Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by ¹³C incorporation from [1-¹³C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. ¹³C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.     

deltaC Values in Maize Leaf Correlate with Phosphoenolpyruvate Carboxylase Levels

Values of δ¹³C and levels of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed in segments from the fourth leaf of young maize (Zea mays L.) plants. The δ¹³C values became significantly more negative from the base to the tip of the leaves. Phosphoenolpyruvate carboxylase levels and ribulose bisphosphate carboxylase levels both increased from the base to the tip. The principal effect of phosphoenolpyruvate carboxylase levels or δ¹³C should arise through its effect on the carboxylation/diffusion balance in the mesophyll. In this case, δ¹³C values should become more negative as phosphoenolpyruvate carboxylase levels increase, unless there are offsetting changes in stomatal aperture. The principal effect of ribulose bisphosphate carboxylase/oxygenase on δ¹³C should occur through its effect on the extent of leakage of CO₂ from the bundle sheath cells. In this case, δ¹³C values should become more positive as ribulose bisphosphate carboxylase levels increase. Accordingly, the variation in δ¹³C values seen in maize leaves appears to be the result of variations in the level of phosphoenolpyruvate carboxylase.     

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase

The effect of 5-5′-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V_max is found to be independent of the presence of this thiol reagent. The K_m is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K_m (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K_m for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.     

Control of gluconeogenesis by phosphoenolpyruvate carboxykinase in cotyledons ofCucurbita pepo L.

3-Mercaptopicolinic acid, a non-competitive inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.19) was used to study the control of gluconeogenesis by this enzyme in germinating marrow (Cucurbita pepo) cotyledons. In vitro, phosphoenolpyruvate carboxykinase was inhibited by 3-mercaptopicolinic acid, with aKi of 5.9 M. At 25°C the inhibitor caused an increase in the label incorporated from [2-¹⁴C]acetate into CO₂, and a decrease in the label incorporated into the insoluble and neutral fractions. Phosphoenolpyruvate carboxykinase had a flux control coefficient for gluconeogenesis (C _PEPCK ^J ) of between 0.7 and 1.0. 3-Mercaptopicolinic acid was a less effective inhibitor of phosphoenolpyruvate carboxykinase at lower temperatures (Ki = 8.6 M at 17°C, 13.3 M at 10°C) and had similar effects on the metabolism of [2-¹⁴C]acetate by marrow cotyledons when the temperature was reduced to 17°C and 10°C. The control coefficient for this enzyme did not change with temperature, indicating that phosphoenolpyruvate carboxykinase exerts a high degree of control over gluconeogenesis at all temperatures examined.Abbreviations PEP Phosphoenolpyruvate - PEPCK PEP carboxykinase The authors thank Dr. Ian Woodrow (University of Melbourne, Australia) for helpful discussions. This work was supported by a grant from the Science and Engineering Research Council, U.K. (GR/F 50978).     

Fluorescence Study of Chemical Modification of Phosphoenolpyruvate Carboxylase from Crassula argentea

The chemical modification of phosphoenolpyruvate carboxylase purified from Crassula argentea leaves was studied using the fluorescence of the extrinsic probe 8-anilino-1-naphalenesulfonate. The effects of ligands on kinetic parameters of phosphoenolpyruvate carboxylase activity, and its response to pH and metal cations, were associated with the binding of the ligands to the enzyme as measured by fluorescence. Binding of the ligands phosphoenolpyruvate, malate, and glucose-6-phosphate revealed by fluorescence measurements corresponds to competitive phenomena observed in kinetic studies. The fluorescence measurements also suggest the involvement of specific amino acids in the binding of a given ligand. Arginyl residues modified by 2,3-butanedione appear to be directly involved in the binding of phosphoenolpyruvate and malate to the active and the inhibition sites, respectively. A histidyl residue was involved in the binding of malate, accounting for the lack of inhibition by malate in kinetic studies of the enzyme treated with diethylpyrocarbonate. Although activity was lost, there was no decrease in the ability of the treated enzyme to bind phosphoenolpyruvate, suggesting that additional histidyl residues are essential for activity although not directly involved in the binding of phosphoenolpyruvate. The lysine reagent trinitrobenzenesulfonate caused a loss of activity and a reduction in malate inhibition and glucose-6-phosphate activation, but these modifications were not related to changes in the ability of the enzyme to bind any of the three ligands. This suggests that lysine residues were not directly involved in the binding of these ligands.     

A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium

A one step procedure is presented for the preparation of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using pyruvate kinase. The reaction is carried out at chemical equilibrium and involves only an exchange of isotope between ATP and phosphoenolpyruvate. The initial phosphoenolpyruvate/ATP ratio in the reaction mixture determines the degree of ³²P incorporation into phosphoenolpyruvate when isotopic equilibrium is achieved.     

Short-Term Metabolite Changes during Transient Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum

In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH₄⁺ addition we found that: (a) N accumulated in glutamine while glutamate and α-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH₄⁺ addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru-1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH₄⁺ addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme.     

Stress Induction of Mitochondrial Formate Dehydrogenase in Potato Leaves

The metabolism of [1-¹³C]glucose in Pisolithus tinctorius cv Coker & Couch, in uninoculated seedlings of Eucalyptus globulus bicostata ex Maiden cv Kirkp., and in the E. globulus-P. tinctorius ectomycorrhiza was studied using nuclear magnetic resonance spectroscopy. In roots of uninoculated seedlings, the ¹³C label was mainly incorporated into sucrose and glutamine. The ratio (¹³C3 + ¹³C2)/¹³C4 of glutamine was approximately 1.0 during the time-course experiment, indicating equivalent contributions of phosphoenolpyruvate carboxylase and pyruvate dehydrogenase to the production of α-ketoglutarate used for synthesis of this amino acid. In free-living P. tinctorius, most of the ¹³C label was incorporated into mannitol, trehalose, glutamine, and alanine, whereas arabitol, erythritol, and glutamate were weakly labeled. Amino acid biosynthesis was an important sink of assimilated ¹³C (43%), and anaplerotic CO₂ fixation contributed 42% of the C flux entering the Krebs cycle. In ectomycorrhizae, sucrose accumulation was decreased in the colonized roots compared with uninoculated control plants, whereas ¹³C incorporation into arabitol and erythritol was nearly 4-fold higher in the symbiotic mycelium than in the free-living fungus. It appears that fungal utilization of glucose in the symbiotic state is altered and oriented toward the synthesis of short-chain polyols.     

NO(3) Enhances the Kinase Activity for Phosphorylation of Phosphoenolpyruvate Carboxylase and Sucrose Phosphate Synthase Proteins in Wheat Leaves: Evidence from the Effects of Mannose and Okadaic Acid

The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO₃⁻ uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO₃⁻ leaves) or 40 mm NO₃⁻ solution (high-NO₃⁻ leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO₃⁻-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO₃⁻ and high-NO₃⁻ leaves, indicating that NO₃⁻ had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO₃⁻ compared with low NO₃⁻ leaves showed evidence that NO₃⁻ enhanced the protein kinase activity. These results support the concept that NO₃⁻, or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s).     

Isolation and characterization of the tricarboxylate transporter from pea mitochondria

The tricarboxylate transporter was solubilized from pea (Pisum sativum) mitochondria with Triton X-114, partially purified over a hydroxylapatite column, and reconstituted in phospholipid vesicles. The proteoliposomes exchanged external [¹⁴C]citrate for internal citrate or malate but not for preloaded d,l-isocitrate. Similarly, although external malate, succinate, and citrate competed with [¹⁴C]citrate in the exchange reaction, d,l-isocitrate and phosphoenolpyruvate did not. This tricarboxylate transporter differed from the equivalent activity from animal tissues in that it did not transport isocitrate and phosphoenolpyruvate. In addition, tricarboxylate transport in isolated plant mitochondria, as well as that measured with the partially purified and reconstituted transporter, was less active than the transporter isolated from animal tissues.     

Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [¹⁴C]Arg, [¹⁴C]Orn, and [¹⁴C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [¹⁴C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.     

The disposition of citric acid cycle intermediates by isolated rat heart mitochondria

The mechanism of depletion of tricarboxylic acid cycle intermediates by isolated rat heart mitochondria was studied using hydroxymalonate (an inhibitor of malic enzymes) and mercaptopicolinate (an inhibitor of phosphoenolpyruvate carboxykinase) as tools. Hydroxymalonate inhibited the respiration rate of isolated mitochondria in state 3 by 40% when 2 mM malate was the only external substrate, but no inhibition was found with 2 mM malate plus 0.5 mM pyruvate as substrates. In the prescence od bicarbonate, arsenite and ATP, propionate was converted to pyruvate and malate at the rates of 14.0 ± 2.9 and 2.8 ± 1.8 nmol/mg protein in 5 min, respectively. Under these conditions, 0.1 mM mercaptopicolinate did not affect this conversion, but 2 mM hydroxymalonate inhibited pyruvate formation completely and resulted in an accumulation of malate up to 13.2 ± 2.9 nmol/mg protein. No accumulation of phosphoenolpyruvate was found under any condition tested. It is concluded that malic enzymes but not phosphoenolpyruvate carboxykinase, are involved in conversion of propionate to pyruvate in isolated rat heart mitochondria.     

An in Vitro System from Maize Seedlings for Tryptophan-Independent Indole-3-Acetic Acid Biosynthesis

The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [¹⁴C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [¹⁴C]Trp nor [¹⁴C]serine substituted for [¹⁴C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.     

Glutamate metabolism in relation to glutamate transport in kidney cortex mitochondria of rabbit

1. The metabolism of glutamate was followed by measurements of phosphoenolpyruvate production, aspartate synthesis and ammonia release, whereas the transport of glutamate across the inner membrane of kidney cortex mitochondria was studied using an oxygen electrode and the swelling technique.2. When added separately, avenaciolide and aminooxyacetate only partially inhibited both State 3 and uncoupled respiration of the mitochondria, as studied in the presence of glutamate as substrate. In contrast, the addition of both inhibitors to the reaction medium resulted in an almost complete inhibition of glutamate oxidation.3. Swelling of kidney mitochondria in an isosmotic solution of ammonium glutamate was accelerated by uncoupler and inhibited by avenaciolide, while the swelling of mitochondria in potassium glutamate was stimulated by valinomycin and inhibited by uncoupler.4. When glutamate was used as the sole substrate, inhibition of aspartate formation by aminooxyacetate resulted in a stimulation of both ammonia release and phosphoenolpyruvate production. In contrast, with glutamate plus malate as substrate an elevation of the rate of glutamate deamination on the addition of aminooxyacetate was accompanied by an inhibition of phosphoenolpyruvate synthesis in both State 3 and uncoupled conditions.5. In the presence of valinomycin to induce K⁺-permeability a marked enhancement of glutamate deamination was accompanied by a significant inhibition of glutamate transamination.6. Based on the presented results it was concluded that in rabbit renal mitochondria utilizing glutamate as substrate the rates of ammonia production, phosphoenolpyruvate formation and aspartate synthesis vary in response to different metabolic conditions, in which both the glutamate—H⁺ symport and the glutamate—aspartate exchange systems are functioning to different extents.     

Assaying for pyruvate,orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.Abbreviations G-6-P glucose-6-phosphate - LDH lactate dehydrogenase - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone - PPDK pyruvate, orthophosphate dikinase - U unit of enzyme activity (mol/min)     

Inhibition of phosphoenolpyruvate carboxylase by malate

Malate has been noted to be a `mixed' inhibitor of phosphoenolpyruvate (PEP) carboxylase. The competitive portion of this inhibition appears to be fairly constant regardless of the condition of the enzyme being measured, but the noncompetitive (V-type) inhibition is subject to variation depending on the source of the enzyme, its storage condition, the presence or absence of various ligands, and differences in pH. In the case of the maize (Zea mays L.) phosphoenolpyruvate carboxylase (PEPC), the V-type inhibition by malate is much less pronounced at pH 8 than at pH 7. Examination of the response of the maize PEPC to PEP concentration reveals a pronounced cooperativity at pH 8 which is not present at pH 7, and which results in the disappearance of the V-type inhibition at pH 8. The ability of high concentrations of PEP to convert PEPC from a form readily inhibited by malate to one resistant to malate inhibition has been previously demonstrated and we attribute the cooperativity shown at pH 8 to this response to high levels of PEP. Support for this proposal is provided by studies of the enzyme at pH 7 and pH 8 run in 20% glycerol. In this case there was no V-type inhibition of PEPC at either pH. Treatment with 20% glycerol has been shown to result in the aggregation of maize PEPC.     

Enzymatic Combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from Nematoloma frowardii

The direct involvement of manganese peroxidase (MnP) in the mineralization of natural and xenobiotic compounds was evaluated. A broad spectrum of aromatic substances were partially mineralized by the MnP system of the white rot fungus Nematoloma frowardii. The cell-free MnP system partially converted several aromatic compounds, including [U-¹⁴C]pentachlorophenol ([U-¹⁴C]PCP), [U-¹⁴C]catechol, [U-¹⁴C]tyrosine, [U-¹⁴C]tryptophan, [4,5,9,10-¹⁴C]pyrene, and [ring U-¹⁴C]2-amino-4,6-dinitrotoluene ([¹⁴C]2-AmDNT), to ¹⁴CO₂. Mineralization was dependent on the ratio of MnP activity to concentration of reduced glutathione (thiol-mediated oxidation), a finding which was demonstrated by using [¹⁴C]2-AmDNT as an example. At [¹⁴C]2-AmDNT concentrations ranging from 2 to 120 μM, the amount of released ¹⁴CO₂ was directly proportional to the concentration of [¹⁴C]2-AmDNT. The formation of highly polar products was also observed with [¹⁴C]2-AmDNT and [U-¹⁴C]PCP; these products were probably low-molecular-weight carboxylic acids. Among the aliphatic compounds tested, glyoxalate was mineralized to the greatest extent. Eighty-six percent of the ¹⁴COOH-glyoxalate and 9% of the ¹⁴CHO-glyoxalate were converted to ¹⁴CO₂, indicating that decarboxylation reactions may be the final step in MnP-catalyzed mineralization. The extracellular enzymatic combustion catalyzed by MnP could represent an important pathway for the formation of carbon dioxide from recalcitrant xenobiotic compounds and may also have general significance in the overall biodegradation of resistant natural macromolecules, such as lignins and humic substances.     

Inactivation of maize phosphoenolpyruvate carboxylase by urea

Phosphoenolpyruvate carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 m urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 m urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of phosphoenolpyruvate carboxylase. However, the presence of 20 mm phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 m urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.     

Light Induction and the Effect of Nitrogen Status upon the Activity of Carbonic Anhydrase in Maize Leaves

The regulation of carbonic anhydrase (CA) activity in maize (Zea mays L.) leaves by light and nitrogen nutrition was determined. CA activity increased by more than 100-fold in illuminated leaves and decreased in leaves placed in the dark; low levels of CA activity were observed in leaves illuminated with low light intensities. CA activity was reduced in plants grown under nitrogen deficiency and recovered only slowly when supplemented with nitrate. Parallel studies were conducted to follow the levels of phosphoenolpyruvate carboxylase. Experiments indicate that the level of CA and phosphoenolpyruvate carboxylase present in leaves may be controlled by similar mechanisms.     

More Productive Than Maize in the Midwest: How Does Miscanthus Do It?

In the first side-by-side large-scale trials of these two C₄ crops in the U.S. Corn Belt, Miscanthus (Miscanthus × giganteus) was 59% more productive than grain maize (Zea mays). Total productivity is the product of the total solar radiation incident per unit land area and the efficiencies of light interception (ε_i) and its conversion into aboveground biomass (ε_ca). Averaged over two growing seasons, ε_ca did not differ, but ε_i was 61% higher for Miscanthus, which developed a leaf canopy earlier and maintained it later. The diurnal course of photosynthesis was measured on sunlit and shaded leaves of each species on 26 dates. The daily integral of leaf-level photosynthetic CO₂ uptake differed slightly when integrated across two growing seasons but was up to 60% higher in maize in mid-summer. The average leaf area of Miscanthus was double that of maize, with the result that calculated canopy photosynthesis was 44% higher in Miscanthus, corresponding closely to the biomass differences. To determine the basis of differences in mid-season leaf photosynthesis, light and CO₂ responses were analyzed to determine in vivo biochemical limitations. Maize had a higher maximum velocity of phosphoenolpyruvate carboxylation, velocity of phosphoenolpyruvate regeneration, light saturated rate of photosynthesis, and higher maximum quantum efficiency of CO₂ assimilation. These biochemical differences, however, were more than offset by the larger leaf area and its longer duration in Miscanthus. The results indicate that the full potential of C₄ photosynthetic productivity is not achieved by modern temperate maize cultivars.