NADP+-malate dehydrogenase in C3-plants: Regulation and role of a light-activated enzyme

The thioredoxin-dependent light/dark modulation system of the chloroplast is described as a prerequisite enabling the flexible control of fluxes through the various parts of the CO₂-fixation pathway. Both the rapid turnover of the reduced thiol-containing form of the respective target enzyme, and the metabolite effect upon the reductive enzyme modulation, allow rapid adjustment of the amount of active species to the actual requirements. The structural basis of the regulation of chloroplast NADP⁺-malate dehydrogenase (EC 1.1.1.82) is described in more detail. The modulable plastid enzyme is characterized by two sequence extensions not present in any other known NADP⁺- and/or NAD⁺-specific malate dehydrogenase. The NADP⁺-malate dehydrogenase of C₃-plants is part of the "malate valve", which catalyzes the export of reducing equivalents in the form of malate from the chloroplast only when the NADPH to NADP⁺ ratio is high, thus poising the NADPH to ATP ratio required for optimal carbon reduction in the light. The mode of regulation of other light/dark modulated enzymes is discussed.     

Differential effects of chilling on the activity of C4 enzymes in two ecotypes of Echinochloa crus-galli from sites of contrasting climates

Five-week-old plants of Echinochloa crusgalli (L.) Beauv. from Mississippi and from Québec grown under controlled conditions were subjected to dark chilling for 10 h at 5°C or light chilling treatments for 14 h at 7°C under hight light (1 000 μmol m⁻² s⁻¹). The activities of four C₄ enzymes of Québec plants, measured 4 h after the completion of the cold treatment, were not affected by the chilling treatment in the dark. The activities of pyruvate, P_i dikinase (PPDK; EC 2.7.9.1) and NADP⁺-malic enzyme (NADP⁺-ME; EC 1.1.1.40), were significantly reduced in dark-chilled Mississippi plants. Chilling under high light conditions elicited significant levels of reduction in the activities of the four enzymes from both ecotypes but the reductions were significantly less severe for Québec plants. The recovery of activities of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and PPDK for both ecotypes was completed within 36 to 60 hours following the chilling treatment, but NADP⁺-malate dehydro-genase (NADP⁺-MDH; EC 1.1.1.82) and NADP⁺-ME activities of chilled Mississippi plants remained below that of control plants at the end of the 5-day monitoring period. PPDK was inactivated in vitro at 0 and 10°C and the rates of cold inactivation were significantly higher for PPDK extracted from Mississippi plants. The activity of PEPC of Mississippi extracts was slightly, but significantly reduced by a 60 min treatment at 0°C.     

Peroxidase and catalase activity in leaves of Halimione portulacoides exposed to salinity

The effect of high NaCl concentrations on the activity of catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7) and malate dehydrogenase (NAD⁺-linked; EC 1.1.1.37) from leaves of Halimione portulacoides (L.) Aellen was studied. The plants were exposed to high salinity during growth and enzyme activity was measured either in the absence or in the presence of various concentrations of NaCl. Increasing salinity in vitro induced three types of effects: (1) an increase in activity (peroxidase); (2) a decrease in activity (catalase); (3) stimulation by low salt concentration but inhibition by higher concentrations (malate dehydrogenase). Salinity in vivo induced a marked decrease in catalase and malate dehydrogenase activities. However, peroxidase in vivo showed an optimum curve of activity vs external NaCl concentration, with an optimum at ca 1 M NaCl. Exposure of plants to salinity induced changes in the properties of the enzyme proteins: they precipitated at a higher (NH₄)₂SO₄ concentration, were eluted later during Sephadex G-200 filtration, and showed a shift in the maximal, minimal and optimal temperatures. These data are interpreted as evidence for conformational changes in the enzymes due to prolonged exposure to high salinity stress; such changes could be disruption into monomers (catalase and malate dehydrogenase), or changes in molecular shape (in the peroxidase).     

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [ND_in(NADPH)] in the oxidation of strictly NAD⁺-linked substrates by pea ( Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 μ M diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of ND_in(NADPH) during glycine oxidation (measured both as O₂ uptake and as CO₂ release) was 40–50 nmol mg⁻¹ protein min⁻¹. No significant activity of ND_in(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD⁺-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 μ M , DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, ND_in(NADPH), and ND_in(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD⁺ and NADP⁺, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.     

In vitro and in vivo regulation of chJoroplast glyceraldehyde-3-phosphate dehydrogenase isozymes from Chenopodium rubrum. III. The molecular basis of the aggregation phenomenon: chloroplast glyceraldehyde-3-phosphate dehydrogenase as an ambiquitous enzyme

The nature of the aggregated form of chloroplast glyceraldehyde-3-phosphate dehydrogenase isozymes (GPD, EC 1.2.1.13) from Chenopodium rubrum leaves was investigated. After disaggregation of the isozymes in NADP ⁺ buffer, and resuspension of the disaggregated isozymes in NAD⁺ buffer, complete reaggregation could only be achieved by remixing the enzyme with a high molecular weight fraction, from which the isozymes had dissociated during the NADP⁺ filtration. After separation of the isozymes by inverse ammonium sulphate gradient solubilization, spontaneous extensive reaggregation of each isozyme was observed in NAD⁺ buffer. The high molecular weight material consisted of ribonucleoprotein, and RNase treatment impaired its ability to promote reaggregation of chloroplast GPD. It is proposed that pyridine nucleotide-controlled aggregation and binding to ribonucleoprotein in vitro are artifacts which reflect an in situ binding to cellular components. Since uncontrolled NAD⁺-linked activities of the bifunctional isozymes in the chloroplast would lead to an equalization of the NAD ⁺ and NADP ⁺ redox couples, it is suggested that the reversible binding of the isozymes forms the basis of a regulatory system in vivo.     

The appearance of a new molecular species of phosphoenolpyruvate carboxylase (PEPC) and the rapid induction of CAM in Sedum telephium L.

Abstract. When detached leaves of Sedum telephium are incubated in the absence of water, a rapid switch from C₃ photosynthesis to CAM (as indicated by the onset of day-to-night fluctuations in titratable acidity. ΔH⁺) occurs within the first dark period. The C₃-CAM switch in intact plants occurs within 3 5d. Extractable activity of phospho enol pyruvate carboxylase (PEPC) increases five-fold in intact plants during CAM induction; however, during rapid CAM induction in detached leaves, there is only a very small increase in PEPC activity. Fractionation by anion exchange chromatography of crude extracts from leaves of intact plants subjected to water deficit shows that CAM induction is associated with the appearance of a molecular species of PEPC termed PEPC I. PEPC I is barely detectable in well-watered plants which are not performing CAM. The major form in these plants is termed PEPC II. In leaves from intact plants, there is a significant positive correlation between PEPC I activity and ΔH⁺ during a period of increasing water deficit. PEPC I exhibits day to night fluctuations in malate sensitivity, being less sensitive during the dark period. In contrast, PEPC II is more sensitive to inhibition by malate and has no day to night fluctuation in sensitivity. In detached leaves deprived of water, a small increase in PEPC I capacity is detected at the end of the first dark period (20 h after the start of treatment). The results suggest that PEPC I is required for attainment of maximum nocturnal malic acid synthesis. There is a significant correlation between leaf water status (relative water content), ΔH⁺, total PEPC and PEPC I activity suggesting that the internal water status of the plant may be a trigger for CAM induction. Abscisic acid applied to detached leaves does not cause nocturnal acidification.     

Coordination of photosynthetic and respiratory metabolism in Chlorella vulgaris UAM 101 in the light

In Chlorella vulgaris UAM 101, the presence of glucose altered the photosynthetic and respiratory metabolism in the light. When glucose was added to the growth medium, an increase in the cellular level of enzymes involved in glucose oxidation, namely glucose-6-P dehydrogenase (EC 1.1.1.49) and NAD⁺-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12), was observed. Glucose also enhanced respiratory O₂ consumption. In addition, CO₂ released by glucose oxidation was refixed in photosynthesis. The presence of glucose also affected photosynthesis. Phosphoribulokinase (EC 2.7.1.19) and NADP⁺-dependent glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13), two regulatory enzymes of the reductive pentose phosphate cycle, were increased by glucose. However, Rubisco (EC 4.1.1.39) activity of these cells was lower than that of autotrophic cells. Despite these alterations, the photosynthetic O₂ evolution was not significantly inhibited by glucose. On the other hand, an increase in the cytosolic NADP⁺-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.9) that is involved in obtaining reducing power for anabolic processes was observed. The CO₂ levels in the growth medium did not significantly affect the cellular level of enzymes measured in this work, except those involved in biosynthetic pathways. These data suggest that the effect of glucose on photosynthesis and respiration can be explained by alteration of the cellular level of photosynthetic enzymes and respiratory substrates, respectively.     

Modulation by weak bases or weak acids of the pH of cell sap and phosphoenolpyruvate carboxylase activity in leaf discs of C4 plants

When leaf discs of a C₄ species, Alternanthera pungens (L.) H.B. and K. or Amaranthus hypochondriacus L., were preincubated in 7.5 m M NH₄Cl, the pH of the cell sap increased by nearly 0.3 unit, while the activity of phosphoenolpyruvate carboxylase (PEPC) about doubled compared to the cell sap from control leaf discs (preincubated in 5 m M Tricine‐KOH, pH 8.5). The sensitivity of PEPC to L ‐malate (a feedback inhibitor) decreased marginally as a result of cytosolic alkalization. The pH of the cell sap and PEPC activity decreased by nearly 0.4 unit and 50%, respectively, when leaf discs were incubated in weak organic acids such as propionic, butyric or salicylic acid. Thus, our results demonstrate a marked modulation in vivo of cell sap pH and PEPC activity in leaf discs from C₄ plants by external alkalizing or acidifying reagents. The rise in PEPC activity due to alkalization of leaf discs was not sensitive to cycloheximide, implying that cytosolic protein synthesis was not involved in the activation of PEPC. Despite the marked increase in the PEPC activity due to the base‐loading of leaf discs, the change in malate sensitivity of the enzyme was only marginal, indicating that there was no significant increase in the extent of PEPC‐phosphorylation. Besides the physiological significance, the technique of acid/ base‐loading may be an important tool for studying the regulation of PEPC in leaf discs of C₄ species, since the activity of PEPC could be enhanced apparently without phosphorylation of the enzyme.     

Continuous measurement of oxaloacetate in purified mitochondria from the leaves of Kalanchoë blossfeldiana

A new method for the continuous assay of oxaloacetate released or taken up by plant mitochondria during malate oxidation is described. It is based on the continuous spectrophotometric recording of the reduction level of externally added NAD⁺ (0.4 m M ) to a mitochondrial preparation. In the presence of 20 m M malate and of externally added malate dehydrogenase (EC 1.1.1.37), an equilibrium is reached instantaneously, bringing about a partial reduction of NAD⁺ and the production of a proportional amount of oxaloacetate (OAA). Owing to the presence of a very active OAA carrier on the inner mitochondrial membrane, the concentration at the equilibrium position of the reactants of the external MDH is permanently displaced by the OAA released or taken up by the mitochondria. Therefore, changes in OAA concentration can be followed from the measurement of the reduction level of the external NAD⁺. This method appears as sensitive as the classical enzymatic method, but is much more rapid and requires much less mitochondrial protein. The proposed method was applied to Percoll-purified mitochondria from the leaves of a CAM plant, Kalanchoë blossfeldiana Poelln. cv. Tom Thumb. The simultaneous recording of the change in OAA concentration and of oxygen uptake during malate oxidation emphasizes the major control exerted by OAA on the rate of malate oxidation.     

Nitrogen fixation and carbon metabolism by nodules and bacteroids of pea plants under sodium chloride stress

Acetylene reduction activity (ARA) and leghemoglobin (Lb) content in nodules were sigificantly reduced when pea ( Pisum sativum L. cv. Lincoln) plants were subjected to 50 m M sodium chloride stress for 3 weeks. C₂H₂ reduction activity by bacteriods isolated from pea nodules was drastically inhibited by saline stress, and malate appeared to be a more appropriate substrate than glucose or succinate in maintaining this activity. Salt added directly to the incubation mixture of bacteriods or to the culture medium of plants inhibited O₂ uptake by bacteroids. Nodule cytosolic phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and bacteriod malate dehydrogenase (MDH; EC 1.1.1.37) activities were strongly enhanced by salt stress. Under these conditions, malate concentration was depressed in bacteroids and cytosol, whereas total soluble sugar (TSS)content slightly increased in both fractions. The effect of salt stress on TSS and malate content suggests that the utilization of carbohydrate within nodules could be inhibited during salt stress. The inhibitory effect of NaCl on N₂ fixation activity of bacteroids and to the decrease in bacteroid respiration. The stimulation of fermentative metabolism induced by salinity suggests some reduction in O₂ availability within the nodule. Salt stress was also responsible for a decrease of the cytosolic protein content, specifically of leghemoglobin, in the nodules.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis

Abstract The bradyzoite and tachyzoite forms of Toxoplasma gondii , purified from infected animals, were analysed for their activities of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NAD⁺- and NADH-linked isocitrate dehydrogenases, and succinic dehydrogenase. Both developmental stages contained high activities of phosphofructokinase (specific for pyrophosphate rather than ATP), pyruvate kinase and lactate dehydrogenase, suggesting that energy metabolism in both forms may centre around a high glycolytic flux linked to lactate production. The markedly higher activity of the latter two enzymes in bradyzoites suggests that lactate production is particularly important in this developmental form. NAD⁺-specific isocitrate dehydrogenase was not detectable in either stage of the parasite (and proved useful as a measure of the purity of the bradyzoite preparation), whereas both parasite forms contained low activities of NADP⁺-linked isocitrate dehydrogenase. The results are consistent with the bradyzoites lacking a functional TCA cycle and respiratory chain and are suggestive of a lack of susceptibility of this developmental stage to atovaquone.     

Capacity for hexose respiration in symbiotic Frankia from Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules containing a local source of Frankia by an improved homogenization-filtration procedure. The capacity of the vesicle clusters to metabolize hexoses was investigated by respirometric and enzymological studies. The vesicle clusters could utilize glucose, glucose-6-phosphate and 6-phosphogluconate provided that appropriate cofactors were added to the preparations. The enzymes hexokinase (EC 2.7.1.1), NADP⁺: glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and NAD⁺;6-phosphogluconate dehydrogenase (EC 1.1.1.44) were found in cell-free extracts of the vesicle clusters and kinetic constants for the enzymes were determined. Hexokinase had a lower K_m for glucose than for fructose. Extracts from both symbiotic and propionate grown Frankia AvcII also showed activity of these hexose-degrading enzymes, indicating that their presence is not necessarily dependent on sugars as carbon source. The NAD⁺- dependent 6-phosphogluconate dehydrogenase was only present in Frankia cells and not in alder root cells, which makes this enzyme a useful Frankia -specific marker in these symbiotic systems.     

Mitochondria from Zea mays leaf tissues: Differentiation of carbon assimilation and photorespiratory activity between mesophyll and bundle sheath cells

A procedure was developed to obtain intact and purified mitochondria from mesophyll and bundle sheath tissues of Zea mays L. cv. I.N.R.A. 180, an NADP⁺-malic enzyme type C₄ plant. There was little cross-contamination between the two mitochondrial fractions.
Both types of mitochondria oxidized NADH, succinate and malate with respiratory control. In mesophyll mitochondria malate oxidation was highly sensitive to KCN (85–90% inhibition of first state 3) and showed good respiratory control. In bundle sheath mitochondria malate oxidation was less sensitive to cyanide (75-80% inhibition) and showed poor respiratory control. Malate and NADH appeared to be the best substrates for respiratory activity. Mesophyil mitochondria could not oxidize glycine, whereas bundle sheath mitochondria could.
The results indicate that mesophyll and bundle sheath mitochondria of Zea mays are differentiated, not only with respect to the decarboxylation of malate but also with respect to the decarboxylation phase of photorespiration.     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

The redox levels and subcellular distribution of pyridine nucleotides in illuminated barley leaf protoplasts studied by rapid fractionation

The redox level and compartmentation of pyridine nucleotides was studied under photorespiratory and non-photorespiratory conditions using rapid fractionation of barley ( Hordeum vulgare L. cv. Gunilla, Svalöv) leaf protoplasts. From comparative measurements of the NADPH/NADP⁺ ratio and the ATP/ADP ratio one acidic and one alkaline extraction medium was chosen which quenched the metabolism very efficiently. The mitochondrial NADH/NAD⁺ was higher under photorespiratory conditions than under non-photorespiratory conditions. Aminoacetonitrile, an inhibitor of the photorespiratory conversion of glycine to serine, lowered the mitochondrial NADH/NAD⁺ ratio. This supports the hypothesis that glycine oxidation is coupled to oxidative phosphorylation to provide ATP to the cytosol. The chloroplastic NADPH/NADP⁺ as well as the NADH/NAD⁺ ratios were quite stable in saturating and limiting CO₂ as well as in the presence of aminoacetonitrile, although the triosephosphate/phosphoglycerate ratios changed. Thus, the redox level in the stroma seems to be tightly regulated.     

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O₂ limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O₂-limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO₂ fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O₂ regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.     

Activity of glyceraldehyde-3-phosphate dehydrogenase isozymes during photoperiodic floral induction in spinach leaves

The activity of NAD⁺ and NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase isozymes (EC 1.2.1.12 and EC 1.2.1.13, respectively) were measured in spinach ( Spinacia oleracea L. cv. Nobel) leaves grown under different photoperiodic treatments in order to discriminate between the early events of floral induction and processes related to acclimation. Glycolysis-linked isozyme activities were increased not only during floral induction and acclimation, but also during acclimation alone, suggesting that the changes in cytosolic activities were most probably associated with acclimation. In contrast, the chloroplast-linked isozyme activities only increased during flower induction and appeared to be specifically associated with the initiation of the flowering process. The relative activity changes in the chloroplast and cytosol compartments may thus be supposed to be among the first signs of translation of the photoperiodic signal into cytosolic and cellular metabolic adaptation, whereby the leaf moves rapidly into a new metabolic state.     

Seasonal differences in photosynthesis between the C3 and C4 subspecies of Alloteropsis semialata are offset by frost and drought

The regional abundance of C₄ grasses is strongly controlled by temperature, however, the role of precipitation is less clear. Progress in elucidating the direct effects of photosynthetic pathway on these climate relationships is hindered by the significant genetic divergence between major C₃ and C₄ grass lineages. We addressed this problem by examining seasonal climate responses of photosynthesis in Alloteropsis semialata , a unique grass species with both C₃ and C₄ subspecies. Experimental manipulation of rainfall in a common garden in South Africa tested the hypotheses that: (1) photosynthesis is greater in the C₄ than C₃ subspecies under high summer temperatures, but this pattern is reversed at low winter temperatures; and (2) the photosynthetic advantage of C₄ plants is enhanced during drought events. Measurements of leaf gas exchange over 2 years showed a significant photosynthetic advantage for the C₄ subspecies under irrigated conditions from spring through autumn. However, the C₄ leaves were killed by winter frost, while photosynthesis continued in the C₃ plants. Unexpectedly, the C₄ subspecies also lost its photosynthetic advantage during natural drought events, despite greater water-use efficiency under irrigated conditions. This study highlights previously unrecognized roles for climatic extremes in determining the ecological success of C₃ and C₄ grasses.     

The seven NAD(H)-glutamate dehydrogenase isoenzymes exhibit similar anabolic and catabolic activities

The specific activities of aminating NADH- and deaminating NAD⁺-glutamate dehydrogenase (GDH, EC 1.4.1.2) varied considerably in crude extracts of grapevine ( Vitis vinifera L. cv. Sultanina) callus and were dependent on the nitrogen source of the culture medium. However, dialysis of the enzyme preparations resulted in a significant decrease in the deaminating GDH specific activity while the aminating activity was not affected. The presence of malate in the crude extract resulted in erroneous overestimation of the NAD⁺-GDH activity through the malate dehydrogenase reaction. Thus, in dialysed extracts, the ratio of the NADH-GDH/NAD⁺-GDH specific activities remained relatively constant irrespective of the nitrogen source. In view of this evidence, we now have modified methods for staining both the NADH-GDH and NAD⁺-GDH activities on gels in order to compare the aminating and deaminating activities of each of the 7 GDH isoenzymes. The results from the staining of NADH-GDH and NAD⁺-GDH activity of enzyme preparations from calluses revealed the same isoenzyme profile. Furthermore, separated leaf isoenzymes showed similar activity ratios and kinetic properties. These results may suggest that each one of the 7 isoenzymes have similar in vitro anabolic and catabolic activities.