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Background: Salt stress is one of the main constraints determining crop productivity, and therefore one of the main limitations for food production. The aim of this study was to characterize the salt stress response at the physiological and molecular level of different Broccoli (Brassica oleracea L. var. Italica Plenck) cultivars that were previously characterized in field and greenhouse trials as salt sensitive or salt tolerant. This study aimed to identify functional and molecular traits capable of predicting the ability of uncharacterized lines to cope with salt stress. For this purpose, this study measured different physiological parameters, hormones and metabolites under control and salt stress conditions. Results: This study found significant differences among cultivars for stomatal conductance, transpiration, methionine, proline, threonine, abscisic acid, jasmonic acid and indolacetic acid. Salt tolerant cultivars were shown to accumulate less sodium and potassium in leaves and have a lower sodium to potassium ratio under salt stress. Analysis of primary metabolites indicated that salt tolerant cultivars have higher concentrations of several intermediates of the Krebs cycle and the substrates of some anaplerotic reactions. Conclusions: This study has found that the energetic status of the plant, the sodium extrusion and the proline content are the limiting factors for broccoli tolerance to salt stress. Our results establish physiological and molecular traits useful as distinctive markers to predict salt tolerance in Broccoli or to design novel biotechnological or breeding strategies for improving broccoli tolerance to salt stress. [ABSTRACT FROM AUTHOR]

Dark, that is, nonphototrophic, microbial CO2 fixation occurs in a large range of soils. However, it is still not known whether dark microbial CO2 fixation substantially contributes to the C balance of soils and what factors control this process. Therefore, the objective of this study was to quantitate dark microbial CO2 fixation in temperate forest soils, to determine the relationship between the soil CO2 concentration and dark microbial CO2 fixation, and to estimate the relative contribution of different microbial groups to dark CO2 fixation. For this purpose, we conducted a 13C‐CO2 labeling experiment. We found that the rates of dark microbial CO2 fixation were positively correlated with the CO2 concentration in all soils. Dark microbial CO2 fixation amounted to up to 320 µg C kg−1 soil day−1 in the Ah horizon. The fixation rates were 2.8–8.9 times higher in the Ah horizon than in the Bw1 horizon. Although the rates of dark microbial fixation were small compared to the respiration rate (1.2%–3.9% of the respiration rate), our findings suggest that organic matter formed by microorganisms from CO2 contributes to the soil organic matter pool, especially given that microbial detritus is more stable in soil than plant detritus. Phospholipid fatty acid analyses indicated that CO2 was mostly fixed by gram‐positive bacteria, and not by fungi. In conclusion, our study shows that the dark microbial CO2 fixation rate in temperate forest soils increases in periods of high CO2 concentrations, that dark microbial CO2 fixation is mostly accomplished by gram‐positive bacteria, and that dark microbial CO2 fixation contributes to the formation of soil organic matter. [ABSTRACT FROM AUTHOR]

Pyruvate carboxylase of Corynebacterium glutamicum serves as anaplerotic enzyme when cells are growing on carbohydrates and plays an important role in the industrial production of metabolites derived from the tricarboxylic acid cycle, such as l-glutamate or l-lysine. Previous studies suggested that the enzyme from C. glutamicum is very labile, as activity could only be measured in permeabilized cells, but not in cell-free extracts. In this study, we established conditions allowing activity measurements in cell-free extracts of C. glutamicum and purification of the enzyme by avidin affinity chromatography and gel filtration. Using a coupled enzymatic assay with malate dehydrogenase, Vmax values between 20 and 25 μmol min−1 mg−1 were measured for purified pyruvate carboxylase corresponding to turnover numbers of 160 – 200 s−1 for the tetrameric enzyme. The concentration dependency for pyruvate and ATP followed Michaelis-Menten kinetics with Km values of 3.76 ± 0.72 mM and 0.61 ± 0.13 mM, respectively. For bicarbonate, concentrations ≥5 mM were required to obtain activity and half-maximal rates were found at 13.25 ± 4.88 mM. ADP and aspartate inhibited PCx activity with apparent Ki values of 1.5 mM and 9.3 mM, respectively. Acetyl-CoA had a weak inhibitory effect, but only at low concentrations up to 50 μM. The results presented here enable further detailed biochemical and structural studies of this enzyme. [ABSTRACT FROM AUTHOR]

A gluconeogenic precursor is a biochemical compound acted on by a gluconeogenic pathway enabling the net synthesis of glucose. Recognized gluconeogenic precursors in fasting placental mammals include glycerol, lactate/pyruvate, certain amino acids, and odd-chain length fatty acids. Each of these precursors is capable of contributing net amounts of carbon to glucose synthesis via the tricarboxylic acid cycle (TCA cycle) because they are anaplerotic, that is, they are able to increase the pools of TCA cycle intermediates by the contribution of more carbon than is lost via carbon dioxide. The net synthesis of glucose from even-chain length fatty acids (ECFAs) in fasting placental mammals, via the TCA cycle alone, is not possible because equal amounts of carbon are lost via carbon dioxide as is contributed from fatty acid oxidation via acetyl-CoA. Therefore, ECFAs do not meet the criteria to be recognized as a gluconeogenic precursor via the TCA cycle alone. ECFAs are gluconeogenic precursors in organisms with a functioning glyoxylate cycle, which enables the net contribution of carbon to the intermediates of the TCA cycle from ECFAs and the net synthesis of glucose. The net conversion of ECFAs to glucose in fasting placental mammals via C3 metabolism of acetone may be a competent though inefficient metabolic path by which ECFA could be considered a gluconeogenic precursor. Defining a substrate as a gluconeogenic precursor requires careful articulation of the definition, organism, and physiologic conditions under consideration. [ABSTRACT FROM AUTHOR]

Aims: Detailed knowledge about the enzymes responsible for conversion of C3 and C4 compounds will be helpful to establish the bacterial strain Ralstonia eutropha as platform for the production of biotechnologically interesting compounds. Although various studies about these enzymes were accomplished in the past, some contradicting information about the enzyme pattern in this bacterium still exists. To resolve these discrepancies, the C3/C4 metabolism was reinvestigated after the genome sequence of this bacterium became available. Methods and Results: In silico analysis of genome sequence revealed putative genes coding for NAD(P)+-dependent malic enzymes (Mae), phoshoenolpyruvate carboxykinase (Pck), phosphoenolpyruvate carboxylase (Ppc), phosphoenolpyruvate synthase (Pps) and pyruvate carboxylase (Pyc). Reverse transcription PCR revealed constitutive expression of mae and pck genes, whereas no transcripts of pyc and ppc were found. Expression of active NADP+-dependent MaeB and Pck and absence of Pyc and Ppc was confirmed by spectrophotometric enzyme assays. Conclusions: The data reported in this study suggest that two enzymes, (i) MaeB and (ii) Pck, mediate between the C3 and C4 intermediates in R. eutropha H16. The enzymatic conversion of pyruvate into phosphoenolpyruvate (PEP) is catalysed by Pps, and an NADH+-dependent Mdh mediates the reversible conversion of malate and oxaloacetate. Significance and Impact of the Study: An increased knowledge of the enzymes mediating between C3 and C4 intermediates in R. eutropha will facilitate metabolic engineering. [ABSTRACT FROM AUTHOR]

Soils are the largest terrestrial organic carbon pool and the largest terrestrial source of atmospheric CO 2. Non-phototrophic CO 2 fixation by microbes re-fixes and recycles CO 2 respired in soils. Our previous study showed that in temperate deciduous forest soil profiles, rates of dark CO 2 fixation were proportional to microbial biomass, irrespective of soil depth. However, the amount and quality of organic matter entering different soil depths vary for different temperate forest types and these influence microbial communities with unknown consequences for CO 2 fixation rates. To test whether differences in the amount and quality of SOC caused by tree species affect dark CO 2 fixation rates with depth, we conducted a study using acidic soils from two forest plots from the Schorfheide-Chorin Exploratory, Germany. These soils, dominated by either beech (deciduous) or pine (coniferous) tree stands differ in their SOC content and quality. We traced the incorporation of 2% (v:v) 13C–CO 2 label into microbial biomass and estimated the CO 2 fixation rates relative to microbial biomass carbon content (in μg C g MBC−1 d−1) across the soil profiles. The rates of dark CO 2 fixation per g MBC were similar across the beech soil profiles, but significantly lower in the pine soils at the B2 and BC horizons, suggesting that while dark CO 2 fixation rates are linked to MBC in deciduous forest soils, other factors influence dark CO 2 fixation rates in coniferous forest soils. The pine subsoils had low SOC content and quality, with a microbial community enriched with heterotrophic fermenters like Chloroflexi that are predicted to have a lower potential for heterotrophic CO 2 fixation compared to the other dominant bacteria phyla. In contrast, the beech soil profiles, characterized by higher SOC inputs, featured higher fractions of copiotrophs like Proteobacteria, Acidobacteria, and Actinobacteria predicted to have high heterotrophic CO 2 fixation potential. We thus speculate that in contrast to the beech soils, lower SOC inputs in the pine subsoils affected the community composition, leading to lower CO 2 fixation rates. We further made comparisons to soils dominated by mixed deciduous trees and featuring a higher MBC and SOC content. Over this range of temperate forest soils, CO 2 fixation rates were highest in the mixed deciduous forest soils, with MBC and Shannon index (used as a proxy parameter for community composition) showing the strongest correlations with the varying CO 2 fixation rates. Our study suggests that predictions of dark CO 2 fixation rates need to consider tree-species specific or site-specific conditions, as these may alter root carbon inputs and affect microbial community composition and their metabolic CO 2 fixation potentials in soil. • Dark CO 2 fixation rates correlate with MBC in beech soil profiles. • In pine subsoils, CO 2 fixation rates per gram MBC are lower than in beech soils. • Lower rates in pine subsoils reflect distinct subsoil communities and SOC inputs. • Comparing 3 temperate forests, microbial communities control CO 2 fixation rates. • SOC & vegetation effects on the community explain their control on fixation rates. [ABSTRACT FROM AUTHOR]

Our aim in this commentary is to provide evidence that certain oxoacids formed in anaplerotic reactions control cell proliferation/apoptosis. In tumour cells with impaired Krebs cycle enzymes, some anaplerotic reactions do compensate for the deficit in oxoacids. One of these, oxaloacetate, derived from the transamination of asparagine but not of aspartate, is decarboxylated 4-fold more efficiently in polyoma-virus transformed cells than in their non-transformed counterparts. The deamidation of asparagine, in the cell culture medium, to aspartate by asparaginase decreases asparagine transamination and inhibits concomitantly the growth of asparaginase-sensitive lymphoma cells, suggesting a causal relationship between asparagine transamination and growth. Another oxoacid that can provide ATP when metabolised in mitochondria, but by the branched-chain oxoacid dehydrogenase complex (BCOADC), is 2-oxobutanoate. It has two origins: (a) deamination of threonine, and (b) cleavage of cystathionine, a metabolite derived from methionine. 2-Oxobutanoate in the presence of insulin promotes growth in G1/S arrested cells. But methionine also gives rise to another substrate of BCOADC, 4-methylthio-2-oxobutanoate (MTOB), which is synthesised exclusively from methylthioadenosine (MTA) by the action of MTA phosphorylase. In Met-dependent tumour cells with defective MTA phosphorylase, 2-oxobutanoate production would exceed that of MTOB. Further, BCOADC also has 3-fold greater affinity for 2-oxobutanoate than for MTOB; hence, the deficiency in 3-methylthio propionyl CoA, the final product of MTOB decarboxylation, would be exacerbated. Methional, the transient metabolic precursor in 3-methylthio propionyl CoA biosynthesis, is apoptogenic for both normal and bcl2-negative transformed cells in culture. Investigations of other causal relationships between the genes/enzymes mediating the homeostasis of anaplerotic oxoacids and cell growth/death may be worthwhile. [Copyright &y& Elsevier]

Anaplerotic reactions replenish TCA cycle intermediates during growth. In Saccharomyces cerevisiae , pyruvate carboxylase and the glyoxylate cycle have been experimentally identified to be the main anaplerotic routes during growth on glucose (C6) and ethanol (C2), respectively. The current study investigates the importance of the two isoenzymes of pyruvate carboxylase (PYC1 and PYC2) and one of the key enzymes of the glyoxylate cycle (ICL1) for growth on glycerol (C3) as a sole carbon source. As the wild-type strains of the CEN.PK family are unable to grow in pure synthetic glycerol medium, a reverse engineered derivative showing a maximum specific growth rate of 0.14 h−1 was used as the reference strain. While the deletion of PYC1 reduced the maximum specific growth rate by about 38%, the deletion of PYC2 had no significant impact, neither in the reference strain nor in the pyc1 Δ mutant. The deletion of ICL1 only marginally reduced growth of the reference strain but further decreased the growth rate of the pyc1 deletion strain by 20%. Interestingly, the triple deletion (pyc1 Δ pyc2 Δ icl1 Δ) did not show any growth. Therefore, both the pyruvate carboxylase and the glyoxylate cycle are involved in anaplerosis during growth on glycerol. [ABSTRACT FROM AUTHOR]

Glycerol is an interesting alternative carbon source in industrial bioprocesses due to its higher degree of reduction per carbon atom compared to sugars. During the last few years, significant progress has been made in improving the well-known industrial platform organism Saccharomyces cerevisiae with regard to its glycerol utilization capability, particularly in synthetic medium. This provided a basis for future metabolic engineering focusing on the production of valuable chemicals from glycerol. However, profound knowledge about the central carbon catabolism in synthetic glycerol medium is a prerequisite for such incentives. As a matter of fact, the current assumptions about the actual in vivo fluxes active on glycerol as the sole carbon source have mainly been based on omics data collected in complex media or were even deduced from studies with other non-fermentable carbon sources, such as ethanol or acetate. A number of uncertainties have been identified which particularly regard the role of the glyoxylate cycle, the subcellular localization of the respective enzymes, the contributions of mitochondrial transporters and the active anaplerotic reactions under these conditions. The review scrutinizes the current knowledge, highlights the necessity to collect novel experimental data using cells growing in synthetic glycerol medium and summarizes the current state of the art with regard to the production of valuable fermentation products from a carbon source that has been considered so far as 'non-fermentable' for the yeast S. cerevisiae. [ABSTRACT FROM AUTHOR]