Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Changes in energy status of leaf cells as a consequence of mitochondrial genome rearrangement

The MSC16 cucumber (Cucumis sativus L.) mutant with lower activity of mitochondrial Complex I was used to study the influence of mitochondrial metabolism on whole cell energy and redox state. Mutant plants had lower content of adenylates and NADP(H) whereas the NAD(H) pool was similar as in wild type. Subcellular compartmentation of adenylates and pyridine nucleotides were studied using the method of rapid fractionation of protoplasts. The data obtained demonstrate that dysfunction of mitochondrial respiratory chain decreased the chloroplastic ATP pool. No differences in NAD(H) pools in subcellular fractions of mutated plants were observed; however, the cytosolic fraction was highly reduced whereas the mitochondrial fraction was more oxidized in MSC16, as compared to WTc. The NADP(H) pool in MSC16 protoplasts was greatly decreased and the chloroplastic NADP(H) pool was more reduced, whereas the extrachloroplastic pool was much more oxidized, than in WTc protoplast. Changes in nucleotides distribution in cucumber MSC16 mutant were compared to changes found in tobacco (Nicotiana sylvestris) CMS II mitochondrial mutant. In contrast to MSC16 cucumber, the content of adenylates in tobacco mutant was much higher than in tobacco wild type. The differences were more pronounced in leaf tissue collected after darkness than in the middle of the photoperiod. Results obtained after tobacco protoplast fractionating showed that the increase in CMS II adenylate content was mainly due to a higher level in extrachloroplast fraction. Both mutations have a negative effect on plant growth through perturbation of chloroplast/mitochondrial interactions.     

Hexokinase Binding to Mitochondria: A Basis for Proliferative Energy Metabolism

Current thought is that proliferating cells undergo a shift from oxidative to glycolytic metabolism, where the energy requirements of the rapidly dividing cell are provided by ATP from glycolysis. Drawing on the hexokinase–mitochondrial acceptor theory of insulin action, this article presents evidence suggesting that the increased binding of hexokinase to porin on mitochondria of cancer cells not only accelerates glycolysis by providing hexokinase with better access to ATP, but also stimulates the TCA cycle by providing the mitochondrion with ADP that acts as an acceptor for phosphoryl groups. Furthermore, this acceleration of the TCA cycle stimulates protein synthesis via two mechanisms: first, by increasing ATP production, and second, by provision of certain amino acids required for protein synthesis, since the amino acids glutamate, alanine, and aspartate are either reduction products or partially oxidized products of the intermediates of glycolysis and the TCA cycle. The utilization of oxygen in the course of the TCA cycle turnover is relatively diminished even though TCA cycle intermediates are being consumed. With partial oxidation of TCA cycle intermediates into amino acids, there is necessarily a reduction in formation of CO₂ from pyruvate, seen as a relative diminution in utilization of oxygen in relation to carbon utilization. This has been assumed to be an inhibition of oxygen uptake and therefore a diminution of TCA cycle activity. Therefore a switch from oxidative metabolism to glycolytic metabolism has been assumed (the Crabtree effect). By stimulating both ATP production and protein synthesis for the rapidly dividing cell, the binding of hexokinase to mitochondrial porin lies at the core of proliferative energy metabolism. This article further reviews literature on the binding of the isozymes of hexokinase to porin, and on the evolution of insulin, proposing that intracellular insulin-like proteins directly bind hexokinase to mitochondrial porin.     

Investigation of the dark metabolism of acetate in photoheterotrophically grown cells ofRhodospirillum rubrum

The mechanism of the aerobic dark assimilation of acetate in the photoheterotrophically grown purple nonsulfur bacteriumRhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation inRsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C₄-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle inRsp. rubrum cells can function as an anaplerotic pathway under aerobic dark conditions. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO₂ in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicated that citramalate and mesaconate were intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts ofRsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function inRsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Plasma membrane-generated ROS and their possible contribution to leaf cell growth of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) MSC16 mitochondrial mutant

Reactive oxygen species (ROS) generally regarded as harmful products of oxygenic metabolism causing oxidative stress and cell damage are also important for control and regulation of biological processes. ROS can be generated by various enzymatic activities and removed by an array of ROS-scavenging molecules in the cell. In plants, the generation of ROS initiated by the plasma membrane NADPH oxidase can be used for controlled polymer breakdown leading to cell wall loosening during extension growth. The mosaic (MSC16) mitochondrial mutant of cucumber (Cucumis sativus L.) has marked phenotypic changes, including a slower growth rate which partially may result from disturbed leaf carbon and energy metabolism and ROS/antioxidants equilibrium. Cytochemical localization of H₂O₂ in leaf cells showed lower total level of H₂O₂ particularly in the apoplast of MSC16 leaf cells as compared to WT. The activity of plasma membrane NADPH oxidase (EC 1.6.3.1) was about 30% lower in plasmalemma vesicles isolated from MSC16 leaf tissue as compared to WT. The total foliar ascorbate pool (reduced and oxidized) was about 35% higher in MSC16 compared to WT leaves due to an increased content of the oxidized form. About 3% of the whole-leaf ascorbate was localized in the apoplast but in MSC16 it was considerably more reduced. We conclude that the lower apoplastic ROS content caused by decreased activity of plasma membrane NADPH oxidase and lower amounts of H₂O₂ in the apoplast may also contribute to altered growth of the MSC16 cucumber mutant.     

Mitochondrial Respiration Can Support NO(3) and NO(2) Reduction during Photosynthesis : Interactions between Photosynthesis, Respiration, and N Assimilation in the N-Limited Green Alga Selenastrum minutum

Mass spectrometric analysis shows that assimilation of inorganic nitrogen (NH₄⁺, NO₂⁻, NO₃⁻) by N-limited cells of Selenastrum minutum (Naeg.) Collins results in a stimulation of tricarboxylic acid cycle (TCA cycle) CO₂ release in both the light and dark. In a previous study we have shown that TCA cycle reductant generated during NH₄⁺ assimilation is oxidized via the cytochrome electron transport chain, resulting in an increase in respiratory O₂ consumption during photosynthesis (HG Weger, DG Birch, IR Elrifi, DH Turpin [1988] Plant Physiol 86: 688-692). NO₃⁻ and NO₂⁻ assimilation resulted in a larger stimulation of TCA cycle CO₂ release than did NH₄⁺, but a much smaller stimulation of mitochondrial O₂ consumption. NH₄⁺ assimilation was the same in the light and dark and insensitive to DCMU, but was 82% inhibited by anaerobiosis in both the light and dark. NO₃⁻ and NO₂⁻ assimilation rates were maximal in the light, but assimilation could proceed at substantial rates in the light in the presence of DCMU and in the dark. Unlike NH₄⁺, NO₃⁻ and NO₂⁻ assimilation were relatively insensitive to anaerobiosis. These results indicated that operation of the mitochondrial electron transport chain was not required to maintain TCA cycle activity during NO₃⁻ and NO₂⁻ assimilation, suggesting an alternative sink for TCA cycle generated reductant. Evaluation of changes in gross O₂ consumption during NO₃⁻ and NO₂⁻ assimilation suggest that TCA cycle reductant was exported to the chloroplast during photosynthesis and used to support NO₃⁻ and NO₂⁻ reduction.     

Cytochrome and Alternative Pathway Respiration during Transient Ammonium Assimilation by N-Limited Chlamydomonas reinhardtii

Mass spectrometric analysis of gas exchange in light and dark by N-limited cells of Chlamydomonas reinhardtii indicated that ammonium assimilation was accompanied by an increase in respiratory carbon flow to provide carbon skeletons for amino acid synthesis. Tricarboxylic acid (TCA) cycle carbon flow was maintained by the oxidation of TCA cycle reductant via the mitochondrial electron transport chain. In wild-type cells, inhibitor studies and ¹⁸O₂ discrimination experiments indicated that respiratory electron flow was mediated entirely via the cytochrome pathway in both the light and dark, despite a large capacity for the alternative pathway. In a cytochrome oxidase deficient mutant, or in wild-type cells in the presence of cyanide, the alternative pathway could support the increase in TCA cycle carbon flow. These different mechanisms of oxidation of TCA cycle reductant were reflected by the much greater SHAM sensitivity of ammonium assimilation by cytochrome oxidase-deficient cells as compared to wild type.     

Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H₂O₂) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H₂O₂ derives from superoxide (O₂^˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O₂^˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O₂^˙̄ and H₂O₂ may provide feedback signals to modulate mitochondrial metabolism.     

Photosynthetic Carbon Fixation Characteristics of Fruiting Structures of Brassica campestris L

Activities of key enzymes of the Calvin cycle and C₄ metabolism, rates of CO₂ fixation, and the initial products of photosynthetic ¹⁴CO₂ fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv `Toria.' Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5-phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C₄ metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwall and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of ¹⁴CO₂ assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO₂ during light. However, respiratory losses were very high during the dark period.     

Comparative study of metabolism of the purple photosynthetic bacteria grown in the light and in the dark under anaerobic and aerobic conditions

For three species of anoxygenic phototrophic alphaproteobacteria differing in their reaction to oxygen and light, physiological characteristics (capacity for acetate assimilation, activity of the tricarboxylic acid (TCA) cycle enzymes, respiration, and the properties of the oxidase systems) were studied. Nonsulfur purple bacteria Rhodobacter sphaeroides, Rhodobaca bogoriensis, and aerobic anoxygenic phototrophic bacteria Roseinatronobacter thiooxidans were the subjects of investigation. All of these organisms were able to grow under aerobic conditions in the dark using the respiratory system with cytochrome aa ₃ as the terminal oxidase. They differed, however, in their capacity for growth in the light, bacteriochlorophyll synthesis, and regulation of activity of the TCA cycle enzymes. Oxygen suppressed bacteriochlorophyll synthesis by Rha. sphaeroides and Rbc. bogoriensis both in the dark and in the light. Bacteriochlorophyll synthesis in Rna. thiooxidans occurred only in the dark and was suppressed by light. The results on acetate assimilation by the studied strains reflected the degree of their adaptation to aerobic growth in the dark. Acetate assimilation by light-grown Rha. sphaeroides was significantly higher than by the dark-grown ones. Unlike Rha. sphaeroides, acetate assimilation by Rbc. bogoriensis in the light under anaerobic and aerobic conditions was much less dependent on the growth conditions. Aerobic acetate assimilation by all studied bacteria was promoted by light. In Rha. sphaeroides, activity of the TCA cycle enzymes increased significantly in the cells grown aerobically in the dark. In Rbc. bogoriensis, activity of most of the TCA cycle enzymes under aerobic conditions either decreased or remained unchanged. Our results confirm the origin of modern chemoorganotrophs from anoxygenic phototrophic bacteria. The evolution from anoxygenic photoorganotrophs to aerobic chemoorganotrophs included several stages: nonsulfur purple bacteria → nonsulfur purple bacteria similar to Rbc. bogoriensis → aerobic anoxygenic phototrophs → chemoorganotrophs.     

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB₁ receptors peripherally modulate energy metabolism. Here, we investigated the role of CB₁ receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB₁ receptor antagonist AM251 (3 mg kg^-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB₁ receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB₁ receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB₁ receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB ₁ ^-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C₂C₁₂ myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB₁ receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.     

Methionine supplementation stimulates mitochondrial respiration

Mitochondria play essential metabolic functions in eukaryotes. Although their major role is the generation of energy in the form of ATP, they are also involved in maintenance of cellular redox state, conversion and biosynthesis of metabolites and signal transduction. Most mitochondrial functions are conserved in eukaryotic systems and mitochondrial dysfunctions trigger several human diseases.By using multi-omics approach, we investigate the effect of methionine supplementation on yeast cellular metabolism, considering its role in the regulation of key cellular processes. Methionine supplementation induces an up-regulation of proteins related to mitochondrial functions such as TCA cycle, electron transport chain and respiration, combined with an enhancement of mitochondrial pyruvate uptake and TCA cycle activity. This metabolic signature is more noticeable in cells lacking Snf1/AMPK, the conserved signalling regulator of energy homeostasis. Remarkably, snf1Δ cells strongly depend on mitochondrial respiration and suppression of pyruvate transport is detrimental for this mutant in methionine condition, indicating that respiration mostly relies on pyruvate flux into mitochondrial pathways.These data provide new insights into the regulation of mitochondrial metabolism and extends our understanding on the role of methionine in regulating energy signalling pathways.     

Oxidation of carbon sources via the tricarboxylic acid cycle during calcium-induced conidation of Penicillium notatum

The TCA cycle was examined during Ca²⁺-induced conidiation in Penicillium notatum over the 12-h period after addition of Ca²⁺ to vegetative cultures. Conidiation was independent of Ca²⁺ when certain intermediates and derivatives of the TCA cycle served as sole carbon sources. Arsenite and malonate augmented the effect of Ca²⁺ on conidiation but did not substitute for it. Mitochondria from vegetative cells had low rates of oxidation of TCA cycle intermediates and, with the exception of pyruvate, aconitate and glutamate, these were poorly linked to phosphorylation processes. Calcium ions affected mitochondrial function causing reduced oxidation of oxoglutarate, elimination of pyruvate oxidation and a decline in respiratory control of these substrates with increased oxidation of NADH and NADPH. Radiorespirometric studies and enzyme searches revealed a complete but weakly oxidative TCA cycle in vegetative cells. In Ca²⁺-induced cells oxoglutarate dehydrogenase activity was deleted within 6.5 h of Ca²⁺ addition and this was accompanied by establishment of an incomplete Krebs cycle. Calcium-induced conidiation was associated with increased capacity for acetate and glutamate metabolism involving an activated glyoxylate shunt which may be related to enhanced biosynthetic demand. The metabolic basis of the Ca²⁺ effect on conidiation is discussed in connection with previous findings.     

Derivatization of the tricarboxylic acid intermediates with O-benzylhydroxylamine for liquid chromatography–tandem mass spectrometry detection

The tricarboxylic acid (TCA) cycle is an interface among glycolysis, lipid metabolism, and amino acid metabolism. Increasing interest in cancer metabolism has created a demand for rapid and sensitive methods for quantifying the TCA cycle intermediates and related organic acids. We have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to quantify the TCA cycle intermediates in a 96-well format after O-benzylhydroxylamine (O-BHA) derivatization under aqueous conditions. This method was validated for quantitation of all common TCA cycle intermediates with good sensitivity, including α-ketoglutarate, malate, fumarate, succinate, 2-hydroxyglutarate, citrate, oxaloacetate, pyruvate, isocitrate, and lactate using a 8-min run time in cancer cells and tissues. The method was used to detect and quantify changes in metabolite levels in cancer cells and tumor tissues treated with a pharmacological inhibitor of nicotinamide phosphoribosyl transferase (NAMPT). This method is rapid, sensitive, and reproducible, and it can be used to assess metabolic changes in cancer cells and tumor samples.     

Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using ¹³C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival.     

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (F _v/F _m), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism.     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Concentrations of CO(2) in Maize Leaves

The rate of CO₂ assimilation and levels of metabolites of the C₄ cycle and reductive pentose phosphate pathway in an attached leaf of maize (Zea mays L) were measured over a range of intercellular CO₂ concentration (Ci) of 10 to 190 microliters per liter. The CO₂ assimilation rate was saturated at a Ci of around 175 microliters per liter. The levels of ribulose 1,5-bisphosphate and fructose 1,6-bisphosphate decreased substantially with increasing Ci. The levels of 3-phosphoglycerate, phosphoenolpyruvate (PEP), and pyruvate increased with increasing Ci. The level of dihydroxyacetone phosphate increased moderately from Ci of 10 microliters per liter to 20 to 50 microliters per liter and stayed almost constant over the rest of the range of Ci investigated. The levels of fructose 6-phosphate did not show any significant changes over the range of Ci. The levels of glucose 6-phosphate decreased slightly with increasing Ci. Although photosynthetically inactive pools of malate, asparate, and alanine could mask real changes in levels of the photosynthetically active pools of these compounds, the apparent levels of these compounds and the total amount of intermediates in the C₄ cycle (malate, aspartate, pyruvate, PEP, and alanine) increased with increasing Ci. The results suggest that there is carbon input into the C₄ cycle from the reductive pentose phosphate pathway which increases the level of total intermediates of the C₄ cycle with increasing Ci.     

Carbon Metabolism Transitions during the Development of Marine Macroalga Gracilaria verrucosa

The changes in the rate of photosynthetic and dark CO₂ assimilation and the activity of key enzymes of carboxylation were studied during the main developmental stages (shoots, juvenile plants, and mature plants) of red macroalga Gracilaria verrucosa (Huds.) Papenf. Changes in the direction of primary carbon metabolism were also investigated. It was estimated that the transition of metabolism related to the shift in the pathways of carboxylation did not occur during development of G. verrucosa. During all developmental stages, the level of dark CO₂ assimilation was by at least one order of magnitude lower than that of photosynthetic assimilation The predominant pathway of CO₂ assimilation was ribulosobisphosphate carboxylation. At the same time, the transition of metabolism related to the changes in the type of phosphoglyceric acid utilization was found. At the early developmental stages, a substantial part of phosphoglyceric acid was directed into the amino acid metabolism via the anaplerotic pathway of photosynthesis similar to that in higher plants.     

Carboxylation and anaplerosis in neurons and glia

Anaplerosis, or de novo formation of intermediates of the tricarboxylic acid (TCA) cycle, compensates for losses of TCA cycle intermediates, especially α-ketoglutarate, from brain cells. Loss of α-ketoglutarate occurs through release of glutamate and GABA from neurons and through export of glutamine from glia, because these amino acids are α-ketoglutarate derivatives. Anaplerosis in the brain may involve four different carboxylating enzymes: malic enzyme, phosphoenopyruvate carboxykinase (PEPCK), propionyl-CoA carboxylase, and pyruvate carboxylase. Anaplerotic carboxylation was for many years thought to occur only in glia through pyruvate carboxylase; therefore, loss of transmitter glutamate and GABA from neurons was thought to be compensated by uptake of glutamine from glia. Recently, however, anaplerotic pyruvate carboxylation was demonstrated in glutamatergic neurons, meaning that these neurons to some extent can maintain transmitter synthesis independently of glutamine. Malic enzyme, which may carboxylate pyruvate, was recently detected in neurons. The available data suggest that neuronal and glial pyruvate carboxylation could operate at as much as 30% and 40–60% of the TCA cycle rate, respectively. Cerebral carboxylation reactions are probably balanced by decarboxylation reactions, because cerebral CO₂ formation equals O₂ consumption. The finding of pyruvate carboxylation in neurons entails a major revision of the concept of the glutamine cycle.