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.     

Antioxidants in the apoplast and symplast of beech (Fagus sylvatica L.) leaves: seasonal variations and responses to changing ozone concentrations in air

Concentrations of the antioxidants ascorbate and glutathione were measured in the apoplast of beech (Fagus sylvatica L.) leaves and in leaf tissue. During early leaf development, reduced ascorbate (ASC) was almost absent from the apoplast, whereas levels of oxidized ascorbate (DHA) were high. Less than 20% of the apoplastic ascorbate was reduced. ASC increased towards midsummer, reaching top levels of about 4molm^?3 apoplast volume in July and August. Reduction increased to 60–75% in summer. Neither DHA reductase nor glutathione was detected in the apoplast of beech leaves. Levels of apoplastic ascorbate were compared with ambient concentrations of ozone in air. Statistical analysis indicated a significant interrelation between atmospheric ozone and apoplastic ascorbate. In midsummer of 1993, contents of DHA were increased in the apoplast when ozone concentrations were high. Apoplastic ASC was also positively correlated with ambient ozone concentrations, but with a delay of 3 to 7d. In leaf tissue, levels of ascorbate were between 17 and 21 μmolg^?1 FW in summer. Except for late April and November, more than 95% of the intracellular ascorbate was reduced. Glutathione contents were lowest during the summer. Oxidation was increased in spring and autumn, when apoplastic ascorbate was also largely oxidized. Usually, 80 to 90% of the glutathione was reduced. During the summer, intracellular concentrations of oxidized glutathione (GSSG) were increased, with a delay of about 1d following periods of high ambient ozone concentrations. The transitory accumulation of GSSG may be explained by slow enzymatic regeneration of glutathione.     

Role of Ascorbate in Detoxifying Ozone in the Apoplast of Spinach (Spinacia oleracea L.) Leaves

Both reduced and oxidized ascorbate (AA and DHA) are present in the aqueous phase of the extracellular space, the apoplast, of spinach (Spinacia oleracea L.) leaves. Fumigation with 0.3 [mu]L L-1 of ozone resulted in ozone uptake by the leaves close to 0.9 pmol cm-2 of leaf surface area s-1. Apoplastic AA was slowly oxidized by ozone. The initial decrease of apoplastic AA was <0.1 pmol cm-2 s-1. The apoplastic ratio of AA to (AA + DHA) decreased within 6 h of fumigation from 0.9 to 0.1. Initially, the concentration of (AA + DHA) did not change in the apoplast, but when fumigation was continued, DHA increased and AA remained at a very low constant level. After fumigation was discontinued, DHA decreased very slowly in the apoplast, reaching control level after 70 h. The data show that insufficient AA reached the apoplast from the cytosol to detoxify ozone in the apoplast when the ozone flux into the leaves was 0.9 pmol cm-2 s-1. The transport of DHA back into the cytosol was slower than AA transport into the apoplast. No dehydroascorbate reductase activity could be detected in the apoplast of spinach leaves. In contrast to its extracellular redox state, the intracellular redox state of AA did not change appreciably during a 24-h fumigation period. However, intracellular glutathi-one became slowly oxidized. At the beginning of fumigation, 90% of the total glutathione was reduced. Only 10% was reduced after 24-h exposure of the leaves to 0.3 [mu]L L-1 of ozone. Necrotic leaf damage started to become visible when fumigation was extended beyond a 24-h period. A close correlation between the extent of damage, on the one hand, and the AA content and the ascorbate redox state of whole leaves, on the other, was observed after 48 h of fumigation. Only the youngest leaves that contained high ascorbate concentrations did not exhibit necrotic leaf damage after 48 h.     

Ozone detoxification in the apoplasm and symplasm of spinach,broad bean and beech leaves at ambient and elevated concentrations of ozone in air

Spinach (Spinacia oleracea L.), broad bean (Vicia faba L.) and beech (Fagus sylvatica L.) plants were exposed to ozone at concentrations often measured in air during the summer months (120–300 g·m^–3) and antioxidants were determined in the leaf tissue and in the aqueous phase of the cell wall, the apoplasm. Concentrations of both reduced ascorbate (AA) and its oxidized form, dehydroascorbate (DHA), showed the tendency to increase transiently in the apoplasm of spinach leaves 6–24 h after starting fumigation with ozone. In beech leaves, apoplasmic AA and DHA increased 3–7 d after beginning of treatment. At the very high concentration of 1600 g O₃·m^–3, an increase of apoplasmic AA was already measured after 1 d in beech leaves. Apparently, spinach and beech leaves respond to oxidative stress by increasing AA transport into the apoplasm and by accelerating DHA export. In contrast to these observations, DHA accumulated during 3 d of fumigation with only 120 g O₃·m^–3 in the apoplasm of broad bean leaves, while AA contents did not increase. After termination of fumigation, the extracellular redox state of ascorbate normalized within 1 d. Glutathione could not be detected in the apoplasm of any of the three leaf species. Intracellular AA changed its redox state in response to exposure to elevated concentrations of ozone. After 4–6 weeks of fumigation with 200–300 g O₃·m^–3 an increase of intracellular DHA was measured in beech leaves. At the same time, chlorophyll contents decreased and characteristic symptoms of ozone damage could be observed. However, no significant change in the redox state of apoplasmic ascorbate could be detected in beech leaves. Evidently, detoxification of ozone by apoplasmic AA was insufficient to protect the leaf tissue. Fumigation with a high ozone concentration (1600 g·m^–3) caused an appreciable increase in the cellular contents of the oxidized forms of ascorbate and glutathione in beech leaves. Whereas in spinach leaves intracellular antioxidant contents and redox states were not altered during fumigation with 120–240 g O₃·m^–3, in broad bean leaves the intracellular DHA concentration increased and intracellular ascorbate became more oxidized after fumigation of the plants with 120 g O₃·m^–3. Apparently, broad bean leaves are more sensitive to ozone than beech and spinach leaves.Abbreviations AA ascorbate, reduced form - DHA ascorbate, oxidized form (dehydroascorbate) - FW fresh weight - GSH glutathione, reduced form - GSSG glutathione, oxidized form - IWF intercellular washing fluid - V_air intercellular air space volume of leaves - V_apo apoplasmic water volume of leaves This work was supported within the Sonderforschungsbereich 251 of the University of Würzburg.     

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.     

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     

Symplastic Transfer of Fluorescent Dyes from Mesophyll to Sieve Tube in Stripped Leaf Tissue and Partly Isolated Minor Veins of Commelina benghalensis

We have stripped small (3 × 3 mm) fields of the upper and the opposite lower epidermis of Commelina benghalensis leaves. Pectinase treatment of the resulting chlorenchyma windows produced free-lying viable minor veins with small lumps of mesophyll cells attached. These veins were still connected with the intact remainder of the leaf. Fluorescent dyes were injected into mesophyll cells or mestome sheath cells. Continuous following of the dye from the moment of injection and use of the simple vein system allowed an unhindered and precise assessment of the cell-to-cell route of dye transfer. Disodium fluorescein and Lucifer Yellow CH injected into mesophyll or mestome sheath cells readily moved to the sieve tube. This symplastic dye transfer from mesophyll to sieve tube was also observed after injection into unmacerated stripped leaf tissue. The displacement of fluorescent dyes substantiates a symplastic continuity between mesophyll and sieve tube and therefore supports the possibility of symplastic phloem loading.     

In vivo occurrence of carbonyl residues in Phaseolus vulgaris proteins as a direct consequence of a chronic ozone stress

We aimed to show that a chronic and realistic ozone stress could induce in vivo formation of carbonyl groups in leaf proteins of bean (Phaseolus vulgaris L. cv Bergamo). Plants were grown in three open-top chambers with increasing ozone concentrations: non-filtered air (NF), NF+40 nL·L^–1, NF+80 nL·L^–1 ozone 7 h·d^–1 for 22 d. Carbonyl contents in proteins, evaluation of Rubisco (EC 4.1.1.39) amounts and visible damages were systematically investigated in primary and first trifoliate leaves. Visible ozone injuries clearly reflected the total external ozone dose (expressed as AOT40) that the leaf had suffered. Ozone was effective at inducing aldehydes and ketones formation in bean proteins. This production of carbonyl groups increased with ozone concentration, the most relevant difference being observed on the Rubisco small subunit (Rubisco SSU). Contrary to young first trifoliate leaves, older primary leaves from O₃-enriched atmospheres exhibited a two-fold decrease in Rubisco level. Carbonyl group formation in Rubisco SSU and decrease in Rubisco level were not necessarily linked. Depending on ozone concentration, exposure time and leaf age, these two effects were observed either together or separately for an almost similar external dose of ozone. To conclude, leaf symptoms, loss of Rubisco and oxidized Rubisco SSU could participate in the assessment of the impact of a chronic ozone stress.     

Long-term exposure to enhanced ultraviolet-B radiation in the sub-arctic does not cause oxidative stress in Vaccinium myrtillus

The aim of this work was to assess whether or not oxidative stress had developed in a dwarf shrub bilberry ( Vaccinium myrtillus L.) under long-term exposure to enhanced levels of ultraviolet-B (u.v.-B) radiation. The bilberry plants were exposed to increased u.v.-B representing a 15% stratospheric ozone depletion for seven full growing seasons (1991–1997) at Abisko, Swedish Lapland (68°N). The oxidative stress was assessed on leaves and stems by analysing ascorbate and glutathione concentrations, and activities of the closely related enzymes ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The affects of autumnal leaf senescence and stem cold hardening on these variables were also considered. The results showed that the treatment caused scarcely any response in the studied variables, indicating that u.v.-B flux representing a 15% ozone depletion under clear sky conditions is not sufficient to cause oxidative stress in the bilberry. It is suggested that no strain was evoked since adaptation was possible under such u.v.-B increases. The studied variables did, however, respond significantly to leaf senescence and especially to stem cold hardening.     

Species- and age-dependent sensitivity to ozone in young plants of pea,wheat and spinach: Effects on acyl lipid and pigment content and metabolism

Acyl lipids and pigments were analyzed in young plants of garden pea, spring wheat and spinach exposed to < 5 or 65 nl l^?1 ozone 12 h per day for 6 days. In one set of experiments, the plants were exposed to ¹⁴CO₂ for 2 h 3 days prior to ozone exposure. The plants responded differently to the moderately enhanced level of ozone used Spinach was not at all sensitive while in both pea and wheat, leaves of different ages differed in ozone sensitivity. In pea, ozone sensitivity increased with leaf age. In the second and third oldest leaves, the amounts of galactolipids per leaf area and the proportions of 18:3 of the total lipid extract and of phosphatidylglycerol decreased. In the second oldest leaf, ozone also caused a decreased proportion of 18:3 of monogalactosyldiacylglycerol. In the fourth oldest leaf, lipid composition and galactolipid unsaturation was unaffected, but ozone caused decreased leaf expansion resulting in increased acyl lipid content per leaf area. In both the first and second leaves of wheat, ozone fumigation caused a marked decrease in the content of monogalactosyldiacylglycerol and in the first leaf, the contents of phosphatidylcholine and phosphatidylethanolamine increased. The proportion of 18:3 in phosphatidylcholine was larger in ozone-fumigated than in control plants, while the reverse applied for phosphatidylglycerol. In the oldest sampled leaves of pea and wheat, ozone caused an increase in the radioactivity associated with β-carotene, indicating increased turnover. Thus, while spinach was unaffected, in both pea and wheat ozone caused a decrease in the proportion of chloroplast membrane lipids to non-chloroplast membrane lipids in older leaves while younger leaves were less sensitive.     

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.     

Cultivar-specific impairment of strawberry growth,photosynthesis, carbohydrate and nitrogen accumulation by ozone

In a 2-year study, fruiting plants of strawberry (Fragaria × ananassa Duch.) cv. ‘Korona’ and ‘Elsanta’ were exposed for 2 months to 78 ppb ozone on average or filtered air without ozone in controlled environment chambers. Plant growth, photosynthesis, carbohydrate accumulation, and macronutrient concentrations were investigated in order to demonstrate cultivar-specific differences in the ozone sensitivity of ‘Korona’ and ‘Elsanta’ on the whole plant level. Moreover, the hypothesis was tested whether properties of the root system in strawberry were involved in ozone tolerance, for example, the roots’ ability to store or make available carbohydrates and their capacity to secure plants’ supply with nitrogen during a stress situation. In strawberry, ozone reduced leaf area by reducing leaf number. Moreover, specific leaf area (SLA) and relative leaf water content were reduced. Net photosynthesis was only slightly impaired, but activity of Rubisco and chlorophyll content in older leaves of cv. ‘Elsanta’ were significantly reduced. The most important, indirect impairment of photosynthesis was the reduction of plants’ total leaf area, which resulted in a decrease in plant biomass. The reduction of root biomass, the root/shoot ratio, and also the distribution of carbohydrates indicated a partitioning priority of the shoot at expense of the root system. Cultivar ‘Elsanta’ was characterized by significantly lower carbohydrate levels in ozone-exposed leaves, whereas levels remained fairly stable in ‘Korona’ leaves. In addition, nitrogen concentrations in leaves and roots decreased significantly in ‘Elsanta’, not in ‘Korona’. The reduced nitrogen concentration in leaves may be related with the more distinct reduction in Rubisco activity and chlorophyll content in older leaves of ‘Elsanta’.     

Molecular size limit for movement in the symplast of the Elodea leaf

A range of water-soluble fluorescent dyes and dye conjugates have been injected into cells in Elodea canadensis Michx. leaves. All compounds are unable to cross the plasmalemma between living cells and the external solution, are not degraded to other fluorescent compounds by tissue homogenates, and do not affect cytoplasmic streaming. Despite being unable to cross the plasmalemma, molecules up to 874 dalton pass from cell to cell, smaller molecules showing greater mobility. The conjugate of fluorescein isothiocyanate and leucyl-diglutamylleucine (874 dalton) appears to be close to the limit for movement: in only three out of 17 injections was any movement visible; this movement was only to adjacent cells and was close to the limit of detection. Dye molecules of 1678 dalton and larger did not pass from cell to cell. From the relationship between the size of the dye molecules, measured using molecular models, and their intercellular mobility, the equivalent pore diameter of the Elodea leaf plasmodesmata has been estimated to lie within the range 3.0–5.0 nm.     

Nutrition and the ozone sensitivity of birch (Betula pendula)

 Cuttings of a single birch clone (Betula pendula) were grown in field fumigation chambers throughout the growing season in either filtered air (control) or 90/40 nl O₃ l^–1 (day/night). Both regimes were split into plants under high and low nutrient supply (macro- and micronutrients). The stomatal density of leaves was increased by ozone but was lowered at high nutrition, while the inner air space was hardly affected by the treatments. Ozone induced macroscopic leaf injury regardless of nutrition, but leaf shedding was delayed in the low-fertilized plants, despite O₃ uptake being similar to that in high-fertilized plants. The leaf turn-over was enhanced in the O₃-exposed high-fertilized plants, but length growth and leaf formation of stems were not affected by ozone in either nutrient regime. Leaves of high-fertilized plants showed O₃-caused decline in photosynthetic capacity, water-use efficiency, apparent carbon uptake efficiency and quantum yield earlier as compared with low-fertilized plants, whereas chlorophyll fluorescence (F_V/F_M) and leaf nitrogen concentration were rather stable. CO₂ uptake rate and rubisco activity of young leaves compensated for the O₃ injury in the ageing leaves of the low-fertilized plants. In 8-week-old leaves, however, the O₃-induced decline in CO₂ uptake did not differ between the nutrient regimes and was associated with increased dark respiration rather than changed photorespiration. The balance between CO₂ supply and demand was lost, as was stomatal limitation on CO₂ uptake. High nutrition did not help leaves to maintain a high photosynthetic capacity and life span under O₃ stress. Received: 6 July 1996 / Accepted: 4 June 1997     

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.     

The influence of ambient and elevated ozone concentrations on photosynthesis in Populus nigra

Light-saturated net leaf photosynthesis (A_sat), CO₂ response curves (A/C_i), current photochemical capacity (F_v/F_m) and pigment contents were measured in leaves of Populus nigra (Clone T107) which had been exposed to ozone stress in open-top chambers for the entire growth period. Surprisingly, not only elevated (ao⁺, i.e. ambient air + 50 mm³ m^?3 ozone) but also ambient (aa) ozone concentrations led to a reduction in A_sat, in comparison with leaves exposed to air containing almost no ozone (cf^?, i.e. charcoal filtered ambient air). The very small change in leaf conductance (g₁) indicated that the decrease in A_sat was not due to stomatal limitation. This finding was supported by the fact that, a decrease in carboxylation efficiency (CE) correlated with a loss in A_sat. In comparison to cf^? leaves, aa leaves showed no change in current photochemical capacity (F_v/F_m) throughout the whole experiment. However, a marked decline in F_v/F_m in ao⁺ leaves was observed at a time when A_sat and CE were already decreased by about 45% and 60% respectively. As the chlorophyll b content of leaves is known to correlate with the amount of LHC and PSII centres, it was used to normalize fluorescence parameters in relation to PSII centres present. The normalized values for F_m and F₀ increased with the dosage of ozone in ao⁺ leaves but not in aa leaves, indicating a change of the pigment content of PSII in the former, but not in the latter. These data led to the conclusion that ozone interacts primarily with components of the Calvin cycle, which results in a decrease in A_sat, with subsequent feedback on the current photochemical capacity of PSII centres.     

Spectral and fluorescent study of the interaction of squarylium dyes,derivatives of 3<Emphasis Type="Italic">H</Emphasis>-indolium,with albumins

Noncovalent interaction of intraionic squarylium dyes, derivatives of 3H-indolium, as well as the structurally analogous ionic indodicarbocyanine dye with serum albumins (human, bovine, rat) and, for comparison, with ovalbumin has been studied by spectral and fluorescent methods. The hydrophilic squarylium dye with sulfonate groups was found to interact with albumins more efficiently, which is probably due to the double negative charge on the dye molecule at the expense of the sulfonate groups and the ability to form hydrogen bonds with albumin. The hydrophilic indodicarbocyanine dye without the squarylium group in its structure binds to albumins much weaker than the structurally analogous squarylium dye. The dyes bind to ovalbumin less efficiently than to serum albumins. Along with the binding of monomeric dye molecules, the aggregation of the dyes on albumins is also observed. The hydrophobic squarylium dye without sulfonate groups tends to form aggregates in aqueous solutions, which partially decompose upon the introduction of albumin into the solution. The hydrophilic squarylium dye with sulfonate groups can be recommended for tests as a spectral-fluorescent probe for serum albumins in extracellular media of living organisms.     

Factors that affect leaf extracellular ascorbic acid content and redox status

Leaf ascorbic acid content and redox status were compared in ozone-tolerant (Provider) and ozone-sensitive (S156) genotypes of snap bean ( Phaseolus vulgaris L.). Plants were grown in pots for 24 days under charcoal-filtered air (CF) conditions in open-top field chambers and then maintained as CF controls (29 nmol mol⁻¹ ozone) or exposed to elevated ozone (71 nmol mol⁻¹ ozone). Following a 10-day treatment, mature leaves of the same age were harvested early in the morning (06:00–08:00 h) or in the afternoon (13:00–15:00 h) for analysis of ascorbic acid (AA) and dehydroascorbic acid (DHA). Vacuum infiltration methods were used to separate leaf AA into apoplast and symplast fractions. The total ascorbate content [AA + DHA] of leaf tissue averaged 28% higher in Provider relative to S156, and Provider exhibited a greater capacity to maintain [AA + DHA] content under ozone stress. Apoplast [AA + DHA] content was 2-fold higher in tolerant Provider (360 nmol g⁻¹ FW maximum) relative to sensitive S156 (160 nmol g⁻¹ FW maximum) regardless of sampling period or treatment, supporting the hypothesis that extracellular AA is a factor in ozone tolerance. Apoplast [AA + DHA] levels were significantly higher in the afternoon than early morning for both genotypes, evidence for short-term regulation of extracellular ascorbate content. Total leaf ascorbate was primarily reduced with AA/[AA + DHA] ratios of 0.81–0.90. In contrast, apoplast AA/[AA + DHA] ratios were 0.01–0.60 and depended on genotype and ozone treatment. Provider exhibited a greater capacity to maintain extracellular AA/[AA + DHA] ratios under ozone stress, suggesting that ozone tolerance is associated with apoplast ascorbate redox status.     

Seasonal Changes of the Antioxidative Systems in Foliar Buds and Leaves of Field-grown Beech Trees (Fagus sylvatica,L.) in a Stressful Climate

The activities of superoxide dismutase, ascorbate peroxidase, monodehydroascorbate radical reductase, and dehydroascorbate reductase and the contents of ascorbate, chlorophyll and soluble protein were determined in beech (Fagus sylvatica, L.) foliage over two or three seasons. Four important stages of leaf development were distinguished: resting buds, emerging, mature and senescent leaves. Foliar buds in spring, prior to the emergence of new leaves, contained a lower chlorophyll content but a higher protein content and higher activities of ascorbate peroxidase and monodehydroascorbate radical reductase than mature leaves in summer. By contrast, superoxide dismutase and glutathione reductase activities and ascorbate contents were higher in mature leaves than in swollen foliar buds. Dehydroascorbate reductase activity was low in all developmental stages. Resting buds in winter contained activities of superoxide dismutase, ascorbate peroxidase and monodehydroascorbate radical reductase that were similar to those found in mature leaves in summer, whereas the contents of total and reduced ascorbate were 6- and 20-times lower, respectively, in buds than in mature leaves. The low foliar concentration of reduced ascorbate in resting buds, despite high monodehydroascorbate radical reductase activity, suggests that the regeneration of ascorbate might be limited by the availability of reductant. High antioxidative capacity was conferred by mature beech leaves and may be an important protection measure for coping with the large fluctuations in temperature and exposure to elevated ozone concentrations in summer.     

Ozone sensitivity in Triticum durum and T. aestivum with respect to leaf injury, photosynthetic activity and free radical content

Sensitivity to ozone is highly variable in cultivars of different wheat species, leading to differences in leaf injury and yield. Not much is known about the physiological background of these differences. The objective of this study was to compare the effects of ozone on photosynthetic parameters in Triticum aestivum L. (spring wheat cv. Nandu, winter wheat cv. Perlo) and Triticum durum Desf. (cv. Extradur). Plants cultivated in pots were exposed to 80 nmol mol^?1 ozone, or were used as control plants in a greenhouse. Stages of growth and senescence of single leaves were recorded. Light-saturated net photosynthesis, leaf conductance for water vapour, and chlorophyll fluorescence were measured. Stomatal limitation was calculated from CO₂ response curves, and the free radical content of whole leaves measured by EPR spectroscopy. Senescence of single leaves was enhanced by the ozone-treatment in all three cultivars, in the order Nandu > Perlo > Extradur. Development of whole plants was slightly delayed in Perlo and Nandu, but was accelerated significantly in Extradur. The rate of net photosynthesis under light saturation (A_sat) decreased significantly in older, ozone-fumigated leaves of Perlo and Nandu but not of Extradur. Leaf conductance (g₁) showed a similar behaviour, but stomatal limitation (l) was similar between ozone-treated and control plants. Thus, an ozone-induced closure of stomata was not the reason for the observed difference in A_sat. Perlo and Nandu showed a significant, only partly reversible decrease in F_v/F_m in ozone-fumigated leaves, whereas in Extradur the decrease was fully reversible only in older leaves. Whole leaves of Extradur, in contrast to Perlo and Nandu, showed no increase in EPR free radical signals. The higher ozone tolerance of Extradur was thus not caused by decreased ozone uptake via the stomata, but by a better ability of photosynthetically active mesophyll cells to cope with photooxidative stress.