Both reduced and oxidized ascorbate (AA and DHA) are present in the aqueous phase of the extracellular space, the apoplast, of spinach (Spinacia oleracea 1.) leaves. Fumigation with 0.3 pL 1-' of ozone resulted in ozone uptake by the leaves close to 0.9 pmol cm-' of leaf surface area s-'. Apoplastic AA was slowly oxidized by ozone. The initial decrease of apoplastic AA was ~0 . 1 pmol cm-2 -1 s . 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-'s-'. 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 glutathione 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 pL 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.

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~-' 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 /.imolg"' FW in summer. Except for late April and November, more than 95% of the intracellular ascorbate was reduced. Glutatbione 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 1 d following periods of high ambient ozone concentrations. The transitory accumulation of GSSG may be explained by slow enzymatic regeneration of glutathione.

Abstract. Spinach (Spinacia oleracea L.), broad bean (Viciafaba L.) and beech (Fagus sylvatica L.) plants were exposed to ozone at concentrations often measured in air during the summer months (120-300 pg. 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 lagO3.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 lag 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, lntracellular AA changed its redox state in response to exposure to elevated concentrations of ozone. After 4-6 weeks of fumigation with 200-300 lag 03" 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 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; Va~r = intercellular air space volume of leaves; V,po = apoplasmic water volume of leavesCorrespondence to: U. Heber; Fax: 49 (931)71446; E-mail: heber@botanik.uni-wuerzburg.de concentration (1600 jag-m-a) 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 lag 03" m-3, in broad bean leaves the intracellular DHA concentration increased and intracellular ascorbate became more oxidized after fumigation of the plants with 120 lag 03" m-3. Apparently, broad bean leaves are more sensitive to ozone than beech and spinach leaves.

In a beech (Fagus sylvatica L.) stand in north-west Germany vegetation of two transects (25m: lm and 20m: lm) was mapped and contents of macronutrients (Ca, Mg and K), micronutrients (Fe, Mn, Zn and Cu), and potentially phytotoxic metals (Pb, Cd, Ni and A1) were measured in different soil compartments and in roots, rhizomes, stems and leaves of two forest floor plant species (Mercurialis perennis L. and Polygonatum multiflorum L.). NH4C1 extractable cation contents, pH and other soil variables were also determined.The highest macronutrient contents could be found in the leaves ofM. perennis and P. multiflorum. Heavy metals and AI accumulated in the roots. Correlation analysis suggests a considerable translocation of Zn and Cd between below-and above-ground organs of both investigated forest floor plants. No significant correlation was found between the contents of the other elements in the below-and above-ground parts.Available data indicate a considerable uptake by the plants not only of nutrients, but also of heavy metals from the upper mineral soil. Amounts of heavy metals and A1 solubilized in the presence of NH4CI increased with decreasing pH, whereas levels of soluble Ca and Mg were maximal at high pH-values of the extracts. It can be concluded that element uptake in the investigated plants is indirectly controlled by the pH of the upper mineral soil.

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