Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules

Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cytosolic fraction of N₂-fixing soybean ( Glycine max [L.] Merr.) root nodules. The partially purified PK preparation was completely free of contamination by phospho enol pyruvate carboxylase (PEPC, EC 4.1.1.31), the other major phospho enol pyruvate (PEP)-utilizing enzyme in legume root nodules. Latency experiments with sonicated nodule extracts showed that Bradyrhizobium japonicum bacteroids do not express either PK or PEPC activity in symbiosis. In contrast, free-living B. japonicum bacteria expressed PK activity, but not PEPC activity. Antibodies specific for the cytosolic isoform of PK from castor bean endosperm cross-reacted with a 52-kDa polypeptide in the partially purified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extracts was 2.6 times the corresponding PK activity. This would tend to favour PEP metabolism by PEPC over PEP metabolism by PK. However, at pH 7.0 in the presence of 5 m M malate, PEPC activity was strongly inhibited, but PK activity was unaffected. Thus, we propose that PK and PEPC activity in legume root nodules may be coordinately regulated by fluctuations in malate concentration in the plant cytosolic fraction of the bacteroid-containing cells. Reduced uptake of malate by the bacteroids, as a result of reduced rates of N₂ fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.     

Nitrogen fixation and carbon metabolism by nodules and bacteroids of pea plants under sodium chloride stress

Acetylene reduction activity (ARA) and leghemoglobin (Lb) content in nodules were sigificantly reduced when pea ( Pisum sativum L. cv. Lincoln) plants were subjected to 50 m M sodium chloride stress for 3 weeks. C₂H₂ reduction activity by bacteriods isolated from pea nodules was drastically inhibited by saline stress, and malate appeared to be a more appropriate substrate than glucose or succinate in maintaining this activity. Salt added directly to the incubation mixture of bacteriods or to the culture medium of plants inhibited O₂ uptake by bacteroids. Nodule cytosolic phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and bacteriod malate dehydrogenase (MDH; EC 1.1.1.37) activities were strongly enhanced by salt stress. Under these conditions, malate concentration was depressed in bacteroids and cytosol, whereas total soluble sugar (TSS)content slightly increased in both fractions. The effect of salt stress on TSS and malate content suggests that the utilization of carbohydrate within nodules could be inhibited during salt stress. The inhibitory effect of NaCl on N₂ fixation activity of bacteroids and to the decrease in bacteroid respiration. The stimulation of fermentative metabolism induced by salinity suggests some reduction in O₂ availability within the nodule. Salt stress was also responsible for a decrease of the cytosolic protein content, specifically of leghemoglobin, in the nodules.     

Nitrogen fixation and carbon metabolism in legume nodules

A large amount of energy is utilized by legume nodules for the fixation of nitrogen and assimilation of fixed nitrogen (ammonia) into organic compounds. The source of energy is provided in the form of photosynthates by the host plant. Phosphoenol pyruvate carboxylase (PEPC) enzyme, which is responsible for carbon dioxide fixation in C4 and crassulacean acid metabolism plants, has also been found to play an important role in carbon metabolism in legume root nodule. PEPC-mediated CO2 fixation in nodules results in the synthesis of C4 dicarboxylic acids, viz. aspartate, malate, fumarate etc. which can be transported into bacteroids with the intervention of dicarboxylate transporter (DCT) protein. PEPC has been purified from the root nodules of few legume species. Information on the relationship between nitrogen fixation and carbon metabolism through PEPC in leguminous plants is scanty and incoherent. This review summarizes the various aspects of carbon and nitrogen metabolism in legume root nodules.     

Phosphoenolpynivate carboxylase, malate and alcohol dehydrogenase activities in alfalfa (Medicago sativa) nodules under water stress

The effect of drought upon phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), malate ddiydrogenase (MDH; EC 1.1.1.37), alcohol dehydrogenase (ADH; EC 1.1.1.1) and β -hydroxybulyrate dehydrogenase ( β -OH-BDH; EC 1.1.1.30) enzyme activities as well as the leghemoglobin (Lb), malate and ethanol contents of alfalfa nodules ( Medicago sativa L. cv. Aragon) were examined. Both the ieghemoglobin (Lb) content and the Lb/soluble protein ratio were significantly reduced at a nodule water potential (Ψ_nod) of—1.3 MPa. At lower Ψ_nod, Lb content decreased further, but the ratio remained unchanged. Slight stress (—1.3 MPa) drastically affected acetylene reduction activity (ARA; 60% reduction) whereas in vitro PEPC activity was main-tained at relatively constant values. As stress progressed (—2.0 MPa), a simultaneous reduction in both activities was observed. Severe stress (Ψ_nod lower than —2.0 MPa) stimulated in vitro PEPC. Bacteroid β -J-OH-BDH activity was stimulated by slight (—1.3 MPa) and moderate (—2.0 MPa) drought. MDH activity rose in slightly stressed nodules (Ψ_nod—1.3 MPa). Greater water deficits sharply decreased MDH activity to values significantly lower than those found in control nodules. Nodule malate content followed the same pattern as MDH. The plant fraction of the nodule showed constitutive ADH activity and contained ethanol. ADH was stimulated at slight (— 1.3 MPa) and moderate drought levels (—2.0 MPa). Ethanol content showed similar behavior to ADH activity. Inhibition of ARA, reduction of Lb content and stimulation of the fermentative metabolism induced by water stress suggest some reduction ira O₂ availability within the nodule.     

Organic acid accumulation may inhibit N2 fixation in phosphorus-stressed lupin nodules.

Nodulated lupins (Lupinus angustifolius cv. Wonga) were hydroponically grown under conditions of low phosphate (LP) or adequate phosphate (HP) to assess the effect of phosphoenolpyruvate carboxylase (PEPC)-derived organic acids on nitrogen assimilation in LP nodules. LP conditions are linked to altered organic acid metabolism, by the engagement of PEP metabolism via PEPC. In LP nodules, the enhanced organic acid synthesis may reduce the available organic carbon for nitrogen assimilation. The diversion of carbon between the organic acid- and amino acid pools was assessed through key nodular enzymes and (14)CO(2) metabolism. Under LP conditions, increased rates of organic acid synthesis via PEPC and malate dehydrogenase (MDH), coincided with reduced nitrogen assimilation via aspartate aminotransferase (AAT), aspartate synthetase (AS) and glutamine synthetase (GS)/glutamate synthase (GOGAT) activities. There was a preferential metabolism of nodular (14)CO(2) into organic acids and particularly into malate. High malate levels were associated with reduced N(2) fixation and synthesis of amino acids. These results indicate that phosphorus deficiency can enhance malate synthesis in nodules, but that excessive malate accumulation may inhibit N(2) fixation and nitrogen assimilation.     

Nodule phosphoenolpyruvate carboxylase: a review

Recent data concerning the fixation of CO₂ and the functioning of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in legume, nodules are reviewed. The activites of N₂ fixation (acctylene reduction) and PEP carboxylase are correlated Activities of PEP carboxylase are always higher in nodules than in roots. PEP carboxylase is located in the cytosol of the plant part of the nodules. When nodules are fed with ¹⁴CO₂, radioactivity appears predominantly in malate and aspartate. The resolution of isoenzymes of PEP carboxylase shows one more band in nodules than in related roots. The role of PEP carboxylase in nodule metabolism is discussed.     

Developmental changes of enzymes of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation in peach leaves

The developmental profile of the activities of some enzymes involved in malate metabolism, namely phosphoenolpyruvate carboxylase (PEPC; EC 4. 1. 1. 31), NAD⁺-linked (EC 1. 1. 1. 37) and NADP⁺-linked (EC 1. 1. 1. 82) malate dehydrosenase (MDH), NAD⁺linked (EC 1. 1. 1. 39) and NADP⁺-linked (EC 1. 1. 1. 40) malic enzyme (ME), has been determined in leaves of peach [ Prunus persica (L.) Batsch cv. Maycrest], a woody C₃ species. In order to study the role of these enzymes, their activities were related to developmental changes of photosynthesis, respiration, and capacity for N assimilation. Activities of PEPC, NAD(P)⁺-MDH and NADP⁺-ME were high in young expanding leaves and decreased 2- to 3-fold in mature ones, suggesting that such enzymes play some role during the early stages of leaf expansion. In leaves of peach, such a role did not seem to be linked to C₃ photosynthesis or nitrate assimilation, in that photosynthetic O₂ evolution and activities of nitrate reductase (EC 1. 6. 6. 1) and glutamine synthetase (EC 6. 3. 1. 2) increased during leaf development. In contrast, leaf respiration strongly decreased with increasing leaf age. We suggest that in expanding leaves of this woody species the enzymes associated with malate metabolism have anaplerotic functions, and that PEPC may also contribute to the recapture of respiratory CO₂.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

Nitrate metabolism in soybean root nodules

The nitrate metabolism in nodules induced by Bradyrhizobium japonicum strain PJ17 on roots of soybean [ Glycine max (L.) Merr. cv. Hodgson] has been characterized by the nitrate reductase (NR; EC 1.6.6.1 and EC 1.6.6.3) activity of both partners of the symbiosis. NR activities of bacteroids and nodular cytosol were comparable and significantly higher than those of the roots. Nitrate reduction led to nitrite accumulation in root nodules, which was maximum after pod filling. The nodule had the capacity to metabolize nitrite via nitrite reductase (NiR; EC 1.6.6.4), at least in the cytosolic fraction. This activity was partly inhibited by the low content of free O₂ in the nodule. Indeed, nitrite accumulation decreased in the presence of an increased external pressure of O₂.     

Alteration of N nutrition in Myrica gale induces changes in nodule growth, nodule activity and amino acid composition

Changes in nodule growth and activity and in the concentrations of soluble N compounds in nodules, leaves and xylem sap under conditions of altered N nutrition in the actinorhizal plant Myrica gale L. are reported. Altering the N nutrition of symbiotic plants may alter the internal regulation of combined N which in turn may regulate nodule growth and activity. Flushing nodules daily with 100% O₂ caused a decline in amide concentration and an increase in nodule growth although plants had recovered some nitrogenase activity within 4 h of exposure to O₂. Samples of nodules, leaves and xylem sap were derivatized and amino acids identified and quantified using either reverse phase high performance liquid chromatography or gas chromatography-mass spectrometry in single ion monitoring mode. The ratio of asparagine in the nodules to that in the xylem was much higher in plants fed N (6.7 for NH⁺₄-fed and 8.3 for NO⁻₃-fed plants) than for N₂-fixing plants (2.5). Significant amounts of ¹⁵N added as ¹⁵NH⁺₄ or ¹⁵NO⁻₃ accumulated in nodules following accumulation in the shoot which is consistent with the translocation of N to the nodules via the phloem. The uptake of ¹⁵NH⁺₄ led to the synthesis and subsequent translocation of glutamine in the xylem sap. These results are discussed in terms of the feedback mechanisms that may regulate nitrogen fixation in Myrica root nodules.     

Continuous CO2 enrichment leads to increased nodule biomass, carbon availability to nodules and activity of carbon-metabolising enzymes but does not enhance specific nitrogen fixation in pea

Recent research has shown that nodule nitrogen fixation is limited under a wide range of environmental constraints by lowered carbon flux within the nodule due to down-regulation of sucrose synthase activity. The aim of this work was to elucidate whether an increase in both carbon flux and activity of enzymes of carbon metabolism in nodules may lead to an increased nitrogen fixation. We report the effects caused by a continuous exposure to atmospheric CO₂ enrichment in nodulated pea plants. CO₂ enrichment led to an enhanced whole-plant growth and increased nodule biomass. Moreover, nodules of plants grown at increased CO₂ showed a higher sugar content as well as enhancement of some activities related to nodule carbon metabolism, such as sucrose synthase, UDP glucose pyrophosphorylase and phosphoenolpyruvate carboxylase. Indeed, acetylene reduction activity, measured by the classical technique, was increased more than four times. However, when specific nitrogen fixation was determined as hydrogen evolution, no significant differences were detected, consistent with the lack of changes of enzymes involved in nitrogen metabolism such as glutamate synthase and aspartate aminotransferase. These results are discussed in the context of the regulation of nitrogen fixation and nodule metabolism.     

Accumulation of γ‐aminobutyric acid in nodulated soybean in response to drought stress

Nitrogen fixation and nodule permeability to O₂ diffusion are decreased by drought stress. Since γ‐aminobutyric acid (GABA) synthesis is rapidly stimulated by a variety of stress conditions including hypoxia, it was hypothesized that decreased O₂ availability in nodules stimulates glutamate decarboxylase (GAD) activity (EC 4.1.1.15), thereby resulting in GABA accumulation. First, the amino acid composition of xylem sap was determined in plants subjected to soil water deficits. While the xylem sap concentration of several amino acids increased when the plant was subjected to a water deficit, the greatest increase was in GABA. GABA accumulation was examined in response to stress induced by hypoxia or the addition of polyethylene glycol (PEG) to the nutrient solution. The exposure of soybean nodules to hypoxia for 6 h enhanced the GABA concentration by 6‐fold, but there was no change in GABA concentration in response to the PEG treatment. No major changes in the in vitro GAD activity were measured in nodule cytosol or bacteroids. The present data do not support the hypothesis that decreased nodule O₂ permeability and a resulting O₂ deprivation inside nodules may stimulate in vitro GAD activity and thus GABA accumulation. However, the data could indicate a possible effect of hypoxia and drought stress on the in vivo activity of GAD.     

Regulation of soybean nodule phosphoenolpyruvate carboxylase in vivo

The sensitivity of soybean ( Glycine max L. Merr, cv. PS47) nodule phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) to inhibition by L-malate in vitro increased when well-nodulated plants were subjected to decapitation (shoot removal). There was no effect of decapitation on the apparent K_m of the enzyme for its substrate PEP but the I₅₀ (L-malate) decreased from 4.2 to 1.7 m M. The total amount of PEP doubled and that of malate decreased by half in the nodules of decapitated plants relative to the control plants. This observation was consistent with a decrease in the activity of PEPC in vivo as a result of the increased malate sensitivity of the enzyme observed in vitro. Sucrose levels in the nodules declined in response to decapitation but there were no effects on the levels of glucose, fructose, pyruvate, 2-oxoglutarate, glutamine or glutamate. The results are discussed in terms of the role of protein phosphorylation in the regulation of PEPC activity in legume nodules.     

Salt stress induces a decrease in the oxygen uptake of soybean nodules and in their permeability to oxygen diffusion

The effects of short-term NaCl-salinity on nodules of soybean ( Glycine max L. cv. Kingsoy) were studied on hydroponically-grown plants. Both acetylene reducing activity (ARA) and nodule respiration (O₂ uptake and CO₂ evolution) were immediately inhibited, and the stimulation of them by rising the external partial pressure of O₂ (pO₂) was diminished by the application of 0.1 M NaCl in the nutrient solution. The permeability of the nodule to O₂ diffusion, estimated by O₂ consumption or CO₂ evolution, was significantly lower in the stressed nodules than in the cootrol ones. The respiratory quotient of intact nodules and the ethanol production of excised nodules were increased by low pO₂ and by salt stress. These data confirm that in salt-stressed soybean nodules, O₂ availability is reduced and fermentative pathways are stimulated.     

Changes in ethanol, lactate and malate content in Acer pseudoplatanus cells: effects of fusicoccin and O2 availability

Changes in the contents of ethanol, lactate and malate were determined at different activities of the plasma membrane H⁺ pump [in the presence and absence of fusicoccin (FC)] and at different O₂ availability in cultured cells of Acer pseudoplatanus L. FC induced acidification of the medium under all tested conditions of O₂ availability. At low O₂ concentrations both ethanolic and lactic fermentations occurred, and FC markedly stimulated lactate production but had no effect on ethanol production. There was also a small, stimulating effect of FC on malate production. At high O₂ concentrations no ethanol production was observed and lactate production was reduced. Under these conditions the stimulating effect of FC on lactate production decreased, while that on malate production increased. FC-induced synthesis of lactate and malate is interpreted as depending on the activation of lactate dehydrogenase (EC 1.1.1.27) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) (alkaline pH optima), respectively, due to the alkalinization of the cytoplasmic pH resulting from the stimulation of the H⁺ pump by FC. These results suggest that the balance between the two pH stat systems depends on the availability of O₂.     

Kinetic properties of phosphoenolpyruvate carboxylase from lupin nodules and roots

The kinetic properties of two forms of phosphoenolpyruvate carboxylase (PEPC I and PEPC II, EC 4.1, 1.31) from lupin ( Lupinus luteus L. cv. Ventus) nodules and one enzyme form (PEPC III) from roots were studied. The Michaelis constant (K_m) values for PEP, Mg²⁺ and especially HCO₃⁻were lower for PEPC I. Kinetic studies showed that aspartate is a competitive inhibitor at pH 7.2 and inhibitor constant (K_i) values are different for the three forms of PEPC. Malate is a competitive inhibitor for PEPC I and PEPC III and shows mixed-type inhibition for PEPC II. Malate inhibition is dependent upon the pH of the assay. Different effect of several metabolites was also observed. The temperature optimum was near 39°C for PEPC I and around 43°C for PEPC II and PEPC III. PEPC I appeared to be the most thermolabile. It is suggested that PEPC I from lupin nodules is closely associated with N₂ fixation.     

Reactive oxygen species and antioxidants in legume nodules

Reactive oxygen species are a ubiquitous danger for aerobic organisms. This risk is especially elevated in legume root nodules due to the strongly reducing conditions, the high rates of respiration, the tendency of leghemoglobin to autoxidize, the abundance of nonprotein Fe and the presence of several redox proteins that leak electrons to O₂. Consequently, nodules are particularly rich in both quantity and diversity of antioxidant defenses. These include enzymes such as superoxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.11) and metabolites such as ascorbate and thiol tripeptides. Nodule antioxidants have been the subject of intensive molecular, biochemical and functional studies that are reviewed here. The emerging theme is that antioxidants are especially critical for the protection and optimal functioning of N₂ fixation. We hypothesize that this protection occurs at least at two levels: the O₂ diffusion barrier in the nodule parenchyma (inner cortex) and the infected cells in the central zone.     

Effects of exogenous application and stem infusion of ascorbate on soybean (Glycine max) root nodules

Numerous biochemical and physiological studies have demonstrated the importance of ascorbate (ASC) as a reducing agent and antioxidant in higher plant metabolism. Of special note is the capacity of ASC to eliminate damaging activated oxygen species (AOS) including O₂^−· and H₂O₂. N₂-fixing legume nodules are especially vulnerable to oxidative damage because they contain large amounts of leghaemoglobin which produces AOS through spontaneous autoxidation; thus, ASC and other components of the ascorbate–reduced glutathione (ASC–GSH) pathway are critical antioxidants in nodules. In order to establish a meaningful correlation between concentrations of ASC and capacity for N₂ fixation in legume root nodules, soybean ( Glycine max ) plants were treated with excess ASC via exogenous irrigation or continuous intravascular infusion through needles inserted directly into plant stems. Treatment with ASC led to striking increases in nitrogenase activity (acetylene reduction), nodule leghaemoglobin content, and activity of ASC peroxidase, a key antioxidant enzyme. The concentration of lipid peroxides, which are indicators of oxidative damage and onset of senescence, was decreased in ASC-treated nodules. These results support the conclusion that ASC is critical for N₂ fixation and that elevated ASC allows nodules to maintain a greater capacity to fix N₂ over longer periods.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.