Phosphoenolpyruvate Carboxylase Isoforms Research Articles

Prokaryotic Expression of Phosphoenolpyruvate Carboxylase Fragments from Peanut and Analysis of Osmotic Stress Tolerance of Recombinant Strains.

Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme that catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) in presence of HCO3− to produce oxaloacetate (OAA) during carbon fixation and photosynthesis. It is well accepted that PEPC genes are expressed in plants upon stress. PEPC also supports the biosynthesis of biocompatible osmolytes in many plant species under osmotic stress. There are five isoforms of PEPC found in peanut (Arachis hypogaea L.), namely, AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the gene expression patterns of these AhPEPC genes were different in mature seeds, stems, roots, flowers, and leaves. The expression of all the plant type PEPC (PTPCs) (AhPEPC1, AhPEPC2, AhPEPC3, and AhPEPC4) was relatively high in roots, while the bacterial type PEPC (BTPC) (AhPEPC5) showed a remarkable expression level in flowers. Principal component analysis (PCA) result showed that AhPEPC3 and AhPEPC4 are correlated with each other, indicating comparatively associations with roots, and AhPEPC5 have a very close relationship with flowers. In order to investigate the function of these AhPEPCs, the fragments of these five AhPEPC cDNA were cloned and expressed in Escherichia coli (E. coli). The recombinant proteins contained a conserved domain with a histidine site, which is important for enzyme catalysis. Results showed that protein fragments of AhPEPC1, AhPEPC2, and AhPEPC5 had remarkable expression levels in E. coli. These three recombinant strains were more sensitive at pH 9.0, and recombinant strains carrying AhPEPC2 and AhPEPC5 fragments exhibited more growth than the control strain with the presence of PEG6000. Our findings showed that the expression of the AhPEPC fragments may enhance the resistance of transformed E. coli to osmotic stress.

Rapidly evolving phosphoenolpyruvate carboxylase Gmppc1 and Gmppc7 are highly expressed in the external seed coat of immature soybean seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon fixation enzyme which probably plays crucial roles in seed development. A greater number of PEPC isoforms are encoded in the soybean genome, while most of the PEPC isoforms are functionally unknown. In this study, we investigated on soybean PEPC expressed in the external layer of seed coat (ELSC) during seed formation. PEPC activity in ELSC ranged from 0.24 to 1.0U/g F.W., which could be comparable to those in whole seeds at U per dry matter. Public RNA-Seq data in separated soybean seed tissues revealed that six plant-type PEPC isogenes were substantially expressed in ELSC, and Gmppc1 and Gmppc7 were highly expressed in hourglass cells of ELSC. Gene Ontology enrichment of co-expressed genes with Gmppc1 and Gmppc7 implicated a role of these isogenes in assisting energy production and cellulose biosynthesis. Comparison of PEPC sequences from 16 leguminous species hypothesized adaptive evolution of the Gmppc1 and Gmppc7 lineage after divergence from the other plant-type PEPC lineages. Molecular diversification of these plant-type PEPC was possibly accomplished by adaptation to the functions of the soybean seed tissues. This study indicates that energy demand in immature seeds may be a driving force for the molecular evolution of PEPC.

The plant-type phosphoenolpyruvate carboxylase Gmppc2 is developmentally induced in immature soy seeds at the late maturation stage: a potential protein biomarker for seed chemical composition.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon-fixing enzyme with critical roles in seed development. Previously we observed a positive correlation between PEPC activity and protein content in mature seeds among soybean cultivars and varietal differences of PEPC activity in immature seeds, which is concordant with seed protein accumulation. Here, we report a PEPC isoform (Gmppc2) which is preferentially expressed in immature soybean seeds at the late maturation stage. Gmppc2 was co-expressed with enzyme genes involved in starch degradation: α-amylase, hexokinase, and α-glucan phosphorylase. Gmppc2 was developmentally induced in the external seed coats, internal seed coats, hypocotyls, and cotyledons at the late maturation stage. The expression of Gmppc2 protein was negatively regulated by the application of a nitrogen fertilizer, which suppressed nodule formation. These results imply that Gmppc2 is involved in the metabolism of nitrogen originated from nodules into seeds, and Gmppc2 might be applicable as a biomarker of seed protein content.Abbreviations: PEP: phosphoenolpyruvate; PEPC: phosphoenolpyruvate carboxylase; RNA-Seq: RNA sequencing; PCA: principal component analysis; SE: standard error.

Diffusion of CO2 across the Mesophyll-Bundle Sheath Cell Interface in a C4 Plant with Genetically Reduced PEP Carboxylase Activity.

Phosphoenolpyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C4 photosynthetic pathway. Here, we used RNA interference to target the cytosolic photosynthetic PEPC isoform in Setaria viridis and isolated independent transformants with very low PEPC activities. These plants required high ambient CO2 concentrations for growth, consistent with the essential role of PEPC in C4 photosynthesis. The combination of estimating direct CO2 fixation by the bundle sheath using gas-exchange measurements and modeling C4 photosynthesis with low PEPC activity allowed the calculation of bundle sheath conductance to CO2 diffusion (gbs ) in the progeny of these plants. Measurements made at a range of temperatures suggested no or negligible effect of temperature on gbs depending on the technique used to calculate gbs Anatomical measurements revealed that plants with reduced PEPC activity had reduced cell wall thickness and increased plasmodesmata (PD) density at the mesophyll-bundle sheath (M-BS) cell interface, whereas we observed little difference in these parameters at the mesophyll-mesophyll cell interface. The increased PD density at the M-BS interface was largely driven by an increase in the number of PD pit fields (cluster of PDs) rather than an increase in PD per pit field or the size of pit fields. The correlation of gbs with bundle sheath surface area per leaf area and PD area per M-BS area showed that these parameters and cell wall thickness are important determinants of gbs It is intriguing to speculate that PD development is responsive to changes in C4 photosynthetic flux.

Effect of Nitrogen Source and Concentration on Growth and Activity of Nitrogen Assimilation Enzymes in Roots of a Moroccan Sorghum Ecotype

Carbon and nitrogen metabolism pathways are regulated by complex mechanisms in order to optimize growth and development of plants. This study was conducted to contribute to the determination of roles of some key enzymes of carbon and nitrogen metabolism in sorghum roots. Sorghum were grown under nitrate (NO3-), ammonium (NH 4+) or nitrate combined to ammonium at different concentrations. Growth parameters and protein content were evaluated. In addition, we analyzed key enzymes activities involved in nitrogen and carbon metabolism such as: nitrate reductase (NR), glutamine synthetase (GS), aspartate aminotransferase (AAT) and phosphoenolpyruvate carboxylase (PEPC). At high nitrogen levels (ammonium or nitrate), the sorghum roots showed an increase in biomass accompanied by an increase in protein content. Likewise, high concentrations induced accumulation of the non-photosynthetic isoform of phosphoenolpyruvate carboxylase. Glutamine synthetase and aspartate aminotransferase activities were also increased, especially at high levels of ammonium. These results showed that sorghum plants have an efficient system for nitrogen assimilation in their roots, allowing this plant to tolerate excessive concentration of this nutrient.

Analysis and elucidation of phosphoenolpyruvate carboxylase in cyanobacteria.

Phosphoenolpyruvate carboxylase (PEPC) a cytosolic enzyme of higher plants is also found in bacteria and cyanobacteria. Genetic and biochemical investigations have indicated that there are several isoforms of PEPC belonging to C3; C3/C4 and C4 groups but, the evolution of PEPC in cyanobacteria is not yet understood. The present study opens up an opportunity to understand the isoforms and functions of PEPC in cyanobacteria. The variations observed in PEPC among lower and higher orders of cyanobacteria, suggests convergent evolution of PEPC. There is a specific PEPC phosphorylation residue 'serine' at the N-terminus and PEPC determinant residue 'serine' at the C-terminal that facilitates high affinity for substrate binding. These residues were unique to higher orders of cyanobacteria, but, not in lower orders and other prokaryotes. The different PEPC forms of cyanobacteria were investigated for their kinetic properties with phosphoenolpyruvate as the substrate and the findings corroborated well with the in silico findings. In vitro enzymatic study of cyanobacteria belonging to three different orders demonstrated the role of aspartate as an allosteric effector, which inhibited PEPC by interacting with the highly conserved residues in the active site. The differences in mode of inhibition among the different order, thus, give a fair picture about the cyanobacterial PEPCs. The higher orders appear to possess the sequence coordinates and functionally conserved residues similar to isoforms of C4 type higher plants, whereas isoforms of PEPC of the lower orders did not resemble either that of C3 or C4 plants.

Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii.

The regulation of lipid biosynthesis is important in photosynthetic eukaryotic cells. This regulation is facilitated by the direct synthesis of fatty acids and triacylglycerol (TAG), and by other controls of the main carbon metabolic pathway. In this study, knockdown of the mRNA expression of the Chlamydomonas phosphoenolpyruvate carboxylase isoform 1 (CrPEPC1) gene by RNA interference increased TAG level by 20% but decreased PEPC activities in the corresponding transgenic algae by 39-50%. The decrease in CrPEPC1 expression increased the expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, CrPEPC1 over-expression decreased TAG level by 37% and increased PEPC activities by 157-184%. These observations suggest that the lipid content of algal cells can be controlled by regulating the CrPEPC1 gene.

Multiple isoforms of phosphoenolpyruvate carboxylase in the Orchidaceae (subtribe Oncidiinae): implications for the evolution of crassulacean acid metabolism.

Phosphoenolpyruvate carboxylase (PEPC) catalyses the initial fixation of atmospheric CO2 into oxaloacetate and subsequently malate. Nocturnal accumulation of malic acid within the vacuole of photosynthetic cells is a typical feature of plants that perform crassulacean acid metabolism (CAM). PEPC is a ubiquitous plant enzyme encoded by a small gene family, and each member encodes an isoform with specialized function. CAM-specific PEPC isoforms probably evolved from ancestral non-photosynthetic isoforms by gene duplication events and subsequent acquisition of transcriptional control elements that mediate increased leaf-specific or photosynthetic-tissue-specific mRNA expression. To understand the patterns of functional diversification related to the expression of CAM, ppc gene families and photosynthetic patterns were characterized in 11 closely related orchid species from the subtribe Oncidiinae with a range of photosynthetic pathways from C3 photosynthesis (Oncidium cheirophorum, Oncidium maduroi, Rossioglossum krameri, and Oncidium sotoanum) to weak CAM (Oncidium panamense, Oncidium sphacelatum, Gomesa flexuosa and Rossioglossum insleayi) and strong CAM (Rossioglossum ampliatum, Trichocentrum nanum, and Trichocentrum carthagenense). Phylogenetic analysis revealed the existence of two main ppc lineages in flowering plants, two main ppc lineages within the eudicots, and three ppc lineages within the Orchidaceae. Our results indicate that ppc gene family expansion within the Orchidaceae is likely to be the result of gene duplication events followed by adaptive sequence divergence. CAM-associated PEPC isoforms in the Orchidaceae probably evolved from several independent origins.

Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C4 photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C4 pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C4 PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower μM range and a strong preference for C4 PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C4 weeds.

Greater efficiency of photosynthetic carbon fixation due to single amino-acid substitution.

The C4-photosynthetic carbon cycle is an elaborated addition to the classical C3-photosynthetic pathway, which improves solar conversion efficiency. The key enzyme in this pathway, phosphoenolpyruvate carboxylase, has evolved from an ancestral non-photosynthetic C3 phosphoenolpyruvate carboxylase. During evolution, C4 phosphoenolpyruvate carboxylase has increased its kinetic efficiency and reduced its sensitivity towards the feedback inhibitors malate and aspartate. An open question is the molecular basis of the shift in inhibitor tolerance. Here we show that a single-point mutation is sufficient to account for the drastic differences between the inhibitor tolerances of C3 and C4 phosphoenolpyruvate carboxylases. We solved high-resolution X-ray crystal structures of a C3 phosphoenolpyruvate carboxylase and a closely related C4 phosphoenolpyruvate carboxylase. The comparison of both structures revealed that Arg884 supports tight inhibitor binding in the C3-type enzyme. In the C4 phosphoenolpyruvate carboxylase isoform, this arginine is replaced by glycine. The substitution reduces inhibitor affinity and enables the enzyme to participate in the C4 photosynthesis pathway.

Kinetic analyses of recombinant isoforms of phospho enolpyruvate carboxylase from Hydrilla verticillata leaves and the impact of substituting a C 4-signature serine

Hydrilla verticillata (L.f.) Royle is a single-cell C 4 NADP-malic enzyme species in which the C 4 and Calvin cycles operate in the same cell; phospho enolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) is the initial carboxylase. The cDNAs of two Hydrilla PEPC isoforms were sequenced; one encodes the photosynthetic form, HVPEPC4, and the other an anaplerotic, HVPEPC3. Both lack the C 4-signature Ser. Recombinant proteins were generated and the C 3-Ala was replaced by the C 4-Ser using site-directed mutagenesis. Kinetic analyses of the recombinant isoforms indicated that rHVPEPC4 had a higher K 0 .5 (PEP) (0.3 mM), more typical of C 4 PEPC, despite the lack of a signature Ser. Replacing Ala with Ser in rHVPEPC4 further increased the K 0.5(PEP) to 0.78 mM, but reduced the V max by 25%. However, a similar Ser substitution in rHVPEPC3 not only increased the K 0.5(PEP) from 0.15 to 0.92 mM but also increased the I 50(MAL), V max and k cat. Thus, with a single amino acid substitution rHVPEPC3 kinetically became similar to a C 4 PEPC. In all cases, glucose-6-phosphate lowered the K 0.5(PEP) values and reduced malate inhibition. Although, the C 4-Ser had an impact, overall the C 4 characteristics of HVPEPC4 are conferred by other parts of its primary structure.

Evolution of the C4 phosphoenolpyruvate carboxylase promoter of the C4 species Flaveria trinervia: the role of the proximal promoter region

BackgroundThe key enzymes of photosynthetic carbon assimilation in C4 plants have evolved independently several times from C3 isoforms that were present in the C3 ancestral species. The C4 isoform of phosphoenolpyruvate carboxylase (PEPC), the primary CO2-fixing enzyme of the C4 cycle, is specifically expressed at high levels in mesophyll cells of the leaves of C4 species. We are interested in understanding the molecular changes that are responsible for the evolution of this C4-characteristic PEPC expression pattern, and we are using the genus Flaveria (Asteraceae) as a model system. It is known that cis-regulatory sequences for mesophyll-specific expression of the ppcA1 gene of F. trinervia (C4) are located within a distal promoter region (DR).ResultsIn this study we focus on the proximal region (PR) of the ppcA1 promoter of F. trinervia and present an analysis of its function in establishing a C4-specific expression pattern. We demonstrate that the PR harbours cis-regulatory determinants which account for high levels of PEPC expression in the leaf. Our results further suggest that an intron in the 5' untranslated leader region of the PR is not essential for the control of ppcA1 gene expression.ConclusionThe allocation of cis-regulatory elements for enhanced expression levels to the proximal region of the ppcA1 promoter provides further insight into the regulation of PEPC expression in C4 leaves.

Species having C4 single-cell-type photosynthesis in the Chenopodiaceae family evolved a photosynthetic phosphoenolpyruvate carboxylase like that of Kranz-type C4 species.

Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C(4) photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C(4) photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C(4) photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO(2) via PEPC from C(4) acid decarboxylation and CO(2) donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C(4) species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C(3) (Suaeda linifolia) chenopods. It was found that PEPC from both types of C(4) chenopods displays higher specific activity than that of the C(3) species and shows kinetic and regulatory characteristics similar to those of C(4) species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C(4) species possesses a Kranz-type C(4) isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C(4) species are in a clade with the C(3) and Kranz C(4) Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C(4)-type PEPC similar to that in Kranz C(4) plants, which likely is required for effective function of C(4) photosynthesis.

Research note: Large gene family of phosphoenolpyruvate carboxylase in the crassulacean acid metabolism plant Kalanchoe pinnata (Crassulaceae) characterised by partial cDNA sequence analysis.

Clones coding for a 1100-bp cDNA sequence of phosphoenolpyruvate carboxylase (PEPC) of the constitutive crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers., were isolated by reverse transcription-polymerase chain reaction (RT-PCR) and characterised by restriction fragment length polymorphism analysis and DNA sequencing. Seven distinct PEPC isogenes were recovered, four in leaves and three in roots (EMBL accession numbers: AJ344052-AJ344058). Sequence similarity comparisons and distance neighbour-joining calculations separate the seven PEPC isoforms into two clades, one of which contains the three PEPCs found in roots. The second clade contains the four isoforms found in leaves and is divided into two branches, one of which contains two PEPCs most similar with described previously CAM isoforms. Of these two isoforms, however, only one exhibited abundant expression in CAM-performing leaves, but not in very young leaves, which do not exhibit CAM, suggesting this isoform encodes a CAM-specific PEPC. Protein sequence calculations suggest that all isogenes are likely derived from a common ancestor gene, presumably by serial gene duplication events. To our knowledge, this is the most comprehensive identification of a PEPC gene family from a CAM plant, and the greatest number of PEPC isogenes reported for any vascular plant to date.

Cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene.

C(4) photosynthesis depends on the strict compartmentalization of CO(2) assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C(4) isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C(4) dicot Flaveria trinervia. To elucidate and understand the anatomy of the C(4) ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C(4) species F. bidentis, revealing that the C(4) promoter contains two regions, a proximal segment up to -570 and a distal part from -1566 to -2141, which are necessary but also sufficient for high mesophyll-specific expression of the beta-glucuronidase reporter gene. The distal region behaves as an enhancer-like expression module that can direct mesophyll-specific expression when inserted into the ppcA1 promoter of the C(3) plant F. pringlei. Mesophyll expression determinants were restricted to a 41-bp segment, referred to as mesophyll expression module 1 (Mem1). Evolutionary and functional studies identified the tetranucleotide sequence CACT as a key component of Mem1.

Molecular evolution of C4 phosphoenolpyruvate carboxylase in the genus Flaveria--a gradual increase from C3 to C4 characteristics.

In order to elucidate the discrete steps in phospho enolpyruvate carboxylase (PEPC) evolution concerning K(m)-PEP and malate tolerance a comparison was made between C3, C3-C4 and C4 species of the dicot genus Flaveria. The PEPCs of this genus are encoded by a gene family comprising three classes: ppcA, ppcB and ppcC [J. Hermans and P. Westhoff (1990) Mol Gen Genet 224:459-468, (1992) Mol Gen Genet 234:275-284]. The ppcA of F trinervia (C4) codes for the C4 PEPC isoform but other plants of the genus contain ppcA orthologues too. The C3 plant F. pringlei showed the lowest levels of ppcA PEPC mRNA followed by F. pubescens (C3-C4) while the C4-like plant F. brownii displayed RNA amounts close to the C4 species F. trinervia. In contrast to the similar expression profiles of F. brownii (C4-like) and F. trinervia (C4) the PEPC amino acid sequence of F. brownii was more similar to the C3 and C3-C4 ppcA PEPCs than to the C4 PEPC. Similarly, the C3, C3-C4 and C4-like ppcA PEPCs showed almost identical PEP saturation kinetics when activated by glucose-6-phosphate ( K(m)-PEP: 17-20 microM) while the K(m)-PEP for the C4 PEPC was determined to be 53 microM. However, without activation the ppcA PEPCs of F. pubescens and F. brownii displayed C3-C4 intermediate values. A similar picture was obtained when the malate sensitivities were compared. In the non-activated state the F. trinervia (C4) enzyme was 10 times more tolerant to malate than the F. pringlei counterpart. The ppcA enzymes of F. pubescens (C3-C4) and F. brownii (C4-like) displayed intermediate values. In contrast, the inclusion of 5 mM glucose-6-phosphate in the reaction mixture changed the order totally. Interestingly, the activation rendered the C4 enzyme about 50% less tolerant to malate than the C3 PEPC. The activation had a positive effect on malate tolerance of the F. pubescens (C3-C4) PEPC while the ppcA PEPC of F. brownii (C4-like) was almost unaffected.

Evolution of C4 phospho enolpyruvate carboxylase

C4 plants are known to be of polyphyletic origin and to have evolved independently several times during the evolution of angiosperms. This implies that the C4 isoform of phospho enolpyruvate carboxylase (PEPC) originated from a nonphotosynthetic PEPC gene that was already present in the C3 ancestral species. To meet the special requirements of the C4 photosynthetic pathway the expression program of the C4 PEPC gene had to be changed to achieve a strong and selective expression in leaf mesophyll cells. In addition, the altered metabolite concentrations around C4 PEPC in the mesophyll cytoplasm necessitated changes in the enzyme’s kinetic and regulatory properties. To obtain insight into the evolutionary steps involved in these altered enzyme characteristics, and even the order of these steps, the dicot genus Flaveria (Asteraceae) appears to be the experimental system of choice. Flaveria contains closely related C3, C3-C4, and C4 species that can be ordered by their gradual increase in C4 photosynthetic traits. The C4 PEPC of F. trinervia, which is encoded by the ppcA gene class, possesses typical kinetic and regulatory features of a C4-type PEPC. Its nearest neighbor is the orthologous ppcA gene of the C3 species F. pringlei. This latter gene encodes a typical nonphotosynthetic C3-type PEPC which is believed to be similar to the C3 ancestral PEPC. This pair of orthologous PEPCs has been used to map C4-specific molecular determinants for the kinetic and regulatory characteristics of C4 PEPCs. The most notable finding from these investigations was the identification of a C4 PEPC invariant site-specific mutation from alanine (C3) to serine (C4) at position 774 that was a necessary and late step in the evolution of C3 to C4 PEPC. The C3-C4 intermediate ppcA PEPCs are used to identify the sequence of events leading from a C3- to a C4-type PEPC.

Structural and Kinetic Properties of High and Low Molecular Mass Phosphoenolpyruvate Carboxylase Isoforms from the Endosperm of Developing Castor Oilseeds

Phosphoenolpyruvate carboxylase (PEPC) is believed to play an important role in producing malate as a substrate for fatty acid synthesis by leucoplasts of the developing castor oilseed (COS) endosperm. Two kinetically distinct isoforms of COS PEPC were resolved by gel filtration chromatography and purified. PEPC1 is a typical 410-kDa homotetramer composed of 107-kDa subunits (p107). In contrast, PEPC2 exists as an unusual 681-kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated 64-kDa polypeptide (p64) that is structurally and immunologically unrelated to p107. Relative to PEPC1, PEPC2 demonstrated significantly enhanced thermal stability and a much lower sensitivity to allosteric activators (Glc-6-P, Glc-1-P, Fru-6-P, glycerol-3-P) and inhibitors (Asp, Glu, malate) and pH changes within the physiological range. Nondenaturing PAGE of clarified extracts followed by in-gel PEPC activity staining indicated that the ratio of PEPC1:PEPC2 increases during COS development such that only PEPC1 is detected in mature COS. Dissimilar developmental profiles and kinetic properties support the hypotheses that (i) PEPC1 functions to replenish dicarboxylic acids consumed through transamination reactions required for storage protein synthesis, whereas (ii) PEPC2 facilitates PEP flux to malate in support of fatty acid synthesis. Interestingly, the respective physical and kinetic properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low M(r) Class 1 and heteromeric high M(r) Class 2 PEPC isoforms of unicellular green algae.

Restriction accessibility in isolated nuclei reveals light-induced chromatin reorganization at the PEPC promoter in maize.

Expression of genes necessary to perform C4 photosynthesis in maize is activated by light. It is not known how this activation is regulated on the chromatin level in vivo. We analysed alterations in the chromatin structure of the promoter of the C4-specific isoform of phosphoenolpyruvate carboxylase (PEPC) after illumination of seedlings. A protocol was established that facilitates the preparation of nuclei from maize leaves with intact chromatin structure and resistance to DNA degradation during prolonged incubation at high temperatures. The presence of non-spliced transcripts from the C4-PEPC gene in the nuclei was demonstrated by RT-PCR. The chromatin was partially digested with restriction endonucleases. Quantitative PCR analyses revealed a clear increase in the accessibility of the promoter chromatin to restriction dependent on illumination of the seedlings. The data indicate chromatin reorganization at the C4-PEPC promoter during activation.

Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single-cell, facultative C(4) system of Hydrilla verticillata.

The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C(4) plant. It typically exhibits C(3) photosynthetic characteristics, but exposure to low [CO(2)] induces a C(4) system in which the C(4) and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C(4) induction. This and enzyme kinetic data were consistent with it being the C(4) photosynthesis isoform. However, the C(4) signature serine of terrestrial plant C(4) isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C(3) sequences, was present. Western analyses of C(3) and C(4) leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C(3), non-graminaceous C(4), and Crassulacean acid metabolism PEPCs but not with the graminaceous C(4), and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C(4) angiosperms.