Tissue- and time-dependent metabolite profiles during early grain development under normal and high night-time temperature conditions.

BACKGROUND: Wheat grain development in the first few days after pollination determines the number of endosperm cells that influence grain yield potential and is susceptible to various environmental conditions, including high night temperatures (HNTs). Flag leaves and seed-associated bracts (glumes, awn, palea, and lemma) provide nutrients to the developing seed. However, the specific metabolic roles of these tissues are uncertain, especially their dynamics at different developmental stages and the time in a day. Tissue- and time-dependent metabolite profiling may hint at the metabolic roles of tissues and the mechanisms of how HNTs affect daytime metabolic status in early grain development. RESULTS: The metabolite profiles of flag leaf, bract, seed (embryo and endosperm), and entire spike were analyzed at 12:00 (day) and 23:00 (night) on 2, 4, and 6 days after fertilization under control and HNT conditions. The metabolite levels in flag leaves and bracts showed day/night oscillations, while their behaviors were distinct between the tissues. Some metabolites, such as sucrose, cellobiose, and succinic acid, showed contrasting oscillations in the two photosynthetic tissues. In contrast, seed metabolite levels differed due to the days after fertilization rather than the time in a day. The seed metabolite profile altered earlier in the HNT than in the control condition, likely associated with accelerated grain development caused by HNT. HNT also disrupted the day/night oscillation of sugar accumulation in flag leaves and bracts. CONCLUSIONS: These results highlight distinct metabolic roles of flag leaves and bracts during wheat early seed development. The seed metabolite levels are related to the developmental stages. The early metabolic events in the seeds and the disruption of the day/night metabolic cycle in photosynthetic tissues may partly explain the adverse effects of HNT on grain yield.

Vitamin E biofortification: Maximizing oilseed tocotrienol and total vitamin E tocochromanol production by use of metabolic bypass combinations.

Vitamin E tocochromanols are generated in plants by prenylation of homogentisate using geranylgeranyl diphosphate (GGDP) for tocotrienol biosynthesis and phytyl diphosphate (PDP) for tocopherol biosynthesis. Homogentisate geranylgeranyl transferase (HGGT), which uses GGDP for prenylation, is a proven target for oilseed tocochromanol biofortification that effectively bypasses the chlorophyll-linked pathway that limits PDP for vitamin E biosynthesis. In this report, we explored the feasibility of maximizing tocochromanol production in the oilseed crop camelina (Camelina sativa) by combining seed-specific HGGT expression with increased biosynthesis and/or reduced homogentisate catabolism. Plastid-targeted Escherichia coli TyrA-encoded chorismate mutase/prephenate dehydrogenase and Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) cDNA were co-expressed in seeds to bypass feedback-regulated steps and increase flux into homogentisate biosynthesis. Homogentisate catabolism was also suppressed by seed-specific RNAi of the gene for homogentisate oxygenase (HGO), which initiates homogentisate degradation. In the absence of HGGT expression, tocochromanols were increased by ∼2.5-fold with HPPD/TyrA co-expression, and ∼1.4-fold with HGO suppression compared to levels in non-transformed seeds. No further increase in tocochromanols was observed in HPPD/TyrA lines with the addition of HGO RNAi. HGGT expression alone increased tocochromanol concentrations in seeds by âˆ¼four-fold to ≤1400 µg/g seed weight. When combined with HPPD/TyrA co-expression, we obtained an additional three-fold increase in tocochromanol concentrations indicating that homogentisate concentrations limit HGGT's capacity for maximal tocochromanol production. The addition of HGO RNAi further increased tocochromanol concentrations to 5000 µg/g seed weight, an unprecedented tocochromanol concentration in an engineered oilseed. Metabolomic data obtained from engineered seeds provide insights into phenotypic changes associated with "extreme" tocochromanol production.

The Role of Abscisic Acid Signaling in Maintaining the Metabolic Balance Required for Arabidopsis Growth under Nonstress Conditions.

Abscisic acid (ABA) is a plant hormone that regulates a diverse range of cellular and molecular processes during development and in response to osmotic stress. In Arabidopsis (Arabidopsis thaliana), three Suc nonfermenting-1-related protein kinase2s (SnRK2s), SRK2D, SRK2E, and SRK2I, are key positive regulators involved in ABA signaling whose substrates have been well studied. Besides reduced drought-stress tolerance, the srk2d srk2e srk2i mutant shows abnormal growth phenotypes, such as an increased number of leaves, under nonstress conditions. However, it remains unclear whether, and if so how, SnRK2-mediated ABA signaling regulates growth and development. Here, we show that the primary metabolite profile of srk2d srk2e srk2i grown under nonstress conditions was considerably different from that of wild-type plants. The metabolic changes observed in the srk2d srk2e srk2i were similar to those in an ABA-biosynthesis mutant, aba2-1, and both mutants showed a higher leaf emergence rate than wild type. Consistent with the increased amounts of citrate, isotope-labeling experiments revealed that respiration through the tricarboxylic acid cycle was enhanced in srk2d srk2e srk2i These results, together with transcriptome data, indicate that the SnRK2s involved in ABA signaling modulate metabolism and leaf growth under nonstress conditions by fine-tuning flux through the tricarboxylic acid cycle.

Chloroplast competition is controlled by lipid biosynthesis in evening primroses.

In most eukaryotes, organellar genomes are transmitted preferentially by the mother, but molecular mechanisms and evolutionary forces underlying this fundamental biological principle are far from understood. It is believed that biparental inheritance promotes competition between the cytoplasmic organelles and allows the spread of so-called selfish cytoplasmic elements. Those can be, for example, fast-replicating or aggressive chloroplasts (plastids) that are incompatible with the hybrid nuclear genome and therefore maladaptive. Here we show that the ability of plastids to compete against each other is a metabolic phenotype determined by extremely rapidly evolving genes in the plastid genome of the evening primrose Oenothera Repeats in the regulatory region of accD (the plastid-encoded subunit of the acetyl-CoA carboxylase, which catalyzes the first and rate-limiting step of lipid biosynthesis), as well as in ycf2 (a giant reading frame of still unknown function), are responsible for the differences in competitive behavior of plastid genotypes. Polymorphisms in these genes influence lipid synthesis and most likely profiles of the plastid envelope membrane. These in turn determine plastid division and/or turnover rates and hence competitiveness. This work uncovers cytoplasmic drive loci controlling the outcome of biparental chloroplast transmission. Here, they define the mode of chloroplast inheritance, as plastid competitiveness can result in uniparental inheritance (through elimination of the "weak" plastid) or biparental inheritance (when two similarly "strong" plastids are transmitted).

Assessing durum wheat ear and leaf metabolomes in the field through hyperspectral data.

Hyperspectral techniques are currently used to retrieve information concerning plant biophysical traits, predominantly targeting pigments, water, and nitrogen-protein contents, structural elements, and the leaf area index. Even so, hyperspectral data could be more extensively exploited to overcome the breeding challenges being faced under global climate change by advancing high-throughput field phenotyping. In this study, we explore the potential of field spectroscopy to predict the metabolite profiles in flag leaves and ear bracts in durum wheat. The full-range reflectance spectra (visible (VIS)-near-infrared (NIR)-short wave infrared (SWIR)) of flag leaves, ears and canopies were recorded in a collection of contrasting genotypes grown in four environments under different water regimes. GC-MS metabolite profiles were analyzed in the flag leaves, ear bracts, glumes, and lemmas. The results from regression models exceeded 50% of the explained variation (adj-R2 in the validation sets) for at least 15 metabolites in each plant organ, whereas their errors were considerably low. The best regressions were obtained for malate (82%), glycerate and serine (63%) in leaves; myo-inositol (81%) in lemmas; glycolate (80%) in glumes; sucrose in leaves and glumes (68%); γ-aminobutyric acid (GABA) in leaves and glumes (61% and 71%, respectively); proline and glucose in lemmas (74% and 71%, respectively) and glumes (72% and 69%, respectively). The selection of wavebands in the models and the performance of the models based on canopy and VIS organ spectra and yield prediction are discussed. We feel that this technique will likely to be of interest due to its broad applicability in ecophysiology research, plant breeding programmes, and the agri-food industry.

Metabolic profiles of six African cultivars of cassava (Manihot esculenta Crantz) highlight bottlenecks of root yield.

Cassava is an important staple crop in sub-Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C3 mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin-Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse-grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.

The transcription factor bZIP14 regulates the TCA cycle in the diatom Phaeodactylum tricornutum.

Diatoms are amongst the most important marine microalgae in terms of biomass, but little is known concerning the molecular mechanisms that regulate their versatile metabolism. Here, the pennate diatom Phaeodactylum tricornutum was studied at the metabolite and transcriptome level during nitrogen starvation and following imposition of three other stresses that impede growth. The coordinated upregulation of the tricarboxylic acid (TCA) cycle during the nitrogen stress response was the most striking observation. Through co-expression analysis and DNA binding assays, the transcription factor bZIP14 was identified as a regulator of the TCA cycle, also beyond the nitrogen starvation response, namely in diurnal regulation. Accordingly, metabolic and transcriptional shifts were observed upon overexpression of bZIP14 in transformed P. tricornutum cells. Our data indicate that the TCA cycle is a tightly regulated and important hub for carbon reallocation in the diatom cell during nutrient starvation and that bZIP14 is a conserved regulator of this cycle.

MADS78 and MADS79 Are Essential Regulators of Early Seed Development in Rice.

MADS box transcription factors (TFs) are subdivided into type I and II based on phylogenetic analysis. The type II TFs regulate floral organ identity and flowering time, but type I TFs are relatively less characterized. Here, we report the functional characterization of two type I MADS box TFs in rice (Oryza sativa), MADS78 and MADS79 Transcript abundance of both these genes in developing seed peaked at 48 h after fertilization and was suppressed by 96 h after fertilization, corresponding to syncytial and cellularized stages of endosperm development, respectively. Seeds overexpressing MADS78 and MADS 79 exhibited delayed endosperm cellularization, while CRISPR-Cas9-mediated single knockout mutants showed precocious endosperm cellularization. MADS78 and MADS 79 were indispensable for seed development, as a double knockout mutant failed to make viable seeds. Both MADS78 and 79 interacted with MADS89, another type I MADS box, which enhances nuclear localization. The expression analysis of Fie1, a rice FERTILIZATION-INDEPENDENT SEED-POLYCOMB REPRESSOR COMPLEX2 component, in MADS78 and 79 mutants and vice versa established an antithetical relation, suggesting that Fie1 could be involved in negative regulation of MADS78 and MADS 79 Misregulation of MADS78 and MADS 79 perturbed auxin homeostasis and carbon metabolism, as evident by misregulation of genes involved in auxin transport and signaling as well as starch biosynthesis genes causing structural abnormalities in starch granules at maturity. Collectively, we show that MADS78 and MADS 79 are essential regulators of early seed developmental transition and impact both seed size and quality in rice.

Phytochromes control metabolic flux, and their action at the seedling stage determines adult plant biomass.

Phytochrome photoreceptors are known to regulate plastic growth responses to vegetation shade. However, recent reports also suggest an important role for phytochromes in carbon resource management, metabolism, and growth. Here, we use 13CO2 labelling patterns in multiallele phy mutants to investigate the role of phytochrome in the control of metabolic fluxes. We also combine quantitative data of 13C incorporation into protein and cell wall polymers, gas exchange measurements, and system modelling to investigate why biomass is decreased in adult multiallele phy mutants. Phytochrome influences the synthesis of stress metabolites such as raffinose and proline, and the accumulation of sugars, possibly through regulating vacuolar sugar transport. Remarkably, despite their modified metabolism and vastly altered architecture, growth rates in adult phy mutants resemble those of wild-type plants. Our results point to delayed seedling growth and smaller cotyledon size as the cause of the adult-stage phy mutant biomass defect. Our data signify a role for phytochrome in metabolic stress physiology and carbon partitioning, and illustrate that phytochrome action at the seedling stage sets the trajectory for adult biomass production.

A novel seed plants gene regulates oxidative stress tolerance in Arabidopsis thaliana.

Oxidative stress can lead to plant growth retardation, yield loss, and death. The atr7 mutant of Arabidopsis thaliana exhibits pronounced tolerance to oxidative stress. Using positional cloning, confirmed by knockout and RNA interference (RNAi) lines, we identified the atr7 mutation and revealed that ATR7 is a previously uncharacterized gene with orthologs in other seed plants but with no homology to genes in lower plants, fungi or animals. Expression of ATR7-GFP fusion shows that ATR7 is a nuclear-localized protein. RNA-seq analysis reveals that transcript levels of genes encoding abiotic- and oxidative stress-related transcription factors (DREB19, HSFA2, ZAT10), chromatin remodelers (CHR34), and unknown or uncharacterized proteins (AT5G59390, AT1G30170, AT1G21520) are elevated in atr7. This indicates that atr7 is primed for an upcoming oxidative stress via pathways involving genes of unknown functions. Collectively, the data reveal ATR7 as a novel seed plants-specific nuclear regulator of oxidative stress response.

Biochemical and functional characterization of a mitochondrial citrate carrier in Arabidopsis thaliana.

A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5'-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.

The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in Arabidopsis thaliana.

Nicotinamide adenine dinucleotide (NAD+ ) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD+ biosynthesis is exclusively cytosolic. Hence, NAD+ must be imported into organelles to support their metabolic functions. Three NAD+ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T-DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD+ homeostasis for both metabolic and developmental processes in plants.

Downregulation of a Mitochondrial NAD+ Transporter (NDT2) Alters Seed Production and Germination in Arabidopsis.

Despite the fundamental importance of nicotinamide adenine dinucleotide (NAD+) for metabolism, the physiological roles of NAD+ carriers in plants remain unclear. We previously characterized the Arabidopsis thaliana gene (At1g25380), named AtNDT2, encoding a protein located in the mitochondrial inner membrane, which imports NAD+ from the cytosol using ADP and AMP as counter-exchange substrates for NAD+. Here, we further investigated the physiological roles of NDT2, by isolating a T-DNA insertion line, generating an antisense line and characterizing these genotypes in detail. Reduced NDT2 expression affected reproductive phase by reducing total seed yield. In addition, reduced seed germination and retardation in seedling establishment were observed in the mutant lines. Moreover, remarkable changes in primary metabolism were observed in dry and germinated seeds and an increase in fatty acid levels was verified during seedling establishment. Furthermore, flowers and seedlings of NDT2 mutants displayed upregulation of de novo and salvage pathway genes encoding NAD+ biosynthesis enzymes, demonstrating the transcriptional control mediated by NDT2 activity over these genes. Taken together, our results suggest that NDT2 expression is fundamental for maintaining NAD+ balance amongst organelles that modulate metabolism, physiology and developmental processes of heterotrophic tissues.

NTRC Plays a Crucial Role in Starch Metabolism, Redox Balance, and Tomato Fruit Growth.

NADPH-thioredoxin reductase C (NTRC) forms a separate thiol-reduction cascade in plastids, combining both NADPH-thioredoxin reductase and thioredoxin activities on a single polypeptide. While NTRC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NTRC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NTRC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NTRC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NTRC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NTRC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NTRC regulates the osmotic balance of developing fruits. These results indicate that NTRC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality.

Appropriate Thiamin Pyrophosphate Levels Are Required for Acclimation to Changes in Photoperiod.

Thiamin pyrophosphate (TPP) is the active form of vitamin B1 and works as an essential cofactor for enzymes in key metabolic pathways, such as the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. Although its action as a coenzyme has been well documented, the roles of TPP in plant metabolism are still not fully understood. Here, we investigated the functions of TPP in the regulation of the metabolic networks during photoperiod transition using previously described Arabidopsis (Arabidopsis thaliana) riboswitch mutant plants, which accumulate thiamin vitamers. The results show that photosynthetic and metabolic phenotypes of TPP riboswitch mutants are photoperiod dependent. Additionally, the mutants are more distinct from control plants when plants are transferred from a short-day to a long-day photoperiod, suggesting that TPP also plays a role in metabolic acclimation to the photoperiod. Control plants showed changes in the amplitude of diurnal oscillation in the levels of metabolites, including glycine, maltose, and fumarate, following the photoperiod transition. Interestingly, many of these changes are not present in TPP riboswitch mutant plants, demonstrating their lack of metabolic flexibility. Our results also indicate a close relationship between photorespiration and the TCA cycle, as TPP riboswitch mutants accumulate less photorespiratory intermediates. This study shows the potential role of vitamin B1 in the diurnal regulation of central carbon metabolism in plants and the importance of maintaining appropriate cellular levels of thiamin vitamers for the plant's metabolic flexibility and ability to acclimate to an altered photoperiod.

Enhanced N-metabolites, ABA and IAA-conjugate in anthers instigate heat sensitivity in spring wheat.

Unraveling the metabolic and phytohormonal changes in anthers exposed to heat stress would help identify mechanisms regulating heat stress tolerance during the sensitive reproductive stage. Two spring wheat genotypes contrasting for heat tolerance were exposed to heat stress during heading in controlled environment chambers. Anthers were collected from main and primary spikes for metabolic and phytohormonal profiling. A significant reduction in seed set (38%), grain number (54%) and grain weight (52%) per plant was recorded in the sensitive (KSG1177) but not in the tolerant (KSG1214) genotype under heat stress compared to control. Anther metabolite accumulation did not vary quantitatively between main and primary spikes. Hierarchical clustering of the genotypes and treatments using metabolites and phytohormones revealed a distinct cluster for KSG1177 under heat stress from that of control and KSG1214. A significant increase in N-based amino acids, ABA, IAA-conjugate and a decrease in polyamines and organic acids were observed in wheat anthers exposed to heat stress. Unlike KSG1214, a significantly higher accumulation of amino acids, ABA and IAA-conjugate in anthers of the sensitive KSG1177 was recorded under heat stress. These findings provide the rationale and direction towards developing molecular markers for enhancing heat stress tolerance in wheat.

Correction: Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation.

[This corrects the article DOI: 10.1371/journal.pgen.1006490.].

Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate, and dicarboxylates.

The Arabidopsis thaliana genome contains 58 members of the solute carrier family SLC25, also called the mitochondrial carrier family, many of which have been shown to transport specific metabolites, nucleotides, and cofactors across the mitochondrial membrane. Here, two Arabidopsis members of this family, AtUCP1 and AtUCP2, which were previously thought to be uncoupling proteins and hence named UCP1/PUMP1 and UCP2/PUMP2, respectively, are assigned with a novel function. They were expressed in bacteria, purified, and reconstituted in phospholipid vesicles. Their transport properties demonstrate that they transport amino acids (aspartate, glutamate, cysteine sulfinate, and cysteate), dicarboxylates (malate, oxaloacetate, and 2-oxoglutarate), phosphate, sulfate, and thiosulfate. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. AtUCP1 and AtUCP2 catalyzed a fast counterexchange transport as well as a low uniport of substrates, with transport rates of AtUCP1 being much higher than those of AtUCP2 in both cases. The aspartate/glutamate heteroexchange mediated by AtUCP1 and AtUCP2 is electroneutral, in contrast to that mediated by the mammalian mitochondrial aspartate glutamate carrier. Furthermore, both carriers were found to be targeted to mitochondria. Metabolite profiling of single and double knockouts shows changes in organic acid and amino acid levels. Notably, AtUCP1 and AtUCP2 are the first reported mitochondrial carriers in Arabidopsis to transport aspartate and glutamate. It is proposed that the primary function of AtUCP1 and AtUCP2 is to catalyze an aspartateout/glutamatein exchange across the mitochondrial membrane and thereby contribute to the export of reducing equivalents from the mitochondria in photorespiration.

The Lack of Mitochondrial Thioredoxin TRXo1 Affects In Vivo Alternative Oxidase Activity and Carbon Metabolism under Different Light Conditions.

The alternative oxidase (AOX) constitutes a nonphosphorylating pathway of electron transport in the mitochondrial respiratory chain that provides flexibility to energy and carbon primary metabolism. Its activity is regulated in vitro by the mitochondrial thioredoxin (TRX) system which reduces conserved cysteines residues of AOX. However, in vivo evidence for redox regulation of the AOX activity is still scarce. In the present study, the redox state, protein levels and in vivo activity of the AOX in parallel to photosynthetic parameters were determined in Arabidopsis knockout mutants lacking mitochondrial trxo1 under moderate (ML) and high light (HL) conditions, known to induce in vivo AOX activity. In addition, 13C- and 14C-labeling experiments together with metabolite profiling were performed to better understand the metabolic coordination between energy and carbon metabolism in the trxo1 mutants. Our results show that the in vivo AOX activity is higher in the trxo1 mutants at ML while the AOX redox state is apparently unaltered. These results suggest that mitochondrial thiol redox systems are responsible for maintaining AOX in its reduced form rather than regulating its activity in vivo. Moreover, the negative regulation of the tricarboxylic acid cycle by the TRX system is coordinated with the increased input of electrons into the AOX pathway. Under HL conditions, while AOX and photosynthesis displayed similar patterns in the mutants, photorespiration is restricted at the level of glycine decarboxylation most likely as a consequence of redox imbalance.

The Extra-Pathway Interactome of the TCA Cycle: Expected and Unexpected Metabolic Interactions.

The plant tricarboxylic acid (TCA) cycle provides essential precursors for respiration, amino acid biosynthesis, and general nitrogen metabolism; moreover, it is closely involved in biotic stress responses and cellular redox homeostasis. To further understand the in vivo function of the TCA cycle enzymes, we combined affinity purification with proteomics to generate a comprehensive extra-pathway protein-protein interaction network of the plant TCA cycle. We identified 125 extra-pathway interactions in Arabidopsis (Arabidopsis thaliana) mostly related to the mitochondrial electron transport complex/ATP synthesis and amino acid metabolism but also to proteins associated with redox stress. We chose three high-scoring and two low-scoring interactions for complementary bimolecular fluorescence complementation and yeast two-hybrid assays, which highlighted the reliability of our approach, supported the intimate involvement of TCA cycle enzymes within many biological processes, and reflected metabolic changes reported previously for the corresponding mutant lines. To analyze the function of a subset of these interactions, we selected two mutants of mitochondrial glutaredoxin S15 and Amidase, which have not yet been analyzed with respect to their TCA cycle function, and performed metabolite profiling and flux analysis. Consistent with their interactions identified in this study, TCA cycle metabolites and the relative TCA flux of the two mutants were altered significantly.