysis, leading to drastic changes in both the proteome and the free amino acid pool. Thus, we selected progressive drought stress in Arabidopsis (Arabidopsis thaliana) as a model to investigate on a quantitative level the balance between protein and free amino acid homeostasis. We analyzed the mass composition of the leaf proteome based on proteomics datasets, and estimated how many protein molecules are present in a plant cell and its subcellular compartments. In addition, we calculated stress-induced changes in the distribution of individual amino acids between the free and protein-bound pools. Under control conditions, an average Arabidopsis mesophyll cell contains about 25 billion protein molecules, of which 80% are localized in chloroplasts. Severe water deficiency leads to degradation of more than 40% of the leaf protein mass, and thus causes a drastic shift in distribution toward the free amino acid pool. Stress-induced proteolysis of just half of the 340 million RubisCO hexadecamers present in the chloroplasts of a single mesophyll cell doubles the cellular content of free amino acids. A major fraction of the amino acids released from proteins is channeled into synthesis of proline, which is a compatible osmolyte. Complete oxidation of the remaining fraction as an alternative respiratory substrate can fully compensate for the lack of photosynthesis-derived carbohydrates for several hours.

The function of glutaredoxin GRXS15 is required for lipoyl-dependent dehydrogenases in mitochondria.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol, and mitochondria. A single monothiol glutaredoxin (GRX) is involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homolog GRXS15 has only partially been characterized. Arabidopsis (Arabidopsis thaliana) grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to the knockdown line GRXS15amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain, and aconitase in the tricarboxylic acid (TCA) cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine, and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain α-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs. In wild-type plants, pyruvate, glycine, and BCKAs are all metabolized through decarboxylation by mitochondrial lipoyl cofactor (LC)-dependent dehydrogenase complexes. These enzyme complexes are very abundant, comprising a major sink for LC. Because biosynthesis of LC depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why LC-dependent processes are most sensitive to restricted Fe-S supply in grxs15 mutants.

Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics.

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.

The role of amino acid metabolism in signaling and metabolic adaptation to stress-induced energy deficiency in plants.

The adaptation of plant metabolism to stress-induced energy deficiency involves profound changes in amino acid metabolism. Anabolic reactions are suppressed, whereas respiratory pathways that use amino acids as alternative substrates are activated. This review highlights recent progress in unraveling the stress-induced amino acid oxidation pathways, their regulation, and the role of amino acids as signaling molecules. We present an updated map of the degradation pathways for lysine and the branched-chain amino acids. The regulation of amino acid metabolism during energy deprivation, including the coordinated induction of several catabolic pathways, is mediated by the balance between TOR and SnRK signaling. Recent findings indicate that some amino acids might act as nutrient signals in TOR activation and thus promote a shift from catabolic to anabolic pathways. The metabolism of the sulfur-containing amino acid cysteine is highly interconnected with TOR and SnRK signaling. Mechanistic details have recently been elucidated for cysteine signaling during the abscisic acid-dependent drought response. Local cysteine synthesis triggers abscisic acid production and, in addition, cysteine degradation produces the gaseous messenger hydrogen sulfide, which promotes stomatal closure via protein persulfidation. Amino acid signaling in plants is still an emerging topic with potential for fundamental discoveries.

The role of amino acid metabolism during abiotic stress release.

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.

Synthesis versus degradation: directions of amino acid metabolism during Arabidopsis abiotic stress response.

KEY MESSAGE: 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.

Dealing with the sulfur part of cysteine: four enzymatic steps degrade l-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria.

Amino acid catabolism is essential for adjusting pool sizes of free amino acids and takes part in energy production as well as nutrient remobilization. The carbon skeletons are generally converted to precursors or intermediates of the tricarboxylic acid cycle. In the case of cysteine, the reduced sulfur derived from the thiol group also has to be oxidized in order to prevent accumulation to toxic concentrations. Here we present a mitochondrial sulfur catabolic pathway catalyzing the complete oxidation of l-cysteine to pyruvate and thiosulfate. After transamination to 3-mercaptopyruvate, the sulfhydryl group from l-cysteine is transferred to glutathione by sulfurtransferase 1 and oxidized to sulfite by the sulfur dioxygenase ETHE1. Sulfite is then converted to thiosulfate by addition of a second persulfide group by sulfurtransferase 1. This pathway is most relevant during early embryo development and for vegetative growth under light-limiting conditions. Characterization of a double mutant produced from Arabidopsis thaliana T-DNA insertion lines for ETHE1 and sulfurtransferase 1 revealed that an intermediate of the ETHE1 dependent pathway, most likely a persulfide, interferes with amino acid catabolism and induces early senescence.

The mitochondrial sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 is required for amino acid catabolism during carbohydrate starvation and embryo development in Arabidopsis.

The sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) catalyzes the oxidation of persulfides in the mitochondrial matrix and is essential for early embryo development in Arabidopsis (Arabidopsis thaliana). We investigated the biochemical and physiological functions of ETHE1 in plant metabolism using recombinant Arabidopsis ETHE1 and three transfer DNA insertion lines with 50% to 99% decreased sulfur dioxygenase activity. Our results identified a new mitochondrial pathway catalyzing the detoxification of reduced sulfur species derived from cysteine catabolism by oxidation to thiosulfate. Knockdown of the sulfur dioxygenase impaired embryo development and produced phenotypes of starvation-induced chlorosis during short-day growth conditions and extended darkness, indicating that ETHE1 has a key function in situations of high protein turnover, such as seed production and the use of amino acids as alternative respiratory substrates during carbohydrate starvation. The amino acid profile of mutant plants was similar to that caused by defects in the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex and associated dehydrogenases. Thus, in addition to sulfur amino acid catabolism, ETHE1 also affects the oxidation of branched-chain amino acids and lysine.

Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III.

We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.

Modulation of sulfide oxidation and toxicity in rat mitochondria by dehydroascorbic acid.

Hydrogen sulfide is enzymatically produced in mammalian tissues and functions as a gaseous transmitter. However, H(2)S is also highly toxic as it inhibits mitochondrial respiration at the level of cytochrome c oxidase, which additionally is involved in sulfide oxidation. The accumulation of toxic sulfide levels contributes to the pathology of some diseases. This paper demonstrates that sulfide toxicity can be modified, and dehydroascorbic acid functions as an effector in this process. It significantly reduces the inhibitory effect of sulfide on cytochrome c oxidase, resulting in higher rates of respiration and sulfide oxidation in rat mitochondria. After the addition of dehydroascorbic acid mitochondria maintained more than 50% of the oxygen consumption and ATP production rates with different substrates in the presence of high concentrations of sulfide that would normally lead to complete inhibition. Dehydroascorbic acid significantly increased the sulfide concentration necessary to cause half maximal inhibition of mitochondrial respiration and thus completely prevented inhibition at low, physiological sulfide concentrations. In addition, sulfide oxidation was stimulated and led to ATP production even at high concentrations. The decrease in sulfide toxicity was more pronounced when analyzing supermolecular functional units of the respiratory chain than in isolated cytochrome c oxidase activity. Furthermore, the protective effect of dehydroascorbic acid at high sulfide concentrations was completely abolished by quantitative solubilization of mitochondrial membrane proteins with dodeclymaltoside. These results suggest that binding of cytochrome c oxidase to other proteins probably within respiratory chain supercomplexes is involved in the modulation of sulfide oxidation and toxicity by dehydroascorbic acid.

Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria.

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 membrane-bound 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.

Extended darkness induces internal turnover of glucosinolates in Arabidopsis thaliana leaves.

Prolonged darkness leads to carbohydrate starvation, and as a consequence plants degrade proteins and lipids to oxidize amino acids and fatty acids as alternative substrates for mitochondrial ATP production. We investigated, whether the internal breakdown of glucosinolates, a major class of sulfur-containing secondary metabolites, might be an additional component of the carbohydrate starvation response in Arabidopsis thaliana (A. thaliana). The glucosinolate content of A. thaliana leaves was strongly reduced after seven days of darkness. We also detected a significant increase in the activity of myrosinase, the enzyme catalyzing the initial step in glucosinolate breakdown, coinciding with a strong induction of the main leaf myrosinase isoforms TGG1 and TGG2. In addition, nitrilase activity was increased suggesting a turnover via nitriles and carboxylic acids. Internal degradation of glucosinolates might also be involved in diurnal or developmental adaptations of the glucosinolate profile. We observed a diurnal rhythm for myrosinase activity in two-week-old plants. Furthermore, leaf myrosinase activity and protein abundance of TGG2 varied during plant development, whereas leaf protein abundance of TGG1 remained stable indicating regulation at the transcriptional as well as post-translational level.

The Role of Persulfide Metabolism During Arabidopsis Seed Development Under Light and Dark Conditions.

The sulfur dioxygenase ETHE1 oxidizes persulfides in the mitochondrial matrix and is involved in the degradation of L-cysteine and hydrogen sulfide. ETHE1 has an essential but as yet undefined function in early embryo development of Arabidopsis thaliana. In leaves, ETHE1 is strongly induced by extended darkness and participates in the use of amino acids as alternative respiratory substrates during carbohydrate starvation. Thus, we tested the effect of darkness on seed development in an ETHE1 deficient mutant in comparison to the wild type. Since ETHE1 knock-out is embryo lethal, the knock-down line ethe1-1 with about 1% residual sulfur dioxygenase activity was used for this study. We performed phenotypic analysis, metabolite profiling and comparative proteomics in order to investigate the general effect of extended darkness on seed metabolism and further define the specific function of the mitochondrial sulfur dioxygenase ETHE1 in seeds. Shading of the siliques had no morphological effect on embryogenesis in wild type plants. However, the developmental delay that was already visible in ethe1-1 seeds under control conditions was further enhanced in the darkness. Dark conditions strongly affected seed quality parameters of both wild type and mutant plants. The effect of ETHE1 knock-down on amino acid profiles was clearly different from that found in leaves indicating that in seeds persulfide oxidation interacts with alanine and glycine rather than branched-chain amino acid metabolism. Sulfur dioxygenase deficiency led to defects in endosperm development possibly due to alterations in the cellularization process. In addition, we provide evidence for a potential role of persulfide metabolism in abscisic acid (ABA) signal transduction in seeds. We conclude that the knock-down of ETHE1 causes metabolic re-arrangements in seeds that differ from those in leaves. Putative mechanisms that cause the aberrant endosperm and embryo development are discussed.

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.

Sulfide detoxification in plant mitochondria.

In contrast to animals, which release the signal molecule sulfide in small amounts from cysteine and its derivates, phototrophic eukaryotes generate sulfide as an essential intermediate of the sulfur assimilation pathway. Additionally, iron-sulfur cluster turnover and cyanide detoxification might contribute to the release of sulfide in mitochondria. However, sulfide is a potent inhibitor of cytochrome c oxidase in mitochondria. Thus, efficient sulfide detoxification mechanisms are required in mitochondria to ensure adequate energy production and consequently survival of the plant cell. Two enzymes have been recently described to catalyze sulfide detoxification in mitochondria of Arabidopsis thaliana, O-acetylserine(thiol)lyase C (OAS-TL C), and the sulfur dioxygenase (SDO) ethylmalonic encephalopathy protein 1 (ETHE1). Biochemical characterization of sulfide producing and consuming enzymes in mitochondria of plants is fundamental to understand the regulatory network that enables mitochondrial sulfide homeostasis under nonstressed and stressed conditions. In this chapter, we provide established protocols to determine the activity of the sulfide releasing enzyme ß-cyanoalanine synthase as well as sulfide-consuming enzymes OAS-TL and SDO. Additionally, we describe a reliable and efficient method to purify OAS-TL proteins from plant material.

Amino Acid Catabolism in Plants.

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.