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The Omp85 proteins form a large membrane protein family in bacteria and eukaryotes. Omp85 proteins are composed of a C-terminal β-barrel-shaped membrane domain and one or more N-terminal polypeptide transport-associated (POTRA) domains. However, Arabidopsis thaliana contains two genes coding for Omp85 proteins without a POTRA domain. One gene is designated P39, according to the molecular weight of the encoded protein. The protein is targeted to plastids and it was established that p39 has electrophysiological properties similar to other Omp85 family members, particularly to that designated as Toc75V/Oep80. We analysed expression of the gene and characterised two T-DNA insertion mutants, focusing on alterations in photosynthetic activity, plastid ultrastructure, global expression profile and metabolome. We observed pronounced expression of P39, especially in veins. Mutants of P39 show growth aberrations, reduced photosynthetic activity and changes in plastid ultrastructure, particularly in the leaf tip. Further, they display global alteration of gene expression and metabolite content in leaves of mature plants. We conclude that the function of the plastid-localised and vein-specific Omp85 family protein p39 is important, but not essential, for maintenance of metabolic homeostasis of full-grown A. thaliana plants. Further, the function of p39 in veins influences the functionality of other plant tissues. The link connecting p39 function with metabolic regulation in mature A. thaliana is discussed., (© 2018 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

The process of dark-induced senescence in plants is relatively poorly understood, but a functional electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports respiration during carbon starvation, has recently been identified. Here, we studied the responses of Arabidopsis thaliana mutants deficient in the expression of isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase to extended darkness and other environmental stresses. Evaluations of the mutant phenotypes following carbon starvation induced by extended darkness identify similarities to those exhibited by mutants of the ETF/ETFQO complex. Metabolic profiling and isotope tracer experimentation revealed that isovaleryl-CoA dehydrogenase is involved in degradation of the branched-chain amino acids, phytol, and Lys, while 2-hydroxyglutarate dehydrogenase is involved exclusively in Lys degradation. These results suggest that isovaleryl-CoA dehydrogenase is the more critical for alternative respiration and that a series of enzymes, including 2-hydroxyglutarate dehydrogenase, plays a role in Lys degradation. Both physiological and metabolic phenotypes of the isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase mutants were not as severe as those observed for mutants of the ETF/ETFQO complex, indicating some functional redundancy of the enzymes within the process. Our results aid in the elucidation of the pathway of plant Lys catabolism and demonstrate that both isovaleryl-CoA dehydrogenase and 2-hydroxyglutarate dehydrogenase act as electron donors to the ubiquinol pool via an ETF/ETFQO-mediated route.

Plants possess acclimation responses in which structural reconfigurations adapt the photosynthetic apparatus to fluctuating illumination. Long-term acclimation involves changes in plastid and nuclear gene expression and is controlled by redox signals from photosynthesis. The kinetics of these signals and the adjustments of energetic and metabolic demands to the changes in the photosynthetic apparatus are currently poorly understood. Using a redox signaling system that preferentially excites either photosystem I or II, we measured the time-dependent impact of redox signals on the transcriptome and metabolome of Arabidopsis thaliana. We observed rapid and dynamic changes in nuclear transcript accumulation resulting in differential and specific expression patterns for genes associated with photosynthesis and metabolism. Metabolite pools also exhibited dynamic changes and indicate readjustments between distinct metabolic states depending on the respective illumination. These states reflect reallocation of energy resources in a defined and reversible manner, indicating that structural changes in the photosynthetic apparatus during long-term acclimation are additionally supported at the level of metabolism. We propose that photosynthesis can act as an environmental sensor, producing retrograde redox signals that trigger two parallel adjustment loops that coordinate photosynthesis and metabolism to adapt plant primary productivity to the environment.

Background and Aims: Oxygen can fall to low concentrations within plant tissues, either because of environmental factors that decrease the external oxygen concentration or because the movement of oxygen through the plant tissues cannot keep pace with the rate of oxygen consumption. Recent studies document that plants can decrease their oxygen consumption in response to relatively small changes in oxygen concentrations to avoid internal anoxia. The molecular mechanisms underlying this response have not been identified yet. The aim of this study was to use transcript and metabolite profiling to investigate the genomic response of arabidopsis roots to a mild decrease in oxygen concentrations., Methods: Arabidopsis seedlings were grown on vertical agar plates at 21, 8, 4 and 1 % (v/v) external oxygen for 0.5, 2 and 48 h. Roots were analysed for changes in transcript levels using Affymetrix whole genome DNA microarrays, and for changes in metabolite levels using routine GC-MS based metabolite profiling. Root extension rates were monitored in parallel to investigate adaptive changes in growth., Key Results: The results show that root growth was inhibited and transcript and metabolite profiles were significantly altered in response to a moderate decrease in oxygen concentrations. Low oxygen leads to a preferential up-regulation of genes that might be important to trigger adaptive responses in the plant. A small but highly specific set of genes is induced very early in response to a moderate decrease in oxygen concentrations. Genes that were down-regulated mainly encoded proteins involved in energy-consuming processes. In line with this, root extension growth was significantly decreased which will ultimately save ATP and decrease oxygen consumption. This was accompanied by a differential regulation of metabolite levels at short- and long-term incubation at low oxygen., Conclusions: The results show that there are adaptive changes in root extension involving large-scale reprogramming of gene expression and metabolism when oxygen concentration is decreased in a very narrow range.

Superoxide dismutases (SODs) are key components of the plant antioxidant defense system. While plastidic and cytosolic isoforms have been extensively studied, the importance of mitochondrial SOD at a cellular and whole-plant level has not been established. To address this, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated in which expression of AtMSD1, encoding the mitochondrial manganese (Mn)SOD, was suppressed by antisense. The strongest antisense line showed retarded root growth even under control growth conditions. There was evidence for a specific disturbance of mitochondrial redox homeostasis in seedlings grown in liquid culture: a mitochondrially targeted redox-sensitive green fluorescent protein was significantly more oxidized in the MnSOD-antisense background. In contrast, there was no substantial change in oxidation of cytosolically targeted redox-sensitive green fluorescent protein, nor changes in antioxidant defense components. The consequences of altered mitochondrial redox status of seedlings were subtle with no widespread increase of mitochondrial protein carbonyls or inhibition of mitochondrial respiratory complexes. However, there were specific inhibitions of tricarboxylic acid (TCA) cycle enzymes (aconitase and isocitrate dehydrogenase) and an inhibition of TCA cycle flux in isolated mitochondria. Nevertheless, total respiratory CO2 output of seedlings was not decreased, suggesting that the inhibited TCA cycle enzymes can be bypassed. In older, soil-grown plants, redox perturbation was more pronounced with changes in the amount and/or redox poise of ascorbate and glutathione. Overall, the results demonstrate that reduced MnSOD affects mitochondrial redox balance and plant growth. The data also highlight the flexibility of plant metabolism with TCA cycle inhibition having little effect on overall respiratory rates.

We evaluated the application of gas chromatography-mass spectrometry metabolic fingerprinting to classify forward genetic mutants with similar phenotypes. Mutations affecting distinct metabolic or signaling pathways can result in common phenotypic traits that are used to identify mutants in genetic screens. Measurement of a broad range of metabolites provides information about the underlying processes affected in such mutants. Metabolite profiles of Arabidopsis (Arabidopsis thaliana) mutants defective in starch metabolism and uncharacterized mutants displaying a starch-excess phenotype were compared. Each genotype displayed a unique fingerprint. Statistical methods grouped the mutants robustly into distinct classes. Determining the genes mutated in three uncharacterized mutants confirmed that those clustering with known mutants were genuinely defective in starch metabolism. A mutant that clustered away from the known mutants was defective in the circadian clock and had a pleiotropic starch-excess phenotype. These results indicate that metabolic fingerprinting is a powerful tool that can rapidly classify forward genetic mutants and streamline the process of gene discovery.

To cope with oxidative stress, the metabolic network of plant cells must be reconfigured either to bypass damaged enzymes or to support adaptive responses. To characterize the dynamics of metabolic change during oxidative stress, heterotrophic Arabidopsis (Arabidopsis thaliana) cells were treated with menadione and changes in metabolite abundance and (13)C-labeling kinetics were quantified in a time series of samples taken over a 6 h period. Oxidative stress had a profound effect on the central metabolic pathways with extensive metabolic inhibition radiating from the tricarboxylic acid cycle and including large sectors of amino acid metabolism. Sequential accumulation of metabolites in specific pathways indicated a subsequent backing up of glycolysis and a diversion of carbon into the oxidative pentose phosphate pathway. Microarray analysis revealed a coordinated transcriptomic response that represents an emergency coping strategy allowing the cell to survive the metabolic hiatus. Rather than attempt to replace inhibited enzymes, transcripts encoding these enzymes are in fact down-regulated while an antioxidant defense response is mounted. In addition, a major switch from anabolic to catabolic metabolism is signaled. Metabolism is also reconfigured to bypass damaged steps (e.g. induction of an external NADH dehydrogenase of the mitochondrial respiratory chain). The overall metabolic response of Arabidopsis cells to oxidative stress is remarkably similar to the superoxide and hydrogen peroxide stimulons of bacteria and yeast (Saccharomyces cerevisiae), suggesting that the stress regulatory and signaling pathways of plants and microbes may share common elements.

Amino acids are not only fundamental protein constituents but also serve as precursors for many essential plant metabolites. Although amino acid biosynthetic pathways in plants have been identified, pathway regulation, catabolism, and downstream metabolite partitioning remain relatively uninvestigated. Conversion of Thr to Gly and acetaldehyde by Thr aldolase (EC 4.1.2.5) was only recently shown to play a role in plant amino acid metabolism. Whereas one Arabidopsis thaliana Thr aldolase (THA1) is expressed primarily in seeds and seedlings, the other (THA2) is expressed in vascular tissue throughout the plant. Metabolite profiling of tha1 mutants identified a >50-fold increase in the seed Thr content, a 50% decrease in seedling Gly content, and few other significant metabolic changes. By contrast, homozygous tha2 mutations cause a lethal albino phenotype. Rescue of tha2 mutants and tha1 tha2 double mutants by overproduction of feedback-insensitive Thr deaminase (OMR1) shows that Gly formation by THA1 and THA2 is not essential in Arabidopsis. Seed-specific expression of feedback-insensitive Thr deaminase in both tha1 and tha2 Thr aldolase mutants greatly increases seed Ile content, suggesting that these two Thr catabolic enzymes compete for a common substrate pool.

In mammals, the electron transfer flavoprotein (ETF) is a heterodimeric protein composed of two subunits, alpha and beta, that is responsible for the oxidation of at least nine mitochondrial matrix flavoprotein dehydrogenases. Electrons accepted by ETF are further transferred to the main respiratory chain via the ETF ubiquinone oxide reductase (ETFQO). Sequence analysis of the unique Arabidopsis homologues of two subunits of ETF revealed their high similarity to both subunits of the mammalian ETF. Yeast two-hybrid experiments showed that the Arabidopsis ETFalpha and ETFbeta can form a heteromeric protein. Isolation and characterization of two independent T-DNA insertional Arabidopsis mutants of the ETFbeta gene revealed accelerated senescence and early death compared to wild-type during extended darkness. Furthermore in contrast to wild-type, the etfb mutants demonstrated a significant accumulation of several amino acids, isovaleryl CoA and phytanoyl CoA during dark-induced carbohydrate deprivation. These phenotypic characteristics of etfb mutants are broadly similar to those that we observed previously in Arabidopsis etfqo mutants, suggesting functional association between ETF and ETFQO in Arabidopsis, and confirming the essential roles of the ETF/ETFQO electron transfer complex in the catabolism of leucine and involvement in the chlorophyll degradation pathway activated during dark-induced carbohydrate deprivation.

In mammals, electron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO) and electron-transfer flavoprotein (ETF) are functionally associated, and ETF accepts electrons from at least nine mitochondrial matrix flavoprotein dehydrogenases and transfers them to ubiquinone in the inner mitochondrial membrane. In addition, the mammalian ETF/ETFQO system plays a key role in beta-oxidation of fatty acids and catabolism of amino acids and choline. By contrast, nothing is known of the function of ETF and ETFQO in plants. Sequence analysis of the unique Arabidopsis thaliana homologue of ETFQO revealed high similarity to the mammalian ETFQO protein. Moreover, green fluorescent protein cellular localization experiments suggested a mitochondrial location for this protein. RNA gel blot analysis revealed that Arabidopsis ETFQO transcripts accumulated in long-term dark-treated leaves. Analysis of three independent insertional mutants of Arabidopsis ETFQO revealed a dramatic reduction in their ability to withstand extended darkness, resulting in senescence and death within 10 d after transfer, whereas wild-type plants remained viable for at least 15 d. Metabolite profiling of dark-treated leaves of the wild type and mutants revealed a dramatic decline in sugar levels. In contrast with the wild type, the mutants demonstrated a significant accumulation of several amino acids, an intermediate of Leu catabolism, and, strikingly, high-level accumulation of phytanoyl-CoA. These data demonstrate the involvement of a mitochondrial protein, ETFQO, in the catabolism of Leu and potentially of other amino acids in higher plants and also imply a novel role for this protein in the chlorophyll degradation pathway activated during dark-induced senescence and sugar starvation.

An Arabidopsis (Arabidopsis thaliana) L. Heynh mutant deficient in an isoform of adenylate kinase (ADK; At2g37250) was isolated by reverse genetics. It contains a T-DNA insertion 377 bp downstream of the start point of transcription. The mutant lacks At2g37250 transcripts and has a mild reduction in total cellular ADK activity. Green fluorescent protein-fusion based cellular localization experiments, carried out with the full-length At2g37250, suggested a plastidial localization for this isoform. In keeping with this observation, organelle isolation experiments revealed that the loss in ADK activity was confined to the inner plastid. This plastid stroma ADK gene was found to be expressed tissue constitutively but at much higher levels in illuminated leaves. Phenotypic and biochemical analyses of the mutant revealed that it exhibited higher amino acid biosynthetic activity in the light and was characterized by an enhanced root growth. When the mutant was subjected to either continuous light or continuous dark, growth phenotypes were also observed in the shoots. While the levels of adenylates were not much altered in the leaves, the pattern of change observed in the roots was consistent with the inhibition of an ATP-consuming reaction. Taken together, these data suggest a role for the plastid stromal ADK in the coordination of metabolism and growth, but imply that the exact importance of this isoform is tissue dependent.