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Physiology of young Norway spruce
Environmental Pollution 68 (1990)319-330
Physiology of Young Norway Spruce
E. B e r m a d i n g e r , H. G u t t e n b e r g e r & D. Grill Institut ffir Pflanzenphysiologieder Karl-Franzens-Universit~itGraz, Schubertstr. 51 8010 Graz, Austria
ABSTRACT In the Hohenheim experiment young spruce (Picea abies L. Karst.) were exposed to low levels of SO 2 and~or 03 and acid precipitation. A t the end of a five-year experimental period (1983-88) the following physiological parameters were examined: water soluble thiols, ascorbic acid, glutathionereductase activity and pigment content. Exposure to SO 2, leads to an increase in thiol content, to a slight decrease of ascorbic acid and to a pronounced decrease of pigments. 0 3 exposure increases the content of ascorbic acid and decreases the thiols and the glutathione-reductase activity with no change in the pigment content. The combined exposure to SO 2, and 0 3 results in the most distinct deviations compared to the control chamber response. These needles show the highest increase of ascorbic acid and thiols, the dry weight is decreased as is the glutathione-reductase activity and the pigment content is reduced. Consequences of these physiologieal alterations for the plant's health are discussed.
INTRODUCTION Current interest of research on the impact of air pollutants on trees is focussed on long-term low-level effects (Darral & J~iger, 1984; Darral, 1989; Mehlhorn et al., 1986). Determining impact in the field is very complex, because many different stress factors are involved. Alterations of plant physiology and biochemistry under specific low-level pollution concentrations in a defined environment is possible with long-term open-top chamber experiments (Arndt et al, 1985; Seufert et ak, this volume). 319 Environ. Pollut. 0269-7491/90/$03"50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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Life in an oxygen atmosphere is inevitably connected with the exposure of the cells to radicals even without pollutants (Rennenberg, 1988). Plant cells have evolved special detoxification systems to cope with radicals, which include the antioxidants ascorbic acid and glutathione, the carotenoids and various enzymes (Foyer & Halliwell, 1976; Goodwin, 1980; Rennenberg, 1988). The high reactivity of photo-oxidative compounds and also of SO2 can produce free radicals and/or enhance the natural formation of radicals (J/iger & Klein, 1980; Rennenberg, 1984; Jfiger et al., 1986). Alterations in these scavenging systems may serve as early indicators of air pollution damage to vegetation (Rennenberg, 1984, 1988; Kunert & Ederer, 1985; Jfiger et al., 1986; Mehlhorn et al., 1987; Osswald et al., 1987; Grill et al., 1988). At the end of a five-year exposure period (1983-88) with low pollutant concentrations in open-top chambers (Seufert et al., this volume) the influence of SO2 and/or 03 combined with acidic rain on various physiological reactions of young spruce was investigated. The investigations include the contents of water soluble thiols, ascorbic acid, pigments and glutathione-reductase activity.
M A T E R I A L A N D METHODS In open-top chambers 13-year-old spruce (Picea abies L. Karst.) were exposed to charcoal-filtered air or to charcoal-filtered air supplemented with SO 2 (30 pg cm - 3) and/or 0 3 (50-180 pg cm - 3). Additionally simulated acidic precipitation (pH 4.0) was applied weekly to all gaseous pollutant treatments (control/pH 4.0, SO2/pH 4.0, O J p H 4.0, SO2 + O3/PH 4.0), one control chamber was watered with rain pH 5"6 (control/pH 5.6). For detailed information about the open-top chambers, the experimental design, the pollutant concentrations and additional information about the plant material see Seufert et al. (1990). The samples were harvested in March 1988 from two trees from each chamber. For pigment analysis the needles (needle age classes 1987-85) were cut off the branches immediately after harvesting, 1 g was weighed, frozen and transported in liquid nitrogen to Graz, Austria, for analysis. The other harvested material was immediately transported to Graz without special cooling; there was no danger of transport damage because the weather was very cold and cloudy. In the laboratory in Graz current and previous year's needles (needle year 1987 and 1986) were cut off the branches, 1 g was weighed, frozen in liquid nitrogen and stored at - 25°C until determination. The following parameters were determined:
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(1)
Glutathione-reductase activity: acetone dry powder was prepared from 1 g fresh needles and stored until determination at - 25°C. The enzyme activity was measured photometrically by following the decrease of NADPH-absorbance at 340 nm according to the method of Esterbauer & Grill (1978). (2) The total water soluble thiols were determined photometrically with D T N B (Ellmann reagent) at 412 nm as described in detail by Grill & Esterbauer (1973) and Grill et aL (1982). (3) Ascorbic acid was determined with the isocratic HPLC method according to Bui-Nguyen (1980) and Wimanlasiri & Wills (1983). The ascorbic acid was extracted from the needle homogenate with citric acid and the separation was performed on a column packed with Spherisorb S 5-NH 2 (Forschungszentrum Seibersdorf). The eluent was methanol/0.03M NaH2PO 4 (3/1) and detection occurred at 268 nm. (4) Pigment analysis was performed with a gradient HPLC method according to Pfeifhofer (1989) on a 5/~m Spherisorb ODS-2 column (Forschungszentrum Seibersdorf). Solvent A consisted of acetonitrile/water/methanol = 100/10/5 and solvent B of acetone/ethyl acetate = 2/1. A linear gradient from 10% B to 70% B was performed within 18 min. Detection occurred at 440nm.
The values presented are the average of duplicate analyses, therefore no statistical analyses were performed.
RESULTS The results are presented in Tables 1 and 2. The needles of the chamber with the combined SO2 + 03 treatment have a slight tendency to decreased dry weights. The first-year needles average 90% and the second-year needles average 94 % of control/pH 5.6. The youngest needles of the control/pH 4.0 show a slightly decreased dry weight too (94% of control/pH 5.6), whereas there are no differences between the other samples (Table 1). The content of water soluble thiols is shown in Table 1. As a rule, the water soluble thiols of spruce needles consist of 95-100% glutathione and only a little cysteine (Grill & Esterbauer, 1973; Esterbauer & Grill, 1978; Grill et al., 1982). Both the one- and the two-year-old needles of the ozone exposure show a distinct decrease in the thiol content compared to the control/pH 5-6 (83 % for the first needle year and 68 % for the second needle year). In contrast to this decrease the needles of the SO2 treatment exhibit a clear increase in both needle years (145 % and 138 % respectively). In the combined exposure to
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TABLE 1 Biochemical D a t a of the Different T r e a t m e n t s (Based o n Dry Weight)
Treatments
C o n t r o l / p H 5'6 C o n t r o l / p H 4-0 S O z / p H 4.0 O3/pH 4.0 SO z + O 3 / p H 4.0
Needle )'ear
Water soluble thiols (t~mol g- l)
Ascorbic acid (rag g- 1)
Glutathione reductase (units g- 1)
Dry weight of 1 g fresh weight (g)
1 2 1 2 1 2 1 2 1 2
1.054 0.860 1.027 0.739 1.526 1.185 0.878 0.587 1.463 1.507
-6.61 -7.44
0'98 0-78 1.04 0"46 0'19 0.86 0"54 0-43 0-30 0-19
0.49 0"50 0.46 0"49 0"48 0-50 0.48 0.51 0.44 0.47
5.94 -8"90 -13.11
ozone and SO2 the youngest needles show with 139% (as compared to control/pH 5"6) a similar increase as those of the SO2 exposure, whereas with 175 % the increase of the thiol content of the second-year needles is more pronounced. Both controls do not exhibit differences for either pH treatment. The glutathione-reductase activity is shown in Table 1. In the 03 and the SO2 + 03 treatment both needle years show a reduction in enzyme activity. The 03 treatment was 55% of control/pH 5"6 for each needle year, the SO2 + 0 3 treatment was 31% for the first and 24% for the second year needles. With SO2 the first year needles show an even sharper decrease with only 19% of control/pH 5"6, whereas the enzyme activity of the second needle year is slightly increased (110% as compared to control/pH 5"6). For the two year-old needles of the SO2 treatment a high thiol-content (138 % as compared to control/pH 5"6) is combined with a slightly increased glutathione-reductase activity (110% as compared to control/pH 5.6), whereas needles of both age classes of the combined exposure to ozone + SO2 show a pronounced increase of the thiols with 139% and 175% as compared to control/pH 5.6, but a distinct decrease of the enzyme-activity to 31% and 24% as compared to control/pH 5.6. Ascorbic acid content is shown in Table 1. Because of shortage of material only the two year-old needles were examined. A slight decrease in ascorbic acid content of 90% as compared to the control/pH 5"6 is seen in the needles of the SO 2 exposure, whereas those of the control/pH 4.0 show a slight increase of 113 %. The 03 treatment results in an increase to 135 %, which is even more pronounced in the needles of the SO2 + 03 treatment where ascorbic acid content was 198% that of the control/pH 5"6.
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TABLE2 PigmentContentoftheNeedlesoftheVariousTreatments(ggg -~ Dry Weight) Treatments
Control/pH 5"6
Control/pH 4.0
SO2/PH 4"0
O3/PH 4"0
SO2 + O3/pH 4"0
Needle Ch~rophyll Carotin XanthophyH Viola- Anthera- Zeayear xanthm xanthm xanthin 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
3 886 4640 4 275 3548 4 771 4385 3163 3890 2984 3393 4789 4 525 3206 4136 3 351
162 205 210 141 237 221 155 165 156 136 223 222 179 225 179
874 947 835 778 904 873 802 805 594 744 978 924 795 847 719
45 49 49 43 61 63 55 53 40 55 72 69 78 76 61
60 57 50 46 60 60 60 68 48 56 69 68 70 80 59
135 118 77 115 89 59 108 82 41 96 78 64 90 21 33
Treatments
Needle year
Xanthophyll/ carotin
~-/flcarotin
Chlorophyll a/b
Chlorophyll/ carotinoids
Control/pH 5.6
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
5.84 4.67 3.99 6'08 4.07 3.94 5'76 4-97 3"92 5.70 4.41 4'20 4.49 3.76 3.86
0"22 0-31 0-34 0"23 0"31 0'38 0"20 0'28 0'28 0"35 0-37 0"41 0'19 0"33 0.32
3.43 3'23 3.05 3-47 3' 10 3.00 2'98 3-18 3-13 3"53 3'10 3"04 3"45 3.14 3"08
3.77 4"03 4.10 3'88 4"01 4-02 3'37 4.01 3.96 3-86 3'99 3'96 3"53 3"86 3-88
Control/pH 4.0
SO2/pH 4'0
O3/pH 4'0
SO2 + Os/pH 4.0
Pigment concentration is shown in Table 2. The pigment concentrations of the first year needles (1987) are only of limited use because pigment levels of the youngest needles vary depending on light available. The results of the pigment analyses indicate differing light conditions existing for the trees from the same chamber. For example, when considering the xanthophyll/ carotin-ratio (nondimensional) the average difference between those parallel determinations amounts to 2.37 for the first needle year, 0.91 for the second
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needle year and only 0-32 for the third one. However, these differences are not due to differences between the various chambers as can be seen when comparing to Table 2. The same is true of the other quotients and pigment classes. Due to this lack of consistency only the two-year-old and the threeyear-old needles are evaluated. The analysis of total chlorophyll content (chlorophyll a + b) shows a clear decrease in the content of the second and third year needles from the SO2 exposure as compared to the control/pH 5"6. The chlorophyll content of the two-year-old needles decreases to 84%, that of the three-year-old needles to 70 % as compared to the chlorophyll content of the needles from the control treatment. The combined treatment of SO 2 + 03 also leads to a decrease with levels at 89% and 78% respectively of the controls for the two needle years analysed. But this decrease was not as pronounced as in the needles from the SO2 treatment. No changes in the chlorophyll content were noted in the 0 3 or the varying rain pH treatments. With the carotins (~- and fl-carotin) the results of the analyses are more confusing. SO 2 leads to a clear decrease of 80% and 74% respectively for the two needle years as compared to control/pH 5"6, which is similar to the decrease of the chlorophyll content. The needles taken from the O 3 treatment show a slight increase in the carotin content of 109% and 106% respectively. The combined exposure to SO 2 and ozone also leads to a slight increase in the second-year needles (110% as compared to control/pH 5"6) whereas the third-year needles show a slight decrease of 85%. The evaluation of xanthophyll content (sum of neoxanthin, violaxanthin, antheraxanthin, lutein and zeaxanthin) yields results similar to that of the chlorophyll analyses. SO 2 leads to a distinct decrease of the xanthophyll content in the second and the third year needles with 85% and 71% respectively. The degree of the decrease is the same that is found in the chlorophylls. The combined treatment also decreases the xanthophyll content. But with 89 % and 86% for the two needle years this decrease is not as distinct as SO 2 alone does. The ozone exposure does not change the xanthophyll content of the two-year-old needles and slightly increases the content of the three-year-old needles to 111% as compared to control/pH 5.6. The components of the xanthophyll-cycle are listed separately in Table 2. The two-year-old needles of the ozone treatment and of the combined exposure to SO2 and ozone reveal a higher content of violaxanthin (03: 147%, SO 2 + 03: 155%) and antheraxanthin (O3: 121%, SO2 + 03: 140%) as compared to the control/pH 5.6. In comparison, a sharp decrease of the zeaxanthin content to 66% for the ozone treatment and to 18% of the combined treatment as compared to the control is observable. The same is true for the three-year-old needles, but not as distinct as for the two-year-old needles. Consequently there are pronounced
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differences in the violaxanthin/zeaxanthin-ratios. For the second-year needles in the control/pH 5.6 chamber this quotient amounts 0.42, for the needles from the SO2 treatment 0.65, for the needles from the ozone exposure 0.92 and for the two year-old needles from the combined exposure to SO2 + 03 it is 3.62. The ratios chlorophyll/carotinoids, chlorophyll a/b and xanthophyll/carotin show no distinct differences between the various treatments. But there are changes concerning the quotient ~- to//-carotin. SO2 decreases this quotient in the two-year-old and three-year-old needles to 90% and 82% respectively as compared to control/pH 5.6, whereas 0 3 exposure results in a slight increase to 119% and 121% respectively. The combined exposure to ozone and SO2 does not affect this ratio.
DISCUSSION Because of shortage of available material it was not possible to perform a statistical analysis. Therefore the presented results can only be indications. However, according to Seufert et al. (1989) the presented results of the opentop chamber experiments should be interpreted in such a way that they could give an initial idea of principles of action and help to understand observations in the forest. The acidic rain treatment alone does not apparently alter the physiological parameters examined. However, it is known that acidic precipitation leads to a destruction of the epicuticular waxes (Kazda & Glatzel, 1986; Rinallo et al., 1986; Schmitt et al., 1987). Scanning electron microscopical investigations were performed in this study but an interpretation was not possible because of pronounced mechanical damage and fungal infections, which influence the surface structures. Mechanical damage is a considerable problem in such experiments because the trees stand relatively close together and are repeatedly handled promoting mechanical injury. The pigment analyses were also performed by Siefermann-Harms (this volume) and the results are consistent in the degree of pigment reduction due to the different treatments. The differences in the absolute pigment content may be due to slight differences in the light conditions of the harvested needles. Siefermann-Harms (this volume) found a rather high violaxanthin/ zeaxanthin ratio compared to the other samples in the first-year needles from the combined SO2 + 03 treatment, i.e. these needles contain more violaxanthin compared to zeaxanthin. The presented study also shows a highly decreased zeaxanthin content in the needles from the combined SO2 + 03 exposure, although the results from these analyses are not directly comparable to those of Siefermann-Harms. At the time the spruce samples
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were harvested for this study the one- to three-year-old needles were adapted to daylight under a cloudy sky, whereas the needles analysed by SiefermannHarms were adapted to dark or exposed to strong artificial light. A detailed discussion is provided in Siefermann-Harms (this volume). SO 2 and the combination of SO2 + 03 lead to a decrease in the pigment content, where all pigment groups (chlorophylls, carotins and xanthophylls) are affected. This overall decrease is consistent with Horsman & Wellburn (1976) and Pfeifhofer & Grill (1987). On the contrary an exposure to ozone alone does not seem to influence the pigment content, at least at these low gaseous concentrations used in this experiment. However, at much higher ozone concentrations (up to 2000pgm-3), Senser et al. (1987) observed pronounced alterations in the contents of pigment concentrations. Lichtenthaler & Buschmann (1984) found a reduced ratio of chlorophyll a to chlorophyll b in photo-oxidatively damaged needles. No alterations of this ratio with any treatment were observed in this study. But, as Lichtenthaler & Buschmann (1984) also observed, a slight decrease of/%carotin compared to e-carotin in the needles exposed to ozone was measured resulting in an increase of the e-//~-carotin quotient. The xanthophyll/carotin-quotient did not differ between the various treatments which is consistent with the observation that none of the samples was yellowed, for an increased xanthophyll/carotin-quotient is an indication of yellowing (Pfeifhofer & Grill, 1987). Alterations of photosynthetically active pigments mean alterations of photosynthetic processes, as a consequence of which disturbances of the anabolic metabolism of the plant may occur. However, a decrease in pigment content is an unspecific response to the impact of various stresses and therefore alterations in the pigments are not stressspecific (Pfeifhofer & Grill, 1987), perhaps with the exception of photooxidatively induced stronger decrease of/~-carotin as compared to e-carotin. Therefore pigment analyses are useful for diagnostic assays only in combination with other physiological examinations, i.e. content of antioxidants or enzyme activities (Pfeifhofer & Grill, 1987). It is well known that SO z leads to an increase in the content of water soluble thiols (Grill & Esterbauer, 1973; Grill et al., 1982; Rennenberg, 1984; Mehlhorn et al., 1986; Kunert & Hofer, 1987), which is again demonstrated by these results. Glutathione, the main component of water soluble thiols (Grill & Esterbauer, 1973; Esterbauer & Grill, 1978; Grill et aL, 1982), appears to be the storage form of reduced sulphur in higher plants (Rennenberg, 1984). Consistently, the needle sulphur content increased due to the SO 2 fumigation indicating uptake and incorporation of the gaseous pollutant (Bender et al., 1990). On the contrary, the ozone treatment results in a decreased thiol content and an even more pronounced decrease of the
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activity of the glutathione-reductase. As this enzyme appears to be very sensitive to photo-oxidative influences (A. Schmidt, pers. comm.) this may indicate disturbances in the glutathione-reductase dependent process of regenerating reduced glutathione (GSH) from the oxidized glutathione (GSSG), a process which is necessary for the maintenance of the radical detoxifying system in chloroplasts (Jfiger et al., 1976; Halliwell, 1984). After exposure to ozone a slight increase in ascorbic acid content was found in the presented study, though these results are not statistically provided. This is consistent with Barnes (1972), Lee et ak (1984) and Mehlhorn et ak (1986), who found a slight increase after exposure to slight ozone concentrations, whereas Kunert & Hofer (1987) did not find significant alterations of the ascorbic acid content due to ozone. Contrary to the results presented here which reveal even a slight decrease of ascorbic acid due to SO 2 Mehlhorn et al. (1986) and Kunert & Hofer (1987) observed a distinct increase of ascorbic acid due to SO2. Grill et al. (1979) also observed a decrease of ascorbic acid content caused by the impact of SO2. The combined exposure to SO 2 and ozone resulted in the greatest increase of the antioxidants glutathione and ascorbic acid, consistent with the results of Mehlhorn et al. (1986), Osswald & Elstner (1986), Kunert & Hofer (1987), Osswald et al. (1987). Contrary to the increase of the thiol content the activity of the glutathione-reductase is extremely low which is in contrast to the observations of Kunert & Hofer (1987) and Mehlhorn et al. (1987), who found a strong increase in the enzyme activity after similar treatments. This contrasting behavior of thiols and glutathione-reductase may indicate that the enhancement of the thiols reflects the SO2 impact as thiols serve as storage for reduced sulphur (Rennenberg, 1984). Therefore, the increased thiol content is not mainly due to an enhancement of the glutathione of the detoxifying system in chloroplasts. On the other hand the strong decrease of the enzyme activity reflects the ozone impact. These results may possibly indicate that an increased level of glutathione observed in field investigations (e.g. Osswald & Elstner, 1986; Osswald et al., 1987; Grill et al., 1988; Bermadinger et aL,1989) is due to the synergistic effect of ozone and SO 2 but is not due to ozone alone. An increase in the content of the antioxidant ascorbic acid means an enhancement of the detoxifying system in the plant cell, due to the potency of these pollutants in inducing oxidative processes in cells (J~iger et al., 1986; Mehlhorn et al., 1986, 1987; Osswald et aL, 1987; Grill et al., 1988). According to Rennenberg (1988) the influence of pollutants, such as SO2 and ozone, results in metabolic alterations and the onset of detoxification and balancing mechanisms, i.e. increase of antioxidants. As a consequence a new equilibrium is developed. If this new developed equilibrium is exceeded by additional stress severe cell damage will occur (Rennenberg, 1988).
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Contradictions to former investigations within the scope of the Hohenheim open-top chamber experiments (Kunert & Hofer, 1987; Mehlhorn et al., 1986, 1987) can be possibly explained by the occurrence of different equilibrium states according to Rennenberg (1988). Pronounced seasonal variations of those detoxifying systems occurring in spruce (Bermadinger et al., 1989) may also be responsible for contradictory results if the samples were not harvested during comparable physiologically developmental states. Bender et al. (this volume) observed significantly higher peroxidase activities in needles from the combined treatment, which is an early but unspecific metabolic response to stress situations in plants. According to H a m p p et al. (this volume) increased A T P / A D P and redox ratios in needles from the combination experiment could be due to both repair mechanisms or increased rates of leaf senescence. Siefermann-Harms (this volume) also observed alterations in the pigment contents. To sum up, the needles from the combined exposure exhibit the greatest deviation from the control indicating pronounced metabolic alterations. However, it is extremely difficult to distinguish between observations still reflecting protection and those already indicating damage (Elstner, 1982). Results from this open-top chamber experiment are consistent with field investigations which were performed in different regions of Austria. In studying a profile in Western Styria (Grill et al., 1988) and in Tirol (Bermadinger et al., 1989) it was possible to differentiate between samples collected from lower elevations dominated by SO 2 and higher elevations dominated by ozone (Western Styria) or by a combination o f SO 2 and ozone (Tirol). REFERENCES Arndt, U., Seufert, G., Bender, J. & J/iger, H. J. (1985). Untersuchungen zum Stoffhaushalt von Waldbfiumen aus belasteten Modell6kosystemen in Opentop Kammern. VDI-Berichte, 560, 783-803. Barnes, R. L. (1972). Effects of chronic exposure to ozone on soluble sugar and ascorbic acid contents of pine seedlings. Can. J. Bot., 50, 215-9. Bender, J., Manderscheid, R. & J/iger, H. J. (1990). Analyses of enzyme activities and other metabolic criteria after five years of fumigation. Environ. Pollut. (this volume). Bermadinger, E., Grill, D. & Guttenberger, H. (1989). Thiole, Ascorbins/iure, Pigmente und Epikutikularwachse in Fichtennadeln aus dem 'H6henprofil Zillertal'. Phyton (Austria), 29(3), 163-85. Bui-Nguyen, M . H . (1980). Application of high-performance liquid chromatography to the separation of ascorbic acid from isoascorbic acid. J. Chromatogr., 196, 163-5. Darral, N. M. (1989). The effects of air pollutants on physiological processes in plants. Plant, Cell and Environ., 12, 1-30.
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Darral, N. M. & J~iger, H. J. (1984). Biochemical diagnostic tests for the effects of air pollution on plants. In Gaseous Air Pollutants and Plant Metabolism, ed. M. J. Koziol & F. R. Whatley. Butterworth, London, pp. 333-49. Elstner, E. F. (1982). Oxygen activation and oxygen activity. Ann. Rev. Plant Physiol., 33, 73-96. Esterbauer, H. & Grill, D. (1978). Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiol., 61, 119-21. Foyer, C. H., Hailiwell, B. (1976). The presence of glutathione and glutathione reductase in chloroplasts: A proposed role in ascorbic acid metabolism. Planta, 133, 21-5. Goodwin, T. W. (1980). The Biochemistry of the Carotenoids, Vol. l: Plants, Chapman and Hall, London-New York. Grill, D. & Esterbauer, H. (1973). Quantitative Bestimmung wasseri6slicher Sulfhydrilverbindungen in gesunden und SO2-gesch/idigten Nadeln von Picea abies. Phyton (Austria), 15, 87-101. Grill, D., Esterbauer, H. & Hellig, K. (1982). Further studies on the effect of SO2pollution on the sulfhydril-systems of plants. Phytopath. Z., 104, 264-71. Grill, D., Esterbauer, H. & Welt, R. (1979). EinfluB von SO 2 auf das Ascorbins/iuresystem der Fichtennadeln. Phytopath. Z., 96, 361-8. Grill, D., Kern, T., Bermadinger, E. & J~iger, H. J. (1988). Physiologische Reaktionen yon Fichten in Inversionszonen. GSF-Bericht, 17, 391-9. Halliwell, B. (1984). Chloroplast Metabolism, Clarendon Press, Oxford. Hampp, R., Einig, W. & Egger, B. (1990). Energy and redox status and carbon allocation in one- to three-year-old spruce needles. Environ. Pollut. (this volume). Horsman, D. C. & Wellburn, A. R. (1976). Guide to the metabolic and biochemical effects of air pollutants on higher plants. In Effects of Air Pollutants on Plants, ed. T. A. Mansfield. Cambridge University Press, London, pp. 185-99. J~ger, H. J. & Klein, H. (1980). Biochemical and physiological effects of SO 2 on plants. Angew. Bot., 54, 337-48. Jfiger, H. J., Weigel, H. J. & Grfinhage, L. (1986). Physiologische und biochemische Aspekte der Wirkung von Immissionen auf Waldb~ume Eur. J. For. Path., 16, 98-109. Kazda, M. & Glatzel, G. (1986). Schadstoffbelasteter Nebel f6rdert die Infektion yon Fichtennadeln durch pathogene Pilze. AFZ, 18, 436-8. Kunert, K. J. & Ederer, M. (1985). Leaf aging and lipid peroxidation: The role of the antioxidants vitamin C and E. Physiol. Plant, 65, 85-8. Kunert, K. J. & Hofer, G. (1987). Geben Ver/inderungen des antioxidativen Systems yon Pflanzen Hinweise auf die Wirkung yon Luftschadstoffen? AFZ, 2712gl29, 697-9. Lee, E. H., Jersey, J. A., Gifford, C. & Bennett, J. (1984). Differential ozone tolerance in soybean and snapbeans: Analysis of ascorbic acid in O3-susceptible and O aresistant cultivars by high-performance liquid chromatography. Environ. Exp. Bot., 24, 331-41. Lichtenthaler, H. K. & Buschmann, C. (1984). Das Waldsterben-Verlauf, Ursachen und Konsequenzen. Fridericiana, 33, 39-60. Mehlhorn, H., Cottam, D. A., Lucas, P. W. & Wellburn, A. R. (1987). Induction of ascorbate peroxidase and glutathione reductase activities by interaction of mixtures of air pollutants. Free Rad. Res. Comms., 3, 193-7.
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Mehlhorn, H., Seufert, G., Schmidt, A. & Kunert, K. J. (1986). Effects o f SO 2 and 0 3 on production of antioxidants in conifers. Plant Physiol., 82, 336--8. Osswald, W. F. & Elstner, E. F. (1986). Fichtenerkrankungen in den Hochlagen des Bayerischen Mittelgebirges. Bet. Deutsch. Bot. Ges., 99, 313-39. Osswald, W. F., Senger, H. & Elstner, E. F. (1987). Ascorbic acid and glutathione content of spruce needles from different locations in Bavaria. Z. Naturforsch., 42c, 879-84. Pfeifhofer, H. (1989). Evidence for chlorophyll b and lack of lutein in Neottia nidusavis plastids. Biochem. Physiol. Pfl., 184, 55-61. Pfeifhofer, H. & Grill, D. (1987). Pigmentuntersuchungen an Fichten mit 'klassischen' und 'neuartigen' Waldschfiden. In FIW-Bericht, ed. Bundesministerium f/Jr Wissenschaft und Forschung, Wien, pp. 124-34. Rennenberg, H. (1984). The fate of excess sulfur in higher plants. Ann. Rev. Plant Physiol., 35, 121-53. Rennenberg, H. (1988). Wirkungen yon Photooxidantien aufPflanzen. GSF-Bericht, 17, 360-70. Rinalio, C., Raddi, P., Gellini, R. & DiLonardo, V. (1986). Effects of simulated acid deposition on the surface structure of Norway spruce and silver fir needles. Eur. J. For. Path., 16, 440-6. Schmitt, U., Ruetze, M. & Liese, W. (1987). Rasterelektronenmikroskopische Untersuchungen an Stomata von Fichten- und Tannennadeln nach Begasung und saurer Beregnung. Eur. J. For. Path., 17, 118-24. Senser, M., H6pker, K.-A. & Peuker, A. (1987). Wirkungen extremer Ozonkonzentrationen auf Koniferen. AFZ, 27/28[29, 709-14. Seufert, G., Hoyer, V., W611mer, H. S. & Arndt, U. (1990). General methods and materials. Environ. Pollut. (this volume). Seufert, G., Arndt, U., J/iger, H. J. & Bender, J. (1989). Long-term effects of air pollutants on forest trees in open-top chambers. I: Experimental approach and results in mineral cycling. In Air Pollution andForest Decline, ed. J. Bucher & I. Bucher-Wallin. Birmensdorf, pp. 159-65. Siefermann-Harms, D. (1990). Chlorophyll, carotenoids and the activity of the xanthophyll cycle. Environ. Pollut. (this volume). Wimanlasiri, P. & Wills, R. B. H. (1983). Simultaneous analysis of ascorbic acid and dehydroascorbic acid in fruit and vegetables by high performance liquid chromatography. J. Chromat., 256, 368-71.
Physiology of Young Norway Spruce
E. B e r m a d i n g e r , H. G u t t e n b e r g e r & D. Grill Institut ffir Pflanzenphysiologieder Karl-Franzens-Universit~itGraz, Schubertstr. 51 8010 Graz, Austria
ABSTRACT In the Hohenheim experiment young spruce (Picea abies L. Karst.) were exposed to low levels of SO 2 and~or 03 and acid precipitation. A t the end of a five-year experimental period (1983-88) the following physiological parameters were examined: water soluble thiols, ascorbic acid, glutathionereductase activity and pigment content. Exposure to SO 2, leads to an increase in thiol content, to a slight decrease of ascorbic acid and to a pronounced decrease of pigments. 0 3 exposure increases the content of ascorbic acid and decreases the thiols and the glutathione-reductase activity with no change in the pigment content. The combined exposure to SO 2, and 0 3 results in the most distinct deviations compared to the control chamber response. These needles show the highest increase of ascorbic acid and thiols, the dry weight is decreased as is the glutathione-reductase activity and the pigment content is reduced. Consequences of these physiologieal alterations for the plant's health are discussed.
INTRODUCTION Current interest of research on the impact of air pollutants on trees is focussed on long-term low-level effects (Darral & J~iger, 1984; Darral, 1989; Mehlhorn et al., 1986). Determining impact in the field is very complex, because many different stress factors are involved. Alterations of plant physiology and biochemistry under specific low-level pollution concentrations in a defined environment is possible with long-term open-top chamber experiments (Arndt et al, 1985; Seufert et ak, this volume). 319 Environ. Pollut. 0269-7491/90/$03"50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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Life in an oxygen atmosphere is inevitably connected with the exposure of the cells to radicals even without pollutants (Rennenberg, 1988). Plant cells have evolved special detoxification systems to cope with radicals, which include the antioxidants ascorbic acid and glutathione, the carotenoids and various enzymes (Foyer & Halliwell, 1976; Goodwin, 1980; Rennenberg, 1988). The high reactivity of photo-oxidative compounds and also of SO2 can produce free radicals and/or enhance the natural formation of radicals (J/iger & Klein, 1980; Rennenberg, 1984; Jfiger et al., 1986). Alterations in these scavenging systems may serve as early indicators of air pollution damage to vegetation (Rennenberg, 1984, 1988; Kunert & Ederer, 1985; Jfiger et al., 1986; Mehlhorn et al., 1987; Osswald et al., 1987; Grill et al., 1988). At the end of a five-year exposure period (1983-88) with low pollutant concentrations in open-top chambers (Seufert et al., this volume) the influence of SO2 and/or 03 combined with acidic rain on various physiological reactions of young spruce was investigated. The investigations include the contents of water soluble thiols, ascorbic acid, pigments and glutathione-reductase activity.
M A T E R I A L A N D METHODS In open-top chambers 13-year-old spruce (Picea abies L. Karst.) were exposed to charcoal-filtered air or to charcoal-filtered air supplemented with SO 2 (30 pg cm - 3) and/or 0 3 (50-180 pg cm - 3). Additionally simulated acidic precipitation (pH 4.0) was applied weekly to all gaseous pollutant treatments (control/pH 4.0, SO2/pH 4.0, O J p H 4.0, SO2 + O3/PH 4.0), one control chamber was watered with rain pH 5"6 (control/pH 5.6). For detailed information about the open-top chambers, the experimental design, the pollutant concentrations and additional information about the plant material see Seufert et al. (1990). The samples were harvested in March 1988 from two trees from each chamber. For pigment analysis the needles (needle age classes 1987-85) were cut off the branches immediately after harvesting, 1 g was weighed, frozen and transported in liquid nitrogen to Graz, Austria, for analysis. The other harvested material was immediately transported to Graz without special cooling; there was no danger of transport damage because the weather was very cold and cloudy. In the laboratory in Graz current and previous year's needles (needle year 1987 and 1986) were cut off the branches, 1 g was weighed, frozen in liquid nitrogen and stored at - 25°C until determination. The following parameters were determined:
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(1)
Glutathione-reductase activity: acetone dry powder was prepared from 1 g fresh needles and stored until determination at - 25°C. The enzyme activity was measured photometrically by following the decrease of NADPH-absorbance at 340 nm according to the method of Esterbauer & Grill (1978). (2) The total water soluble thiols were determined photometrically with D T N B (Ellmann reagent) at 412 nm as described in detail by Grill & Esterbauer (1973) and Grill et aL (1982). (3) Ascorbic acid was determined with the isocratic HPLC method according to Bui-Nguyen (1980) and Wimanlasiri & Wills (1983). The ascorbic acid was extracted from the needle homogenate with citric acid and the separation was performed on a column packed with Spherisorb S 5-NH 2 (Forschungszentrum Seibersdorf). The eluent was methanol/0.03M NaH2PO 4 (3/1) and detection occurred at 268 nm. (4) Pigment analysis was performed with a gradient HPLC method according to Pfeifhofer (1989) on a 5/~m Spherisorb ODS-2 column (Forschungszentrum Seibersdorf). Solvent A consisted of acetonitrile/water/methanol = 100/10/5 and solvent B of acetone/ethyl acetate = 2/1. A linear gradient from 10% B to 70% B was performed within 18 min. Detection occurred at 440nm.
The values presented are the average of duplicate analyses, therefore no statistical analyses were performed.
RESULTS The results are presented in Tables 1 and 2. The needles of the chamber with the combined SO2 + 03 treatment have a slight tendency to decreased dry weights. The first-year needles average 90% and the second-year needles average 94 % of control/pH 5.6. The youngest needles of the control/pH 4.0 show a slightly decreased dry weight too (94% of control/pH 5.6), whereas there are no differences between the other samples (Table 1). The content of water soluble thiols is shown in Table 1. As a rule, the water soluble thiols of spruce needles consist of 95-100% glutathione and only a little cysteine (Grill & Esterbauer, 1973; Esterbauer & Grill, 1978; Grill et al., 1982). Both the one- and the two-year-old needles of the ozone exposure show a distinct decrease in the thiol content compared to the control/pH 5-6 (83 % for the first needle year and 68 % for the second needle year). In contrast to this decrease the needles of the SO2 treatment exhibit a clear increase in both needle years (145 % and 138 % respectively). In the combined exposure to
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TABLE 1 Biochemical D a t a of the Different T r e a t m e n t s (Based o n Dry Weight)
Treatments
C o n t r o l / p H 5'6 C o n t r o l / p H 4-0 S O z / p H 4.0 O3/pH 4.0 SO z + O 3 / p H 4.0
Needle )'ear
Water soluble thiols (t~mol g- l)
Ascorbic acid (rag g- 1)
Glutathione reductase (units g- 1)
Dry weight of 1 g fresh weight (g)
1 2 1 2 1 2 1 2 1 2
1.054 0.860 1.027 0.739 1.526 1.185 0.878 0.587 1.463 1.507
-6.61 -7.44
0'98 0-78 1.04 0"46 0'19 0.86 0"54 0-43 0-30 0-19
0.49 0"50 0.46 0"49 0"48 0-50 0.48 0.51 0.44 0.47
5.94 -8"90 -13.11
ozone and SO2 the youngest needles show with 139% (as compared to control/pH 5"6) a similar increase as those of the SO2 exposure, whereas with 175 % the increase of the thiol content of the second-year needles is more pronounced. Both controls do not exhibit differences for either pH treatment. The glutathione-reductase activity is shown in Table 1. In the 03 and the SO2 + 03 treatment both needle years show a reduction in enzyme activity. The 03 treatment was 55% of control/pH 5"6 for each needle year, the SO2 + 0 3 treatment was 31% for the first and 24% for the second year needles. With SO2 the first year needles show an even sharper decrease with only 19% of control/pH 5"6, whereas the enzyme activity of the second needle year is slightly increased (110% as compared to control/pH 5"6). For the two year-old needles of the SO2 treatment a high thiol-content (138 % as compared to control/pH 5"6) is combined with a slightly increased glutathione-reductase activity (110% as compared to control/pH 5.6), whereas needles of both age classes of the combined exposure to ozone + SO2 show a pronounced increase of the thiols with 139% and 175% as compared to control/pH 5.6, but a distinct decrease of the enzyme-activity to 31% and 24% as compared to control/pH 5.6. Ascorbic acid content is shown in Table 1. Because of shortage of material only the two year-old needles were examined. A slight decrease in ascorbic acid content of 90% as compared to the control/pH 5"6 is seen in the needles of the SO 2 exposure, whereas those of the control/pH 4.0 show a slight increase of 113 %. The 03 treatment results in an increase to 135 %, which is even more pronounced in the needles of the SO2 + 03 treatment where ascorbic acid content was 198% that of the control/pH 5"6.
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TABLE2 PigmentContentoftheNeedlesoftheVariousTreatments(ggg -~ Dry Weight) Treatments
Control/pH 5"6
Control/pH 4.0
SO2/PH 4"0
O3/PH 4"0
SO2 + O3/pH 4"0
Needle Ch~rophyll Carotin XanthophyH Viola- Anthera- Zeayear xanthm xanthm xanthin 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
3 886 4640 4 275 3548 4 771 4385 3163 3890 2984 3393 4789 4 525 3206 4136 3 351
162 205 210 141 237 221 155 165 156 136 223 222 179 225 179
874 947 835 778 904 873 802 805 594 744 978 924 795 847 719
45 49 49 43 61 63 55 53 40 55 72 69 78 76 61
60 57 50 46 60 60 60 68 48 56 69 68 70 80 59
135 118 77 115 89 59 108 82 41 96 78 64 90 21 33
Treatments
Needle year
Xanthophyll/ carotin
~-/flcarotin
Chlorophyll a/b
Chlorophyll/ carotinoids
Control/pH 5.6
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
5.84 4.67 3.99 6'08 4.07 3.94 5'76 4-97 3"92 5.70 4.41 4'20 4.49 3.76 3.86
0"22 0-31 0-34 0"23 0"31 0'38 0"20 0'28 0'28 0"35 0-37 0"41 0'19 0"33 0.32
3.43 3'23 3.05 3-47 3' 10 3.00 2'98 3-18 3-13 3"53 3'10 3"04 3"45 3.14 3"08
3.77 4"03 4.10 3'88 4"01 4-02 3'37 4.01 3.96 3-86 3'99 3'96 3"53 3"86 3-88
Control/pH 4.0
SO2/pH 4'0
O3/pH 4'0
SO2 + Os/pH 4.0
Pigment concentration is shown in Table 2. The pigment concentrations of the first year needles (1987) are only of limited use because pigment levels of the youngest needles vary depending on light available. The results of the pigment analyses indicate differing light conditions existing for the trees from the same chamber. For example, when considering the xanthophyll/ carotin-ratio (nondimensional) the average difference between those parallel determinations amounts to 2.37 for the first needle year, 0.91 for the second
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needle year and only 0-32 for the third one. However, these differences are not due to differences between the various chambers as can be seen when comparing to Table 2. The same is true of the other quotients and pigment classes. Due to this lack of consistency only the two-year-old and the threeyear-old needles are evaluated. The analysis of total chlorophyll content (chlorophyll a + b) shows a clear decrease in the content of the second and third year needles from the SO2 exposure as compared to the control/pH 5"6. The chlorophyll content of the two-year-old needles decreases to 84%, that of the three-year-old needles to 70 % as compared to the chlorophyll content of the needles from the control treatment. The combined treatment of SO 2 + 03 also leads to a decrease with levels at 89% and 78% respectively of the controls for the two needle years analysed. But this decrease was not as pronounced as in the needles from the SO2 treatment. No changes in the chlorophyll content were noted in the 0 3 or the varying rain pH treatments. With the carotins (~- and fl-carotin) the results of the analyses are more confusing. SO 2 leads to a clear decrease of 80% and 74% respectively for the two needle years as compared to control/pH 5"6, which is similar to the decrease of the chlorophyll content. The needles taken from the O 3 treatment show a slight increase in the carotin content of 109% and 106% respectively. The combined exposure to SO 2 and ozone also leads to a slight increase in the second-year needles (110% as compared to control/pH 5"6) whereas the third-year needles show a slight decrease of 85%. The evaluation of xanthophyll content (sum of neoxanthin, violaxanthin, antheraxanthin, lutein and zeaxanthin) yields results similar to that of the chlorophyll analyses. SO 2 leads to a distinct decrease of the xanthophyll content in the second and the third year needles with 85% and 71% respectively. The degree of the decrease is the same that is found in the chlorophylls. The combined treatment also decreases the xanthophyll content. But with 89 % and 86% for the two needle years this decrease is not as distinct as SO 2 alone does. The ozone exposure does not change the xanthophyll content of the two-year-old needles and slightly increases the content of the three-year-old needles to 111% as compared to control/pH 5.6. The components of the xanthophyll-cycle are listed separately in Table 2. The two-year-old needles of the ozone treatment and of the combined exposure to SO2 and ozone reveal a higher content of violaxanthin (03: 147%, SO 2 + 03: 155%) and antheraxanthin (O3: 121%, SO2 + 03: 140%) as compared to the control/pH 5.6. In comparison, a sharp decrease of the zeaxanthin content to 66% for the ozone treatment and to 18% of the combined treatment as compared to the control is observable. The same is true for the three-year-old needles, but not as distinct as for the two-year-old needles. Consequently there are pronounced
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differences in the violaxanthin/zeaxanthin-ratios. For the second-year needles in the control/pH 5.6 chamber this quotient amounts 0.42, for the needles from the SO2 treatment 0.65, for the needles from the ozone exposure 0.92 and for the two year-old needles from the combined exposure to SO2 + 03 it is 3.62. The ratios chlorophyll/carotinoids, chlorophyll a/b and xanthophyll/carotin show no distinct differences between the various treatments. But there are changes concerning the quotient ~- to//-carotin. SO2 decreases this quotient in the two-year-old and three-year-old needles to 90% and 82% respectively as compared to control/pH 5.6, whereas 0 3 exposure results in a slight increase to 119% and 121% respectively. The combined exposure to ozone and SO2 does not affect this ratio.
DISCUSSION Because of shortage of available material it was not possible to perform a statistical analysis. Therefore the presented results can only be indications. However, according to Seufert et al. (1989) the presented results of the opentop chamber experiments should be interpreted in such a way that they could give an initial idea of principles of action and help to understand observations in the forest. The acidic rain treatment alone does not apparently alter the physiological parameters examined. However, it is known that acidic precipitation leads to a destruction of the epicuticular waxes (Kazda & Glatzel, 1986; Rinallo et al., 1986; Schmitt et al., 1987). Scanning electron microscopical investigations were performed in this study but an interpretation was not possible because of pronounced mechanical damage and fungal infections, which influence the surface structures. Mechanical damage is a considerable problem in such experiments because the trees stand relatively close together and are repeatedly handled promoting mechanical injury. The pigment analyses were also performed by Siefermann-Harms (this volume) and the results are consistent in the degree of pigment reduction due to the different treatments. The differences in the absolute pigment content may be due to slight differences in the light conditions of the harvested needles. Siefermann-Harms (this volume) found a rather high violaxanthin/ zeaxanthin ratio compared to the other samples in the first-year needles from the combined SO2 + 03 treatment, i.e. these needles contain more violaxanthin compared to zeaxanthin. The presented study also shows a highly decreased zeaxanthin content in the needles from the combined SO2 + 03 exposure, although the results from these analyses are not directly comparable to those of Siefermann-Harms. At the time the spruce samples
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were harvested for this study the one- to three-year-old needles were adapted to daylight under a cloudy sky, whereas the needles analysed by SiefermannHarms were adapted to dark or exposed to strong artificial light. A detailed discussion is provided in Siefermann-Harms (this volume). SO 2 and the combination of SO2 + 03 lead to a decrease in the pigment content, where all pigment groups (chlorophylls, carotins and xanthophylls) are affected. This overall decrease is consistent with Horsman & Wellburn (1976) and Pfeifhofer & Grill (1987). On the contrary an exposure to ozone alone does not seem to influence the pigment content, at least at these low gaseous concentrations used in this experiment. However, at much higher ozone concentrations (up to 2000pgm-3), Senser et al. (1987) observed pronounced alterations in the contents of pigment concentrations. Lichtenthaler & Buschmann (1984) found a reduced ratio of chlorophyll a to chlorophyll b in photo-oxidatively damaged needles. No alterations of this ratio with any treatment were observed in this study. But, as Lichtenthaler & Buschmann (1984) also observed, a slight decrease of/%carotin compared to e-carotin in the needles exposed to ozone was measured resulting in an increase of the e-//~-carotin quotient. The xanthophyll/carotin-quotient did not differ between the various treatments which is consistent with the observation that none of the samples was yellowed, for an increased xanthophyll/carotin-quotient is an indication of yellowing (Pfeifhofer & Grill, 1987). Alterations of photosynthetically active pigments mean alterations of photosynthetic processes, as a consequence of which disturbances of the anabolic metabolism of the plant may occur. However, a decrease in pigment content is an unspecific response to the impact of various stresses and therefore alterations in the pigments are not stressspecific (Pfeifhofer & Grill, 1987), perhaps with the exception of photooxidatively induced stronger decrease of/~-carotin as compared to e-carotin. Therefore pigment analyses are useful for diagnostic assays only in combination with other physiological examinations, i.e. content of antioxidants or enzyme activities (Pfeifhofer & Grill, 1987). It is well known that SO z leads to an increase in the content of water soluble thiols (Grill & Esterbauer, 1973; Grill et al., 1982; Rennenberg, 1984; Mehlhorn et al., 1986; Kunert & Hofer, 1987), which is again demonstrated by these results. Glutathione, the main component of water soluble thiols (Grill & Esterbauer, 1973; Esterbauer & Grill, 1978; Grill et aL, 1982), appears to be the storage form of reduced sulphur in higher plants (Rennenberg, 1984). Consistently, the needle sulphur content increased due to the SO 2 fumigation indicating uptake and incorporation of the gaseous pollutant (Bender et al., 1990). On the contrary, the ozone treatment results in a decreased thiol content and an even more pronounced decrease of the
Physiology of young Norway spruce
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activity of the glutathione-reductase. As this enzyme appears to be very sensitive to photo-oxidative influences (A. Schmidt, pers. comm.) this may indicate disturbances in the glutathione-reductase dependent process of regenerating reduced glutathione (GSH) from the oxidized glutathione (GSSG), a process which is necessary for the maintenance of the radical detoxifying system in chloroplasts (Jfiger et al., 1976; Halliwell, 1984). After exposure to ozone a slight increase in ascorbic acid content was found in the presented study, though these results are not statistically provided. This is consistent with Barnes (1972), Lee et ak (1984) and Mehlhorn et ak (1986), who found a slight increase after exposure to slight ozone concentrations, whereas Kunert & Hofer (1987) did not find significant alterations of the ascorbic acid content due to ozone. Contrary to the results presented here which reveal even a slight decrease of ascorbic acid due to SO 2 Mehlhorn et al. (1986) and Kunert & Hofer (1987) observed a distinct increase of ascorbic acid due to SO2. Grill et al. (1979) also observed a decrease of ascorbic acid content caused by the impact of SO2. The combined exposure to SO 2 and ozone resulted in the greatest increase of the antioxidants glutathione and ascorbic acid, consistent with the results of Mehlhorn et al. (1986), Osswald & Elstner (1986), Kunert & Hofer (1987), Osswald et al. (1987). Contrary to the increase of the thiol content the activity of the glutathione-reductase is extremely low which is in contrast to the observations of Kunert & Hofer (1987) and Mehlhorn et al. (1987), who found a strong increase in the enzyme activity after similar treatments. This contrasting behavior of thiols and glutathione-reductase may indicate that the enhancement of the thiols reflects the SO2 impact as thiols serve as storage for reduced sulphur (Rennenberg, 1984). Therefore, the increased thiol content is not mainly due to an enhancement of the glutathione of the detoxifying system in chloroplasts. On the other hand the strong decrease of the enzyme activity reflects the ozone impact. These results may possibly indicate that an increased level of glutathione observed in field investigations (e.g. Osswald & Elstner, 1986; Osswald et al., 1987; Grill et al., 1988; Bermadinger et aL,1989) is due to the synergistic effect of ozone and SO 2 but is not due to ozone alone. An increase in the content of the antioxidant ascorbic acid means an enhancement of the detoxifying system in the plant cell, due to the potency of these pollutants in inducing oxidative processes in cells (J~iger et al., 1986; Mehlhorn et al., 1986, 1987; Osswald et aL, 1987; Grill et al., 1988). According to Rennenberg (1988) the influence of pollutants, such as SO2 and ozone, results in metabolic alterations and the onset of detoxification and balancing mechanisms, i.e. increase of antioxidants. As a consequence a new equilibrium is developed. If this new developed equilibrium is exceeded by additional stress severe cell damage will occur (Rennenberg, 1988).
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Contradictions to former investigations within the scope of the Hohenheim open-top chamber experiments (Kunert & Hofer, 1987; Mehlhorn et al., 1986, 1987) can be possibly explained by the occurrence of different equilibrium states according to Rennenberg (1988). Pronounced seasonal variations of those detoxifying systems occurring in spruce (Bermadinger et al., 1989) may also be responsible for contradictory results if the samples were not harvested during comparable physiologically developmental states. Bender et al. (this volume) observed significantly higher peroxidase activities in needles from the combined treatment, which is an early but unspecific metabolic response to stress situations in plants. According to H a m p p et al. (this volume) increased A T P / A D P and redox ratios in needles from the combination experiment could be due to both repair mechanisms or increased rates of leaf senescence. Siefermann-Harms (this volume) also observed alterations in the pigment contents. To sum up, the needles from the combined exposure exhibit the greatest deviation from the control indicating pronounced metabolic alterations. However, it is extremely difficult to distinguish between observations still reflecting protection and those already indicating damage (Elstner, 1982). Results from this open-top chamber experiment are consistent with field investigations which were performed in different regions of Austria. In studying a profile in Western Styria (Grill et al., 1988) and in Tirol (Bermadinger et al., 1989) it was possible to differentiate between samples collected from lower elevations dominated by SO 2 and higher elevations dominated by ozone (Western Styria) or by a combination o f SO 2 and ozone (Tirol). REFERENCES Arndt, U., Seufert, G., Bender, J. & J/iger, H. J. (1985). Untersuchungen zum Stoffhaushalt von Waldbfiumen aus belasteten Modell6kosystemen in Opentop Kammern. VDI-Berichte, 560, 783-803. Barnes, R. L. (1972). Effects of chronic exposure to ozone on soluble sugar and ascorbic acid contents of pine seedlings. Can. J. Bot., 50, 215-9. Bender, J., Manderscheid, R. & J/iger, H. J. (1990). Analyses of enzyme activities and other metabolic criteria after five years of fumigation. Environ. Pollut. (this volume). Bermadinger, E., Grill, D. & Guttenberger, H. (1989). Thiole, Ascorbins/iure, Pigmente und Epikutikularwachse in Fichtennadeln aus dem 'H6henprofil Zillertal'. Phyton (Austria), 29(3), 163-85. Bui-Nguyen, M . H . (1980). Application of high-performance liquid chromatography to the separation of ascorbic acid from isoascorbic acid. J. Chromatogr., 196, 163-5. Darral, N. M. (1989). The effects of air pollutants on physiological processes in plants. Plant, Cell and Environ., 12, 1-30.
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