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.     

Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C₄ PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C₄ plant species. Neither antiserum reacted strongly with any C₃ leaf proteins. The molecular weight of the PEPC polypeptide from C₄ leaves and legume nodules appears to be similar.     

Expression of a soybean nodule‐enhanced phosphoenolpyruvate carboxylase gene that shows striking similarity to another gene for a house‐keeping isoform

Three different cDNAs for phosphoenolpyruvate carboxylase (PEPC) were isolated from soybean root nodules. The full-length cDNA of the most abundant isoform (GmPEPC7) was very similar to another one (GmPEPC15), the nucleotide sequence of which is identical to that of a reported clone (gmppc1) (Vazquez-Tello, A., Whittier, R.F., Kawasaki, T., Sugimoto, T., Kawamura, Y., Shibata, D. (1993) Plant Physiol. 103, 1025–1026). In the coding region, the newly isolated GmPEPC7 and the previously reported were gmppc1 99% and 98% identical at the amino acid and nucleotide levels, respectively. In contrast, they exhibited only 39% identity in the 3′ non-coding region, indicating that they are encoded by distinct genes. Northern blot analysis with 3′ non-coding regions as isoform-specific probes showed that GmPEPC7 is nodule-enhanced whereas GmPEPC15 (gmppc1) is expressed in most soybean tissues. The third clone (GmPEPC4) was much less homologous to the above two clones and thus was not further characterized. It was also shown by in situ hybridization that the nodule-enhanced isoform is expressed in all cell types in nodules, including in Bradyrhizobium-infected and uninfected cells and cortical cells. A relatively strong hybridization signal was detected in the vascular bundle pericycle. Southern blot analysis indicated that there are only two PEPC genes exhibiting a high degree of similarity in the soybean genome, one for the nodule-enhanced GmPEPC7 and the other for the constitutively expressed gmppc1. A phylogenetic tree based on the amino acid sequences of soybean PEPCs and nodule-enhanced PEPCs of alfalfa and pea suggested that the soybean nodule-enhanced isoform evolved from the housekeeping PEPC gene after the ureid-translocating and amide-translocating legumes diverged from each other.     

Localization of carbonic anhydrase in legume nodules

Extracts of the central infected zone and the surrounding cortex of nodules from Lupinus angustifolius L., Vigna unguiculata L. (Walp), Pisum sativum L., Phaseolus vulgaris L., Vicia faba L. and Medicago sativa L. contained significant activities of carbonic anhydrase (CA). Immunoassay of extracts using antisera to a putative nodule CA (Msca1) cloned from M. sativa also indicated expression in both tissue types. Quantitative confocal microscopy using laser scanning imaging and a fluorescent CA‐specific probe (5‐dimethylaminonaphthalene‐1‐sulfonamide [DNSA]) localized expression to the infected cells in the central zone tissue and a narrow band of 2–3 files of cells in the cortical tissue that corresponded to the inner cortex. In the infected cells, the enzyme activity was distributed evenly in the cytosol, but in the inner cortical cells, it was restricted to the periphery – possibly to the plasma membrane or cell wall. The functions of CA in these two tissues are considered in relation to the carbon metabolism of nodules and the participation of the inner cortex in the regulation of gaseous diffusive resistance.     

Specialization of the inner cortex for ureide production in soybean root nodules

Summary The possibility that cells in the inner cortex of determinate root nodules participate in ureide production from recently fixed N₂, as do the uninfected (interstitial) cells of the infected central region, has been investigated in soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. Like the interstitial cells, cells of the three innermost cortical layers produce enlarged peroxisomes and a meshwork of tubular ER during differentiation. These changes are most pronounced in the innermost cortical layer, are successively less so in the 2nd and 3rd layers, and are usually undetectable in more distant layers. Peroxisomes in the inner three layers are stained in the DAB (3,3-diaminobenzidine) test for uricase (EC 1.7.3.3) activity, indicative of the potential for ureide formation, but peroxisomes in more distant cortical cells are not stained. A nodulespecific uricase also is demonstrable in the inner three cortical layers by immunogold labeling enhanced with silver for visualization in the light microscope. The observations suggest that with respect to ureide production the cells of the inner layers of the cortex are functionally similar to the interstitial cells of the infected region despite the apparent distinctiveness of the two regions anatomically.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

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.     

Glutamine Induces the N-Dependent Accumulation of mRNAs Encoding Phosphoenolpyruvate Carboxylase and Carbonic Anhydrase in Detached Maize Leaf Tissue

We have used detached leaves to study the N-dependent control of expression of phosphoenolpyruvate carboxylase (PEPC) and carbonic anhydrase (CA) genes in maize (Zea mays L. cv Golden Cross Bantam T51). Following supplementation with an N-source and zeatin, PEPC and CA mRNA levels increased in leaves detached from N-deficient maize plants. Addition of methionine sulfoximine (MSX), a specific inhibitor of glutamine synthetase, inhibited the nitrate-dependent increase of PEPC and CA mRNA but did not affect the glutamine-dependent increase of PEPC and CA mRNA levels. Glutamine levels in detached maize leaves treated with various N sources in the presence or absence of MSX correlated with the levels of PEPC and CA mRNA. We conclude that glutamine is the most likely effector for controlling the N-dependent expression of PEPC and CA in maize plants.     

Immunocytochemical localization of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in soybean nodules

Polyclonal antibodies raised against NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase [phosphorylating], EC 1.2.1.12) from the plant cytosolic fraction of soybean [ Glycine max (L.) Merr. cv. Williams] nodules were used to study the subcellular location of the enzyme and its relative distribution between infected, interstitial and cortical cells of soybean (cv. Lincoln) nodules. Post-embedding immunogold labelling was carried out on nodules harvested 5, 12, 19 and 25 days after the first sign of nodulation. Labelling for glyceraldehyde-3-phosphate dehydrogenase was observed over the cytoplasm and nuclei of infected and uninfected cells, as well as over the nucleoid regions of bacteroids. In 5-day-old nodules, label also bound adjacent to the peribacteroid membranes. Statistical analysis of the number of gold particles per cell area indicated that in 5-day-old nodules, glyceraldehyde-3-phosphate dehydrogenase was distributed equally between infected, interstitial and cortical cells, but in older nodules the enzyme was more prominent in the interstitial and cortical cells than in infected cells.     

Detopping causes production of intercellular space occlusions in both the cortex and infected region of soybean nodules

A structural analysis was conducted to determine whether glycoprotein‐containing intercellular space occlusions are involved in medium‐term regulation of O₂ diffusion in soybean (Glycine max) nodules. Alterations in O₂ diffusion were induced by a 3 h detopping treatment, and glycoprotein was immunolocalized with the monoclonal antibodies MAC236 and MAC265. Western blots of unstressed nodules revealed that these antibodies recognize antigens with two different molecular weights in soybean nodules. Tissue printing of halved nodules showed that both antigens were present in fresh nodules from control and 3 h detopped plants. The main localization appeared to be the inner cortex, but some immunolabelling also occurred in the infected region. ELISAs demonstrated a significant increase in total nodule concentration of intercellular glycoprotein following detopping, and cryosections of fresh nodules from this treatment also showed localization of antigens within the intercellular spaces of the infected region. The production of intercellular space occlusions in both the mid‐cortex and infected regions after 3 h detopping was confirmed by light microscopy and silver‐enhanced immunolabelling; cortical changes were quantified by image analysis techniques. Electron microscopy revealed that the occlusions within the infected region were less dense and less heavily labelled than those in the cortex. These results are discussed in relation to O₂ diffusion regulation in soybean nodules     

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.     

Seryl-phosphorylation of soybean nodule sucrose synthase (nodulin-100) by a Ca-dependent protein kinase

Sucrose synthase (SS; EC 2.4.1.13) was radiolabeled in situ by incubating detached soybean nodules with ³²Pi. Phosphoamino acid analysis indicated that SS was phosphorylated on a serine residue(s). In-vitro phosphorylation of purified nodule SS by desalted nodule extracts was Ca²⁺-dependent. This SS-kinase was partially purified (2200-fold) from nodules harvested from illuminated plants. The molecular mass of the SS-kinase was about 55 000 on a Superdex 75 size-exclusion column or in a denaturing autophosphorylation gel. With either purified nodule SS or Syntide 2 as substrate, exogenous calmodulin and phosphatidylserine showed little or no effect on the in-vitro activity of this partially purified protein kinase. However, its activity was inhibited by W-7. The purified nodule SS-kinase (or CDPK) phosphorylated nodule PEP carboxylase (PEPC; EC 4.1.1.31) in the presence of Ca²⁺. In contrast, a partially purified nodule PEPC-kinase preparation was incapable of phosphorylating nodule SS. Unlike nodule PEPC [Zhang et al. (1995) Plant Physiol. 108, 1561–1568], the phosphorylation state of SS is not likely modulated in planta by photosynthate supply from the shoots.     

Relationship between NH(4) Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity : Regulation of Anaplerotic Carbon Flow in the Green Alga Selenastrum minutum

The rate of NH₄⁺ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH₄⁺ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H¹⁴CO⁻₃ into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C₃ organism.     

Oxygen regulation of a nodule-located carbonic anhydrase in alfalfa

Control of the permeability to oxygen is critical for the function of symbiotic nitrogen fixation in legume nodules. The inner cortex (IC) seems to be a primary site for this regulation. In alfalfa (Medicago sativa) nodules, expression of the Msca1 gene encoding a carbonic anhydrase (CA) was previously found to be restricted to the IC. We have now raised antibodies against recombinant Msca1 protein and used them, together with antibodies raised against potato leaf CA, to demonstrate the presence of two forms of CA in mature nodules. Each antibody recognizes a different CA isoform in nodule tissues. Immunolocalization revealed that leaf-related CAs were localized primarily in the nitrogen-fixing zone, whereas the Msca1 protein was restricted exclusively to the IC region, in indeterminate and determinate nodules. In alfalfa nodules grown at various O(2) concentrations, an inverse correlation was observed between the external oxygen pressure and Msca1 protein content in the IC, the site of the putative diffusion barrier. Thus Msca1 is a molecular target of physiological processes occurring in the IC cells involved in gas exchange in the nodule.     

Metabolite regulation of partially purified soybean nodule phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was purified 40-fold from soybean (Glycine max L. Merr.) nodules to a specific activity of 5.2 units per milligram per protein and an estimated purity of 28%. Native and subunit molecular masses were determined to be 440 and 100 kilodaltons, respectively, indicating that the enzyme is a homotetramer. The response of enzyme activity to phosphoenolpyruvate (PEP) concentration and to various effectors was influenced by assay pH and glycerol addition to the assay. At pH 7 in the absence of glycerol, the K_m (PEP) was about twofold greater than at pH 7 in the presence of glycerol or at pH 8. At pH 7 or pH 8 the K_m (MgPEP) was found to be significantly lower than the respective K_m (PEP) values. Glucose-6-phosphate, fructose-6-phosphate, glucose-1-phosphate, and dihydroxyacetone phosphate activated PEPC at pH 7 in the absence of glycerol, but had no effect under the other assay conditions. Malate, aspartate, glutamate, citrate, and 2-oxoglutarate were potent inhibitors of PEPC at pH 7 in the absence of glycerol, but their effectiveness was decreased by raising the pH to 8 and/or by adding glycerol. In contrast, 3-phosphoglycerate and 2-phosphoglycerate were less effective inhibitors at pH 7 in the absence of glycerol than under the other assay conditions. Inorganic phosphate (up to 20 millimolar) was an activator at pH 7 in the absence of glycerol but an inhibitor under the other assay conditions. The possible significance of metabolite regulation of PEPC is discussed in relation to the proposed functions of this enzyme in legume nodule metabolism.