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Biochemical response of Norway spruce (Picea abies (L.) karst.) towards 14-month exposure to ozone and acid mist: Part II—Effects on protein biosynthesis
Environmental Pollution 64 (1990) 367-373
Biochemical Response of Norway Spruce (Picea abies (L.) Karst.) towards 14-Month Exposure to Ozone and Acid Mist: Part II Effects on Protein Biosynthesis
R. S c h m i t t & H. S a n d e r m a n n Jr Institute ffir BiochemischePflanzenpathologie,GSF Miinchen, lngolstfidter LandstraBe 1, D-8042 Neuherberg, FRG ABSTRACT Four-year-old clonal Picea abies ( L. ) Karst. plants were treated with ozone ( 100 I~g m - 3 plus peaks of 130 to 360 #g m - 3 ) and acid mist (pH 3"0) during two vegetation periods. Pulse labelling experiments on shoots were performed with [35 S]methionine in the second year of exposure. Extraction of soluble needle proteins in citric acid buffer of pH 2"8 revealed protein patterns on SDS polyacrylamide gels that differed ,from those of control needles Jumigated with ambient levels of ozone (50 Itg m - 3 ) and mist of pH 5"6. New proteins of M W 16000 and 32000 were synthesized only in ozone-exposed needles and could not be detected in the controls.
INTRODUCTION Plants synthesize different sets of proteins during stress conditions, such as heat shock, heavy metals, water stress and fungal or virus infection (Sachs & Ho, 1986; Hahlbrock & Scheel, 1987). The induction of stress proteins is particularly well documented as a plant response in host-pathogen interactions (van Loon, 1985). The alteration of de novo protein synthesis in conifers with regard to gaseous air pollutant stress has so far not been investigated. In the present report, methods of in vivo pulse labelling and gel electrophoretic separation of proteins were applied in order to search for newly synthesized proteins. The needle material used originated from Norway spruce subjected to a 14-month exposure to elevated levels of ozone and acid mist. This work is a contribution to the joint experiments described 367 Environ. Pollut. 0269-7491/90/$03"50 (c~ 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
368
R. Schmitt, H. Sandermann Jr
in this issue (Blank et al., 1990). Some of the results have been briefly communicated (Sandermann et al., 1989).
MATERIALS A N D METHODS
Pulse labelling experiments Trees of clone No. 14 potted in calcareous soil (No. 2) were chosen for in vivo pulse labelling. The first experiment was performed immediately after a peak of ozone treatment (Blank et al., 1990) on 18 July 1987, 3 weeks after budbreak. Subsequent labelling experiments were performed on 8 August, 29 August and 5 September 1987. According to the schedule of treatments, these experiments were performed with trees from chambers 2 and 4 immediately after exposure to acid mist (pH 3.0) plus ozone peaks of 220-260pg m-3. At the same time 'control' trees from chambers 1 and 3 treated with 'neutral' mist of pH 5"6 plus low ozone levels of 50/~g m - 3 were studied for comparison. For details of the treatment protocol see Blank et al. (1990). For application of radioactivity, two shoots of the current year from the second whorl were cut under water. Each shoot was transferred into an Eppendorf tube to take up 200/~Ci (9.25MBq, 46TBq/mmol) [35S]methionine over the cut end in a volume of 100/zl H20 for 2h. Within the next 4h three further 50/~1 portions of H20 were added to allow transpiration and to promote the translocation of the labelled amino acid to the needles.
Extraction procedure The shoots were immersed into liquid nitrogen, whereupon the needles became detached. The needles (500-600 mg per shoot) were homogenized after addition of polyvinyl-polypyrrolidone (50%, w/w) and Dowex 1 × 2 (5%, w/w) in precooled teflon tubes in a ball mill (Retsch, Haan, FRG). The powder was extracted 2-3 times with 100% acetone at - 2 0 ° C to remove chlorophyll and excess non-incorporated [35S]methionine. The colourless dry acetone powder was then extracted at 4°C with 2 x 3.5 ml 84 mra sodium citrate, pH 2.8, containing 5 mM EDTA, 5 mM dithiothreitol, 10mM 2-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride, 1 m ~ benzamidine for 10 min in a sonication bath (Bandelin, Berlin, FRG). After centrifugation for 30min at 12000g, proteins from the combined supernatants were precipitated by trichloroacetic acid (6% final concentration). The pellet was dissolved in SDS-containing sample buffer
Biochemical response of Norway Spruce to ozone and acid mist: Part H
369
according to Laemmli (1970; 10min, 60°C) and adjusted to pH 6-8 for gel electrophoresis. For more complete solubilization of needle proteins, the buffer described by Zedler et aL (1986) was employed in the extraction procedure of Ziegler & Kandler (1980) in the following way. Borate buffer (50mM sodium tetraborate containing 5mM EDTA, 5mM dithiothreitol, 42mM 2mercaptoethanol, 5% SDS (w/v), final pH brought to 8.0 with NaOH; 2 × 2ml) was added to the dry needle acetone powder at 25°C and the resulting suspensions were treated in the sonic bath for 10 min, as described above. After centrifugation for 30 min at 48 000g, the clear supernatants were combined and aliquots were employed for gel electrophoresis after addition of equal amounts of sample buffer.
SDS-polyacrylamide gel electrophoresis and autoradiography SDS gel electrophoresis was performed in a vertical casting system (Pharmacia-LKB, Freiburg, FRG), as described in the manufacturer's manual. Equal amounts of radioactivity (100 000 dpm in 10-40/~1 of sample buffer) were applied to each lane. Marker proteins carrying a (14C-methyl)label, were from Amersham-Buchler (Braunschweig, FRG). The separation gel contained 15 % polyacrylamide. Dried slab gels were exposed to Kodak X-Omat AR films with intensifying screen for 7 days.
RESULTS h~ vivo pulse labelling of proteins in shoots of Norway spruce showed that more than 95 % of the [-35S]methionine applied was taken up within 2 h. The bulk of radioactivity was located in the stem even after prolonged transpiration periods. The needle proteins extracted with citrate buffer of pH2.8 contained 0-8-2"8% of the applied radioactivity. In the buffer described by Zedler et al. (1986), 9-14% of the applied radioactivity was solubilized. The experiment performed on 18 July I987, is illustrated in Fig. 1A, where an autoradiogram of the protein profiles is shown. Needles from four control and four exposed trees show reproducible protein banding patterns. Newly synthesized proteins of MW 16000 and 32000 which occur in all samples from exposed trees are marked by arrows. These protein bands are absent in control needles. No difference in banding patterns could be observed on the autoradiograms between control and exposed trees when needle proteins were solubilized in borate buffer of pH 8"0 (Fig. 1B), or in SDS-sample buffer of
370
R. Schmitt, H. Sandermann Jr
pH 8"8 (data not shown). The needle protein profiles from the eight trees tested were almost identical and no alteration of any of the separated proteins had occurred as far as the present resolution could reveal. The second set of experiments performed on 8 August 1987, revealed the same induced protein band of MW 16000 extracted at pH 2.8 exclusively in needles from exposed trees. Time intervals of 0.5 h and 16 h between ozone treatments and incubation with [35S]methionine both led to the selective labelling of the 16 kDa protein (data not shown). Forty and 48 days after the first experiment the protein patterns of needles from polluted and control trees were highly variable and differed from those
A +
+
+
+
M
~!
i¸¸
69
i i ' ~ ¸~¸,I¸¸' 4 6 ~ ~i~~?~?~ il 30
~i~!~i~i~i~iiii ~;i!i~
14.3
124
109
107
126
130
125
117
140
Fig. 1. (A) autoradiogram of needle proteins extracted in citric acid buffer of pH 2"8 and separated by SDS-polyacrylamide gel electrophoresis (18 July 1987, experiment). (B) autoradiogram of total needle proteins solubilized in borate buffer ofpH 8'0 and separated by SDS-polyacrylamide gel electrophoresis (18 July 1987, experiment). (C) autoradiogram of needle proteins extracted in citric buffer of pH 2.8 and separated by SDS-polyacrylamide gel electrophoresis (29 August 1987, experiment). The following symbols appear at the top of the various gel lanes: (-), Norway spruce exposed to mist of pH 5"6 plus background levels of ozone (50 #g m - 3). (+), Norway spruce exposed to mist ofpH 3'0 plus elevated levels of ozone (100 pg m-3) plus seasonally varying ozone peaks of 130-360pg m-3. (M). [~4C]methylated protein markers, bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30kDa) and lysozyme (14-3kDa). Numbers appearing at the bottom of the gel lanes correspond to the individual clone 14 trees employed.
Biochemical response of Norway Spruce to ozone and acid mist: Part lI
B --
m
+
+
+
+
M
69 46
30
14.3
124
109
107
126
130
125
117
140
C .
.
.
.
+
+
+
+
M
69 N
46
30
14.3
124
109 107 126
130
125
117
Fig. l--contd,
140
371
372
R. Schmitt, H. Sandermann Jr
of Fig. 1A. In particular, the 16 kDa-protein was not observable. An example is shown in Fig. 1C.
DISCUSSION Norway spruce subjected to acid mist plus elevated levels of ozone showed incorporation of [35S]methionine into newly induced proteins of 16 and 32 kDa. Under the conditions employed, ozone and acid mist treatment did not cause visible injury of the needles. Stress related proteins of agricultural plants are known to be selectively extracted by citric acid buffer of pH 2.8 (van Loon, 1985). Among various extraction methods tested, only this procedure was successful in revealing the induction of the present 16 kDa and 32 kDa proteins. However, the function of the two new needle proteins is as yet unknown. Borate buffer of pH 8"6 has previously been found to be an effective solvent for spruce needle proteins (Ziegler & Kandler, 1980). However, in agreement with Zedler et al. (1986), we observed no differences in protein profiles with respect to needle age or ozone treatment when borate buffer was employed for extraction. Moreover, Fedderau-Himme et al. (1984) failed to detect alterations in total protein profiles in needles from differently damaged trees, using twenty different clones of Norway spruce. In summary, protein biosynthesis was affected by treatment with ozone and acid mist. The 16 kDa and 32 kDa proteins appeared most clearly at a certain time interval of the present long-term study. It is, therefore, not clear at present whether these stress proteins can be developed into an early diagnostic marker. In a subsequent independent fumigation experiment, the 16kDa protein was again specifically induced by ozone under different experimental conditions (Langebartels et al., 1989).
ACKNOWLEDGEMENT The technical assistance of Mrs E. Mattes is gratefully acknowledged. This work has been supported by GSF Mfinchen.
REFERENCES Blank, L. W., Payer, H. D., Pfirrmann, T., Gnatz, G., Kloos, M., Runkel, K. H., Schmolke, W., Strube, D. & Rehfuess, K. E. (1990). Effects of ozone, acid mist and soil characteristics on clonal Norway spruce. An introduction to the joint
Biochemical response of Norway Spruce to ozone and acid mist: Part II
373
14 month tree exposure experiment in closed chambers. Environ. Pollution, 64(3/4) this issue. Fedderau-Himme, B., Feig, R., Herms, A., Rosenpl~inter, K., Grothey, V. & Hiittermann, A. (1984). Untersuchungen von physiologischen Parametern von Altfichten. Ein Beitrag zur Quantifizierung von Hypothesen zur Ursache des Waldsterbens. Ber. Forschungsz. Walddkosysteme/Waldsterben GSttingen, 4, 1-126. Hahlbrock, K. & Scheel, D. (1987). Biochemical responses of plants to pathogens. In Innovative Approaches to Plant Disease Control, ed. by I. Chet, J. Wiley, New York, pp. 229-54. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680-5. Langebartels, C., Fiihrer, G., H~ickl, B., Heller, W., Kloos, M., Payer, H.-D., Schmitt, R. & Sandermann, Jr, H. (1989). Dose-dependent biochemical reactions of Norway spruce to ozone fumigation. In Air Pollution and Forest Decline, ed. J. B. Bucher & I. Bucher-Wallin, Proc. 14. IUFRO meeting, Birmensdorf, pp. 466-9. van Loon, L. C. (1985). Pathogenesis-related proteins. Plant Mol. Biol., 4, 111-16. Sachs, M. M. & Ho, T. D. (1986). Alteration of gene expression during environmental stress in plants. Ann. Rev. Plant Physiol., 37, 363-76. Sandermann, H., Schmitt, R., Heller, W., Rosemann, D. & Langebartels, C. (1989t. Ozone-induced early biochemical reactions in conifers. In Acid Deposition. Sources, Effects and Controls, ed. J. W. S. Longhurst, British Library, London, pp. 243-54. Zedler, B., Plarre, R. & Rothe, G. M. (1986). Impact of atmospheric pollution on the protein and amino acid metabolism of spruce Picea abies trees. Environ. Pollution (Series A), 40, 193-212. Ziegler, P. & Kandler, O. (1980). Tonoplast stability as a critical factor in frost injury and hardening of spruce needles. Z. Pflanzenphysiol., 99, 393-410.
Biochemical Response of Norway Spruce (Picea abies (L.) Karst.) towards 14-Month Exposure to Ozone and Acid Mist: Part II Effects on Protein Biosynthesis
R. S c h m i t t & H. S a n d e r m a n n Jr Institute ffir BiochemischePflanzenpathologie,GSF Miinchen, lngolstfidter LandstraBe 1, D-8042 Neuherberg, FRG ABSTRACT Four-year-old clonal Picea abies ( L. ) Karst. plants were treated with ozone ( 100 I~g m - 3 plus peaks of 130 to 360 #g m - 3 ) and acid mist (pH 3"0) during two vegetation periods. Pulse labelling experiments on shoots were performed with [35 S]methionine in the second year of exposure. Extraction of soluble needle proteins in citric acid buffer of pH 2"8 revealed protein patterns on SDS polyacrylamide gels that differed ,from those of control needles Jumigated with ambient levels of ozone (50 Itg m - 3 ) and mist of pH 5"6. New proteins of M W 16000 and 32000 were synthesized only in ozone-exposed needles and could not be detected in the controls.
INTRODUCTION Plants synthesize different sets of proteins during stress conditions, such as heat shock, heavy metals, water stress and fungal or virus infection (Sachs & Ho, 1986; Hahlbrock & Scheel, 1987). The induction of stress proteins is particularly well documented as a plant response in host-pathogen interactions (van Loon, 1985). The alteration of de novo protein synthesis in conifers with regard to gaseous air pollutant stress has so far not been investigated. In the present report, methods of in vivo pulse labelling and gel electrophoretic separation of proteins were applied in order to search for newly synthesized proteins. The needle material used originated from Norway spruce subjected to a 14-month exposure to elevated levels of ozone and acid mist. This work is a contribution to the joint experiments described 367 Environ. Pollut. 0269-7491/90/$03"50 (c~ 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
368
R. Schmitt, H. Sandermann Jr
in this issue (Blank et al., 1990). Some of the results have been briefly communicated (Sandermann et al., 1989).
MATERIALS A N D METHODS
Pulse labelling experiments Trees of clone No. 14 potted in calcareous soil (No. 2) were chosen for in vivo pulse labelling. The first experiment was performed immediately after a peak of ozone treatment (Blank et al., 1990) on 18 July 1987, 3 weeks after budbreak. Subsequent labelling experiments were performed on 8 August, 29 August and 5 September 1987. According to the schedule of treatments, these experiments were performed with trees from chambers 2 and 4 immediately after exposure to acid mist (pH 3.0) plus ozone peaks of 220-260pg m-3. At the same time 'control' trees from chambers 1 and 3 treated with 'neutral' mist of pH 5"6 plus low ozone levels of 50/~g m - 3 were studied for comparison. For details of the treatment protocol see Blank et al. (1990). For application of radioactivity, two shoots of the current year from the second whorl were cut under water. Each shoot was transferred into an Eppendorf tube to take up 200/~Ci (9.25MBq, 46TBq/mmol) [35S]methionine over the cut end in a volume of 100/zl H20 for 2h. Within the next 4h three further 50/~1 portions of H20 were added to allow transpiration and to promote the translocation of the labelled amino acid to the needles.
Extraction procedure The shoots were immersed into liquid nitrogen, whereupon the needles became detached. The needles (500-600 mg per shoot) were homogenized after addition of polyvinyl-polypyrrolidone (50%, w/w) and Dowex 1 × 2 (5%, w/w) in precooled teflon tubes in a ball mill (Retsch, Haan, FRG). The powder was extracted 2-3 times with 100% acetone at - 2 0 ° C to remove chlorophyll and excess non-incorporated [35S]methionine. The colourless dry acetone powder was then extracted at 4°C with 2 x 3.5 ml 84 mra sodium citrate, pH 2.8, containing 5 mM EDTA, 5 mM dithiothreitol, 10mM 2-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride, 1 m ~ benzamidine for 10 min in a sonication bath (Bandelin, Berlin, FRG). After centrifugation for 30min at 12000g, proteins from the combined supernatants were precipitated by trichloroacetic acid (6% final concentration). The pellet was dissolved in SDS-containing sample buffer
Biochemical response of Norway Spruce to ozone and acid mist: Part H
369
according to Laemmli (1970; 10min, 60°C) and adjusted to pH 6-8 for gel electrophoresis. For more complete solubilization of needle proteins, the buffer described by Zedler et aL (1986) was employed in the extraction procedure of Ziegler & Kandler (1980) in the following way. Borate buffer (50mM sodium tetraborate containing 5mM EDTA, 5mM dithiothreitol, 42mM 2mercaptoethanol, 5% SDS (w/v), final pH brought to 8.0 with NaOH; 2 × 2ml) was added to the dry needle acetone powder at 25°C and the resulting suspensions were treated in the sonic bath for 10 min, as described above. After centrifugation for 30 min at 48 000g, the clear supernatants were combined and aliquots were employed for gel electrophoresis after addition of equal amounts of sample buffer.
SDS-polyacrylamide gel electrophoresis and autoradiography SDS gel electrophoresis was performed in a vertical casting system (Pharmacia-LKB, Freiburg, FRG), as described in the manufacturer's manual. Equal amounts of radioactivity (100 000 dpm in 10-40/~1 of sample buffer) were applied to each lane. Marker proteins carrying a (14C-methyl)label, were from Amersham-Buchler (Braunschweig, FRG). The separation gel contained 15 % polyacrylamide. Dried slab gels were exposed to Kodak X-Omat AR films with intensifying screen for 7 days.
RESULTS h~ vivo pulse labelling of proteins in shoots of Norway spruce showed that more than 95 % of the [-35S]methionine applied was taken up within 2 h. The bulk of radioactivity was located in the stem even after prolonged transpiration periods. The needle proteins extracted with citrate buffer of pH2.8 contained 0-8-2"8% of the applied radioactivity. In the buffer described by Zedler et al. (1986), 9-14% of the applied radioactivity was solubilized. The experiment performed on 18 July I987, is illustrated in Fig. 1A, where an autoradiogram of the protein profiles is shown. Needles from four control and four exposed trees show reproducible protein banding patterns. Newly synthesized proteins of MW 16000 and 32000 which occur in all samples from exposed trees are marked by arrows. These protein bands are absent in control needles. No difference in banding patterns could be observed on the autoradiograms between control and exposed trees when needle proteins were solubilized in borate buffer of pH 8"0 (Fig. 1B), or in SDS-sample buffer of
370
R. Schmitt, H. Sandermann Jr
pH 8"8 (data not shown). The needle protein profiles from the eight trees tested were almost identical and no alteration of any of the separated proteins had occurred as far as the present resolution could reveal. The second set of experiments performed on 8 August 1987, revealed the same induced protein band of MW 16000 extracted at pH 2.8 exclusively in needles from exposed trees. Time intervals of 0.5 h and 16 h between ozone treatments and incubation with [35S]methionine both led to the selective labelling of the 16 kDa protein (data not shown). Forty and 48 days after the first experiment the protein patterns of needles from polluted and control trees were highly variable and differed from those
A +
+
+
+
M
~!
i¸¸
69
i i ' ~ ¸~¸,I¸¸' 4 6 ~ ~i~~?~?~ il 30
~i~!~i~i~i~iiii ~;i!i~
14.3
124
109
107
126
130
125
117
140
Fig. 1. (A) autoradiogram of needle proteins extracted in citric acid buffer of pH 2"8 and separated by SDS-polyacrylamide gel electrophoresis (18 July 1987, experiment). (B) autoradiogram of total needle proteins solubilized in borate buffer ofpH 8'0 and separated by SDS-polyacrylamide gel electrophoresis (18 July 1987, experiment). (C) autoradiogram of needle proteins extracted in citric buffer of pH 2.8 and separated by SDS-polyacrylamide gel electrophoresis (29 August 1987, experiment). The following symbols appear at the top of the various gel lanes: (-), Norway spruce exposed to mist of pH 5"6 plus background levels of ozone (50 #g m - 3). (+), Norway spruce exposed to mist ofpH 3'0 plus elevated levels of ozone (100 pg m-3) plus seasonally varying ozone peaks of 130-360pg m-3. (M). [~4C]methylated protein markers, bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30kDa) and lysozyme (14-3kDa). Numbers appearing at the bottom of the gel lanes correspond to the individual clone 14 trees employed.
Biochemical response of Norway Spruce to ozone and acid mist: Part lI
B --
m
+
+
+
+
M
69 46
30
14.3
124
109
107
126
130
125
117
140
C .
.
.
.
+
+
+
+
M
69 N
46
30
14.3
124
109 107 126
130
125
117
Fig. l--contd,
140
371
372
R. Schmitt, H. Sandermann Jr
of Fig. 1A. In particular, the 16 kDa-protein was not observable. An example is shown in Fig. 1C.
DISCUSSION Norway spruce subjected to acid mist plus elevated levels of ozone showed incorporation of [35S]methionine into newly induced proteins of 16 and 32 kDa. Under the conditions employed, ozone and acid mist treatment did not cause visible injury of the needles. Stress related proteins of agricultural plants are known to be selectively extracted by citric acid buffer of pH 2.8 (van Loon, 1985). Among various extraction methods tested, only this procedure was successful in revealing the induction of the present 16 kDa and 32 kDa proteins. However, the function of the two new needle proteins is as yet unknown. Borate buffer of pH 8"6 has previously been found to be an effective solvent for spruce needle proteins (Ziegler & Kandler, 1980). However, in agreement with Zedler et al. (1986), we observed no differences in protein profiles with respect to needle age or ozone treatment when borate buffer was employed for extraction. Moreover, Fedderau-Himme et al. (1984) failed to detect alterations in total protein profiles in needles from differently damaged trees, using twenty different clones of Norway spruce. In summary, protein biosynthesis was affected by treatment with ozone and acid mist. The 16 kDa and 32 kDa proteins appeared most clearly at a certain time interval of the present long-term study. It is, therefore, not clear at present whether these stress proteins can be developed into an early diagnostic marker. In a subsequent independent fumigation experiment, the 16kDa protein was again specifically induced by ozone under different experimental conditions (Langebartels et al., 1989).
ACKNOWLEDGEMENT The technical assistance of Mrs E. Mattes is gratefully acknowledged. This work has been supported by GSF Mfinchen.
REFERENCES Blank, L. W., Payer, H. D., Pfirrmann, T., Gnatz, G., Kloos, M., Runkel, K. H., Schmolke, W., Strube, D. & Rehfuess, K. E. (1990). Effects of ozone, acid mist and soil characteristics on clonal Norway spruce. An introduction to the joint
Biochemical response of Norway Spruce to ozone and acid mist: Part II
373
14 month tree exposure experiment in closed chambers. Environ. Pollution, 64(3/4) this issue. Fedderau-Himme, B., Feig, R., Herms, A., Rosenpl~inter, K., Grothey, V. & Hiittermann, A. (1984). Untersuchungen von physiologischen Parametern von Altfichten. Ein Beitrag zur Quantifizierung von Hypothesen zur Ursache des Waldsterbens. Ber. Forschungsz. Walddkosysteme/Waldsterben GSttingen, 4, 1-126. Hahlbrock, K. & Scheel, D. (1987). Biochemical responses of plants to pathogens. In Innovative Approaches to Plant Disease Control, ed. by I. Chet, J. Wiley, New York, pp. 229-54. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227, 680-5. Langebartels, C., Fiihrer, G., H~ickl, B., Heller, W., Kloos, M., Payer, H.-D., Schmitt, R. & Sandermann, Jr, H. (1989). Dose-dependent biochemical reactions of Norway spruce to ozone fumigation. In Air Pollution and Forest Decline, ed. J. B. Bucher & I. Bucher-Wallin, Proc. 14. IUFRO meeting, Birmensdorf, pp. 466-9. van Loon, L. C. (1985). Pathogenesis-related proteins. Plant Mol. Biol., 4, 111-16. Sachs, M. M. & Ho, T. D. (1986). Alteration of gene expression during environmental stress in plants. Ann. Rev. Plant Physiol., 37, 363-76. Sandermann, H., Schmitt, R., Heller, W., Rosemann, D. & Langebartels, C. (1989t. Ozone-induced early biochemical reactions in conifers. In Acid Deposition. Sources, Effects and Controls, ed. J. W. S. Longhurst, British Library, London, pp. 243-54. Zedler, B., Plarre, R. & Rothe, G. M. (1986). Impact of atmospheric pollution on the protein and amino acid metabolism of spruce Picea abies trees. Environ. Pollution (Series A), 40, 193-212. Ziegler, P. & Kandler, O. (1980). Tonoplast stability as a critical factor in frost injury and hardening of spruce needles. Z. Pflanzenphysiol., 99, 393-410.