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.     

NaCl treatment markedly enhances H2O2-scavenging system in leaves of halophyte Suaeda salsa

The C₃ halophyte Suaeda salsa L. grown under the high concentration of NaCl (200 m M ) was used to investigate the role of the hydrogen peroxide (H₂O₂)-scavenging system [catalase, ascorbate peroxidase, glutathione reductase (GR), ascorbic acid, and glutathione (GSH)] in removal of reactive oxygen species. The activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and GR (EC 1.6.4.2) increased significantly after 7 days of NaCl treatment. The isoform patterns of CAT and GR were not affected, but the staining intensities were significantly increased by NaCl treatment. Activities of both the thylakoid-bound APX or GR and stromal APX (S-APX) or GR in the chloroplasts were markedly enhanced under high salinity. Fifty percent of APX in the chloroplasts is thylakoid-bound APX. S-APX and GR activity represented about 74–78 and 64–71% of the total soluble leaf APX and GR activity, respectively. Salt treatment increased the contents of ascorbic acid and GSH. By contrast, a decreased content of H₂O₂ was found in the leaves of NaCl-treated S . salsa . The level of membrane lipid peroxidation decreased slightly after NaCl treatment. The plants grew well with high rate of net photosynthesis under high salinity. These data suggest that upregulation of the H₂O₂-scavenging system in plant cells, especially in the chloroplasts, is at least one component of the tolerance adaptations of halophytes to high salinity.     

The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2

Changes in antioxidant metabolism because of the effect of salinity stress (0, 80, 160 or 240 m M NaCl) on protective enzyme activities under ambient (350 μmol mol⁻¹) and elevated (700 μmol mol⁻¹) CO₂ concentrations were investigated in two barley cultivars ( Hordeum vulgare L., cvs Alpha and Iranis). Electrolyte leakage, peroxidation, antioxidant enzyme activities [superoxide dismutase (SOD), EC 1.15.1.1; ascorbate peroxidase (APX), EC 1.11.1.11; catalase (CAT), EC 1.11.1.6; dehydroascorbate reductase (DHAR), EC 1.8.5.1; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; glutathione reductase (GR), EC 1.6.4.2] and their isoenzymatic profiles were determined. Under salinity and ambient CO₂, upregulation of antioxidant enzymes such as SOD, APX, CAT, DHAR and GR occurred. However, this upregulation was not enough to counteract all ROS formation as both ion leakage and lipid peroxidation came into play. The higher constitutive SOD and CAT activities together with a higher contribution of Cu,Zn-SOD 1 detected in Iranis might possibly contribute and make this cultivar more salt-tolerant than Alpha. Elevated CO₂ alone had no effect on the constitutive levels of antioxidant enzymes in Iranis, whereas in Alpha it induced an increase in SOD, CAT and MDHAR together with a decrease of DHAR and GR. Under combined conditions of elevated CO₂ and salinity the oxidative damage recorded was lower, above all in Alpha, together with a lower upregulation of the antioxidant system. So it can be concluded that elevated CO₂ mitigates the oxidative stress caused by salinity, involving lower ROS generation and a better maintenance of redox homeostasis as a consequence of higher assimilation rates and lower photorespiration, being the response dependent on the cultivar analysed.     

Salt-induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria

The effect in vivo of salt stress on the activated oxygen metabolism of mitochondria, was studied in leaves from two NaCl-treated cultivars of Pisum sativum L. with different sensitivity to NaCl. In mitochondria from NaCl-sensitive plants, salinity brought about a significant decrease of Mn-SOD (EC 1. 15. 1. 1) Cu, Zn-SOD I (EC 1. 15. 1. 1) and fumarase (EC 4. 2. 1. 2) activities. Conversely, in salt-tolerant plants NaCl treatment produced an increase in the mitochondrial Mn-SOD activity and, to a lesser extent, in fumarase activity. In mitochondria from both salt-treated cultivars, the internal H₂O₂ concentration remained unchanged. The NADH- and succinate-dependent generation of O₂^.−radicals by submitochondrial particles and the lipid peroxidation of mitochondrial membranes, increased as a result of salt treatment, and these changes were higher in NaCl-sensitive than in NaCl-tolerant plants. Accordingly, the enhanced rates of superoxide production by mitochondria from salt-sensitive plants were concomitant with a strong decrease in the mitochondrial Mn-SOD activity, whereas NaCl-tolerant plants appear to have a protection mechanism against salt-induced increased O₂^.− production by means of the induction of the mitochondrial Mn-SOD activity. These results indicate that in the subcellular toxicity of NaCl in pea plants, at the level of mitochondria, an oxidative stress mechanism mediated by superoxide radicals is involved, and also imply a function for mitochondrial Mn-SOD in the molecular mechanisms of plant tolerance to NaCl.     

Activity and properties of ribulose-1,5-bisphosphate carboxylase of sugarbeet plants grown under saline conditions

Activity and properties of sugar beet ( Beta vulgaris var. Polyrave) leaf ribulose-1,5-bisphosphate (RuBP) carboxylase were investigated following the exposure of plants to NaCl in the range of 45 to 270 m M for 7 days. An enhancement in RuBP carboxylase activity was found both in crude extracts and in purified preparations following plant exposure to 180 m M NaCl. Kinetic properties of the enzyme were significantly affected by salinity as determined by a 4.5 fold increase in K_m [HCO^-₃] and K_m [CO₂], and a V_max increase of 50%. Data based on polyacrylamide-gel-electrophoresis suggest that the molecular weight of the small subunit of RuBP carboxylase was reduced from 15,500 to 12,500 in plants grown under salinity. The large subunit was much less affected and no change was found in the whole enzyme. The enzyme isolated from plants exposed to salinity contained about 50% fewer titratable SH groups as compared with the control. The results indicate that in this plant, mild salt concentrations induced conformational changes in RuBP carboxylase which may be responsible for its tolerance to semi-salinity.     

Peroxidase and polyphenol oxidase activities in Cyperus esculentus leaves following glyphosate applications

Following a 2-week treatment with glyphosate [N-(phosphonomethyl)glycine] changes in peroxidase (EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.14.18.1) activities of yellow nutsedge ( Cyperus esculentus L.) plants, were determined. Glyphosate caused significant increases of both activities. Isoelectric focusing gave 3 species (F₁, F₂ and F₃) of peroxidase activity, at pl 3.8, 4.4 and 4.8, and 4 species (F_a, F_b, F_c and F_d) of PPO activity at pl 7.0, 7.5, 7.8 and 9.5. The activity of the 4 active forms of PPO increased with increasing glyphosate dose up to 10⁻² M . The effect of the herbicide on the 3 fractions with peroxidase activity was to change their relative activities. Highest F₁ activity was found in control plants whereas the F₂ fraction was the predominant form in the plants treated with glyphosate at 10⁻² M and the highest F₃ activity occurred in plants treated with 5 × 10⁻³ M glyphosate. The increased PPO activity could produce phytotoxic o -quinones, and variations in peroxidase isoenzymes activity could enhance isoperoxidases with lignin biosynthetic activity.     

Fermentation and malate metabolism in response to elevated CO2 concentrations in two strawberry cultivars

Concentrations of acetaldehyde, ethanol, ethyl acetate (EA), organic acids and activities and gene expression of alcohol dehydrogenase (ADH; EC 1.1.1.1), pyruvate decarboxylase (PDC; EC 4.1.1.1), alcohol acyltransferase (AAT; EC 1.4.1.14), malate dehydrogenase (MDH; EC 1.1.1.37), malic enzyme (ME; EC 1.1.1.40) and glutamate dehydrogenase (EC 1.4.1.14) were investigated in two strawberry ( Fragaria × ananassa Duch) cultivars with different responses to CO₂ during storage. 'Jewel' fruit treated with CO₂ accumulated acetaldehyde and ethanol but little EA, while 'Cavendish' accumulated little acetaldehyde or ethanol but accumulated EA. In CO₂-treated fruit, PDC activity was positively correlated with EA accumulation in 'Jewel' but not in 'Cavendish', while no differential effect of atmosphere was observed on its gene expression. ADH activity and gene expression show a correlation with ethanol accumulation in 'Cavendish'. In 'Jewel', there was a positive correlation between ADH gene expression and enzyme activity; however, this correlation does not explain ethanol accumulation in this cultivar. EA accumulation did not show any correlation with AAT activity and gene expression in any of the cultivars. Succinate concentrations were highest and those of malate lowest in CO₂-treated fruit of both cultivars, but MDH and ME activities were not affected by CO₂. Gene expression of MDH and ME were not affected by atmosphere in 'Cavendish', although in 'Jewel' the MDH expression was slightly lower in CO₂- than air-treated fruit. The results of this study show that differences in fermentation products and malate accumulation in CO₂-treated strawberry fruit are not consistently correlated with enzyme activities and gene expression.     

Salt induced responses of chloroplast activities in species of differing salt tolerance. Photosynthetic electron transport in Aster tripolium and Pisum sativum

A comparative study was made of the effects of high concentrations of NaCl, KCl and MgCl₂ on two electron transport reactions of thylakoids isolated from a mesophyte, Pisum sativum and a halophyte, Aster tripolium . The rate of photosystem I mediated electron transport from reduced N, N, N', N'-tetramethyl- p -phenylenediamine (TMPD) to methyl viologen was determined polarographically, and photosystem II mediated electron flow from water to 2,6-dichlorophenolindophenol (DCPIP) was monitored spectrophotometrically. The response of photosystem II to increasing in vitro salt concentrations was similar for thylakoids isolated from both A. tripolium and P. sativum , but differences in the response of photosystem I to salinity changes were observed for the two species. Increasing NaCl, KCl and MgCl₂ concentrations produced similar patterns of response of photosystem I activity in P. sativum thylakoids, whilst for A. tripolium KCl induced a completely different response pattern compared to NaCl and MgCl₂. The salinity of the culture medium in which A. tripolium was grown also had an effect on both the absolute in vitro activities of photosystems I and II and their response to changes in salt concentration of the reaction media.     

Kinetic changes with temperature of phosphoenolpyruvate carboxylase from a CAM plant

Abstract. Purified and crude phosphoenolpyruvate carboxylase from the CAM plant Kalanchoë daigremontiana Hamet et Perrier ( Bryophyllum diagremontianum ) was assayed at temperatures between 10 and 45° C. The optimum temperature of the enzyme activity changed with substrate availability and effector concentration in the assay. l -malate inhibited the enzyme activity and lowered the optimum temperature. Glucose-6-phosphate raised the optimum temperature to 43°C. K _m values for phosphoenolpyruvate increased with assay temperature from 0.12 mol m^-3 at 15° C to 0.36 mol^m−3 at 35° C. Inhibition by malate increased with temperature and acidity of the assay. In the crude enzyme 50% of control activity was inhibited by 1.65 mol m^-3 malate at 15° C and by 0.5 mol m^-3 at 35° C (at pH 7.0). With purification malate sensitivity was lost ( K _i values for malate at least 10 times higher). The shift in optimum temperatures for PEP-carboxylase activity thus results from changes in the kinetic parameters with temperature and allosteric effectors. The often low optimum temperatures for CO₂ fixation observed in nature may thus be the result of substrate and effector concentrations in the cytoplasm and the antagonistic effect of temperature on substrate affinity and effector efficiency on phosphoenolpyruvate carboxylase.     

Nitrate reductase and molybdenum cofactor in annual ryegrass as affected by salinity and nitrogen source

The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass ( Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m⁻¹, containing nitrogen (0.5 or 4.5 m M ) in the form of NH₄NO₃ or NaNO₃ Salinity-treated (11.2 dS m⁻¹) plants produced less biomass and more organic nitrogen while accumulating more NO⁻₃ than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO⁻₃ accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 m M NH₄NO₃ or NaNO₃. Similar effects were observed in roots of plants grown in 4.5 m M NaNO₃. Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors.     

Supplemental manganese improves the relative growth, net assimilation and photosynthetic rates of salt-stressed barley

Previous results in our laboratory indicated that a reduced Mn concentration in the leaves of barley was highly correlated with the reduced relative growth and net assimilation rates of salt-stressed plants. If Mn deficiency limits the growth of salt-stressed barley, then increasing leaf Mn concentrations should increase growth. In the present study, the effect of supplemental Mn on the growth of salt-stressed barley ( Hordeum vulgare L. cv. CM 72) was tested to determine if a salinity-induced Mn deficiency was limiting growth. Plants were salinized with 125 mol m⁻³ NaCl and 9.6 mol m⁻³ CaCl₂. Supplemental Mn was applied in 2 ways: 1) by increasing the Mn concentration in the solution culture and 2) by spraying Mn solutions directly onto the leaves. Growth was markedly inhibited at this salinity level. Dry matter production was increased 100% in salt-stressed plants treated with supplemental Mn to about 32% of the level of nonsalinized controls. The optimum solution culture concentration was 2.0 mmol m⁻³, and the optimum concentration applied to the leaves was 5.0 mol m⁻³. Supplemental Mn did not affect the growth of control plants. Further experiments showed that supplemental Mn increased Mn concentrations and uptake to the shoot. Supplemental Mn increased the relative growth rate of salt-stressed plants and this increase was attributed to an increase in the net assimilation rate; there were no significant effects on the leaf area ratio. Supplemental Mn also increased the net photosynthetic rate of salt-stressed plants. The data support the hypothesis that salinity induced a Mn deficiency in the shoot, which partially reduced photosynthetic rates and growth.     

Response of antioxidant systems to NaCl stress in the halophyte Cakile maritima

The effects of salt stress on antioxidative activities were investigated in a coastal halophyte, Cakile maritima . Two Tunisian accessions, Jerba and Tabarka, were compared. Plants were subjected to 100, 200, or 400 m M NaCl for 20 days. Parameters of oxidative stress [malondialdehyde (MDA), electrolyte leakage (EL), and hydrogen peroxide (H₂O₂) concentration], activities of several enzymes [superoxide dismutase (SOD), catalase (CAT), peroxydase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR)], and antioxidant molecules (ascorbate, ASC, and glutathione, GSH) were determined. Growth of Jerba plants was improved at 100 m M NaCl as compared to that of control. Tabarka growth was inhibited by salt at all NaCl concentrations. The relative salt tolerance of Jerba was associated with high antioxidant enzyme activities and glutathione content, together with low MDA content, EL, and H₂O₂ concentration. Lower antioxidant activities and higher MDA content, EL, and H₂O₂ concentration were found in Tabarka. As a whole, these data suggest that the capacity to limit oxidative damage is important for salt tolerance of C. maritima .     

Antioxidant enzyme activities in embryologic and early larval stages of turbot

The antioxidant enzymes superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6), selenium-dependent glutathione peroxidase (SeGPX; EC 1.11.1.9), glutathione reductase (EC 1.6.4.2) and DT-diaphorase (EC 1.6.99.2), plus total GPX activity (sum of SeGPX and Se-independent GPX activities), were studied in 13 500 g supernatants of embryos and 3-day and 11-day post-hatch larvae of turbot Scophthalmus maximus L. SOD activity decreased progressively during development from embryos to 11-day-old larvae, indicative of a decreased need to detoxify superoxide anion radical (O₂⁻). In contrast, catalase, SeGPX and glutathione reductase activities increased progressively from embryos to 11-day-old larvae, indicative of an increased need to metabolize hydrogen peroxide (H₂O₂) and organic peroxides. Consistent with the latter changes, levels of lipid peroxides (i.e. thiobarbituric acid reactive substances) increased 13-fold from embryos to 3-day-old larvae, whilst total peroxidizable lipid was indicated to decrease. Increases were seen for NADPH-dependent DT-diaphorase (after hatching) and total GPX (between 3 and 11 days post-hatch) activities, whilst no change was found in NADH-dependent DT-diaphorase activity. Overall, the results demonstrate a capacity for early life-stages of S. maximus to detoxify reactive oxygen species (O₂⁻ and H₂O₂) and other pro-oxidant compounds (organic peroxides, redox cycling chemicals). Furthermore, qualitative and quantitative antioxidant changes occur during hatching and development, possibly linked to such events as altered respiration rates (SOD changes) and tissue reorganization and development (catalase, SeGPX, lipid peroxidation).     

The Effect of Irradiance on Carboxylating/Decarboxylating Enzymes and Fumarase Activities in Mesembryanthemum crystallinum L. Exposed to Salinity Stress

Abstract: In Mesembryanthemum crystallinum plants, treated for 9 days with 0.4 M NaCl at low light intensities (80 - 90 or 95 - 100 μE m^-2 s^-1; λ = 400 - 700 nm), no day/night malate level differences (Δmalate) were detected. At high light (385 - 400 μE m^-2 s^-1) strong stimulation of PEPC activity, accompanied by a Δmalate of 11.3 mM, demonstrated the presence of CAM metabolism. This indicates that, to evolve day/night differences in malate concentration, high light is required. Salt treatment at low light induces and increases the activity of NAD- and NADP-malic enzymes by as much as 3.7- and 3.9-fold, while at high light these values reach 6.4- and 17.7-fold, respectively. The induction of activity of both malic enzymes and PEPC (phospo enol pyruvate carboxylase) take place before Δmalate is detectable. An increase in SOD (superoxide dismutase) was observed in plants cultivated at high light in both control and salt-treated plants. However, in salt-treated plants this effect was more pronounced. Carboxylating and decarboxylating enzymes seem to be induced by a combination of different signals, i.e., salt and light intensity. Plants performing CAM, after the decrease of activity of both the decarboxylating enzymes at the beginning of the light period, showed an increase in these enzymes in darkness when the malate pool reaches higher levels. In CAM plants the activity of fumarase (Krebs cycle) is much lower than that in C₃ plants. The role of mitochondria in CAM plants is discussed.     

Effects of chloride and sodium on gas exchange parameters and water relations of Citrus plants

Gas exchange parameters, water relations and Na⁺/Cl^- content were measured on leaves of one-year-old sweet orange ( Citrus sinensis [L.] Osbeck cv. Hamlin) seedlings grown at increasing levels of salinity. Different salts (NaCl, KCl and NaNO₃) were used to separate the effects of Cl⁻ and Na⁺ on the investigated parameters. The chloride salts reduced plant dry weight and increased defoliation. Accumulation of Cl⁻ in the leaf tissue caused a sharp reduction in photosynthesis and stomatal conductance. By contrast, these parameters were not affected by leaf Na⁺ concentrations of up to 478 m M in the tissue water. Leaf water potentials reached values near −1.8 MPa at high NaCl and KCl supplies. This reduction was offset by a decrease in the osmotic potential so that turgor was maintained at or above control values. The changes in osmotic potential were closely correlated with changes in leaf proline concentrations. Addition of Ca²⁺ (as calcium acetate) increased growth and halved defoliation of salt stressed plants. Furthermore, calcium acetate decreased the concentration of Cl⁻ and Na⁺ in the leaves, and increased photosynthesis and stomatal conductance. Calcium acetate also counteracted the reductions in leaf water and osmotic potentials induced by salinity. In addition, calcium acetate inhibited the accumulation of proline in the leaves which affected the reduction in osmotic potential. These results indicate that adverse effects of salinity in Citrus leaves are caused by accumulation of chloride.     

Changes in ethanol, lactate and malate content in Acer pseudoplatanus cells: effects of fusicoccin and O2 availability

Changes in the contents of ethanol, lactate and malate were determined at different activities of the plasma membrane H⁺ pump [in the presence and absence of fusicoccin (FC)] and at different O₂ availability in cultured cells of Acer pseudoplatanus L. FC induced acidification of the medium under all tested conditions of O₂ availability. At low O₂ concentrations both ethanolic and lactic fermentations occurred, and FC markedly stimulated lactate production but had no effect on ethanol production. There was also a small, stimulating effect of FC on malate production. At high O₂ concentrations no ethanol production was observed and lactate production was reduced. Under these conditions the stimulating effect of FC on lactate production decreased, while that on malate production increased. FC-induced synthesis of lactate and malate is interpreted as depending on the activation of lactate dehydrogenase (EC 1.1.1.27) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) (alkaline pH optima), respectively, due to the alkalinization of the cytoplasmic pH resulting from the stimulation of the H⁺ pump by FC. These results suggest that the balance between the two pH stat systems depends on the availability of O₂.     

Stomatal limitation of photosynthesis and reduced growth of the halophyte, Plantago maritima L., at high salinity

Abstract. Plantago maritima L. was grown at three levels of salinity, 50, 200, 350 mol m⁻³ NaCl, and the effects on growth, ion content and photosynthetic capacity were studied. Shoot and root dry weight, leaf production and leaf length were all substantially reduced in plants grown at high salinity. Total leaf area of plants grown at 350 mol m⁻³ NaCl was only 20% of that in plants at low salinity. Both the Na⁺ and K⁺ content of leaves and roots increased with external salinity. There was no change in the Na⁺/K⁺ ratio of leaves or roots at different salinity levels. Despite the large reductions in growth and high accumulation of Na⁺ ions, leaf photosynthetic rate was only slightly reduced by salinity stress. The reduction in photosynthesis was not caused by reduced biochemical capacity as judged by photosynthetic response to intercellular CO₂ and by ribulose-1,5-bisphosphate carboxylase activity, but was due to reduced leaf conductance and low intercellular CO₂ concentration. The increased stomatal limitation of photosynthesis resulted in higher water-use efficiency of plants grown at high salinity.     

Partial purification and characterization of stomatal phosphoenolpyruvate carboxylase from Vicia faba

Stomatal phosphoenolpyruvate carboxylase (PEPCase EC 4.1.1.31), extracted from abaxial epidermal peels of Vicia faba L. cv. Frühe Weiβkeimige, was partially purified by ammoniumsulfate precipitation, and molecular sieve (Sepharosc S-400) and ion exchange (DEAE-Sepharose) chromatography. The partially purified enzyme, essentially free of a PEPCase isoform existing in mesophyll and epidermal cells, had a specific activity of 300 nkat mg-¹ protein at 25°C. Western immunoblot analysis revealed that the stomatal enzyme had two bands (M_: of 110000 and 112000), crossreacting with PEPCase antibodies raised against PEPCase from Ka-lanchoe daigremontiana . The native molecular mass of the enzyme (467000) points to a tetrameric subunit structure. The temperature optimum was found to be 35°C; cold treatments of PEPCase before assaying were accompanied by inactivation. The energy of activation was calculated to 51 kJ mol-¹. The kinetic behaviour of the enzyme at fixed MgCl₂ concentrations is characterized by a pH optimum between pH 8.0–8.2 with or without 1 m M malate or 5 m M glucose-6-phosphate (Glc-6-P), but a combination of both effectors resulted in a shift of the optimum to pH 7.6. The enzyme showed a pH sensitive inhibition by 1 m M malate and an activation by Glc-6-P. At low pH (6–7), Glc-6-P was able to compensate for the malate induced inhibition of the enzyme. Malate and Glc-6-P both affected K_m(PEP), drastically and influenced V_max at pH 7, but not at pH 8.3. The inhibition constant of malate was determined to be 1.2 m M at pH 7. From the Dixon plot, a competitive inhibition of malate was assumed under defined assay conditions.     

Antioxidant defense in the leaves of C3 and C4 plants under salinity stress

The effect of salt stress (50, 100 and 150 m M of NaCl) on the activity of superoxide dismutase (SOD, EC. 1.15.1.1), ascorbate peroxidase (APX, EC. 1.11.1.11), glutathione reductase (GR, EC. 1.6.4.2) enzymes and also on the rate of lipid peroxidation in terms of thiobarbituric acid-reactive substances (TBARS) content and photosynthetic capacity in two wheat (C3 plants) and two maize (C4 plants) varieties was studied. In the non-salined control plants, the antioxidant enzymes activities were significantly higher for maize than for wheat. Adding salt to the nutrient solution increased the level of antioxidants in leaves of both maize and wheat. The first substantial response to salinity was found for SOD on the 2nd day, whereas changes occurred for APX on the 4th day and for GR on the 4th/5th day of salt treatment. Although SOD activity increased considerably more in wheat (C3), it never reached as high levels as in maize (C4) grown in the same treatment combinations. The total increase in APX activity was similar for wheat and maize, whereas GR activity was higher in leaves of maize. Lipid peroxidation analyses showed an increase in TBARS contents in both plants' species grown under salinity that corresponded to the damage that occurred in secondary oxidative stress. However, as a result of advanced antioxidant defense in maize, the TBARS quantities did not elevate to as high level as in wheat. Chlorophyll fluorescence measurements revealed a considerable decrease in the efficiency of PS II and electron-transport chain (ETC). Assimilation rate of CO₂ decreased in both plant groups; however, in C4 maize, we observed a much better capacity to preserve the photosynthetic apparatus against overproduction of ROS. Results suggest that efficient antioxidant defense plays an important role in maize, the C₄ plant, resistance to environmental stresses like salinity or drought.     

Changes in catalase activity and hydrogen peroxide concentration in plants in response to low temperature

Taxicity of oxygen species such as free radicals and H₂O₂ has been invoked to explain a number of degradative processes in plants, most involving photo-oxidation. Since catalase is a major protectant against accumulation and toxicity of H₂O₂, we examined alterations in catalase activity in several plant species ( Pisum sativum L. cv. Greenfeast, Vigna radiata (L.) R. Wilcz, Cucumis sativus L. cv. Heinz Pickling, and Passiflora spp.) during chilling, and compared this change to change in H₂O₂ content. Catalase activity was reduced in a range of chilling sensitive and tolerant species by exposure to low temperature. This reduction in catalase activity correlated better with the onset of visible symptoms than with the treatment itself. Visible injury in turn was dependent on light and temperature differences. Hydrogen peroxide concentrations invariably decreased with low temperatures.
Reduction in catalase activity therefore does not necessarily imply accumulation of H₂O₂ to damaging levels. The absence of a clear inverse relationship between catalase activity and H₂O₂ concentration suggests the continued activity of other reactions that remove H₂O₂ and these may be important in the tolerance of plants to oxidative attack. Loss of catalase activity may result from the inability of damaged peroxisomal membranes to transport catalase precursors into the peroxisome.