Enzyme regulation in C4 photosynthesis: Mechanism of activation of NADP-malate dehydrogenase by reduced thioredoxin

The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using ¹⁴C-iodoacetate and ¹⁴C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.     

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH₄)₂SO₄, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; K_m values at pH 8.5 are oxaloacetate, 18 μm; malate, 24 mm; NADPH, 50 μm; and NADP, 45 μm. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, K_i = 50 μm). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of M_r 38,000. Active enzyme is much more sensitive (>50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10² from the binding affinities for active NADP-malate dehydrogenase and 10⁴ for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.     

Regulation of Photosynthetic Electron Transport in Intact Spinach Chloroplasts: II. MECHANISM OF SALT-INDUCED INCREASE IN OXALOACETATE PHOTOREDUCTION

The main focus of this study was to determine the mechanism by which certain exogenous monovalent salts stimulate rates of net O₂ evolution linked to oxaloacetate reduction in intact spinach chloroplasts. The influence of salts on the dicarboxylate translocator involved in the transport of oxaloacetate and on the activity and activation of the chloroplast enzyme NADP-malate dehydrogenase, which mediates electron transport to oxaloacetate, was examined. High concentrations of KCl (155 millimolar) increased the apparent K_m for oxaloacetate but did not significantly alter the maximal velocity of uptake. Likewise, external salts (KCl, MgCl₂, or KH₂PO₄) had minimal effects on the magnitude of light activation of NADP-malate dehydrogenase. In contrast, measurements of chloroplast NADP-malate dehydrogenase activity (after release by osmotic shock) showed a marked dependence on salt concentration. Rates were stimulated approximately 2-fold by both monovalent (optimally 75 millimolar) and divalent (optimally 20 millimolar) salts. It was inferred that the salt-induced increase in net rates of O₂ evolution linked to oxaloacetate reduction is due, at least in part, to stimulation of NADP-malate dehydrogenase caused by monovalent cation permeability of the chloroplast inner envelope membrane.     

Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase

Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP⁺ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653–658) the total sequence is now known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713–722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme ss characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain.     

NADP-malate dehydrogenase from Zea mays leaves: Amino acid composition and thiol content of active and inactive forms

NADP-malate dehydrogenase (L-malate: NADP⁺ oxidoreductase, E.C. 1.1.1.82) was purified from the leaves of Zea mays L. and its subunit molecular weight, amino acid composition and the changes in number of thiol groups during activation were determined. The amino acid composition we found differed from that reported earlier for the Z. mays enzyme but was very similar to that reported for the enzyme isolated from pea leaves. The maize enzyme contains fewer methionine residues (3 compared to 5 in pea) but a greater total number of cysteine residues (6 compared to 3 in pea). In its inactive form (oxidised) the enzyme contained 2 thiols per subunit of which only 1 reacts with 5,5′-dithiobis(2-nitrobenzoic acid) when the enzyme is in its native form. During activation by dithiothreitol two disulphide bonds are reduced per subunit to give 4 new thiol groups. We conclude that NADP-malate dehydrogenase from leaves of the C₄ plant Z. mays is very similar to the enzyme from the C₃ plant pea. However, apparently two disulphide bonds are reduced during the reductive activation of the Z. mays enzyme in vitro compared with 1 disulphide bond for the pea enzyme.     

THE RELATIVE SIGNIFICANCE OF CO2-FIXING ENZYMES IN THE METABOLISM OF RAT BRAIN

To evaluate the relative significance of CO₂-fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase, as well as the fixation of H¹⁴CO₃^? by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol-pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H¹⁴CO₃^? at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H¹⁴CO₃^? in the presence of α-ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H¹⁴CO₃^? at a rate of about 7 nmol/g of tissue/min and it was increased by two-fold when pyruvate was added to the incubation medium. The carboxylation of α-ketoglutarate and pyruvate by the reversal of the cytosolic NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO₂-fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α-ketoglutarate by a sequential action of aconitate hydratase and NADP-isocitrate dehydrogenase. The operation of the citrate-cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO₂-fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO₂-fixation in the brain.     

Gas exchange of two CAM species of the genus Cissus (vitaceae) differing in morphological features

NADP-malate dehydrogenase extracted from darkened leaves of the C₃ plants pea, barley, wheat and spinach was activated by reduced glutathione, a monothiol, as well as by dithiothreitol (DTT). However, in the C₄ plants maize and Flaveria trinervia, only dithiothreitol could effectively activate the enzyme. There was no activation of the maize enzyme and little or no activation of the F. trinervia enzyme by glutathione. The failure of glutathione to activate NADP-MDH in leaf extracts of maize and F. trinervia may indicate there is some difference in disulfide groups of the protein compared to the C₃ plant enzyme. Both DTT and glutathione could activate NADP-malate dehydrogenase in a partially purified enzyme preparation from pea leaves with or without addition of partially purified thioredoxin. However, the required concentration of reductant was lower with addition of thioredoxin than in its absence. In extracts of C₃ species and the partially purified pea enzyme the level of activation after 40 to 60 min under aerobic conditions was higher (up to twofold) with DTT than with glutathione. Under anaerobic conditions, the initial rate of activation was about twice as high with DTT as with glutathione, but the total activation after 40 to 60 min was similar. Ascorbate was totally ineffective as a reducing agent in activating NADP-MDH from C₃ or C₄ plants, possibly due to its more positive redox potential.Abbreviations Chl Chlorophyll - DTT Dithiothreitol - GSH Reduced Glutathione - NADP-MDH NADP-malate Dehydrogenase     

Plants under Climatic Stress: VI. Chilling and Light Effects on Photosynthetic Enzymes of Sorghum and Maize

The activity of several photosynthetic enzymes was unaltered by exposure of sorghum or maize to low temperatures (10 C) and light (170 w m⁻²). Two light-activated C₄-pathway enzymes, NADP-malate dehydrogenase and pyruvate Pi dikinase, were reduced in activity, and this was largely attributable to a loss of enzyme rather than to incomplete enzyme activation. Loss of NADP-malate dehydrogenase was more marked in sorghum than in maize, and in both species no loss occurred at 10 C when light levels were reduced from 170 to 50 w m⁻². A light-dependent, low temperature-induced loss of catalase activity was also observed in maize leaves.     

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O₂ was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O₂ was only slightly lower than that at 21% O₂. The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O₂.

The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17°C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32°C and 45 minutes at 17°C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32°C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

     

Regulation of C4 photosynthesis: characterization of a protein factor mediating the activation and inactivation of NADP-malate dehydrogenase.

The leaf NADP-malate dehydrogenase of Zea mays is rapidly activated when leaves are illuminated and inactivated in the dark. The present studies show that inactive enzyme isolated from darkened leaves was activated by dithiothreitol and that the active enzyme was rapidly inactivated by oxygen in dithiothreitol-free solutions. Following the fractionation of leaf extracts, both the activation and inactivation of NADP-malate dehydrogenase in vitro were partially or totally dependent upon a separate small molecular weight protein factor. Activation and inactivation were largely or solely dependent upon this factor at pH 8.0 or less, but apparently only partially factor dependent at pH 9.0. The factor was heat stable, inactivated by incubation with trypsin, and had a molecular weight of about 10,000. It was mostly associated with the chloroplasts of mesophyll cells.     

Relationship between Photosynthetic Electron Transport and Stromal Enzyme Activity in Pea Leaves : Toward an Understanding of the Nature of Photosynthetic Control

The responses of the quantum efficiencies of photosystem (PS) II and PSI measured in vivo simultaneously with estimations of the activities and activation states of NADP-malate dehydrogenase, chloroplast fructose-1,6-bisphosphatase, and ribulose-1,5-bisphosphate carboxylase were used to study the relationship between electron transport and carbon metabolism. The effects of varying irradiance and CO₂ partial pressure on the relationship between the quantum efficiencies of PSI and II, and the activity of these enzymes shows that the interrelationships vary according to the limitations placed on the system. The relationship between the quantum efficiencies of PSII and PSI was linear in most situations. In response to increasing irradiance, the activity of all three enzymes increased. In the case of NADP-malate dehydrogenase this increase was well correlated with the estimated flux of electrons through PSI and PSII. The other two enzymes showed a more complex relationship with the estimated flux of electrons through both photosystems. These relationships are consistent with the known interactions between these stromal enzymes and the thylakoids. The response to varying CO₂ partial pressure is more complex. The efficiencies of PSI and II declined with decreasing CO₂ partial pressure and the activity of each enzyme varied uniquely. However, there are clear correlations between the activities of the enzymes and the flux of electrons through the photosystems. In contrast to the data obtained under conditions of varying irradiance, there is clear evidence of photosynthetic control of electron transport when the CO₂ concentration is varied.     

Enzyme regulation in C4 photosynthesis: purification, properties, and activities of thioredoxins from C4 and C3 plants

Procedures are described for the purification to homogeneity of chloroplast thioredoxins f and m from leaves of corn (Zea mays, a C4 plant) and spinach (Spinacea oleracea, a C3 plant). The C3 and C4f thioredoxins were similar immunologically and biochemically, but differed in certain of their physiochemical properties. The f thioredoxins from the two species were capable of activating both NADP-malate dehydrogenase (EC 1.1.1.37) and fructose-1,6-bisphosphatase (EC 3.1.3.11) when tested in standard thioredoxin assays. Relative to its spinach counterpart, corn thioredoxin f showed a greater molecular mass (15.0-16.0 kDa vs 10.5 kDa), lower isoelectric point (ca. 5.2 vs 6.0), and lower ability to form a stable noncovalent complex with its target fructose bisphosphatase enzyme. The C3 and C4 m thioredoxins were similar in their specificity (ability to activate NADP-malate dehydrogenase, and not fructose-1,6-bisphosphatase) and isoelectric points (ca. 4.8), but differed slightly in molecular mass (13.0 kDa for spinach vs 13.5 kDa for corn) and substantially in their immunological properties. Results obtained in conjunction with these studies demonstrated that the thioredoxin m-linked activation of NADP-malate dehydrogenase in selectively enhanced by the presence of halide ions (e.g., chloride) and by an organic solvent (e.g., 2-propanol). The results suggest that in vivo NADP-malate dehydrogenase interacts with thylakoid membranes and is regulated to a greater extent by thioredoxin m than thioredoxin f.     

Relationships between the Efficiencies of Photosystems I and II and Stromal Redox State in CO(2)-Free Air : Evidence for Cyclic Electron Flow in Vivo

The responses of the efficiencies of photosystems I and II, stromal redox state (as indicated by NADP-malate dehydrogenase activation state), and activation of the Benson-Calvin cycle enzymes ribulose 1,5-bisphosphate carboxylase and fructose 1,6-bisphosphatase to varying irradiance were measured in pea (Pisum sativum L.) leaves operating close to the CO₂ compensation point. A comparison of the relationships among these parameters obtained from leaves in air was made with those obtained when the leaves were maintained in air from which the CO₂ had been removed. P700 was more oxidized at any measured irradiance in CO₂-free air than in air. The relationship between the quantum efficiencies of the photosystems in CO₂-free air was distinctly curvilinear in contrast to the predominantly linear relationship obtained with leaves in air. This nonlinearity may be consistent with the operation of cyclic electron flow around photosystem I because the quantum efficiency of photosystem II was much more restricted than the quantum efficiency of photosystem I. In CO₂-free air, measured NADP-malate dehydrogenase activities varied considerably at low irradiances. However, at high irradiance the activity of the enzyme was low, implying that the stroma was oxidized. In contrast, fructose-1,6-bisphosphatase activities tended to increase with increasing electron flux through the photosystems. Ribulose-1,5-bisphosphate carboxylase activity remained relatively constant with respect to irradiance in CO₂-free air, with an activation state 50% of maximum. We conclude that, at the CO₂ compensation point and high irradiance, low redox states are favored and that cyclic electron flow may be substantial. These two features may be the requirements necessary to trigger and maintain the dissipative processes in the thylakoid membrane.     

Modulation of NADP-Malate Dehydrogenase Activity in Maize Mesophyll Chloroplasts

When intact mesophyll chloroplasts of Zea mays var Kelvedon Glory were illuminated, activation of NADP-malate dehydrogenase occurred. Activity declined rapidly on darkening. Light activation of the enzyme was very much greater in the presence of pyruvate (~10- to 20-fold) than with the electron acceptors 3-phosphoglycerate or oxaloacetate present (~2-fold). Following preillumination in the presence of pyruvate, addition of 3-phosphoglycerate, oxaloacetate, or nitrite substantially diminished the activity of NADP-malate dehydrogenase. In these circumstances, with pyruvate and 3-phosphoglycerate present, activity could be restored by the addition of nigericin or dihydroxyacetone phosphate. Nigericin also restored activity with both oxaloacetate and pyruvate present. The effect of nitrite was more marked in the presence of low concentrations of DCMU.

These observations are discussed in terms of the dependence of enzyme activity upon the redox state of ferredoxin and electron carriers; the redox state of the latter was estimated by analysis of the DCMU-induced relaxation kinetics of chlorophyll fluorescence quenching in the presence of different substrates.

     

Properties and regulation of leaf nicotinamide–adenine dinucleotide phosphate–malate dehydrogenase and `malic'' enzyme in plants with the C4-dicarboxylic acid pathway of photosynthesis

1. NADP-malate dehydrogenase and ;malic' enzyme in maize leaf extracts were separated from NAD-malate dehydrogenase and their properties were examined. 2. The NADP-malate dehydrogenase was nicotinamide nucleotide-specific but otherwise catalysed a reaction comparable with that with the NAD-specific enzyme. By contrast with the latter enzyme, a thiol was absolutely essential for maintaining the activity of the NADP-malate dehydrogenase, and the initial velocity in the direction of malate formation, relative to the reverse direction, was faster. 3. For the ;malic' enzyme reaction the K(m) for malate was dependent on pH and the pH optimum varied with the malate concentration. At their respective optimum concentrations the maximum velocity for this enzyme was higher with Mg(2+) than with Mn(2+). 4. The NADP-malate dehydrogenase in green leaves was rapidly inactivated in the dark and was reactivated when plants were illuminated. Reactivation of the enzyme extracted from darkened leaves was achieved simply by adding a thiol compound. 5. The activity of both enzymes was low in etiolated leaves of maize plants grown in the dark but increased 10-20-fold, together with chlorophyll, when leaves were illuminated. 6. The activity of these enzymes in different species with the C(4)-dicarboxylic acid pathway was compared and their possible role in photosynthesis was considered.     

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxin_m and _f (Td_m, Td_f) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m^–2 under nitrogen and 50 W·m^–2 under air, while NADP photoreduction was saturated at 240 W·m^–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N₂ by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O₂ was located at the level of Fd.Abbreviations Fd ferredoxin - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - LEM light effect mediator - NADP-MDH NADP-malate dehydrogenase - Td thioredoxin     

Subcellular Localization of Oxaloacetate Decarboxylase and Its Isolation from Maize Leaves

The activity of oxaloacetate decarboxylase was revealed in leaves of a C₄ plant, maize (Zea mays L.). This activity was unrelated to decarboxylase activities of other enzymes, e.g., NAD-malate dehydrogenase (EC 1.1.1.38) or NADP-malate dehydrogenase (EC 1.1.1.40), and was located in chloroplasts (83.1%). Using a four-step purification procedure, an electrophoretically pure enzyme preparation of oxaloacetate decarboxylase was obtained from maize leaves. The specific activity of the enzyme was 3.150 EU/mg protein, the factor of purification was 40.4, and the yield was 11.0%. The enzyme exhibited Michaelis–Menten kinetics with K _m for oxaloacetate 30 ± 5 M and pH optimum 7.1 ± 0.5. The metabolite-mediated regulation of oxaloacetate decarboxylase activity has been investigated. It is found that sodium chloride (1.0 mM) activates the enzyme, whereas ATP inhibits the enzyme activity.     

Anatomical and enzymic studies of leaves of a C3 × C4Flaveria F1 hybrid exhibiting reduced photorespiration

Flaveria pringlei exhibits C₃ CO₂ compensation concentration (Г) values averaging 53 μl CO₂/l at 21% (v/v) O₂ and 25 ± 2°C. When this species is hybridized with the C₄ species, F. brownii (male) ($\text{Г = 6 μ}\text{l CO}{}_2\text{/}\text{l}$), the F₁ hybrid plants exhibit an average Г value of 31 μl CO₂/l at 21% O₂.Although light micrographs of leaf cross-sections show that the leaves of the hybrid plants possess the mesophyll arrangement characteristic of F. pringlei leaves, the hybrid plants have some bundle-sheath chloroplasts. However, the numbers of these organelles do not appear to be intermediate with respect to the numbers in the parents and are closest to the small number present in the bundle-sheath cells of F. pringlei leaves. The activities of key C₄ enzymes (in μmol · mg Chl^?1 · h^?1) are: phosphoenolpyruvate (PEP) carboxylase, 121; pyruvate, orthophosphate (P_i) dikinase, 26; NADP-malate dehydrogenase, 2529; and NADP-malic enzyme, 82. All of these activities are substantially higher than in F. pringlei, but are only 7–10% of those in F. brownii (with the exception of the NADP-malate dehydrogenase activity). These data suggest that a C₄ cycle might be operating to a limited extent in the hybrid plants resulting in reduced photorespiration.Whether or not C₄ photosynthesis occurs in these hybrid plants, they represent the first reported C₃ × C₄ F₁ hybrids to exhibit reduced Γ-values. This cross and its reciprocal should be useful models for studying the anatomical and biochemical factors determining the development of limited C₄ photosynthesis in C₃ species.     

Haloxyfop Inhibition of the Pyruvate and the alpha-Ketoglutarate Dehydrogenase Complexes of Corn (Zea mays L.) and Soybean (Glycine max [L.] Merr.)

The grass-specific herbicide haloxyfop, ((±)-2-[4-((3-chloro-5-(trifluoromethyl)-2-pyridinyl)oxy)-phenoxy] propionic acid) has been shown to inhibit lipid synthesis and respiration, to cause the accumulation of amino acids, and not to affect cellular sugar or ATP levels. Thus studies were carried out with enzyme activities from corn (Zea mays L.) (haloxyfop sensitive) and soybean (Glycine max [L.] Merr.) (haloxyfop tolerant) to locate the possible inhibition sites among the glycolytic and tricarboxylic acid (TCA) cycle enzymes. Following along the oxidative metabolism pathway of sugars, the pyruvate dehydrogenase complex (PDC) was the first enzyme among the glycolytic enzymes that demonstrated noticeable inhibition by 1 millimolar haloxyfop. Kinetic studies with corn and soybean PDC from both purified etioplasts and mitochondria gave K_i values of from 1 to 10 millimolar. Haloxyfop also inhibited the activity of the TCA cycle enzyme, the α-ketoglutarate dehydrogenase complex (α-KGDC) which carries out the same reaction as PDC except for the substitution of α-ketoglutarate for pyruvate as one of the substrates. The K_i values were somewhat lower in this case (near 1 millimolar). The relatively high K_i values for both enzyme complexes would indicate that these may not be the herbicidal sites of inhibition, but it is possible that the herbicide could be concentrated in compartments and/or the substrate concentrations may be well below optimal. Likewise little difference was seen in the haloxyfop inhibition of the enzyme activities from the sensitive species, corn, and from the tolerant species, soybean, so the selectivity of the herbicide is not evident from these results. The inhibition of the PDC and α-KGDC as the mode of action of haloxyfop is, however, consistent with the observed physiological effects of the herbicide, and these are the only enzymic activities so far found to be sensitive to haloxyfop.     

Regulation of C4 photosynthesis: regulation of activation and inactivation of NADP-malate dehydrogenase by NADP and NADPH

Inactive NADP-malate dehydrogenase (disulfide form) from chloroplasts of Zea mays is activated by reduced thioredoxin while the active enzyme (dithiol form) is inactivated by incubation with oxidized thioredoxin. This reductive activation of NADP-malate dehydrogenase is inhibited by over 95% in the presence of NADP and the Kd for this interaction of NADP with the inactive enzyme is about 3 microM. Other substrates of the enzyme (malate, oxaloacetate, or NADPH) do not effect the rate of enzyme activation but NADPH can reverse the inhibitory effect of NADP. It appears that NADPH (Kd = 250 microM) and NADP (Kd = 3 microM) compete for the same site, presumably the coenzyme-binding site at the active centre. Apparently the enzyme . NADP binary complex cannot be reduced by thioredoxin whereas the enzyme . NADPH complex is reduced at the same rate as is the free enzyme. Similarly the oxidative inactivation of reduced NADP-malate dehydrogenase is inhibited by up to 85% by NADP and NADPH completely reverses this inhibition. The Kd values of the active-reduced enzyme for NADP and NADPH were both estimated to be 30 microM. From these data a model was constructed which predicts how changing NADPH/NADP levels in the chloroplast might change the steady-state level of NADP-malate dehydrogenase activity. The model indicates that at any fixed ratio of reduced to oxidized thioredoxin high proportions of active NADP-malate dehydrogenase and, hence, high rates of oxaloacetate reduction, can only occur with very high NADPH/NADP ratios.