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Investigation on the Nitrogen Metabolism of Spruce Needles in Relation to the Occurrence of Novel Forest Decline
J. PlantPhysiol. Vol. 132. pp. 491-498(1988)
Investigation on the Nitrogen Metabolism of Spruce Needles in Relation to the Occurrence of Novel Forest Decline SYLVIA DUBALL
and
ALOYSIUS WILD
Institute of General Botany of the Johannes Gutenberg-Universitat, Saarstr. 21, D-6500 Mainz, Federal Republic of Germany Received June 24, 1987 . Accepted October 29, 1987
Summary In order to obtain information on the nitrogen metabolism of undamaged and damaged spruce (Picea abies), investigations were performed in 1985 and 1986 on the activity of glutamine synthetase and on the content of free ammonium and soluble proteins in spruce needles. In addition, the chlorophyll content was determined. Needles from the second needle year and the seventh whorl were used. The investigations were carried out in pair comparison on a 20-year-old spruce plantation and in comparison with damage classes on an 80-year-old spruce plantation. Both the pair comparison and the contrasting of different damage groups show an unequivocal decrease of the chlorophyll content correlated with increasing damage to the trees. The chlorophyll content proves to be a reliable indicator of the overall extent of damage to the tree, even in apparently green needles. A rise in the content of soluble proteins in the green needles of the damaged trees was shown at both locations. A breakdown of soluble proteins was only observed in definitely yellow needles. The accumulation of soluble proteins in the damaged trees is correlated with the measured activities of ribulose bisphosphate carboxylase and glutamine synthetase. The needles of the damaged trees contain reduced levels of ammonium. Conversely the activity of glutamine synthetase showed a major rise, especially in 1985. The results contradict the assumption that the increase in activity of glutamine synthetase is associated with an elevated nitrogen supply. The lower content of free ammonium as well as the increase in soluble proteins associated with an increase of the glutamine synthetase activity may indicate a high protein turnover and a shift in the protein composition. This can be interpreted as a response to environmental stress and membrane damage in the cells.
Key words: Picea abies, air pollution, chlorophyll content, ammonium, glutamine synthetase, novel forest decline, soluble proteins. Abbreviations: GS - glutamine synthetase; GS-GOGAT - glutamine synthetase-glutamate synthase; RubP - ribulose-1,5-bisphosphate.
Introduction For several years «novel forest decline» has been occurring over wide areas in Central Europe. This class of forest diseases cannot be subsumed under the general category of classical smoke damage alone, because it is manifest also in areas far away from major polluters or industrial centres and even in so-called «clean air» regions with low S02 pollution. Symptoms of novel forest decline were first observed on European silver fir in the early 1970's; they have subse© 1988 by Gustav Fischer Verlag, Stuttgart
quently been described in the early 1980's for Norway spruce and recently for broad-leaved trees. The damage has increased in severity and extent over the past six years. Damage severity increases with altitude, although symptoms can now be observed at lower-elevation forests. The damage, which now affects all economically-important tree species of the forests and which is mainly visible in older plantations, already comprises about 54% (damage categories 1-4) of the entire forest area of West Germany today; of this a total of about 19 % - represented by the damage categories 2 - 4 -
492
SYLVIA DOBALL and ALOYSIUS WILD
shows medium to severe damage (d. Waldschadenserhebung 1986). There are numerous hypotheses on the causes of novel forest decline. The major factors discussed are natural factors or anthropogenic pollutants. Nevertheless, it is not known whether primarily natural or primarily anthropogenic factors (especially air pollutants) are responsible for the tree diseases. An involvement of climatic, edaphic, silvicultural, mycological or microbial factors in the causal complex is undisputed and may be of crucial significance for damage in restricted areas (Kowalski and Lang 1984, Rehfuess and Rodenkirchen 1984, Butin and Wagner 1985). However, these factors cannot be the sole cause of such massive and widespread damage to the forest ecosystem. There is increasing evidence that anthropogenic pollutants are crucial for the novel forest decline over large regions (Elstner 1983, Mohr 1984, Prinz 1984, Ulrich 1984, Wentzel 1985, Krause et al. 1986). The investigations described here were carried out in the context of our research project on the physiological, biochemical and cytomorphological characterization of spruce trees growing in natural habitats and showing different degrees of damage. Photosynthesis, the water balance, the content of chemical elements and light- and electron-microscopic structure were previously examined (Wild and Bode 1986, Benner and Wild 1987, Jung and Wild 1988, Wild 1987 a, b). Here we report on aspects of nitrogen assimilation. On the basis of analysis of xylem sap, it is thought that in gymnosperms nitrate reduction takes place predominantly in the roots (Barnes 1962, Pate 1971, 1980, Smirnoff et al. 1984, Wingsle et al. 1987). The xylem sap contains only minor amounts of nitrate (Martin et al. 1981). Nitrogen is mainly transported from the root into the shoot largely in the form of organic compounds such as amino acids and amides (Bollard 1956, Barnes 1962, Pate 1973, Martin et al. 1981). In the needles, assimilation and reassimilation of ammonium takes place via the GS-GOGA T pathway (Miflin and Lea 1976). In order to obtain an insight into the nitrogen metabolism of spruce with damage of varying severity, we investigated the activity of glutamine synthetase as well as the content of free ammonium and soluble proteins in needles at two sample locations with different categories of damage defined in the context of our research program. In addition, chlorophyll content and fresh and dry weights of the needles were determined.
Materials and Methods The locations and the trees were selected with the assistance of professional foresters.
Locations
Two locations in the Hunsriick and in the Taunus mountains were chosen for the investigations. These locations were comparable with regard to altitude and thus e.g. duration of the vegeta-
tion period which is shortened by two to three weeks on the slopes compared to valley locations. Location in the Taunus mountains
The location is on a southwest slope of the Feldberg (Konigstein Forestry Office, Glashiitten Forest District, Division 43/44) with an altitude of 660 -760 m above sea level. The annual precipitation is about 850mmm- 2 a- 1 (Hessische Landesanstalt fiir Umwelt 1986). The soil conditions are characterized by highly acid typical brown earth consisting of mica sandstone and T aunus quartzite. Location in the HunsrUck mountains
This area is on a moderately steep south slope on the west side of the Hunsriick at about 660 m above sea level (Idar-Oberstein Forestry Office, Hattgenstein Forest District, Division 257). The annual precipitation is 800 - 1000 mm m - 2 a -I. The soil consists mainly of quartzite and brown earth (pH3.5-4.3). The investigations presented were carried out between April and October in 1985 and in 1986. In 1986 only the Hunsriick location was further investigated. During the trial period, a relatively cool climate with very little sunshine and much precipitation prevailed at both locations from April to June 1985. From July to October, the weather was dry and warm, with a relatively high proportion of sunny days. During the 1986 vegetation period, a warm, dry, sunny climate prevailed at both locations (Zimen 1985, 1986).
Material Experimental spruce at the location in the Taunus mountains
This is a spruce [Picea abies (L.) Karst.] plantation which is about 80 - 90 years old and which displays damage in all categories. In spring 1985, 16 trees of different damage classes were selected with the assistance of the Hessian Forest Research Institute for the investigations. A first selection was made on the basis of visible characteristics, above all needle discoloration and degree of needle loss. A more detailed classification was made after harvesting (see results). Experimental spruce at the location in the HunsrUck mountains
This plantation allowed the investigations to be carried out as a pair comparison (according to Schiitt et al. 1983), since slightly damaged trees and trees of the same age (about 20 years) with clearly visible damage were growing side by side. Five (1985) and four (1986) tree pairs were selected for the measurements on the basis of visible characteristics in spring. The damage criteria used in selecting the trees at the two locations were the loss of needles and yellow discoloration (Waldschadenserhebung 1986). During the rainy and cool months (May to July) of 1985, differences became less obvious between the partners of a pair. In the warm and dry autumn of 1985, however,' the differences became significant again. During the trial period 1986, no such convergence between the appearance of damaged and largely asymptomatic trees was observed. The investigations at the two locations were carried out on the needles of the second needle year and the seventh whorl of the trial trees.
Methods The investigations were carried out with needle material which was quick-frozen in liquid nitrogen (-196°C) on site, directly after harvesting, and stored at -20°C.
Nitrogen metabolism of spruce needles Preparation of the needle extract
LOg (frwt) needles were homogenized (cutter rod 18N, Ultra Turrax, IKA-Werk Staufen im Breisgau) in 8-10 ml of a O.5M trisHCl buffer, pH 8.5 (modified according to Sanderson and Cocking 1964). To protect the glutamine synthetase, the buffer contained ImM cysteine, 10mM of DTE, 0.5mM MgS0 4, 0.5 mM EDTA and 2 % soluble PVP (polyvinylpyrrolidone 25), which largely inactivates the phenols released during extraction. The phenols could be completely absorbed by insoluble PVP (Polyclar AT), but PVP interfered with the extraction of the proteins. Thus the presence of a part of the phenols had to be tolerated when using soluble PVP. The optimum concentration of PVP was 2 % in our experiments with the protein assay method used (BIO-RAD, Bradford 1976). Homogenization and subsequent centrifugation (30 min at 20,000 g) were performed with precooled equipment at 4 0c. The supernatant obtained after centrifugation (crude extract) was used immediately to determine the concentrations of soluble proteins and to assay the glutamine synthetase activity. Detennination of the activity ofglutamine synthetase
After reaction of a defined amount of the crude extract for a specific time with a given amount of the substrate of glutamine synthetase (sodium glutamate) the enzyme activity can be calculated from the concentration of the end product of the reaction (gammaglutamyl-hydroxamate). Final composition of the incubation medium (total 2 ml): 60 mM imidazole-HCl buffer, pH7.3, 50mM MgS04, 10mM EDTA (Titriplex III), 15mM ATP, 10mM NH2 0H, 80mM sodiumglutamate, 200 JLl extract. The reaction proceeded for fifteen min at 37°C. It was started by addition of the extract and stopped by addition of 1.5 ml of an acid FeCh solution (0.37 M FeCh, 0.67 M HCl, 0.2 M CChCOOH) to the reaction mixture, which results in precipitation of the proteins and formation of a brown colored complex of gamma-glutamyl-hydroxamate with the ferric chloride. The measurement was carried out spectrophotometrically at a wavelength of 560 nm. A reaction stopped immediately after addition of the extract served as timezero control. In order to preclude interference with the enzyme assay by the phenols contained in the crude extract, which also react to form a black brown colored complex with ferrous chloride, the extinction of the controls was determined against air at 540 nm. The extinction values determined fluctuate only within the range of error in the measurements. Therefore, an error occurring in this way can be neglected.
troprusside as catalyzer intensifies this reaction. Since the color complex is only formed in acid medium (pH 3.5), the needles were homogenized in sulfosalicylic acid at pH 3.5. The extracts obtained were mixed with 1-3 g insoluble Polyclar, centrifuged and diluted 1 : 20 for the reaction.
Results Characterization of the spruce trees investigated at the location in the Taunus mountains
The individual spruce trees were classified in different damage groups at the Taunus location mainly on the basis of the description of the samples of the needles which were later used for the physiological and biochemical investigations. The trees were classified on the basis of the different portions of undamaged green, green and bright green needles with symptoms of damage (such as point necroses, gold tips and other discolorations), yellow and brown needles in the respective mixed samples. Further criteria were the needle cover of the branch harvested, as well as the overall impression of color. The physiological and biochemical investigations as well as the cytomorphological investigations of the mixed needle samples carried out in parallel proved to be largely consistent with this classification (see also Benner and Wild 1987, Hasemann and Wild, in preparation). Chlorophyll content
A significant decrease of the total chlorophyll was shown at both locations in the spruce with more severe symptoms (Fig. 1, Table 1). The annual mean value of the more damaged trees in the Hunsriick location is about 27 % lower (statistical probability P = 0.01). The trees of group III at the Taunus location show a decrease of 35 % (P = 0.01) com-
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Detennination of the content of soluble proteins
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Detennination of the chlorophyll content
The amount of chlorophyll (chlorophyll a and b) was determined in accordance with the method of Ziegler and Egle (1965) with 80 % acetone. Detennination of the content offree ammonium
The assay reaction is based on a method developed by Berthelot, and is a reaction between NH4 + and phenol in the presence of sodium hypochlorite with formation of a blue colored complex. The concentration can be determined spectrophotometrically at a wavelength of 625nm (e.g. Wild et al. 1987b). Addition of sodium ni-
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Fig. 1: Content of chlorophyll in relation to the dry weight (drwt). Location in the Hunsriick: comparison of five tree pairs, D = slightly damaged partner, • = more severely damaged partner. Location in the Taunus: D = four slightly damaged, I2:J = eight severely damaged, • = four very severely damaged spruce trees. MV = mean values of the 1985 investigation period. f------i = standard deviation.
494
SYLVIA DUBALL
and ALOYSIUS
WILD
pared to the slightly damaged trees in group 1. These data were obtained in 1985. In 1986 only the Hunsriick location Table 1: Annual mean values (MY) of several parameters measured during the 1985 vegetation period. Location in the Hunsriick: harvest dates on May 28th, July 29th, August 23rd and October 2nd. Comparison of five tree pairs (1 = slightly damaged, 2 = severely damaged partner). Location in the Taunus: harvest dates on April 26th, July 1st and October 7th in the damage groups I, II, and III. parameter
Taunus location Tree MY 1985 group I 2.25± 0.04 II 2.20± 0.02 III 2.10± 0.05
frwt/dr wt g.g-I
Hunsriick location Tree MY 1985 group 2.3l± 0.01 1 2 2.28± 0.02
Chlldrwt mg·g- I
1 2
2.5l± 0.17 1.84± 0.20
I II III
2.91± 0.35 2.32± 0.46 1.89± 0.26
Solubl: rroteinsl dr wt mg·g
1 2
13.2l± 4.57 16.44± 2.26
I II III
17.36± 8.80 20.69± 3.91 21.78± 3.42
free NHt I dr wt /Lg. g-I
1 2
232.70±41.66 218.52±45.30
I II III
268.55±31.47 172.81 ±26.21 173.8l±35.41
abs. activity of GS /Lmol . h - I . g - I dr wt
1 2
106.12±32.20 159.75±37.78
I II III
106.65 ± 17.79 180.25±44.47 188.53 ±45.55
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Free ammonium Overall, a decrease of the ammonium content was found with increasing damage to the trees at the two locations: about 6 % at the Hunsriick location (P = 0.1) and 35 % (P = 0.1) at the Taunus location (Fig.3, Table 1). The same trend was observed in the subsequent vegetation period at the Hunsriick location (17%, P = 0.2).
Activity of glutamine synthetase The absolute enzyme activity (units g-I dry weight) of glutamine synthetase was shown to be raised in the severely
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was further investigated. Similar results were obtained: a decrease of about 25 % (P = 0.01). The rise in chlorophyll content during the summer months of 1985 (Fig. 1) could also be observed in 1986 on the trial trees in the Hunsriick. This is consistent with the function of the chloroplasts within a vegetation period (Senser et al. 1975).
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Fig.2: Content of soluble proteins in relation to the dry weight. Location in the Hunsriick: pair comparison with five tree pairs, 0 = slightly damaged, • = more severely damaged partner. Location in the Taunus: 0 = four slightly damaged spruce (group I), [{J = eight severely damaged •spruce (group II), • = four very severely damaged spruce (group III). MY = mean values of the 1985 investigation period. 1-----1 = standard deviation.
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Fig.3: Content of free ammonium in relation to the dry weight. Location in the Hunsriick: comparison of five tree pairs, 0 = slightly damaged, • = more severely damaged partner. Location in the Taunus: o = four trees of group I, [{J = eight trees of group II, • = four trees of group III. 1985 investigation period.
Nitrogen metabolism of spruce needles
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Fig. 4: The absolute activity of glutamine synthetase in relation to the dry weight. Location in the Hunsrtick: pair comparison with five tree pairs, 0 = slightly damaged, • = more severely damaged partner. Location in the Taunus: 0 = group I, IT::J = group II, • = group III. 1985 investigation period.
495
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damaged spruce trees at both locations compared to the relatively asymptomatic control trees (Fig.4, Table 1). The increase in activity in samples collected at the Hunsriick location was about 51 % in 1985 (P = 0.05); in 1986 a consistent trend was found (9 %, P = 0.1). 85 % increase (P = 0.1) was measured in the trees of group III (very severely damaged) at the Taunus location. Less clear-cut statements can be made with regard to the specific activity of glutamine synthetase (units mg -1 soluble protein), because the protein content and the enzyme activity changed with the degree of damage. In severely and irreversibly damaged needles, however, an unchanged and in some cases even an elevated specific activity of the enzyme occurs compared to apparently green needles of a tree. These severely damaged needles show intense yellowing and a decline in protein content, as well as a decrease in the volume activity of the glutamine synthetase. Discussion Both the pair comparison at the Hattgenstein location in the Hunsriick mountains and the comparison of the different damage groups at Konigstein in the Taunus mountains showed an unequivocal decrease in the chlorophyll content of the more severely damaged trees at the different harvest dates. The chlorophyll content thus proves to be a reliable indicator of the overall damage situation of the tree even in needle groups that appear visibly green and healthy (Bode et al. 1985, Wild and Bode 1986, Benner and Wild 1987). Fig. 1 shows that the chlorophyll content of the 1984 needle generation is without exception much lower in the trees classified as damaged. This does not only apply to the mean values of all five tree pairs used in Fig. 1, but also to each individual pair comparison. The mean value of all data is 2.5 mg ChI a + b per g dry weight for the controls and 1.8 mg for the damaged partners in 1985. This is noteworthy because the differences in the damage appeared to be slight. There were often, at least superficially, no differences visible between the needle groups of the 1984 needle generation;
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this was particularly clear during the rainy summer months of 1985 (Zimen 1985). When harvesting the material, attention should be paid to similar light exposure of the branches. It is known that the photosynthetic apparatus adapts to the light conditions (e.g. low light and high light adaptation: Wild 1979, Ruhle and Wild 1985). In addition, it is remarkable that the chlorophyll contents of the needles rose markedly from the harvesting date in May to that in June (see also Senser and Beck 1979, Grill et a1. 1983). Corresponding results were obtained during the investigations of the spruce trees more than 80 years old at Konigstein in the Taunus mountains. The content of ChI a + b decreased with the increasing damage to the trees. It was actually 35 % lower in the severely diseased spruce trees of damage group III than in the damage group I. A lower chlorophyll content is always found in the needles of the damaged trees, even in needle groups with a green appearance, and with no yellowing or other visible characteristics of damage. This, however, applies only to plant material of similar age growing in the same environment and harvested at the same time, as done in the present study. Similar comparisons were made with regard to the nitrogen metabolism. The highly symptomatic trees from the location in the T aunus, which were classified in the damage groups II and III had 35 % less free ammonium in the needles than the control spruce trees of the damage group I. In pair comparisons of the younger spruce trees at the Hunsriick mountains, the difference is less pronounced. A rise in the content of soluble proteins was shown at the two locations. The needles from the trees of damage class III had an average of 25 % more soluble proteins compared to those of damage class I. A consistent trend was found in the pair comparison at the Hunsriick location. There was evidently a degradation of soluble proteins only in needles with an intense yellow discoloration; this is evidently associated with irreversible cellular damage (Hasemann et a1., submitted). There was a definite rise in the activity of glutamine synthetase in the damaged trees, especially in 1985. An increase of the activity by an average of 85 % was found in severely
496
SYLVIA DtiBALL
and ALOYSIUS WILD
damaged trees in the Taunus. An increase averaging 51 % was observed in the Hunsrlick pair comparison in 1985, whereas in the following year 1986, the difference was only 9 %. Considering the reduced NH3 content of the tissue and the pair comparison it appears as if any assumption that the increase of the activity of glutamine synthetase is causally connected with an excessive nitrogen supply (N0 3-, NH4 +, NOx) from the air or from the soil is not necessarily correct. In addition, the rate of pollution at the Konigstein location (Environmental Research Plan of the Federal Ministry of Environmental Protection and Reactor Safety, Concluding Report A, Hessen 1986) showed that the nitrogen influx reached maximum values in the summer months. The ammonium contents in the needles showed the lowest values at this time. The increased content of soluble proteins in the damaged trees is in good agreement with the measured activities of glutamine synthetase and RubP carboxylase. We did not detect any reduction in the in vitro activity of RubP carboxylase in the more severely damaged spruce trees at the two locations (Wild and Bode 1986, Wild 1987 a). This enzyme accounts for a major proportion of the soluble proteins. However, it is surprising that the damaged spruce needles, which are impaired in their photosynthetic activity (Benner and Wild 1987), synthesize large amounts of soluble proteins; this requires large amounts of energy. The low content of free ammonia as well as the increase of soluble proteins in connection with a substantial increase in the activity of glutamine synthetase indicates a high rate of metabolism of nitrogenous compounds. This could be a defense reaction of the organism to stress conditions and to the primary damage. An increase of glutamine synthetase activity has also been observed in gassing experiments with S02 and NO x in dicotyledonous herbaceous plants (Pahlich et al. 1972, Wallace and Spedding 1976, Wellburn et al. 1981). In addition, a surprisingly high specific activity of glutamine synthetase is found in yellow needles which already display a substantial loss of protein and which have suffered irreversible damage. These findings are an additional indication that the activation of glutamine synthetase should be considered as a general stress symptom. The organism responds to stress with specific and nonspecific reactions (adaptation syndrome) in order to eliminate or reduce the stresses and the primary damage elicited. The organism mobilizes defense mechanisms which can be subdivided into different phases (Schlee 1986). All stress-inducing factors have in common the activation of the biochemical and physiological apparatus of the organism. Glutamine synthetase, the actual N-assimilating enzyme of the GS-GOGAT pathway, thus has an absolutely crucial role in the assimilation and reassimilation of NH3. This enzyme accordingly has a particularly clear functional significance and physiological relevance for the activation of protein turnover and modification of the protein composition. The accumulation of the amino acid proline, which is regarded as a general stress metabolite with protective function, also indicates metabolic alterations at the protein level. In the trees recently investigated, the content of free proline was generally higher than in the control partners (Bode et al. 1985, Wild and Bode 1986). The findings showed proline ac-
cumulation in the damaged spruce at different ages, at different locations and in different seasons. There may be a connection with the elevated glutamine synthetase activity, because the biosynthesis of proline in plant tissues starts from glutamate (Shevyakowa 1983). Together with the chlorophyll content, the proline content can serve at least as an indicator for damage to the spruce (Wild 1987 a). Benner and Wild (1987) found a marked damage to photorespiration in the diseased spruce trees. By inhibition of glutamine synthetase with glufosinate (phosphinothricin) it was shown that in Sinapis and Valerianella plants the photorespiratory nitrogen cycle is the most important ammoniumforming and -consuming process in photosynthetically active leaves and thus the central point of redistribution of NH4 + (Wild et al. 1987 b, Sauer et al. 1987). The decline of the ammonium content in the needles of diseased spruce trees might therefore be associated with e.g. the reduced photorespiratory turnover of ~ + . The investigations of the chlorophyll content and the alterations in nitrogen metabolism described here were carried out in the context of an extensive research project on physiological, biochemical and cytomorphological characterization of damaged spruce trees growing in natural habitats. The results for different components of the photosynthetic apparatus (Benner and Wild 1987, Wild 1987 a, b, Wild et al. 1987, Wild 1988) and the electron microscopic analyses Gung and Wild 1988) showed consistently that early damage occurs especially at the cell membranes. This membrane damage is apparent even in the green needles of damaged spruce. Anthropogenic air pollutants appear to playa major role in the genesis of these effects. The loss of chlorophyll in the damaged spruce needles may therefore be associated with destabilization and damage to the thylakoid membranes. Furthermore, the decline of the ammonium content, the increase of soluble proteins and the marked increase in the activity of glutamine synthetase constitute metabolic reactions and responses which may be caused by the damage to the biomembranes and by an inhibition of photorespiration which may lead to the synthesis of stress metabolites. At both locations high concentrations of ozone were measured during the vegetation period from April to September: at the Hunsrlick location monthly mean values of 90-150p.gm- 3 air (Zimen 1985, 1986) and at the Taunus location of 50 -70 p.g m - 3 air (Hessische Landesanstalt fUr Umwelt, Wiesbaden, personal communication). Therefore, it appears very possible that especially ozone (and probably also further photooxidants and activated oxygen species and different organic compounds such as chloroethenes) is directly involved in the early damage to the biomembranes. The combined action of anthropogenic and cellular noxae as well as of natural stressors can disturb the equilibrium between the processes of membrane degradation and repair synthesis, leading to irreparable cell damage, even though a diverse system of protective mechanisms is present in the cells. The altered membrane structure might, for instance, impair the compartmentalization in cells, the transport processes and the function of stomata and thus lead to loss of function not only of the cells but also of the needles, and lead ultimately to the death of the whole tree.
Nitrogen metabolism of spruce needles Acknowledgements We gratefully acknowledge the support by the Federal Environmental Office (Umweltbundesamt Berlin) and the Commission of the European Communities (Brussels).
References BARNES, R. L.: Glutamine synthesis and translocation in pine. Plant PhysioI. 37, 323-327 (1962). BENNER, P. and A. WILD: Measurement of photosynthesis and transpiration in spruce trees with various degrees of damage. J. Plant PhysioI. 129, 59-72 (1987). BODE, J., H.-P. KUHN, and A. WILD: The accumulation of proline in needles of damaged spruce. Forstw. CbI. 104, 353-360 (1985). BOLLARD, E. G.: Nitrogenous compounds in plant xylem sap. Nature 178, 1189-1190 (1956). BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of a protein-dye binding. Analytical Chern. 72,248-554 (1976). BUTIN, H. and CH. WAGNER: Mykologische Untersuchungen zur «Nadelri:ite» der Fichte. Forstw. CbI. 104, 178 -186 (1985). ELSTNER, E.: Baumkrankheiten und Baumsterben. Naturwiss. Rundsch.36, 381-387 (1983). GRILL, D., I. POLZ, and W. PFEIFHOFER: Chlorophyll and Chlorophyllabbau in Fichtennadeln. Phyton 23, 79-90 (1983). HASEMANN, G., G. JUNG, and A. WILD: Losses of structural resistance in damaged spruce needles of locations exposed to air pollution. I. Mesophyll and central cylinder. European J. For. Path., submitted. - - - Losses of structural resistance in damaged spruce needles of locations exposed to air pollution. II. Epidermis and stomata (dermal tissue). European. J. For. Path., submitted. Hessische Landesanstalt fiir Umwelt: Waldbelastungen durch Immissionen - Immissionserfassung. 3. Zwischenbericht, Wiesbaden (1986). JUNG, G. and A. WILD: Electron microscopic studies of spruce needles in connection with the occurrence of new kinds of tree damage. I. Investigation of the mesophylI. J. Phytopath., in press (1988). KOWALSKI, T. and K. J. LANG: Die Pilzflora von Nadeln, Trieben und Asten unterschiedlich alter Fichten [Picea abies (L.) Karst.] mit besonderer Beriicksichtigung vom Fichtensterben betroffener Altbaume: Forstw. CbI. 103, 349-360 (1984). KRAUSE, G. H. M., U. ARNDT, C. J. BRANDT, J. BUCHER, G. KENK, and E. MATZNER: Forest decline in Europe: Development and possible causes. Water, Air, and Soil Pollution 31, 647-668 (1986). MARTIN, F., M. CHEMARDIN, and P. GADAL: Nitrate assimilation and nitrogen circulation in Austrian pine. PhysioI. Plant. 53, 105-110 (1981). MIFLIN, B. J. and P. J. LEA: The pathway of nitrogen assimilation in plants. Phytochemistry 15,873-885 (1976). MOHR, H.: «Baumsterben» als pflanzenphysiologisches Problem. BioI. in unserer Zeit 14, 103 -110 (1984). PAHLICH, E., H.-J. JAGER, and L. STEUBING: Beeinflussung der Aktivitaten von Glutamat-Dehydrogenase und Glutaminsynthetase aus Erbsenkeimlingen durch S02. Angew. Botanik 46, 183 -197 (1972). PATE, J. S.: Movement of nitrogenous solutes in plants. In: Nitrogen -15 in Soil Plant Studies. Int. Atom. Energy Ag. Vienna, 165 -187 (1971). - Uptake, assimilation and transport of nitrogen compounds by plants. Soil. BioI. Biochem. 5, 109 -119 (1973).
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- Transport and partitioning of nitrogenous solutes. Ann. Rev. Plant PhysioI. 31,313 -340 (1980). PRINZ, B.: Woran sterben unsere Walder? Umschau 18, 544-549 (1984). REHFUESS, K. E. and H. RODENKIRCHEN: Uber die Nadelri:ite-Erkrankung der Fichte (Picea abies Karst.) in Siiddeutschland. Forstw. CbI. 104, 381-390 (1984). RUHLE, W. and A. WILD: Die Anpassung des Photosyntheseapparates hi:iherer Pflanzen an die Lichtbedingungen. Naturwiss. 72, 10-16 (1985). SANDERSON, G. W. and E. C. COCKING: Enzymic assimilation of nitrate in tomato plants. I. Reduction of nitrate to nitrite. II. Reduction of nitrite to ammonia. Plant PhysioI. 39, 416-431 (1964). SAUER, H., A. WILD, and W. RUHLE: A study of the effect of phosphinothricin (glufosinate) on photosynthesis. II. The causes of inhibition of photosynthesis. Z. N aturforsch. 42 c, 270 - 278 (1987). SCHLEE, D.: Wirkung von abiogenen Stressoren auf Proteinabbau und Proteinsynthese in hi:iheren Pflanzen. BioI. Rundschau 24, 293-313 (1986). SENSER, M., F. SCHOTZ, and E. BECK: Seasonal damages in structure and function of spruce chloroplasts. Planta 126, 1-10 (1975). SENSER, M. and E. BECK: Kalteresistenz der Fichte. II. EinfluB von Photoperiode und Temperatur auf die Struktur und photochemischen Reaktionen von Chloroplasten. Ber. Deutsch. Bot. Ges. 92,243-249 (1979). SCHUTT, P., H. BLASCHKE, E. HOQUE, W. KOCH, K. J. LANG, and H. J. SCHUCK: Erste Ergebnisse einer botanischen Inventur des «Fichtensterbens». Forstw. CbI. 102, 158-166 (1983). SHEVYAKOWA, N. I.: Metabolism and the physiological role of proline in plants under conditions of water and salt stress. Soviet Plant PhysioI. 30, 597 -608 (1983). SMIRNOFF, N., P. TODD, and G. R. STEWART: The occurrence of nitrate reduction in the leaves of woody plants. Ann. Bot. 54, 363 -374 (1984). ULRICH, B.: Waldsterben durch saure Niederschlage. Umschau 11, 348-355 (1984). Waldschadenserhebung 1986: Bundesministerium fiir Ernahrung, Landwirtschaft und Forsten, Bonn, 1986. WALLACE, R. G. and D. J. SPEDDING: The biochemical basis of plant damage by atmospheric sulphur dioxide. Clean Air 10, 61-64 (1976). WELLBURN, A. R., C. HIGGINSON, D. ROBINSON, and M. WALMSLEY: Biochemical explanations of more than additive inhibitory effects of low atmospheric levels of sulphur dioxide plus nitrogen dioxide upon plants. New Phytologist 88, 223 -237 (1981). WENTZEL, K. F.: Hypothesen und Theorien zum Waldsterben. Forstarchiv 56, 51- 56 (1985). WILD, A.: Physiology of photosynthesis in higher plants. The adaptation to light intensity and light quality. Ber. Deutsch. Bot. Ges.92, 341-364 (1979). - Physiologische und cytomorphologische Charakterisierung von immissionsbelasteten Fichten. In: Beitrage der Pflanzenphysiologie zur Waldschadensforschung. Allg. Forstz. (AFZ) 27/28/ 29,734-737 (1987 a). - Licht als StreBfaktor bei Holzgewachsen. In: Klima und Witterung im Zusammenhang mit den neuartigen Waldschaden. Proceedings Symposium 13./14. Oktober 1986 (PBWU, Herausgeber). GSF-Bericht 10/87, 100-113, Miinchen-Neuherberg (1987 b). Licht als StreBfaktor bei Waldbaumen. Naturwiss. Rundschau 41(3),93-96 (1988).
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WILD, A. and J. BODE: Physiologische, biochemische und anatomische Untersuchungen von immissionsbelasten Fichten verschiedener Standorte. In: Wirkung von Luftverunreinigungen auf Waldbaume und Waldb6den. Proceedings Statusseminar Julich 2.-4. Dez. 1985. Spezielle Berichte der Kernforschungsanlage Jiilich - Nr. 369 (ISSN 0343-7639), pp. 116-181 (1986). WILD, A., B. DIETZ, U. FLAMMERSFELD, and 1. MOORS: Comparative investigations on the photosynthetic electron transport chain of spruce (Picea abies) with different degrees of damage in the open air. In: Effects of Air Pollution on Terrestrial and Aquatic Ecosystems. Proceedings Symposium Commission of the European Communities, Grenoble 18th-22nd May (1987). WILD, A., H. SAUER, and W. RUHLE: A study of the effect of phosphinothricin(glufosinate) on photosynthesis. 1. Inhibition of
photosynthesis and accumulation of ammonia. Z. Naturforsch. 42c, 263-269 (1987 b). WINGSLE, G., T. NXSHOLM, T. LUNDMARK, and A. ERICSSON: Induction of nitrate reductase in needles of Scots pine seedlings by NOx and NO:!. Physiol. Plant arum 70, 399-403 (1987). ZIEGLER, R. and K. EGLE: Zur guantitativen Analyse der Chloroplastenpigmente. I. Kritische Uberprlifung der spektralphotometrischen Chlorophyllbestimmung. Beitr. BioI. Pfl. 41, 11- 37 (1965). ZIMEN: Monatsberichte uber die MeBergebnisse des Zentralen Immissionsnetzes - ZIMEN - von Rheinland-Pfalz. Landesamt fur Umweltschutz und Gewerbeaufsicht (ed.), Rheinallee 97, D-6500 Mainz.
Investigation on the Nitrogen Metabolism of Spruce Needles in Relation to the Occurrence of Novel Forest Decline SYLVIA DUBALL
and
ALOYSIUS WILD
Institute of General Botany of the Johannes Gutenberg-Universitat, Saarstr. 21, D-6500 Mainz, Federal Republic of Germany Received June 24, 1987 . Accepted October 29, 1987
Summary In order to obtain information on the nitrogen metabolism of undamaged and damaged spruce (Picea abies), investigations were performed in 1985 and 1986 on the activity of glutamine synthetase and on the content of free ammonium and soluble proteins in spruce needles. In addition, the chlorophyll content was determined. Needles from the second needle year and the seventh whorl were used. The investigations were carried out in pair comparison on a 20-year-old spruce plantation and in comparison with damage classes on an 80-year-old spruce plantation. Both the pair comparison and the contrasting of different damage groups show an unequivocal decrease of the chlorophyll content correlated with increasing damage to the trees. The chlorophyll content proves to be a reliable indicator of the overall extent of damage to the tree, even in apparently green needles. A rise in the content of soluble proteins in the green needles of the damaged trees was shown at both locations. A breakdown of soluble proteins was only observed in definitely yellow needles. The accumulation of soluble proteins in the damaged trees is correlated with the measured activities of ribulose bisphosphate carboxylase and glutamine synthetase. The needles of the damaged trees contain reduced levels of ammonium. Conversely the activity of glutamine synthetase showed a major rise, especially in 1985. The results contradict the assumption that the increase in activity of glutamine synthetase is associated with an elevated nitrogen supply. The lower content of free ammonium as well as the increase in soluble proteins associated with an increase of the glutamine synthetase activity may indicate a high protein turnover and a shift in the protein composition. This can be interpreted as a response to environmental stress and membrane damage in the cells.
Key words: Picea abies, air pollution, chlorophyll content, ammonium, glutamine synthetase, novel forest decline, soluble proteins. Abbreviations: GS - glutamine synthetase; GS-GOGAT - glutamine synthetase-glutamate synthase; RubP - ribulose-1,5-bisphosphate.
Introduction For several years «novel forest decline» has been occurring over wide areas in Central Europe. This class of forest diseases cannot be subsumed under the general category of classical smoke damage alone, because it is manifest also in areas far away from major polluters or industrial centres and even in so-called «clean air» regions with low S02 pollution. Symptoms of novel forest decline were first observed on European silver fir in the early 1970's; they have subse© 1988 by Gustav Fischer Verlag, Stuttgart
quently been described in the early 1980's for Norway spruce and recently for broad-leaved trees. The damage has increased in severity and extent over the past six years. Damage severity increases with altitude, although symptoms can now be observed at lower-elevation forests. The damage, which now affects all economically-important tree species of the forests and which is mainly visible in older plantations, already comprises about 54% (damage categories 1-4) of the entire forest area of West Germany today; of this a total of about 19 % - represented by the damage categories 2 - 4 -
492
SYLVIA DOBALL and ALOYSIUS WILD
shows medium to severe damage (d. Waldschadenserhebung 1986). There are numerous hypotheses on the causes of novel forest decline. The major factors discussed are natural factors or anthropogenic pollutants. Nevertheless, it is not known whether primarily natural or primarily anthropogenic factors (especially air pollutants) are responsible for the tree diseases. An involvement of climatic, edaphic, silvicultural, mycological or microbial factors in the causal complex is undisputed and may be of crucial significance for damage in restricted areas (Kowalski and Lang 1984, Rehfuess and Rodenkirchen 1984, Butin and Wagner 1985). However, these factors cannot be the sole cause of such massive and widespread damage to the forest ecosystem. There is increasing evidence that anthropogenic pollutants are crucial for the novel forest decline over large regions (Elstner 1983, Mohr 1984, Prinz 1984, Ulrich 1984, Wentzel 1985, Krause et al. 1986). The investigations described here were carried out in the context of our research project on the physiological, biochemical and cytomorphological characterization of spruce trees growing in natural habitats and showing different degrees of damage. Photosynthesis, the water balance, the content of chemical elements and light- and electron-microscopic structure were previously examined (Wild and Bode 1986, Benner and Wild 1987, Jung and Wild 1988, Wild 1987 a, b). Here we report on aspects of nitrogen assimilation. On the basis of analysis of xylem sap, it is thought that in gymnosperms nitrate reduction takes place predominantly in the roots (Barnes 1962, Pate 1971, 1980, Smirnoff et al. 1984, Wingsle et al. 1987). The xylem sap contains only minor amounts of nitrate (Martin et al. 1981). Nitrogen is mainly transported from the root into the shoot largely in the form of organic compounds such as amino acids and amides (Bollard 1956, Barnes 1962, Pate 1973, Martin et al. 1981). In the needles, assimilation and reassimilation of ammonium takes place via the GS-GOGA T pathway (Miflin and Lea 1976). In order to obtain an insight into the nitrogen metabolism of spruce with damage of varying severity, we investigated the activity of glutamine synthetase as well as the content of free ammonium and soluble proteins in needles at two sample locations with different categories of damage defined in the context of our research program. In addition, chlorophyll content and fresh and dry weights of the needles were determined.
Materials and Methods The locations and the trees were selected with the assistance of professional foresters.
Locations
Two locations in the Hunsriick and in the Taunus mountains were chosen for the investigations. These locations were comparable with regard to altitude and thus e.g. duration of the vegeta-
tion period which is shortened by two to three weeks on the slopes compared to valley locations. Location in the Taunus mountains
The location is on a southwest slope of the Feldberg (Konigstein Forestry Office, Glashiitten Forest District, Division 43/44) with an altitude of 660 -760 m above sea level. The annual precipitation is about 850mmm- 2 a- 1 (Hessische Landesanstalt fiir Umwelt 1986). The soil conditions are characterized by highly acid typical brown earth consisting of mica sandstone and T aunus quartzite. Location in the HunsrUck mountains
This area is on a moderately steep south slope on the west side of the Hunsriick at about 660 m above sea level (Idar-Oberstein Forestry Office, Hattgenstein Forest District, Division 257). The annual precipitation is 800 - 1000 mm m - 2 a -I. The soil consists mainly of quartzite and brown earth (pH3.5-4.3). The investigations presented were carried out between April and October in 1985 and in 1986. In 1986 only the Hunsriick location was further investigated. During the trial period, a relatively cool climate with very little sunshine and much precipitation prevailed at both locations from April to June 1985. From July to October, the weather was dry and warm, with a relatively high proportion of sunny days. During the 1986 vegetation period, a warm, dry, sunny climate prevailed at both locations (Zimen 1985, 1986).
Material Experimental spruce at the location in the Taunus mountains
This is a spruce [Picea abies (L.) Karst.] plantation which is about 80 - 90 years old and which displays damage in all categories. In spring 1985, 16 trees of different damage classes were selected with the assistance of the Hessian Forest Research Institute for the investigations. A first selection was made on the basis of visible characteristics, above all needle discoloration and degree of needle loss. A more detailed classification was made after harvesting (see results). Experimental spruce at the location in the HunsrUck mountains
This plantation allowed the investigations to be carried out as a pair comparison (according to Schiitt et al. 1983), since slightly damaged trees and trees of the same age (about 20 years) with clearly visible damage were growing side by side. Five (1985) and four (1986) tree pairs were selected for the measurements on the basis of visible characteristics in spring. The damage criteria used in selecting the trees at the two locations were the loss of needles and yellow discoloration (Waldschadenserhebung 1986). During the rainy and cool months (May to July) of 1985, differences became less obvious between the partners of a pair. In the warm and dry autumn of 1985, however,' the differences became significant again. During the trial period 1986, no such convergence between the appearance of damaged and largely asymptomatic trees was observed. The investigations at the two locations were carried out on the needles of the second needle year and the seventh whorl of the trial trees.
Methods The investigations were carried out with needle material which was quick-frozen in liquid nitrogen (-196°C) on site, directly after harvesting, and stored at -20°C.
Nitrogen metabolism of spruce needles Preparation of the needle extract
LOg (frwt) needles were homogenized (cutter rod 18N, Ultra Turrax, IKA-Werk Staufen im Breisgau) in 8-10 ml of a O.5M trisHCl buffer, pH 8.5 (modified according to Sanderson and Cocking 1964). To protect the glutamine synthetase, the buffer contained ImM cysteine, 10mM of DTE, 0.5mM MgS0 4, 0.5 mM EDTA and 2 % soluble PVP (polyvinylpyrrolidone 25), which largely inactivates the phenols released during extraction. The phenols could be completely absorbed by insoluble PVP (Polyclar AT), but PVP interfered with the extraction of the proteins. Thus the presence of a part of the phenols had to be tolerated when using soluble PVP. The optimum concentration of PVP was 2 % in our experiments with the protein assay method used (BIO-RAD, Bradford 1976). Homogenization and subsequent centrifugation (30 min at 20,000 g) were performed with precooled equipment at 4 0c. The supernatant obtained after centrifugation (crude extract) was used immediately to determine the concentrations of soluble proteins and to assay the glutamine synthetase activity. Detennination of the activity ofglutamine synthetase
After reaction of a defined amount of the crude extract for a specific time with a given amount of the substrate of glutamine synthetase (sodium glutamate) the enzyme activity can be calculated from the concentration of the end product of the reaction (gammaglutamyl-hydroxamate). Final composition of the incubation medium (total 2 ml): 60 mM imidazole-HCl buffer, pH7.3, 50mM MgS04, 10mM EDTA (Titriplex III), 15mM ATP, 10mM NH2 0H, 80mM sodiumglutamate, 200 JLl extract. The reaction proceeded for fifteen min at 37°C. It was started by addition of the extract and stopped by addition of 1.5 ml of an acid FeCh solution (0.37 M FeCh, 0.67 M HCl, 0.2 M CChCOOH) to the reaction mixture, which results in precipitation of the proteins and formation of a brown colored complex of gamma-glutamyl-hydroxamate with the ferric chloride. The measurement was carried out spectrophotometrically at a wavelength of 560 nm. A reaction stopped immediately after addition of the extract served as timezero control. In order to preclude interference with the enzyme assay by the phenols contained in the crude extract, which also react to form a black brown colored complex with ferrous chloride, the extinction of the controls was determined against air at 540 nm. The extinction values determined fluctuate only within the range of error in the measurements. Therefore, an error occurring in this way can be neglected.
troprusside as catalyzer intensifies this reaction. Since the color complex is only formed in acid medium (pH 3.5), the needles were homogenized in sulfosalicylic acid at pH 3.5. The extracts obtained were mixed with 1-3 g insoluble Polyclar, centrifuged and diluted 1 : 20 for the reaction.
Results Characterization of the spruce trees investigated at the location in the Taunus mountains
The individual spruce trees were classified in different damage groups at the Taunus location mainly on the basis of the description of the samples of the needles which were later used for the physiological and biochemical investigations. The trees were classified on the basis of the different portions of undamaged green, green and bright green needles with symptoms of damage (such as point necroses, gold tips and other discolorations), yellow and brown needles in the respective mixed samples. Further criteria were the needle cover of the branch harvested, as well as the overall impression of color. The physiological and biochemical investigations as well as the cytomorphological investigations of the mixed needle samples carried out in parallel proved to be largely consistent with this classification (see also Benner and Wild 1987, Hasemann and Wild, in preparation). Chlorophyll content
A significant decrease of the total chlorophyll was shown at both locations in the spruce with more severe symptoms (Fig. 1, Table 1). The annual mean value of the more damaged trees in the Hunsriick location is about 27 % lower (statistical probability P = 0.01). The trees of group III at the Taunus location show a decrease of 35 % (P = 0.01) com-
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Detennination of the content of soluble proteins
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The amount of chlorophyll (chlorophyll a and b) was determined in accordance with the method of Ziegler and Egle (1965) with 80 % acetone. Detennination of the content offree ammonium
The assay reaction is based on a method developed by Berthelot, and is a reaction between NH4 + and phenol in the presence of sodium hypochlorite with formation of a blue colored complex. The concentration can be determined spectrophotometrically at a wavelength of 625nm (e.g. Wild et al. 1987b). Addition of sodium ni-
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Fig. 1: Content of chlorophyll in relation to the dry weight (drwt). Location in the Hunsriick: comparison of five tree pairs, D = slightly damaged partner, • = more severely damaged partner. Location in the Taunus: D = four slightly damaged, I2:J = eight severely damaged, • = four very severely damaged spruce trees. MV = mean values of the 1985 investigation period. f------i = standard deviation.
494
SYLVIA DUBALL
and ALOYSIUS
WILD
pared to the slightly damaged trees in group 1. These data were obtained in 1985. In 1986 only the Hunsriick location Table 1: Annual mean values (MY) of several parameters measured during the 1985 vegetation period. Location in the Hunsriick: harvest dates on May 28th, July 29th, August 23rd and October 2nd. Comparison of five tree pairs (1 = slightly damaged, 2 = severely damaged partner). Location in the Taunus: harvest dates on April 26th, July 1st and October 7th in the damage groups I, II, and III. parameter
Taunus location Tree MY 1985 group I 2.25± 0.04 II 2.20± 0.02 III 2.10± 0.05
frwt/dr wt g.g-I
Hunsriick location Tree MY 1985 group 2.3l± 0.01 1 2 2.28± 0.02
Chlldrwt mg·g- I
1 2
2.5l± 0.17 1.84± 0.20
I II III
2.91± 0.35 2.32± 0.46 1.89± 0.26
Solubl: rroteinsl dr wt mg·g
1 2
13.2l± 4.57 16.44± 2.26
I II III
17.36± 8.80 20.69± 3.91 21.78± 3.42
free NHt I dr wt /Lg. g-I
1 2
232.70±41.66 218.52±45.30
I II III
268.55±31.47 172.81 ±26.21 173.8l±35.41
abs. activity of GS /Lmol . h - I . g - I dr wt
1 2
106.12±32.20 159.75±37.78
I II III
106.65 ± 17.79 180.25±44.47 188.53 ±45.55
01
Free ammonium Overall, a decrease of the ammonium content was found with increasing damage to the trees at the two locations: about 6 % at the Hunsriick location (P = 0.1) and 35 % (P = 0.1) at the Taunus location (Fig.3, Table 1). The same trend was observed in the subsequent vegetation period at the Hunsriick location (17%, P = 0.2).
Activity of glutamine synthetase The absolute enzyme activity (units g-I dry weight) of glutamine synthetase was shown to be raised in the severely
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was further investigated. Similar results were obtained: a decrease of about 25 % (P = 0.01). The rise in chlorophyll content during the summer months of 1985 (Fig. 1) could also be observed in 1986 on the trial trees in the Hunsriick. This is consistent with the function of the chloroplasts within a vegetation period (Senser et al. 1975).
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Fig.2: Content of soluble proteins in relation to the dry weight. Location in the Hunsriick: pair comparison with five tree pairs, 0 = slightly damaged, • = more severely damaged partner. Location in the Taunus: 0 = four slightly damaged spruce (group I), [{J = eight severely damaged •spruce (group II), • = four very severely damaged spruce (group III). MY = mean values of the 1985 investigation period. 1-----1 = standard deviation.
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Fig.3: Content of free ammonium in relation to the dry weight. Location in the Hunsriick: comparison of five tree pairs, 0 = slightly damaged, • = more severely damaged partner. Location in the Taunus: o = four trees of group I, [{J = eight trees of group II, • = four trees of group III. 1985 investigation period.
Nitrogen metabolism of spruce needles
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damaged spruce trees at both locations compared to the relatively asymptomatic control trees (Fig.4, Table 1). The increase in activity in samples collected at the Hunsriick location was about 51 % in 1985 (P = 0.05); in 1986 a consistent trend was found (9 %, P = 0.1). 85 % increase (P = 0.1) was measured in the trees of group III (very severely damaged) at the Taunus location. Less clear-cut statements can be made with regard to the specific activity of glutamine synthetase (units mg -1 soluble protein), because the protein content and the enzyme activity changed with the degree of damage. In severely and irreversibly damaged needles, however, an unchanged and in some cases even an elevated specific activity of the enzyme occurs compared to apparently green needles of a tree. These severely damaged needles show intense yellowing and a decline in protein content, as well as a decrease in the volume activity of the glutamine synthetase. Discussion Both the pair comparison at the Hattgenstein location in the Hunsriick mountains and the comparison of the different damage groups at Konigstein in the Taunus mountains showed an unequivocal decrease in the chlorophyll content of the more severely damaged trees at the different harvest dates. The chlorophyll content thus proves to be a reliable indicator of the overall damage situation of the tree even in needle groups that appear visibly green and healthy (Bode et al. 1985, Wild and Bode 1986, Benner and Wild 1987). Fig. 1 shows that the chlorophyll content of the 1984 needle generation is without exception much lower in the trees classified as damaged. This does not only apply to the mean values of all five tree pairs used in Fig. 1, but also to each individual pair comparison. The mean value of all data is 2.5 mg ChI a + b per g dry weight for the controls and 1.8 mg for the damaged partners in 1985. This is noteworthy because the differences in the damage appeared to be slight. There were often, at least superficially, no differences visible between the needle groups of the 1984 needle generation;
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MV
I
this was particularly clear during the rainy summer months of 1985 (Zimen 1985). When harvesting the material, attention should be paid to similar light exposure of the branches. It is known that the photosynthetic apparatus adapts to the light conditions (e.g. low light and high light adaptation: Wild 1979, Ruhle and Wild 1985). In addition, it is remarkable that the chlorophyll contents of the needles rose markedly from the harvesting date in May to that in June (see also Senser and Beck 1979, Grill et a1. 1983). Corresponding results were obtained during the investigations of the spruce trees more than 80 years old at Konigstein in the Taunus mountains. The content of ChI a + b decreased with the increasing damage to the trees. It was actually 35 % lower in the severely diseased spruce trees of damage group III than in the damage group I. A lower chlorophyll content is always found in the needles of the damaged trees, even in needle groups with a green appearance, and with no yellowing or other visible characteristics of damage. This, however, applies only to plant material of similar age growing in the same environment and harvested at the same time, as done in the present study. Similar comparisons were made with regard to the nitrogen metabolism. The highly symptomatic trees from the location in the T aunus, which were classified in the damage groups II and III had 35 % less free ammonium in the needles than the control spruce trees of the damage group I. In pair comparisons of the younger spruce trees at the Hunsriick mountains, the difference is less pronounced. A rise in the content of soluble proteins was shown at the two locations. The needles from the trees of damage class III had an average of 25 % more soluble proteins compared to those of damage class I. A consistent trend was found in the pair comparison at the Hunsriick location. There was evidently a degradation of soluble proteins only in needles with an intense yellow discoloration; this is evidently associated with irreversible cellular damage (Hasemann et a1., submitted). There was a definite rise in the activity of glutamine synthetase in the damaged trees, especially in 1985. An increase of the activity by an average of 85 % was found in severely
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damaged trees in the Taunus. An increase averaging 51 % was observed in the Hunsrlick pair comparison in 1985, whereas in the following year 1986, the difference was only 9 %. Considering the reduced NH3 content of the tissue and the pair comparison it appears as if any assumption that the increase of the activity of glutamine synthetase is causally connected with an excessive nitrogen supply (N0 3-, NH4 +, NOx) from the air or from the soil is not necessarily correct. In addition, the rate of pollution at the Konigstein location (Environmental Research Plan of the Federal Ministry of Environmental Protection and Reactor Safety, Concluding Report A, Hessen 1986) showed that the nitrogen influx reached maximum values in the summer months. The ammonium contents in the needles showed the lowest values at this time. The increased content of soluble proteins in the damaged trees is in good agreement with the measured activities of glutamine synthetase and RubP carboxylase. We did not detect any reduction in the in vitro activity of RubP carboxylase in the more severely damaged spruce trees at the two locations (Wild and Bode 1986, Wild 1987 a). This enzyme accounts for a major proportion of the soluble proteins. However, it is surprising that the damaged spruce needles, which are impaired in their photosynthetic activity (Benner and Wild 1987), synthesize large amounts of soluble proteins; this requires large amounts of energy. The low content of free ammonia as well as the increase of soluble proteins in connection with a substantial increase in the activity of glutamine synthetase indicates a high rate of metabolism of nitrogenous compounds. This could be a defense reaction of the organism to stress conditions and to the primary damage. An increase of glutamine synthetase activity has also been observed in gassing experiments with S02 and NO x in dicotyledonous herbaceous plants (Pahlich et al. 1972, Wallace and Spedding 1976, Wellburn et al. 1981). In addition, a surprisingly high specific activity of glutamine synthetase is found in yellow needles which already display a substantial loss of protein and which have suffered irreversible damage. These findings are an additional indication that the activation of glutamine synthetase should be considered as a general stress symptom. The organism responds to stress with specific and nonspecific reactions (adaptation syndrome) in order to eliminate or reduce the stresses and the primary damage elicited. The organism mobilizes defense mechanisms which can be subdivided into different phases (Schlee 1986). All stress-inducing factors have in common the activation of the biochemical and physiological apparatus of the organism. Glutamine synthetase, the actual N-assimilating enzyme of the GS-GOGAT pathway, thus has an absolutely crucial role in the assimilation and reassimilation of NH3. This enzyme accordingly has a particularly clear functional significance and physiological relevance for the activation of protein turnover and modification of the protein composition. The accumulation of the amino acid proline, which is regarded as a general stress metabolite with protective function, also indicates metabolic alterations at the protein level. In the trees recently investigated, the content of free proline was generally higher than in the control partners (Bode et al. 1985, Wild and Bode 1986). The findings showed proline ac-
cumulation in the damaged spruce at different ages, at different locations and in different seasons. There may be a connection with the elevated glutamine synthetase activity, because the biosynthesis of proline in plant tissues starts from glutamate (Shevyakowa 1983). Together with the chlorophyll content, the proline content can serve at least as an indicator for damage to the spruce (Wild 1987 a). Benner and Wild (1987) found a marked damage to photorespiration in the diseased spruce trees. By inhibition of glutamine synthetase with glufosinate (phosphinothricin) it was shown that in Sinapis and Valerianella plants the photorespiratory nitrogen cycle is the most important ammoniumforming and -consuming process in photosynthetically active leaves and thus the central point of redistribution of NH4 + (Wild et al. 1987 b, Sauer et al. 1987). The decline of the ammonium content in the needles of diseased spruce trees might therefore be associated with e.g. the reduced photorespiratory turnover of ~ + . The investigations of the chlorophyll content and the alterations in nitrogen metabolism described here were carried out in the context of an extensive research project on physiological, biochemical and cytomorphological characterization of damaged spruce trees growing in natural habitats. The results for different components of the photosynthetic apparatus (Benner and Wild 1987, Wild 1987 a, b, Wild et al. 1987, Wild 1988) and the electron microscopic analyses Gung and Wild 1988) showed consistently that early damage occurs especially at the cell membranes. This membrane damage is apparent even in the green needles of damaged spruce. Anthropogenic air pollutants appear to playa major role in the genesis of these effects. The loss of chlorophyll in the damaged spruce needles may therefore be associated with destabilization and damage to the thylakoid membranes. Furthermore, the decline of the ammonium content, the increase of soluble proteins and the marked increase in the activity of glutamine synthetase constitute metabolic reactions and responses which may be caused by the damage to the biomembranes and by an inhibition of photorespiration which may lead to the synthesis of stress metabolites. At both locations high concentrations of ozone were measured during the vegetation period from April to September: at the Hunsrlick location monthly mean values of 90-150p.gm- 3 air (Zimen 1985, 1986) and at the Taunus location of 50 -70 p.g m - 3 air (Hessische Landesanstalt fUr Umwelt, Wiesbaden, personal communication). Therefore, it appears very possible that especially ozone (and probably also further photooxidants and activated oxygen species and different organic compounds such as chloroethenes) is directly involved in the early damage to the biomembranes. The combined action of anthropogenic and cellular noxae as well as of natural stressors can disturb the equilibrium between the processes of membrane degradation and repair synthesis, leading to irreparable cell damage, even though a diverse system of protective mechanisms is present in the cells. The altered membrane structure might, for instance, impair the compartmentalization in cells, the transport processes and the function of stomata and thus lead to loss of function not only of the cells but also of the needles, and lead ultimately to the death of the whole tree.
Nitrogen metabolism of spruce needles Acknowledgements We gratefully acknowledge the support by the Federal Environmental Office (Umweltbundesamt Berlin) and the Commission of the European Communities (Brussels).
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