Phosphoenolpyruvate Carboxylase Research Articles

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.

Changes in physiological performance and protein expression in the larvae of the coral Pocillopora damicornis and their symbionts in response to elevated temperature and acidification

Climate change causes ocean warming and acidification, which threaten coral reef ecosystems. Ocean warming and acidification cause bleaching and mortality, and decrease calcification in adult corals, leading to changes in the composition of coral communities; however, their interactive effects on coral larvae are not comprehensively understood. To examine the underlying molecular mechanisms of larval responses to elevated temperature and pCO2, we examined the physiological performance and protein expression profiles of Pocillopora damicornis at two temperatures (29 and 33 °C) and pCO2 levels (500 and 1000 μatm) for 5 d. Extensive physiological and proteomic changes were observed in coral larvae. The results indicated a significant decrease in net photosynthesis (PNET) and autotrophic capability (PNET/RD) of larvae exposed to elevated temperature but a marked increase in PNET and PNET/RD of larvae exposed to high pCO2 levels. Elevated temperature significantly reduced endosymbiont densities by 70% and photochemical efficiency, indicating that warming impaired host-symbiont symbiosis. Expression of photosynthesis-related proteins, the photosystem (PS) I reaction center subunits IV and XI as well as oxygen-evolving enhancer 1, was downregulated at higher temperatures in symbionts, whereas expression of the PS I iron‑sulfur center protein was increased under high pCO2 conditions. Furthermore, expression of phosphoribulokinase (involved in the Calvin cycle) and phosphoenolpyruvate carboxylase (related to the C4 pathway) was downregulated in symbionts under thermal stress; this finding suggests reduced carbon fixation at high temperatures. The abundance of carbonic anhydrase-associated proteins, which are predicted to exert biochemical roles in dissolved inorganic carbon transport in larvae, was reduced in coral host and symbionts at high temperatures. These results elucidate potential mechanisms underlying the responses of coral larvae exposed to elevated temperature and acidification and suggest an important role of symbionts in the response to warming and acidification.

Lipidomic profiling reveals biosynthetic relationships between phospholipids and diacylglycerol ethers in the deep-sea soft coral Paragorgia arborea

The cold-water gorgonian coral Paragorgia arborea is considered as a foundation species of deep-sea ecosystems in the northern Atlantic and Pacific oceans. To advance lipidomic studies of deep-sea corals, molecular species compositions of diacylglycerol ethers (DAGE), which are specific storage lipids of corals, and structural glycerophospholipids (GPL) including ethanolamine, choline, inositol and serine GPL (PE, PC, PI, and PS, respectively) were analyzed in P. arborea by HPLC and tandem mass spectrometry. In DAGE molecules, alkyl groups (16:0, 14:0, and 18:1), polyunsaturated fatty acids (PUFA), and monounsaturated FA are mainly substituted the glycerol moiety at position sn-1, sn-2, and sn-3, respectively. The ether form (1-O-alkyl-2-acyl) predominates in PE and PC, while PI is comprised of the 1,2-diacyl form. Both ether and diacyl forms were observed in PS. At position sn-2, C20 PUFA are mainly attached to PC, but C24 PUFA, soft coral chemotaxonomic markers, concentrate in PS, PI, and PE. A comparison of non-polar parts of molecules has shown that DAGE, ether PE, and ether PC can originate from one set of 1-O-alkyl-2-acyl-sn-glycerols. Ether PE may be converted to ether PS by the base-exchange reaction. A diacylglycerol unit generated from phosphatidic acid can be a precursor for diacyl PS, PC, and PI. Thus, a lipidomic approach has confirmed the difference in biosynthetic origins between ether and diacyl lipids of deep-sea gorgonians.

Limitation of C4 photosynthesis by low carbonic anhydrase activity increases with temperature but does not influence mesophyll CO2 conductance.

The CO2-concentrating mechanism (CCM) in C4 plants is initiated by the uptake of bicarbonate (HCO3-) via phosphoenolpyruvate carboxylase (PEPC). Generation of HCO3- for PEPC is determined by the interaction between mesophyll CO2 conductance and the hydration of CO2 to HCO3- by carbonic anhydrase (CA). Genetic reduction of CA was previously shown not to limit C4 photosynthesis under ambient atmospheric partial pressures of CO2 (pCO2). However, CA activity varies widely across C4 species and it is unknown if there are specific environmental conditions (e.g. high temperature) where CA may limit HCO3- production for C4 photosynthesis. Additionally, CA activity has been suggested to influence mesophyll conductance, but this has not been experimentally tested. We hypothesize that CA activity can limit PEPC at high temperatures, particularly at low pCO2, but does not directly influence gm. Here we tested the influence of genetically reduced CA activity on photosynthesis and gm in the C4 plant Zea mays under a range of pCO2 and temperatures. Reduced CA activity limited HCO3- production for C4 photosynthesis at low pCO2 as temperatures increased, but did not influence mesophyll conductance. Therefore, high leaf CA activity may enhance C4 photosynthesis under high temperature when stomatal conductance restricts the availability of atmospheric CO2.

Prediction of Genes That Function in Methanogenesis and CO2 Pathways in Extremophiles.

Gaet’ale (GAL) and Mud’ara (MUP) are two hypersaline ponds located in the Danakil Depression recharged by underground water from the surrounding highlands. These two ponds have different pH, salinity, and show variation in the concentration of many ionic components. Metagenomic analysis concludes that GAL is dominated by bacteria as in the case of the other hypersaline and acidic ponds in the Danakil Depression. However, Archaea dominated the ponds of MUP. In the current study, the application of SEED and KEGG helped to map the ordered steps of specific enzyme catalyzed reaction in converting CO2 into cell products. We predict that highly efficient and light-independent carbon fixation involving phosphoenolpyruvate carboxylase takes place in MUP. On the contrary, genes encoding enzymes involved in hydrogenotrophic and acetoclastic methanogenesis appeared solely in ponds of GAL, implying the biological source of the hazardous methane gas in that environment. Based on the investigation of the sources of the genes of interest, it is clear that cooperative interactions between members of the two communities and syntrophic metabolism is the main strategy adapted to utilize inorganic carbon as a carbon source in both MUP and GAL. This insight can be used to design biotechnological applications of microbial communities in production of methane biogas or to minimize CO2 emissions.

Improve gamma-aminobutyric acid production in Corynebacterium glutamicum by optimizing the metabolic flux

Gamma-aminobutyric acid is an important nonprotein amino acid and has been extensively applied in pharmaceuticals, livestock, food additives, and so on. It is important to develop Corynebacterium glutamicum strains that can efficiently produce gamma-aminobutyric acid from glucose. In this study, production of gamma-aminobutyric acid in C. glutamicum CGY700 was improved by construction of CO2 anaplerotic reaction and overexpression of citrate synthase. The co-expression of ppc encoding phosphoenolpyruvate carboxylase and gltA encoding citrate synthase was constructed and optimized in the chromosome to compensate carbon loss and conquer metabolic bottleneck. The expression of ppc and gltA were controlled by promoters Ptac and PtacM, and the optimal mode of PtacM-ppc-Ptac-gltA was determined. Simultaneously, the genes pknG encoding serine/threonine protein kinase G and ldh encoding l-lactate dehydrogenase were deleted, and glnA2 encoding glutamine synthase was overexpressed in the chromosome. The final strain CGY-PG-304 constructed in this study could produce 41.17 g/L gamma-aminobutyric acid in shake flask cultivation and 58.33 g/L gamma-aminobutyric acid via Fed-Batch fermentation with a yield of 0.30 g/g glucose. CGY-PG-304 was constructed by genome editing; therefore, it is stable and not necessary to add any antibiotics and inducer during fermentation.

Novel QTL identification and candidate gene analysis for enhancing salt tolerance in soybean (Glycine max (L.) Merr.)

Soybean, a glycophyte that is sensitive to salt stress, is greatly affected by salinity at all growth stages. A mapping population derived from a cross between a salt-sensitive Korean cultivar, Cheongja 3, and a salt-tolerant landrace, IT162669, was used to identify quantitative trait loci (QTLs) conferring salt tolerance in soybean. Following treatment with 120 mM NaCl for 2 weeks, phenotypic traits representing physiological damage, leaf Na+ content, and K+/Na+ ratio were characterized. Among the QTLs mapped on a high-density genetic map harboring 2,630 single nucleotide polymorphism markers, we found two novel major loci, qST6, on chromosome 6, and qST10, on chromosome 10, which controlled traits related to ion toxicity and physiology in response to salinity, respectively. These loci were distinct from the previously known salt tolerance allele on chromosome 3. Other QTLs associated with abiotic stress overlapped with the genomic regions of qST6 and qST10, or with their paralogous regions. Based on the functional annotation and parental expression differences, we identified eight putative candidate genes, two in qST6 and six in qST10, which included a phosphoenolpyruvate carboxylase and an ethylene response factor. This study provides additional genetic resources to breed soybean cultivars with enhanced salt tolerance.

Transcriptomic and Biochemical Analysis Reveal Integrative Pathways Between Carbon and Nitrogen Metabolism in Guzmania monostachia (Bromeliaceae) Under Drought.

Most epiphytes are found in low-nutrient environments with an intermittent water supply. To deal with water limitation, many bromeliads perform crassulacean acid metabolism (CAM), such as Guzmania monostachia, which shifts from C3 to CAM and can recycle CO2 from the respiration while stomata remain closed during daytime and nighttime (CAM-idling mode). Since the absorbing leaf trichomes can be in contact with organic (urea) and inorganic nutrients (NO3−, NH4+) and the urea hydrolysis releases NH4+ and CO2, we hypothesized that urea can integrate the N and C metabolism during periods of severe drought. Under this condition, NH4+ can be assimilated into amino acids through glutamine synthetase (GS), while the CO2 can be pre-fixated by phosphoenolpyruvate carboxylase (PEPC). In this context, we evaluated the foliar transcriptome of G. monostachia to compare the relative gene expression of some genes involved with CAM and the N metabolism when bromeliads were submitted to 7days of drought. We also conducted a controlled experiment with an extended water deficit period (21days) in which bromeliads were cultivated in different N sources (urea, NH4+, and NO3−). Our transcriptome results demonstrated an increment in the expression of genes related to CAM, particularly those involved in the carboxylation metabolism (PEPC1, PPCK, and NAD-MDH), the movement of malate through vacuolar membrane (ALMT9), and the decarboxylation process (PEPCK). Urea stimulated the expression of PEPC1 and ALMT9, while Urease transcripts increased under water deficit. Under this same condition, GS1 gene expression increased, indicating that the NH4+ from urea hydrolysis can be assimilated in the cytosol. We suggest that the link between C and N metabolism occurred through the supply of carbon skeleton (2-oxoglutarate, 2-OG) by the cytosolic isocitrate dehydrogenase since the number of NADP-ICDH transcripts was also higher under drought conditions. These findings indicate that while urea hydrolysis provides NH4+ that can be consumed by glutamine synthetase-cytosolic/glutamate synthase (GS1/GOGAT) cycle, the CO2 can be used by CAM, maintaining photosynthetic efficiency even when most stomata remain closed 24h (CAM-idling) as in the case of a severe water deficit condition. Thus, we suggest that urea could be used by G. monostachia as a strategy to increase its survival under drought, integrating N and C metabolism.

Fluorescent g-C3N4 nanosheets enhanced photosynthetic efficiency in maize

Nano-enabled agriculture becomes a new and rapidly evolving area of research, particularly, nanomaterials (NMs) with light-harvesting capacities for enhancing photosynthesis. However, mechanisms for the interactions between these NMs and plants are not fully understood. Herein, fluorescent and water-soluble graphitic carbon nitride (g-C3N4) nanosheets were prepared and used as artificial antenna to amplify light harvesting ability and enhance photosynthesis in maize. Upon root exposure to 10 mg·L−1 g-C3N4 nanosheets, the g-C3N4 can be taken up and distributed in leaves. Also, the nutrients (Mg, P, Fe, and Mn), chlorophyll content, electron transfer rate, net photosynthetic rate, and carbohydrates content in maize were increased significantly by 1.1%, 51.8%, 44.6%, 121.8%, 12.1%, 44.5%, 30.0% and 32.3%, respectively. In addition, the gene expressions of psbA (photosystem II reaction center protein A) and psaA (photosystem I P700 chlorophyll A apoprotein A1) were up-regulated by 56.3% and 26.8%, respectively. Moreover, the activities of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were significantly increased by 242.3% and 156.3%, respectively. This study provides a new perspective on the use of g-C3N4 nanosheets to promote plant growth and develop nano-enabled agricultural technology.

Multiplex modification of Escherichia coli for enhanced β-alanine biosynthesis through metabolic engineering

β-Alanine is the only naturally occurring β-amino acid, widely used in the fine chemical and pharmaceutical fields. In this study, metabolic design strategies were attempted in Escherichia coli W3110 for enhancing β-alanine biosynthesis. Specifically, heterologous L-aspartate-α-decarboxylase was used, the aspartate kinase I and III involved in competitive pathways were down-regulated, the β-alanine uptake system was disrupted, the phosphoenolpyruvate carboxylase was overexpressed, and the isocitrate lyase repressor repressing glyoxylate cycle shunt was delete, the glucose uptake system was modified, and the regeneration of amino donor was up-regulated. On this basis, a plasmid harboring the heterologous panD and aspB was constructed. The resultant strain ALA17/pTrc99a-panDBS-aspBCG could yield 4.20 g/L β-alanine in shake flask and 43.94 g/L β-alanine (a yield of 0.20 g/g glucose) in 5-L bioreactor via fed-batch cultivation. These modification strategies were proved effective and the constructed β-alanine producer was a promising microbial cell factory for industrial production of β-alanine.

Establishment of a GC-MS-based 13 C-positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism.

13 C-Metabolic flux analysis (13 C-MFA) has greatly contributed to our understanding of plant metabolic regulation. However, the generation of detailed in vivo flux maps remains a major challenge. Flux investigations based on nuclear magnetic resonance have resolved small networks with high accuracy. Mass spectrometry (MS) approaches have broader potential, but have hitherto been limited in their power to deduce flux information due to lack of atomic level position information. Herein we established a gas chromatography (GC) coupled to MS-based approach that provides 13 C-positional labelling information in glucose, malate and glutamate (Glu). A map of electron impact (EI)-mediated MS fragmentation was created and validated by 13 C-positionally labelled references via GC-EI-MS and GC-atmospheric pressure chemical ionization-MS technologies. The power of the approach was revealed by analysing previous 13 C-MFA data from leaves and guard cells, and 13 C-HCO3 labelling of guard cells harvested in the dark and after the dark-to-light transition. We demonstrated that the approach is applicable to established GC-EI-MS-based 13 C-MFA without the need for experimental adjustment, but will benefit in the future from paired analyses by the two GC-MS platforms. We identified specific glucose carbon atoms that are preferentially labelled by photosynthesis and gluconeogenesis, and provide an approach to investigate the phosphoenolpyruvate carboxylase (PEPc)-derived 13 C-incorporation into malate and Glu. Our results suggest that gluconeogenesis and the PEPc-mediated CO2 assimilation into malate are activated in a light-independent manner in guard cells. We further highlight that the fluxes from glycolysis and PEPc toward Glu are restricted by the mitochondrial thioredoxin system in illuminated leaves.

Differential Expression, Tissue-Specific Distribution, and Posttranslational Controls of Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme, which is crucial for plant carbon metabolism. PEPC participates in photosynthesis by catalyzing the initial fixation of atmospheric CO2 and is abundant in both C4 and crassulacean acid metabolism leaves. PEPC is differentially expressed at different stages of plant development, mostly in leaves, but also in developing seeds. PEPC is known to show tissue-specific distribution in leaves and in other plant organs, such as roots, stems, and flowers. Plant PEPC undergoes reversible phosphorylation and monoubiquitination, which are posttranslational modifications playing important roles in regulatory processes and in protein localization. Phosphorylation activates the PEPC enzyme, making it more sensitive to glucose-6-phosphate and less sensitive to malate or aspartate. PEPC phosphorylation is known to be diurnally regulated and delicately changed in response to various environmental stimuli, in addition to light. PEPCs belong to a small gene family encoding several plant-type and distantly related bacterial-type PEPCs. This paper provides a minireview of the general information on PEPCs in both C4 and C3 plants.

Genome-Wide Identification and Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Suaeda aralocaspica, an Annual Halophyte With Single-Cellular C4 Anatomy

Phosphoenolpyruvate carboxylase (PEPC) plays pivotal roles in the carbon fixation of photosynthesis and a variety of metabolic and stress pathways. Suaeda aralocaspica belongs to a single-cellular C4 species and carries out a photosynthetic pathway in an unusually elongated chlorenchyma cell, which is expected to have PEPCs with different characteristics. To identify the different isoforms of PEPC genes in S. aralocaspica and comparatively analyze their expression and regulation patterns as well as the biochemical and enzymatic properties in this study, we characterized a bacterial-type PEPC (BTPC; SaPEPC-4) in addition to the two plant-type PEPCs (PTPCs; SaPEPC-1 and SaPEPC-2) using a genome-wide identification. SaPEPC-4 presented a lower expression level in all test combinations with an unknown function; two SaPTPCs showed distinct subcellular localizations and different spatiotemporal expression patterns but positively responded to abiotic stresses. Compared to SaPEPC-2, the expression of SaPEPC-1 specifically in chlorenchyma cell tissues was much more active with the progression of development and under various stresses, particularly sensitive to light, implying the involvement of SaPEPC-1 in a C4 photosynthetic pathway. In contrast, SaPEPC-2 was more like a non-photosynthetic PEPC. The expression trends of two SaPTPCs in response to light, development, and abiotic stresses were also matched with the changes in PEPC activity in vivo (native) or in vitro (recombinant), and the biochemical properties of the two recombinant SaPTPCs were similar in response to various effectors while the catalytic efficiency, substrate affinity, and enzyme activity of SaPEPC-2 were higher than that of SaPEPC-1 in vitro. All the different properties between these two SaPTPCs might be involved in transcriptional (e.g., specific cis-elements), posttranscriptional [e.g., 5′-untranslated region (5′-UTR) secondary structure], or translational (e.g., PEPC phosphorylation/dephosphorylation) regulatory events. The comparative studies on the different isoforms of the PEPC gene family in S. aralocaspica may help to decipher their exact role in C4 photosynthesis, plant growth/development, and stress resistance.

A conserved C-terminal peptide of sorghum phosphoenolpyruvate carboxylase promotes its proteolysis, which is prevented by Glc-6P or the phosphorylation state of the enzyme

Main conclusionA synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect.Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.

English

Nitrogen (N), one of the most important elements for plant growth, is needed by plants in large quantities. However, this nutrient has limited supply in the soil. Arbuscular mycorrhizal fungi (AMF) are known for their ability to form symbiotic association with plants and transfer the mineral nutrients to the host plants. To validate this hypothesis on sorghum plants, three ecotypes of this cereal (3p4, 3p9 and 4p11) were cultivated with and without AMF under low nitrogen concentration (0.5 mM NH4+). Growth parameters were determined and key enzymes responsible for nitrogen and carbon metabolisms such as glutamine synthetase (GS), glutamate dehydrogenase (GDH), phosphoenolpyruvate carboxylase (PEPC), isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH) and asparate aminotransferase (AAT) were measured. For the three sorghum ecotypes, mycorrhizal plants showed a higher plant growth compared to the control plants. The biochemical parameters revealed a significant increase in the nitrogen assimilatory enzymes; GS and GDH in the leaves and roots of mycorrhizal plants. Furthermore, mycorrhizal fungi also appear to have a significant effect on carbon assimilatory enzymes. These enzymes are known to have a cardinal role in the provision of carbon skeletons essential for the assimilation of ammonium and thus, amino acids synthesis. Our study indicates clearly that AMF can be an efficient way to optimize nitrogen uptake and/or assimilation by plants and thus improve the crop yields with lower amount of nitrogen fertilizers. © 2021 Friends Science Publishers

Efficient Regulation of CO2 Assimilation Enables Greater Resilience to High Temperature and Drought in Maize.

Increasing temperatures and extended drought episodes are among the major constraints affecting food production. Maize has a relatively high temperature optimum for photosynthesis compared to C3 crops, however, the response of this important C4 crop to the combination of heat and drought stress is poorly understood. Here, we hypothesized that resilience to high temperature combined with water deficit (WD) would require efficient regulation of the photosynthetic traits of maize, including the C4–CO2 concentrating mechanism (CCM). Two genotypes of maize with contrasting levels of drought and heat tolerance, B73 and P0023, were acclimatized at high temperature (38°C versus 25°C) under well-watered (WW) or WD conditions. The photosynthetic performance was evaluated by gas exchange and chlorophyll a fluorescence, and in vitro activities of key enzymes for carboxylation (phosphoenolpyruvate carboxylase), decarboxylation (NADP-malic enzyme), and carbon fixation (Rubisco). Both genotypes successfully acclimatized to the high temperature, although with different mechanisms: while B73 maintained the photosynthetic rates by increasing stomatal conductance (gs), P0023 maintained gs and showed limited transpiration. When WD was experienced in combination with high temperatures, limited transpiration allowed water-savings and acted as a drought stress avoidance mechanism. The photosynthetic efficiency in P0023 was sustained by higher phosphorylated PEPC and electron transport rate (ETR) near vascular tissues, supplying chemical energy for an effective CCM. These results suggest that the key traits for drought and heat tolerance in maize are limited transpiration rate, allied with a synchronized regulation of the carbon assimilation metabolism. These findings can be exploited in future breeding efforts aimed at improving maize resilience to climate change.

Metabolic engineering of Escherichia coli for quinolinic acid production by assembling L-aspartate oxidase and quinolinate synthase as an enzyme complex

Quinolinic acid (QA) is a key intermediate of nicotinic acid (Niacin) which is an essential human nutrient and widely used in food and pharmaceutical industries. In this study, a quinolinic acid producer was constructed by employing comprehensive engineering strategies. Firstly, the quinolinic acid production was improved by deactivation of NadC (to block the consumption pathway), NadR (to eliminate the repression of L-aspartate oxidase and quinolinate synthase), and PtsG (to slow the glucose utilization rate and achieve a more balanced metabolism, and also to increase the availability of the precursor phosphoenolpyruvate). Further modifications to enhance quinolinic acid production were investigated by increasing the oxaloacetate pool through overproduction of phosphoenolpyruvate carboxylase and deactivation of acetate-producing pathway enzymes. Moreover, quinolinic acid production was accelerated by assembling NadB and NadA as an enzyme complex with the help of peptide-peptide interaction peptides RIAD and RIDD, which resulted in up to 3.7 g/L quinolinic acid being produced from 40 g/L glucose in shake-flask cultures. A quinolinic acid producer was constructed in this study, and these results lay a foundation for further engineering of microbial cell factories to efficiently produce quinolinic acid and subsequently convert this product to nicotinic acid for industrial applications.

Updating the steady-state model of C4 photosynthesis.

C4 plants play a key role in world agriculture. For example, C4 crops such as maize and sorghum are major contributors to food production in both developed and developing countries, and the C4 grasses sugarcane, miscanthus, and switchgrass are major plant sources of bioenergy. In the challenge to manipulate and enhance C4 photosynthesis, steady-state models of leaf photosynthesis provide an important tool for gas exchange analysis and thought experiments that can explore photosynthetic pathway changes. Here a previous C4 photosynthetic model developed by von Caemmerer and Furbank has been updated with new kinetic parameterization and temperature dependencies added. The parameterization was derived from experiments on the C4 monocot, Setaria viridis, which for the first time provides a cohesive parameterization. Mesophyll conductance and its temperature dependence have also been included, as this is an important step in the quantitative correlation between the initial slope of the CO2 response curve of CO2 assimilation and in vitro phosphoenolpyruvate carboxylase activity. Furthermore, the equations for chloroplast electron transport have been updated to include cyclic electron transport flow, and equations have been added to calculate the electron transport rate from measured CO2 assimilation rates.

Transition From Proto-Kranz-Type Photosynthesis to HCO3 - Use Photosynthesis in the Amphibious Plant Hygrophila polysperma.

Hygrophila polysperma is a heterophyllous amphibious plant. The growth of H. polysperma in submerged conditions is challenging due to the low CO2 environment, increased resistance to gas diffusion, and bicarbonate ion (HCO3–) being the dominant dissolved inorganic carbon source. The submerged leaves of H. polysperma have significantly higher rates of underwater photosynthesis compared with the terrestrial leaves. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate (DIDS), an anion exchanger protein inhibitor, and ethoxyzolamide (EZ), an inhibitor of internal carbonic anhydrase, repressed underwater photosynthesis by the submerged leaves. These results suggested that H. polysperma acclimates to the submerged condition by using HCO3– for photosynthesis. H. polysperma transports HCO3– into the leaf by a DIDS-sensitive HCO3– transporter and converted to CO2 by carbonic anhydrase. Additionally, proteome analysis revealed that submerged leaves accumulated fewer proteins associated with C4 photosynthesis compared with terrestrial leaves. This finding suggested that H. polysperma is capable of C4 and C3 photosynthesis in the terrestrial and submerged leaves, respectively. The ratio of phosphoenol pyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the submerged leaves was less than that in the terrestrial leaves. Upon anatomical observation, the terrestrial leaves exhibited a phenotype similar to the Kranz anatomy found among C4 plants; however, chloroplasts in the bundle sheath cells were not located adjacent to the vascular bundles, and the typical Kranz anatomy was absent in submerged leaves. These results suggest that H. polysperma performs proto-Kranz type photosynthesis in a terrestrial environment and shifts from a proto-Kranz type in terrestrial leaves to a HCO3– use photosynthesis in the submerged environments.

Photosynthetic acclimation during low-light-induced leaf senescence in post-anthesis maize plants.

Low light conditions not only induce leaf senescence, but also photosynthetic acclimation. This study aimed to determine whether plants exhibit photosynthetic acclimation during low-light-induced leaf senescence. The influences of shading on leaf senescence and photosynthetic acclimation were explored in post-anthesis maize plants. The results showed that whole shading (WS) of maize plants accelerated leaf senescence, whereas partial shading (PS) slowed leaf senescence. WS led to larger decreases in the photosynthetic rate (Pn) and stomatal conductance (Gs) compared to those of the PS treatment. Interestingly, chlorophyll afluorescence (ChlF) demonstrated that the absorption flux (ABS/CSo) and trapped energy flux (TRo/CSo) per cross section in leaves remained relatively stable under WS, whereas significant decreases in the active PSII reaction centers (RC/CSo) resulted in considerable increases in absorption (ABS/RC) and trapped energy flux (TRo/RC) per reaction center. ABS/CSo, TRo/CSo, ABS/RC, and TRo/RC increased markedly under PS, whereas there were slight decreases in RC/CSo and electron transport activity. These results suggest that the PS treatment resulted in obvious improvements in the absorption and capture of light energy in shaded leaves. Further analysis demonstrated that both the WS and PS treatments resulted in a greater decrease in the activity of Rubisco compared to that of phosphoenolpyruvate carboxylase (PEPC). Moreover, PEPC activity in PS was maintained at a high level. Consequently, the current study proposed that the improvement of the absorption and capture of light energy and the maintenance of PEPC activity of mesophyll cells were due to photosynthetic acclimation of low-light-induced leaf senescence in maize plants. In addition, the rate of senescence of vascular bundle cells in maize leaves exceeded that of mesophyll cells under low light, showing obvious tissue specificity.