Nodule Phosphoenolpyruvate Carboxylase Research Articles

Effect of iron deficiency on the localization of phosphoenolpyruvate carboxylase in common bean nodules

Phosphoenolpyruvate carboxylase (PEPC) plays an important role in nodules, when there is an increase in the demand for energy. This enzyme provides carbon skeletons to sustain amino acid synthesis and malate to support energy required to fix nitrogen. Since PEPC is important for nodules, and there is lack of information about the effect of some nutrient deficiency in the expression and localization of this enzyme in legume nodules, this work focused on the localization of PEPC in nodules under iron deficiency of two common bean cultivars: Flamingo tolerant and Coco blanc sensitive to iron (Fe) deficiency. The results of immunolocalization using polyclonal antibody showed that this enzyme was detected in all regions of nodule sections; but the signal intensity was increased in Fe-deficient nodules as compared to Fe-sufficient ones in the tolerant cultivar, whereas the intensity was less pronounced in nodules of Fe-deficient plants than in those of Fe-sufficient plants for the sensitive cultivar Coco blanc. This work showed that the symbiotic tolerance of Flamingo to iron deficiency was linked to the increase of PEPC enzymes expression. However, the activity of these enzymes supported the energy required in bacteroids to maintain the nitrogenase activity. Keywords: Common bean, immunolocalization, iron deficiency, nodules, phosphoenol pyruvate carboxylase

Inhibition of alfalfa root nodule phosphoenolpyruvate carboxylase through an antisense strategy impacts nitrogen fixation and plant growth

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) plays a paramount role in nodule metabolism and several reports have shown that PEPC provides substantial carbon for N2-fixation and N assimilation. To study the short- and long-term implications of reduced nodule CO2 fixation for N2 fixation in alfalfa, PEPC enzyme expression was reduced through an antisense strategy. The full-length root nodule-enhanced PEPC cDNA in antisense orientation driven by the nodule-enhanced AAT-2 promoter was transformed into alfalfa. Out of 105 transformed plants, 14 showed reduced in vitro nodule PEPC activity. Three plants were selected for further evaluation. RNA and protein blots showed reduced PEPC transcript and protein. Nodules of these plants also displayed reducedin vivo CO2 fixation. Total nitrogenase activity as measured by H2 evolution was reduced, although there was no change in apparent nitrogenase. The nodule electron allocation coefficient of antisense plants was reduced. All antisense plants accumulated less dry matter and nitrogen in a 6-week growing period under controlled conditions. The data confirm a strong interdependence of nodule PEPC and nitrogenase activity.

Phosphoenolpyruvate Carboxylase Protein Kinase from Soybean Root Nodules: Partial Purification, Characterization, and Up/Down-Regulation by Photosynthate Supply from the Shoots

Phosphoenolpyruvate carboxylase (PEPC) kinase was partially purified about 3000-fold from soybean root nodules by a fast-protein liquid chromatography protocol. This protein-serine kinase has an apparent native molecular mass of about 30,000 as estimated by size-exclusion chromatography. Following electrophoresis of this partially purified PEPC-kinase preparation in a denaturing gel containing dephospho maize leaf PEPC as substrate, thein siturenaturation and assay of protein kinase activity revealed two, PEPC-dependent kinase polypeptides with molecular masses of about 32 and 37 kDa. The ∼32-kDa polypeptide was significantly more active than the ∼37-kDa catalytic subunit. The activity of this partially purified PEPC kinase, and a less purified sample, was Ca2+-insensitive. This protein kinase preparation was able to phosphorylate purified PEPCs from soybean nodules, maize leaves, and a sorghum recombinant C4PEPC. In contrast, this PEPC kinase was unable to phosphorylate a phosphorylation-site mutant form of sorghum C4PEPC (S8Y), two other soybean nodule phosphoproteins [nodulin-26 and nodulin-100 (sucrose synthase)], bovine serum albumin, and histone III-S. Followingin vitrophosphorylation of purified dephospho soybean nodule PEPC from stem-girdled plants by the partially purified nodule PEPC kinase, the former's activity and sensitivity tol-malate inhibition increased and decreased, respectively. Notably, the Ca2+-independent PEPC kinase activity in nodules from illuminated plants was markedly greater than that in nodules harvested from plants subjected to stem girdling or prolonged darkness. Furthermore, the kinase activity in nodules was controlled reversibly by illumination and extended darkness pretreatments of the parent plants, suggesting that photosynthate supply from the shoots is likely responsible for these striking changes in PEPC kinase activity observedin plantain the legume nodule.

Isolation of a cDNA for nodule-enhanced phosphoenolpyruvate carboxylase from pea and its expression in effective and plant-determined ineffective pea nodules

A cDNA clone for phosphoenolpyruvate carboxylase (PEPC) was isolated from nodules of pea (Pisum sativum L.). The levels of activity, the PEPC polypeptide and the mRNA for PEPC in effective nodules and in ineffective nodules induced on the pea mutant E135 (sym 13) were compared to evaluate the regulation of PEPC activity in pea nodules. The cDNA was 3 222 bp long and contained an open reading frame of 2 901 bp, capable of encoding a polypeptide of 967 amino acids. The deduced amino acid sequence was similar to those of other plant PEPCs. The expression of this gene was enhanced in nitrogen-fixing effective pea nodules. The activity of PEPC in the effective nodules increased during development of the plants and the activity was maintained until senescence. This increase was associated with increased levels of the 110 kDa polypeptide and the mRNA for PEPC. By contrast, roots always had lower levels of activity, the polypeptide and the mRNA for PEPC than effective nodules. In ineffective E135 nodules, the activity of PEPC increased similarly to that in effective nodules, but then it decreased to the level in roots after 2.5 weeks post-planting. However, the levels of the 110 kDa polypeptide and the mRNA for PEPC in ineffective nodules were higher than those in roots, even though ineffective nodules had reduced levels of the polypeptide and mRNA for PEPC as compared with those in effective nodules. These findings indicate that after nodule initiation, the activity of PEPC in pea nodules may be regulated at the transcriptional level and after translation by a putative factor(s) that governs the effectiveness of the nodules.

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 Km of the enzyme for its substrate PEP but the I50 (L‐malate) decreased from 4.2 to 1.7 mM. 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.

Regulation of soybean nodule phosphoenolpyruvate carboxylase in vivo

Regulation of soybean nodule phosphoenolpyruvate carboxylase in vivo

In Vivo Regulatory Phosphorylation of Soybean Nodule Phosphoenolpyruvate Carboxylase

In this report we provide evidence that cytosolic phosphoenolpyruvate carboxylase (PEPC) in soybean (Glycine max L.) root nodules is regulated in vivo by a seryl-phosphorylation cycle, as with the C4, Crassulacean acid metabolism, and C3 leaf isoforms. Pretreatment of parent plants by stem girdling for 5 or 14 h caused a significant decrease in the apparent phosphorylation state of nodule PEPC, as indicated by the 50% inhibition constant (L-malate) and specific activity values assayed at suboptimal conditions, whereas short-term darkness alone was without effect. However, extended (26 h) darkness led to the formation of a relatively dephosphorylated nodule PEPC, an effect that was reversed by illuminating the darkened plants for 3 h. This reversal of the apparent phosphorylation state in the light was prevented by concomitant stem girdling. In contrast, the optimal activity of nodule PEPC and its protein level showed little or no change in all pretreated plants. These results suggest that the phosphorylation state of PEPC in soybean root nodules is possibly modulated by photosynthate transported recently from the shoots. In situ [32P]orthophosphate labeling, immunoprecipitation, and phosphoamino acid analyses confirmed directly that PEPC in detached intact soybean nodules is phosphorylated on a serine residue(s).

Phosphoenolpyruvate carboxylase activity, fixation of<sup>14</sup>C in amino acids and nitrogen transport in stem nodules ofSesbania rostrata

Thein vivo 14CO2 fixation assay and xylem sap analysis showed that inSesbania rostrata the transport of fixed nitrogen from stem nodules was in the amide form. The majority of nitrogen was transported as asparagine. The close relationship between nodule phosphoenolpyruvate carboxylase and nitrogenase activities suggested that nodule CO2 fixation contributed directly to nitrogen assimilation in stem nodules ofS. rostrata.

Phosphorylation of Soybean (Glycine max L.) Nodule Phosphoenolpyruvate Carboxylase in Vitro Decreases Sensitivity to Inhibition by L-Malate.

Phosphoenolpyruvate carboxylase (PEPC) from soybean (Glycine max L.Merr.) nodules was purified 187-fold to a final specific activity of 56 units mg-1 of protein. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed one major polypeptide band, with a molecular mass of 110 kD, after the final purification step. Two-dimensional PAGE resolved four isoelectric forms of the purified enzyme. Antibodies raised against the purified enzyme immunoprecipitated PEPC activity from a desalted nodule extract. Two cross-reacting bands were obtained when protein immunoblots of crude nodule extracts subjected to SDS-PAGE were probed with the antiserum. One of these corresponded to the 110-kD subunit of PEPC, and the other had a molecular mass of about 60 kD. PEPC was shown to be activated in a time-dependent manner when desalted soybean nodule extracts were preincubated with Mg.ATP in vitro. Activation was observed when PEPC was assayed at pH 7 in the absence of glycerol but not at pH 8 in the presence of glycerol. When o.5 mM L-malate was included in the assay, activation was much more pronounced than without malate. Maximal activation was 30% in the absence of L-malate and 200% in its presence. The L-malate concentrations producing 50% inhibition of PEPC activity were o.35 and 1.24 mM, respectively, before and after preincubation with Mg.ATP. The antiserum against soybean nodule PEPC was used to immunoprecipitate PEPC from a desalted nodule extract that had been preincubated with Mg.[[gamma]-32P]ATP. The immunoprecipitate was then subjected to SDS-PAGE, followed by autoradiography. The autoradiograph revealed intense labeling of the 110-kD subunit of PEPC following preincubation with [[gamma]-32P]ATP. The data suggest that soybean nodule PEPC becomes phosphorylated by an endogenous protein kinase, resulting in decreased sensitivity of the enzyme to inhibition by L-malate in vitro. The results are discussed in relation to the proposed functions of PEPC in legume nodules.

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N(2)-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N(2)-fixing) alfalfa recessive for the in(1) gene were compared to determine the effects of the in(1) gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in(1) effective nodules.

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.

Variation for Activity of Nodule Nitrogen and Carbon Assimilating Enzymes in Alfalfa1

The nodule enzymes glutamate synthase (GOGAT) and phosphoenolpyruvate carboxylase (PEPC) are important in the assimilation of symbiotically fixed N in alfalfa (Medicago sativa L.). The objectives of this study were: to evaluate several alfalfa germplasm sources for variability in nodule GOGAT and PEPC activities, and to determine the relationships between N2 fixation and plant yield traits with nodule GOGAT and PEPC enzyme activities. Approximately 160 plants from each of six alfalfa germplasms were grown in sand culture in the glasshouse and assayed for nodule GOGAT and PEPC enzyme activities. Twenty‐four genotypes (four from each germplasm) were selected to represent a range in GOGAT and PEPC activities. These genotypes were evaluated in glasshouse and field clonal studies for GOGAT and PEPC activities per gram nodule fresh weight, acetylene reduction activity (ARA), nodule mass, nodule color, nodule soluble protein concentration, and plant yield. Variability for nodule GOGAT and PEPC activities within each of the six germplasms was large and normally distributed. The 12 high GOGAT genotypes (2 from each germplasm) were 44% greater GOGAT activity than the 12 low genotypes. The 12 high PEPC genotypes were 27% greater in PEPC activity than the 12 low genotypes. Both groups of high enzyme activity genotypes were significantly greater than the low genotypes in ARA, nodule soluble protein concentration, and field shoot yield. Both GOGAT and PEPC activities were positively correlated (r=0.61**, significant at the 0.01 probability level). Also, GOGAT and PEPC were each correlated with ARA (r=0.43*, significant at the 0.05 probability level, and 0.45*, respectively), nodule color score (r=0.46** and 0.73**) soluble protein concentration (r= 0.77** and 0.90**). Plant yield and N2 fixation measured in the field were not correlated with nodule GOGAT and PEPC activities measured in the glasshouse. The relationships between GOGAT and PEPC with other plant traits indicated that these enzymes are associated with the host‐Rhizobium meliloti Dang. symbiosis and should be explored as possible selection criteria to include in an alfalfa breeding program.

Alfalfa Root Nodule Carbon Dioxide Fixation

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 C(4) 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 C(4) plant species. Neither antiserum reacted strongly with any C(3) leaf proteins. The molecular weight of the PEPC polypeptide from C(4) leaves and legume nodules appears to be similar.

Alfalfa Root Nodule Carbon Dioxide Fixation

In vivo CO(2) fixation activity and in vitro phosphoenolpyruvate carboxylase activity were demonstrated in effective and ineffective nodules of alfalfa (Medicago sativa L.) and in the nodules of four other legume species. Phosphoenolpyruvate carboxylase activity was greatly reduced in nodules from both host and bacterially conditioned ineffective alfalfa nodules as compared to effective alfalfa nodules.Forage harvest and nitrate application reduced both in vivo and in vitro CO(2) fixation activity. By day 11, forage harvest resulted in a 42% decline in in vitro nodule phosphoenolpyruvate carboxylase activity while treatment with either 40 or 80 kilograms nitrogen per hectare reduced activity by 65%. In vitro specific activity of phosphoenolpyruvate carboxylase and glutamate synthase were positively correlated with each other and both were positively correlated with acetylene reduction activity.The distribution of radioactivity in the nodules of control plants (unharvested, 0 kilograms nitrogen per hectare) averaged 73% into the organic acid and 27% into the amino acid fraction. In nodules from harvested plants treated with nitrate, near equal distribution of radioactivity was observed in the organic acid (52%) and amino acid (48%) fractions by day 8. Recovery to control distribution occurred only in those nodules whose in vitro phosphoenolpyruvate carboxylase activity recovered.The results demonstrate that CO(2) fixation is correlated with nitrogen fixation in alfalfa nodules. The maximum rate of CO(2) fixation for attached and detached alfalfa nodules at low CO(2) concentrations (0.13-0.38% CO(2)) were 18.3 and 4.9 nanomoles per hour per milligram dry weight, respectively. Nodule CO(2) fixation was estimated to provide 25% of the carbon required for assimilation of symbiotically fixed nitrogen in alfalfa.

Plant determined ineffective nodules in alfalfa (Medicago sativa): structural and biochemical comparisons

Nodule structure, nodule enzymes of ammonia assimilation, nodule phosphoenolpyruvate carboxylase, nitrogenase-dependent acetylene reduction, and soluble protein were studied in four plant-determined ineffective genotypes and in one effective genotype of alfalfa (Medicago sativa L.) grown in a glasshouse. Ineffective nodules that formed on genotypes MnAg(In) and MnSa(In) were similar to effective nodules in early development. However, these nodules had smaller bacteroids and senesced much more rapidly than effective nodules. In contrast, nodules that formed on ineffective genotypes MnNC-3226(In) and MnNC-3811(In) were tumorlike and had few infection threads and bacteroids, and host cells were filled with starch. Nodules from plant-determined ineffective genotypes had reduced glutamine synthetase, glutamate synthase, phosphoenolpyruvate carboxylase, and nodule soluble protein when compared with effective nodules. There were differences between genotypes for all biochemical parameters assayed.