Changes in ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase activities and 14CO2 fixation during the rooting of strawberry shoots in vitro

1994 ◽  
Vol 74 (4) ◽  
pp. 827-831 ◽  
Author(s):  
Chafik Hdider ◽  
Yves Desjardins
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Time Course ◽  
Carbon Fixation ◽  
Acid Synthesis ◽  
Rubisco Activity ◽  
Amino Acid Synthesis ◽  
Bisphosphate Carboxylase ◽  
Rooting Medium ◽  
Rapid Incorporation

The potential for carbon fixation was investigated in in vitro strawberry (Fragaria × ananassa Duch. Kent) shoots 5, 10 and 28 d after transfer to a rooting medium. The activities of ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) and the time course of 14CO2 fixation were investigated. Five days after transfer, Rubisco activity was low but was increased two-fold after 28 d. In contrast PEPC activity was highest at 5 d and declined to about 0.4-fold by day 28. The rate of 14CO2 fixation was similar at 5, 10 and 28 d after transfer. However, a more rapid incorporation of 14CO2 into amino acids was observed at 5 than at 10 or 28 d after transfer. These results suggest that strawberry shoots undergo a progressive transition from heterotrophic to autotrophic carbon fixation during their rooting and that PEPC plays an important role in sustaining carbon fixation and amino acid synthesis during the first few days after their transfer to rooting medium. Key words: In vitro culture, phosphoenolpyruvate carboxylase, ribulose-1,5-bisphosphate carboxylase

The inorganic carbon requirements for nitrogen assimilation

1991 ◽  
Vol 69 (5) ◽  
pp. 1139-1145 ◽  
Author(s):  
David H. Turpin ◽  
Greg C. Vanlerberghe ◽  
Alan M. Amory ◽  
Robert D. Guy
Keyword(s):  
Amino Acid ◽  
Phosphoenolpyruvate Carboxylase ◽  
Nitrogen Assimilation ◽  
Inorganic Carbon ◽  
Dissolved Inorganic Carbon ◽  
Sufficient Conditions ◽  
Acid Synthesis ◽  
Amino Acid Synthesis ◽  
Bisphosphate Carboxylase ◽  
N Assimilation

In the green alga Selenastrum minutum (Naeg.) Collins the assimilation of NH4+ into the full suite of protein amino acids requires at least three separate and distinct inorganic carbon fixing reactions, catalyzed by the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPC), and carbamoyl phosphate synthetase. In this paper we examine the requirements for CO2 fixation of NH4+ assimilation in this organism. When grown under N-sufficient conditions, NH4+ assimilation is directly dependent upon photosynthetic CO2 fixation to provide carbon skeletons for amino acid synthesis. When cultured under N-limited conditions, the cells accumulate starch, which is then available for amino acid synthesis. This alleviates the requirement of photosynthetic CO2 fixation for NH4+ assimilation. N-limited cells, however, still exhibit a nonphotosynthetic CO2 requirement for N assimilation that is mediated through PEPC. This activity of PEPC increases during N assimilation to replenish TCA cycle intermediates consumed during amino acid synthesis. The in vivo activity of this enzyme is tightly regulated so that there are ~0.3 moles C fixed per mole N assimilated. In S. minutum PEPC is regulated primarily by the ratio of glutamine/glutamate, thus providing a mechanism by which primary NH4+ assimilation modulates the supply of carbon for amino acid biosynthesis. Activation of PEPC during NH4+ assimilation occurs in both the light and the dark. Key words: dissolved inorganic carbon, nitrogen assimilation, phosphoenolpyruvate carboxylase, photosynthesis, amino acid synthesis, respiration.

scholarly journals Design and in vitro realization of carbon-conserving photorespiration

2018 ◽  
Vol 115 (49) ◽  
pp. E11455-E11464 ◽  
Author(s):  
Devin L. Trudeau ◽  
Christian Edlich-Muth ◽  
Jan Zarzycki ◽  
Marieke Scheffen ◽  
Moshe Goldsmith ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Calvin Cycle ◽  
Computational Design ◽  
Carbon Loss ◽  
Fixation Rate ◽  
Bisphosphate Carboxylase ◽  
Stoichiometric Model ◽  
Synthetic Routes ◽  
Coa Reductase

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO2. Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO2. Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO2 release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic–stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO2, even if most of their enzymes operate at a tenth of Rubisco’s maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD+, thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbon-conserving photorespiration pathway.

Phosphoenolpyruvate carboxylase during grape berry development: protein level, enzyme activity and regulation

2000 ◽  
Vol 27 (3) ◽  
pp. 221 ◽  
Author(s):  
Paraskevi Diakou ◽  
Laurence Svanella ◽  
Philippe Raymond ◽  
Jean-Pierre Gaudillère ◽  
Annick Moing
Keyword(s):  
Malic Acid ◽  
Protein Level ◽  
Phosphoenolpyruvate Carboxylase ◽  
Phosphorylation Site ◽  
Acid Synthesis ◽  
Grape Berry ◽  
Vitis Vinifera L ◽  
Normal Acid

The protein level and regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, involved in malic acid synthesis) was studied during the fruit development of two grape (Vitis vinifera L.) varieties, ‘Cabernet Sauvignon’ and ‘Gora Chirine’, with berries of normal and low organic acid content, respectively. The protein level and in vitro activity were higher in the low-acid variety than in the normal-acid variety for most stages. In vivo PEPC activity, measured using 14 CO2 labelling, was significantly higher in the low-acid variety than in the normal-acid variety about 1 week before and 1 week after veraison (the day which corresponds to the onset of ripening). However, partitioning into malate was the same for both varieties. Antibodies raised against the N-terminal part of SorghumPEPC recognised the grape berry PEPC, indicating the presence of the consensus phosphorylation site involved in PEPC regulation. PEPC phosphorylation status was estimated by studying sensitivity to pH and malate. Grape berry PEPC appeared more sensitive to low pH and malate during ripening (IC50 malate, 0.2–0.7 mM) compared to during the earlier stages of development (IC50 malate, 1.2–2 mM) for both varieties. Therefore, in the normal-acid variety, PEPC seems to participate in controlling malic acid accumulation but does not seem to control the differences in malic acid concentration observed between the two varieties.

scholarly journals Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

1999 ◽  
Vol 181 (4) ◽  
pp. 1088-1098 ◽  
Author(s):  
Castor Menendez ◽  
Zsuzsa Bauer ◽  
Harald Huber ◽  
Nasser Gad’on ◽  
Karl-Otto Stetter ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Carbon Fixation ◽  
Coenzyme A ◽  
Autotrophic Growth ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed forC. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus andAcidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula,S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

scholarly journals Photometric Measurements of Rubisco Activity in Leaves of Deciduous Fruit Crops

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 531A-531
Author(s):  
Lailiang Cheng ◽  
Leslie H. Fuchigami
Keyword(s):  
Net Photosynthesis ◽  
Photometric Method ◽  
Ribulose Bisphosphate Carboxylase ◽  
Fruit Crops ◽  
Rubisco Activity ◽  
Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate Carboxylase Oxygenase ◽  
Deciduous Fruit ◽  
Photometric Measurements

Ribulose bisphosphate carboxylase/oxygenase (Rubisco) initiates the photosynthetic carbon metabolism;therefore, its activity has been measured in many physiological studies. However, information on in vitro Rubisco activity from leaves of deciduous fruit crops is very limited and the reported activities are suspiciously low. We measured Rubisco activity in crude extracts of leaves of apple, pear, peach, cherry, and grape by using a photometric method in which RuBP carboxylation was enzymically coupled to NADH oxidation. Replacing polyvinylpyrrolidone with polyvinylpolypyrrolidone in the extraction solution significantly increased extractable Rubisco activity. Depending on species, freezing leaf discs in liquid nitrogen followed by storage at –80°C for only 24 hr reduced both initial and total Rubisco activity to 5% to 50% of that obtained from fresh leaves. Initial Rubisco activity from fresh leaf tissues of all species was well correlated with maximum Rubisco activity (Vcmax) estimated from gas exchange; an exception was pear, where initial Rubisco activity was higher than Vcmax. In most cases, initial Rubisco activity was approximately two to three times higher than net photosynthesis.

scholarly journals The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate–citrate exchange

2021 ◽  
Author(s):  
Chun Pong Lee ◽  
Marlene Elsässer ◽  
Philippe Fuchs ◽  
Ricarda Fenske ◽  
Markus Schwarzländer ◽  
...  
Keyword(s):  
Systems Approach ◽  
Acid Synthesis ◽  
Amino Acid Synthesis ◽  
Mitochondrial Carrier ◽  
Cell Compartments ◽  
Organic Acid Metabolism ◽  
In Organello ◽  
Mitochondrial Carrier Proteins

Abstract Malate and citrate underpin the characteristic flexibility of central plant metabolism by linking mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. However, the identity of mitochondrial carrier proteins that influence both processes has remained elusive. Here we show by a systems approach that DICARBOXYLATE CARRIER 2 (DIC2) facilitates mitochondrial malate–citrate exchange in vivo in Arabidopsis thaliana. DIC2 knockout (dic2-1) retards growth of vegetative tissues. In vitro and in organello analyses demonstrate that DIC2 preferentially imports malate against citrate export, which is consistent with altered malate and citrate utilization in response to prolonged darkness of dic2-1 plants or a sudden shift to darkness of dic2-1 leaves. Furthermore, isotopic glucose tracing reveals a reduced flux towards citrate in dic2-1, which results in a metabolic diversion towards amino acid synthesis. These observations reveal the physiological function of DIC2 in mediating the flow of malate and citrate between the mitochondrial matrix and other cell compartments.

scholarly journals Increasing metabolic potential: C-fixation

2018 ◽  
Vol 62 (1) ◽  
pp. 109-118 ◽  
Author(s):  
P. John Andralojc ◽  
Elizabete Carmo-Silva ◽  
Gustaf E. Degen ◽  
Martin A.J. Parry
Keyword(s):  
Carbon Fixation ◽  
Crop Yields ◽  
World Population ◽  
Rubisco Activity ◽  
Metabolic Potential ◽  
Bisphosphate Carboxylase ◽  
Changing Environments ◽  
Photosynthetic Carbon Fixation ◽  
Photosynthetic Carbon ◽  
Breeding Programmes

Due to the growing world population, crop yields must increase to meet the rising demand. Crop plants also require adaptation to optimize performance in the changing environments caused by climate change. Improving photosynthetic carbon fixation is a promising, albeit technically challenging, strategy whose potential has only just begun to be considered in breeding programmes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a fundamental enzyme of carbon fixation, is extremely inefficient and many strategies to improve photosynthesis focus on overcoming the limitations of this enzyme, either by improving Rubisco activity and regulation or by improving the supply of substrates. Although progress is being made, the need to tailor solutions for each crop and their respective environments has been highlighted. Even so, continuing research will be required to achieve these objectives and to grow crops more sustainably in the future.

scholarly journals Folates and Anti-folates � Chemical Perspectives, in vitro and in ovo Assessments

2020 ◽  
Vol 71 (7) ◽  
pp. 481-490
Author(s):  
Bogdan Sorop ◽  
Vlad Laurentiu David ◽  
Alina Heghes ◽  
Delia Berceanu-Vaduva ◽  
Lavinia Balan ◽  
...  
Keyword(s):  
Human Fibroblasts ◽  
Acid Synthesis ◽  
Enzymatic Reactions ◽  
Dependent Manner ◽  
Cam Assay ◽  
In Ovo ◽  
Amino Acid Synthesis ◽  
A Cell ◽  
Dose Dependent

Folates participate in DNA replication reactions, act as a substrate in enzymatic reactions related to amino acid synthesis and vitamin metabolism while antifolates participate in reactions that inhibit the formation of tetrahydrofolate with consequences on protein and nucleic acid synthesis and implicitly on growth and development both types of cells, healthy and diseased. In the present study, the viability of healthy cells, keratinocytes and human fibroblasts was evaluated in the presence of three folates (folic, dihydrofolic and tetrahydrofolic acids), one antifolate (methotrexate) and combinations between them by Alamar blue assay. The antiangiogenic potential was also evaluated by in ovo technique, CAM assay. Cell viability was influenced in a cell-dependent and dose-dependent manner, fibroblasts being more sensitive to the action of the test compounds, especially the combination of metrotrexate and dihydrofolate. Data related to CAM assay showed that methotrexate revealed a slightly higher vessel density, but without inducing toxicity on vascular architecture and functionality. The data obtained highlight the greater sensitivity of the viability of fibroblasts in the presence of metrotrexate and its combinations with folates used in the study.

scholarly journals Metals enable a non-enzymatic acetyl CoA pathway

2017 ◽  
Author(s):  
Sreejith J. Varma ◽  
Kamila B. Muchowska ◽  
Paul Chatelain ◽  
Joseph Moran
Keyword(s):  
Carbon Fixation ◽  
Acid Synthesis ◽  
Last Universal Common Ancestor ◽  
Amino Acid Synthesis ◽  
Evolutionary Origins ◽  
Universal Common Ancestor ◽  
Wide Range ◽  
Biological Conversion ◽  
Acetyl Coa Pathway ◽  
Late Invention

The evolutionary origins of carbon fixation, the biological conversion of CO2to metabolites, remain unclear. Phylogenetics indicates that the AcCoA pathway, the reductive fixation of CO2to acetyl and pyruvate, was a key biosynthetic route used by the Last Universal Common Ancestor (LUCA) to build its biochemistry. However, debate exists over whether CO2fixation is a relatively late invention of pre-LUCA evolution or whether it dates back to prebiotic chemistry. Here we show that zero-valent forms of the transition metals known to act as co-factors in the AcCoA pathway (Fe0, Ni0, Co0) fix CO2on their surface in a manner closely resembling the biological pathway, producing acetate and pyruvate in near mM concentrations following cleavage from the surface. The reaction is robust over a wide range of temperatures and pressures with acetate and pyruvate constituting the major products in solution at 1 bar of CO2and 30 °g;C. The discovered conditions also promote 7 of the 11 steps of the rTCA cycle and amino acid synthesis, providing a stunning direct connection between simple inorganic chemistry and ancient CO2-fixation pathways. The results strongly sup-port the notion that CO2-fixation pathways are an outgrowth of spontaneous geochemistry.

Fatty acid synthesis and triacylglycerol accumulation in rat liver after chronic ethanol consumption

1987 ◽  
Vol 73 (2) ◽  
pp. 159-163 ◽  
Author(s):  
S. Venkatesan ◽  
R. J. Ward ◽  
T. J. Peters
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Time Course ◽  
Fatty Acid Synthase ◽  
Acid Synthesis ◽  
Hepatic Fatty Acid ◽  
Chronic Ethanol Consumption ◽  
Triacylglycerol Content ◽  
Triacylglycerol Accumulation

1. Liver slices from chronically alcohol-fed rats incubated with 3H2O showed less than half the fatty acid synthesis rates of pair-fed controls. Addition of 50 mmol/l ethanol or of 10 mmol/l lactate and 1 mmol/l pyruvate to the incubation medium did not alter the fatty acid synthesis rates in either groups. Hepatic fatty acid synthesis rates measured in vivo with 3H2O were also significantly reduced in alcohol-fed rats. 2. Time-course experiments showed that after 1 week on the ethanol diet hepatic fatty acid synthesis rates in vitro were similar to control rats, although the liver triacylglycerol content was significantly increased. From the second week of feeding, fatty acid synthesis rates were significantly lower in alcohol-fed rats and the liver triacylglycerol content progressively increased compared with controls. 3. Fatty acid synthase activity in liver cytosolic fractions were similar to controls in the alcohol-fed group after 1 week of feeding but were significantly lower in alcohol-fed rats from the second week onwards. 4. These results indicate that hepatic triacylglycerol accumulation after alcohol feeding is not due to increased fatty acid synthesis. The reduced fatty acid synthesis observed is a consequence of triacylglycerol accumulation.

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