Quantification of ozone influx and apoplastic ascorbate content in needles of Norway spruce trees (Picea abies L., Karst) at high altitude

The objective of the present study was to investigate whether peak concentrations of ozone can deplete the apoplastic ascorbate pool of needles from Norway spruce trees (Picea abies L. Karst.) and, thereby, contribute to damage to forest trees. Twigs of forest trees grown at high altitude (1950m above sea level; Mt Patscherkofel, Austria) were enclosed in situ in chambers and fumigated for 5-5 or 17 h with ozone concentrations ranging from 60 to 798 nmol mol^?1. Adjacent branches were fumigated with filtered air. Ozone influx into the foliage ranging from 1-7 to 17nmolm^?2s^?1 had little effect on whole-needle ascorbate or glutathione contents. However, apoplastic ascorbate decreased by about 30% when the needles were exposed to environmentally relevant ozone concentrations and increased about 3-fold at higher ozone concentrations. This response suggests the induction of ascorbate as a protective system and may also be important under field conditions. Needles of spruce trees from high altitude that were exposed to chronically increased ozone concentrations contained significantly higher apoplastic ascorbate concentrations than needles from spruce trees from lower altitudes with lower mean atmospheric ozone concentrations. The results show that peak concentrations of ozone do not act in spruce via a depletion of the apoplastic ascorbate pool.     

Responses of citrus plants to ozone: leaf biochemistry, antioxidant mechanisms and lipid peroxidation.

The effects of ozone upon 3-year-old trees of Clementina mandarin (Citrus clementina Hort. ex Tan.) cv. Marisol exposed for 12 months to ambient (10 nl l(-1)) and high (30 and 65 nl l(-1)) concentrations in open top chambers (OTCs) were investigated. The data showed that in leaves, ozone reduced total chlorophylls, carotenoid and carbohydrate concentration, and increased 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production. In treated plants, the ascorbate leaf pool was decreased, while lipid peroxidation and solute leakage were significantly higher than in ozone-free controls. The data indicated that ozone triggered protective mechanisms against oxidative stress in citrus.     

Response of Spinach Leaves (Spinacia oleracea L.) to Ozone Measured by Gas Exchange,Chlorophyll a Fluorescence,Antioxidant Systems,and Lipid Peroxidation

Spinach (Spinacia oleracea L. cv. Clermont) leaves grown in open-top chambers and exposed to three different concentrations of ozone were measured for gas exchange, chlorophyll a fluorescence, antioxidant systems, and lipid peroxidation at the end of growing season. High O₃ concentration reduced F_v/F_m, indicating that the efficiency in the energy conversion of photosystem 2 (PS2) was altered. The rate of non-cyclic electron transport rate and the capacity to reduce the quinone pool were also affected. The development of non-photochemical quenching was not high enough to decrease the photon excess in the PS2. The limitation of photosynthetic activity was probably correlated with stomata closure and with an increase in intercellular CO₂ concentration. Under oxidative stress, superoxide dismutase (SOD) activity was stimulated in parallel with lipid peroxidation. We did not find any differences in the ascorbate (AsA) pool and ascorbate peroxidase (APX) or glutathione reductase (GR) activities between air qualities. Small, but similar responses were observed in spinach leaves exposed to ambient ozone concentration.     

The control of ascorbic acid synthesis and turnover in pea seedlings

The rate of ascorbate synthesis and turnover in pea seedling embryonic axes was investigated in relation to its pool size. Ascorbate accumulated in embryonic axes of germinating pea seeds which has been supplied with ascorbate. Incorporation of [U-14C]glucose into ascorbate after a 2 h labelling period was reduced by ascorbate loading for 3 h and 20 h, providing evidence that ascorbate biosynthesis is inhibited by endogenous ascorbate. Ascorbate turnover was estimated by following the metabolism of [1-14C]ascorbate over 2 h after ascorbate loading and by the rate of decrease of the ascorbate pool size after ascorbate loading. Ascorbate turnover rate, determined by [1-14C]ascorbate metabolism, increased as a linear function of pool size. The absolute turnover rate was higher in ascorbate-loaded embryonic axes but was always about 13% of the pool per hour. The initial rate of ascorbate turnover, estimated from the net decrease in pool size after ascorbate loading, also showed a similar turnover rate to that estimated from [1-14C]ascorbate metabolism. Ascorbate loading had no effect on ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase activity. Ascorbate oxidase activity decreased after ascorbate loading.     

Effects of ozone and mild drought stress on gas exchange, antioxidants and chloroplast pigments in current-year needles of young Norway spruce [Picea abies (L.) Karst.]

 To investigate the effects of ozone exposure and soil drought, singly and in combination, on gas exchange, antioxidant contents and pigments in current-year needles of Norway spruce [Picea abies (L.) Karst.] 4-year-old seedlings were fumigated in growth chambers with either charcoal-filtered air or with 100 nl l^–1 ozone for 106 days. After 3 weeks a 20% reduction in gas exchange was observed in ozone-treated seedlings. However, no further decrease occurred in spite of continued ozone exposure. Whole needle ascorbate and apoplastic ascorbate increased until the end of the experiment and contents were 62% and 82%, respectively, higher than in ozone-free controls. This increase in ascorbate might have protected net photosynthesis from further decline. Ozone pre-treated plants and ozone-free controls were subjected to soil drought for 38 days which caused stomatal narrowing. Thereby ozone uptake was reduced when compared to well watered seedlings. At the end of the experiment drought alone, and even more in combination with ozone, had also caused an increase in ascorbate. Glutathione increased only in drought-stressed seedlings. The redox states of the ascorbate and the glutathione pools were not affected by any treatment. Superoxide dismutase activity declined under both stresses but was most reduced by ozone alone. While chlorophyll and neoxanthin contents remained unchanged, carotenes were significantly decreased upon drought. The combination of O₃ and drought induced increased lutein contents, an increased pool size of the xanthophyll cycle as well as an increased epoxidation status of the xanthophyll cycle. These results suggest that spruce needles seem to be able to acclimate to ozone stress but also to drought stress by increasing their ascorbate pools and protecting pigments. Received: 15 September 1997 / Accepted: 24 March 1998     

Changes in Chlorophyll a Fluorescence,Lipid Peroxidation,and Detoxificant System in Potato Plants Grown under Filtered and Non-Filtered Air in Open-Top Chambers

Its high oxidant capacity and ability to generate reactive oxygen species cause ozone toxicity. We studied the effect of ambient ozone on chlorophyll (Chl) a fluorescence, antioxidant enzymes, ascorbate contents, and lipid peroxidation in potatoes grown in open-top chambers in the field. In plants grown in non-filtered air (NFA), the development of non-photochemical quenching brought about a decrease in photosystem 2 (PS2) photochemical efficiency. Also the ability of PS2 to reduce the primary acceptor Q_A was lower than in charcoal-filtered, ozone-free air (CFA). Changes in Chl fluorescence yield were associated with changes in the thylakoid membrane. Ozone altered chloroplast membrane properties, as indicated by an increase in membrane lipid peroxidation in FNA-leaves compared to CFA plants. The ascorbate pool and activities of antioxidant enzymes were used for an indication of the detoxification system state in NFA and CFA leaves, whereby ozone affects the ascorbate concentration and decreases the antioxidant enzymes activities. The capacity of both detoxifying systems together was not high enough to protect potato plants against ambient ozone concentrations which reduced the photosynthetic yield in this potato cultivar.     

Hierarchy of ozone scavenging reactions in the plant cell wall

To estimate protection of the plasmalemma against ozone by the cell apoplast, the decomposition network of ozone in the mesophyll cell wall is analysed in consideration of data on published bimolecular reaction rate constants and concentrations of the reactants involved. The effect of dimerization of ascorbate free radicals (AFR) on the stoichiometric ratio of ozone reduction by ascorbate is quantified over the range of cell wall acidity, pH = 5.0‐6.5. As the disproportionation of AFR decreases sharply towards higher pH, the flow of AFR through dimerization is low over the pH range 5.5‐6.5, allowing abstraction of the second electron from AFR and formation of dehydroascorbate with a nearly 1:1 stoichiometric ratio in relation to ozone. The direct reaction between ozone and ascorbate (AA) in cell walls 0.3‐0.5 µm thick and at an AA concentration of 0.5 m M is able to detoxify 50‐70% of the O₃ that impinges on the wall surface. Generation of singlet oxygen and the hydroxyl radical, which are more reactive to AA than O₃, decreases markedly the O₃ flow to the plasmalemma. The question is raised whether cell wall alkalinization under ozone may hasten the decomposition of the pollutant due to the more rapid generation of hydroxyl radical by phenolic compounds.     

Changes of net photosynthesis,antioxidant enzyme activities,and antioxidant contents of <Emphasis Type="Italic">Liriodendron tulipifera</Emphasis> under elevated ozone

Liriodendron tulipifera was exposed to gradually elevated ozone concentrations of 100–300 μg kg⁻¹ in the naturally irradiated environment chamber. During 15 d of exposure to O₃, net photosynthetic rate (P _N) decreased and there was large difference between the control (C) and treatment with ozone (OT), while there was no significant difference in water use efficiency. Total chlorophyll content as well as the value of fluorescence parameter F_v/F_m decreased, while antioxidant enzyme activities related to ascorbate-glutathione cycle increased after 15 d of OT. Unchanged contents of ascorbate and glutathione indirectly suggest that the species hastened the antioxidant’s oxidization/reduction cycle using enzymes instead of expanding their pool against oxidative stress.     

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.     

LEAF ONTOGENY AS A FACTOR IN SUSCEPTIBILITY TO OZONE: AMINO ACID AND CARBOHYDRATE CHANGES DURING EXPANSION

During development (expansion), the cotton leaf passes through a stage in which it is highly susceptible to ozone. This period of susceptibility occurs after the maximum expansion rate but before complete expansion occurs. The period of maximum susceptibility corresponds to a minimum concentration of soluble sugars and free pool amino acids. Specific amino acids such as aspartate, serine, threonine, glutamate, asparagine, alanine, glycine, valine, isoleucine, leucine, histidine, and tryptophan tended to reach a minimum concentration at about the same time leaves were susceptible to ozone. Ozone exposure during the susceptible period is manifested by visible leaf flecking of the upper surface and a dramatic (up to 2 fold) increase in total free pool amino acids. Most individual amino acids tended to increase except for phenylalanine, alanine, phosphoserine, phosphoethanolamine, and ethanolamine. Soluble protein is decreased but not to the same extent that the free pool amino acids increase. Because there is ample evidence that ozone enters the leaf during nonsusceptible periods as well as susceptible periods, it is postulated that ozone damage results because of the depletion of soluble reserves (carbohydrates and amino acids). Perhaps repair of damage cannot occur.     

Effects of 2-month ozone exposure in spinach leaves on photosynthesis,antioxidant systems and lipid peroxidation

The photosynthesis response, antioxidant systems and lipid peroxidation were studied in leaves from spinach plants (Spinacia oleracea L.) in response to ozone fumigation, ambient air and charcoal filtered air treatments. The photosynthetic activity was tested through gas exchange and chlorophyll a fluorescence measurements. Ambient air and ozone fumigation caused a decrease in the photosynthetic rate (25% and 63%, respectively) mainly due to a reduced mesophyll activity, as evidenced by the increased intercellular CO₂ concentration. These data agree with a large reduction in the non-cyclic electron flow (7% and 16%), a lower capacity to reduce the quinone pool and a higher development of non-photochemical quenching upon high O₃ concentration. The results suggest that the oxidative stress produced, together with the stimulation of superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11) activities and the increase in lipid peroxidation (20% and 36%, respectively), generated an alteration of the membrane properties.     

Antioxidative responses in Vitis vinifera infected by grapevine fanleaf virus

The antioxidative response of grapevine leaves (Vitis vinifera cv. Trebbiano) affected by the presence of grapevine fanleaf virus was studied during the summer of 2010 at three different harvest times (July 1st and 26th, and August 30th). At the first and second harvest, infected leaves showed increases in the concentration of superoxide radical and hydrogen peroxide, the latter increasing for enhanced activity of superoxide dismutase. In contrast, at the last harvest time, increases in the ascorbate pool and ascorbate peroxidase activity maintained hydrogen peroxide to control levels. The glutathione pool was negatively affected as summer progressed, showing a decrease in its total and reduced form amounts. At the same time, increases in the ascorbate pool were observed, making antioxidant defenses of grapevine effective also at the last harvest time. Increases in phenolic acids, and in particular in p-hydroxybenzoic acid, at the first and second harvest might have enhanced the efficiency of the antioxidant system through an interrelation between a peroxidase/phenol/ascorbate system and the NADPH/glutathione/ascorbate cycle. The lack of increase in p-hydroxybenzoic acid at the third harvest could be due instead to the enhanced utilization of this acid for hydrogen peroxide detoxification. With time, grapevine plants lost their capacity to contrast the spread of grapevine fanleaf virus, but acquired a greater ability to counteract pathogen-induced oxidative stress, being endowed with more reduced antioxidant pools.     

Ozone-induced inactivation of antioxidant enzymes

Ozone is an air pollutant that damages a variety of biomolecules. We investigated ozone-induced inactivation of three major antioxidant enzymes. Cu/Zn superoxide dismutase was inactivated by ozone in a concentration-dependent manner. The concentration of ozone for 50% inactivation was approximately 45 microM when 10 microM Cu/Zn superoxide dismutase was incubated for 30 min in the presence of ozone. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was randomly fragmented. Both ascorbate and glutathione were very effective in protecting Cu/Zn superoxide dismutase from ozone-induced inactivation. The other two enzymes, catalase and glutathione peroxidase, were much more resistant to ozone than Cu/Zn superoxide dismutase. The ozone concentrations for 50% inactivation of 10 microM catalase and glutathione peroxidase were 500 and 240 microM, respectively. SDS-PAGE demonstrated that ozone caused formation of high molecular weight aggregates in catalase and dimerization in glutathione peroxidase. Glutathione protected catalase and glutathione peroxidase from ozone but the effective concentrations were much higher than that for Cu/Zn superoxide dismutase. Ascorbate was almost ineffective. The result suggests that, among the three antioxidant enzymes, Cu/Zn superoxide dismutase is a major target for ozone-induced inactivation and both glutathione and ascorbate are very effective in protecting the enzyme from ozone.     

大气臭氧浓度升高对银杏叶片活性氧代谢及相关基因表达的影响

采用开顶式气室熏蒸法,设置自然条件下臭氧(O₃)浓度(对照,约40 nmol·mol^-1)、80、160及200 nmol·mol^-14个臭氧浓度,观测了不同浓度臭氧条件下银杏叶片可见伤害、活性氧生成量、抗氧化酶活性及相关基因表达变化情况,分析大气臭氧浓度升高对植物活性氧代谢的影响.结果表明: 160和200 nmol·mol^-1 O₃熏蒸明显伤害银杏叶片,80 nmol·mol^-1与对照无差异,无可见伤害.O₃处理20 d后,160和200 nmol·mol^-1条件下银杏叶片的超氧自由基(O₂^-·)产生速率显著高于80 nmol·mol^-1和对照,而80 nmol·mol^-1与对照无差异;O₃处理40 d后,160和200 nmol·mol^-1熏蒸下叶片过氧化氢(H₂O₂)含量显著高于80 nmol·mol^-1和对照,而过氧化氢酶(CAT)活性显著高于80 nmol·mol^-1和对照,各臭氧处理抗坏血酸过氧化物酶(APX)活性均低于对照.熏蒸40 d后,CAT、APX基因的转录表达持续加强;防御素(GbD)的表达强度则随着臭氧浓度的增加及熏蒸时间的延长而呈显著加强.高浓度臭氧胁迫可使银杏叶片活性氧生成量增加、抗氧化酶活性下降、相关基因表达水平上调,有明显可见叶片伤害.     

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.     

近地层臭氧对小麦抗氧化酶活性变化动态的影响

摘要:利用开顶式气室（TOCs）模拟了地表O3浓度升高的大田试验条件,测定了小麦各生长期(返青期、拔节期、孕穗期、抽穗期、灌浆期、成熟期)叶片抗氧化酶的活性。通过两年的重复试验,得到相似结果。试验表明,过氧化氢酶（CAT）和过氧化物酶（POD）活性在小麦的生长进程中,先升高后降低。O3浓度升高使CAT酶和POD酶活性的峰值期提前,POD酶峰值期明显提前所需的O3浓度比CAT酶高,O3诱导小麦生长前期的CAT、POD酶活性升高,后期则起抑制作用。POD酶对低浓度O3的胁迫响应时间较晚。当O3浓度提高到150 nL?L-1时,超氧化物歧化酶（SOD）活性随小麦生长期的变化,由原来的双峰曲线变为单峰曲线,高浓度O3使SOD酶活性在抽穗期后持续显著降低。O3不明显改变抗坏血酸过氧化物酶（APX）对胁迫的响应时间,但一定浓度范围内O3能诱导APX酶活性升高。不同浓度的O3对SOD酶和APX酶的作用效应和强度显著不同, CAT酶和POD酶对O3比较敏感。 因此,近地层O3浓度增加,改变了CAT、POD和SOD酶活性变化的时序特征,抗氧化酶对O3胁迫的响应特征取决于小麦的生育期、O3的浓度和薰气时间。     

Ascorbate degradation in tomato leads to accumulation of oxalate,threonate and oxalyl threonate

Ascorbate content in plants is controlled by its synthesis from carbohydrates, recycling of the oxidized forms and degradation. Of these pathways, ascorbate degradation is the least studied and represents a lack of knowledge that could impair improvement of ascorbate content in fruits and vegetables as degradation is non‐reversible and leads to a depletion of the ascorbate pool. The present study revealed the nature of degradation products using [¹⁴C]ascorbate labelling in tomato, a model plant for fleshy fruits; oxalate and threonate are accumulated in leaves, as is oxalyl threonate. Carboxypentonates coming from diketogulonate degradation were detected in relatively insoluble (cell wall‐rich) leaf material. No [¹⁴C]tartaric acid was found in tomato leaves. Ascorbate degradation was stimulated by darkness, and the degradation rate was evaluated at 63% of the ascorbate pool per day, a percentage that was constant and independent of the initial ascorbate or dehydroascorbic acid concentration over periods of 24 h or more. Furthermore, degradation could be partially affected by the ascorbate recycling pathway, as lines under‐expressing monodehydroascorbate reductase showed a slight decrease in degradation product accumulation.     

Carbohydrate Accumulation Affects the Redox State of Ascorbate in Detached Tobacco Leaves

A decreased utilization of NADPH for CO₂ fixation as a result of carbohydrate accumulation in chlorotic leaves is generally believed to be associated with an increase in oxidative stress. Molecular oxygen may serve as an alternative electron acceptor of photosynthesis under these conditions. In order to test this hypothesis mature leaves of tobacco plants (Nicotiana tabacum) were detached, fed with glucose (50 mM) via the petiole, and used to study the effect of carbohydrate accumulation on the pigment content and the components of the superoxide dismutase-ascorbate-glutathione cycle. During a period of five days the concentration of total soluble carbohydrates increased substantially in leaves supplied with glucose in comparison with control leaves supplied only with water. This increase was accompanied by a twofold decrease in the chlorophyll content. In detached water-fed leaves the levels of most of the antioxidative components increased, whereas glucose feeding had no or only little additional effect on the activities of the protective enzymes, but caused a 1.6- and 4-fold increase in the contents of glutathione and ascorbate, respectively. In relation to the total foliar ascorbate pool, the amount of reduced ascorbate increased from about 30 % to 60 % upon feeding with glucose. These results do not support the idea that the accumulation carbohydrates per se cause an increased rate of superoxide production which necessitates increased activities of antioxidative enzymes. It rather appears that glucose-fed leaves have an increased reductive capacity that can be released via glutathione into the ascorbate pool, thereby, regulating the redox state of ascorbate.     

Ascorbate and glutathione contents in duckweed, Lemna minor, as biomarkers of the stress generated by copper, folpet and diuron

Glutathione and ascorbate are essential components of the general antioxidative strategy to overcome oxidative stress due to environmental constraints such as pollution. The variation of glutathione and ascorbate contents in duckweed (Lemna minor) was investigated after a 48 h exposure to copper, diuron and folpet under laboratory conditions in order to determine whether changes in their level could serve as suitable and early biomarkers of pollution. One could observe that diuron and folpet caused the glutathione level to increase, its redox status remaining unchanged, while copper led to a depletion of this antioxidant and to an increase in its oxidation rate. When duckweed was contaminated by folpet and the metal, an increase of the ascorbate pool size occurred from concentrations as low as 1 mg l^-1 and 50 μg l^-1 respectively. While the ascorbate pool became more oxidized because of exposure to copper concentrations ≤ 200 μg l^-1, folpet caused an increase in its reduction rate. Diuron was responsible for depletion of ascorbate, the redox status of which remained unchanged. Because it is an adaptation to stress and a defence process, the increase in the antioxidant pool size was proposed as a biomarker of exposure to an unsafe environment. Since depletion of antioxidant and an increase in its oxidation rate weakened cellular defences and indicated a precarious state, they could constitute early indicators of toxicity. So they were proposed as potential biomarkers of toxicity. It was concluded that the antioxidant content in duckweed might serve as a useful biomarker for monitoring water quality.     

Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone

Transgenic tobacco ( Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O(3)). Three homozygous transgenic lines, chosen on the basis of a preliminary screen of AO activity in the leaves of 29 lines, revealed up to a 380-fold increase in AO activity, with expression predominantly associated with leaf cell walls. Over-expression of AO resulted in no change in the total ascorbate content recovered in apoplast washing fluid, but the redox state of ascorbate was reduced from 30% in wild-type leaves to below the threshold for detection in transgenic plants. Levels of ascorbic acid and glutathione in the symplast were not affected by AO over-expression, but the redox state of ascorbate was reduced, while that of glutathione was increased. AO over-expressing plants exposed to 100 nmol mol(-1) ozone for 7 h day(-1) exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO(2) assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation, compared with wild-type plants. Transgenic plants also exhibited a greater decline in CO(2) assimilation rate when exposed to a brief ozone episode (300 nmol mol(-1) for 8 h). Stomatal conductance, hence O(3) uptake, was unaffected by AO over-expression. Our findings illustrate the important role played by ascorbate redox state and sub-cellular compartmentation in mediating the tolerance of plants to ozone-induced oxidative stress.