Phosphoenolpyruvate Carboxylase Research Articles

Potassium ameliorates cotton (Gossypium hirsutum L.) fiber length by regulating osmotic and K+ /Na+ homeostasis under salt stress.

Potassium (K) application can alleviate cotton salt stress, but the regulatory mechanisms affecting cotton fiber elongation and ion homeostasis are still unclear. A two-year field experiment was conducted to explore the effects of K on the osmolyte contents (soluble sugar, K+ content, and malate) and related enzyme activities during the fiber elongation of two cotton cultivars with contrasting salt sensitivity (CCRI-79; salt tolerant cultivar, and Simian 3; salt-sensitive cultivar). Three K application treatments (0, 150, and 300 kg K2 O ha-1 ) were applied at three soil salinity levels (low salinity, EC=1.73 ± 0.05 dS m-1 ; medium salinity, EC=6.32 ± 0.10 dS m-1 ; high salinity, EC=10.84 ± 0.24 dS m-1 ). K application improved fiber length and alleviated salt stress by increasing the maximum velocity of fiber elongation (Vmax ). The increase rate of K on fiber length decreased with elevating salt stress, and the increase rate of K on Vmax of CCRI-79 was greater than that of Simian3. K application can increase the enzyme activities (phosphoenolpyruvate carboxylase, PEPC, E.C. 4.1.1.31; pyrophosphatase, PPase, E.C. 3.6.1.1; and plasma membrane H+ -ATPase, PM H+ -ATPase, E.C. 3.6.3.14) as well as the content of osmolytes associated with the enzymes mentioned above. K increased the osmolyte contents under salt stress, and the increase in the K+ content of the fibers was much higher than that of soluble sugar and malate. The results of this study indicated K fertilizer application rates regulate the metabolism of osmolytes in cotton fiber development under salt stress, K+ is more critical to fiber elongation.

C4 Phosphoenolpyruvate Carboxylase: Evolution and transcriptional regulation.

Photosynthetic phosphoenolpyruvate carboxylase (PEPC) catalyses the irreversible carboxylation of phosphoenolpyruvate (PEP), producing oxaloacetate (OAA). This enzyme catalyses the first step of carbon fixation in C4 photosynthesis, contributing to the high photosynthetic efficiency of C4 plants. PEPC is also involved in replenishing tricarboxylic acid cycle intermediates, such as OAA, being involved in the C/N balance. In plants, PEPCs are classified in two types: bacterial type (BTPC) and plant-type (PTPC), which includes photosynthetic and non-photosynthetic PEPCs. During C4 evolution, photosynthetic PEPCs evolved independently. C4 PEPCs evolved to be highly expressed and active in a spatial-specific manner. Their gene expression pattern is also regulated by developmental cues, light, circadian clock as well as adverse environmental conditions. However, the gene regulatory networks controlling C4 PEPC gene expression, namely its cell-specificity, are largely unknown. Therefore, after an introduction to the evolution of PEPCs, this review aims to discuss the current knowledge regarding the transcriptional regulation of C4 PEPCs, focusing on cell-specific and developmental expression dynamics, light and circadian regulation, as well as response to abiotic stress. In conclusion, this review aims to highlight the evolution, transcriptional regulation by different signals and importance of PEPC in C4 photosynthesis and its potential as tool for crop improvement.

Stepwise metabolic engineering of Corynebacterium glutamicum for the production of phenylalanine.

Corynebacterium glutamicum was metabolically engineered to produce phenylalanine, a valuable aromatic amino acid that can be used as a raw material in the food and pharmaceutical industries. First, a starting phenylalanine-producer was constructed by overexpressing tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and phenylalanine- and tyrosine-insensitive bifunctional enzyme chorismate mutase prephenate dehydratase from Escherichia coli, followed by the inactivation of enzymes responsible for the formation of dihydroxyacetone and the consumption of shikimate pathway-related compounds. Second, redirection of the carbon flow from tyrosine to phenylalanine was attempted by deleting of the tyrA gene encoding prephenate dehydrogenase, which catalyzes the committed step for tyrosine biosynthesis from prephenate. However, suppressor mutants were generated, and two mutants were isolated and examined for phenylalanine production and genome sequencing. The suppressor mutant harboring an amino acid exchange (L180R) on RNase J, which was experimentally proven to lead to a loss of function of the enzyme, showed significantly enhanced production of phenylalanine. Finally, modifications of phosphoenolpyruvate-pyruvate metabolism were investigated, revealing that the inactivation of either phosphoenolpyruvate carboxylase or pyruvate carboxylase, which are enzymes of the anaplerotic pathway, is an effective means for improving phenylalanine production. The resultant strain, harboring a phosphoenolpyruvate carboxylase deficiency, synthesized 50.7 mM phenylalanine from 444 mM glucose. These results not only provided new insights into the practical mutations in constructing a phenylalanine-producing C. glutamicum but also demonstrated the creation of a potential strain for the biosynthesis of phenylalanine-derived compounds represented by plant secondary metabolites.

Response of waxy maize (Zea mays L. var. ceratina Kulesh) leaf photosynthesis to low temperature during the grain-filling stage.

Low temperature (LT) during the grain-filling stage is an important factor that affects the source-sink relationship and leads to yield loss in maize (Zea mays L). In this study, field and pot trials were conducted to investigate the effects of LT during the grain-filling stage on leaf photosynthesis, antioxidant system, hormones, and grain yield of waxy maize cultivars Suyunuo 5 (S5) and Yunuo 7 (Y7). The results showed that LT treatment inhibited the chlorophyll biosynthesis and reduced the photosynthetic pigment levels during grain-filling stage. Ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase activities, photosynthetic rate, transpiration rate, and stomatal conductance decreased under LT treatment during the grain-filling stage. Furthermore, LT treatment increased the contents of malondialdehyde and reactive oxygen species, and decreased the activities of catalase, superoxide dismutase, peroxidase, and ascorbate peroxidase in the ear leaves, which accelerated the oxidative damage of leaf. The LT treatment also raised abscisic acid content and reduced indole acetic acid content in the ear leaves during grain-filling stage. The results of field and pot trials were verified by each other, but the field effect was greater than that of pot. Overall, LT treatment reduced the waxy maize dry matter accumulation after silking by affecting the physiological and biochemical processes of leaves, and ultimately decreased grain yield.

Photosynthesis and leaf structure of F1 hybrids between Cymbidium ensifolium (C3) and C. bicolor subsp. pubescens (CAM).

The introduction of crassulacean acid metabolism (CAM) into C3 crops has been considered as a means of improving water-use efficiency. In this study, we investigated photosynthetic and leaf structural traits in F1 hybrids between Cymbidium ensifolium (female C3 parent) and C. bicolor subsp. pubescens (male CAM parent) of the Orchidaceae. Seven F1 hybrids produced through artificial pollination and in vitro culture were grown in a greenhouse with the parent plants. Structural, biochemical and physiological traits involved in CAM in their leaves were investigated. Cymbidium ensifolium accumulated very low levels of malate without diel fluctuation, whereas C. bicolor subsp. pubescens showed nocturnal accumulation and diurnal consumption of malate. The F1s also accumulated malate at night, but much less than C. bicolor subsp. pubescens. This feature was consistent with low nocturnal fixation of atmospheric CO2 in the F1s. The δ13C values of the F1s were intermediate between those of the parents. Leaf thickness was thicker in C. bicolor subsp. pubescens than in C. ensifolium, and those of the F1s were more similar to that of C. ensifolium. This was due to the difference in mesophyll cell size. The chloroplast coverage of mesophyll cell perimeter adjacent to intercellular air spaces of C. bicolor subsp. pubescens was lower than that of C. ensifolium, and that of the F1s was intermediate between them. Interestingly, one F1 had structural and physiological traits more similar to those of C. bicolor subsp. pubescens than the other F1s. Nevertheless, all F1s contained intermediate levels of phosphoenolpyruvate carboxylase but as much pyruvate, Pi dikinase as C. bicolor subsp. pubescens. CAM traits were intricately inherited in the F1 hybrids, the level of CAM expression varied widely among F1 plants, and the CAM traits examined were not necessarily co-ordinately transmitted to the F1s.

Hypoglycemic effect of the Phellinus baumii extract with α-glucosidase-inhibited activity and its modulation to gut microbiota in diabetic patients

Phellinus baumii extract (PBE) possesses considerable α-glucosidase-inhibited activity. This study investigated the hypoglycemic effect in vitro and in vivo using a glucose consumption assay in HepG2 cells, intragastric administration for ten weeks in STZ-induced mice, and intestinal flora fermentation in patients with type 2 diabetes to reveal the possible underlying mechanisms. PBE was prepared, including α-glucosidase-inhibited ethanol extract (EE) and aqueous extract (AE). In vitro, PBE promoted glucose consumption and enhanced glycogen content and hexokinase activity but lowered phosphoenolpyruvate carboxylase kinase activity in HepG2 cells. In vivo, PBE treatment significantly reduced the body weight (p < 0.05) and fasting blood glucose levels of diabetic mice (p < 0.01), with the lowest blood glucose level observed in the EE+AE group. Furthermore, the serum insulin levels and insulin resistance index (HOMA) of PBE-treated groups decreased significantly (p < 0.01). Moreover, gene expression levels of the IRS-1/PI3K/AKT pathway were significantly upregulated by PBE treatment (p < 0.01). In vitro fermentation demonstrated that EE significantly inhibited the production of H2S and NH3 in the intestinal flora fermentation model in diabetic patients (p < 0.05). In addition, the ratio of Firmicutes to Bacteroidetes was reduced, the growth of Lactobacillus and Prevotella 9 was promoted, and Pseudomonas aeruginosa was inhibited. This study provides new insights and clues for using PBE as a functional food and clinical drug for glycemic control.

Integrated multi-omics reveals the molecular mechanisms underlying efficient phosphorus use under phosphate deficiency in elephant grass (Pennisetum purpureum).

Phosphorus (P) is an essential macronutrient element for plant growth, and deficiency of inorganic phosphate (Pi) limits plant growth and yield. Elephant grass (Pennisetum purpureum) is an important fodder crop cultivated widely in tropical and subtropical areas throughout the world. However, the mechanisms underlying efficient P use in elephant grass under Pi deficiency remain poorly understood. In this study, the physiological and molecular responses of elephant grass leaves and roots to Pi deficiency were investigated. The results showed that dry weight, total P concentration, and P content decreased in Pi-deprived plants, but that acid phosphatase activity and P utilization efficiency (PUE) were higher than in Pi-sufficient plants. Regarding Pi starvation-responsive (PSR) genes, transcriptomics showed that 59 unigenes involved in Pi acquisition and transport (especially 18 purple acid phosphatase and 27 phosphate transporter 1 unigenes) and 51 phospholipase unigenes involved in phospholipids degradation or Pi-free lipids biosynthesis, as well as 47 core unigenes involved in the synthesis of phenylpropanoids and flavonoids, were significantly up-regulated by Pi deprivation in leaves or roots. Furthermore, 43 unigenes related to Pi-independent- or inorganic pyrophosphate (PPi)-dependent bypass reactions were markedly up-regulated in Pi-deficient leaves, especially five UDP-glucose pyrophosphorylase and 15 phosphoenolpyruvate carboxylase unigenes. Consistent with PSR unigene expression changes, metabolomics revealed that Pi deficiency significantly increased metabolites of Pi-free lipids, phenylpropanoids, and flavonoids in leaves and roots, but decreased phospholipid metabolites. This study reveals the mechanisms underlying the responses to Pi starvation in elephant grass leaves and roots, which provides candidate unigenes involved in efficient P use and theoretical references for the development of P-efficient elephant grass varieties.

Inorganic carbon utilization: A target of silver nanoparticle toxicity on a submerged macrophyte

Submerged macrophytes play an important role in the global carbon cycle through diversified pathways of inorganic carbon (Ci) utilization distinct from terrestrial plants. However, the effects of silver nanoparticles (AgNPs), an emerging contaminant, were unknown on the Ci utilization of submerged macrophytes. In Ottelia alismoides, the only known submerged macrophyte with three pathways of Ci utilization, before absorption, AgNPs inhibited the external carbonic anhydrase activity thus reducing the capacity of the plant to use HCO3−. After entering the plant, AgNPs mainly aggregated at the cell wall and in the chloroplast. The internalized AgNPs inhibited ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) activity blocking CO2 fixation and disturbed C4 and crassulacean acid metabolism (CAM) by inhibiting phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphate dikinase (PPDK), and NAD-dependent malic enzyme (NAD-ME) activities to alter intracellular malate biosynthesis and decarboxylation. Overall, our findings indicate that the Ci utilization of the submerged macrophyte is a target of AgNPs toxicity that might affect the carbon cycle in aquatic systems.

Optimized potassium application rate increases foxtail millet grain yield by improving photosynthetic carbohydrate metabolism.

Photosynthetic carbohydrate metabolism is an important biochemical process related to crop production and quality traits. Potassium (K) critically contributes to the process of photosynthetic carbon assimilation and carbohydrate metabolism. We explored the effects of potassium fertilization on physiological mechanisms including carbohydrate metabolism in foxtail millet and its yield. Field experiments were performed using two foxtail millet (Setaria italica L.) cultivars: 1) Jingu 21 (low-K sensitive); and 2) Zhangza 10 (low-K tolerant). Effect of five different potassium fertilizer (K2O) rates (0, 60, 120, 180, and 240 kg·hm-2) were tested in two consecutive years, 2020 and 2021. We found that potassium application significantly increases the K content, dry matter accumulation and yield. Jingu 21 and Zhangza 10 had maximum yields at 180 kg·hm-2 K application, which were 29.91% and 31.51% larger than without K application, respectively. Excessive K application (K240) did not further improve their yields. The suitable K fertilizer application of Jingu 21 and Zhangza 10 are 195.25-204.27 and 173.95-175.87 kg·K2O·hm-2, respectively. The net photosynthetic rate (Pn), ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC), and fructose-1,6-bisphosphatase (FBPase) were positively correlated with the potassium content. Potassium application improved the availability of carbon sources for carbohydrate synthesis. Compared with the K0 treatment, variations in the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) in potassium-treated Jingu 21 (K60, K120, K180, and K240) were 17.94%-89.93% and 22.48%-182.10%, respectively, which were greater than those of Zhangza 10 (11.34%-71.12% and 16.18%-109.13%, respectively) and indicate that Jingu 21 is more sensitive to potassium application. The sucrose contents in the Jingu 21 and Zhangza 10 grains were 0.97%-1.15% and 1.04%-1.23%, respectively. The starch contents were 28.99%-37.75% and 24.81%-34.62%, respectively. The sucrose: ratio of Jingu 21 was smaller than that of Zhangza 10, indicating that Jingu 21 utilized nutrients better than Zhangza 10. Stepwise regression and path analysis showed that leaf and grain SuSy activity, by coordinating the source-sink relationship, have the greatest direct effect on Jingu 21 yield, whereas leaf SuSy activity, by promoting the generation of photosynthates at the source leaf, plays a leading role in Zhangza 10 yield increase. In conclusion, optimized K application can increase foxtail millet grain yield by improving photosynthesis and promoting carbohydrate accumulation and distribution.

Metabolic and Microbial Community Engineering for Four-Carbon Dicarboxylic Acid Production from CO2-Derived Glycogen in the Cyanobacterium Synechocystis sp. PCC6803.

The four-carbon (C4) dicarboxylic acids, fumarate, malate, and succinate, are the most valuable targets that must be exploited for CO2-based chemical production in the move to a sustainable low-carbon future. Cyanobacteria excrete high amounts of C4 dicarboxylic acids through glycogen fermentation in a dark anoxic environment. The enhancement of metabolic flux in the reductive TCA branch in the Cyanobacterium Synechocystis sp. PCC6803 is a key issue in the C4 dicarboxylic acid production. To improve metabolic flux through the anaplerotic pathway, we have created the recombinant strain PCCK, which expresses foreign ATP-forming phosphoenolpyruvate carboxykinase (PEPck) concurrent with intrinsic phosphoenolpyruvate carboxylase (Ppc) overexpression. Expression of PEPck concurrent with Ppc led to an increase in C4 dicarboxylic acids by autofermentation. Metabolome analysis revealed that PEPck contributed to an increase in carbon flux from hexose and pentose phosphates into the TCA reductive branch. To enhance the metabolic flux in the reductive TCA branch, we examined the effect of corn-steep liquor (CSL) as a nutritional supplement on C4 dicarboxylic acid production. Surprisingly, the addition of sterilized CSL enhanced the malate production in the PCCK strain. Thereafter, the malate and fumarate excreted by the PCCK strain are converted into succinate by the CSL-settling microorganisms. Finally, high-density cultivation of cells lacking the acetate kinase gene showed the highest production of malate and fumarate (3.2 and 2.4 g/L with sterilized CSL) and succinate (5.7 g/L with non-sterile CSL) after 72 h cultivation. The present microbial community engineering is useful for succinate production by one-pot fermentation under dark anoxic conditions.

Adaptive laboratory evolution and metabolic regulation of genetic Escherichia coli W3110 toward low-carbon footprint production of 5-aminolevulinic acid

BackgroundThe 5-aminolevulinic acid (5-ALA) is a prodrug that has been approved by FDA for photodynamic therapy of cancer. 5-ALA is intracellularly metabolized to protoporphyrin IX, which upon illumination with red light results in photooxidative damage to cells. However, microbial 5-ALA production is challenged by strain tolerance and limited metabolic engineering, while carbon capture is rarely conducted. In this study, E. coli W3110 was equipped with recombinant ALAS production and concomitant CO2 assimilation capacity for low-carbon footprint 5-ALA production. MethodsA stable E. coli strain expressing recombinant ALAS was further improved by adaptive laboratory evolution (ALE). Glycine distribution between biomass and 5-ALA was deciphered using C13 isotope analysis based on C13–2-glycine. Metabolic flux was enforced by overexpressing phosphoenolpyruvate carboxylase (ppc) and knockout of phosphate acetyltransferase (pta). The ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) were added to confer CO2 assimilation ability in RSSL strain. Significant findingsThe adaptive RL8 strain demonstrated 129% and 205% increase in biomass and 5-ALA, respectively. C13 isotope analysis revealed that RSSL assimilated CO2 into biomass and generated glycine from glucose for maximal 5-ALA production. Overexpression of ppc and knockout of pta led to energy conservation, reduced acetate production, thus increasing 5-ALA accumulation (9.53 g/L). Carbon uptake via RuBisCO/PRK significantly reduced CO2 emission to -2.32 g-CO2/g-DCW.

Overexpression of Setaria italica phosphoenolpyruvate carboxylase gene in rice positively impacts photosynthesis and agronomic traits

C4 plants have the inherent capacity to concentrate atmospheric CO2 in the vicinity of RuBisCo, thereby increasing carboxylation, and inhibiting photorespiration. Carbonic anhydrase (CA), the first enzyme of C4 photosynthesis, converts atmospheric CO2 to HCO3−, which is utilized by PEPC to produce C4 acids. Bioengineering of C4 traits into C3 crops is an attractive strategy to increase photosynthesis and water use efficiency. In the present study, we isolated the PEPC gene from the C4 plant Setaria italica and transferred it to C3 rice. Overexpression of SiPEPC resulted in a 2-6-fold increment in PEPC enzyme activity in transgenic lines with respect to non-transformed control. Photosynthetic efficiency was enhanced in transformed plants, which was associated with increased ФPSII, ETR, lower NPQ, and higher chlorophyll accumulation. Water use efficiency was increased by 16–22% in PEPC transgenic rice lines. Increased PEPC activity enhanced quantum yield and carboxylation efficiency of PEPC transgenic lines. Transgenic plants exhibited higher light saturation photosynthesis rate and lower CO2 compensation point, as compared to non-transformed control. An increase in net photosynthesis increased the yield by (23–28.9%) and biomass by (24.1–29%) in transgenic PEPC lines. Altogether, our findings indicate that overexpression of C4-specific SiPEPC enzyme is able to enhance photosynthesis and related parameters in transgenic rice.

Comparative analysis of farmer practices and high yield experiments: Farmers could get more maize yield from maize-soybean relay intercropping through high density cultivation of maize

Intercropping is a high-yield, resource-efficient planting method. There is a large gap between actual yield and potential yield at farmer's field. Their actual yield of intercropped maize remains unclear under low solar radiation-area, whether this yield can be improved, and if so, what are the underlying mechanism for increasing yield? In the present study, we collected the field management and yield data of intercropping maize by conducting a survey comprising 300 farmer households in 2016-2017. Subsequently, based on surveyed data, we designed an experiment including a high density planting (Dense cultivation and high N fertilization with plough tillage; DC) and normal farmer practice (Common cultivation; CC) to analyze the yield, canopy structure, light interception, photosynthetic parameters, and photosynthetic productivity. Most farmers preferred rotary tillage with a low planting density and N fertilization. Survey data showed that farmer yield ranged between 4-6 Mg ha-1, with highest yield recorded at 10-12 Mg ha-1, suggesting a possibility for yield improvement by improved cropping practices. Results from high density experiment showed that the two-years average yield for DC was 28.8% higher than the CC. Compared to CC, the lower angle between stem and leaf (LA) and higher leaf area index (LAI) in DC resulted in higher light interception in middle canopy and increased the photosynthetic productivity under DC. Moreover, in upper and lower canopies, the average activity of phosphoenolpyruvate (PEP) carboxylase was 70% higher in DC than CC. Briefly, increase in LAI and high Pn improved both light interception and photosynthetic productivity, thereby mediating an increase in the maize yield. Overall, these results indicated that farmer's yields on average can be increased by 2.1 Mg ha-1 by increasing planting density and N fertilization, under plough tillage.

Responses to aluminum and cadmium of a RNAi sorghum line with decreased levels of phosphoenolpyruvate carboxylase 3 (PPC3)

Phosphoenolpyruvate carboxylase (PEPC) is an enzyme family with central roles in carbon and nitrogen metabolisms. In order to obtain knowledge about the function of specific PEPC isoenzymes, we have characterized RNAi sorghum lines with decreased level of PPC3 (Ppc3 lines). PPC3 is the main root PEPC, and participates in responses to salinity and ammonium stress. This prompted us to study the responses of Ppc3 lines to Al and Cd stress. Both Al and Cd treatments decreased germination rate, growth rate, and shoot and root biomass production. These detrimental effects were higher in Ppc3 lines than in Wt. Root PPC3 expression and PEPC activity was increased by Al and Cd in Wt, but not in Ppc3 lines. The treatments also increased PPC2 and PPCK gene expression, and PEPCk activity, but these changes did not substitute the lack of PPC3. The production and secretion of exudates by plant roots play an important role in plant tolerance to Al and Cd toxicity. Al (2 days of treatment) caused a 40-fold increase of the citrate content of root exudates, without differences between Wt and Ppc3 lines. On the contrary, Cd (7 days of treatment) caused a 400-fold increase of citrate in Wt exudates, but only a 30-fold increase in Ppc3 lines. These results indicate that although PPC3 is not necessary for early production of organic acids in root exudates, it is crucial for maintaining high level of synthesis and accumulation of citrate, and that PPC3 is the main PEPC isoenzyme responsible for this response to heavy metal stress.

Effective 5-aminolevulinic acid production via T7 RNA polymerase and RuBisCO equipped Escherichia coli W3110.

Chromosome-based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site-specific integration. Herein, we performed the 5-aminolevulinic acid (5-ALA) production in four T7RNAP-equipped W3110 strains using recombinant 5-aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5-ALA production reached to 5.5 g/L; thus, the least by-product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site. Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5-ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α-ketoglutaric acid dominating 5-ALA production. Finally, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low-carbon assimilation and high-level of 5-ALA production up to 11.2 g/L in fed-batch fermentation was established.

Response and acclimatization of a CAM orchid, Dendrobium Sonia ‘Earsakul’ to drought, heat, and combined drought and heat stress

Response and the acclimation to abiotic stress, including drought, heat (40–45°C), and combined drought and heat stress for 30 days, have been clarified in a crassulacean acid metabolism (CAM)-orchid, Dendrobium Sonia ‘Earsakul’. The drought stress alone caused a more severe effect than the heat stress only. In the drought stress, at 30 days after stress treatments (DAT), the stomatal conductance (gs) and transpiration rate (TR) rapidly dropped after the peak time (6 pm) then the stomata were completely closed to maintain the leaf's relative water content (RWC) at 95.6%, resulting in a drop in the carbon exchange rate (CER). However, the phosphoenolpyruvate carboxylase (PEPC) activity, malondialdehyde (MDA), and antioxidant activity increased at 30 DAT. In heat stress, CER dropped significantly at 7 DAT and was retrieved to the same as the control's level during 14–30 DAT.Moreover, the gs and TR of the heat treatment were significantly increased during 14–30 DAT. In addition, we also found the presence of aerenchyma in leaf sheath in response to heat stress. The combined drought and heat stress caused the severest symptom. The significant loss of leaf RWC at the back pseudobulb was detected from 7 to 30 DAT, resulting in decreasing leaf CER, gs, and TR. However, an increase in PEPC, MDA, and antioxidant activity at 30 DAT was observed after facing both stress combinations.

Attenuating the toxicity of isoproturon to maize by priming in ascorbate, glutathione or thiourea

Maize grains were primed in ascorbate (AsA), glutathione (GSH) or thiourea (TU) for 24 h, sown in pots for 10 days and then treated with Ipu whose residues persisted in shoots from the next day and increased over 8 days and then decreased continuously. The herbicide significantly reduced growth parameters, pigments, photosynthetic activity parameters, AsA, GSH and GSH/oxidized glutathione (GSSG) ratio and inhibited the activities of δ-aminolevulinate dehydratase (ALA-D), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEP-C), malate dehydrogenase (MDH), pyruvate phosphate dikinase (PPDK), superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) but significantly increased malondialdehyde (MDA), H 2 O 2 , superoxide radicals (O 2 ∙−) and GSSG. The priming mostly improved the tested parameters and overcame the deficiency in enzymatic and non-enzymatic antioxidants as well as in the photosynthetic parameters and enzyme activities; AsA or GSH were more efficient than TU. Besides, in vitro tests showed that the inhibition constant (IC50) for Ipu was lowest for CAT but highest for PPDK indicating that CAT was the most inhibited but PPDK was the least while the others were moderate. Furthermore, calculations showed that Ipu competitively inhibited ALA-D, Rubisco, APX, PEP-C, MDH and PPDK but caused non-competitive inhibition of APX, CAT and SOD. These findings reveal that maize was prone to suffering from Ipu; however, priming attenuated the toxicity of Ipu and overcame the herbicide impacts by repairing, maintaining and improving antioxidants and photosynthetic activities .

Role of C4 photosynthetic enzyme isoforms in C3 plants and their potential applications in improving agronomic traits in crops.

As compared to C3, C4 plants have higher photosynthetic rates and better tolerance to high temperature and drought. These traits are highly beneficial in the current scenario of global warming. Interestingly, all the genes of the C4 photosynthetic pathway are present in C3 plants, although they are involved in diverse non-photosynthetic functions. Non-photosynthetic isoforms of carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), the decarboxylating enzymes NAD/NADP-malic enzyme (NAD/NADP-ME), and phosphoenolpyruvate carboxykinase (PEPCK), and finally pyruvate orthophosphate dikinase (PPDK) catalyze reactions that are essential for major plant metabolism pathways, such as the tricarboxylic acid (TCA) cycle, maintenance of cellular pH, uptake of nutrients and their assimilation. Consistent with this view differential expression pattern of these non-photosynthetic C3 isoforms has been observed in different tissues across the plant developmental stages, such as germination, grain filling, and leaf senescence. Also abundance of these C3 isoforms is increased considerably in response to environmental fluctuations particularly during abiotic stress. Here we review the vital roles played by C3 isoforms of C4 enzymes and the probable mechanisms by which they help plants in acclimation to adverse growth conditions. Further, their potential applications to increase the agronomic trait value of C3 crops is discussed.

Thioredoxins regulate the metabolic fluxes throughout the tricarboxylic acid cycle and associated pathways in a light-independent manner

The metabolic fluxes throughout the tricarboxylic acid cycle (TCAC) are inhibited in the light by the mitochondrial thioredoxin (TRX) system. However, it is unclear how this system orchestrates the fluxes throughout the TCAC and associated pathways in the dark. Here we carried out a13C–HCO3 labelling experiment in Arabidopsis leaves from wild type (WT) and mutants lacking TRX o1 (trxo1), TRX h2 (trxh2), or both NADPH-dependent TRX reductase A and B (ntra ntrb) exposed to 0, 30 and 60 min of dark or light conditions. No 13C-enrichment in TCAC metabolites in illuminated WT leaves was observed. However, increased succinate content was found in parallel to reductions in Ala in the light, suggesting the latter operates as an alternative carbon source for succinate synthesis. By contrast to WT, all mutants showed substantial changes in the content and 13C-enrichment in TCAC metabolites under both dark and light conditions. Increased 13C-enrichment in glutamine in illuminated trxo1 leaves was also observed, strengthening the idea that TRX o1 restricts in vivo carbon fluxes from glycolysis and the TCAC to glutamine. We further demonstrated that both photosynthetic and gluconeogenic fluxes toward glucose are increased in trxo1 and that the phosphoenolpyruvate carboxylase (PEPc)-mediated 13C-incorporation into malate is higher in trxh2 mutants, as compared to WT. Our results collectively provide evidence that TRX h2 and the mitochondrial NTR/TRX system regulate the metabolic fluxes throughout the TCAC and associated pathways, including glycolysis, gluconeogenesis and the synthesis of glutamine in a light-independent manner.

Improving 3-hydroxypropionic acid production in E. coli by in silico prediction of new metabolic targets

3-Hydroxypropionic acid (3-HP) production from renewable feedstocks is of great interest in efforts to develop greener processes for obtaining this chemical platform. Here we report an engineered E. coli strain for 3-HP production through the β-alanine pathway. To obtain a new strain capable of producing 3-HP, the pathway was established by overexpressing the enzymes pyruvate aminotransferase, 3-hydroxyacid dehydrogenase, and L-aspartate-1-decarboxylase. Further increase of the 3-HP titer was achieved using evolutionary optimizations of a genome-scale metabolic model of E. coli containing the adopted pathway. From these optimizations, three non-intuitive targets for in vivo assessment were identified: L-alanine aminotransferase and alanine racemase overexpression, and L-valine transaminase knock-out. The implementation of these targets in the production strain resulted in a 40% increase in 3-HP titer. The strain was further engineered to overexpress phosphoenolpyruvate carboxylase, reaching 0.79 ± 0.02 g/L of 3-HP when grown using glucose. Surprisingly, this strain produced 63% more of the desired product when grown using a mixture of glucose and xylose (1:1, C-mol), and gene expression analysis showed that the cellular adjustment to consume xylose had a positive impact on 3-HP accumulation. Fed-batch culture with xylose feeding led to a final titer of 29.1 g/L. These results reinforce the value of computational methods in strain engineering, enabling the design of more efficient strategies to be assessed. Moreover, higher production of 3-HP under a sugar mixture condition points towards the development of bioprocesses based on renewable resources, such as hemicellulose hydrolysates.