Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Aspartate Aminotransferase in Alfalfa Root Nodules : II. Immunological Distinction between Two Forms of the Enzyme

Aspartate aminotransferase (AAT), a key enzyme in the biosynthesis of aspartate and asparagine, occurs as two forms in alfalfa (Medicago sativa L.), AAT-1 and AAT-2. Both forms were purified to near homogeneity, and high titer polyclonal antibodies produced to the native proteins. Alfalfa AAT-1 was purified from root suspension culture cells, while AAT-2 was purified from effective root nodules. Antibodies prepared to AAT-1 and used as probes for western blots readily recognized native and SDS forms of AAT-1 but did not recognize either native or SDS forms of AAT-2. Conversely, antibodies to AAT-2 readily recognized native and SDS forms of AAT-2 but did not recognize AAT-1. Immunotitrations further confirmed the immunological distinction between AAT-1 and AAT-2. AAT-1 antibodies immunotitrated 100% of the in vitro activity of purified AAT-1 but had no effect on AAT-2 in vitro activity. Likewise, AAT-2 antibodies removed 100% of the in vitro activity of purified AAT-2 but did not affect AAT-1 in vitro activity. Sequential titration of total AAT activity from roots and nodules showed that AAT-1 comprised the major form (62%) of AAT in roots, while AAT-2 was the predominant form (90%) in nodules. Last, SDS-PAGE western blots showed that the molecular masses of AAT-1 and AAT-2 were 42 and 40 kilodaltons, respectively. These data indicate that AAT is under the control of at least two distinct genes in alfalfa.     

Subcellular Distribution of Aspartate Transaminase,Alanine Amino Transferase,Glutamate Dehydrogenases,and Lactate Dehydrogenase in Tetrahymena*

SYNOPSIS. Mitochondria and peroxisomes were isolated from homogenates of Tetrahymena pyriformis by sedimentation through a sucrose gradient. Succinate dehydrogenase was used as a mitochondrial marker; catalase and isocitrate lyase were used to mark the peroxisomal fraction. Lactate dehydrogenase, glutamate dehydrogenase, and alanine aminotransferase were found only in the mitochondrial fraction. Aspartate transaminase was found in both mitochondrial and peroxisomal fractions.     

The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K_m (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K_i of about 6 millimolar. For comparison the K_i for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K_a values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and β-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.     

Effect of Salt Stress on Carbon Metabolism and Bacteroid Respiration in Root Nodules of Common Bean (Phaseolus vulgaris L.)

Abstract: In the present work, we examined the effect of salinity on growth, N fixation and carbon metabolism in the nodule cytosol and bacteroids of Phaseolus vulgaris, and measured the O₂ consumption by bacteroids incubated with or without the addition of exogenous respiratory substrates. The aim was to ascertain whether the compounds that accumulate under salt stress can increase bacteroid respiration and whether this capacity changes in response to salinity in root nodules of Phaseolus vulgaris. The plants were grown in a controlled environment chamber, and 50, 100 mM or no NaCl (control) was added to the nutrient solution. Two harvests were made, at the vegetative growth period and at the beginning of the reproductive period. The enzyme activities in the nodule cytosol were reduced by the salt treatments, while in the bacteroid cytosol the enzyme activities increased at high salt concentrations at the first harvest and for ADH in all treatments. The data presented here confirm that succinate and malate are the preferred substrates for bacteroid respiration in common bean, but these bacteroids may also utilize glucose, either in control or under saline conditions. The addition of proline or lactate to the incubation medium significantly raised oxygen consumption in the bacteroids isolated from plants treated with salt.     

Identification of a Transport Mechanism for NH4+ in the Symbiosome Membrane of Pea Root Nodules

Symbiosome membrane vesicles, facing bacteroid-side-out, were purified from pea (Pisum sativum L.) root nodules and used to study NH4+ transport across the membrane by recording vesicle uptake of the NH4+ analog [14C]methylamine (MA). Membrane potentials ([delta][psi]) were imposed on the vesicles using K+ concentration gradients and valinomycin, and the size of the imposed [delta][psi] was determined by measuring vesicle uptake of [14C]tetraphenylphosphonium. Vesicle uptake of MA was driven by a negative [delta][psi] and was stimulated by a low extravesicular pH. Protonophore-induced collapse of the pH gradient indicated that uptake of MA was not related to the presence of a pH gradient. The MA-uptake mechanism appeared to have a large capacity for transport, and saturation was not observed at MA concentrations in the range of 25 [mu]M to 150 mM. MA uptake could be inhibited by NH4+, which indicates that NH4+ and MA compete for the same uptake mechanism. The observed fluxes suggest that voltage-driven channels are operating in the symbiosome membrane and that these are capable of transporting NH4+ at high rates from the bacteroid side of the membrane to the plant cytosol. The pH of the symbiosome space is likely to be involved in regulation of the flux.     

Rapid Swelling of a CHO-K1 Aspartate/Glutamate Transport Mutant in Hypo-osmotic Medium

Two Chinese hamster ovary cell (CHO-K1) mutants selected for defective glutamate transport via system X⁻ _AG are also highly permeable to small neutral molecules. Light microscopy demonstrated that exposure of one of these mutants, Ed-A1, to hypo-osmotic medium led to extremely rapid swelling, presumably due to increased water flux. When placed in 20% saline, Ed-A1 cells swelled to three times their original volume within 15 sec, a sixfold larger increase than parental CHO-K1. In spite of this rapid volume increase, mutant and wild-type cells remained viable for 20 min in dilute saline. A regulatory volume decrease in Ed-A1, and the continual swelling of CHO-K1, resulted in the two cells achieving equal size after 5 min in 20% saline. The time course of these volume changes permitted analysis of large numbers of cells by a hydrodynamic technique, steric field flow fractionation (FFF). Steric FFF demonstrated the expected inhibition of osmotic swelling of human erythrocytes by the mercurial, p-chloromercuribenzenesulfonic acid (PCMBS). However, PCMBS increased the apparent swelling rate of Ed-A1 and CHO-K1, suggesting that an aquaporin-like molecule is not responsible for any significant fraction of the water fluxes into either line. PCMBS also strongly inhibited aspartate transport by system X⁻ _AG. By taking advantage of their different swelling rates in hypotonic medium, steric FFF can separate mixtures of CHO-K1 and Ed-A1. Received: 2 August 1996/Revised: 25 October 1996     

Participation of ADP/ATP- and Aspartate/Glutamate Antiporters in the Uncoupling Action of Fatty Acids in Liver Mitochondria of the Frog Rana temporaria

Data are presented on molecular mechanisms of uncoupling of oxidative phosphorylation by fatty acids (laurate) in liver mitochondria of one of the poikilothermal animals, the frog Rana temporaria. It has been shown that the uncoupling action of laurate in frog liver mitochondria, like in those of mammals, occurs with participation of protein carriers of anions of the inner mitochondrial membrane, ADP/ATP- and aspartate/glutamate antiporters. At the same time, in frog liver mitochondria the uncoupling activity of laurate is lower than in liver mitochondria of mammals (white mice). Seasonal differences in the laurate uncoupling activity in frog liver mitochondria are revealed: it is much lower in April, than in January, the season of metabolic depression. This difference is due to that in January the degree of participation of the aspartate/glutamate antiporter in the uncoupling is considerably decreased.     

Phytohormones, Rhizobium Mutants, and Nodulation in Legumes : III. Auxin Metabolism in Effective and Ineffective Pea Root Nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied to the apical bud of intact pea (Pisum sativum L. cv Greenfeast) plants. Radioactivity was detected in all tissues after 24 hours. More radioactivity accumulated in the nodules than in the parent root on a fresh weight basis and more in effective (nitrogen-fixing) nodules than in ineffective nodules (which do not fix nitrogen).

For most samples, thin layer chromatography revealed major peaks of radioactivity at the R_F values of IAA and indole-3-acetylaspartic acid (IAAsp) and further evidence of the identity of these compounds was obtained by chromatography in other systems. Disintegrations per minute due to IAA per unit fresh weight were significantly greater for root than for nodule tissue, but were not significantly different for effective and ineffective nodules. Radioactivity due to IAAsp, expressed both on a percentage basis and per unit fresh weight, was significantly greater for nodule than for root tissue and significantly greater for the effective nodules than for the ineffective nodules. When [³H]IAA was applied to effective nodules, IAAsp was the dominant metabolite in the nodule. The data suggest that metabolism of auxins may be important for the persistence of a functional root nodule.

     

The Mitochondrial Aspartate/Glutamate Carrier AGC1 and Calcium Homeostasis: Physiological Links and Abnormalities in Autism

Autism spectrum disorder (ASD) is a severe, complex neurodevelopmental disorder characterized by impairments in reciprocal social interaction and communication, and restricted and stereotyped patterns of interests and behaviors. Recent evidence has unveiled an important role for calcium (Ca²⁺) signaling in the pathogenesis of ASD. Post-mortem studies of autistic brains have pointed toward abnormalities in mitochondrial function as possible downstream consequences of altered Ca²⁺ signaling, abnormal synapse formation, and dysreactive immunity. SLC25A12, an ASD susceptibility gene, encodes the Ca²⁺-regulated mitochondrial aspartate–glutamate carrier, isoform 1 (AGC1). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and adenosine triphosphate (ATP) production. Here, we review the physiological roles of AGC1, its links to calcium homeostasis, and its involvement in autism pathogenesis.     

The Effect of Darkness on the Leghemoglobin Content and Amino Acid Levels in the Root Nodules of Pea Plants

The dependence of the nitrogen fixing system in the root nodules of pea plants (Pisum sativum) L. cv. Torsdag II) on light induced reactions was studied. The pots of the inoculated pea plants, after the nolules had fixed nitrogen for a fornight, were transferred to a dark room. The control plants were kept under normal lighting conditions. The decay of leghemoglobin was measured after photosynthesis had ceased. In the dark the red nodules turned green in three days, when about half of the haem had been broken down. The plants in normal lighting conditions had maintained the red nodules. The appearence of leghemoglobin and bacteroids was simultaneouos. In normal lighting conditions the number of bacteroids was about 1.6 × 10⁸ per g fresh nodules. The appearance of leghemoglobin and bacteroids was simultaneous. In normal lighting conditons the number of bacteroids was bout 1.6 × 10⁸ per g fresh nodules. At the same time as the nodules turned green in the dark most of the bacteroids disappeared and the number of rod-shaped bacteria increased. After five days int the dark thenumber of bacteria of the green nodules was 2.2 × 10⁸ per g fresh nodules. A large increase of of bacteria in the nodules is one of the results after the termination of effective symbiosis. Quantitative estimations were made with an automatic amino acid analysator of the amino acid composition in the root nodules of pea plants grown in the light and of pea plants grown in the dark. Altogether 27 amino acids and amides and 3 unknown ninhydrin positive compounds were found in the free amino acid fraction. In the red N-fixing nodules asparagine, the amide of aspartic acid, was the most prominent (more than 50 per cent of the total amino acid fraction), indicating the energy charge of the nitrogen fixation. 5 days in the dark affected the proportions of the amino acids as follows. Asparagine, homoserine, γ-aminobutyric acid and ethanolamine were decreased and the most of the others increased. In the hydrolysate of the non-soluble protein fraction 25 amino acids could be detected. The proportions of the amino acids in the root nodules of light-grown and dark-grown pea plants were very similar. Hydroxyproline and α, γ-diaminopimelic acid (DAP) were found in these fraction. Most of the DAP was contained in the peptide fraction. Also hydroxyproline was found to a small extent. It was assumed that the amino acids in this fraction were derived from the peptides of both plant cells and rhizobia.     

A Relationship between Length of Basis and Adventitious Root Formation in Pea Cuttings

A positive correlation between the length of the basis and the ability of the cuttings to form adventitious roots was observed in pea cuttings. Plants with a different basis length (the third internode) were obtained in different ways: Regulation by the level of irradiance, dark treatment or gibberellic acid. The length of the basis was also regulated by excision of the cuttings at different places on the stock plants. With increasing basis length an increase was found in the number of roots subsequently formed. The results were similar in cuttings from plants grown at different levels of irradiance or from dark treated plants. Optimal rooting was obtained by cutting the plants just above the second scale leaf. Cuttings from plants treated with 10^?3M GA₃ showed the same correlation between the length of the third internode and root formation as found in the other experiments, but the number of roots were at a lower level.     

Hippocampal Glutamate Level and Glutamate Aspartate Transporter (GLAST) are Up-Regulated in Senior Rat Associated with Isoflurane-Induced Spatial Learning/Memory Impairment

Postoperative cognitive decline is a clinical concern especially for senior patients. It is generally recognized that glutamatergic system plays a crucial role in the physiopathologic process of neurocognitive deterioration. However, alterations of glutamatergic system in prolonged isoflurane-induced learning/memory decline are still unclear. This study investigates the question whether glutamate concentration and corresponding transporters or receptors display any alternations in aged rat suffering from isoflurane-induced learning/memory impairment. 111 male Sprague–Dawley rats (>18 months) were randomly divided into two main groups: hippocampal microdialysis group (n = 38) and western blotting group (n = 73). Each group was subdivided into three subgroups including (1) control subgroup (n = 6 and 10, receiving no behavioral trial, anesthesia or air exposure); (2) air-exposed subgroup (n = 7 and 15, receiving behavioral trial and air exposure but not anesthesia); (3) isoflurane anesthesia subgroup (n = 25 and 48, receiving both behavioral trial and anesthesia). The isoflurane-exposed rats were further divided into a learning/memory-impaired subgroup and a non-learning/memory-impaired subgroup according to their behavioral performance, which was measured using Morris water maze. Hippocampal glutamate concentrations in microdialysates were determined by high-performance liquid chromatography. Expression levels of GLAST, GLT-1, NMDAR1, NMDAR2A/B, AMPAR and tau in hippocampus were assessed via quantitative Western blotting. The incidences of learning/memory impairment of isoflurane-exposed rats in hippocampal microdialysis group and western blotting group were 12.0 (3/25) and 10.4 % (5/48) respectively. The intra-anesthesia hippocampal glutamate levels were significantly lower than those of non-anesthesized rats. The learning/memory-impaired rats showed a long-lasting increased glutamate level from 24 h after isoflurane exposure to the end of the study, but the other 22 isoflurane-exposed rats did not. The learning/memory-impaired subgroup displayed a significantly higher GLAST level than the other three subgroups (p = 0.026, 0.02 and 0.032 respectively). The expression levels of GLT-1, NMDAR1, NMDAR2A/B and AMPAR of every subgroup were comparable. We found a continuous raised hippocampal glutamate and an up-regulation of GLAST rather than GLT-1, NMDAR1, NMDAR2A/B, AMPAR or tau in hippocampus of aged rats associated with isoflurane-induced learning/memory impairment.     

Two Isoenzymes of NADH-dependent Glutamate Synthase in Root Nodules of Phaseolus vulgaris L: Purification, Properties and Activity Changes during Nodule Development

The specific activity of plant NADH-dependent glutamate synthase (NADH-GOGAT) in root nodules of Phaseolus vulgaris L. is over threefold higher than the specific activity of ferredoxin-dependent GOGAT. The NADH-GOGAT is composed of two distinct isoenzymes (NADH-GOGAT I and NADH-GOGAT II) which can be separated from crude nodule extracts by ion-exchange chromatography. Both NADH-GOGAT isoenzymes have been purified to apparent homogeneity and shown to be monomeric proteins with similar M_rs of about 200,000. They are both specific for NADH as reductant. An investigation of their kinetic characteristics show slight differences in their K_ms for l-glutamine, 2-oxoglutarate, and NADH, and they have different pH optima, with NADH-GOGAT I exhibiting a broad pH optimum centering at pH 8.0 whereas NADH-GOGAT II has a much narrower pH optimum of 8.5. The specific activity of NADH-GOGAT in roots is about 27-fold lower than in nodules and consists almost entirely of NADH-GOGAT I. During nodulation both isoenzymes increase in activity but the major increase is due to NADH-GOGAT II which increases over a time course similar to the increase in nitrogenase activity. This isoenzyme is twice as active as NADH-GOGAT I in mature nodules. The roles and regulation of these two isoenzymes in the root nodule are discussed.     

Comparison of the Protein Composition and Enzymatic Activities during Development between Effective and Plant-Determined Ineffective Nodules in Pea

The protein composition and enzymatic activities during developmentof ineffective nodules, produced by mutant E135 (sym 13) ofpea (Pisum sativum L.), were compared with those of the nitrogen-fixingnodules of the normal parent, the Sparkle cultivar. The proteincomposition of 3-week-old E135 nodules, as determined by SDS-polyacrylamidegel electrophoresis, was quite similar to that of Sparkle nodules.After 4 weeks, however, the intensities of bands of 15-, 38-,and 87-kDa polypeptides were lower in the case of E135 nodules.Western blot analysis using a "nodule-specific" antiserum revealedthat most nodulins could be detected in 3-week-old E135 nodules,but a 35.5-kDa nodulin disappeared after 5 weeks and severalnovel peptides ranging in molecular weight from 26 to 31 kDaappeared after 6 weeks in E135 nodules. The activities of glutaminesynthetase, glutamate synthase, alanine-pyruvate aminotransferase,sucrose synthase, and phosphoenolpyruvate carboxylase increasedduring development of Sparkle nodules, but such increases werenot found in E135 nodules after 5 weeks. These results showthat the nodules of E135 begin to develop normally but differfrom those of Sparkle within 4 weeks, indicating that, duringearly stages of nodule development, the protein compositionand activities of enzymes involved in carbon and nitrogen metabolismare not regulated by the presence or absence of nitrogenaseactivity. (Received February 26, 1993; Accepted May 19, 1993)     

Import of Aspartate and Malate by DcuABC Drives H2/Fumarate Respiration to Promote Initial Salmonella Gut-Lumen Colonization in Mice

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