Kinetic Properties of Phosphoenolpyruvate Carboxylase in Peperomia camptotricha

Kinetic characteristics of phosphoenolpyruvate carboxylase (PEPC) from the epiphytic C₃ or C₄: CAM intermediate plant, Peperomia camptotricha, were investigated. Few day versus night differences in V_max,K_m(PEP)), or malate inhibition were observed, even in extracts from water-stressed plants which characteristically perform CAM, regardless of efforts to stabilize day/night forms. The PEPC extracted from plants during the light period remained stable, without much of an increase or decrease in activity for at least 22 hours at 0 to 4°C. Extracts from mature, fully developed leaves had slightly greater PEPC activity than from very young, developing leaves. Generally, however, the kinetic properties of PEPC extracted from mature leaves of plants grown under short day (SD), long day (LD), or 1-week water-stress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, l-malate, decreased the V_max and increased the K_m(PEP) for all treatments. Under specific conditions, malate did not inhibit PEPC rates in the dark extracts as much as the light. The PEPC activator, glucose-6-phosphate (G-6-P), lowered the K_m(PEP) for all treatments. At saturating PEP concentrations, PEPC activity was independent of pH in the range of 7.5 to 9.0. At subsaturating PEP concentrations, the pH optimum was 7.8. The rates of PEPC activity were lower in the light period extracts than the dark, at pH 7.1, but day/night PEPC was equally active at pH 7.8. At pH 7.5 and a subsaturating PEP concentration, G-6-P significantly activated PEPC. At pH 8, however, only slight activation by G-6-P was observed. The lower pH of 7.5 combined with l-malate addition, greatly inhibited PEPC, particularly in extracts from young, developing leaves which were completely inhibited at an l-malate concentration of 1 millimolar. However, malate did not further inhibit PEPC activity in mature leaves when assayed at pH 7.1. The fairly constant day/night kinetic and regulatory properties of PEPC from P. camptotricha are unlike those of PEPC from CAM or C₄ species studied, and are consistent with the photosynthetic metabolism of this plant.     

Light modulation of maize leaf phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH₄)₂SO₄ fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.     

A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C4 Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable1

We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C₄ phosphoenolpyruvate carboxylase (PEPC) by its Ca²⁺-insensitive protein-serine/threonine kinase (PEPC kinase) in the C₄ mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 μmol m⁻² s⁻¹ photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO₂-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.     

Characterization of Early Morning Crassulacean Acid Metabolism in Opuntia erinacea var Columbiana (Griffiths) L. Benson

The nature and sequence of metabolic events during phase II (early morning) Crassulacean acid metabolism in Opuntia erinacea var columbiana (Griffiths) L. Benson were characterized. Gas exchange measurements under 2 and 21% O₂ revealed increased O₂ inhibition of CO₂ fixation with progression of phase II. Malate and titratable acidity patterns indicated continued synthesis of C₄ acids for at least 30 minutes into the light period. Potential activities of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme exhibited little change during phase II, while light activation of NADP-malate dehydrogenase, pyruvate, orthophosphate dikinase, and ribulose-1,5-bisphosphate carboxylase was apparent. Short-term ¹⁴CO₂ fixation experiments showed that the per cent of ¹⁴C incorporated into C₄ acids decreased while incorporation into other metabolites increased with time. PEPC exhibited increased sensitivity to 2 millimolar malate, and the K_i(malate) for PEPC decreased markedly with time. Sensitivity of PEPC to malate inhibition was considerably greater at pH 7.5 than at 8.0. The results indicate that decarboxylation and synthesis of malate occur simultaneously during the early morning period, and that phase II acid metabolism is not limited by CO₂ diffusion through stomata. With progression of phase II, CO₂ fixation by PEPC decreases while fixation by ribulose-1,5-bisphosphate carboxylase increases.     

Posttranslational regulation of phosphoenolpyruvate carboxylase in c(4) and crassulacean Acid metabolism plants

Control of C₄ photosynthesis and Crassulacean acid metabolism (CAM) is, in part, mediated by the diel regulation of phosphoenolpyruvate carboxylase (PEPC) activity. The nature of this regulation of PEPC in the leaf cell cytoplasm of C₄ and CAM plants is both metabolite-related and posttranslational. Specificially, the regulatory properties of the enzyme vary in accord with the physiological activity of C₄ photosynthesis and CAM: PEPC is less sensitive to feedback inhibition by l-malate under light (C₄ plants) or at night (CAM plants) than in darkness (C₄) or during the day (CAM). While the view that a light-induced change in the aggregation state of the holoenzyme is a general mechanism for the diel regulation of PEPC activity in CAM plants is currently in dispute, there is no supportive in vivo evidence for such a tetramer/dimer interconversion in C₄ plants. In contrast, a wealth of in vitro and in vivo data has accumulated in support of the view that the reversible phosphorylation of a specific, N-terminal regulatory serine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize or sorghum enzymes, respectively) plays a key, if not cardinal, role in the posttranslational regulation of the carboxylase by light/dark or day/night transitions in both C₄ and CAM plants, respectively.     

Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants

Phosphoenolpyruvate carboxylase (PEPC) was overproduced in the leaves of rice plants by introducing the intact maize C₄-specific PEPC gene. Maize PEPC in transgenic rice leaves underwent activity regulation through protein phosphorylation in a manner similar to endogenous rice PEPC but contrary to that occurring in maize leaves, being downregulated in the light and upregulated in the dark. Compared with untransformed rice, the level of the substrate for PEPC (phosphoenolpyruvate) was slightly lower and the product (oxaloacetate) was slightly higher in transgenic rice, suggesting that maize PEPC was functioning even though it remained dephosphorylated and less active in the light. ¹⁴CO₂ labeling experiments indicated that maize PEPC did not contribute significantly to the photosynthetic CO₂ fixation of transgenic rice plants. Rather, it slightly lowered the CO₂ assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower O₂ concentrations. It was concluded that overproduction of PEPC does not directly affect photosynthesis significantly but it suppresses photosynthesis indirectly by stimulating respiration in the light. We also found that while the steady-state stomatal aperture remained unaffected over a wide range of humidity, the stomatal opening under non-steady-state conditions was destabilized in transgenic rice. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparative studies of phosphoenolpyruvate carboxylase from c(3) and c(4) plants

Phosphoenolpyruvate carboxylase (PEPC) from several C₃ plants was compared to maize PEPC by immunoblotting using an antibody against maize PEPC and by peptide mapping. In C₃ gramineous plants, PEPCs of slightly different monomeric sizes were detected as two bands for wheat and barley leaves, as three bands for etiolated maize leaves and as four bands for rice leaves by SDS-polyacrylamide gel electrophoresis and immunoblotting, whereas only one PEPC band was detected for maize leaves, a C₄ plant, or tobacco leaves, a dicotyledonous C₃ plant. The peptide fragment patterns of the lower molecular weight PEPC (major band in immunoblotting) in wheat leaves was similar to that of maize PEPC in peptide mapping by protein staining or by immunological detection, but the upper one (minor band) had a different pattern from the lower one in peptide mapping by immunological detection and few peptide fragments from this were recognized by the anti-(maize) PEPC antibody. These results suggest that there are multiple forms of PEPC subunits in the gramineous plants tested, and the major PEPC has a primary structure similar to that of maize PEPC. To obtain information about the expression of PEPCs in C₃ plants, changes in the amount of PEPC protein were investigated during the greening of rice and wheat seedlings. Judging from the regulation by light, there were two types of PEPCs in greening rice seedlings, one induced by light and the other reduced by it. Greening wheat seedlings also show a PEPC band induced by light. These findings indicate that some PEPCs in C₃ gramineous plants not only have structures similar to that of maize PEPC, but also are regulated by light in a similar manner.     

In vivo regulatory phosphorylation site in c(4)-leaf phosphoenolpyruvate carboxylase from maize and sorghum

Reversible seryl-phosphorylation contributes to the light/dark regulation of C₄-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malate-sensitivity of the target enzyme has been recently identified as Ser-15 in ³²P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [³²P]phosphopeptides were isolated and purified from in vivo ³²P-labeled maize and sorghum leaf PEPC and subjected to automated Edman degradation analysis. The results show that purified light-form maize PEPC contains 14-fold more ³²P-radioactivity than the corresponding dark-form enzyme on an equal protein basis and, more notably, only a single N-terminal serine residue (Ser-15 in maize PEPC and its structural homolog, Ser-8, in the sorghum enzyme) was found to be ³²P-phosphorylated in the light or dark. These in vivo observations, combined with the results from our previous in vitro phosphorylation studies (J-A Jiao, R Chollet [1989] Arch Biochem Biophys 269: 526-535; [1990] Arch Biochem Biophys 283: 300-305), demonstrate that an N-terminal seryl residue in C₄ PEPC is, indeed, the regulatory site that undergoes light/dark changes in phosphorylation-status and, thus, plays a major, if not cardinal role in the light-induced changes in catalytic and regulatory properties of this cytoplasmic C₄-photosynthesis enzyme in vivo.     

Stimulation by abscisic acid of the activity of phosphoenolpyruvate carboxylase in leaf disks of <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> L., C<Subscript>4</Subscript> 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 20 μM 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.     

Light activation of maize phosphoenolpyruvate carboxylase protein-serine kinase activity is inhibited by mesophyll and bundle sheath-directed photosynthesis inhibitors

C₄ phosphoenolpyruvate carboxylase (PEPC) is post-translationally regulated by reversible phosphorylation of a specific N-terminal seryl residue in response to light/dark transitions of the parent leaf tissue. The protein-serine kinase (PEPC-PK) that phosphorylates/activates this mesophyll-cytoplasm target enzyme is slowly, but strikingly, activated by high light and inactivated in darkness in vivo by a mechanism involving cytoplasmic protein synthesis/degradation as a primary component. In this report, evidence is presented indicating that the inhibition of Calvin cycle activity by a variety of mesophyll (3-(3,4-dichlorophenyl)-1,1-dimethylurea, isocil, methyl viologen) and bundle sheath (dl-glyceraldehyde)-directed photosynthesis inhibitors blocks the light activation of maize (Zea mays L.) PEPC-PK and the ensuing regulatory phosphorylation of its target enzyme in vivo. Based on these and related observations, we propose that the Calvin cycle supplies the C₄ mesophyll cell with (a) a putative signal (e.g. phosphorylated metabolite, amino acid) that interacts with the cytoplasmic protein synthesis event to effect the light activation of PEPC-PK and the concomitant phosphorylation of PEPC, and (b) high levels of known positive effectors (e.g. triose-phosphate, glucose-6-phosphate) that interact directly with the carboxylase. The combined result of this complex regulatory cascade is to effectively desensitize PEPC to feedback inhibition by the millimolar levels of l-malate required for rapid diffusive transport to the bundle sheath during high rates of C₄ photosynthesis.     

Relationship between NH(4) Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity : Regulation of Anaplerotic Carbon Flow in the Green Alga Selenastrum minutum

The rate of NH₄⁺ assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH₄⁺ assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H¹⁴CO⁻₃ into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C₃ organism.     

Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C₄ PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C₄ plant species. Neither antiserum reacted strongly with any C₃ leaf proteins. The molecular weight of the PEPC polypeptide from C₄ leaves and legume nodules appears to be similar.     

Patterns of phosphoenolpyruvate carboxylase activity and cytosolic pH during light activation and dark deactivation in C3 and C4 plants

The rate and extent of light activation of PEPC may be used as another criterion to distinguish C₃ and C₄ plants. Light stimulated phosphoenolypyruvate carboxylase (PEPC) in leaf discs of C₄ plants, the activity being three times greater than that in the dark but stimulation of PEPC was limited about 30% over the dark-control in C₃ species. The light activation of PEPC in leaves of C₃ plants was complete within 10 min, while maximum activation in C₄ plants required illumination for more than 20 min, indicating that the relative pace of PEPC activation was slower in C₄ plants than in C₃ plants. Similarly, the dark-deactivation of the enzyme was also slower in leaves of C₄ than in C₃ species. The extent of PEPC stimulation in the alkaline pH range indicated that the dark-adapted form of the C₄ enzyme is very sensitive to changes in pH. The pH of cytosol-enriched cell sap extracted from illuminated leaves of C₄ plants was more alkaline than that of dark-adapted leaves. The extent of such light-dependent alkalization of cell sap was three times higher in C₄ leaves than in C₃ plants. The course of light-induced alkalization and dark-acidification of cytosol-enriched cell sap was markedly similar to the pattern of light activation and dark-deactivation of PEPC in Alternanthera pungens, a C₄ plant. Our report provides preliminary evidence that the photoactivation of PEPC in C₄ plants may be mediated at least partially by the modulation of cytosolic pH.Abbreviations CAM Crassulacean acid metabolism - G-6-P glucose-6-phosphate - PMSF phenylmethylsulfonyl fluoride - PEPC phosphoenolpyruvate carboxylase - PEPC-PK phosphoenolpyruvate ca carboxylase-protein kinase     

Reduced Glutathione as an Effector of Phosphoenolpyruvate Carboxylase of the Crassulacean Acid Metabolism Plant Sedum praealtum D.C

Reduced glutathione, but not mercaptoethanol or dithiothreitol, inhibits phosphoenolpyruvate carboxylase (PEPC) in desalted leaf extracts from Sedum praealtum D.C. The inhibition is more evident at low pH values (< 7.2) and becomes increasingly smaller at higher pH. In the presence of the inhibitor, the hyperbolic rate curve of night PEPC is transformed to sigmoid and the S_0.5 is increased. When the enzyme is extracted during the day, the rate curve is sigmoid and it is not changed by the inhibitor, though the S_0.5 is further increased. Oxidized glutathione is completely inactive. Levels of reduced glutathione in leaf tissue are distinctly higher in the light. A role of photosynthetically reduced glutathione in the regulation of PEPC in Crassulacean acid metabolism species appears probable.     

Effect of drought stress on net CO2 uptake by Zea leaves

The net CO₂ assimilation by leaves of maize (Zea mays L. cv. Adonis) plants subjected to slow or rapid dehydration decreased without changes in the total extractable activities of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (ME). The phosphorylation state of PEPC extracted from leaves after 2–3 h of exposure to light was not affected by water deficit, either. Moreover, when plants which had been slowly dehydrated to a leaf relative water content of about 60% were rehydrated, the net CO₂ assimilation by leaves increased very rapidly without any changes in the activities of MDH, ME and PEPC or phosphorylation state of PEPC. The net CO₂-dependent O₂ evolution of a non-wilted leaf measured with an oxygen electrode decreased as CO₂ concentration increased and was totally inhibited when the CO₂ concentration was about 10%. Nevertheless, high CO₂ concentrations (5–10%) counteracted most of the inhibitory effect of water deficit that developed during a slow dehydration but only counteracted a little of the inhibitory effect that developed during a rapid dehydration. In contrast to what could be observed during a rapidly developing water deficit, inhibition of leaf photosynthesis by cis-abscisic acid could be alleviated by high CO₂ concentrations. These results indicate that the inhibition of leaf net CO₂ uptake brought about by water deficit is mainly due to stomatal closure when a maize plant is dehydrated slowly while it is mainly due to inhibition of non-stomatal processes when a plant is rapidly dehydrated. The photosynthetic apparatus of maize leaves appears to be as resistant to drought as that of C₃ plants. The non-stomatal inhibition observed in rapidly dehydrated leaves might be the result of either a down-regulation of the photosynthetic enzymes by changes in metabolite pool sizes or restricted plasmodesmatal transport between mesophyll and bundle-sheath cells.     

Over-expression of phosphoenolpyruvate carboxylase cDNA from C4 millet (Seteria italica) increase rice photosynthesis and yield under upland condition but not in wetland fields

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the initial fixation of CO₂ in C₄ plants. Under the control of the rice Rubisco small subunit promoter, cDNA of a C₄ SiPPC gene cloned from Seteria italica was introduced into Japonica rice by Agrobacterium-mediated transformation. Integration of the gene was confirmed by PCR analysis. RT-PCR showed expression of the gene at the RNA level in transgenic plants, and enzyme activity measurements confirmed the increase in PEPC protein. The transformants showed improvements in both photosynthesis rate and yield only under upland field cultivation. The possible function of PEPC in rice stress tolerance is discussed.     

Nitrogen enhanced photosynthesis of Miscanthus by increasing stomatal conductance and phosphoenolpyruvate carboxylase concentration

Miscanthus is one of the most promising bioenergy crops with high photosynthetic nitrogen-use efficiency (PNUE). It is unclear how nitrogen (N) influences the photosynthesis in Miscanthus. Among three Miscanthus genotypes, the net photosynthetic rate (P _N) under the different light intensity and CO₂ concentration was measured at three levels of N: 0, 100, and 200 kg ha^?1. The concentrations of chlorophyll, soluble protein, phosphoenolpyruvate carboxylase (PEPC), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, leaf anatomy and carbon isotope discrimination (Δ) in the leaf were analyzed to probe the response of photosynthesis in Miscanthus genotypes to N levels. P _N in all genotypes rose significantly as N application increased. The initial slope of response curves of P _N to C _i was promoted by N application in all genotypes. Both stomatal conductance and C _i were increased with increased N supply, indicating that stomatal factors played an important role in increasing P _N. At a given C _i, P _N in all genotypes was enhanced by N, implying that nonstomatal factors might also play an important role in increasing P _N. Miscanthus markedly regulated N investment into PEPC rather than the Rubisco large subunit under higher N conditions. Bundle sheath leakiness of CO₂ was constant at about 0.35 for all N levels. Therefore, N enhanced the photosynthesis of Miscanthus mainly by increasing stomatal conductance and PEPC concentration.