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.

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.

The role of the electron-transfer flavoprotein: ubiquinone oxidoreductase following carbohydrate starvation in Arabidopsis cell cultures.

KEY MESSAGE: The functional absence of the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) directly impacts electrons donation to the mitochondrial electron transport chain under carbohydrate-limiting conditions without major impacts on the respiration of cell cultures. Alternative substrates (e.g., amino acids) can directly feed electrons into the mitochondrial electron transport chain (mETC) via the electron transfer flavoprotein/electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports plant respiration during stress situations. By using a cell culture system, here we investigated the responses of Arabidopsis thaliana mutants deficient in the expression of ETFQO (etfqo-1) following carbon limitation and supplied with amino acids. Our results demonstrate that isovaleryl-CoA dehydrogenase (IVDH) activity was induced during carbon limitation only in wild-type and that these changes occurred concomit with enhanced protein content. By contrast, neither the activity nor the total amount of IVDH was altered in etfqo-1 mutants. We also demonstrate that the activities of mitochondrial complexes in etfqo-1 mutants, display a similar pattern as in wild-type cells. Our findings suggest that the defect of ETFQO protein culminates with an impaired functioning of the IVDH, since no induction of IVDH activity was observed. However, the functional absence of the ETFQO seems not to cause major impacts on plant respiration under carbon limiting conditions, most likely due to other alternative electron entry pathways.

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 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.

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.

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.

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.

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.

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.

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.

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.

Comparative analysis of salt-induced changes in the root proteome of two accessions of the halophyte Cakile maritima.

NaCl stress is a major abiotic stress factor limiting the productivity and the geographical distribution of many plant species. Although halophytes are able to withstand and even to require salt in the rhizosphere, roots are the most sensitive organs to salinity. Here, we investigate the variability of salt tolerance in two Tunisian accessions of the halophyte Cakile maritima (Raoued and Djerba, harvested from the semi-arid and arid Mediterranean bioclimatic stages, respectively) with a special emphasis on the proteomic changes in roots. Seedlings were hydroponically grown for one month under salt-free conditions and subsequently at three salinities (0, 100, and 300 mM NaCl). Physiological parameters (plant growth, water content, Na+, K+ contents) and root protein profiles were analyzed. Plant biomass was higher in Raoued than in Djerba but the latter was impacted to a lesser extent by salinity, notably due to lower sodium accumulation and higher selectivity for K+. 121 and 97 salt-responsive proteins were identified in Djerba and Raoued accessions, respectively. These proteins can be assigned to several different functional categories: protein metabolism, nucleotide metabolism, amino acid metabolism, glutathione metabolism, translation and ribosome biogenesis, carbohydrate and energy metabolism, and reactive oxygen species regulation and detoxification. The comparative proteome analysis revealed that 33 proteins were salt-responsive in both accessions, while 88 and 64 proteins were salt-responsive only in the Djerba or Raoued accessions, respectively. Our results give deeper insights into the plasticity of salt-stress response of C. maritima in its native ecosystems.

Differential impact of amino acids on OXPHOS system activity following carbohydrate starvation in Arabidopsis cell suspensions.

Plant respiration mostly depends on the activity of glycolysis and the oxidation of organic acids in the tricarboxylic acid cycle to synthesize ATP. However, during stress situations plant cells also use amino acids as alternative substrates to donate electrons through the electron-transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (ETF/ETFQO) complex to the mitochondrial electron transport chain (mETC). Given this, we investigated changes of the oxidative phosphorylation (OXPHOS) system in Arabidopsis thaliana cell culture under carbohydrate starvation supplied with a range of amino acids. Induction of isovaleryl-CoA dehydrogenase (IVDH) activity was observed under carbohydrate starvation which was associated with increased amounts of IVDH protein detected by immunoblotting. Furthermore, activities of the protein complexes of the mETC were reduced under carbohydrate starvation. We also observed that OXPHOS system activity behavior is differently affected by different amino acids and that proteins associated with amino acids catabolism are upregulated in cells following carbohydrate starvation. Collectively, our results support the contention that ETF/ETFQO is an essential pathway to donate electrons to the mETC and that amino acids are alternative substrates to maintain respiration under carbohydrate starvation.

The mitochondrial complexome of Arabidopsis thaliana.

Mitochondria 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_leaves) to deposit the data and to allow straightforward access and custom data analyses.

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.

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.

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.