Phosphoenolpyruvate Carboxylase Research Articles

A pathogen-specific isotope tracing approach reveals metabolic activities and fluxes of intracellular Salmonella.

Pathogenic bacteria proliferating inside mammalian host cells need to rapidly adapt to the intracellular environment. How they achieve this and scavenge essential nutrients from the host has been an open question due to the difficulties in distinguishing between bacterial and host metabolites in situ. Here, we capitalized on the inability of mammalian cells to metabolize mannitol to develop a stable isotopic labeling approach to track Salmonella enterica metabolites during intracellular proliferation in host macrophage and epithelial cells. By measuring label incorporation into Salmonella metabolites with liquid chromatography-mass spectrometry (LC-MS), and combining it with metabolic modeling, we identify relevant carbon sources used by Salmonella, uncover routes of their metabolization, and quantify relative reaction rates in central carbon metabolism. Our results underline the importance of the Entner-Doudoroff pathway (EDP) and the phosphoenolpyruvate carboxylase for intracellularly proliferating Salmonella. More broadly, our metabolic labeling strategy opens novel avenues for understanding the metabolism of pathogens inside host cells.

Gene duplications facilitate C4-CAM compatibility in common purslane.

Common purslane (Portulaca oleracea) integrates both C4 and crassulacean acid metabolism (CAM) photosynthesis pathways and is a promising model plant to explore C4-CAM plasticity. Here, we report a high-quality chromosome-level genome of nicotinamide adenine dinucleotide (NAD)-malic enzyme (ME) subtype common purslane that provides evidence for 2 rounds of whole-genome duplication (WGD) with an ancient WGD (P-β) in the common ancestor to Portulacaceae and Cactaceae around 66.30 million years ago (Mya) and another (Po-α) specific to common purslane lineage around 7.74 Mya. A larger number of gene copies encoding key enzymes/transporters involved in C4 and CAM pathways were detected in common purslane than in related species. Phylogeny, conserved functional site, and collinearity analyses revealed that the Po-α WGD produced the phosphoenolpyruvate carboxylase-encoded gene copies used for photosynthesis in common purslane, while the P-β WGD event produced 2 ancestral genes of functionally differentiated (C4- and CAM-specific) beta carbonic anhydrases involved in the C4 + CAM pathways. Additionally, cis-element enrichment analysis in the promoters showed that CAM-specific genes have recruited both evening and midnight circadian elements as well as the Abscisic acid (ABA)-independent regulatory module mediated by ethylene-response factor cis-elements. Overall, this study provides insights into the origin and evolutionary process of C4 and CAM pathways in common purslane, as well as potential targets for engineering crops by integrating C4 or CAM metabolism.

Critical taste substances and regulatory pathways of fresh lotus seed pulps at different ripeness stages

Previous reports about measuring flavor substances in lotus seeds focus on the nutritional quality. Currently, no research is devoted to the taste quality of lotus seeds. In this study, fresh lotus seeds' pulp at different ripeness stages was used to explore the critical taste substances and related regulatory pathways affecting the suitability of lotus seeds for fresh eating. Sweetness and umami were the basic flavors of low maturity, and astringency mainly existed in high maturity fresh lotus seeds. Fructose is a crucial factor affecting the suitability of fresh lotus seeds. During the ripening of lotus seeds, the increase of fructokinase (FRK) activity and related gene expression caused the decrease in fructose content. In contrast, the decrease in phosphoenolpyruvate carboxylase (PEPC) and NAD-malate dehydrogenase (NAD-MDH) activity was related to the decline of malic acid content in low maturity lotus seeds. The present study revealed the crucial taste substances and regulatory pathways of lotus seeds, which laid a theoretical foundation for the study of the taste formation mechanism of fresh lotus seeds.

Elucidating the molecular responses to waterlogging stress in onion (Allium cepa L.) leaf by comparative transcriptome profiling.

Waterlogging is a major stress that severely affects onion cultivation worldwide, and developing stress-tolerant varieties could be a valuable measure for overcoming its adverse effects. Gathering information regarding the molecular mechanisms and gene expression patterns of waterlogging-tolerant and sensitive genotypes is an effective method for improving stress tolerance in onions. To date, the waterlogging tolerance-governing molecular mechanism in onions is unknown. This study identified the differentially expressed genes (DEGs) through transcriptome analysis in leaf tissue of two onion genotypes (Acc. 1666; tolerant and W-344; sensitive) presenting contrasting responses to waterlogging stress. Differential gene expression analysis revealed that in Acc. 1666, 1629 and 3271 genes were upregulated and downregulated, respectively. In W-344, 2134 and 1909 genes were upregulated and downregulated, respectively, under waterlogging stress. The proteins coded by these DEGs regulate several key biological processes to overcome waterlogging stress such as phytohormone production, antioxidant enzymes, programmed cell death, and energy production. The clusters of orthologous group pathway analysis revealed that DEGs contributed to the post-translational modification, energy production, and carbohydrate metabolism-related pathways under waterlogging stress. The enzyme assay demonstrated higher activity of antioxidant enzymes in Acc. 1666 than in W-344. The differential expression of waterlogging tolerance related genes, such as those related to antioxidant enzymes, phytohormone biosynthesis, carbohydrate metabolism, and transcriptional factors, suggested that significant fine reprogramming of gene expression occurs in response to waterlogging stress in onion. A few genes such as ADH, PDC, PEP carboxylase, WRKY22, and Respiratory burst oxidase D were exclusively upregulated in Acc. 1666. The molecular information about DEGs identified in the present study would be valuable for improving stress tolerance and for developing waterlogging tolerant onion varieties.

B2 O3 nanoparticles alleviate salt stress in maize leaf growth zones by enhancing photosynthesis and maintaining mineral and redox status.

Salt stress induces significant loss in crop yield worldwide. Although the growth-stimulating effects of micronutrient nanoparticles (NPs) application under salinity have been studied, the molecular and biochemical mechanisms underlying these effects are poorly understood. The large size of maize leaf growth zones provides an ideal model system to sample and investigate the molecular and physiological bases of growth at subzonal resolution. Using kinematic analysis, our study indicated that salinity at 150 mM inhibited maize leaf growth by decreasing cell division and expansion in the meristem and elongation zones. Consistently, salinity downregulated cell cycle gene expression (wee1, mcm4, and cyclin-B2-4). B2 O3 NP (BNP) mitigated the stress-induced growth inhibition by reducing the decrease in cell division and expansion. BNP also enhanced the photosynthesis-related parameters. Simultaneously, chlorophyll, phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase were stimulated in the mature zone. Concomitant with growth stimulation by BNP, mineral homeostasis, particularly for B and Ca, was monitored. BNP reduced oxidative stress (e.g., lessened H2 O2 generation along the leaf zones and reduced lipid peroxidation in the mature zone) induced by salinity. This resulted from better maintenance of the redox status, that is, increased the glutathione-ascorbate cycle in the meristem and elongation zones, and flavonoids and tocopherol levels in the mature zone. Our study has important implications for assessing the salinity stress impact mitigated by BNP on maize growth, providing a basis to improve the resilience of crop species under salinity stress conditions.

Protein-Protein Interactions and Quantitative Phosphoproteomic Analysis Reveal Potential Mitochondrial Substrates of Protein Phosphatase 2A-B'ζ Holoenzyme.

Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B', and B" non-related families in land plants. In Arabidopsis, the close homologs B'η, B'θ, B'γ, and B'ζ are further classified into a subfamily of B' called B'η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B'ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B'ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B'ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B'ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B'ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.

The molecular mechanism of propionate-regulating gluconeogenesis in bovine hepatocytes.

Cows that are nursing get around 80% of their glucose from liver gluconeogenesis. Propionate, a significant precursor of liver gluconeogenesis, can regulate the key genes involved in hepatic gluconeogenesis expression, but its precise effects on the activity of enzymes have not yet been fully elucidated. Therefore, the aim of this study was to investigate the effects of propionate on the activity, gene expression, and protein abundance of the key enzymes involved in the gluconeogenesis of dairy cow hepatocytes. The hepatocytes were cultured and treated with various concentrations of sodium propionate (0, 1.25, 2.50, 3.75, and 5.00 mM) for 12 h. Glucose content in the culture media was determined by an enzymatic coloring method. The activities of gluconeogenesis related enzymes were determined by enzyme linked immunosorbent assay kits, and the levels of gene expression and protein abundance of the enzymes were detected by real-time quantitative polymerase chain reaction and Western blot, respectively. Propionate supplementation considerably increased the amount of glucose in the culture medium compared to the control (p<0.05); while there was no discernible difference among the various treatment concentrations (p>0.05). The activities of cytoplasmic phosphoenolpyruvate carboxylase (PEPCK1), mitochondrial phosphoenolpyruvate carboxylase (PEPCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC) were increased with the addition of 2.50 and 3.75 mM propionate; the gene expressions and protein abundances of PEPCK1, PEPCK2, PC, and G6PC were increased by 3.75 mM propionate addition. Propionate encouraged glucose synthesis in bovine hepatocytes, and 3.75 mM propionate directly increased the activities, gene expressions and protein abundances of PC, PEPCK1, PEPCK2, and G6PC in bovine hepatocytes, providing a theoretical basis of propionate-regulating gluconeogenesis in bovine hepatocytes.

Silencing of SbPPCK1-3 Negatively Affects Development, Stress Responses and Productivity in Sorghum.

Phosphoenolpyruvate carboxylase (PEPC) plays central roles in photosynthesis, respiration, amino acid synthesis, and seed development. PEPC is regulated by different post-translational modifications. Between them, the phosphorylation by PEPC-kinase (PEPCk) is widely documented. In this work, we simultaneously silenced the three sorghum genes encoding PEPCk (SbPPCK1-3) by RNAi interference, obtaining 12 independent transgenic lines (Ppck1-12 lines), showing different degrees of SbPPCK1-3 silencing. Among them, two T2 homozygous lines (Ppck-2 and Ppck-4) were selected for further evaluation. Expression of SbPPCK1 was reduced by 65% and 83% in Ppck-2 and Ppck-4 illuminated leaves, respectively. Expression of SbPPCK2 was higher in roots and decreased by 50% in Ppck-2 and Ppck-4 in this tissue. Expression of SbPPCK3 was low and highly variable. Despite the incomplete gene silencing, it decreased the degree of phosphorylation of PEPC in illuminated leaves, P-deficient plants, and NaCl-treated plants. Both leaves and seeds of Ppck lines had altered metabolic profiles and a general decrease in amino acid content. In addition, Ppck lines showed delayed flowering, and 20% of Ppck-4 plants did not produce flowers at all. The total amount of seeds was lowered by 50% and 36% in Ppck-2 and Ppck-4 lines, respectively. The quality of seeds was lower in Ppck lines: lower amino acid content, including Lys, and higher phytate content. These data confirm the relevance of the phosphorylation of PEPC in sorghum development, stress responses, yield, and quality of seeds.

Unveiling the dark side of guard cell metabolism

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a13C–HCO3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.

Transcriptome Analysis of Vicia villosa in Response to Low Phosphorus Stress at Seedling Stage

Vicia villosa, a high-quality green manure crop, helps to increase the content of soil phosphorus (P) by returning to the field. Soil P deprivation is a severe constraint on crop productivity that triggers the low P stress response in plants, which is controlled by various transcriptional regulatory network pathways. Identifying key genes from these transcriptional regulatory networks can help in developing low P-tolerant crops. In this study, we performed physiological observations and transcriptome sequencing of seedlings from the two Vicia villosa varieties, Xu Shao 3 and Soviet Vicia villosa, under P starvation conditions. The results showed that the main root length, plant height, shoot dry weight, root acid phosphatase activity, and inorganic P content of Xu Shao 3 were significantly higher than those of Soviet Vicia villosa under low P conditions. Based on transcriptome data analysis, 183 (shoot) + 144 (root) differential genes (DEGs) between the two varieties were identified; 144 (shoot) + 79 (root) were upregulated, and 69 (shoot) + 65 (root) were downregulated. KEGG analysis found that DEGs in shoots were significantly enriched in photosynthesis pathways, such as vitamin B6 and riboflavin metabolism. Meanwhile, DEGs in roots were enriched in plant signal transduction, fatty acid degradation, citric acid cycle, pentose, glucuronic acid conversion, etc. GO enrichment analysis suggested that DEGs in shoots were significantly enriched in biological processes, including cell response to P stress, intracellular ion homeostasis, etc., and molecular functions, including phosphate ester hydrolase, phosphatase, acid phosphatase activity, etc. Furthermore, DEGs associated with low P tolerance included three acid phosphatases, a phosphoesterase, a sulfoquinovosyl diacylglycerol synthase, a phosphoenolpyruvate carboxylase, six phosphate transporters and glycerol-3-phosphate transporters, eight SPX, and two PHL genes. In conclusion, Xu Shao 3 exhibited stronger inorganic P accumulation ability and a lesser effect on growth than Soviet Vicia villosa under low P conditions, which might result from photosynthesis, sugar, and P metabolism differences between the two varieties. Acid phosphatase, phosphoesterase, phosphoenolpyruvate carboxylase, sulfoquinovosyl diacylglycerol synthase, phosphate transporter, glycerol-3-phosphate transporter, and SPX were key DEGs leading to the difference in low P stress tolerance between the two varieties.

Enhanced carbon capture, lipid and lutein production in Chlamydomonas reinhardtii under meso-thermophilic conditions using chaperone and CRISPRi system

Microalgae are widely recognized as a promising bioresource for producing renewable fuels and chemicals. Microalgal biorefinery has tremendous potential for incorporation into circular bioeconomy, including sustainability, cascading use, and waste reduction. In this study, genetic engineering was used to enhance the growth, lipid and lutein productivity of Chlamydomonas reinhardtii including strains of CC400, PY9, pCHS, and PG. Notably, CRISPRi mediated on phosphoenolpyruvate carboxylase (PEPC1) gene to down-regulate the branch pathway from glycolysis to partitioning more carbon flux to lipid was explored under meso-thermophilic condition. The best chassis PGi, which has overexpressed chaperone GroELS and applied CRISPRi resulting in the highest biomass of 2.56 g/L and also boosted the lipids and lutein with 893 and 23.5 mg/L, respectively at 35°C. Finally, all strains with CRISPRi exhibited higher transcriptional levels of the crucial genes from photosynthesis, starch, lipid and lutein metabolism, thus reaching a CO2 assimilation of 1.087 g-CO2/g-DCW in mixotrophic condition.

Effects of the ammonium stress on photosynthesis and ammonium assimilation in submerged leaves of Ottelia cordata - an endangered aquatic plant

Although ammonium (NH4+-N) is an important nutrient for plants, increases in soil nitrogen (N) input and atmospheric deposition have made ammonium toxicity a serious ecological problem. In this study, we explored the effects of NH4+-N stress on the ultrastructure, photosynthesis, and NH4+-N assimilation of Ottelia cordata (Wallich) Dandy, an endangered heteroblastic plant native to China. Results showed that 15 and 50 mg L−1 NH4+-N damaged leaf ultrastructure and decreased the values of maximal quantum yield (Fv/Fm), maximal fluorescence (Fm), and relative electron transport rate (rETR) in the submerged leaves of O. cordata. Furthermore, when NH4+-N was ≥ 2 mg L−1, phosphoenolpyruvate carboxylase activity (PEPC) and soluble sugar and starch contents decreased significantly. The content of dissolved oxygen in the culture water also decreased significantly. The activity of the NH4+-N assimilation enzyme glutamine synthetase (GS) significantly increased when NH4+-N was ≥ 10 mg L−1 and NADH-glutamate synthase (NADH-GOGAT) and Fd-glutamate synthase (Fd-GOGAT) increased when NH4+-N was at 50 mg L−1. However, the activity of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase (NADH-GDH) and nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADPH-GDH) did not change, indicating that GS/GOGAT cycle may play an important role in NH4+-N assimilation in the submerged leaves of O. cordata. These results show that short-term exposure to a high concentration of NH4+-N is toxic to O. cordata.

Screening and identification of genes involved in β-alanine biosynthesis in Bacillus subtilis

β-alanine is the only naturally occurring β-amino acid, which is widely used in medicine, food, and feed fields, and generally produced through synthetic biological methods based on engineered strains of Escherichia coli or Corynebacterium glutamicum. However, the β-alanine biosynthesis in Bacillus subtilis, a traditional industrial model microorganism of food safety grade, has not been thoroughly explored. In this study, the native l-aspartate-α-decarboxylase was overexpressed in B. subtilis 168 to obtain an increase of 842% in β-alanine production. A total of 16 single-gene knockout strains were constructed to block the competitive consumption pathways to identify a total of 6 genes (i.e., ptsG, fbp, ydaP, yhfS, mmgA, and pckA) involved in β-alanine synthesis, while the multigene knockout of these 6 genes obtained an increased β-alanine production by 40.1%. Ten single-gene suppression strains with the competitive metabolic pathways inhibited revealed that the inhibited expressions of genes glmS, accB, and accA enhanced the β-alanine production. The introduction of heterologous phosphoenolpyruvate carboxylase increased the β-alanine production by 81.7%, which was 17-fold higher than that of the original strain. This was the first study using multiple molecular strategies to investigate the biosynthetic pathway of β-alanine in B. subtilis and to identify the genetic factors limiting the excessive synthesis of β-alanine by microorganisms.

Weighted gene coexpression correlation network analysis reveals the potential molecular regulatory mechanism of citrate and anthocyanin accumulation between postharvest ‘Bingtangcheng’ and ‘Tarocco’ blood orange fruit

BackgroundOrganic acids and anthocyanins are the most important compounds for the flavor and nutritional quality of citrus fruit. However, there are few reports on the involvement of co-regulation of citrate and anthocyanin metabolism. Here, we performed a comparative transcriptome analysis to elucidate the genes and pathways involved in both citrate and anthocyanin accumulation in postharvest citrus fruit with ‘Tarocco’ blood orange (TBO; high accumulation) and ‘Bingtangcheng’ sweet orange (BTSO; low accumulation).ResultsA robust core set of 825 DEGs were found to be temporally associated with citrate and anthocyanin accumulation throughout the storage period through transcriptome analysis. Further according to the results of weighted gene coexpression correlation network analysis (WGCNA), the turquoise and brown module was highly positively correlated with both of the content of citrate and anthocyanin, and p-type ATPase (PH8), phosphoenolpyruvate carboxylase kinase (PEPCK), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3’-hydroxylase (F3’H) and glutathione S transferase (GST) were considered key structural genes. Moreover, MYB family transcription factor (PH4), Zinc finger PHD-type transcription factor (CHR4, HAC12), Zinc finger SWIM-type transcription factor (FAR1) and Zinc finger C3H1-type transcription factor (ATC3H64) were considered hub genes related to these structural genes. Further qRT-PCR analysis verified that these transcription factors were highly expressed in TBO fruit and their expression profiles were significantly positively correlated with the structural genes of citrate and anthocyanin metabolism as well as the content of citrate and anthocyanin content.ConclusionsThe findings suggest that the CHR4, FAR1, ATC3H64 and HAC12 may be the new transcription regulators participate in controlling the level of citrate and anthocyanin in postharvest TBO fruit in addition to PH4. These results may providing new insight into the regulation mechanism of citrate and anthocyanin accumulation in citrus fruit.

Effects of Citric Acid and Humic-like Substances on Yield, Enzyme Activities, and Expression of Genes Involved in Iron Uptake in Tomato Plants

Iron (Fe) deficiency is a common abiotic stress on plants growing in calcareous soils where low organic matter content, high carbonate–bicarbonate concentration, and high pH precipitate Fe in unavailable forms. Enzymatic activity is a mechanism for plants to access soil nutrients; enzymes such as H+-ATPase, phosphoenolpyruvate carboxylase (PEPC), and the intracellular enzyme ferric reduction oxidase (FRO) are involved in Fe absorption. The effects of the application of citric acid (CA) and humic-like substances (HLS) on the yield, H+-ATPase, PEPC, and FRO enzyme activity, and expression of LeHA1, LePEPC1, and LeFRO1 genes in tomato plants grown under calcareous soil were studied. CA and HLS improved the SPAD units and increased the number of harvested fruits and yield per plant. Temporary alterations in enzyme activity, which reduced PEPC and FRO activity in roots, were documented. In leaf tissue, CA resulted in lower expression of LeHA1 and LePEPC1 and the induction of LeFRO1 expression, whereas HLS application resulted in higher expression of LePEPC1 and LeFRO1. In roots, LeHA1 expression increased with HLS, whereas LePEPC1 and LeFRO1 showed lower expression with CA and HLS, respectively. The application of CA and HLS through a nutrient solution in combination with Fe-chelate can improve Fe nutrition in tomato plants potted in calcareous soil by inducing temporal alterations in PEPC and FRO enzyme activity and LeFRO1 and LeHA1 gene expression.

Immobilized Phosphoenolpyruvate Carboxylase Beads for Catalytic Carboxylation in Carbon Dioxide-Containing Gas–Liquid Flow

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the Mg2+-dependent carboxylation reaction in aqueous phase with a carbon source. Performing enzymatic reactions in a bioreactor with gas–liquid contacting is challenging because proteins potentially undergo conformational change at the gas–liquid interface. In the present work, immobilized phosphoenolpyruvate carboxylase polystyrene beads (PEPC-PSBs) were prepared and applied to catalyze the carboxylation of phosphoenolpyruvate (PEP) producing oxaloacetate (OAA) in an external loop airlift bubble column (ELBC). For a typical reaction, ELBC was charged with 10 mL of a 0.2 M potassium phosphate buffer solution (pH = 7.5) initially containing 3.1 mM PEP and 9.7 mM MgSO4. When ELBC was operated at 25 °C with 20 PEPC-PSBs ([PEPC] = 2.0 μg/mL) by continuously introducing CO2 gas bubbles at the superficial gas velocity of 1.0 cm/s, the conversion of PEP of 77% was obtained at the reaction time of 300 min. After the reaction, PEPC-PSBs could be recovered from the reaction mixture followed by washing for reuse. PEPC-PSBs were storable at 4 °C and physically robust enough for handling. The catalytic activity of PEPC-PSBs was observed for the total reaction time of 900–1080 min in the gas–liquid flow. On the other hand, no detectable amount of OAA was produced with free PEPC in ELBC operated under the comparable condition to the PEPC-PSB-catalyzed reaction. Adsorption of free PEPC to CO2 gas bubbles was indicated to cause a rapid decrease in the concentration of biologically active PEPC in ELBC. The polystyrene beads with rough surface were suggested to offer an environment for the immobilized enzyme to stably catalyze the prolonged carboxylation of PEP with continuously supplied CO2-containing gas bubbles.

Differences between tree stem CO2 efflux and O2 influx rates cannot be explained by internal CO2 transport or storage in large beech trees.

Tree stem respiration (RS ) is a substantial component of the forest carbon balance. The mass balance approach uses stem CO2 efflux and internal xylem fluxes to sum up RS , while the oxygen-based method assumes O2 influx as a proxy of RS . So far, both approaches have yielded inconsistent results regarding the fate of respired CO2 in tree stems, a major challenge for quantifying forest carbon dynamics. We collected a data set of CO2 efflux, O2 influx, xylem CO2 concentration, sap flow, sap pH, stem temperature, nonstructural carbohydrates concentrationand potential phosphoenolpyruvate carboxylase (PEPC)capacity on mature beech trees to identify the sources of differences between approaches. The ratio of CO2 efflux to O2 influx was consistently below unity (0.7) along a 3-m vertical gradient, but internal fluxes did not bridge the gap between influx and efflux, nor did we find evidence for changes in respiratory substrate use. PEPC capacity was comparable with that previously reported in green current-year twigs. Although we could not reconcile differences between approaches, results shed light on the uncertain fate of CO2 respired by parenchyma cells across the sapwood. Unexpected high values of PEPC capacity highlight its potential relevance as a mechanism of local CO2 removal, which merits further research.

Analysis of organic acid metabolism reveals citric acid and malic acid play major roles in determining acid quality during the development of kiwifruit (Actinidia eriantha).

Actinidia eriantha is one of the most important kiwifruit species in Actinidia. The relative high accumulation of organic acids in fruit of A. eriantha is an unfavorable factor for organoleptic quality. To identify key metabolic enzymes and genes involved in organic acids accumulation during fruit development, physiological, biochemical, and molecular experiments were conducted for the dynamic fruit samples of a new kiwifruit cultivar, A. eriantha 'Ganlv 1'. The contents of citric acid and malic acid increased greatly during fruit development, while quinic acid content decreased obviously. Significant positive correlations were observed between fruit titratable acidity and the contents of both citric acid and malic acid, and a significant negative correlation was found between fruit titratable acidity and the quinic acid content. The high accumulation of citric acid was found to be caused by the increased activity of citrate synthase (CS), and the decreased activities of two degradation-related enzymes, mitochondrial aconitase and nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase. In addition, the accumulation of malic acid depended mainly on the increased synthesis catalyzed by NAD-dependent malate dehydrogenase (NAD-MDH) and phosphoenolpyruvate carboxylase. Further analysis suggested that AeCS2 and AeMDH2 played pivotal roles in controlling the activities of CS and NAD-MDH respectively. The high accumulation level of citric acid relied on both the strong synthesis ability and the weak degradation ability. The accumulation level of malic acid was mainly affected by the synthesis. The novel information would be helpful for our understanding of the formation of fruit acidity quality. © 2023 Society of Chemical Industry.

Phosphorus-induced greater enhancement in carbon supply and storage for oil synthesis during the crucial period made cottonseed kernel oil yield have a higher increment than protein

Cottonseed has a high utilization value due to its luxuriant oil and protein, but low phosphorus (P) in cropland reduces its yield and quality. A limited understanding of the physiological mechanism underlying these results restricted the exploration of P efficient management in cotton cultivation. A 3-year experiment was performed with Lu 54 (low-P sensitive) and Yuzaomian 9110 (low-P tolerant) under 0 (deficient-P), 100 (critical-P), and 200 (excessive-P) kg P2O5 ha−1 in a field having 16.9 mg kg−1 available P to explore the key pathway for P to regulate cottonseed oil and protein formation. P application markedly increased cottonseed oil and protein yields, with the enhanced acetyl-CoA and oxaloacetate contents during 20–26 days post anthesis being a vital reason. Notably, during the crucial period, decreased phosphoenolpyruvate carboxylase activity weakened the carbon allocation to protein, making malonyl-CoA content increase greater than free amino acid; Meanwhile, P application accelerated the carbon storage in oil but retarded that in protein. Consequently, cottonseed oil yield increased more than protein. Oil and protein synthesis in Lu 54 was more susceptible to P, resulting in greater increments in oil and protein yields than Yuzaomian 9110. Based on acetyl-CoA and oxaloacetate contents (the key substrates), the critical P content in the subtending leaf to cotton boll needed by oil and protein synthesis in Lu 54 (0.35%) was higher than Yuzaomian 9110 (0.31%). This study provided a new perception of the regulation of P on cottonseed oil and protein formation, contributing to the efficient P management in cotton cultivation.

CO2 recycling by phosphoenolpyruvate carboxylase enables cassava leaf metabolism to tolerate low water availability.

Cassava is a staple crop that acclimatizes well to dry weather and limited water availability. The drought response mechanism of quick stomatal closure observed in cassava has no explicit link to the metabolism connecting its physiological response and yield. Here, a genome-scale metabolic model of cassava photosynthetic leaves (leaf-MeCBM) was constructed to study on the metabolic response to drought and stomatal closure. As demonstrated by leaf-MeCBM, leaf metabolism reinforced the physiological response by increasing the internal CO2 and then maintaining the normal operation of photosynthetic carbon fixation. We found that phosphoenolpyruvate carboxylase (PEPC) played a crucial role in the accumulation of the internal CO2 pool when the CO2 uptake rate was limited during stomatal closure. Based on the model simulation, PEPC mechanistically enhanced drought tolerance in cassava by providing sufficient CO2 for carbon fixation by RuBisCO, resulting in high production of sucrose in cassava leaves. The metabolic reprogramming decreased leaf biomass production, which may lead to maintaining intracellular water balance by reducing the overall leaf area. This study indicates the association of metabolic and physiological responses to enhance tolerance, growth, and production of cassava in drought conditions.