Plant-type Phosphoenolpyruvate Carboxylase Research Articles

Over-expression of a plant-type phosphoenolpyruvate carboxylase derails Arabidopsis stamen formation

We examined whether plant-type phosphoenolpyruvate carboxylase (PEPC) is involved in flower organ formation or not by over-expression in Arabidopsis. A wheat PEPC isogene Tappc3A, belonging to the ppc3 group, was targeted due to its preferential expression pattern in pistils and stamens. Transgenic Arabidopsis over-expressing Tappc3A exhibited irregular stamen formation, i.e., a lesser number of stamens per flower and shorter filaments in T2 and T3 generations. Irregular stamens were frequently observed in homozygous T4 lines, but no morphological change was observed in other floral organs. High-degree gene co-expression of Tappc3 isogenes with wheat SEEDSTICKs but not with other homeotic transcription factor genes for flower formation implicates that Tappc3 is under control by the class D genes of the ABCDE model to flower development. In addition, the conservation of CArG box sequences on the Tappc3 promoters supported the developmentally programmed gene expression of ppc3 in wheat flowering organs. Thus, this study provides the first experimental evidence for the critical regulation of plant-type PEPC for flower formation.

Phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase isoenzymes play an important role in the filling and quality of Arabidopsis thaliana seed

Three plant-type phosphoenolpyruvate carboxylase (PPC1 to PPC3) and two phosphoenolpyruvate carboxylase kinase (PPCKs: PPCK1 and 2) genes are present in the Arabidopsis thaliana genome. In seeds, all PPC genes were found to be expressed. Examination of individual ppc mutants showed little reduction of PEPC protein and global activity, with the notable exception of PPC2 which represent the most abundant PEPC in dry seeds. Ppc mutants exhibited moderately lower seed parameters (weight, area, yield, germination kinetics) than wild type. In contrast, ppck1-had much altered (decreased) yield. At the molecular level, ppc3-was found to be significantly deficient in global seed nitrogen (nitrate, amino-acids, and soluble protein pools). Also, N-deficiency was much more marked in ppck1-, which exhibited a tremendous loss of 95% and 90% in nitrate and proteins, respectively. The line ppck2-had accumulated amino-acids but lower levels of soluble proteins. Regarding carboxylic acid pools, Krebs cycle intermediates were found to be diminished in all mutants; this was accompanied by a consistent decrease in ATP. Lipids were stable in ppc mutants, however ppck1-seeds accumulated more lipids while ppck2-seeds showed high level of polyunsaturated fatty acid oleic and linolenic (omega 3). Altogether, the results indicate that the complete PEPC and PPCK family are needed for normal C/N metabolism ratio, growth, development, yield and quality of the seed.

Convergent molecular evolution of phosphoenolpyruvate carboxylase gene family in C4 and crassulacean acid metabolism plants.

Phosphoenolpyruvate carboxylase (PEPC), as the key enzyme in initial carbon fixation of C4and crassulacean acid mechanism (CAM) pathways, was thought to undergo convergent adaptive changes resulting in the convergent evolution of C4 and CAM photosynthesis in vascular plants. However, the integral evolutionary history and convergence of PEPC in plants remain poorly understood. In the present study, we identified the members of PEPC gene family across green plants with seventeen genomic datasets, found ten conserved motifs and modeled three-dimensional protein structures of 90 plant-type PEPC genes. After reconstructing PEPC gene family tree and reconciled with species tree, we found PEPC genes underwent 71 gene duplication events and 16 gene loss events, which might result from whole-genome duplication events in plants. Based on the phylogenetic tree of the PEPC gene family, we detected four convergent evolution sites of PEPC in C4 species but none in CAM species. The PEPC gene family was ubiquitous and highly conservative in green plants. After originating from gene duplication of ancestral C3-PEPC, C4-PEPC isoforms underwent convergent molecular substitution that might facilitate the convergent evolution of C4 photosynthesis in Angiosperms. However, there was no evidence for convergent molecular evolution of PEPC genes between CAM plants. Our findings help to understand the origin and convergent evolution of C4 and CAM plants and shed light on the adaptation of plants in dry, hot environments.

Genome-Wide Identification and Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Suaeda aralocaspica, an Annual Halophyte With Single-Cellular C4 Anatomy

Phosphoenolpyruvate carboxylase (PEPC) plays pivotal roles in the carbon fixation of photosynthesis and a variety of metabolic and stress pathways. Suaeda aralocaspica belongs to a single-cellular C4 species and carries out a photosynthetic pathway in an unusually elongated chlorenchyma cell, which is expected to have PEPCs with different characteristics. To identify the different isoforms of PEPC genes in S. aralocaspica and comparatively analyze their expression and regulation patterns as well as the biochemical and enzymatic properties in this study, we characterized a bacterial-type PEPC (BTPC; SaPEPC-4) in addition to the two plant-type PEPCs (PTPCs; SaPEPC-1 and SaPEPC-2) using a genome-wide identification. SaPEPC-4 presented a lower expression level in all test combinations with an unknown function; two SaPTPCs showed distinct subcellular localizations and different spatiotemporal expression patterns but positively responded to abiotic stresses. Compared to SaPEPC-2, the expression of SaPEPC-1 specifically in chlorenchyma cell tissues was much more active with the progression of development and under various stresses, particularly sensitive to light, implying the involvement of SaPEPC-1 in a C4 photosynthetic pathway. In contrast, SaPEPC-2 was more like a non-photosynthetic PEPC. The expression trends of two SaPTPCs in response to light, development, and abiotic stresses were also matched with the changes in PEPC activity in vivo (native) or in vitro (recombinant), and the biochemical properties of the two recombinant SaPTPCs were similar in response to various effectors while the catalytic efficiency, substrate affinity, and enzyme activity of SaPEPC-2 were higher than that of SaPEPC-1 in vitro. All the different properties between these two SaPTPCs might be involved in transcriptional (e.g., specific cis-elements), posttranscriptional [e.g., 5′-untranslated region (5′-UTR) secondary structure], or translational (e.g., PEPC phosphorylation/dephosphorylation) regulatory events. The comparative studies on the different isoforms of the PEPC gene family in S. aralocaspica may help to decipher their exact role in C4 photosynthesis, plant growth/development, and stress resistance.

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.

Molecular Cloning of Novel-Type Phosphoenolpyruvate Carboxylase Isoforms in Pitaya (Hylocereus undatus).

Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme involved in the initial CO2 fixation of crassulacean acid metabolism (CAM) photosynthesis. To understand the cultivation characteristics of a CAM plant pitaya, it is necessary to clarify the characteristics of PEPC in this species. Here, we cloned three PEPC cDNAs in pitaya, HuPPC1, HuPPC2, and HuPPC3, which encode 942, 934, and 966 amino acid residues, respectively. Phylogenetic analysis indicated that these PEPC belonged to plant-type PEPC (PTPC), although HuPPC1 and HuPPC2 have no Ser-phosphorylation motif in N-terminal region, which is a crucial regulation site in PTPC and contributes to CAM periodicity. HuPPC1 and HuPPC2 phylogenetically unique to the Cactaceae family, whereas HuPPC3 was included in a CAM clade. Two isoforms were partially purified at the protein level and were assigned as HuPPC2 and HuPPC3 using MASCOT analysis. The most distinct difference in enzymatic properties between the two was sensitivity to malate and aspartate, both of which are allosteric inhibitors of PEPC. With 2 mM malate, HuPPC3 was inhibited to 10% of the initial activity, whereas HuPPC2 activity was maintained at 70%. Aspartate inhibited HuPPC3 activity by approximately 50% at 5 mM; however, such inhibition was not observed for HuPPC2 at 10 mM. These results suggest that HuPPC3 corresponds to a general CAM-related PEPC, whereas HuPPC1 and HuPPC2 are related to carbon and/or nitrogen metabolism, with a characteristic regulation mechanism similar to those of Cactaceae plants.

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.

Transcriptional and post-translational upregulation of phosphoenolpyruvate carboxylase in Arabidopsis thaliana (L. Heynh) under cadmium stress

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme that replenishes organic acids that have been withdrawn from the tricarboxylic acid cycle. Organic acid anions are important chelators of toxic metals, including cadmium (Cd). To determine the Cd-stress responses of two isozymes of PEPC in Arabidopsis (AtPPC1 and AtPPC3), we compared wild type (Col-0) with two T-DNA insertional mutant lines (atppc1 and atppc3). Plant mass decreased in response to Cd in all lines, but atppc3 was the most sensitive to Cd even though its roots contained 30% lower concentrations of Cd. Treatment with 1 or 5 μM CdCl2 resulted in up to 400% higher expression of genes encoding three isozymes of plant-type PEPCs and up to 200% higher expression of genes encoding two isozymes of PEPC kinase, especially in roots. The amount of PEPC and its phosphorylation status also increased by up to 40% with Cd treatment, as did enzyme activity (up to 200%). AtPPC1 and AtPPC3 transcript levels in the mutant lines were as much as 50% lower than in wild type plants, even in control (no Cd) plants. AtPPC2 expression was unaffected in both mutant lines. In general, concentrations of organic acid anions increased or remained unchanged in response to Cd treatment in each plant line, although roots of atppc1 and atppc3 mutants under control conditions had 25–50% less malate and citrate. Our results confirm the importance of root-specific (i) AtPPC3 expression for the normal development of Arabidopsis, as well as (ii) AtPPC1 and AtPPC3 upregulation for acclimation to Cd stress.

Structural and biochemical characterization of citrate binding to AtPPC3, a plant-type phosphoenolpyruvate carboxylase from Arabidopsis thaliana

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated cytosolic enzyme situated at a crucial branch point of central plant metabolism. The structure of AtPPC3, a C3 PEPC isozyme of the model plant Arabidopsis thaliana, in complex with the inhibitors aspartate and citrate was solved at 2.2-Å resolution. This represents the first PEPC structure with citrate bound. Aspartate and citrate binding sites are in close proximity (5.1–5.3 Å) and interactions between citrate and specific residues were identified. Citrate functions as a mixed (allosteric) inhibitor as it reduced AtPPC3′s Vmax while increasing Km(PEP) values. The PEP saturation data gave an excellent fit to the mixed inhibition model, yielding Ki and Ki’ (citrate) values of 9.3 and 42.5 mM, respectively. Citrate and aspartate inhibition of AtPPC3 was non-additive, likely due to their closely positioned binding sites, their similar negative charge, and type of binding residues. Fewer interactions and lower affinity for citrate support its observed weaker inhibition of AtPPC3 relative to aspartate. Citrate does not appear to induce further conformational change beyond aspartate owing to the similar structural mechanism of inhibition. AtPPC3 largely exhibits root-specific expression in Arabidopsis, where it is markedly upregulated during stresses such as excessive salinity or nutritional Pi deprivation that necessitate large increases in anaplerotic PEP carboxylation. The cytosolic citrate concentration of potato tubers suggests that AtPPC3′s inhibition by citrate may be physiologically relevant. Our results provide novel insights into the structural basis of allosteric PEPC control and the kinetic effects brought about upon inhibitor binding.

Transcript profiling indicates a widespread role for bacterial-type phosphoenolpyruvate carboxylase in malate-accumulating sink tissues.

Phosphoenolpyruvate carboxylase (PEPC) is an important regulatory enzyme situated at a key branch point of central plant metabolism. Plant genomes encode several plant-type PEPC (PTPC) isozymes, along with a distantly related bacterial-type PEPC (BTPC). BTPC is expressed at high levels in developing castor oil seeds where it tightly interacts with co-expressed PTPC polypeptides to form unusual hetero-octameric Class-2 PEPC complexes that are desensitized to allosteric inhibition by L-malate. Analysis of RNA-Seq and microarray transcriptome datasets revealed two distinct patterns of tissue-specific BTPC expression in vascular plants. Species such as Arabidopsis thaliana, strawberry, rice, maize, and poplar mainly exhibited pollen- or floral-specific BTPC expression. By contrast, BTPC transcripts were relatively abundant in developing castor, cotton, and soybean seeds, cassava tubers, as well as immature tomato, cucumber, grape, and avocado fruit. Immunoreactive 118 kDa BTPC polypeptides were detected on immunoblots of cucumber and tomato fruit extracts. Co-immunoprecipitation established that as in castor, BTPCs physically interact with endogenous PTPCs to form Class-2 PEPC complexes in tomato and cucumber fruit. We hypothesize that Class-2 PEPCs simultaneously maintain rapid anaplerotic PEP carboxylation and respiratory CO2 refixation in diverse, biosynthetically active sinks that accumulate high malate levels.

Molecular characterization and expression profiling of the phosphoenolpyruvate carboxylase genes in peanut (Arachis hypogaea L.)

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled enzyme located at the core of plant carbohydrate metabolism. Plant PEPCs belong to a small multigene family encoding several plant-type PEPC genes, along with at least one distantly related bacterial-type PEPC gene. The PEPC genes have been intensively studied in Arabidopsis, but not in peanut (Arachis hypogaea L.). Previously, we isolated five PEPC genes (AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4 and AhPEPC5) from peanut. Here, due to the sequencing of the peanut genome, we analyzed the complexity of its PEPC gene family, including phylogenetic relationships, gene structure and chromosome mapping. The results showed that AhPEPC1, AhPEPC2, AhPEPC3 and AhPEPC4 encoded typical plant-type enzymes, while AhPEPC5 was a bacterial-type PEPC. The recombinant proteins of these genes were expressed in Escherichia coli, and the calculated molecular weights of the recombinant proteins were 110.8 kD (AhPEPC1), 110.7 kD (AhPEPC2), 110.3 kD (AhPEPC3), 110.8 kD (AhPEPC4), and 116.4 kD (AhPEPC5). The expression patterns of AhPEPC1-5 were analyzed under cold, salt and drought conditions. Our results indicated that the expression of AhPEPC3 was rapidly and substantially enhanced under abiotic stress, whereas the expression of AhPEPC1 and AhPEPC2 was slightly enhanced under certain stress conditions. Some genes were down-regulated in leaves under stress: AhPEPC1, AhPEPC4 and AhPEPC5 under salt stress and AhPEPC4 and AhPEPC5 under drought stress. These results suggest that peanut PEPC proteins may differ in their functions during acclimation to abiotic stresses.

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 μM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS.

Role of nitrogen-responsive plant-type phosphoenolpyruvate carboxylase in the accumulation of seed storage protein in ancient wheat (spelt and kamut)

ABSTRACTAncient wheat such as Triticum spelta L. and T. turanicum is a possible genetic material for improving wheat seed quality due to its grain characteristics, which differ from those of common wheat. Ancient wheat grains accumulate proteins with fewer allergens and higher levels of essential amino acids, antioxidants, minerals and vitamins. The identification of a genetic factor that controls seed protein content would be useful for breeding wheat varieties in accordance with various purposes. Previous studies implied that phosphoenolpyruvate carboxylase (PEPC) is involved in nitrogen accumulation in crop seeds. However, multiple types of PEPC genes are present in plant genomes, and it has still not been clarified yet which type of PEPC is relevant to seed nitrogen metabolism. In this study, to determine which PEPC is involved in seed protein accumulation in ancient wheat, we examined the effects of different levels of nitrogen application at the flowering stage on PEPC activity on developing seeds and on the protein content in mature seeds of three ancient wheat varieties. The responses of the PEPC activity and seed protein content were observed, but their patterns differed among the varieties. Among the experimental plots, PEPC activity and seed protein content showed a high correlation (r = 0.757, p value = 1.09E−3). Immunoblot analysis revealed that plant-type PEPC seemed to underlie the response of PEPC activity to nitrogen application, while no bacterial-type PEPC was detected by a peptide-antigen antibody. These results indicate that plant-type PEPC plays an important role in storage protein accumulation in ancient wheat seeds under nitrogen application at the flowering stage.Abbreviations: PEPC, phosphoenolpyruvate carboxylase; 2-ME, 2-mercapto ethanol; EDTA, ethylenediaminetetraacetic acid; DAF, days after flowering; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Phosphoenolpyruvate carboxylase (PEPC) and PEPC-kinase (PEPC-k) isoenzymes in Arabidopsis thaliana: role in control and abiotic stress conditions

Arabidopsis ppc3 mutant has a growth-arrest phenotype and is affected in phosphate- and salt-stress responses, showing that this protein is crucial under control or stress conditions. Phosphoenolpyruvate carboxylase (PEPC) and its dedicated kinase (PEPC-k) are ubiquitous plant proteins implicated in many physiological processes. This work investigates specific roles for the three plant-type PEPC (PTPC) and the two PEPC-k isoenzymes in Arabidopsis thaliana. The lack of any of the PEPC isoenzymes reduced growth parameters under optimal growth conditions. PEPC activity was decreased in shoots and roots of ppc2 and ppc3 mutants, respectively. Phosphate starvation increased the expression of all PTPC and PPCK genes in shoots, but only PPC3 and PPCK2 in roots. The absence of any of these two proteins was not compensated by other isoforms in roots. The effect of salt stress on PTPC and PPCK expression was modest in shoots, but PPC3 was markedly increased in roots. Interestingly, both stresses decreased root growth in each of the mutants except for ppc3. This mutant had a stressed phenotype in control conditions (reduced root growth and high level of stress molecular markers), but was unaffected in their response to high salinity. Salt stress increased PEPC activity, its phosphorylation state, and L-malate content in roots, all these responses were abolished in the ppc3 mutant. Our results highlight the importance of the PPC3 isoenzyme for the normal development of plants and for root responses to stress.

New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.

Comparison of Plant-Type Phospho enol pyruvate Carboxylases from Rice: Identification of Two Plant-Specific Regulatory Regions of the Allosteric Enzyme

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae and vascular plants, and it undergoes allosteric regulation by various metabolic effectors. Rice (Oryza sativa) has five plant-type PEPCs, four cytosolic and one chloroplastic. We investigated their kinetic properties using recombinant proteins and found that, like most plant-type PEPCs, rice cytosolic isozymes were activated by glucose 6-phosphate and by alkaline pH. In contrast, no such activation was observed for the chloroplastic isozyme, Osppc4. In addition, Osppc4 showed low affinity for the substrate phosphoenolpyruvate (PEP) and very low sensitivities to allosteric inhibitors aspartate and glutamate. By comparing the isozyme amino acid sequences and three-dimensional structures simulated on the basis of the reported crystal structures, we identified two regions where Osppc4 has unique features that can be expected to affect its kinetic properties. One is the N-terminal extension; replacement of the extension of Osppc2a (cytosolic) with that from Osppc4 reduced the aspartate and glutamate sensitivities to about one-tenth of the wild-type values but left the PEP affinity unaffected. The other is the N-terminal loop, in which a conserved lysine at the N-terminal end is replaced with a glutamate-alanine pair in Osppc4. Replacement of the lysine of Osppc2a with glutamate-alanine lowered the PEP affinity to a quarter of the wild-type level (down to the Osppc4 level), without affecting inhibitor sensitivity. Both the N-terminal extension and the N-terminal loop are specific to plant-type PEPCs, suggesting that plant-type isozymes acquired these regions so that their activity could be regulated properly at the sites where they function.

Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient harsh hakea.

Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al(3+), Fe(3+), and Ca(2+) phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme's activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced Km [PEP] coupled with elevated I50 [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots.

Bacterial‐ and plant‐type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria

Phosphoenolpyruvate carboxylase (PEPC) from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical class-1 PEPC homotetramer consists of 107-kDa plant-type PEPC (PTPC) subunits, whereas the allosterically desensitized 910-kDa class-2 PEPC hetero-octamer arises from the association of class-1 PEPC with 118-kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension-cultured cells. The BTPC-FP mainly localized to cytoplasmic punctate/globular structures, identified as mitochondria by co-immunostaining of endogenous cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP, respectively. The FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal, NLS) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. Transmission electron microscopy of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as the cytosol. Moreover, thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Overall, our results demonstrate that: (i) COS BTPC and PTPC interact in vivo as a class-2 PEPC complex that associates with the surface of mitochondria, (ii) BTPC's unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (iii) the PTPC-containing class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated class-2 PEPC facilitates rapid refixation of respiratory CO(2) while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.

The bacterial-type phosphoenolpyruvate carboxylase isozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate-metabolism. In developing castor oil seeds (COS) a novel allosterically-densensitized 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between 107-kDa plant-type PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. Mass spectrometry and immunoblotting with anti-phosphoSer451 specific antibodies established that COS BTPC is in vivo phosphorylated at Ser451, a highly conserved target residue that occurs within an intrinsically disordered region. This phosphorylation was enhanced during COS development or in response to depodding. Kinetic characterization of a phosphomimetic (S451D) mutant indicated that Ser451 phosphorylation inhibits the catalytic activity of BTPC subunits within the Class-2 PEPC complex.