Regulation of phosphoenolpyruvate carboxylase in Pinus halepensis needles submitted to ozone and water stress

Effects of ozone and/or drought stresses on phosphoenolpyruvate carboxylase (PEPc, EC 4.1.1.31) regulation in Pinus halepensis Mill. needles were assessed over 3 months in controlled conditions. Whereas moderated water stress applied to Aleppo pine had no effect on PEPc activity compared to the control, which was probably related to the high tolerance of this species to drought, ozone stress induced a dramatic increase of PEPc activity in pine needles. This stimulation of the anaplerotic pathway could provide substrates to repair processes, well known for being enhanced upon ozone exposure. The ozone-increased PEPc activity was related, to a certain extent, to an increase in protein and mRNA levels. The possible role of the stimulation of the phosphorylation status of the enzyme in the increased PEPc activity under ozone was also investigated. Following the demonstration of the existence of the phosphorylation site at the N terminal part of Aleppo pine PEPc, it was shown that, under ozone treatment, the light/dark PEPc activity ratio and the Ki (malate) for PEPc were increased. This strengthens the hypothesis of an ozone-related post-translational process, which could be part of an adaptation of the plants to prolonged stress. When ozone and water stress were applied in combination, the enhancement in PEPc activity was only related to changes in gene expression. This difference in PEPc regulation, compared to the effect of single stress, could be the consequence of the specific action of each stress on the enzyme. This study brings new insights into the regulation of PEPc in a C₃ plant, Aleppo pine under these stresses. A different regulatory mechanism of PEPc is occurring according to the stress. The physiological implications of the increase in PEPc activity in response to ozone and/or water stress are discussed.     

Effects of ozone and elevated atmospheric carbon dioxide on carbohydrate metabolism of spruce needles. Catabolic and detoxification pathways

We have studied the effects of ozone, carbon dioxide and ozone combined with carbon dioxide fumigations on catabolic and detoxification pathways in spruce ( Picea abies [L.] Karst.) needles. The results obtained showed an increase in the activities of three enzymes involved in the detoxification pathway, superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (AscPOD, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) when trees were exposed to ozone and to ozone‐carbon dioxide treatments. In these two treatments, the fraction of SOD activity due to the chloroplastic isoform was increased (1.5‐fold). In the needles of trees exposed to ozone and to ozone‐carbon dioxide fumigation, an increase in the activities of glucose‐6‐phosphate dehydrogenase (G‐6‐PDH, EC 1.1.1.49) showed that the cell had the capacity to produce more NADPH necessary for the detoxification. Stimulation of other enzymes of catabolic pathways (fumarase [EC 4.2.1.2], phosphofructokinase [PFK, EC 2.7.1.1] and phosphoenolpyruvate carboxylase [PEPC, EC 4.1.1.31]), was also observed making it possible for the cell to provide the reducing power necessary for detoxification as well as energy and carbon skeletons involved in the repair processes.
When carbon dioxide alone was applied, no effects could be detected on these enzyme activities. However, when carbon dioxide was combined with ozone, the effect of ozone on trees was less than that induced by ozone alone, suggesting that elevated atmospheric carbon dioxide concentrations may to some extent protect plants from ozone injury.     

The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants

NADPH is an important molecule in the redox balance of the cell. In this paper, using olive tissue cultures as a model of the function of the NADPH-generating dehydrogenases in the mechanism of oxidative stress induced by severe salinity conditions was studied. When olive (Olea europaea) plants were grown with 200 mM NaCl, a 40% reduction in leaf fresh weight was produced. The content of non-enzymatic antioxidants such as ascorbate and glutathione was diminished between 20% to 39%, whereas the H2O2 content was increased threefold. In contrast, the analysis of the activity and protein contents of the main antioxidative enzymes showed a significant increase of catalase, superoxide dismutase and glutathione reductase. Overall, these changes strongly suggests that NaCl induces oxidative stress in olive plants. On the other hand, while the content of glucose-6-phosphate was increased almost eightfold in leaves of plants grown under salt stress, the content of NAD(P)H (reduced and oxided forms) did not show significant variations. Under salt stress conditions, the activity and protein contents of the main NADPH-recycling enzymes, glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICDH), malic enzyme (ME) and ferrodoxin-NADP reductase (FNR) showed an enhancement of 30-50%. In leaves of olive plants grown with 200 mM NaCl, analysis of G6PDH by immunocytochemistry and confocal laser scanning microscopy showed a general increase of this protein in epidermis, palisade and spongy mesophyll cells. These results indicate that in olive plants, salinity causes reactive oxygen species (ROS)-mediated oxidative stress, and plants respond to this situation by inducing different antioxidative enzymes, especially the NADPH-producing dehydrogenases in order to recycle NADPH necessary for the protection against oxidative damages. These NADP-dehydrogenases appear to be key antioxidative enzymes in olive plants under salt stress conditions.     

Ascorbate in the leaf apoplast is a factor mediating ozone resistance in Plantago major

The role of ascorbate in mediating ozone resistance was examined in Plantago major L. Seedlings of eleven populations which exhibited differential resistance to ozone were fumigated in controlled environment chambers with charcoal/Purafil®-filtered air (CFA) or CFA plus 15 nmol·mol^–1 ozone overnight rising to a maximum between 12:00–16:00 hours of 75 nmol·mol^–1 for 14 d. Measurements of ascorbate content were made on apoplastic and symplastic extracts. Populations differed in their constitutive level of ascorbate in youngest fully expanded leaves, and regression analysis revealed a significant correlation between ascorbate content in ozone-treated leaves and the ozone resistance of the populations. The relationship was stronger using apoplastic ascorbate levels than with corresponding symplastic measurements. The ascorbate content of the youngest fully expanded leaf of an ozone sensitive population was increased by foliar application of ascorbate. No significant difference in stomatal conductance was found between control and ascorbate-treated plants. Following spraying, plants were fumigated with 400 nmol·mol^–1 ozone for 7 h. In control plants, ozone exposure resulted in extensive visible leaf damage (20–70 % at the end of the fumigation period) and decreased rates of CO₂ assimilation (–57 %). However, ascorbate treatment prevented the appearance of visible injury, and ameliorated the decline in photosynthesis induced by ozone (–26 %). Modelled data estimating the extent of protection afforded by apoplastic ascorbate against ozone supported the experimental observations. The results suggested that although apoplastic ascorbate plays an important role, other factors must also contribute to the mediation of ozone resistance in P. major.     

Influence of ammonium and nitrate nutrition on the pyridine and adenine nucleotides of soybean and sunflower

Total pyridine nucleotide concentration of root tissue for young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var. Mammoth Russian) plants is the same with either ammonium or nitrate, but nitrate results in an increased proportion of total oxidized plus reduced NADP (NADP[H]) seemingly at the expense of NAD. The activity of NADH- and NADPH-dependent forms of glutamic acid dehydrogenase is correlated with the ratio of total oxidized plus reduced NAD to NADP(H). The low NAD: NADH ratio maintained in nitrate roots despite active NADH utilization via nitrate reductase and glutamic acid dehydrogenase may be the result of nitrate-stimulated glycolysis. Nitrate roots also maintain a high level of NADPH, presumably by the stimulatory effect of nitrate utilization on glucose-6-phosphate dehydrogenase activity. In the presence of nitrate rather than ammonium, the highly active nitrate-reducing leaves of soybean show a greater proportion of total pyridine nucleotide in the form of NADP(H) than do the inactive leaves of sunflower.     

Characterization of recombinant Aspergillus fumigatus mannitol-1-phosphate 5-dehydrogenase and its application for the stereoselective synthesis of protio and deuterio forms of D-mannitol 1-phosphate

A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpressed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a approximately 40-kDa monomer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of d-mannitol 1-phosphate and d-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 degrees C. NADP(H) showed a marginal activity. Hydrogen transfer from formate to d-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of d-mannitol 1-phosphate or, by applying an analogous procedure using deuterio formate, the 5-[2H] derivative thereof. Following the precipitation of d-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%, based on 200 mM of d-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of d-5-[2H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD+, producing (4S)-[2H]-NADH. Comparison of maximum initial rates for NAD+-dependent oxidation of protio and deuterio forms of D-mannitol 1-phosphate at pH 7.1 and 25 degrees C revealed a primary kinetic isotope effect of 2.9+/-0.2, suggesting that the hydride transfer was strongly rate-determining for the overall enzymatic reaction under these conditions.     

Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalent cations.

Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of O2, Fe2+, and ascorbate or dihydroxyfumarate. It appears that O2 and the reducing agent formed H2O2 and that H2O2 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD (but not glycerol) and could no longer be activated by Mn2+ which increased the Vmax of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.     

Carbohydrate and carbon metabolite accumulation responses in leaves of ozone tolerant and ozone susceptible spinach plants after acute ozone exposure

The objective of this study was to determine whether exposure of plants to ozone (O₃) increased the foliar levels of glucose, glucose sources, e.g., sucrose and starch, and glucose-6-phosphate (G6P), because in leaf cells, glucose is the precursor of the antioxidant, L-ascorbate, and glucose-6-phosphate is a source of NADPH needed to support antioxidant capacity. A further objective was to establish whether the response of increased levels of glucose, sucrose, starch and G6P in leaves could be correlated with a greater degree of plant tolerance to O₃. Four commercially available Spinacia oleracea varieties were screened for tolerance or susceptibility to detrimental effects of O₃ employing one 6.5 hour acute exposure to 25O nL O₃ L^-1 air during the light. One day after the termination of ozonation (29 d post emergence), leaves of the plants were monitored both for damage and for gas exchange characteristics. Cultivar Winter Bloomsdale (cv Winter) leaves were least damaged on a quantitative grading scale. The leaves of cv Nordic, the most susceptible, were approximately 2.5 times more damaged. Photosynthesis (Pn) rates in the ozonated mature leaves of cv Winter were 48.9% less, and in cv Nordic, 66.2% less than in comparable leaves of their non-ozonated controls. Stomatal conductance of leaves of ozonated plants was found not to be a factor in the lower Pn rates in the ozonated plants. At some time points in the light, leaves of ozonated cv Winter plants had significantly higher levels of glucose, sucrose, starch, G6P, G1P, pyruvate and malate than did leaves of ozonated cv Nordic plants. It was concluded that leaves of cv Winter displayed a higher tolerance to ozone mediated stress than those of cv Nordic, in part because they had higher levels of glucose and G6P that could be mobilized during diminished photosynthesis to generate antioxidants (e.g., ascorbate) and reductants (e.g., NADPH). Elevated levels of both pyruvate and malate in the leaves of ozonated cv Winter suggested an increased availability of respiratory substrates to support higher respiratory capacity needed for repair, growth, and maintenance.Abbreviations ADPG-PPiase ADPglucose pyrophosphorylase - ASC L-ascorbic acid - APX ascorbate peroxidase - Ce CO₂ concentration in air in the measuring cuvette during photosynthesis measurements - Ci CO₂ concentration in the leaf intercellular spaces during photosynthesis measurement - Chl chlorophyll - DHA dehydroascorbic acid - DHA reductase dehydroascorbate reductase - DHAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - Gluc glucose - GR glutathione reductase - G_sw stomatal conductance with units as mmol H₂O m^-2 s^-1 - GSSG oxidized glutathione - GSH reduced glutathione - G1P glucose-1-phosphate - G6P glucose-6-phosphate - G6P dehydrogenase glucose-6-phosphate dehydrogenase - 6PG 6-phosphogluconate - 6PG dehydrogenase 6-phosphogluconate dehydrogenase - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - MAL malate - MDHA reductase monodehydroascorbate reductase - PE post-emergence - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - PYR pyruvate - Pn net CO₂ photoas-similation in leaves - PPFD photosynthetic photon flux density with units of mol photons m^-2 s^-1 - PPRC pentose phosphate reductive cycle - RuBP ribulose-1,5-bisphosphate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SLW specific leaf weight - TCA cycle tricarboxylic acid cycle - Triose-P DHAP+GAP     

Hydroponically cultivated radish fed L-galactono-1,4-lactone exhibit increased tolerance to ozone

Leaf L-ascorbate content of an ozone (O3)-sensitive radish genotype (Raphanus sativus L. cv. Cherry Belle) was increased 2-fold by feeding hydroponically cultivated plants L-galactono- 1,4-lactone (GalL). Plants were grown in controlled-environment chambers ventilated with charcoal/Purafil-filtered air, and administered one of two O3 fumigation regimes: chronic exposure (75 nmol O3 mol(-1) for 7 h day(-1) for 21 days) and acute exposure (180 nmol O3 mol(-1) for 9 h). Chronic O3 exposure decreased root growth by 11% in plants maintained in pure nutrient solution (-GalL), but resulted in no change in root growth in GalL-fed plants (+GalL). Similarly, GalL-feeding counteracted the negative effects of O3 on CO2 assimilation rate observed in control plants (-GalL). Under acute O3 exposure, GalL-fed plants showed none of the visible symptoms of injury, which were extensive in plants not fed GalL. Leaf CO2 assimilation rate was decreased by acute 03 exposure in both GalL treatments, but the extent of the decline was less marked in GalL-fed plants. No significant changes in stomatal conductance resulted from GalL treatment, so O3 Uptake into leaves was equivalent in + GalL and -GalL plants. Feeding GalL, on the other hand, enhanced the level of ascorbate, and resulted in the maintenance of the redox state of ascorbate under acute O3 fumigation, in both the leaf apoplast and symplast. The effect of GalL treatment on ascorbate pools was consistent with the reduction in O3 damage observed in GalL-fed plants. Attempts to model O3 interception by the ascorbate pool in the leaf apoplast suggested a greater capacity for O3 detoxification in GalL-fed plants, which corresponded with the increase in O3 tolerance observed. However, modelled data for GalL-fed plants suggested that additional constituents of the leaf apoplast may play an important role in the attenuation of environmentally-relevant O3 fluxes.     

Interaction of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase with pyridoxal 5'-diphospho-5'-adenosine. Affinity labeling of Lys-21 and Lys-343

Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides competitively with respect to glucose 6-phosphate and noncompetitively with respect to NAD+ or NADP+, with Ki = 40 microM in the NADP-linked and 34 microM in the NAD-linked reaction. Incubation of glucose-6-phosphate dehydrogenase with [3H]PLP-AMP followed by borohydride reduction shows that incorporation of 0.85 mol of PLP-AMP per mol of enzyme subunit is required for complete inactivation. Both glucose 6-phosphate and NAD+ protect against this covalent modification. The proteolysis of the modified enzyme and isolation and sequencing of the labeled peptides revealed that Lys-21 and Lys-343 are the sites of PLP-AMP interaction and that glucose 6-phosphate and NAD+ protect both lysyl residues against modification. Pyridoxal 5'-phosphate (PLP) also modifies Lys-21 and probably Lys-343. Lys-21 is part of a highly conserved region that is present in all glucose-6-phosphate dehydrogenases that have been sequenced. Lys-343 corresponds to an arginyl residue in other glucose-6-phosphate dehydrogenases and is in a region that is less homologous with those enzymes. PLP-AMP and PLP are believed to interact with L. mesenteroides glucose-6-phosphate dehydrogenase at the glucose 6-phosphate binding site. Simultaneous binding of NAD+ induces conformational changes (Kurlandsky, S. B., Hilburger, A. C., and Levy, H. R. (1988) Arch. Biochem. Biophys. 264, 93-102) that are postulated to interfere with Schiff's-base formation with PLP or PLP-AMP. One or both of the lysyl residues covalently modified by PLP or PLP-AMP may be located in regions of the enzyme undergoing the NAD(+)-induced conformational changes.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

L-galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.     

Targeted inactivation of the plastid ndhB gene in tobacco results in an enhanced sensitivity of photosynthesis to moderate stomatal closure

The ndh genes encoding for the subunits of NAD(P)H dehydrogenase complex represent the largest family of plastid genes without a clearly defined function. Tobacco (Nicotiana tabacum) plastid transformants were produced in which the ndhB gene was inactivated by replacing it with a mutant version possessing translational stops in the coding region. Western-blot analysis indicated that no functional NAD(P)H dehydrogenase complex can be assembled in the plastid transformants. Chlorophyll fluorescence measurements showed that dark reduction of the plastoquinone pool by stromal reductants was impaired in ndhB-inactivated plants. Both the phenotype and photosynthetic performance of the plastid transformants was completely normal under favorable conditions. However, an enhanced growth retardation of ndhB-inactivated plants was revealed under humidity stress conditions causing a moderate decline in photosynthesis via stomatal closure. This distinctive phenotype was mimicked under normal humidity by spraying plants with abscisic acid. Measurements of CO(2) fixation demonstrated an enhanced decline in photosynthesis in the mutant plants under humidity stress, which could be restored to wild-type levels by elevating the external CO(2) concentration. These results suggest that the plastid NAD(P)H:plastoquinone oxidoreductase in tobacco performs a significant physiological role by facilitating photosynthesis at moderate CO(2) limitation.     

Metabolic response to treatment with cold, paraquat, or 3-amino-1,2,4-triazole in leaves of winter wheat.

We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.     

Metabolic Response to Treatment with Cold,Paraquat, or 3-Amino-1,2,4-triazole in Leaves of Winter Wheat

We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.     

The impact of ozone on juvenile maize (Zea mays L.) plant photosynthesis: effects on vegetative biomass, pigmentation, and carboxylases (PEPc and Rubisco)

The impact of ozone on crops was more studied in C (3) than in C (4) species. In C (3) plants, ozone is known to induce a photosynthesis impairment that can result in significant depressions in biomass and crop yields. To investigate the impact of O (3) on C (4) plant species, maize seedlings ( ZEA MAYS L. cv. Chambord) were exposed to 5 atmospheres in open-top chambers: non-filtered air (NF, 48 nL L (-1) O (3)) and NF supplied with 20 (+ 20), 40 (+ 40), 60 (+ 60), and 80 (+ 80) nL L (-1) ozone. An unchambered plot was also available. Leaf area, vegetative biomass, and leaf dry mass per unit leaf area (LMA) were evaluated 33 days after seedling emergence in OTCs. At the same time, photosynthetic pigments as well as carboxylase (PEPc and Rubisco) activities and amounts were also examined in the 5th leaf. Ozone enhanced visible symptoms characterizing foliar senescence. Across NF, + 20, + 40, and + 60 atmospheres, both chlorophylls and carotenoids were found to be linearly decreased against increasing AOT40 ( CA. - 50 % in + 60). No supplementary decrease was observed between + 60 and + 80. Total above-ground biomass was reduced by 26 % in + 80 atmosphere; leaf dry matter being more depressed by ozone than leaf area. In some cases, LMA index was consistent to reflect low negative effects caused by a moderate increase in ozone concentration. PEPc and Rubisco were less sensitive to ozone than pigments: only the two highest external ozone doses reduced their activities by about 20 - 30 %. These changes might be connected to losses in PEPc and Rubisco proteins that were decreased by about one-third. The underlying mechanisms for these results were discussed with special reference to C (3) species. To conclude, we showed that both light and dark reactions of C (4) photosynthesis can be impaired by realistic ozone doses.