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ABSTRACT Malate is an important dicarboxylic acid produced from fumarate in the tricarboxylic acid cycle. Deficiencies of fumarate hydrolase (FH) and malate dehydrogenase (MDH), responsible for malate formation and metabolism, respectively, are known to cause recessive forms of neurodevelopmental disorders (NDDs). The malic enzyme isoforms, malic enzyme 1 (ME1) and 2 (ME2), are required for the conversion of malate to pyruvate. To date, there have been no reports linking deficiency of either malic enzyme isoforms to any Mendelian disease in humans. We report a patient presenting with NDD, subtle dysmorphic features, resolved dilated cardiomyopathy, and mild blood lactate elevation. Whole exome sequencing (WES) revealed a homozygous frameshift variant (c.1379_1380delTT, p.Phe460fs*22) in the malic enzyme 2 (ME2) gene resulting in truncated and unstable ME2 protein in vitro. Subsequent deletion of the yeast ortholog of human ME2 (hME2) resulted in growth arrest, which was rescued by overexpression of hME2, strongly supporting an important role of ME2 in mitochondrial function. Our results also support the pathogenicity and candidacy of the ME2 gene and variant in association with NDD. To our knowledge, this is the first report of a Mendelian human disease resulting from a biallelic variant in the ME encoding gene. Future studies are warranted to confirm ME2‐associated recessive NDD. [ABSTRACT FROM AUTHOR]

Vascular plants develop diverse root system architectures and exudates to facilitate acquisition of soil nutrients (nitrogen and phosphorus). Plant species have inherently specific properties of root system architectures and exudates, but some plants exhibit high plasticity to respond to spatiotemporal variations in soil nutrient forms and availability. This paper synthesizes evidence on how plant species diversity and root plasticity contribute to soil nutrient mobilization and uptake in plants from the tropics to the Arctic with varying plant species diversity. The development of finer roots in the surface soil is a well‐known strategy for the acquisition of limited nutrients (especially phosphorus), but the allocation of roots foraging "nutrient hotspots" in deeper soil (podzolic soil or permafrost‐affected soils) is an alternative strategy for coniferous trees tested in the Arctic and boreal forests. This contrasts with findings in tropical forests, where diverse plant species with different capacities of organic acid exudation coexist and species composition shifts in response to phosphorus deficiency. In particular, high malate exudation from roots and rhizosphere microbes stimulates phosphorus solubilization, aluminum detoxification, and lignin degradation in acidic soils. The diversity and plasticity of the root system architecture, root exudation, and the flexibility of nutrient sources mitigate nutrient limitation in soil. Root plasticity facilitating soil nutrient acquisition has a large impact on biogeochemistry and soil formation, such as podzolization, in the long term. [ABSTRACT FROM AUTHOR]

Low-molecular-weight organic acids (LMWOAs) are essential O-containing metal-binding ligands involved in maintaining metal homeostasis, various metabolic processes, and plant responses to biotic and abiotic stress. Malate, citrate, and oxalate play a crucial role in metal detoxification and transport throughout the plant. This review provides a comparative analysis of the accumulation of LMWOAs in excluders, which store metals mainly in roots, and hyperaccumulators, which accumulate metals mainly in shoots. Modern concepts of the mechanisms of LMWOA secretion by the roots of excluders and hyperaccumulators are summarized, and the formation of various metal complexes with LMWOAs in the vacuole and conducting tissues, playing an important role in the mechanisms of metal detoxification and transport, is discussed. Molecular mechanisms of transport of LMWOAs and their complexes with metals across cell membranes are reviewed. It is discussed whether different endogenous levels of LMWOAs in plants determine their metal tolerance. While playing an important role in maintaining metal homeostasis, LMWOAs apparently make a minor contribution to the mechanisms of metal hyperaccumulation, which is associated mainly with root exudates increasing metal bioavailability and enhanced xylem loading of LMWOAs. The studies of metal-binding compounds may also contribute to the development of approaches used in biofortification, phytoremediation, and phytomining. [ABSTRACT FROM AUTHOR]

In recent studies we have reported on the near-UV light-induced degradation of iron complexes of various pharmaceutical excipients, such as Fe(III)-citrate and Fe(III)-amino acid complexes. Mechanistic studies revealed a common photo-degradation pattern, i.e. the formation of carbon dioxide radical anion, a potent reducing agent, via an alkoxyl/amino radical intermediate generated by light-induced ligand-to-metal charge transfer (LMCT) involving α-hydroxycarboxylates or amino acids. Herein, we confirm the proposed general photo-degradation pathways through the study of the iron complexes of other α-hydroxycarboxylates that may be present in protein formulations, such as lactate and glycolate. The results indicate that lactate generates even higher yields of •CO 2 − as compared to citrate, suggesting a significant potential of lactate for the promotion of photo-degradation in pharmaceutical formulations. [Display omitted] [ABSTRACT FROM AUTHOR]

Summary: Although saline–alkali stress can improve tomato quality, the detailed molecular processes that balance stress tolerance and quality are not well‐understood.Our research links nitric oxide (NO) and γ‐aminobutyric acid (GABA) with the control of root malate exudation and fruit malate storage, mediated by aluminium‐activated malate transporter 9/14 (SlALMT9/14). By modifying a specific S‐nitrosylated site on pyruvate‐dependent GABA transaminase 1 (SlGABA‐TP1), we have found a way to enhance both plant's saline–alkali tolerance and fruit quality.Under saline–alkali stress, NO levels vary in tomato roots and fruits. High NO in roots leads to S‐nitrosylation of SlGABA‐TP1/2/3 at Cys316/258/316, reducing their activity and increasing GABA. This GABA then reduces malate exudation from roots and affects saline–alkali tolerance by interacting with SlALMT14. In fruits, a moderate NO level boosts SlGABA‐TP1 expression and GABA breakdown, easing GABA's block on SlALMT9 and increasing malate storage. Mutants of SlGABA‐TP1C316S that do not undergo S‐nitrosylation maintain high activity, supporting malate movement in both roots and fruits under stress.This study suggests targeting SlGABA‐TP1Cys316 in tomato breeding could significantly improve plant's saline–alkali tolerance and fruit quality, offering a promising strategy for agricultural development. [ABSTRACT FROM AUTHOR]

Bryophyllum pinnatum (BP) is a medicinal plant used to treat many conditions when taken as a leaf juice, leaves in capsules, as an ethanolic extract, and as herbal tea. These preparations have been chemically analyzed except for decoctions derived from boiled green leaves. In preparation for a clinical trial to validate BP tea as a treatment for kidney stones, we used NMR and MS analyses to characterize the saturation kinetics of the release of metabolites. During boiling of the leaves, (a) the pH decreased to 4.8 within 14 min and then stabilized; (b) regarding organic acids, citric and malic acid were released with maximum release time (tmax) = 35 min; (c) for glycoflavonoids, quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (Q-3O-ArRh), myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (M-3O-ArRh), kappinatoside, myricitrin, and quercitrin were released with tmax = 5–10 min; and (d) the total phenolic content (TPC) and the total antioxidant capacity (TAC) reached a tmax at 55 min and 61 min, respectively. In summary, 24 g of leaves boiled in 250 mL of water for 61 min ensures a maximal release of key water-soluble metabolites, including organic acids and flavonoids. These metabolites are beneficial for treating kidney stones because they target oxidative stress and inflammation and inhibit stone formation. [ABSTRACT FROM AUTHOR]

Fruit acidity is an important component of flavor quality in fleshy fruit. The accumulation of malate, the dominant organic acid in the acidity formation of most mature fruit, is highly regulated by metabolism and transportation during fruit development. The knowledge on the mechanism of fruit acidification, as well as the major genes and substances is however still limited. In the present paper, the research advances on the relevance between malate accumulation and the genes associated with malate metabolism and transportation, as well as the transcriptional regulation of malate in fruit was reviewed. Furthermore, positive future research could provide a theoretical reference for optimizing fruit quality and genetic improvement.

To improve the growth and sporulation efficiency of Bacillus subtilis, the effect of malate on the cell growth and metabolism of B. subtilis DF was investigated in a 30 L bioreactor. At 22 h of fermentation, a biomass of 3.68 × 1010 CFU/mL was obtained by malate addition to a final concentration of 1.5% during 16–27 h after the pH reached 8.0 at 16 h, which was 71.9% higher than that of the control group. Meanwhile, the number of spores was 3.63 × 1010 CFU/mL, which was 92.1% higher than that of the control group. Differential transcriptomic analysis showed that malate increased the metabolic efficiency of the gluconeogenesis and pentose phosphate pathways with 1.38 to 2.51-fold up-regulation of the zwf, pckA and fbaA genes, respectively and the metabolic efficiency of the tricarboxylic acid (TCA) cycle with 3.10-fold up-regulation of the citZ gene, reduced cellular oxidative stress with a 72.9% down-regulation of the poxL gene, a 3.88-fold up-regulation of the trxA gene, and thus a 45.9%–51.3% reduction in H2O2 concentration during 19–24 h, and enhanced the phosphorylation efficiency of Spo0A with down-regulation of the kipL, rapAD and abrB genes by 57.5%–75.9%, and up-regulation of the clpX, phrCF, spo0B and sigA genes by 1.73–13.76 folds, significantly improving the growth and sporulation efficiency of B. subtilis DF. These findings provide theoretical supports for the industrial application of B. subtilis.

Low-molecular-weight organic acids (LMWOAs) are essential O-containing metal-binding ligands involved in maintaining metal homeostasis, various metabolic processes, and plant responses to biotic and abiotic stress. Malate, citrate, and oxalate play a crucial role in metal detoxification and transport throughout the plant. This review provides a comparative analysis of the accumulation of LMWOAs in excluders, which store metals mainly in roots, and hyperaccumulators, which accumulate metals mainly in shoots. Modern concepts of the mechanisms of LMWOA secretion by the roots of excluders and hyperaccumulators are summarized, and the formation of various metal complexes with LMWOAs in the vacuole and conducting tissues, playing an important role in the mechanisms of metal detoxification and transport, is discussed. Molecular mechanisms of transport of LMWOAs and their complexes with metals across cell membranes are reviewed. It is discussed whether different endogenous levels of LMWOAs in plants determine their metal tolerance. While playing an important role in maintaining metal homeostasis, LMWOAs apparently make a minor contribution to the mechanisms of metal hyperaccumulation, which is associated mainly with root exudates increasing metal bioavailability and enhanced xylem loading of LMWOAs. The studies of metal-binding compounds may also contribute to the development of approaches used in biofortification, phytoremediation, and phytomining.

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Uncoupling protein 3 (UCP3) belongs to the mitochondrial carrier protein superfamily SLC25 and is abundant in brown adipose tissue (BAT), the heart, and muscles. The expression of UCP3 in tissues mainly dependent on fatty acid oxidation suggests its involvement in cellular metabolism and has drawn attention to its possible transport function beyond the transport of protons in the presence of fatty acids. Based on the high homology between UCP2 and UCP3, we hypothesized that UCP3 transports C4 metabolites similar to UCP2. To test this, we measured the transport of substrates against phosphate (32Pi) in proteoliposomes reconstituted with recombinant murine UCP3 (mUCP3). We found that mUCP3 mainly transports aspartate and sulfate but also malate, malonate, oxaloacetate, and succinate. The transport rates calculated from the exchange of 32Pi against extraliposomal aspartate and sulfate were 23.9 ± 5.8 and 17.5 ± 5.1 µmol/min/mg, respectively. Using site-directed mutagenesis, we revealed that mutation of R84 resulted in impaired aspartate/phosphate exchange, demonstrating its critical role in substrate transport. The difference in substrate preference between mUCP2 and mUCP3 may be explained by their different tissue expression patterns and biological functions in these tissues. [ABSTRACT FROM AUTHOR]

Chromosome aneuploidy is a common phenomenon in industrial yeast. Aneuploidy is considered one of the strategies to enhance the industrial properties of Saccharomyces cerevisiae strains. However, the effects of chromosomal aneuploidy on the brewing properties of sake have not been extensively studied. In this study, sake brewing was performed using a series of genome-wide segmental duplicated laboratory S. cerevisiae strains, and the effects of each segmentally duplicated region on sake brewing were investigated. We found that the duplication of specific chromosomal regions affected the production of organic acids and aromatic compounds in sake brewing. As organic acids significantly influence the taste of sake, we focused on the segmental duplication of chromosome II that alters malate levels. Sake yeast Kyokai No. 901 strains with segmental chromosome II duplication were constructed using a polymerase chain reaction-mediated chromosomal duplication method, and sake was brewed using the resultant aneuploid sake yeast strains. The results showed the possibility of developing sake yeast strains exhibiting low malate production without affecting ethanol production capacity. Our study revealed that aneuploidy in yeast alters the brewing properties; in particular, the aneuploidy of chromosome II alters malate production in sake brewing. In conclusion, aneuploidization can be a novel and useful tool to breed sake yeast strains with improved traits, possessing industrial significance. [ABSTRACT FROM AUTHOR]

The degenerative processes that occur during aging increase the risk of disease and impaired health. Meanwhile, interventions that target aging to promote healthy longevity are gaining interest, both academically and in the public. While nutritional and physical interventions exist, efficacy is often difficult to determine. It is therefore imperative that an aging score measuring the biological aging process is available to the wider public. However, simple, interpret, and accessible biological aging scores are lacking. Here, we developed PhysiAge, a physiological aging score based on five accessible parameters that have influence on or reflect the aging process: (1) average daily step count, (2) blood glucose, (3) systolic blood pressure, (4) sex, and (5) age. Here, we found that compared to calendar age alone, PhysiAge better predicts mortality, as well as established muscle aging markers such as decrease in NAD+ levels, increase in oxidative stress, and decline in physical functioning. In order to demonstrate the usefulness of PhysiAge in identifying relevant factors associated with decelerated aging, we calculated PhysiAges for a cohort of aged individuals and obtained mass spectrometry-based blood plasma metabolomic profiles for each individual. Here, we identified a metabolic signature of decelerated aging, which included components of the TCA cycle, including malate, citrate, and isocitrate. Higher abundance of these metabolites was associated with decelerated aging, in line with supplementation studies in model organisms. PhysiAge represents an accessible way for people to track and intervene in their aging trajectories, and identifies a metabolic signature of decelerated aging in human blood plasma, which can be further studied for its causal involvement in human aging. [ABSTRACT FROM AUTHOR]

Malic acid is the predominant organic acid in grape berries, and its content is affected by abiotic factors such as temperature (fruit zone microclimate) and water (vine water status). The objectives of this study were to explore the potential mechanisms behind the effects of vine water status on the biosynthesis and degradation of berry malic acid and the potential downstream effects on berry metabolism. This study was conducted over two growing seasons in 2021 and 2022, comprising three watering regimes: no water stress (CK), light water stress (LWS), and moderate water stress (MWS). Compared to CK, a significantly higher level of malic acid was found in berries from the MWS treatment when the berry was still hard and green (E-L 33) in both years. However, water stress reduced the malic acid content at the ripe berry harvest (E-L 38) stage. The activities of NAD-malate dehydrogenase (NAD-MDH) and pyruvate kinase (PK) were enhanced by water stress. Except for the E-L 33 stage, the activity of phosphoenolpyruvate carboxylase (PEPC) was reduced by water stress. The highest phosphoenolpyruvate carboxykinase (PEPCK) activity was observed at the berry veraison (E-L 35) stage and coincided with the onset of a decrease in the malate content. Meanwhile, the expression of VvPEPCK was consistent with its enzyme activity. This study showed that water stress changed the content of some free amino acids (GABA, proline, leucine, aspartate, and glutamate), two of which (glutamate and GABA) are primary metabolites of the GABA pathway. [ABSTRACT FROM AUTHOR]

Bryophyllum pinnatum (BP) is a medicinal plant used to treat many conditions when taken as a leaf juice, leaves in capsules, as an ethanolic extract, and as herbal tea. These preparations have been chemically analyzed except for decoctions derived from boiled green leaves. In preparation for a clinical trial to validate BP tea as a treatment for kidney stones, we used NMR and MS analyses to characterize the saturation kinetics of the release of metabolites. During boiling of the leaves, (a) the pH decreased to 4.8 within 14 min and then stabilized; (b) regarding organic acids, citric and malic acid were released with maximum release time (tmax) = 35 min; (c) for glycoflavonoids, quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (Q-3O-ArRh), myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (M-3O-ArRh), kappinatoside, myricitrin, and quercitrin were released with tmax = 5–10 min; and (d) the total phenolic content (TPC) and the total antioxidant capacity (TAC) reached a tmax at 55 min and 61 min, respectively. In summary, 24 g of leaves boiled in 250 mL of water for 61 min ensures a maximal release of key water-soluble metabolites, including organic acids and flavonoids. These metabolites are beneficial for treating kidney stones because they target oxidative stress and inflammation and inhibit stone formation.

NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. We obtained a 50–75% ammonium–sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment. [ABSTRACT FROM AUTHOR]

Ferroptosis has been linked to the pathogenesis of hepatic injury induced by ischemia/reperfusion (I/R). However, the mechanistic basis remains unclear. In this study, by using a mouse model of hepatic I/R injury, it is observed that glutathione (GSH) and cysteine depletion are associated with deficiency of the reducing power of nicotinamide adenine dinucleotide phosphate (NADPH). Genes involved in maintaining NADPH homeostasis are screened, and it is identified that I/R‐induced hepatic ferroptosis is significantly associated with reduced expression and activity of NADP+‐dependent malic enzyme 1 (Me1). Mice with hepatocyte‐specific Me1 gene deletion exhibit aggravated ferroptosis and liver injury under I/R treatment; while supplementation with L‐malate, the substrate of ME1, restores NADPH and GSH levels and eventually inhibits I/R‐induced hepatic ferroptosis and injury. A mechanistic study further reveals that downregulation of hepatic Me1 expression is largely mediated by the phosphatase and tensin homologue (PTEN)‐dependent suppression of the mechanistic target of rapamycin/sterol regulatory element‐binding protein 1 (mTOR/SREBP1) signaling pathway in hepatic I/R model. Finally, PTEN inhibitor, mTOR activator, or SREBP1 over‐expression all increase hepatic NADPH, block ferroptosis, and protect liver against I/R injury. Taken together, the findings suggest that targeting ME1 may provide new therapeutic opportunities for I/R injury and other ferroptosis‐related hepatic conditions. [ABSTRACT FROM AUTHOR]

The response of wheat (Triticum aestivum L.) plants to the soil drought at the metabolome level is still not fully explained. In addition, research focuses mainly on single periods of drought, and there is still a lack of data on the response of plants to short-term cyclical periods of drought. The key to this research was to find out whether wheat shoots are able to resume metabolism after the stress subsides and if the reaction to subsequent stress is the same. Gas chromatography coupled with mass spectrometry (GC-MS) is one of the most valuable and fast methods to discover changes in the primary metabolism of plants. The targeted GC-MS analyses of whole shoots of wheat plants exposed (at the juvenile stage of development) to short-term (five days) mild soil drought/rewatering cycles (until the start of shoot wilting) enabled us to identify 32 polar metabolites. The obtained results revealed an accumulation of sugars (sucrose, fructose, glucose, and 1-kestose), proline, and malic acid. During five days of recovery, shoots regained full turgor and continued to grow, and the levels of accumulated metabolites decreased. Similar changes in metabolic profiles were found during the second drought/rewatering cycle. However, the concentrations of glucose, proline, and malic acid were higher after the second drought than after the first one. Additionally, the concentration of total polar metabolites after each plant rewatering was elevated compared to control samples. Although our results confirm the participation of proline in wheat responses to drought, they also highlight the responsiveness of soluble carbohydrate metabolism to stress/recovery. [ABSTRACT FROM AUTHOR]

Inorganic phosphate (Pi) is often limited in soils due to precipitation with iron (Fe) and aluminum (Al). To scavenge heterogeneously distributed phosphorus (P) resources, plants have evolved a local Pi signaling pathway that induces malate secretion to solubilize the occluded Fe‐P or Al‐P oxides. In this study, we show that Pi limitation impaired brassinosteroid signaling and downregulated BRASSINAZOLE‐RESISTANT 1 (BZR1) expression in Arabidopsis thaliana. Exogenous 2,4‐epibrassinolide treatment or constitutive activation of BZR1 (in the bzr1‐D mutant) significantly reduced primary root growth inhibition under Pi‐starvation conditions by downregulating ALUMINUM‐ACTIVATED MALATE TRANSPORTER 1 (ALMT1) expression and malate secretion. Furthermore, AtBZR1 competitively suppressed the activator effect of SENSITIVITY TO PROTON RHIZOTOXICITY 1 (STOP1) on ALMT1 expression and malate secretion in Nicotiana benthamiana leaves and Arabidopsis. The ratio of nuclear‐localized STOP1 and BZR1 determined ALMT1 expression and malate secretion in Arabidopsis. In addition, BZR1‐inhibited malate secretion is conserved in rice (Oryza sativa). Our findings provide insight into plant mechanisms for optimizing the secretion of malate, an important carbon resource, to adapt to Pi‐deficiency stress. [ABSTRACT FROM AUTHOR]

Abstract Background Malic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for l-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance l-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy. Results In 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations. Conclusions This study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization.

The photon costs of photoreduction/assimilation of nitrate (NO3−) into organic nitrogen in shoots and respiratory driven NO3− and NH4+ assimilation in roots are compared for terrestrial vascular plants, considering associated pH regulation, osmotic and ontogenetic effects. Different mechanisms of neutralisation of the hydroxyl (OH−) ion necessarily generated in shoot NO3− assimilation are considered. Photoreduction/assimilation of NO3− in shoots with malic acid synthesis and either accumulation of malate in leaf vacuoles or transport of malate to roots and catabolism there have a similar cost which is around 35% less than that for root NO3− assimilation and around 20% less than that for photoreduction/assimilation of NO3−, oxalate production and storage of Ca oxalate in leaf vacuoles. The photon cost of root NH4+ assimilation with H+ efflux to the root medium is around 70% less than that of root NO3− assimilation. These differences in photon cost must be considered in the context of the use of a combination of locations of NO3− assimilation and mechanisms of acid–base regulation, and a maximum of 4.9–9.1% of total photon absorption needed for growth and maintenance that is devoted to NO3− assimilation and acid–base regulation. [ABSTRACT FROM AUTHOR]

Abstract Ferroptosis has been linked to the pathogenesis of hepatic injury induced by ischemia/reperfusion (I/R). However, the mechanistic basis remains unclear. In this study, by using a mouse model of hepatic I/R injury, it is observed that glutathione (GSH) and cysteine depletion are associated with deficiency of the reducing power of nicotinamide adenine dinucleotide phosphate (NADPH). Genes involved in maintaining NADPH homeostasis are screened, and it is identified that I/R‐induced hepatic ferroptosis is significantly associated with reduced expression and activity of NADP+‐dependent malic enzyme 1 (Me1). Mice with hepatocyte‐specific Me1 gene deletion exhibit aggravated ferroptosis and liver injury under I/R treatment; while supplementation with L‐malate, the substrate of ME1, restores NADPH and GSH levels and eventually inhibits I/R‐induced hepatic ferroptosis and injury. A mechanistic study further reveals that downregulation of hepatic Me1 expression is largely mediated by the phosphatase and tensin homologue (PTEN)‐dependent suppression of the mechanistic target of rapamycin/sterol regulatory element‐binding protein 1 (mTOR/SREBP1) signaling pathway in hepatic I/R model. Finally, PTEN inhibitor, mTOR activator, or SREBP1 over‐expression all increase hepatic NADPH, block ferroptosis, and protect liver against I/R injury. Taken together, the findings suggest that targeting ME1 may provide new therapeutic opportunities for I/R injury and other ferroptosis‐related hepatic conditions.

Uncoupling protein 3 (UCP3) belongs to the mitochondrial carrier protein superfamily SLC25 and is abundant in brown adipose tissue (BAT), the heart, and muscles. The expression of UCP3 in tissues mainly dependent on fatty acid oxidation suggests its involvement in cellular metabolism and has drawn attention to its possible transport function beyond the transport of protons in the presence of fatty acids. Based on the high homology between UCP2 and UCP3, we hypothesized that UCP3 transports C4 metabolites similar to UCP2. To test this, we measured the transport of substrates against phosphate (32Pi) in proteoliposomes reconstituted with recombinant murine UCP3 (mUCP3). We found that mUCP3 mainly transports aspartate and sulfate but also malate, malonate, oxaloacetate, and succinate. The transport rates calculated from the exchange of 32Pi against extraliposomal aspartate and sulfate were 23.9 ± 5.8 and 17.5 ± 5.1 µmol/min/mg, respectively. Using site-directed mutagenesis, we revealed that mutation of R84 resulted in impaired aspartate/phosphate exchange, demonstrating its critical role in substrate transport. The difference in substrate preference between mUCP2 and mUCP3 may be explained by their different tissue expression patterns and biological functions in these tissues.

Malic acid is the predominant organic acid in grape berries, and its content is affected by abiotic factors such as temperature (fruit zone microclimate) and water (vine water status). The objectives of this study were to explore the potential mechanisms behind the effects of vine water status on the biosynthesis and degradation of berry malic acid and the potential downstream effects on berry metabolism. This study was conducted over two growing seasons in 2021 and 2022, comprising three watering regimes: no water stress (CK), light water stress (LWS), and moderate water stress (MWS). Compared to CK, a significantly higher level of malic acid was found in berries from the MWS treatment when the berry was still hard and green (E-L 33) in both years. However, water stress reduced the malic acid content at the ripe berry harvest (E-L 38) stage. The activities of NAD-malate dehydrogenase (NAD-MDH) and pyruvate kinase (PK) were enhanced by water stress. Except for the E-L 33 stage, the activity of phosphoenolpyruvate carboxylase (PEPC) was reduced by water stress. The highest phosphoenolpyruvate carboxykinase (PEPCK) activity was observed at the berry veraison (E-L 35) stage and coincided with the onset of a decrease in the malate content. Meanwhile, the expression of VvPEPCK was consistent with its enzyme activity. This study showed that water stress changed the content of some free amino acids (GABA, proline, leucine, aspartate, and glutamate), two of which (glutamate and GABA) are primary metabolites of the GABA pathway.

This article is a Commentary on Wang et al. (2023), 239: 1014–1034. [ABSTRACT FROM AUTHOR]

Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of double-stranded RNA (dsRNA) that activates anti-infective innate immunity. The underlying mechanisms are identified as targeting pattern recognition receptors and Th1-inducing. However, whether poly I:C manipulates metabolism to implement this anti-infective function is unknown. Here, GC-MS based metabolomics was used to characterize metabolic profiles induced by different doses of poly I:C. Analysis on the dose-dependent metabolomes shows that elevation of the TCA cycle and malate with the increasing dose of ploy I:C forms the most characteristic feature of the poly I:C stimulation. Exogenous malate activates the TCA cycle and elevates survival of zebrafish infected with Vibrio alginolyticus , which is related to the elevated expression of il-1b, il-6, il-8, tnf-a , and c3b. These results reveal a previously unknown regulation of poly I:C that boosts the TCA cycle to enhance innate immunity against bacterial infection. • Metabolic profiles induced by different doses of poly I:C. was characterized. • Elevated TCA cycle and malate forms the most characteristic feature. • Malate boosts TCA cycle and elevates zebrafish survival post bacterial infection. • This is related to the elevated expression of il-1b, il-6, il-8, tnf-a , and c3b. • These reveal a metabolic modulation of poly I:C against bacterial infection. [ABSTRACT FROM AUTHOR]

Sinapate esters, which are induced in plants under ultraviolet-B (UV-B) irradiation, have important roles not only in the protection against UV-B irradiation but also in the regulation of stomatal closure. Here, we speculated that sinapate esters would function in the stomatal closure of Arabidopsis thaliana in response to UV-B. We measured the stomatal aperture size of the wild-type (WT) and bright trichomes 1 (brt1) and sinapoylglucose accumulator 1 (sng1) mutants under UV-B irradiation; the latter two mutants are deficient in the conversion of sinapic acid to sinapoylglucose (SG) and SG to sinapoylmalate (SM), respectively. Both the brt1 and sng1 plants showed smaller stomatal apertures than the WT under normal light and UV-B irradiation conditions. The accumulation of SM and malate were induced by UV-B irradiation in WT and brt1 plants but not in sng1 plants. Consistently, exogenous malate application reduced UV-B-induced stomatal closure in WT, brt1 and sng1 plants. Nonetheless, levels of reactive oxygen species (ROS), nitric oxide (NO) and cytosolic Ca2+ were higher in guard cells of the sng1 mutant than in those of the WT under normal white light and UV-B irradiation, suggesting that disturbance of sinapate metabolism induced the accumulation of these signaling molecules that promote stomatal closure. Unexpectedly, exogenous sinapic acid application prevented stomatal closure of WT, brt1 and sng1 plants. In summary, we hypothesize that SG or other sinapate esters may promote the UV-B-induced malate accumulation and stomatal closure, whereas sinapic acid inhibits the ROS-NO pathway that regulates UV-B-induced cytosolic Ca2+ accumulation and stomatal closure. [ABSTRACT FROM AUTHOR]

Background: Malic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for l-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance l-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy. Results: In 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations. Conclusions: This study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization. [ABSTRACT FROM AUTHOR]

Summary: Plants secrete malate from guard cells to apoplast under stress conditions and exogenous malate induces stomatal closure. Malate is considered an extracellular chemical signal of stomatal closure. However, the molecular mechanism of malate‐induced stomatal closure is not fully elucidated.We investigated responses of stomatal aperture, ion channels, and cytosolic Ca2+ to malate.A treatment with malate induced stomatal closure in Arabidopsis thaliana wild‐type plants, but not in the mutants deficient in the slow (S‐type) anion channel gene SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1). The treatment with malate increased S‐type anion currents in guard‐cell protoplasts of wild‐type plants but not in the slac1 mutant. In addition, extracellular rather than intracellular application of malate increased the S‐type currents of constitutively active mutants of SLAC1, which have kinase‐independent activities, in a heterologous expression system using Xenopus oocytes. The treatment with malate transiently increased cytosolic Ca2+ concentration in the wild‐type Arabidopsis guard cells and the malate‐induced stomatal closure was inhibited by the Ca2+ channel blocker and the Ca2+ chelator.These results indicate that extracellular malate directly activates SLAC1 and simultaneously stimulates Ca2+ signalling in guard cells, resulting in steady and solid activation of SLAC1 for stomatal closure. [ABSTRACT FROM AUTHOR]

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Programmed cell death (PCD) is a genetically regulated process of cell suicide essential for plant development. The 'malate valve' is a mechanism that ensures redox balance across different subcellular compartments. In broccoli, the BomMDH1 gene encodes malate dehydrogenase in mitochondria, a critical enzyme in the 'malate circulation' pathway. This study investigates the functional role of BomMDH1 in malate (MA)-induced apoptosis in bright yellow-2 (BY-2) suspension cells. Findings revealed that transgenic cells overexpressing BomMDH1 showed enhanced viability under MA-induced oxidative stress compared to wild-type (WT) cells. Overexpression of BomMDH1 also reduced levels of reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2), and malondialdehyde (MDA), while increasing the expression of antioxidant enzyme genes such as NtAPX , NtAOX1a , NtSOD , and NtMDHAR. Additionally, treatment with salicylhydroxamic acid (SHAM), a characteristic inhibitor of mitochondrial respiration, further improved the anti-apoptotic activity of BY-2 cells. Overall, these results highlighted the function of the BomMDH1 gene and the potential of SHAM treatment in mitigating oxidative stress in BY-2 suspension cells. • Malate treatment accelerated the yellowing process of broccoli. • Overexpressing BomMDH1 in BY-2 cells exhibited stronger cell viability. • Confirmed the effective role of SHAM in BY-2 suspension cells exposed to MA. [ABSTRACT FROM AUTHOR]

[Display omitted] • A key target gene ribF responsible for protein stability was identified. • Protein stability regulators based on codon combination subset were developed. • Protein stability was used to enhance the performance of microbial cell factories. • Succinate production was up to 153.36 g/L with its productivity 2.13 g/L/h. Microbial cell factories (MCF) offer an eco-friendliness and cost-effectiveness way for producing high-value chemicals. However, their productive performance is often limited due to the loss of protein stability during microbial fermentation process. Here, protein stability was rationally reprogrammed to build efficient MCF for four-carbon dicarboxylic acids production by developing a codon combination subset-based protein stability regulator (ccsPSR). First, the effect of protein stability on fermentation production was analyzed, and then the targeted genes such as ribF responsible for protein stability were identified by transcriptomic analysis. Next, ccsPSR was designed and constructed by screening and analyzing synonymous codons capable of increasing adenine frequency from 2 to 6 codon positions in the ribF gene. Finally, by ccsPSR to adjust protein stability in E. coli , succinate production was up to 153.36 g/L with its productivity 2.13 g/L/h, and malate production was up to 30.02 g/L with its yield 1.04 g/g, respectively. These results suggest that reprogramming protein stability is an effective strategy for improving the production performance of MCF. [ABSTRACT FROM AUTHOR]

Torreya grandis is a characteristic rare economic tree species in subtropical mountainous areas. The kernels of T. grandis have rich content of organic acids, and malate is the predominant organic acid in T. grandis kernels. However, the contents, biosynthesis/metabolism pathway and transcriptional regulation of malate in developing T. grandis kernels remain completely unknown. Here, the organic acid composition in developing T. grandis kernels was first analyzed. The results showed that the content of malate was increased during the maturation T. grandis kernels. A malate synthase (TgMLS) gene might be involved in the accumulation of malate based on transcriptome data, gene expression and enzyme activity analysis. Transient expression of TgMLS in tobacco resulted in the high malate synthase activity and malate content. Furthermore, a basic helix-loop-helix transcription factor (bHLH), TgbHLH87 was identified to positively regulate the TgMLS expression via directly binding the TgMLS promoter. Our finding contributes to mechanism underlying malate accumulation in T. grandis kernels.

Schizochytrium is one of the few oleaginous microalgae that produce docosahexaenoic acid (DHA)-rich lipids. In this study, global changes in gene expression levels of Schizochytrium sp. FJU-512 cultured with malate in a 15 l-bioreactor was analyzed using comparative transcriptomics. The changes were found mainly in the genes involved in oxidative phosphorylation, β-oxidation, and pentose phosphate pathways. Consequently, the global changes in genes associated with the pathways could lead to an increase in the influx throughputs of pyruvate, branched-chain amino acids, fatty acids, and vitamin B6. Our transcriptome analysis indicated pyruvate dehydrogenase E2 component and acetolactate synthase I/II/III large subunit as major contributors to acetyl-CoA biosynthesis, whereas glucose-6-phosphate dehydrogenase was indicated as the major contributor to the biosynthesis of NADPH. An increase in DHA titer of up to 22% was achieved with the addition of malate to the fed-batch culture of Schizochytrium sp. FJU-512. This study provides an alternate method to enhance DHA production in Schizochytrium sp. FJU-512 through malate induced upregulation of genes responsible for acetyl-CoA and NADPH biosynthesis. [ABSTRACT FROM AUTHOR]

A primary metabolite malate is secreted from guard cells in response to the phytohormone abscisic acid (ABA) and elevated CO2. The secreted malate subsequently facilitates stomatal closure in plants. Here, we investigated the molecular mechanism of malate-induced stomatal closure using inhibitors and ABA signaling component mutants of Arabidopsis thaliana. Malate-induced stomatal closure was impaired by a protein kinase inhibitor, K252a, and also by the disruption of a receptor-like kinase GHR1, which mediates activation of calcium ion (Ca2+) channel by reactive oxygen species (ROS) in guard cells. Malate induced ROS production in guard cells while the malate-induced stomatal closure was impaired by a peroxidase inhibitor, salicylhydroxamic acid, but not by the disruption of Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases, RBOHD and RBOHF. The malate-induced stomatal closure was impaired by Ca2+ channel blockers, verapamil, and niflumic acid. These results demonstrate that the malate signaling is mediated by GHR1 and ROS in Arabidopsis guard cells. [ABSTRACT FROM AUTHOR]

Malate dehydrogenase (MDH), which catalyzes a reversible conversion of L-malate to oxaloacetate, plays essential roles in common metabolic processes, such as the tricarboxylic acid cycle, the oxaloacetate–malate shuttle, and the glyoxylate cycle. MDH2 has lately been recognized as a promising anticancer target; however, the structural information for the human homologue with natural ligands is very limited. In this study, various complex structures of hMDH2, with its substrates and/or cofactors, were solved by X-ray crystallography, which could offer knowledge about the molecular and enzymatic mechanism of this enzyme and be utilized to design novel inhibitors. The structural comparison suggests that phosphate binds to the substrate binding site and brings the conformational change of the active loop to a closed state, which can secure the substate and cofactor to facilitate enzymatic activity. [ABSTRACT FROM AUTHOR]

Simple Summary: The changing climate scenario envisages elevated CO2 (eCO2) and drought in many parts of the world. Elevated CO2 is known to increase yields in C3 crops like rice and wheat, on the other hand, it does not cause a similar increase in C4 crops like maize and sorghum. Drought is known to reduce crop growth and yield. In this mini-review we discuss the combined effects of both eCO2 and drought which is typical of a climate change scenario. We try and explain how C3 and C4 crops are differentially affected by these two manifestations of climate change. We specifically show how eCO2 in addition to its known beneficial effects can also be effective in ameliorating the effects of drought in crops. We have critically analysed the current literature and have come up with some mechanistic explanations in terms of water relations, hormonal regulation, photosynthesis and growth, nutrient uptake, and enzyme dynamics. We present here how these processes operate across a range, from ecosystem to organismal level and from molecular to the whole plant level. The information presented will help researchers to devise strategies for adaptation in crops in agricultural systems. The changing dynamics in the climate are the primary and important determinants of agriculture productivity. The effects of this changing climate on overall productivity in agriculture can be understood when we study the effects of individual components contributing to the changing climate on plants and crops. Elevated CO2 (eCO2) and drought due to high variability in rainfall is one of the important manifestations of the changing climate. There is a considerable amount of literature that addresses climate effects on plant systems from molecules to ecosystems. Of particular interest is the effect of increased CO2 on plants in relation to drought and water stress. As it is known that one of the consistent effects of increased CO2 in the atmosphere is increased photosynthesis, especially in C3 plants, it will be interesting to know the effect of drought in relation to elevated CO2. The potential of elevated CO2 ameliorating the effects of water deficit stress is evident from literature, which suggests that these two agents are brothers in arms protecting the plant from stress rather than partners in crime, specifically for water deficit when in isolation. The possible mechanisms by which this occurs will be discussed in this minireview. Interpreting the effects of short-term and long-term exposure of plants to elevated CO2 in the context of ameliorating the negative impacts of drought will show us the possible ways by which there can be effective adaption to crops in the changing climate scenario. [ABSTRACT FROM AUTHOR]

Guard‐cell‐type aluminium‐activated malate transporters (ALMTs) are involved in stomatal closure by exporting anions from guard cells. However, their physiological and electrophysiological functions are yet to be explored. Here, we analysed the physiological and electrophysiological properties of the ALMT channels in Arabidopsis and tomato (Solanum lycopersicum). SlALMT11 was specifically expressed in tomato guard cells. External malate‐induced stomatal closure was impaired in ALMT‐suppressed lines of tomato and Arabidopsis, although abscisic acid did not influence the stomatal response in SlALMT11‐knock‐down tomato lines. Electrophysiological analyses in Xenopus oocytes showed that SlALMT11 and AtALMT12/QUAC1 exhibited characteristic bell‐shaped current‐voltage patterns dependent on extracellular malate, fumarate, and citrate. Both ALMTs could transport malate, fumarate, and succinate, but not citrate, suggesting that the guard‐cell‐type ALMTs are dicarboxylic anion channels activated by extracellular organic acids. The truncation of acidic amino acids, Asp or Glu, from the C‐terminal end of SlALMT11 or AtALMT12/QUAC1 led to the disappearance of the bell‐shaped current‐voltage patterns. Our findings establish that malate‐activated stomatal closure is mediated by guard‐cell‐type ALMT channels that require an acidic amino acid in the C‐terminus as a candidate voltage sensor in both tomato and Arabidopsis. Summary statement: Apoplastic malate induces stomatal closure mediated by ALMT‐type anion channels (SlALMT11 and AtALMT12/QUAC1) of which voltage gating requires an acidic amino acid at the C‐terminal end. [ABSTRACT FROM AUTHOR]

Cellular metabolism comprises a complex network of biochemical anabolic and catabolic processes that fuel the growth and survival of living organisms. The enzyme malate dehydrogenase (MDH) is most known for its role in oxidizing malate to oxaloacetate (OAA) in the last step of the tricarboxylic acid (TCA) cycle, but it also participates in the malate-aspartate shuttle in the mitochondria as well as the glyoxylate cycle in plants. These pathways and the specific reactions within them are dynamic and must be carefully calibrated to ensure a balance between nutrient/energy supply and demand. MDH structural and functional complexity requires a variety of regulatory mechanisms, including allosteric regulation, feedback, and competitive inhibition, which are often dependent on whether the enzyme is catalyzing its forward or reverse reaction. Given the role of MDH in central metabolism and its potential as a target for therapeutics in both cancer and infectious diseases, there is a need to better understand its regulation. The involvement of MDH in multiple pathways makes it challenging to identify which effectors are critical to its activity. Many of the in vitro experiments examining MDH regulation were done decades ago, and though allosteric sites have been proposed, none to date have been specifically mapped. This review aims to provide an overview of the current knowledge surrounding MDH regulation by its substrate, products, and other intermediates of the TCA cycle while highlighting all the gaps in our understanding of its regulatory mechanisms., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Malate, a precursor in the ruminal propionate production pathway, competes with methanogenesis for metabolic hydrogen, offering a way to reduce ruminal methane (CH 4 ) production in ruminants. However, cost considerations hinder widespread use of malate in ruminant diets. An alternative approach involves utilizing transient malate levels generated during seed germination via the glyoxylate cycle. This study investigated the methane-mitigating potential of malate-containing hydroponic fodder. Fodder samples with peak malate concentrations from alfalfa, forage pea, Italian ryegrass, rye, soybean, triticale, and wheat during germination were subjected to in vitro rumen fermentation using the Hohenheim gas test. The basal diet of in vitro fermentation comprised 40% grass silage, 40% maize silage, 15% hay, and 5% concentrate on a dry matter basis, with nutritional characteristics including 42.1% neutral detergent fiber (NDF), 25.0% acid detergent fiber, 14.0% starch, 12.7% crude protein, and 3.5% ether extract (EE), on a dry matter basis. Experimental treatments were fodder inclusion involved replacing 20% of the basal diet (20R), and additionally, 100% replacement of the silages with alfalfa d 10 and rye d 9 (SR), the 2 high-malate fodders. Reductions in CH 4 production were observed with soybean (20R, 6.7% reduction), alfalfa (20R, 6.6% reduction), and increased with rye (20R, 6.3% increase). In the setup replacing silages with high-malate fodders (SR), alfalfa decreased CH 4 production (17.7%) but increased ammonia (174%), while rye increased CH 4 production (35.8%). Organic matter digestibility increased with SR rye (12.6%). Marginal effects of dietary variables were analyzed in a Generalized Additive Model. A negative relationship between dietary malate content and CH 4 production was observed, whereas dietary NDF and starch content were positive correlated with CH 4 production. In conclusion, malate within the hydroponic fodder could potentially reduce CH 4 emissions in ruminants. However, achieving sufficient efficacy requires high malate content. Additionally, use of hydroponic fodder may increase the risk of nitrogen emissions. Animal studies are required for further investigation., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Malic acid, mainly used as acidulant and taste enhancer in the food industry, is currently produced from fossil resources. In this study, microbial L-malate production with the filamentous fungus A. oryzae using the carbon source acetate was evaluated. Acetate is for example contained in biomass-derived substrates such as lignocellulosic hydrolysates and condensates of fast pyrolysis, thus avoiding competition with food production. Since research on malic acid synthesis from acetate is limited and reported productivities and yields are low, this work aimed to improve the process. First, different cultivation temperatures were tested. This parameter was found to affect the ratio between malic and succinic acid, which is the major by-product of organic acid production with A. oryzae. At 32°C, the malate share was highest (53.7 ± 1.6%), while it was lowest at 38°C (43.3 ± 1.1%) whereas succinate represented the main product (51.5 ± 1.0%). Besides the temperature, the type of nitrogen source was also found to affect malate synthesis as well as biomass production. In the pre-culture, the biomass concentration was increased by a factor of 3.4–3.9, and germination started earlier with the complex nitrogen sources yeast extract, casein hydrolysate and peptone compared to the defined nitrogen source (NH4)2SO4. Especially with yeast extract, malate synthesis in the main culture was accelerated and the titer obtained after 48 h was about 2.6 times higher than that quantified with (NH4)2SO4. To reduce substrate inhibition in acetate medium, fed-batch and repeated-batch processes were evaluated using (NH4)2SO4 or yeast extract as nitrogen source. In the fed-batch process, the period of malate production was extended, and the maximum product concentration was increased to 11.49 ± 1.84 g/L with (NH4)2SO4 and 12.08 ± 1.25 g/L with yeast extract. In the repeated-batch process, the total acid production was highest within the first 240 h of fermentation, but optimization is required to maintain high production rates in later cycles. The lessons learned in this study will help in the development of further process strategies to maximize malate production using acetate as alternative substrate to the commonly used glucose.

Preeclampsia (PE) is a high-prevalence pregnancy disease characterized by placental insufficiency, gestational hypertension, and proteinuria. Overexpression of the A isoform of the STOX1 transcription factor (STOX1A) recapitulates PE in mice, and STOX1A overexpressing trophoblasts recapitulate PE patients hallmarks in terms of gene expression and pathophysiology. STOX1 overexpression induces nitroso-redox imbalance and mitochondrial hyper-activation. Here, by a thorough analysis on cell models, we show that STOX1 overexpression in trophoblasts alters inducible nitric oxide synthase (iNOS), nitric oxide (NO) content, the nitroso-redox balance, the antioxidant defense, and mitochondrial function. This is accompanied by specific alterations of the Krebs cycle leading to reduced l-malate content. By increasing NOS coupling using the metabolite tetrahydrobiopterin (BH4) we restore this multi-step pathway in vitro. Moving in vivo on two different rodent models (STOX1 mice and RUPP rats, alike early onset and late onset preeclampsia, respectively), we show by transcriptomics that BH4 directly reverts STOX1-deregulated gene expression including glutathione metabolism, oxidative phosphorylation, cholesterol metabolism, inflammation, lipoprotein metabolism and platelet activation, successfully treating placental hypotrophy, gestational hypertension, proteinuria and heart hypertrophy. In the RUPP rats we show that the major fetal issue of preeclampsia, Intra Uterine Growth Restriction (IUGR), is efficiently corrected. Our work posits on solid bases BH4 as a novel potential therapy for preeclampsia.

NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. We obtained a 50–75% ammonium–sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment.

The response of wheat (Triticum aestivum L.) plants to the soil drought at the metabolome level is still not fully explained. In addition, research focuses mainly on single periods of drought, and there is still a lack of data on the response of plants to short-term cyclical periods of drought. The key to this research was to find out whether wheat shoots are able to resume metabolism after the stress subsides and if the reaction to subsequent stress is the same. Gas chromatography coupled with mass spectrometry (GC-MS) is one of the most valuable and fast methods to discover changes in the primary metabolism of plants. The targeted GC-MS analyses of whole shoots of wheat plants exposed (at the juvenile stage of development) to short-term (five days) mild soil drought/rewatering cycles (until the start of shoot wilting) enabled us to identify 32 polar metabolites. The obtained results revealed an accumulation of sugars (sucrose, fructose, glucose, and 1-kestose), proline, and malic acid. During five days of recovery, shoots regained full turgor and continued to grow, and the levels of accumulated metabolites decreased. Similar changes in metabolic profiles were found during the second drought/rewatering cycle. However, the concentrations of glucose, proline, and malic acid were higher after the second drought than after the first one. Additionally, the concentration of total polar metabolites after each plant rewatering was elevated compared to control samples. Although our results confirm the participation of proline in wheat responses to drought, they also highlight the responsiveness of soluble carbohydrate metabolism to stress/recovery.

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's lymphoma with considerable heterogeneity and different clinical prognosis. However, plasma metabomics used to forecast occurrence and prognosis of DLBCL are rarely addressed. Method: A total of 65 volunteers including 22 healthy controls (Ctrl), 25 DLBCL patients newly diagnosed (ND), and 18 DLBCL patients achieving complete remission (CR) were enrolled. A gas chromatography mass spectrometry-based untargeted plasma metabolomics analysis was performed. Results: Multivariate statistical analysis displayed distinct metabolic features among Crtl, ND, and CR groups. Surprisingly, metabolic profiles of newly diagnosed DLBCL patients undergoing different prognosis showed clear and distinctive clustering. Based on the candidate metabolic biomarkers (glucose and aspartate) and clinical indicators (lymphocyte, red blood count, and hemoglobin), a distinct diagnostic equation was established showing improved diagnostic performance with an area under curve of 0.936. The enrichment of citric acid cycle, deficiency of branched chain amino acid, methionine, and cysteine in newly diagnosed DLBCL patients was closely associated with poor prognosis. In addition, we found that malate and 2-hydroxy-2-methylbutyric acid were positively correlated with the baseline tumor metabolic parameters (metabolically active tumor volume and total lesion glycolysis), and the higher abundance of plasma malate, the poorer survival. Conclusion: Our preliminary data suggested plasma metabolomics study was informative to characterize the metabolic phenotypes and forecast occurrence and prognosis of DLBCL. Malate was identified as an unfavorable metabolic biomarker for prognosis-prediction of DLBCL, which provided a new insight on risk-stratification and therapeutic targets of DLBCL. More studies to confirm these associations and investigate potential mechanisms are in the process. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus , a Gram-positive bacterium, is an opportunistic pathogen and one of the most frequent causes for community acquired and nosocomial infections that has become a major public health threat due to the increased incidence of its drug resistance. Although being a prominent pathogen, its energetic metabolism is still underexplored, and its respiratory enzymes have been escaping attention. S. aureus can adapt to different environmental conditions by performing both aerobic and anaerobic respirations, which is particularly important as it frequently colonizes niches with different oxygen concentrations. This adaptability is derived from the composition of its respiratory chain, specifically from the presence of terminal electron acceptor reductases. The plasticity of S. aureus energy metabolism is enlarged by the ten quinone reductases encoded in its genome, eight of them being monotopic proteins. The role of these proteins is critical as they connect the different catabolic pathways to the respiratory chain. In this work, we identify, describe, and revise the monotopic quinone reductases present in S. aureus , providing an integrated view of its respiratory chain. [Display omitted] • The plasticity of S. aureus energy metabolism is enlarged by quinone reductases. • S. aureus encodes ten different quinone reductases, eight of which are monotopic proteins. • Monotopic quinone reductases connect different catabolic pathways to the respiratory chain. [ABSTRACT FROM AUTHOR]

Abstract Background Microbial malic acid production is currently not able to compete economically with well-established chemical processes using fossil resources. The utilization of inexpensive biomass-based substrates containing acetate could decrease production costs and promote the development of microbial processes. Acetate is a by-product in lignocellulosic hydrolysates and fast pyrolysis products or can be synthesized by acetogens during syngas fermentation. For the fermentation of these substrates, a robust microorganism with a high tolerance for biomass-derived inhibitors is required. Aspergillus oryzae is a suitable candidate due to its high tolerance and broad substrate spectrum. To pave the path towards microbial malic acid production, the potential of acetate as a carbon source for A. oryzae is evaluated in this study. Results A broad acetate concentration range was tested both for growth and malic acid production with A. oryzae. Dry biomass concentration was highest for acetic acid concentrations of 40–55 g/L reaching values of about 1.1 g/L within 48 h. Morphological changes were observed depending on the acetate concentration, yielding a pellet-like morphology with low and a filamentous structure with high substrate concentrations. For malic acid production, 45 g/L acetic acid was ideal, resulting in a product concentration of 8.44 ± 0.42 g/L after 192 h. The addition of 5–15 g/L glucose to acetate medium proved beneficial by lowering the time point of maximum productivity and increasing malic acid yield. The side product spectrum of cultures with acetate, glucose, and cultures containing both substrates was compared, showing differences especially in the amount of oxalic, succinic, and citric acid produced. Furthermore, the presence of CaCO3, a pH regulator used for malate production with glucose, was found to be crucial also for malic acid production with acetate. Conclusions This study evaluates relevant aspects of malic acid production with A. oryzae using acetate as carbon source and demonstrates that it is a suitable substrate for biomass formation and acid synthesis. The insights provided here will be useful to further microbial malic acid production using renewable substrates.

Aluminum (Al) is the major limiting factor affecting plant productivity in acidic soils. Al3+ ions exhibit increased solubility at a pH below 5, leading to plant root tip toxicity. Alternatively, plants can perceive very low concentrations of Al3+, and Al triggers downstream signaling even at pH 5.7 without causing Al toxicity. The ALUMINUM-ACTIVATED-MALATE-TRANSPORTER (ALMT) family members act as anion channels, with some regulating the secretion of malate from root apices to chelate Al, which is a crucial mechanism for plant Al resistance. To date, the role of the ALMT gene family within the legume Medicago species has not been fully characterized. In this study, we investigated the ALMT gene family in M. sativa and M. truncatula and identified 68 MsALMTs and 18 MtALMTs , respectively. Phylogenetic analysis classified these genes into five clades, and synteny analysis uncovered genuine paralogs and orthologs. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that MtALMT8 , MtALMT9 , and MtALMT15 in clade 2-2b are expressed in both roots and root nodules, and MtALMT8 and MtALMT9 are significantly upregulated by Al in root tips. We also observed that MtALMT8 and MtALMT9 can partially restore the Al sensitivity of Atalmt1 in Arabidopsis. Moreover, transcriptome analysis examined the expression patterns of these genes in M. sativa in response to Al at both pH 5.7 and pH 4.6, as well as to protons, and found that Al and protons can independently induce some Al-resistance genes. Overall, our findings indicate that MtALMT8 and MtALMT9 may play a role in Al resistance, and highlight the resemblance between the ALMT genes in Medicago species and those in Arabidopsis. [ABSTRACT FROM AUTHOR]

Plants acclimate their photosynthetic capacity (Pmax) in response to changing environmental conditions. In Arabidopsis thaliana , photosynthetic acclimation to cold requires the accumulation of the organic acid fumarate, catalysed by a cytosolically localized fumarase, FUM2. However, the role of this accumulation is currently unknown. Here, we use an integrated experimental and modelling approach to examine the role of FUM2 and fumarate across the physiological temperature range. We have studied three genotypes: Col-0; a fum2 mutant in a Col-0 background; and C24, an accession with reduced FUM2 expression. While low temperature causes an increase in Pmax in the Col-0 plants, this parameter decreases following exposure of plants to 30 °C for 7 d. Plants in which fumarate accumulation is partially (C24) or completely (fum2) abolished show a reduced acclimation of Pmax across the physiological temperature range (i.e. Pmax changes less in response to changing temperature). To understand the role of fumarate accumulation, we have adapted a reliability engineering technique, Failure Mode and Effect Analysis (FMEA), to formalize a rigorous approach for ranking metabolites according to the potential risk that they pose to the metabolic system. FMEA identifies fumarate as a low-risk metabolite, while its precursor, malate, is shown to be high risk and liable to cause system instability. We propose that the role of FUM2 is to provide a fail-safe in order to control malate concentration, maintaining system stability in a changing environment. We suggest that FMEA is a technique that is not only useful in understanding plant metabolism but can also be used to study reliability in other systems and synthetic pathways. [ABSTRACT FROM AUTHOR]

Apricot breeding programs could be strongly improved by the availability of molecular markers linked to the main fruit quality traits. Fruit acidity is one of the key factors in consumer acceptance, but despite its importance, the molecular bases of this trait are still poorly understood. In order to increase the genetic knowledge on the fruit acidity, an F1 apricot population ('Lito' × 'BO81604311') has been phenotyped for titratable acidity and juice pH for the three following years. In addition, the contents of the main organic acids of the juice (malate, citrate, and quinate) were also evaluated. A Gaussian distribution was observed for most of the traits in this progeny, confirming their quantitative inheritance. An available simple sequence repeat (SSR)-based molecular map, implemented with new markers in specific genomic regions, was used to perform a quantitative trait loci (QTL) analysis. The molecular map was also anchored to the recently published apricot genome sequence of 'Stella.' Several major QTLs linked to fruit acidity-related traits have been identified both in the 'Lito' (no. 21) and 'BO81604311' (no. 13), distributed in five linkage groups (LG 4, 5, 6, 7, and 8). Some of these QTLs show good stability between years and their linked markers were used to identify candidate genes in specific QTLs genomic regions. [ABSTRACT FROM AUTHOR]