Phenolic Compounds in Needles of Norway Spruce Trees in Relation to Novel Forest Decline I. Studies on Trees from a Site in the Northern Black Forest

Biochem. Physiol. Pflanzen 188, 305 - 320 (1992) Gustav Fischer Verlag Jena

Phenolic Compounds in Needles of Norway Spruce Trees in Relation to Novel Forest Decline I. Studies on Trees from a Site in the Northern Black Forest CHRISTINE M. RICHTER and ALOYSIUS WILD Institute of General Botany of the Johannes Gutenberg University, Mainz, Germany Key Term Index: phenolic compounds, novel forest decline, picein, p-hydroxyacetophenone, catechin, piceatannolglucoside; Picea abies

Summary Contents of selected phenolic compounds in needles of Norway spruce trees (Picea abies) from the Black Forest were measured using a HPLC-technique elaborated for serial studies in forest decline research. Measurements on needles that were harvested on several dates during two growing seasons gave no hint of seasonal variations in the concentrations of the studied phenolic compounds. Values for picein demonstrate an average decrease in the needles of severely damaged trees compared with the still undamaged ones, but the results are impaired by strong individual variations among the single trees. p-Hydroxyacetophenone was found in only very low amounts showing no constant differences between damaged and undamaged trees. In contrast to that catechin, epicatechin, piceatannolglucoside, and two still unidentified compounds show significantly higher contents in needles of the damaged trees.

Introduction The characterization of plant phenolics has often been the subject of wide-spread studies in the fields of chemotaxonomy and the search for new plant raw materials. Because of the toxic and tanning properties of many phenolic compounds they have obtained much interest in the attempts to understand mechanisms of plant disease and disease resistance (CRUICKSHANK and PERRIN 1964; SCHONBECK and SCHLOSSER 1976; OVEREEM 1976; FRIEND 1979; HOQUE 1982; HAHLBROCK and SCHEEL 1987). In many cases an enhancement of the production of phenolic compounds due to both biotic and abiotic stress has been observed (FARKAS and KIRALY 1962; HOWELL 1974; SWAIN 1977; RUBIN et a1. 1983; RHODES 1985; HAHLBROCK and SCHEEL 1987). In consequence metabolism of phenolic compounds also became part of studies on damage caused by anthropogenous stress (i.e. gaseous air pollutants, heavy metals, herbicides, etc.). The studies focussed onto damage exhibited by crop and forestry plants which were reduced in yield and nutritional or useful quality (HOWELL 1970; HOWELL and KREMER 1973; KEEN and TAYLOR 1975; TINGEY et a1. 1975 and 1976; RUBIN et a1. 1983; FLINT et a1. 1985; KATOH et a1. 1989). Abbreviations: d, damaged; DW, dry weight; pHAP, p-hydroxyacetophenone; PTG, piceatannolglucoside; u, undamaged; ym, yearly mean value

BPP 188 (1992) 5

305

Within the scope of novel forest decline research most studies carried out on spruce trees have laid emphasis on a major phenolic component called picein and its aglycone phydroxyacetophenone (HOQUE 1984 and 1985 ; OSSWALD et al. 1986 ; OSSWALD and BENZ 1989; JENSEN and L!2lKKE 1990). But the investigators found different reactions of these substances to stress or damage. HOQUE (1984 and 1985) proposed that the relation pHAP/picein may serve as an indicator for damage since it rose to values bigger than one in the needles of the damaged trees which he and other authors had examined (KICINSKI et al. 1988; JENSEN and L!2lKKE 1990). Only few investigators examined a wider range of phenolic components in spruce needles with regard to forest damage (STRACK 1988; KICINSKI et al. 1988; DITTRICH et al. 1989; HELLER et al. 1990). The goal of the studies presented here was to establish a method for serial measurements of several soluble phenolic compounds including picein and pHAP in spruce needles. Spruce trees of different forestry sites were studied with this method. This work describes the results obtained by the studies of trees from the Black Forest showing acute damage.

Material and Methods Plant material

Studies were carried out with needles from Picea abies (L.) Karst. harvested at a forestry site during the growing seasons of 1989 and 1990. South to south-west exposed twigs were taken from the sixth to eighth whorl. Twig pieces bearing the second and the third needle generation were immediately frozen in liquid nitrogen in order to avoid metabolical changes due to mechanical injury. The deep-frozen twigs became so brittle that the needles could easily be stripped off. They were kept in liquid nitrogen until storage at - 80°C. On several dates in 1989 and 1990 mixed samples composed of needles from six trees of each group (see below) and samples from three single trees each were taken for studies. Freudenstadt site

This site is situated on an unprotected plateau at the Schollkopf mountain, ca. 835 m above sea level and 4 km south of Freudenstadt in the northern Black Forest. It belongs to the Forest District Vordersteinwald (Division IIII12a4 ), Forestry Office Freudenstadt. The soil types below the examined spruce stand are pseudogley-brown earth and stagnogleypseudogley. Oberer Buntsandstein represents the geological formation. Weathering of the sandstone results in the formation of clay layers which are more or less impermeable. The type of humus is mor and raw humus-like mor, respectively. The soil is poor of nutrients and characterized by a marked deficiency of magnesium (v. WILPERT and HILDEBRAND 1992). The studied site is situated in a so-called "Reinluftgebiet" (clean area region) with low immission concentrations of nitric oxides and sulfur dioxide (monthly mean values below 18 and 16 f,tg m- 3 , respectively). But it is characterized by high ozone values that amount to 125 f,tg m- 3 (monthly mean) during summer time. In the course of the considered vegetation periods the maximal half-hour values rose up to 230 f,tg m- 3 (BAUMBACH et al. 1989 and 1990). The immissions and the climatic data were registered by IVD (Institut flir Verfahrenstechnik und Dampfkesselwesen), Department Air Pollution Prevention, University of Stuttgart, at a measuring station that is situated at a distance of 500 m from the stand. The general type of tree damage at this site (and elsewhere in the Black Forest) is called "Montane Vergilbung" (mountainous yellowing of spruce) (SIEFERMANN-HARMS 1990). Three areas of spruce trees were selected for the studies: 306

BPP 188 (1992) 5

1. 20-year-old spruce trees (damage class 0), 2. 40-50-year-old undamaged spruce trees (damage class 0), 3. 40-50-year-old damaged spruce trees (damage class 2-3; for classification parameters see Waldschadenserhebung, 1989). The studies carried out at this site are part of a joint research program on novel forest decline sponsored by the KfK-PEF (Kernforschungszentrum Karlsruhe - Projekt Europiiisches Forschungszentrum flir MaBnahmen zur Luftreinhaltung, 1990). Extraction of phenolic compounds

0 .5 g of deep-frozen needles plus added internal standard (gallic acid, 45 mM in 50% methanol) were homogenized in 80% aqueous methanol, that was chilled to -20°C before (Ultra Turrax TI5, Janke & Kunkel). For transport purposes needles were kept in liquid nitrogen until homogenization. After centrifugation of the homogenate the pellet was washed with 80 % methanol. The combined supernatants were adapted to room-temperature and the volume was made up to 5 ml afterwards. In order to remove remaining particles the extract was filtered (Sartorius membrane filters, regenerated cellulose, 13 mm, 0 .2 !lm). I ml of the filtrate was forced through a single use column filled with a C I8 stationary phase, that had been moistened with 80 % methanol before (SEP-PAK C 18 Cartridges, Waters/Millipore). This procedure plus subsequent elution with 1 ml 80% methanol resulted in retaining the lipids within the column whereas the considered phenolic compounds were completely in the eluate. For following HPLC-analysis I ml of 0 .5 % natrium acetate-buffer, pH 3.3, was added . The extracts could now be subjected to analysis or stored at - 20°C. From each needle sample two (single trees) or three (mixed samples) parallel extracts were prepared. High performance liquid chromatography

The extracted phenolic compounds were separated and quantitatively analysed by reversed phase HPLC (HPLC two-pump-system, LKB; Nucleosil-Cl 8 column, 125 mm X 8 mm X 4 mm, 5 !lm, 100A, Macherey-Nagel; pre-column, LiChrospher RP-18, 5 !lm, LiChroCart 4-4, Merck). Elution was performed at room-temperature with a flow rate of 1 ml min -I. The mobile phase consisted of 0.5 % natrium acetate-buffer, pH 3.3, (eluent A) and methanol (LiChrosolv Gradient grade, Merck ; eluent B). Separation was achieved by a four-step gradient starting at 17 % eluent B: 0-4 min linear 17-22 % B, 4-9 min linear 22-35 % B, 9-14 min linear 35-50% B, 14- 18 min linear 50-80% B, 18-20 min isocratic 80% B. The pressure within the system ranged from 120 bar in the beginning to 180 bar during the course of the separation. The phenolic compounds were detected by absorption of light at a wavelength of 275 nm (Shimadzu, SPD-6AV). The signals were recorded and the peak areas calculated by a Shimadzu Data Processor (Chromatopac CR-3A) . Each needle extract was analysed twice. For identification purposes detection of fluorescence was also applied (Shimadzu RF-530) . Standards

Reference substances were purchased from Serva (gallic acid), Fluka (catechin, epicatechin, 0coumaric acid) and Merck (p-hydroxyacetophenone, p-coumaric acid). Picein was synthesized according to YAMAGUCHI et al. (1990). Isorhapontine was kindly placed at disposal by Dr. W . HELLER, GSF Neuherberg. Calibration was performed with these listed substances dissolved in 50 % aqueous methanol. Values of piceatannol glucoside were calculated using those of isorhapontine according to SANO and SAKAKIBARA (1977). Absorption spectra

Absorption spectra were taken from standards dissolved in 50% methanol (Shimadzu, multipurpose recording spectrophotometer MPS-2000 plus graphic printer PR3). Additionally spectra from BPP 188 (1992) 5

307

components of both the spruce needle extract and the standard solution were recorded by a diodearray-detector (Waters/Millipore, model 991) during analysis by HPLC. Hydrolysis of glycosilated phenolic compounds

Acid hydrolysis of phenolic compounds was performed in 6 % hydrochloric acid at 100 °C according to MABRY et al. (1970). For enzymatic hydrolysis ~-glucosidase (Fluka) was applied (MABRY et al. 1970). An aliquot of spruce needle extract was evaporated until dryness and the residue redissolved with the same volume of 0.5 M natrium acetate-buffer, pH 5. A tip of a spatula of enzyme was added with subsequent incubation at 37 °C over night. Then the enzyme was precipitated by adding a threefold volume of methanol (- 20 0C). After centrifugation an aliquot of the supernatant was analysed by HPLC.

Results Method The method for extraction and analysis of the considered phenolic compounds from spruce needles was elaborated starting from the works of KICINSKI and KETTRUPP (1987), NIEMANN (1977 and 1978), OSSWALD et al. (1986) and COURT (1977). Studies were concentrated on a group of free and glycosilated phenols that can mainly be found in cell vacuoles. Extracts that are prepared with 80 % aqueous methanol do not contain as many lipids as compared with the application of 100% methanol whereas the amount of extracted phenols concerned remains constant. Removal of residual lipids from extracts with SEPPAK single use columns proved to be complete and easy in handling (in contrast to the application of light petrol) which is important for serial extractions. The procedure was consequently carried out at very low temperatures in order to avoid activity of glycosidases and oxidases from the beginning. Addition of the polyphenoloxidases inhibitor K 2 S20 5 (SENSER and BECK 1978) did not show any effect which indicates that inactivation of these enzymes was already achieved. Homogenization of needles in liquid nitrogen with mortar and pestle was not advantageous since the use of an Ultra Turrax was likewise efficient and quicker. Additional ultra sonication at room temperature led to a slight reduction of the yield of phenolic compounds. HPLC-analysis with the described column resulted in a satisfying separation with simultaneously short elution time. Fig. 1 a shows a chromatogram of a needle extract. For separation the pH of eluent A was very important. A pH of 3.3 resulted in the best resolution of the peaks that are eluted after 4.5 to 11 minutes. General identification of spruce needle phenolic compounds was obtained by comparison of their retention times with those of reference substances and co-chromatography. Additionally detection wavelengths were changed according to the spectral properties of each substance in question. Information about those properties was obtained from recorded absorption spectra of reference substances and from data found in literature (ENDRES et al. 1962; DITTRICH 1970). Application of a diode-array-detector confirmed the first findings. Further identification of glucosilated phenols was obtained by hydroly308

BPP 188 (1992) 5

a)

7

3 4

o1'---_ _----'110_ _ _----"20min

b)

6

10

3

~

11

20 10 o,-l_ _ _--IIL-_ _--.JI min

extract detected at 275 nm. Fig. 1. Chromatograms of phenolic compounds from a spruce needle 1 Picein, 2 analyzed idase. ~-glucos with is hydrolys ic Separation a) before, b) after enzymat hin, 6 pHAP, 7 PTG, Epicatec nd,S compou unknown analyzed 4 , Catechin 3 unknown compound, acid. 8 p-Coumaric acid, 9 Isorhapontin, 10 Piceatannol, 11 o-Coumaric

hydrolysis. Here sis. Fig. 1 b shows a chromatogram of a needle extract after enzymatic phenone and the pice in and other peaks are distinctly reduced whereas p-hydroxyaceto by detecting erized charact be ally addition coumaric acids increased. The stilbenes could their fluorescence at 410 nm. BPP 188 (1992) 5

309

Table 1. Selected phenolic compounds given in 1JR101 g-l DW from spruce needles of the second generation taken at Freudenstadt site. Concentrations of the unknown compounds (named after their retention times) are calculated as gallic acid equivalents. a) values of mixed samples for each sampling date; b) single trees, needles sampled on 28-Jun-1989. Standard deviations in brackets. a)

Peak 5.5'

Sampling Date

Sample

Epicatechin

19-Apr-89

young old u old d young old u old d young old u old d young old u old d

23.8 24.5 30.6

4.6 6.0 8.1

3.5 6.4 9.2

17.9 28.6 37.9

5.4 6.3 10.8

29.0 31.5 42.7

7.9 7.9 ll.8

23.0 18.5 37.1

6.5 6.6 ll.8

4.7 5.2 10.4 5.1 5.6 8.9 3.9 4.9 7.4

mean value 1989

young old u old d

23.4 (4.6) 25.8 (5.6) 37.1 (5.0)**

6.1 (1.4) 6.1 (0.8) 10.6 (1.8)***

25-Apr-90

young old u old d young old u old d

27.5 28.7 27.8

5.1 7.5 9.8

4.7 8.0 10.1

21.6 31.7 59.1

5.8 8.5 12.3

4.6 5.8 8.6

young old u old d

24.6 30.2 43.4

5.2 8.0 1l.1

4.6 6.9 9.3

28-Jun-89

30-Aug-89

08-Nov-89

04-Nov-90

mean value 1990

Peak 9.1'

4.3 (0.7) 5.5 (0.6) 8.9 (1.2)***a

b)

Tree-No.

Epicatechin

Peak 5.5'

Peak 9.1'

1u 2u 3u

22.5 19.2 29.6 36.1 37.2 33.0

6.4 4.7 8.5 9.4 12.3 10.3

6.4 5.3 5.3 9.8 11.6 6.5

23.7 (5.3) 35.5 (2.2)*

6.5 (1.9) 10.7. (1.5)*a

1d 2d 3d mean value u

d

5.7 (0.6) 9.3 (2.6)

a Symbols for significance values: * "" p<0.05, ** "" p
BPP 188 (1992) 5

In summary no significant amounts of benzoic acid, chlorogenic acid, catechol, phydroxybenzoic acid, kaempferol and quercetin could be found in the studied spruce needles, whereas picein, pHAP, catechin, epicatechin, piceatannolglucoside, isorhapontin, p- and o-coumaric acid could be localized in the chromatogram. The last mentioned compounds, however, occurred in too little quantities to be worth being evaluated.

Concentrations of phenolic compounds in spruce needles due to severe damage Concentrations of the studied phenolic compounds do not show any seasonal variations in the spruce needles harvested at the Freudenstadt site. This observation is valid for both studied needle generations. The following figures and Table 1 demonstrate the contents of several phenols in spruce needles of the second generation taken on four dates in 1989 and two dates in 1990. In order to support the results obtained from mixed 100 -

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Fig. 2. Contents of picein in spruce needles of the second generation from Freudenstadt site. a) values obtained from mixed samples for various dates in 1989 and 1990. ym = yearly mean value. Significance values according to Student's t-Test for each date (parallel measurements) range from p < 10-5 to p = 0.02; for ym 1989: p = 0.003 (comparison old u with old d). b) values obtained from needles of single selected trees collected in June. mV = mean value, ms = values obtained from the corresponding mixed samples. fZZZl young trees, ~ old undamaged trees, • old damaged trees. BPP 188 (1992) 5

311

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Fig. 3. Contents of pHAP in spruce needles of the second generation from Freudenstadt site. a) values obtained from mixed samples for various dates in 1989 and 1990. b) values obtained from needles of single selected trees collected in June . For explanations see Fig . 2.

samples , concentrations in needles of single selected trees harvested on the early summer date in 1989 were measured , too. Picein a main phenolic compound in spruce, shows significant decreases in needles of damaged spruce trees compared with those of undamaged ones when considering the mixed samples (Fig. 2a). On several dates a pice in reduction can even be stated with proceeding age of the healthy trees . The yearly mean values show a significant difference between damaged and undamaged old trees . Measurements of picein contents starting from single tree samples (Fig. 2b), however, do not support the former result since there are distinct variations among all trees. As a result the mean values of those data do not show any difference as the values of the mixed sample do. p-Hydroxyacetophenone , the aglycone of picein, could be detected only in very small quantities in spruce needles of the Freudenstadt site (Fig . 3). Thus a relation of pHAPI picein amounting to 1 as it has already been discussed as an indicator for damage (HOQUE 1984 and 1985), was never determined here (all values below 0.07). Concentrations of pHAP do not show any constant changes due to damage as far as spruce trees from the 312

BPP 188 (1992) 5

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Tree-No.

Fig. 4. Contents of catechin in spruce needles of the second generation from Freudenstadt site. a) values obtained from mixed samples for various dates in 1989 and 1990. Significance values for each date and for ym 1989 (comparison old u with old d) : p < 10.5 . b) values obtained from needles of single selected trees collected in June. p = 0.02 for mean values . For explanations see Fig. 2.

Freudenstadt site are considered. The general picture is too confusing: differences between needles of damaged and undamaged spruce trees found in spring are quite opposite in their tendencies compared with those found in autumn. Single trees are also showing distinct individual differences. In contrast to pice in and pHAP, catechin, epicatechin, piceatannolglucoside, and even two still unidentified compounds with average retention times of ca. 5.5 and 9.1 minutes occur in distinctly higher concentrations (up to 50%) in spruce needles of the damaged trees than in those of the undamaged ones (Figs. 4 and 5, Table 1). The latter compound may probably be o-coumaric acid glucoside, which may be concluded by Fig. 1 and by the chromatograms published by KICINSKI and KETTRUPP (1987). The data obtained from the mixed samples are now confirmed by those of the single tree measurements, that also show significant differences between undamaged and damaged old trees . Between the two groups of undamaged trees no marked differences or a tendency to an increase of the regarded phenolic compounds due to tree age could be observed. 21

BPP 188 (1992) 5

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Fig. 5. Contents of PTG in spruce needles of the second generation from Freudenstadt site. a) values obtained from mixed samples for various dates in 1989 and 1990. Significance values for each date and for ym 1989 (comparison old u with old d) : p < 10.3 . b) values obtained from needles of single selected trees collected in June . p = 0.03 for mean values. For explanations see Fig. 2.

Needles of the third generation from three selected sampling dates were also subjected to analysis. For each studied compound the results (Table 2) are corresponding to those of the second needle generation. An addition of the concentrations of all investigated phenolic compounds - here called "total phenols" (Fig. 6) - reflects the tendency most single substances show: an increase in needles of damaged spruce trees compared with the undamaged ones. It demonstrates that the decrease in picein concentration is more than compensated by the increase of the other compounds. Thus, excluding picein from the calculation the described increase in "total phenols" is even more pronounced.

Discussion Quantitative analysis of phenolic compounds in spruce needles has mostly been carried out on picein and its aglycone p-hydroxyacetophenone and the results vary considerably in the amounts of these substances. The values we obtained in our studies 314

BPP 188 (1992) 5

Table 2. Phenolic compounds and their sums given in tJlnol g-J DW from spruce needles of the

third generation sampled at Freudenstadt site in 1989 and 1990 (mixed samples). For further explanations see Table 1. young

old u

old d

Compound

Date

Picein

28-Jun-89 25-Apr-90 7-Nov-90

74.4 65.3 87.7

69 .8 47 . 1 64 .5

38.5 43.0 54.6

pHAP

28-Jun-89 25-Apr-90 7-Nov-90

1.3 2.5 1.7

1.1 2.1 1.5

1.9 2.8 2.4

Catechin

28-Jun-89 25-Apr-90 7-Nov-90

40.6 ' 54.9 46.5

45.7 43.3 52.9

88.9 78.8 100.3

Epicatechin

28-Jun-89 25-Apr-90 7-Nov-90

21.0 27.9 27.8

22 .2 21.9 29 .1

38.9 44.0 44.6

PIG

28-Jun-89 25-Apr-90 7-Nov-90

9.7 9.5 11.3

12.3 12.3 12.4

17.4 16.3 18.9

Peak 5.5'

28-Jun-89 25-Apr-90 7-Nov-90

5.5 7.4 7.5

6.3 6.5 9.4

10.1 9.1 14.0

Peak 9.1'

28-Jun-89 25-Apr-90 7-Nov-90

3.9 6.8 5.2

5.2 6.1 5.8

8.1 9.8 12.2

"total phenols" + Picein

28-Jun-89 25-Apr-90 7-Nov-90

158.5 180.5 190.2

164.8 141.8 178.8

206.4 208.5 251.4

- Picein

28-Jun-89 25-Apr-90 7-Nov-90

84.1 115 .2 102.5

95.0 94.7 114.3

167 .9 165 .5 196.8

show comparatively high concentrations for picein and low ones for pHAP and are thus in accordance with the findings of OSSWALD and BENZ (1989), OSSWALD and ELSTNER (1989) and HELLER et al. (1990). For the extraction of phenolic components low temperatures proved to be important in order to avoid enzymic conversions. OSSWALD and BENZ (1989) also pointed out the necessity of needles not being thawed during homogenization. The relations pHAP/picein we obtained remained far below 0.07 for all samples. This result is in contradiction to the findings of HOQUE (1984,1985), KICINSKI et al. (1988) and JENSEN and L0KKE (1990), the former of whom postulated a relation> 1 being an indicator for damage in spruce trees. Only few reports about the other studied compounds in spruce needles could be found. HELLER et al. (1990) measured quantities of catechin and piceatannol glucoside comparable to ours, so did STRACK (1988) for PTG but found extremely higher values for 21*

BPP 188 (1992) 5

315

250

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Fig. 6. Sum of the contents of the analyzed phenolic compounds in spruce needles of the second generation from Freudenstadt site in 1989 and 1990. a) values including picein (p = 0.004 for ym 1989, comparison old u with old d), b) values calculated without picein (p = 0.001 for ym 1989). For explanations see Fig. 2.

catechin. These results were mainly obtained from very young spruce trees exposed in fumigation chambers. Comparing the chromatograms of STRACK (1988) and KICINSKI et al. (1988) with ours the still unknown compound with the longer retention time may be o-coumaric acid glucoside which is confirmed by the enzymatic hydrolysis. The compound with the shorter retention time may be p-coumaroylquinic acid since its peak did not disappear with enzymatic but with acid hydrolysis (last data not shown). The identification of these two substances will be the goal of further research. According to damage no distinct changes could be observed in the contents of pHAP, which shows a general strong variation from sample to sample. In contrast to that picein decreased significantly in the needles of the damaged trees. But the decrease due to damage obtained here with mixed samples was not confirmed by the measurements of single trees that show strong individual variations. Results obtained by other authors for these two substances vary between decrease, increase and no correlation with damage 316

BPP 188 (1992) 5

(HOQUE 1984 and 1985; OSSWALD et a1. 1986; KICINSKI et a1. 1988; STRACK 1988; OSSWALD and ELSTNER 1989; HELLER et a1. 1990; JENSEN and L0KKE 1990). Thus picein does not seem to be a suitable indicator for spruce tree damage at the Freudenstadt site. In contrast to that catechin, PTG, epicatechin, and the still unidentified components show significant increases in needles of damaged spruce trees. These results are supported by single tree measurements and can thus be regarded as a consequence of general stress reactions or damage. The increases are such pronounced that they compensate the reduction of picein as shown by "total phenols". In this connection it should be mentioned that catechin is present in spruce needles in quantities comparable to those of picein and is showing a marked increase due to damage. KICINSKI et a1. (1988) also reported an increase in catechin concentrations due to damage in needles of fumigated young spruce trees. In general no distinct variations during the growing season could be observed for all compounds in the needles of the second generation (third generation needles showing comparable concentrations). The lack of seasonal variations was also reported for total phenols by YEE-MEILER (1974) and ESTERBAUER (1975) and for picein and pHAP by OSSWALD and BENZ (1989). The damaged trees in our studies tend to increase slightly the concentrations of most phenolic components during summer time. Since ozone is the main air pollutant at the Freudenstadt site and reaches its highest immission concentrations during summer the increase of several phenolic compounds due to damage could be connected to the stress situation. A rise of phenolic compounds or of the activities of enzymes of phenol metabolism due to ozone has been observed by many authors with different plants (HOWELL 1974; HOWELL and KREMER 1973; KEEN and TAYLOR 1975; TINGEY et a1. 1975a+b and 1976; RUBIN et a1. 1983; LANGBARTELS et a1. 1989; SANDERMANN et a1. 1990). At Freudenstadt site all examined trees are subjected to a comparable ozone-stress. An explanation for the different reactions to this stress exhibited by the single groups of trees may be found in regarding the soil. For the damaged spruce trees less nutrients are available than for the undamaged ones because the soil below the former shows an extremely hydromorphic character (v. WILPERT and HILDEBRAND 1992). Thus - apart from genetic variations - the greater stress derived from the soil makes the former mentioned trees more susceptible to damage caused by ozone. Other studies within the research program carried out on the same trees (WILD and TIETZ 1991; WILD and TIETZ-SIEMER 1992) show that in the needles of the damaged trees a strong impairment of the photosynthetic apparatus and a shift in metabolism towards degradation processes and an intensified production of protectants like putrescine (TENTER and WILD 1991) and antioxidative substances (SCHMIEDEN et a1. 1992, subm.) has taken place. The changes in the content of various phenolic compounds described in this work fit into the picture of an enhanced production of phenolics as a consequence of stress that causes membrane damage (HOWELL 1974). Ozone, which exerts such a kind of stress, is even more effective if there already exists a membrane instability because of nutrient shortage. Thus, phenolic compounds function as protectants for the still living tissues as well as they take part in senescence processes in a progressed damage state. BPP 188 (1992) 5

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Acknowledgements This study was supported by the KfK-PEF (Kernforschungszentrum Karlsruhe - Projekt Europaisches Forschungszentrum fiir MaBnahmen zur Luftreinhaltung), grant no. : 88/007 II A and by the Umweltbundesamt (Federal Environmental Office) Berlin , grant no.: 10803046/16. We thank Dr. W. HELLER, GSF Neuherberg, for placing at disposal a sample of isorhapontin, Dr. M. WIESNER for synthesizing picein, and HD Dr. CH. WILHELM for critical discussion.

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Authors' address: CHRISTINE M. RICHTER und Prof. Dr. ALOYSIUS WILD, Institut fUr Allgemeine Botanik der Johannes-Gutenberg-Universitat, SaarstraBe 21 , D - W -6500 Mainz 1.

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