C4 PEPC Research Articles

Kinetic Modifications of C4 PEPC Are Qualitatively Convergent, but Larger in Panicum Than in Flaveria.

C4 photosynthesis results from a set of anatomical features and biochemical components that act together to concentrate CO2 within the leaf and boost productivity. This complex trait evolved independently many times, resulting in various realizations of the phenotype, but in all C4 plants the primary fixation of atmospheric carbon is catalyzed by phosphoenolpyruvate carboxylase. Comparisons of C4 and non-C4 PEPC from a few closely related species suggested that the enzyme was modified to meet the demands of the C4 cycle. However, very few C4 groups have been investigated, hampering general conclusions. To test the hypothesis that distant C4 lineages underwent convergent biochemical changes, we compare the kinetic variation between C4 and non-C4 PEPC from a previously assessed young lineage (Flaveria, Asteraceae) with those from an older lineage found within the distantly related grass family (Panicum). Despite the evolutionary distance, the kinetic changes between the non-C4 and C4 PEPC are qualitatively similar, with a decrease in sensitivity for inhibitors, an increased specificity (k cat/K m) for bicarbonate, and a decreased specificity (k cat/K m) for PEP. The differences are more pronounced in the older lineage Panicum, which might indicate that optimization of PEPC for the C4 context increases with evolutionary time.

Stimulation by abscisic acid of the activity of phosphoenolpyruvate carboxylase in leaf disks of Amaranthus hypochondriacus L., C4 plant: role of pH and protein levels.

C4 plants can more efficiently fix carbon in drought, high temperatures, and limitations of nitrogen or CO2. Primary carboxylation is mediated by phosphoenolpyruvate carboxylase (PEPC, 4.1.1.31) in mesophyll cytosol of C4 plants. Studies on hormonal regulation of C4 PEPC have been quite limited. We have examined the activity/regulation of PEPC by abscisic acid (ABA), a plant hormone, in the leaves of Amaranthus hypochondriacus. PEPC activity was enhanced upon 1-h incubation with 20μM ABA by about 30% in dark and more than 2-fold in light. Glucose-6-phosphate activation of PEPC was enhanced, and sensitivity to L-malate was decreased after ABA treatment. Butyric acid (a weak acid) decreased PEPC activity and restricted the stimulation by ABA. In contrast, methylamine (an alkalinizing agent) increased the PEPC activity and enhanced the effect of ABA. ABA increased the levels of PEPC protein as well as its mRNA. Butyric acid/methylamine modulated the changes induced by ABA of PEPC protein and mRNA levels, indicating that acidification/alkalinization of leaf disks was very important. Our results emphasize the marked modulation of PEPC in C4 plants, by ABA. Such modulation by ABA could be significant when C4 plants are under water stress, when ABA is known to accumulate. When present, cycloheximide decreased the PEPC protein levels and restricted the extent of activation by ABA. We conclude that the enhancement by ABA of PEPC activity depends on cellular alkalinization as well as elevated PEPC protein levels.

Phosphoenolpyruvate Carboxylase from C4 Leaves Is Selectively Targeted for Inhibition by Anionic Phospholipids

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an enzyme playing a crucial role in photosynthesis of C4 plants. Here, we identify anionic phospholipids as novel regulators that inhibit C4 PEPC activity and provide evidence that the enzyme partially localizes to membranes.

Reversible phosphorylation of photosynthetic PEP carboxylase: Studies on C4-leaf PP2A and recombinant PEPC-kinase from CAM-induced Mesembryanthemum crystallinum

The activity and allosteric properties of plant PEPC are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved Ser residue near the N-terminus. This up/down-modulation is catalyzed by a transcriptionally regulated, seemingly dedicated Ser/Thr kinase (PpcK) and an opposing Ser/Thr phosphatase (PP2A). We have now partially purified and characterized the native form of this largely neglected PP2A from maize leaves using various chromatographic and affinity matrices, and C4 [32P]PEPC as substrate (Dong et al., 2001, Planta [in press]). The results indicate that the C4-leaf holoenzyme is analogous to yeast and mammalian PP2As in regards to its heterotrimeric structure (~170 kDa), comprised of a ~103-kDa core PP2Ac-A heterodimer complexed with a ~74-kDa B-type subunit, and its sensitivity to free Me2+ and various inhibitors, activators and anionic metabolites. Notably, this native PP2A (a) lacks any strict phosphoprotein specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro, and (b) displays, at best, only modest light/dark effects in vivo on its apparent Mr, component core subunits, and activity against C4 PEPC-SerP. In addition, we will also report new findings on a recombinant form of CAM PpcK from M. crystallinum (Taybi et al., 2000, Plant Physiol.) produced as a highly soluble, active fusion with the ~55-kDa NusA carrier protein in E. coli. This NusA?PpcK fusion protein has been purified by sequential IMAC and FPLC, used for detailed analysis of its target-protein specificity and other kinetic properties, and cleaved on-bead by thrombin to yield free PpcK for antibody production.

Improvement of drought tolerance in maize: towards the functional validation of the Zm-Asr1 gene and increase of water use efficiency by over-expressing C4–PEPC

Water availability is one of the major limiting factors for plant growth. Maize is particularly sensitive to water stress at reproductive stages with a strong impairment of photosynthesis and grain filling. Here, we describe the use of genetic transformation first to assess the role of a candidate gene Asr1—a putative transcription factor—as an explanation for genetically linked drought tolerance Quantitative Trait Loci (QTLs), and second to modify CO 2 fixation rates in leaves through changes of C 4 phosphoenolpyruvate carboxylase (C 4-PEPC) activity. Transgenic Asr1 over-expressing lines show an increase in foliar senescence under drought conditions. The highest C 4-PEPC overexpressing line exhibited an increase (+30%) in intrinsic water use efficiency (WUE) accompanied by a dry weight increase (+20%) under moderate drought conditions. Opposite effects were observed for transgenic plants under-expressing the corresponding proteins. The data presented here indicate the feasibility to increase the level of endogenous biochemical activities related to water economy and/or drought tolerance, and opens a way to develop maize varieties more tolerant to dry growing conditions.

Manipulating PEPC levels in plants.

This review examines the current understanding of the structural, functional and regulatory properties of C4 and C3 forms of higher plant phosphoenolpyruvate carboxylase. The emphasis is on the interactive metabolic and post-translational controls acting on the enzyme in the physiological context of C4 photosynthesis and the anaplerotic pathway. A brief overview is given concerning the recent developments of PEPC-based genetic engineering of C3 plants with the aim of improving photosynthetic performance in normal and limiting environmental conditions. So far, in spite of achieving a considerable increase in PEPC levels, more work needs to be done with respect to the correct dosage and location before that goal is reached. Some unpublished results on the transformation of maize with a sorghum C4 PEPC cDNA are also presented. They show that it is possible to increase photosynthetic PEPC levels in this C4 plant and that the modification in enzyme content has a pleiotropic physiological impact and, notably, an improved water use efficiency when water is limited.

Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosophoenolpyruvate carboxylase.

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.

CDNA cloning and characterization of a C4 PEPC kinase from Flaveria trinervia

PEPC activity in C4 plants is regulated by reversible phosphorylation mainly in response to light. The cDNA for this specific protein kinase (PEPC-PK) has not yet been cloned, though several cDNAs for PEPC-PKs from C3 and CAM plants have been isolated recently. A complete cDNA (~1.4 kb) encoding PEPC-PK was isolated from Flaveria trinervia, a dicot C4 plant, by screening cDNA library with a F. trinervia PEPC-PK fragment prepared by PCR with degenerate primers. The cloned PEPC-PK contained an ORF for a protein of 281 amino acid residues, and it was composed of the kinase domain only. The recombinant PEPC-PK expressed in E. coli phosphorylated a specific serine residue of maize C4-form PEPC near the N-terminus, whereas it did not phosphorylate a mutant PEPC in which the serine residue of the phosphorylation-site have been substituted for aspartate (S15D). The activity of the PEPC-PK was Ca2+-independent. The interesting enzymatic properties will also be presented. Northern blot analysis showed that the transcripts accumulated abundantly in leaves in daytime, but not at night. The transcripts were not detected in roots and stems. Genomic Southern blot analysis under the high-stringency conditions indicated that the F. trinervia genome contained more than two copies. These results suggested that this PEPC-PK plays a major role in the regulatory phosphorylation of PEPC for C4 photosynthesis.

Metabolite control of Sorghum C 4 phospho enol pyruvate carboxylase catalytic activity and phosphorylation state

Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C4 phospho enolpyruvate carboxylase (C4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucose-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C4 PEPC phosphorylation strengthened the effect of the positive effectors thereby decreasing further the enzyme's sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C4 PEPC, a process antagonized by positive metabolite effectors. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C4 PEPC phosphorylation in mesophyll cell protoplasts.

In Vivo and in Vitro Phosphorylation of the Phosphoenolpyruvate Carboxylase from Wheat Seeds during Germination.

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal anti-bodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in addition, a new, 108-kD polypeptide was detected 6 h after imbibition. The use of specific anti-phosphorylation-site immunoglobulin G (APS-IgG) identified the presence of a phosphorylation motif equivalent to that found in other plant PEPCs studied so far. The binding of this APS-IgG to the target protein promoted changes in the properties of seed PEPC similar to those produced by phosphorylation, as previously shown for the recombinant Sorghum leaf C4 PEPC. In desalted seed extracts, an endogenous PEPC kinase activity catalyzed a bona fide phosphorylation of the target protein, as deduced from the immunoinhibition of the in vitro phosphorylation reaction by the APS- IgG. In addition, the major, 103-kD PEPC polypeptide was also shown to be radiolabeled in situ 48 h after imbibition in [32P]orthophosphate. The ratio between optimal (pH 8) and suboptimal (pH 7.3 or 7.1) PEPC activity decreased during germination, thereby suggesting a change in catalytic rate related to an in vivo phosphorylation process. These collective data document that the components needed for the regulatory phosphorylation of PEPC are present and functional during germination of wheat seeds.

Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase (A Cardinal Event Influencing the Photosynthesis Rate in Sorghum and Maize).

C4 leaf phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is subject to a day/night regulatory phosphorylation cycle. By using the cytoplasmic protein synthesis inhibitor cycloheximide (CHX), we previously reported that the reversible in vivo light activation of the C4 PEPC protein-serine kinase requires protein synthesis. In the present leaf gas-exchange study, we have examined how and to what extent the CHX-induced inhibition of PEPC protein kinase activity/PEPC phosphorylation in the light influences C4 photosynthesis. Detached Sorghum vulgare and maize (Zea mays) leaves fed 10 [mu]M CHX showed a gradual but marked decrease in photosynthetic CO2 assimilation capacity. A series of control experiments designed to assess deleterious secondary effects of the inhibitor established that this reduction in C4 leaf CO2 assimilation was not due to (a) an increased stomatal resistance to CO2 diffusion, (b) a decrease in the activation state of other photoactivated C4 cycle enzymes, and (c) a perturbation of the Benson-Calvin C3 cycle, as evidenced by the absence of an inhibitory effect of CHX on leaf photosynthesis by a C3 grass (Triticum aestivum). It is notable that the CHX-induced decrease in CO2 assimilation by illuminated Sorghum leaves was highly correlated with a decrease in the apparent phosphorylation status of PEPC and a concomitant change in carbon isotope discrimination consistent with a shift from a C4 to a C3 mode of leaf CO2 fixation. These collective findings indicate that the light-dependent activation of the PEPC protein-serine kinase and the resulting phosphorylation of serine-8 or serine-15 in Sorghum or maize PEPC, respectively, are fundamental regulatory events that influence leaf C4 photosynthesis in vivo.

Structure, Regulation and Biosynthesis of Phosphoenolpyruvate Carboxylase from C4 Plants

This review attempts to summarize the large body of information on the structure, regulation and biosynthesis of the enzyme phosphoenolpyruvate carboxylase in C4 plants which has accumulated particularly since the appearance of the last review in 1987. Among the major discoveries are the involvement of protein phosphorylation-dephosphorylation cascade in the light activation of the enzyme, extraction and characteristics of PEPC-protein serine kinase, dynamic changes in oligomeric state of the enzyme in response to pH or temperature, isolation of multiple cDNAs encoding different forms of PEPC and cloning and expression of maize/sorghum PEPC in transgenic tobacco or transformed E. coli cells. Further experiments using advanced techniques of biochemistry and molecular biology would help in understanding the molecular mechanism of reaction, regulation of enzyme activity, gene expression and evolutionary pattern of C4 PEPC.