Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N₂ fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.     

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.     

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

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.     

Nodule-specific polypeptides from effective alfalfa root nodules and from ineffective nodules lacking nitrogenase

In addition to leghemoglobin, at least nine nodule-specific polypeptides from the alfalfa (Medicago sativa L.)-Rhizobium meliloti symbiosis were identified by immune assay. Some of these polypeptides may be subunits of larger proteins but none appeared to be subunits of the same multimeric protein. All nine of the nodule-specific polypeptides were localized to within the plant cytosol; they were not found in extracts of bacteroids or in the peribacteroid space. At least one of these nodule-specific polypeptides was found to be antigenically related to nodule-specific polypeptides in pea and/or soybean. Ineffective nodules elicited by R. meliloti strains containing mutations in four different genes required for nitrogenase synthesis contained reduced concentrations of leghemoglobin and of several of the nodule-specific polypeptides. Other nodule-specific polypeptides were unaltered or actually enriched in the ineffective nodules. Many of the differences between the ineffective and effective nodules were apparent in nodules harvested shortly after the nodules became visible. These differences were greatly amplified in older nodules. When the four ineffective nodule types were compared to one another, there were clear quantitative differences in the concentrations of several of the nodule-specific polypeptides. These differences suggest that lack of a functional nitrogenase does not have a single direct effect on nodule development.     

Isolation,characterisation and expression of a cDNA for pea cholinephosphate cytidylyltransferase

In plants, phosphatidylcholine is the major phospholipid in extra-plastid membranes and is synthesised mainly by the CDP-choline pathway. Evidence from studies in animals, as well as in plants, suggests that the intermediate step catalysed by cholinephosphate cytidylyltransferase (CPCT) has a major control in carbon flux to this lipid. We have isolated a full-length CPCT cDNA (designated PCT2) from Pisum sativum cv. Feltham First using an Arabidopsis probe and the polymerase chain reaction (PCR). The deduced amino acid of PCT2 is 48%, 43% and 76% identical to the rat, yeast and Brassica napus amino acid sequences, respectively. Expression of the CPCT protein in Escherichia coli confirmed the activity of the enzyme. Expression of the PCT2 mRNA in pea roots and stems was increased by treatment with 0.1 µM indole-3-acetic acid.     

Sequence analysis of cDNA encoding phosphoenolpyruvate carboxylase from cultured tobacco cells

The complete nucleotide sequence of cDNA encoding phosphoenolpyruvate carboxylase (PEPCase) from cultured tobacco (a C₃ plant) cells was determined and the deduced amino acid sequence was compared with those of PEPCases from other higher plants.     

Isolation, characterization and expression analysis of a leaf-specific phosphoenolpyruvate carboxylase gene in Oryza sativa.

Suppression subtractive hybridization was carried out to enrich gene fragments over-expressed in rice leaves by subtraction to rice roots, from which two identical cDNA fragments were identified to encode putative phosphoenolpyruvate carboxylase. Then the corresponding full-length cDNA (Osppc) is isolated by RT-PCR and sequenced, which indicates an open reading frame of 2895bp is contained. Its deduced protein is encoded in 10 exons and shows high similarity to many other plant PEPCs. Comparing with maize and bacterial PEPCs, it is revealed that OSPPC shares many conserved domains and active sites that responsible for the structure, activity and regulation of this enzyme. Phylogenetic analysis demonstrates that OSPPC is grouped with C3 form PEPCs of wheat, maize and sorghum, which is consistent with the classification of rice. And a putative promoter element is predicted with DOF binding box, CAAT box and TATA box in the 5'-flanking sequence of Osppc gene. Moreover, Quantitative RT-PCR analyses are performed in hybrid rice and its parents, which show that Osppc is specifically expressed in leaf including leaf vein and sheath.     

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.     

Phytohormones, Rhizobium mutants, and nodulation in legumes : v. Cytokinin metabolism in effective and ineffective pea root nodules

[³H]Zeatin riboside was supplied to intact pea (Pisum sativum) plants either onto the leaves or onto the root nodules. When applied directly to nodules, approximately 70% of recovered radioactivity remained in the nodules, approximately 15% was detected in the root system, and 15% was in the shoot. However, when supplied to the leaves, little ³H was transported, with approximately 0.05% of recovered radioactivity being found in the root system and nodules. On a fresh weight basis, nodules accumulated more ³H than the parent root. In both types of studies, metabolites with an intact zeatin moiety were detected in root nodules.

In all experiments, two-dimensional thin layer chromatography revealed that little ³H remained as zeatin riboside in root or nodule tissue at the end of the labeling period. Nodules metabolized [³H]zeatin riboside to the following cytokinins/cytokinin metabolites: zeatin, adenosine, adenine, the O-glucosides of zeatin and zeatin riboside, lupinic acid, nucleotides of adenine and zeatin, and the dihydro derivatives of many of these compounds.

Although a few small differences were observed, there were no major differences between root and nodule tissue in their metabolism of [³H] zeatin riboside. Furthermore, any differences between effective and ineffective nodules were generally minor.

     

Identification and expression analysis of a gene encoding a bacterial-type phosphoenolpyruvate carboxylase from Arabidopsis and rice

Phosphoenolpyruvate carboxylase (PEPC) is distributed in plants and bacteria but is not found in fungi and animal cells. Important motifs for enzyme activity and structure are conserved in plant and bacterial PEPCs, with the exception of a phosphorylation domain present at the N terminus of all plant PEPCs reported so far, which is absent in the bacterial enzymes. Here, we describe a gene from Arabidopsis, stated as Atppc4, encoding a PEPC, which shows more similarity to Escherichia coli than to plant PEPCs. Interestingly, this enzyme lacks the phosphorylation domain, hence indicating that it is a bacterial-type PEPC. Three additional PEPC genes are present in Arabidopsis, stated as Atppc1, Atppc2, and Atppc3, encoding typical plant-type enzymes. As most plant PEPC genes, Atppc1, Atppc2, and Atppc3 are formed by 10 exons interrupted by nine introns. In contrast, Atppc4 gene has an unusual structure formed by 20 exons. A bacterial-type PEPC gene was also identified in rice (Oryza sativa), stated as Osppc-b, therefore showing the presence of this type of PEPC in monocots. The phylogenetic analysis suggests that both plant-type and bacterial-type PEPCs diverged early during the evolution of plants from a common ancestor, probably the PEPC from gamma-proteobacteria. The diversity of plant-type PEPCs in C3, C4, and Crassulacean acid metabolism plants is indicative of the evolutionary success of the regulation by phosphorylation of this enzyme. Although at a low level, the bacterial-type PEPC genes are expressed in Arabidopsis and rice.     

Structure and expression of a sugarcane gene encoding a housekeeping phosphoenolpyruvate carboxylase

A gene (SCPEPCD1) encoding phosphoenolpyruvate carboxylase (PEPC) was isolated from the C-4 monocot sugarcane (Saccharum hybrid var. H32-8560). SCPEPCD1 is ca. 6800 bp long, with 10 exons. The entire gene sequence from –1561 to 262 bp downstream of the putative poly(A) addition signal is reported. A low-level, essentially constitutive pattern of expression, amino acid sequence similarities to other housekeeping PEPC enzymes, and the absence of DNA sequence elements conserved in the upstream region of maize and sorghum C-4-specific PEPC genes indicate that SCPEPCD1 encodes a housekeeping PEPC. Despite this, a motif proposed to act as a phosphorylation site in light-mediated activation of photosynthetic PEPC enzymes [10] is present in the SCPEPCD1 protein; evidence is presented for the presence of this site in other housekeeping PEPC proteins.