Antioxidant status of the potato tuber and Ca2+ deficiency as a physiological stress

The antioxidant status of potato ( Solanum tuberosum L.) tubers of two genotypes, cv. Désirée and clone 10337de40 was investigated in relation to susceptibility to internal rust spot (IRS), a Ca²⁺-related physiological disorder. Concentrations of total calcium within the perimedulla tissue of tubers, grown with a restricted (1 m M CaCl₂) Ca²⁺ supply, were similar in cv. Désirée (IRS resistant) and clone 10337de40 (IRS susceptible). A range of antioxidants was assayed in order to assess antioxidant status in both genotypes under the two Ca²⁺ treatments. Although no appreciable differences were detected between low Ca²⁺ and control treatments, certain antioxidants were present at significantly higher levels in the IRS resistant genotype, cv. Désirée. These included dehydroascorbate reductase (EC 1.8.5.1) activity (more than 100% higher), total glutathione content (ca 40% higher), glutathione reductase (EC 1.6.4.2) activity (almost 50% higher), peroxidase (EC 1.11.1.7) activity (ca 60% higher) and superoxide dismutase (EC 1.15.1.1) activity (almost 80% higher). There was no difference in ascorbate content, ascorbate free radical reductase activity (EC 1.6.5.4), α-tocopherol levels and catalase activity (EC 1.11.1.6) between the two genotypes. The possible relationship between resistance to IRS and a superior antioxidant status, found in cv. Désirée, is discussed.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Properties of glutamate dehydrogenase from developing maize endosperm

Glutamate dehydrogenase (EC 1.4.1.3) activity was assayed in homogenates of maize ( Zea mays L. inbred lines Oh43 and Oh43o₂) endosperm during development. During the period 20–35 days after pollination anabolic (aminative) activities were higher than catabolic (deaminating) ones. In order to study the regulation of GDH activity, glutamine or glutamate were injected into the ear peduncle before sample harvesting. The amination and deamination reactions showed similar behaviour with different nitrogen sources: glutamine increased, whereas glutamate decreased, both aminative and deaminative reactions. Partially purified enzyme was active with NADH and NADPH in a ratio 9:1. In Tris-HCl buffer a broad optimum at pH 7.6–8.9 and pH 6.8–8.9 was observed with NADH and NADPH, respectively, NADH activity was activated by Ca²⁺. Saturation curves for (NH₄)₂SO₄ and NADH showed normal Michaelis-Menten kinetics in the presence of 1 m M Ca²⁺, but substrate inhibition occurred without Ca²⁺. The enzyme was inactivated by EDTA. The effect of EDTA was reversed by Ca²⁺ and Mn²⁺, but not by Cu₂₊ and Mg²⁺.     

Features of the intrageneric Alnus-Frankia specificity

Host specificity between local Frankia strains and native alders [Alnus incana (L.) Moench and A. glutinosa (L.) Gaertn.] was evaluated in inoculation experiments. Pure cultures of Frankia , whether originating from A. incana or A. glutinosa , were infective and effective on both host species. These pure cultures were isolated from spore-negative (Sp⁻) nodules. From spore-positive (Sp⁺) nodules no Frankia isolates were obtained. This strain type resisted all our isolation attempts and therefore crushed nodules had to be used for Sp⁺ type inoculations.
The Sp⁺ type Frankia populations differed in their host specificity. Sp⁺ nodules from A. glutinosa were effective on both alder species, but Sp⁺ nodules from A. incana induced effective nodules only on the original host; on A. glutinosa only small (1-3mm) prenodule-like structures were found. Such A. glutinosa plants died on N-free medium, thus showing that these nodules were ineffective. In the effective nodules the middle cortex was dominated by infected cells filled with vesicle clusters. In the ineffective nodules only a few cortical cells were infected and sporangia predominated in these cells. Surprisingly enough they also contained vesicle-like structures as demonstrated in electron micrographs.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

A comparison of inhibition of French-bean and soybean nitrogen fixation by nitrate, 1% oxygen or direct assimilate deprivation

Inhibition by NO⁻₃ of acetylene reduction in bean ( Phaseolus vulgaris L. cv. Contender) and soybean ( Glycine max L. cv. Amsoy 71) was measured in parallel with nodule carbohydrate and nitrate metabolism. In bean the onset of inhibition of C₂H₂ reduction (6 h) coincided with decreased import of assimilates and a lowering of carbohydrate pools (sucrose, glucose and starch). Nitrate reductase (EC 1.6.6.1) activity was induced in all plant organs after 3 h but no nitrite was detected in the nodules. In soybean, nodule carbohydrate concentrations and import of assimilates into the nodules increased markedly between 6 to 24 h after supply of nitrate when the nitrogenase (EC 1.7.99.2) was progressively inhibited. High nitrate reductase activity was observed in the nodules and nitrites accumulated because of insufficient nitrite reductase activity. The nitrate-induced inhibition of nitrogenase was compared with the inhibition observed with low oxygen around the roots (1% O²) or with direct assimilate deprivation (girdling or decapitation). Soybean and bean appeared equally sensitive to these treatments as regards to acetylene reduction. The results are discussed in relation to the current hypotheses explaining nitrate-induced inhibition of dinitrogen fixation: assimilate deprivation or nitrite poisoning. Present data are in favour of the first for bean and of the second for soybean.     

A metabolic connection between nitrogenase activity and the synthesis of ureides in nodulated soybean

Application of allopurinol (AP; 1H-pyrazolo-[3,5- d ]pyrimidine-4-o1) to intact nodulated roots of ureide-forming legumes causes rapid inhibition of NAD:xanthine dehydrogenase (XDH: EC 1.2.1.37), cessation of ureide synthesis and, subsequently, severe nitrogen deficiency (Atkins et al. 1988. Plant Physiology 88: 1229–1234). Nitrogen deficiency is a result of inhibited nitrogenase (EC 1.7.99.2) activity. Using an open gas exchange system to measure H₂ and CO₂ evolution, short term effects of AP application were examined in a Hup ⁻ soybean symbiosis [ Glycine max (L.) Merr. cv. Harosoy: USDA 16]. The onset of inhibition of nitrogenase was detected after ca 2 h exposure of the roots to AP. At the same time xanthine began to accumulate and ureide levels declined in nodules as a result of inhibition of XDH. The decline in H₂ evolution following AP application was not due to altered electron allocation between N₂ and H⁺ by nitrogenease but was coincident with increased gaseous diffusive resistance of nodules and a decline in intracellular oxygen concentration. A possible scheme for the intermediary metabolism of soybean nodules which might account for a direct connection between nitrogenase activity and ureide synthesis is proposed. The suggested mechanism envisages coupling production of reducing power by cytosolic enzymes of purine oxidation to synthesis of dicarboxylic acid substrates (malate and succinate) required for bacteroid respiration.     

Effects of Fusaric Acid on Reactive Oxygen Species and Antioxidants in Tomato Cell Cultures

Generation of O₂^– and H₂O₂ as well as the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, dehydroascorbate reductase and ascorbate content were studied in tomato cell cultures in response to fusaric acid – a nonspecific toxin of phytopathogenic Fusarium species. Toxin treatment resulted in decreased cell viability which was preceded by culture medium alkalinization up to 0.65 pH unit and enhanced extracellular O₂^– production. The H₂O₂ level was not significantly affected. In toxin-treated cultures, a transient, significant increase occurred in intracellular superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase activities. Fusaric acid-induced ascorbate turnover modulation led to up to a twofold increase in dehydroascorbic acid accumulation, and a decrease in the associated ascorbate redox ratio. It was concomitant with a significant decrease in dehydroascorbate reductase activity. These results support previous observations that the pro- and anti-oxidant systems are involved in response to fusaric acid treatment although differential response of H₂O₂ and its metabolism-related enzymes between the whole leaf and cell culture assays was found.     

Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide

Washed Escherichia coli ATCC11775 cells were killed by (–)-epigallocatechin (EGC) in the presence of a non- lethal concentration of Cu²⁺ (1 μmol l⁻¹) without additional H₂O₂, but not by (–)-epicatechin (EC). EGC alone (< 0·1 mmol l⁻¹) did not reduce the viability of the cells. The survival curve obtained in the presence of EGC and Cu²⁺ was similar to that obtained in the presence of (–)-adrenaline (EN) and Cu²⁺.     

Cytosolic and chloroplastic glutamine synthetase of sugarbeet (Beta vulgaris) respond differently to organ ontogeny and nitrogen source

Changes in the activity and subunit composition of cytosolic glutamine synthetase (GS 1; EC 6.3.1.2) and chloroplastic GS (GS 2) were studied in response to an internal (organ ontogeny) and external signal (N source: NO₃⁻ or NH₄⁺). Maximum GS 1 activity of all organs examined was measured in the fibre roots, irrespective of the N source. The response of GS 1 to the N source was, however, organ specific. In the fibre roots, NH₄⁺ nutrition resulted in a 2- to 7-fold (based on protein or freshweight, respectively) increase of GS 1 activity compared to NO₃⁻-grown plants. In contrast to the roots, GS 1 activity in the leaf blades was 2-fold lower with NH₄⁺ nutrition, whereas only minor changes occurred in the petioles. GS 2 activity was highest in the mature and senescing leaf blade; activity was 2-fold higher with NH₄⁺ than with NO₃⁻ nutrition. Not only activity, but also subunit composition of GS 1 changed during organ ontogeny as well as in response to the N source. In contrast to GS 1, only minor changes were evident in GS 2 subunit composition, despite significant changes in GS 2 activity. Up to 5 different GS 1 subunits of ≈41–43 kDa were separated; they were identical in all organs examined. GS 2 was composed of 4 different subunits of ≈48 kDa.     

Carbohydrate and organic acid composition of effective and ineffective root nodules of Phaseolus vulgaris

Dicarboxylic acid transport mutants of Rhizobium species are usually deficient in their ability to fix atmospheric dinitrogen. We report here a study comparing the physiology of root nodules on Phaseolus vulgaris L. cv. Goldie induced by an effective strain of Rhizobium leguminosarum biovar phaseoli and a C₄-dicarboxylic acid utilization mutant. The mutant, while able to form nodules, was ineffective in N₂ fixation. Carbohydrates and organic acids of roots and nodules formed by the 2 strains were monitored at 3-day intervals from 13 to 34 days after inoculation. Both carbohydrates and organic acids accumulated in ineffective nodules in comparison with the effective nodules. The concentration of malic acid was tenfold higher in ineffective nodules than in effective nodules. Other organic acids, i.e., lactate, malonate, ascorbate and gluconate, were also detected. Lactate and ascorbate were the only other organic acids accumulating in ineffective nodules. The most prevalent carbohydrates found in both types of nodules were sucrose, glucose and fructose. Myo-inositol was the only cyclitol detected in both types of nodules. Carbohydrates and organic acids were present in lower concentration in roots than in nodules, except for lactate. These compounds were not consistently detected in higher concentration in roots from plants inoculated with the mutant strain, as was the case in nodules.     

ACTIVATION OF HEXOSE MONOPHOSPHATE PATHWAY IN BRAIN BY ELECTRICAL STIMULATION IN VITRO

Abstract— The incubation of cerebral cortical slices for 15 min in Krebs-Ringer-tris (pH 7.6) solution at 37°C with [1-¹⁴C]glucose or [6-¹⁴C]glucose as substrates yielded a C-1:C-6 ¹⁴CO₂ ratio of 1.21, whereas this ratio increased to 3.01 after the application of electrical stimulation (ES). Tissue levels of 6-phosphoglu-conate (6PG) and glucose 6-phosphate (G6P), intermediary metabolites of hexose monophosphate (HMP) pathway, were 7 and 180 nmol/g tissue following 15 min incubation, and increased by 33 and 45 per cent respectively following the application of ES. Activities of 6-phosphogluconate dehydrogenase (6PGDH, 6-phospho- d -gluconate: NADP⁺ 2-oxidoreductase, EC 1.1.1.44) and glucose-6-phosphate dehydrogenase (G6PDH, d -glucose-6-phosphate: NADP⁺ 1-oxidoreductase, EC 1.1.1.49), important enzymes in regulating the activity of HMP pathway, in cerebral cortical slices were 689 and 907 pmol/mg protein/min and were increased by 66 and 25 per cent respectively by the application of ES. Synaptosomal G6PDH and 6PGDH activities were maximally activated by the addition of 40 m m -Na⁺ to the reaction mixture, whereas no activation by Na⁺ was observed in microsomal G6PDH and 6PGDH. Amobarbital inhibited more strongly the Embden–Meyerhof (EM) pathway than the HMP pathway, while imipramine had a stronger inhibitory effect on HMP pathway than on EM pathway in the electrically stimulated cerebral tissues.
The present results indicate that the HMP shunt pathway in the cerebral cortex is activated by the application of ES in vitro , possibly at synaptic regions and may play an important metabolic and functional role in the brain.     

Exocytosis of Cortical Alveoli and Its Initiation Time in Medaka Eggs Induced by Microinjection of Various Agents

Various agents were microinjected into the cortical cytoplasm at the animal pole of unfertilized eggs of Oryzias latipes under Ca-free conditions. The agents that triggered a wave of the cortical alveolus exocytosis were Ca²⁺, inositol, 1, 4, 5-trisphosphate (IP₃), Ca-ionophore A23187, cGMP, GMP, GTP and guanosine 5'-0-(2-thio-triphosphate)(GTP-γ-s), while CAMP, ATP, gnanosine 5'-0-(2-thio-triphosphate)(GDP- β-s), inositol monophosphate (IMP) and inositol triphosphate (ITP) were ineffective. Ca²⁺, IP₃ and A231 87 induced the propagative exocytosis after a time lag (5–8 sec), irrespective of the presence of Co²⁺. The time lag was shorter than that (13–28 sec) following microinjection of cGMP or GTP, while were not effective in the presence of Co²⁺. The present data suggest that (1) free cytoplasmic Ca²⁺ participates in both an early and a late step in exocytosis, and (2) cGMP or GTP acts on an early step before initiation of Ca²⁺ release during exocytosis in the medaka egg.     

Effects of cold on NAD+ kinase activity in winter rape plants

Low temperature (2°C) caused an increase in the activity of NAD⁺ kinase (EC 2.7.1.23) in leaves of winter rape plants ( Brassica napus L. var. oleifera L. cv. Górezański). The enzyme activity markedly increased between day 4 and 11 of plant exposure to cold, then tended to decrease. Changes in activity of NAD⁺ kinase coincided with the previously observed changes in the levels of pyridine nucleotides, NADP(H) (U. Maciejewska and A. Kacperska, Physiol. Plant. 69: 687–691, 1987). As a result of cold treatment, Ca²⁺–calmodulin–dependent and Ca²⁺–calmodulin–independent NAD⁺ kinase activities increased to almost the same extent. It seems therefore, that the cold–induced activation of NAD⁺ kinase does not depend on the Ca²⁺–calmodulin complex.     

Physiological effects of Na2SO4 and NaCl on callus cultures of Brassica campestris (Chinese cabbage)

Hypocotyl-derived callus cultures of Brassica campestris L. ssp. pekinensis cv. Kim-jung (Chinese cabbage) were grown on Murashige and Skoog medium containing no additional salt, NaCl or Na₂SO₄. Na₂SO₄ was more than twice as inhibitory in comparison to the same concentration of NaCl when growth and fresh:dry weight ratios of established callus were measured. Levels of protein, starch, sucrose and α-amino nitrogen were not significantly altered in salt-grown callus. Concentrations of reducing sugars and chlorophyll were 2–3 times greater in callus grown on either salt. Proline concentration increased 15–20 fold on the highest levels of salt. Final concentrations (reached in 20–24 days) were closely correlated to the initial Na⁺ concentration of the medium, regardless of salt type. The osmotic potential in callus transferred to NaCl or Na₂SO₄ reached a maximum negative value after 16 days. For both salts, subsequent increases were correlated to increases in fresh:dry weight and growth. On both salts, turgor remained relatively constant (0. 6–0.75 MPa). Changes in Na⁺, K⁺, Mg²⁺ and Ca²⁺ content were correlated to initial Na⁺ concentration in the medium, not salt type. Accumulation of Na⁺ was accompanied by loss of K⁺ and Mg²⁺. Six to seven times less sulfate was measured in callus grown on Na₂SO₄ than chloride in callus grown on similar concentrations of NaCl.     

Ascorbate-Induced Oxidative Inactivation of Zn2+-Glycerophosphocholine Cholinephosphodiesterase

Abstract: Zn²⁺-glycerophosphocholine cholinephosphodiesterase, responsible for the conversion of glycerophosphocholine into glycerol and phosphocholine, was inactivated during incubation with ascorbic acid at 38°C. The inclusion of copper ions or Fe²⁺ accelerated the ascorbate-induced inactivation, with Cu²⁺ or Cu⁺ being much more effective than Fe²⁺, suggestive of ascorbate-mediated oxidation. Dehydroascorbic acid had no effect on the phosphodiesterase, but H₂O₂ inactivated the enzyme in a concentration-dependent manner. Also, the enzyme was inactivated partially by a superoxide anion-generating system but not an HOCl generator. In support of involvement of H₂O₂ in the ascorbate action, catalase and superoxide dismutase expressed a complete and a partial protection, respectively. However, hydroxy radical scavengers such as mannitol, benzoate, or dimethyl sulfoxide were incapable of preventing the ascorbate action, excluding the participation of extraneous ^•OH. Although p -nitrophenylphosphocholine exhibited a modest protection against the ascorbate action, a remarkable protection was expressed by amino acids, especially by histidine. In addition, imidazole, an electron donor, showed a partial protection. Separately, when Cu²⁺-induced inactivation of the phosphodiesterase was compared with the ascorbate-mediated one, the protection and pH studies indicate that the mechanism for the ascorbate action is different from that for the Cu²⁺ action. Here, it is proposed that Zn²⁺-glycerophosphocholine cholinephosphodiesterase is one of brain membrane proteins susceptible to oxidative inactivation.     

Biochemical and symbiotic properties of histidine-requiring mutants of Rhizobium leguminosarum biovar trifolii

A perturbation of the histidine biosynthetic pathway in legume microsymbionts can abolish their symbiotic competence. Twenty-one histidine-requiring (His⁻) mutants were isolated from berseem clover-nodulating, symbiotically-competent (Nod⁺, Fix⁺) Rhizobium leguminosarum bv. ' trifolii ' strain RTH 48 Sm^r by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis followed by enrichment. These mutants were analysed for their biochemical defect and the corresponding effect, if any, on their symbiotic abilities. Cross-feeding, supplementation and enzymatic studies identified three types of mutants. Group 1 mutants, His-2 and His-12, grew with histidine supplementation but not with the addition of either L -histidinol or L -histidinol phosphate to the medium ; they lacked histidinol dehydrogenase (EC 1.1.1.23) activity and consequently formed only ineffective, or 'non-fixing' nodules. Group 2 mutant, His-17, grew when supplemented with either L -histidinol or L -histidine, had low histidinol phosphate phosphatase (EC 3.1.3.15) activity (37% of wild-type), and consequently failed to nodulate berseem clover. Group 3, the remaining 18 mutants, grew when supplemented with L -histidinol phosphate, L -histidinol or histidine, and did not nodulate. Typically, reversion rates were between 10⁻⁷ and 10⁻⁸. Defects in early steps of the pathway abolished nodulating ability, whereas lesions in the last step did not. The last step, however, was required for symbiotic nitrogen fixation. It is hypothesized that histidine may be supplied by the host in sufficient quantity for nodulation by histidinol dehydrogenase mutants to occur, whereas the amount provided in the nodule may be insufficient to support bacteroid development and nitrogen fixation.     

Irreversible Inhibition of Mitochondrial Complex I by 1-Methyl-4-Phenylpyridinium: Evidence for Free Radical Involvement

Abstract: Incubation of 10 m M I-methyl-4-phenylpyridinium (MPP⁺) with sonicated beef heart mitochondria caused an irreversible time-dependent decrease in NADH-ubiquinone-l (CoQ₁) reductase activity (52% inhibition after 1 h). Inclusion of glutathione, ascorbate, or catalase in the incubation mixture protected the NADH-CoQ₁ reductase activity. These results suggest that the interaction of MPP⁺ with complex I induces free radical generation, which in turn leads to the irreversible inhibition of complex I activity. The generation of free radicals by neurotoxin-induced inhibition of complex I has important implications for our interpretation of the increased oxidative stress observed in Parkinson's disease substantia nigra and for our understanding of the cause(s) of dopaminergic cell death in this disorder.     

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.