Effects of Sulphur Dioxide and other Air Pollutants on Acid Phosphatase Activity in Pine Seedlings

Biochem. Physiol. Pflanzen 175, 228-236 (1980)

Effects of Sulphur Dioxide and other Air Pollutants on Acid Phosphatase Activity in Pine Seedlings S. S. MALHOTRA and A. A. KHAN Northern Forest Research Centre, Canadian Forestry Service, Environment Canada Key Term Index: acid phosphatase, isoenzymes, seedling, air pollution, S02 effeets; Pinus banksiana.

Summary Acid phosphat ase (EC 3.1.3.2) activity in 6-month-old needles of jack pine (Pinus banksiana) seedlings was mueh higher than in 3-month-old needles. The soluble and loosely bound (salt-extractable) enzymes produeed the same eleetrophoretie patte;rn, which suggests that the enzymes are very similar in nature. Treatment of pine seedlings with S02 inhibited the acid phosphatase activity. After exposure of the seedlings to 0.35 ppm S02 for 24 h, the inhibition was more pronounced in the older (3-mon) than in the younger (1-mon) tissues from the same seedlings. Upon removal of plants from the S02 environment, the inhibitory effeets of S02 fumigation were completely reversed in plants exposed to S02 for short duration (1 h) but were unchanged in the plants exposed for long duration (24 h). Among the othu pollutants tested, fluoride, zine, arsenate, aluminium and eopper eonsiderably inhibited enzyme aetivity.

Introduction Sulphur dioxide is one of the major air pollutants released by natural gas, coal-powered, and petroleum-related industries. It has been shown to be highly phytotoxic depending upon its concentration, the length of exposure, and the type of vegetation (THOMAS 1951; ZIEGLER 1975; MALHOTRA and HOCKING 1976). The visual symptoms of S02 toxicity on different species of vegetation have been weIl described (BRANDT and HEcK 1968; BARRETT and BENEDICT 1970); however, little information is available about the manner in which S02 dffects the physiological dnd biochemical processes. In pine, a few changes in certain biochemical functions and ultrastructural organization have been observed prior to the development of visual symptoms of S02 phytotoxicity (MALHOTRA 1976; MALHOTRA and KHAN 1978). The impact of low concentrations of air pollutants on plant metabolism over an extended period oi time or of high concentrations over short exposures can result in severe injury to vegetation. Study on the effects oi S02 pollution on acid phosphatase activity (EC 3.1.3.2) was motivated primarily by th~ widespread distribution of acid phosphatase in the plant kingdom, and its implication in key metabolic functions of plants, such as nutrition (HASEGAWA et al. 1976), transportation of stored metabolites (FLINN and SMITH 1967), germination (MEYER et al. 1971), differentiation (HALL 1971), and stomatal opening (MrSHRA and PANDA 1970). Several physiological functions, such as stomatal resistance (MANSFIELD and MAJERNIK 1970; MAJE.RNIK and MANSFIELD 1971; CAPUT et al. 1978),

Effects of 8ulphur Dioxide

229

assimilation, growth, and differentiation (POLLANSHUTZ 1970; HORNTVEDT 1970; KELLER 1976; CONSTANTINIDOU et al. 1976), are influenced by S02 fumigation. Similarly, S02 has also been shown to affect the activity of several other enzyme systems (WELLBURN et al. 1976; MALHOTRA and KHAN 1978). Acid phosphatase from a few species has only recently been examined as a possible indicator of air pollution (SCHMID and KREEB 1975; YEE-MEILER 1975), with varying degrees of success. In the present investigation we report on the response of partially characterized pi ne needle acid phosphatase to different concentrations of gaseous S02' Materials and Methods Growth conditions

Jack pine (Pinus banksiana Lamb.) seedlings were grown in styroblocktrays as described previonsly (MALHoTRA 1976). The seedlings were grown in the greenhouse at 22 -24 oe under approximately 200 f! Einstein S-l • m- 2 supplemented by natural daylight (16 h photoperiod). Needles from either 3- or 6-mon-old seedlings were utilized for experimental purposes. The 6-mon-old seedlings were separa.ted into 3- and 6-mon-old needles, and 3-mon-old seedlings were divided into 1-mon-old (pale green cluster) and 3-mon-old (dark green, fuHy developed) needles and referred to as "young" and "old" tissues, respectively. Fumigation conditions

Approximately 3-mon-old pine seedlings were fumi gated with different 80 2 con centrations in cuvettes using a continuous flow-through system. The cuvettes, measuring 61 x61 x 30.5 cm, were constructed from clear acrylic sheets. Pine seedlings (15-20) were gently pulJed out of the styroblock trays and inserted (basal part of the stern) into neoprene stoppers slitted vertically so tbat a leak-proof barrier could be formed between the foliage and roots. The stoppers holding the seedlings were gently inserted throu gh the holes in the cuvette floor until the slit closed firmly a.round the stern and sealed the holes. The cuvette was then placed on top of another container containing sufficient water to keep the roots moist for tbe duration of the experiment. The access ports for t he air flow were opposite each other on the side walls. Tbe air flow was set at 101/min, and 80 2 flow was adjusted to give tbe desired concentration at the output of tbe cuvette. A Phillips PW-9700 80 2 analyzer was used to monitor 80 2 concentrations at the input and the output of the cuvette; tbis enabled us to determine the amount of 80 2 t aken up per seedling per hour. Tbe mixing of air and 80 2 within the cuvette was achieved by internally mounted fans near the injection port. Two identical cuvette assem blies containing seedlings of the same age, one control and the other for 80 2 treatment, were placed in the controlled environmental chamber. The fumigations for either 1 or 24 h were earried out at 24°C. The light source inside the ehamber was a eombination of high pressure sodium, metal halide, and incandescent lamps (10: 10: 3) with a light intensity of approximately 290 f! Einstein S-l. m- 2 • In 24 h fumigation experiments the photoperiod was 16 h. The relative humidity inside the cuvettes was approximately 65%. After treatment, the needles were harvested and separated into young and old needles as described above. Tissue homogenate preparation

After the tips were removed, the excised needle tissues were homogenized at moderate speed for 1.5 min in a Brinkman Polytron homogenizer (Model PT-lO) with a chilled solution (1: 10 w/v) containing 0.05 M Tris-HCI buffer (pH 7.4), 1 % polyvinyl pyrrolidone (PVP-10), and 1 mM dithioerythreitol (DTE). The homogenate was passed through two layers of cheesecloth, and the filtrate used immediately as "erude enzyme extract."

230

S. S. MALHOTRA and A. A. KHAN

Enzyme purification The crude enzyme extract was centrifuged at 27,000· g for 15 min, the supernatant was saved, and the residue was suspended in the same grinding medium containing 1 M NaCl to release the "loosely bound enzyme." The suspension was again homogenized for 1.5 min, and the homogenate was centrifuged as above. The two supernatant layers were subjected to ammonium sulphate precipitation to give a final concentration of 30 % saturation. The suspensions were centrifuged at 1,200 . g for 10 min. The supernatant layer was brought to 65 % ammonium sulphate saturation and centrifuged at 12,000 . g for 10 min. The protein precipitate thus obtained was dissolved in 5 ml of 0.05 Tris-ROI buffer (pR 7.4) and dialyzed against the same buffer for 3 hat 4 °0. This preparation was then used as "partially purified enzyme."

Enzyme assay A typical reaction mixture contained 0.6 mM p-nitrophenyl phosphate (P-NPP), 0.05 M acetate buffer (pR 5.0), and 25,u1 of appropriately diluted enzyme extract in a final volume of 2 ml. The reaction was run for 30 min in a water bath at 30 °0 and stopped by the addition of 2 ml of 10 % Na2 00 a• A control in which P-NPP was added after the addition of Na2 00 a was always run with the regular reaction set. If necessary, the mixture was clarified by centrifugation, and the activity was determined by measuring the difference in optical density between the control and the reaction solution at 410 nm. In experiments where specificity of the enzyme for different substrates was studied, the re action mixture contained 1 mM of the specified substrate, 0.05 M acetate buffer (pR 5.0), and 0.5 ml of appropriately diluted partially purified enzyme preparation in a final volume of 2 ml. Other re action conditions were the same as described above, except that the enzyme reaction was stopped by the addition of 1 ml of 30 % cold trichloroacetic acid (TOA). The mixture was kept in ice for 15 min and then centrifuged to remove the precipitation. The supernatant layer was used to measure the enzyme activity as determined by the release of inorganic phosphate (FrsKE and SUBBAROW 1925).

Polyacrylamide gel electrophoresis Electrophoresis of enzyme preparations (5-20 mg protein) was carried out by using polyacrylamide gel containing 7 % acrylamide, 0.18 % N, N'-methylenebisacrylamide (BIS) in 0.04 M Trisglycine buffer (pR 8.3). SampIes were prepared for electrophoresis according to the procedure of DAvrs (1964). The electrophoresis was conducted at room temperature for 1 h with a constant current of 2.5 mA per gel. The detection of acid phosphatase pro tein on the gel was carried out according to BARKA (1961).

Other estimations The protein content of the partially purified enzyme preparations was determined according to LOWRY et al. (1951) after precipitation with TOA. Orystalline bovine serum albumin served as the standard. For dry weight determinations, fresh needle sampIes were dried at 80 00 for 24 h.

Results and Discussion

Preliminary experiments with erude and partially purified enzyme extraets showed that the enzyme was more aetive at pR 5, with no deteetable aetivity above pR 6. The rate of reaetion was linear up to 40 min under our reaetion eonditions. Therefore, the assays were routinely run for only 30 min. The enzyme eoneentrations used in the assay were found to be in the limiting range, because a proportional inerease in the enzyme aetivity was observed upon using up to four times more enzyme extraet.

231

Effects of Sulphur Dioxide

When the crude enzyme extract was separated into 27,000· g supernatant and particulate fractions, the major portion of the enzyme activity was found to be associated with the particulate fraction. The activity was best solubilized by extracting the needle tissue in Tris buffer containing 1 M NaCl. Although phosphate buffer alone was found to extract more proteins into solution than the corresponding Tris buffer, it was not suitable for thc enzyme assay. The enzyme extracted with phosphate and citrate phosphate buffers showed considerable reduction in activity compared to the one extracted with Tris buffer. Similar inhibition of pea cotyledon acid phosphatase by inorganic phosphate has been reported by JOHNSON et al. (1973). Therefore, in all our studies, Tris buffer was used for enzyme preparations. On fractionation of the supernatants (obtained after NaCI extraction) with ammonium sulphate, acid phosphatase was found to precipitate at 30-65 % saturation. To avoid any complication due to endogenous levels of test compounds in the crude extract, we used only this fraction for further studies on the characterization of pine needle acid phosphatase.

Substrate specificity A number of phosphate esters were tested for enzyme specificity on the partially purified fraction (Table 1). Among these phosphate esters, the maximum activity was observed with phenol phosphates, followed by phosphocreatina, ADP, ATP, and AMP. High phosphatase activity with regard to adenosine phosphates would suggest a role for acid phosphatase in reactions involving ATP-ases. The sugar phosphates and other related compound'3 were not hydrolyzed. From these results it is evident that dlthough the enzyme from pine needles is relativaly unspecific toward various phosphate esters, it could act preferentidHy on those metabolie su bstrates that are involved in pine tissue physiology. Since pine tissues contain high concentrations of a number of phenolic compounds, a high phosphatase activity with regard to phenol phosphates would imply that acid phosphatase plays a role in the metabolism of aromatic intermediates involved in the biosynthesis of organic and amino acids and ceH wall constituents. Table 1. Substrate specificity of partially purified acid phosphatase frorn jack pine needles* Substrate**

Enzyme activity (% of p-NPP activity)

Substrate

p-nitrophenyl phosphate Phenyl phosphate Phosphocreatine ADP ATP AMP Bis-p-nitrophenyl phosphate

100 74 72 65 60

Glycerophosphate cAMP Glucose-i-phosphate Glucose-6-phosphate Fructose-i, 6-diphosphate Ribose-5-phosphate Phosphoenol pyruvate

M 6

* Needles frorn 3-rnon-old seedlings were used for enzyme isolation. ** All substrates were used at 1 rnM concentration.

Enzyme activity (% of p-NPP activity) 4 4

o o o o o

232

S. S. MALHOTRA and A. A. KHAN

The response of pine needle acid phosphattLse toward various phosphate esters could be due either to (1) one enzyme protein with its preference for certain phosphate esters, or (2) a mixture of different proteins (iso enzymes), each actmg specifically on specific phosphate esters.

Polyacrylamide gel electrophoresis The possibility of there being more than one phosphatase isoenzyme in pine needle extracts was tested by polyacrylamide gel electrophoresis of (1) supunatants (27,000· g) obtained :from Tris buffer and Tri3-NaCI buffer systems and (2) their protrins fractionated between 30 and 65 % ammonium sulphate saturation. Staining of the gels with phosphatase-specific stain showed only one band in both superntLtant and ammonium sulphate :fractions. The band appeared at th~ same position in each gel. On the basis of this electrophore~ic similarity, it is suggested that the soluble (Tris-extractable) and loosely bound (salt-extractable) acid phosphatase activities are similar in nature. This was further supported by the observation that both prepcLTations had the same substrate specificity.

Enzyme response to different compounds It has been observed that there is a good correlation between the heavy metal (Ni++ and Cu++) content of plant tissues and the state of growth of vegetation around mining and smelting industries (BüCKING and BLAUEL 1977). The data in Table 2 show Table 2. Effect of different cornpounds on partially purified acid phosphatase frorn jack pine needles1 ) Compound

Cone. (mM)

NH4 F

1.0 2.0 4.0

Zn acetate

1.0 2.0 4.0 1.0 2.0 4.0

Enzyme activity

(% of contro!) 38 25 15 45 45 45

Al(OHh

0.5 1.0 2.5

Oxalate

1.0 2.0 4.0

57 53 43 98 63 39 81 60 51

CU(N0 3)2

0.5 1.0 2.5

81 77 78

N!L:!HAs0 4

1) Needles from 3-mon-old seedlings were used for enzyme isolation.

Effects of Sulphur Dioxide

233

that acid phosphatase exhibited considerable inhibition in response to NH4 F, Zn acetate, Na 2HAs04 , AI(OH)3' and CU(N0 3)2. HASEGAWA et al. (1976) have reported similar effects of Cu++, Zn++, and F- on acid phosphatase activity from wheat roots. Divalent metals such as Cd++, Mn++, and Mg++ did not ctffect the enzyme activity. Fluoride, zinc, arsenic, aluminium, copper, and cadmium are emitted as airborne pollutants from different industrial operations. Aluminium can be a potent inhibitor of plant growth, especially in areas where soi] pH has become fairly acidic as a result of long-term S02 exposure. The solubility of alliminum in such soils increasea, making aluminum readily available to the vegetation. The typical inhibitors of acid phosphatase, such as fluoride and oxalate, produced varying degrees of inhibition depending upon their concentration. Fluoride was shown to be a more effective inhibitor than oxalate at all thre ~ concentrations tested. It appe ....rs that many airborne compounds released from various industrial activities may have a deleterious effect on vegetation through their action on enzyme systems such as acid phosphatase and possibly other biochemical processes.

Enzyme activity in different-aged needles Acid phosphatase activity varied considerably betwBen 3-mon- and 6-mon-old pine neeelle tissues (Table 3). The enzyme activity in the 6-mon-old needles was found to be Table 3. Acid phosphatase activity in 3- and 6-mon-old needles of p ine seedlings1 ) Experiment 1

2 3

Phosphatase activity (units(g dry wt) 3-mon-old needles

6-mon-old needles

31.7 23.3 36.8

43.9 49.6 56.4

1) A unit of enzyme activity is the amount of enzyme that resulted in a spectral change of 0.1 O.D. unit(min at 410 nm.

almost twice that in the 3-mon-old ones. Simihr results were obtctined by YEE-MEILER (1975), who found that a( id phosphatase activity was higher in older spruce tissues than in younger oues. It appears that the acid phosphatase activity is associated with the stdge of foliar develompent. Other changes, such as increased levels 01 chlorophyll and glycolipids, have also been demonstrated during foliar development (KHAN and MALHOTRA 1977).

--

Effect of 802 on phosphatase activity Under field conditions, vegetation is usually exposed to air pollutants intermittently, which allows the vegetation to recover at least partially from the air pollutant stress. In order to simulate such field con~itions as closely as possible, the plants were fumigated with either low or high S02 concentrations for a known length of time and then placed in an SOz-free environment to determine the percentage of recovery of impaired biochemical functions. Exposure of pine seedlings at 0.35 ppm S02 for 24 h produced a marked inhibition in acid phosphatase activity in both young and old needles (Table 4). Tbe enzyme

234

S. S.

MALHOTRA

and A. A.

KHAN

Table 4. Effats of gaseaus 80 2 on acid phosphatase activity in young and old needles of jack pine seedlings and their ability to recover in an S02-free atmosphere

% of control activity

Fumigation conditions Cone.

Duration

(ppm)

(h)

S02 uptakej Recovery period treejh (ppb) (h)

Ea:pt 1 0.35 0.35

24 24

20 20

0 24

64 54

Expt 2 0.33 0.33

1 1

9 9

0 24

90

82

100

100

Expt3 0.10

24

4

0

80

75

Young needles*

Old needles*

46

36

* Young and old needles were the pale green 1-mon-old top cluster and the dark green 3-monold growth, respectively.

activity was inhibited more in the older needles than in th8 younger ones. Federal ambient air quality standards in Canada allow, on an average, only 0.34 ppm 802 for 1 h exposure time (one of the concentrations used in this stu dy). As is evident from the results, there was a considerable loss in phosphatase activity after fumigating the pine seedlings with 0.35 ppm 802 for 24 h (Table 4, expt. 1). The inhibition of enzyme activity further increased du ring the 24 h recovery period in an 802-free environment. There was very little visual symptom development immediately after the 24 h fumigation, but symptoms became much more obvious the next 24 h period, indicating that a time lag is required for such symptom development. These results therefore suggest that fumigation of pine seedlings under such conditions may nsult in serious impairment of plant metabolism. 8CHMID and KREEB (1975) and RABE and KREEB (1976) reported a similar inhibition of acid phosphatase activity in alfalfa plants and lichens after treatment with 802. The fumigation cf pine seedlings at 0.33 ppm 802 for only 1 h also inhibited the enz)me ac~ivity in both young and old tissues by about 10 %(Table 4, expt. 2). However, this inhibition was completely absent after 24 h in an 802-free atmosphere, and no visual symptoms of 802 phytotoxicity developed. Fumigation with 0.1 ppm 802 for 24 h inhibited the enzyme activity in young nfedles by 20 % and in old needles by 25 % (Table 4, expt. 3). These plants did not produce any visual symptoms of 802 phytotoxicity either. These resplts clearly suggest that 802 concentration and exposure time are important variables afl'ecting the inhibition of acid phosphatase activity in jack pine seedlings. The data in Table 4 also show that the eifect of 802 on acid phosphatase is dependent upon the amount fo 802 taken up per seedling. The variability in the amount of 802 taken up per seedling per hour between expt. 1 and expt. 2 (Table 4) may have been

Effects of Sulphur Dioxide

235

due to increased stomatal resistance brought ab out by lower relative humidity (approx. 50-55%) in expt. 2. It is suggestedthatthestomata in seedlings of expt. 2 were probably only half open, whieh resulted in eonsiderably less S02 uptake per seedling. If the relative specifieity of pine needle acid phosphatase as demonstrated in vitra (Table 1) also exists in viva, an inhibition of this enzyme aetivity by S02 eould create a cellular imbalance in the eoncentrdtion of varioas phosphorylated intermediates. Such ehangEs would idluence the many important metabolie processes in whieh these metabolites dre directly or indirectly involved. As weH, because acid phosphatase has been linked with plant nutrition (HASEGAWA et al. 1976), mtrient transport (FLINN and SMITH 1967) and plant growth (HALL 1971), inhibition of phosphatase dctivity would thenfore result in general impairment of such processes. Our results suggest that acid phosphatase aetivity could be a useful crit8rion of S02 phytotoxicity. The ability of the S02-fumigated plants to recover their enzymatic activities in an S02free environment will have a direct implication in assessing the nature of metabolic injury (reversible vs. irreversible) to vegetation. Acknowledgements We thank Miss E. HARGESHEIMER for technical assistance and Mr. P. HURDLE for construction and operation of S02 facilities. We are grateful to the Alberta Oil Sands Environmental Research Program for financial assistance.

References BARKA, T. J.: Studies of acid phosphatase I. Electrophoretic separation of acid phosphatases of rat liver on polyacrylamide gels. J. Histochem. Cytochem. 9,542-547 (1961). BARRETT, T. W., and BENEDICT, H . .1\1.: Sulfur dioxide. In: "Recognition of air pollution injury to vegetation: A pictorial atlas" (eds. JACOBSEN, J. S. and A. C. HILL), pp. Cl-C5. Air Pollut. Control Assoe., Pittsburgh, Pa. 1970. BRANDT, C. S., and HECK, W. W.: Effects of air pollutants on vegetation. In: "Air pollution" (ed. A. C. STERN), 1, pp. 401-443. Academic Press, New York 1968. CAPUT, C., BELOT, Y., AUCLAIR, D., and DECOURT, N.: Absorption of sulphur dioxide by pine needles leading to acute injury. Environ. Pollut. 16, 3-15 (1978). CONSTANTINIDOU, H., KOZLOWSKI, T. T., and JENSEN, K.: Effects of sulfur dioxide on Pinus resinosa seedlings in the cotyledon stage. J. Environ. Qua!. 5, 141-144 (1976). DAVIS, B. J.: Disc electrophoresis. II, Method and application to human serum proteins. Ann. N. Y. Acad. Sei. 121, 404-427 (1964). FISKE, C. H., and SUBBAROW, Y.: The colorimetric determination of phosphorus. J. Bio!. Chem. 66, 375 -400 (1925). FLINN, A. M., and S~nTH, D. L.: The localization of enzymes in the cotyledons of Pisum arvense L. during germination. Planta 75, 10-22 (1967). HALL, J. L.: Cytochemicallocalization of ATPase activity in plant root cells. J. Microsc. 93,219 to 225 (1971). HASEGAWA, Y., LYNN, K. R., and BROCKBANK, W. J.: Isolation and partial characterization of cytoplasmic and wall-bound acid phosphatases from wheat roots. Can. J. Bot. 54,1163-1169 (1976). Hoc KING, D., and BLAUEL, R. A.: Progressive heavy metal accumulation associated with forest decline ne ar the nickel smelter at Thompson, Manitoba. Fish. Environ. Can., Can. For. Serv., North. For. Res. Cent. Inf. Rep. NOR-X-169 (1977).

236

S. S. MALHOTRA and A. A. KHAN, Effects of Sulphur Dioxide

HORNTVEDT, R.: S02 injury to forests. J. For. Utiliz. 78, 237 -286 (1970). JOHNSON, C. B., HOLLOWAY, B. R., SMITH, H., and GRIERSON, D.: Isoenzymes of acid phosphatase in germinating peas. Planta 115, 1-10 (1973). KELLER, TH.: Der Einfluß von Schwefeldioxid als Luftverunreinigung auf die Assimilation der Fichte. Schweiz. Z. Forstwes. 161, 3-8 (1976). KHAN, A. A., and MALHOTRA, S. S.: Effects of aqueous sulphur dioxide on pine needle glycolipids. Phytochemistry 16, 539 -543 (1977). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RAND ALL, R. J.: Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275 (1951). MAJERNIK, 0., and MANSFIELD, T. A.: Effects of S02 pollution on stomatal movements in Vicia faba. Phytopath. Z. 71, 123-128 (1971). MALHOTRA, S. S.: Effects of sulphur dioxide on biochemical activity and ultrastructural organization of pine needle chloroplasts. New Phytol. 76, 239-245 (1976). and HOCKING, D.: Biochemical and cytological effects of sulphur dioxide on plant metabolism. New Phytol. 76, 227-237 (1976). and KHAN, A. A.: Effects of sulphur dioxide fumigation on lipid biosynthesis in pine needles. Phytochemistry 17,241-244 (1978). MANSFIELD, T. A., and MAJERNIK 0.: Can stomata playapart in protecting plants against air pollutants? Environ. Pollut. 1, 149-154 (1970). MEYER, H., MA YER, E., and HAMEL, E.: Acid phosphatase in germinating lettuce evidence for partial activation. Physiol. Plant. 24, 95-101 (1971). MISHRA, D., and PANDA, K. C.: Acid phosphatase of rice leaves showing diurnal variation and its relation to stomatal opening. Biochem. Physiol. Pflanzen. 161, 532-536 (1970). POLLANSHUTZ, J.: Observations about the susceptibility of various kinds of trees with respect to emission of S02, HF and magnesite dust. In: "Proceedings of 1st European congress on influence of air pollution on plants and animals", pp. 371-377, The Netherlands, Wageningen 1970. RABE, R., and KREEB, K.: Eine Methode zur Laborbegasung von Testpflanzen mit Schwefeldioxide und ihre Anwendung bei Untersuchungen zur Enzymaktivität. Angew. Bot. 50, 71-78 (1976). SCHMID, M. L., and KREEB, K.: Enzymatische Indikation gasgeschädigter Flechten. Angew. Bot. 49, 141-154 (1975). THOMAS, M. D.: Gas damage to plants. Ann. Rev. Plant Physiol. 2,293-322 (1951). WELLBURN, A. R., CAPRON, T. M., CHAN, H. S., and HORSM1\N, D. C.: Biochemical effects of atmospheric pollutants on plants. In: "Effects of air pollutants on plants" (ed. T. A. MANSFIELD), pp. 105 -114. Society for Experimental Biology Seminar Series, Cambridge University Press, New York 1976. YEE-MEILER, VON D.: Über die Eignung von Phosphatase und Esteraseaktivitätsbestimmungen an Fichtennadeln und Birkenblättern zum Nachweis, unsichtbarer physiologischer Fluorimmissionschädigungen. Eur. J. For. Path. 5, 329 -338 (1975). ZIEGLER, 1.: The effect of S02 pollution on plant metabolism. Residue Rev. 56, 79-105 (1975).

Received October 15, 1979. Authors' address: S. S. MALHOTRA and A. A. KHAN, Northern Forest Research Centre, Canadian Forestry Service, Environment Canada, 5320-122 Street, Edmonton, Alberta T6H 3S5, Canada.