Glutathione status and glutathione reductase activity in spruce needles of healthy and damaged trees at two mountain sites

Environmental Pollution 82 (1993) 239-244

GLUTATHIONE STATUS A N D GLUTATHIONE REDUCTASE ACTIVITY IN SPRUCE NEEDLES OF HEALTHY A N D D A M A G E D TREES AT TWO M O U N T A I N SITES U. S c h m i e d e n , S. S c h n e i d e r & A. W i l d * lnstitut ffir Allgemeine Botanik, Johannes Gutenberg-Universitgit, 55099 Mainz, Germany (Received 23 March 1992; accepted 7 September 1992)

Abstract Levels of glutathione, & both reduced and oxidized form, and glutathione reductase activity were monitored in needles of healthy and damaged spruce trees (Picea abies (L.) Karst.) during the course of four vegetation periods at two natural sites. The glutathione content and glutathione reductase activity showed a pronounced annual rhythm in undamaged trees, whereas damaged spruce trees deviated significantly from this course. In comparison with undamaged trees, damaged trees showed markedly increased levels of glutathione during the test period of 1989-1991. However, glutathione reductase activity differed in damaged and undamaged trees, only in 19891990. The ratio of reduced to oxidized glutathione (GSH/GSSG ratio) was slightly higher in damaged trees, and the highest levels were found during the winter months. In the case of damaged trees, a correlation between GSH/GSSG ratio and current ozone levels at the sites could be clearly established The present results indicate that damaged trees suffer from increased oxidative stress, especially in the period from June to October.

detoxification of herbicides and pesticides (Lamourex & Rusness, 1981; SchrOder et al., 1990) and of air pollutants (Alscher & Amthor, 1988). It was assumed that the absorption of gaseous pollutants leads to changes in the antioxidative system and thus to changes in the glutathione--glutathione reductase system. This assumption was based on controlled gas experiments carried out in phytotrons and open-top chambers in connection with the phenomenon of 'novel forest decline' (Mehlhorn et al., 1986; Smith et al., 1989; Bermadinger et al., 1990). According to Hausladen et al. (1990), the relative amount of oxidized glutathione (GSSG) may be enhanced considerably. Changes of the glutathione status can also give hints of strain caused by photooxidants and oxidative stress when measured in ambient conditions. Furthermore, it is expected that the degree of damage to a tree could also influence the type of change that occurs. Therefore, studies were carried out on spruce trees with various degrees of damage at several natural sites in Germany during four vegetation periods. The present study is part of a research project to characterize damaged spruce trees with the help of biochemical, physiological and cytomorphological parameters (Wild & Forschner, 1988; Wild et al., 1990).

INTRODUCTION

The tripeptide y-L-glutamyl-L-cysteinylglycine (glutathione) has several functions in plant cells. In the course of nucleic acid synthesis, glutathione fulfils different purposes in catalysis, metabolism and transport (Meister, 1988). A relationship between glutathione metabolism and the regulation of stress-induced transcriptional processes has been established (Dhindsa, 1987; Wingate et al., 1988; Alscher, 1989, 1990). Apart from that, glutathione serves as a storage and transport metabolite for reduced sulphur compounds (Rennenberg et al., 1979). Together with the enzyme glutathione reductase (EC 1.6.4.2), glutathione plays a role in frost hardening and dormancy of evergreen plants (Esterbauer & Grill, 1978). In addition, glutathione functions as a component of the antioxidative scavenging system (Alscher, 1989; Smith et al., 1989) and seems to play a role in the

MATERIALS A N D M E T H O D S Description of the sites The studies presented here were carried out on needles of spruce trees (Picea abies (U) Karst.) during the vegetation periods 1988 and 1989 at a natural habitat in Rheinland-Pfalz, and from 1989 to 1991 at a natural site in Baden-Wtirttemberg. The selection of the sites and trees was carried out according to the forest damage inquiry criteria (Waldschadenserhebung, 19841991). To evaluate the state of the crown, the needles are allocated to one of five damage categories (0-4). The second important damage characteristic is discoloration of the needles. The needle loss classes and the discoloration classes are plotted against each other to supply the damage class in the damage inventory: 0~undamaged; 1--slightly damaged; 2--moderately damaged; 3--severely damaged; 4--dead. Further

* To whom correspondence should be addressed. Environ. Pollut. 0269-7491/93/$06.00 © 1993 Elsevier Science Publishers Ltd, England. Printed in Great Britain 239

240

U. Schmieden, S. Schneider, A. Wild

descriptions of the sites have been presented by Wild and Forschner (1990), Tenter and Wild (1991) and Tietz and Wild (1991).

Hattgenstein site This site is located on a southeastern slope in the western part of the Hunsr~ck mountains in RheinlandPfalz at about 660 m above sea-level (Idar-Oberstein Forestry Office, Hattgenstein Forest District, Division 257 b:). Climatic and pollution data have been continuously registered by the Leisel measurement station of Z I M E N (Central Network for Measurement of Pollution in Rheinland-Pfalz, Z I M E N , 1985-1991) at a distance of 6 km from the site. During the test period of 1988-1989 the location was characterized by high ozone levels (see Fig. 3 below) whereas SO2 and NOr, played minor roles. A typical feature of the soil is its podzolated, very acidic brown earth (pH 2.7 3.5 for the A-horizon, pH 3.6-3.8 for the B-horizon), based on quartzite. The cation exchange capacity and the nutrient supply----especially Mg 2÷ ions - - are low. The spruce trees of the plantation were between 25 and 30 years old. The research was performed on 10 trees per year; five of those trees were apparently healthy or little damaged (damage classes 0 and 1) and the other five trees were visibly damaged (damage class 2). The damaged trees showed partial needle loss and yellowing of the upper surface of the older needles. Sampling took place on the 26 April, 21 June, 23 August and 4 October in 1988, on 1 February, 2 May, 20 June, 15 August and 11 October in 1989, and on 20 February in 1990. Freudenstadt site The site near Freudenstadt in the northern Black Forest is 820-830 m above sea-level (Freudenstadt Forest Office, Vordersteinwald Forest District, Division III/12 a 4, at the SchOllkopf). Climatic and pollution data were continuously registered by the IVD measurement station (Institut ft~r Verfahrenstechnik und Dampfkesselwesen, IVD, 1990, 1991) at the SchOllkopf about 500 m from the site. The concentrations of SO2 and NO2 were relatively low, whereas the ozone concentration was high, with average monthly levels of 100-150 /~g m 3 during the summer. The soil can be characterized as podzolated brown earth and pseudoglye. The test areas were located on permeable brown earth. Twelve spruce trees, one group of six undamaged (damage class 0) and another group of six clearly damaged trees (damage classes 2 and 3), were selected from two adjoining plantations of 40-50 year-old trees. The damaged trees showed symptoms of 'mountainous yellowing', i.e. a significant yellowing on the upper needle surface can be observed with increasing age of the needles. Mixed samples of needles from each group of trees were examined on 19 April, 28 June, 30 August and 8 November in 1989, on 21 February, 25 April, 27 June, 29 August and 7 November in 1990, and on 13 March, 29 April, 26 June and 28 August in 1991. The Freudenstadt research site is part of a forest decline research project monitoring air pollution, the

analysis of the soil, the genetic constitution and the physiological and biochemical state of trees, and their mycorrhizas, and supervising any biotic damage that may occur; the project is sponsored and co-ordinated by the Kernforschungszentrum Karlsruhe-Projekt Europ~isches Forschungszentrum ft~r Ma[3nahmen zur Luftreinhaltung ( K f K - P E F , 1991). Materials The studies were performed on spruce needles grown in the previous year from the sixth to the eighth whorls. Immediately after harvesting, the needles were removed from the twigs by stirring them in liquid nitrogen, and were then stored in plastic vials at -80°C. To avoid fluctuation of glutathione content during the course of the day, harvesting always took place between 11 a.m. and 1 p.m. Methods

Total and oxidized glutathione Extraction. Frozen needles (1 g) were homogenized (cutter rod 18N, Ultra Turrax) at 4°C for 60 s in 10 ml of 4% (w/v) sulphosalicylic acid containing 5% (w/v) Polyclar AT (insoluble polyvinylpyrrolidone). The homogenate was centrifuged for 10 min at 2°C and 27 000 g and the pellet was resuspended twice in 5 ml of sulphosalicylic acid. All supernatants together were used for measurement of total and oxidized glutathione. Duplicates were run for each extract. Determination of total and oxidized glutathione. The method used employed the specificity of glutathione reductase and was a modification of that of Griffith (1980). An extract volume of 1 ml was neutralized (pH about 6.5) by addition of 1.5 ml of 1 M K-phosphate buffer (pH 8.0). The neutralized extract (150 /A) was added to 700 /A of 0.3 mM N A D P H , 100 /A of 6 mM D T N B (Ellman's reagent), 50 pA of glutatione reductase (10 units ml 1). All reagents were prepared in 125 mM NaH2PO4 buffer, containing 6.3 mM E D T A at pH 7.5. Reaction was followed at 412 nm and the content of total glutathione was calculated from a standard curve. To measure GSSG, an extract volume of 1 ml was neutralized by addition of 1.5 ml of 1 M K-phosphate buffer (pH 7.0). The neutralized extract (1 ml) was added to 75 /~1 of 2-vinylpyridine followed by 100 /~1 of 50% (v/v) triethanolamine, the latter being placed on the side of the tube above the level of the liquid. The solution was vortexed-mixed for 1 min and incubated at 25°C for 1 h. Blanks were prepared using 1 ml of neutralized extraction medium instead of extract. A portion (150 /A) of the resultant solution was assayed as above. Calibration curves were based on GSSG samples treated exactly as above, as triethanolamine slightly alters the rate of colour development. Recovery of reduced glutathione (GSH) and GSSG was more than 85%. The addition of standards to the reaction medium did not lead to any results which indicate a matrix effect.

Glutathione and glutathione reductase in spruce needles Glutathione reductase ( GR) Extraction. Frozen needles (1 g) were homogenized

o

(cutter rod 18N, Ultra Turrax) at 4°C for 60 s in 20 ml of 0.2 M K-phosphate buffer (pH 7.8) containing 1 mM E D T A and 2% (w/v) soluble polyvinylpyrrolidone 25. The homogenate was centrifuged for 25 min at 2°C and 27000 g. The supernatant was mixed with 10% (w/v) Polyclar AT (insoluble polyvinylpyrrolidone) to bind further phenolic compounds, which would impair the enzyme assay. The solution was cleared by centrifugation for 5 min at 4000 g. The supernatant was used for G R assay. Determination of GR activity. G R activity was determined spectrophotometrically by following the decrease of N A D P H absorbance at 340 nm, modified according to the method of Esterbauer and Grill (1978). The enzyme extract (1 ml) was added to the standard incubation mixture containing 500 /zl of 90 mM BSA, 500 /zl of 0.72 mM N A D P H and 500 /zl of 3 mM EDTA. All reagents were prepared in 0.1 M Tris-HC1 buffer at p H 7.5. The reaction was initiated by addition of 500 p.1 of 16 mM G S S G at 25°C. Statistics Student's t-distribution for independent random samples was used for statistical analysis, after the variance homogeneity had been checked by an f-test. (Significance levels were p < 0.05, p _< 0.01 and p < 0.001.)

00I° 200

.]

241

1

5O 0

{.9

12 10

~

8

-r t~ o

6 4

b

2 0 7

c~

C

i Ull 5

Apr

Jun

Aug

Nov

Feb

h o r v e s t dote1989/90

Fig. 2. Freudenstadt site; values of the mixed samples for each harvest date during the test period 1989-1990. (a) Content of glutathione in relation to dry weight; (b) GSH/GSSG ratio; (c) GR activity in relation to dry weight (unshaded columns, undamaged trees; shaded columns, damaged trees). RESULTS The total glutathione content of the needles of spruce trees at the Hattgenstein site (Fig. l(a)) indicates a marked annual rhythm. During the summer months tests showed low levels (63 /zg g ' dry weight in August), and from autumn to early spring maximal levels of up to 300/zg g ' dry weight were measured. In contrast, the severely damaged spruce trees at the Freudenstadt site showed in summer levels of glutathione which were less decreased (Fig. 2(a); Table 1), so that in comparison with the undamaged trees there is no marked annual rhythm.

"

10

(-9

4

"

E

3

2

Table 1. Ratio of fresh weight to dry weight and content of glutathione in relation to dry weight (/zg g-i) in previous year's needles of spruce trees at Freudenstadt site during the test periods 1990 and 1991 Sampling

Fresh/dry weight

Total glutathione content

U n d a m a g e d D a m a g e d U n d a m a g e d D a m a g e d Increase in damaged trees (%)

Apt ~n Aug Oct Feb Moo ion Aug Oct Feb horvest dote

Fig. 1. Hattgenstein site; mean values for each harvest date during the test period 1989-1990. (a) Content of glutathione in relation to dry weight; (b) GSH/GSSG ratio; (c) GR activity in relation to dry weight (unshaded columns, undamaged trees; shaded columns, damaged trees). Percentage standard deviation of the mean values shown (n = 5): (a) and (b) 17.4% for undamaged and 19.5% for damaged trees, (c) 15.6% for undamaged and 18-5% for damaged trees, p < 0.05.

Apr. June Aug. Nov. Mar. Apr. June Aug.

90 90 90 90 91 91 91 91

2.27 2.26 2.31 2.56 2-29 2.16 2.23 2.31

2.31 2.19 2-30 2.41 2.25 2.17 2.31 2.18

184 103 96 192 269 298 158 155

207 202 105 167 345 383 343 254

12.5 96.1 9.4 -13.0 28.3 28.5 117.1 63.9

U. Schmieden, S. Schneider, A. Wild

242

The data for the fresh weight/dry weight ratio shown in Table 1 indicate no significant differences between the values for undamaged and damaged trees. The dry weight did not affect the comparison of data on glutathione content and glutathione reductase activity related to this basic parameter. The comparison between the glutathione content of needles of damaged trees and that of undamaged trees at the Hattgenstein site leads to the following results (Fig. l(a)). In 1988 needles of damaged spruce trees showed a tendency towards higher levels in June and October only. In 1989, however, damaged trees showed markedly higher glutathione levels than undamaged reference trees during the whole year. The increase in the contents of glutathione in needles of damaged trees became most obvious after sprouting and the beginning of the warm season, i.e. when the first high levels of ozone were observed. Results from tests carried out at the Freudenstadt site from 1989 to 1991 lead a similar picture (Fig. 2(a); Table 1). The 2-year-old needles of the damaged trees showed markedly increased levels of glutathione, except for the harvest date in November, with the difference being especially obvious in June, when needles showed an increase in the range of 100-150%. Furthermore, it was noticeable that the glutathione contents of needles of both undamaged and damaged trees, regardless of the annual rhythm, were higher during the test period 1990-1991 (Table 1) than in 1988-1989 (Fig. l(a)). In Table 2 it can be seen that the results from the mixed samples are representative of the chosen single trees of each mixture. The total contents of glutathione of the six undamaged trees, as well as the six damaged trees, were closely similar. Standard deviations of 16.3% for the undamaged group were calculated. The mean value of the single measurements reflects the result of the mixed sample of this sampling date. The difference between the glutathione content of undamaged and damaged trees is statistically significant at p < 0.001. The development of G R activity (Figs l(c) and 2(c)) in the course of 1 year was similar to that of the total glutathione content of the tested needles; however, G R activity was rather lower during the summer months, as well. On the whole, damaged and undamaged trees at Hattgenstein showed almost no significant differences in G R activity. Contrasting results were observed at the Freudenstadt site, where the severely damaged trees showed increased enzyme activities compared with the undamaged trees, except for November. Table 2. Content of glutathione in relation to dry weight (/~g g-l) of the single spruce trees which were constituents of the mixed samples at Frendenstadt site; harvest date 28 August 1991 Trees

Undamaged Damaged

Mean a Mixed sample

1

2

3

4

5

6

180 258

141 254

153 252

120 211

165 226

122 208

147 +_ 24 235_+ 23

aSignificance level of the difference between u n d a m a g e d and damaged trees is p < 0-001.

155 254

10

140 a

120

•

~,

8 O

100

o.• /"

80 60

--o,

o' ' ..o...o\I. • ~

,.. f ~ ,.~ /" -,,

/

~..<

6~ 4

,'-0

Gh

-o..-,)--o

2

20 J FMAMJ

J ASONDJ 1988

FMAMJ

month

J ASOND 1989

12 10 ¸ 8 ~3 U3

o

6

-tin (D

4

r= - 0.706

2

0

1

0

20

/

40

i

i

i

i

60

80

100

120

140

#g / m 3

Fig. 3. (a) GSH/GSSG ratio in needles of damaged trees in comparison with the monthly average of ozone level at the Hattgenstein site during the test period 1988 and 1989 (O, ozone level; t , damaged trees); percentage standard deviation of the mean values shown (n -- 5) is 19.5%. (b) Correlation between GSH/GSSG ratios and monthly average of ozone level at the Hattgenstein and Freudenstadt sites during the test periods in 1988 and 1989. It is striking that for Hattgenstein the G S H / G S S G ratios (Fig. l(b)) do not take the same course within 1 year as the total glutathione content of the tested needles. Especially during the test period of 1988, striking deviations could be observed, and this was true for the Freudenstadt site as well. Above all, these deviations were caused by the annual change of the content of GSSG. The GSSG content of the needles did not show the same marked 'summer depression' as the G S H content. This means that the annual course of the G S H / G S S G ratios is influenced, and results of a comparison of damaged trees with undamaged trees justifies the statement that damaged trees tend towards an increased G S H / G S S G ratio. Air pollutants may constitute another factor which influences the G S H / G S S G ratio. Figure 3(a) depicts the course of the ozone levels at Hattgenstein on the one hand and the development of the G S H / G S S G ratio of the damaged trees of the site on the other. Though the possibility that the effects of ozone appear temporarily delayed cannot be excluded, both parameters describe curves which are to a great extent antiparallel. With increasing ozone levels, a decrease, and with decreasing ozone levels, an increase in the G S H / G S S G ratio could almost always be observed. Taking the corresponding data from the Freudenstadt site into account (Fig. 3(b)) as well, a clear linear correlation (r = -0.706; p = 0.01) between increasing ozone levels and decreasing GSH/

Glutathione and glutathione reductase in spruce needles

GSSG ratios in the needles of damaged spruce trees can be established. In undamaged spruce trees, however, such a linear relation could be observed only in a less pronounced way. DISCUSSION Except for the severely damaged spruce trees at the Freudenstadt site, a pronounced annual rhythm of the total glutathione content and the content of reduced glutathione (GSH) were found at both habitats. The same rhythm, i.e. extremely low summer levels, was observed on all trees for glutathione reductase activities as well. Such a yearly cycle of G S H and G R activity in spruce needles was reported as early as 1978 by Esterbauer and Grill. Similar seasonal variations have been observed in needles of eastern white pine by Anderson et al. (1992). The undamaged trees which were tested by them showed a G R activity and GSH content which described parallel yearly cycles. The increase of the glutathione content and of G R activity during the winter months is generally connected with frost hardening and dormancy (Levitt, 1980; Anderson et al., 1992). At the same time, glutathione serves as a sulphur storage peptide (Rennenberg et al., 1979), and Schupp et al. (1990) were able to prove that the older generations of spruce needles supply reduced sulphur for sprouting in the form of GSH. The results presented here provide evidence that, in damaged trees, the annual rhythm of glutathione content is impaired in comparison to the undamaged trees, and that the connection between glutathione and glutathione reductase is less strong. During the test period 1989-1991, increased glutathione contents were found in needles of damaged spruce trees at both sites, except for two samplings in November. Especially after sprouting and the first extreme ozone levels of the year, which occurred in May (monthly average 113 /xg m 3) and in June (monthly average 120 /zg m 3), damaged trees at the Hattgenstein site showed glutathione levels which were normally 40% higher than those of the undamaged trees. Similar results could be observed at the Freudenstadt site. Here average increases of 151% (1989), 96% (1990) and 117% (1991) were measured in June of each test period. Controlled experiments, in the course of which spruce trees were exposed to SO2 and/or 03, made clear that the glutathione content is an appropriate short-term indicator of oxidative stress (Mehlhorn et al., 1986). In the tests of Senger et al. (1986), Weigel et al. (1989) and Dohmen et al. (1990) exposure to ozone caused a rise in the glutathione content of spruce trees. It was also found that even needles of only slightly damaged spruce trees have reduced photosynthesis rates (Benner et al., 1988) and that their photosynthetic electron transport chain (Dietz et al., 1988; Wild et al., 1988) as well as their ribulose-l,5biphosphate carboxylase activity (Schmieden-Kompalla et al., 1989) are impaired. This means that with increasing damage, trees are put under an increasing strain caused by reactive oxygen species. In the course o f

243

summer, light intensity also becomes a stress factor which puts additional strain on the predisposed plants (Wild, 1988). The increased glutathione content justifies the assumption that these trees were exposed to increased oxidative stress which was obviously strongest in June each year. Moreover, at the Freudenstadt site the rising glutathione content from 1989 to 1991, in both undamaged and damaged trees, indicates an intensified stress caused by reactive oxygen species. The G S H / G S S G ratio is another physiologically important factor. It can be influenced directly by oxidants, by de novo synthesis and reduction of GSH or by activity of the regenerating enzymes such as GR. It is assumed that G S H plays the more important part in its relationship to GSSG, as it is necessary to keep the glutathione system working (Rennenberg, 1982). The spruce needles which were tested in the present study showed G S H / G S S G ratios in the range from 2 to 12, with the highest values being measured in winter. It is a typical feature of this ratio that, from June to October, the GSSG levels do not decrease as much as the G S H levels in both undamaged and damaged trees. Nevertheless, the G S H / G S S G ratio shows certain regularities which indicate oxidative stress. The markedly lower ratios during the summer in particular could indicate increased strain. In addition to endogenous factors, increased ozone levels, light intensity and even water supply could be factors which trigger stress. The antiparallel course of the G S H / G S S G ratio and the ozone level leads to the assumption that, in the case of damaged trees, ozone plays a major role in the generation of oxidative stress. When the activity of glutathione reductase was measured, results varied. Depending on the site and the test period, activities measured in the needles of damaged spruce trees were either similar to those in undamaged trees or were increased. The fact that in 1989-1990 G R activities were almost always increased corresponds to the results found for glutathione content. A typical feature of this test period was the warm, dry weather and the typical stressors (ozone, drought and high light intensity) related to such weather. Apart from that, the levels of G R activity which were measured also give reason to believe that the enzyme activity cannot be the sole cause for the tendency for the GSH/ GSSG ratio to be higher in damaged spruce trees. Glutathione synthesis and degradation somehow influence the ratio as well, though we do not comprehend exactly how. The results found for the glutathione status and the G R activity in spruce needles indicate that damaged spruce trees suffer from increased oxidative stress. The search for the specific reasons for this in natural habitats is made more difficult by other factors, among which are the varying genetic predisposition of the trees and the specific conditions of each habitat. At the Hattgenstein and Freudenstadt sites, for example, the main exogenous factors of stress may be the ozone levels in summer and the reduced Mg 2÷ supply. However, even here one has to assume that various stress

244

U. Schmieden, S. Schneider, A. Wild

factors put a strain on the trees which are predisposed to damage, and that these lead to the effects which are well-known s y m p t o m s o f novel forest decline.

ACKNOWLEDGEMENTS This study was supported by the K f K - P E F (Kernforschungszentrum Karlsruhe-Projekt Europaisches F o r s c h u n g s z e n t r u m ft~r Ma[3nahmen zur Luftreinhaltung), grant 88/007/1A, and by the U m w e l t b u n d e s a m t (Federal E n v i r o n m e n t a l Office) Berlin, grant 108 03046/16.

REFERENCES Alscher, R. G. (1989). Biosynthesis and antioxidant function of glutathione in plants. Physiol. Plant., 77, 457-64. Alscher, R. G. (1990). Response/resistance mechanisms: air pollutants and antioxidant metabolism. Physiol. Plant., 79, 695. Alscher, R. G. & Amthor, J. S. (1988). The physiology of free-radical scavenging: maintenance and repair processes. In Air Pollution and Plant Metabolism, ed. P. SchulteHostede, N. M. Darrall, L. W. Blank, & R. A. Wellburn, Elsevier, New York, pp. 94-115. Anderson, J. V., Chevone, B. I. & Hess, J. L. (1992). Seasonal variation in the antioxidant system of eastern white pine needles. Plant Physiol., 98, 501-8. Benner, P., Sabel, P. & Wild, A. (1988). Photosynthesis and transpiration of healthy and diseased spruce trees in the course of three vegetation periods. Trees, 2, 223-32. Bermadinger, E., Guttenberger, H. & Grill, D. (1990). Physiology of young Norway spruce. Environ. Pollut., 68, 319-30. Dhindsa, R. S. (1987). Glutathione status and protein synthesis during drought and subsequent rehydration in Tortula ruralis. Plant Physiol., 83, 816-19. Dietz, B., Moors, I., Flammersfeld, U., Rt~hle, W. & Wild, A. (1988). Investigation on the photosynthetic membranes of spruce needles in relation to the occurrence of novel forest decline--I. The photosynthetic electron transport. Z. Naturforsch., 43c (Biosciences 43), 581-8. Dohmen, G. P., Koppers, A. & Langebartels, C. (1990). Biochemical response of Norway spruce (Picea abies L. Karst.) towards 14-month exposure to ozone and acid mist: effects on amino acid, glutathione, and polyamine titers. Environ. Pollut., 64, 375-83. Esterbauer, H. & Grill, D. (1978). Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant. Physiol, 61, 119-21. Griffith, O. W. (1980). Determination of glutathione and glutathione disulfide using glutathione reductase and 2vinylpyridine. Anal, Biochem., 106, 207-12. Hausladen, A., Madamanchi, N. R., Fellows, S., Alscher, R. G. & Amundson, R. G. (1990). Seasonal changes in antioxidants in red spruce as affected by ozone. New Phytol., 115, 447--58. KfK PEF (1991). 7. Statuskolloquium des PEF vom 5. 7. Mgirz 1991 im Kernforschungszentrum Karlsruhe. KJKPEF, 80. Kernforschungszentrum Karslruhe, pp. 3948; 93-1-8. Lamoureux, G. L. & Rusness, A. G. (1981). Catabolism of glutathione conjugates of pesticides in higher plants. In Sulfur in Pesticide Action and Metabolism. Am. Chem. Soc. Symp., 152, 132-64. Levitt, J. (1980) Responses of Plants to Environmental Stress. L Chilling, Freezing and High Temperature Stresses. Academic Press, New York, 23-64. Mehlhorn, H., Seufert, G., Schmidt, A. & Kunert, K. J. (1986). Effect of SO2 and O~ on production of antioxidants in conifers. Plant. Physiol., 82, 336-8.

Meister, A. (1988). Glutathione metabolism and its selective modification. J. Biol. Chem., 263, 17 205-8. Rennenberg, H. (1982). Glutathione metabolism and possible biological roles in higher plant. Phytochemistry, 21, 2771-81. Rennenberg, H., Schmitz, K. & Bergmann, L. (1979). Longdistance transport of sulfur in Nicotiana tabacum. Planta, 147, 57-62. Schmieden-Kompalla, U., Hartmann, U., Korthals, S. & Wild, A. (1989). Activity and activation state of ribulose1,5-biphosphate carboxylase of spruce trees with varying degrees of damage. Photosynthesis Res., 21, 161-9. SchrOder, P., Lamoureux, G. L., Rusness, D. & Rennenberg, H. (1990). Glutathione-S-transferase activity in spruce needles. Pesticide Biochem. Physiol., 37, 211-18. Schupp, R., Schatten, Th. & Rennenberg, H. (1990). Untersuchungen zum Langstreckentransport von Glutathion (GSH) in Fichten (Picea abies L. Karst.). Posterbeitrag P1015. Botanikertagung Regensburg, 30 September5 October 1990. Senger, H., Osswald, W., Senser, M., Greim, H. & Elstner, E. F. (1986). Gehalte an Chlorophyll und den Antioxidantien Ascorbat, Glutathion und Tocopherol in Fichtennadeln (Picea abies L. Karst.) in Abhangigkeit yon Mineralstoffern~hrung, Ozon und saurem Nebel. Forstw. Cbl., 105, 264-7. Smith, I. K., Vierheller, T. L. & Thorne, C. A. (1989). Properties and functions of glutathione reductase in plants. Physiol. Plant., 77, 449-56. Tenter, M. & Wild, A. (1991). Investigations on the polyamine content of spruce needles relative to the occurrence of novel forest decline. J. Plant Physiol, 137, 647-54. Tietz, S. & Wild, A. (1991). Phosphoenolpyruvate carboxylase activity and malate content of spruce needles of healthy and damaged trees at three mountain sites. Biochem. Physiol. Pflanzen, 187, 273-82. Waldschadenserhebung (1984-1991). Bundesministerium for Ern~hrung, Landwirtschaft und Forsten, Bonn. Weigel, H. J., Halbwachs, G. & Jager, H. J. (1989). The effects of air pollutants on forest trees from a plant physiological view. Z. Pflanzenkrankheiten Pflanzensehutz, 96, 203-17. Wild, A. (1988). Licht als Stre[3faktor bei Waldb~umen. Naturwiss. Rundschau, 41, 93-6. Wild, A. & Forschner, W. (1988). Physiologische, biochemische und cytomorphologische Charakterisierung immissionsbelasteter Waldbaume von exemplarischen Standorten innerhalb des rheinland-pf'alzischen und des hessischen Immissionsmel3netzes. Forschungszwischenbericht des FE-Vorhabens Nr. 106 07046/16, Umweltbundesamt Berlin. Wild, A. & Forschner, W. (1990). Comparative physiological and biochemical investigations of spruce trees damaged by immissions in context with forest decline in the northern Black Forest. In 6. Statuskolloquium des PEF vom 6.~. Mgirz 1990 im Kernforschungszentrum Karlsruhe. KfK-PEF, 61 Kernforschungszentrum Karlsruhe, pp. 297-309. Wild, A., Flammersfeld, U., Dietz, B., Moors, I. & Rt~hle, W. (1988). Investigation on the photosynthetic membranes of spruce needles in relation to the occurrence of novel forest decline--II. The content of QB-protein, cytochrome f, and P-700. Z. Naturforsch., 43c, (Bioseiences 43), 589-95. Wild, A., Forschner, W. & Schmitt, V. (1990). Physiologische, biochemische und cytomorphologische Untersuchungen an immissionsbelasteten Fichten. Forschungsendbericht des FE-Vorhabens Nr. 108 03 046/1, Umweltbundesamt Berlin. Wingate, V. P. M., Lawton, M. A. & Lamb, C. J. (1988). Glutathione causes a massive and selective induction of plant defense genes. Plant Physiol., 87, 200-10. ZIMEN (1985-91). Monatsberichte tiber die Me[3ergebnisse des Zentralen Immissionsmel3netzes--ZIMEN--von Rheinland-Pfalz. Landesamt ftir Umweltschutz und Gewerbeaufsicht, Mainz.