The Kautsky-effect: A method for the investigation of the actions of air pollutants in chloroplasts

THE KAUTSKY-EFFECT: A METHOD FOR THE INVESTIGATION OF THE ACTIONS OF AIR POLLUTANTS IN CHLOROPLASTS UWE ARNDT

Landesanstalt fiir lmmission~- und Bodennutzungsschutz des Landes N W, 43 Essen, Wallneyer Str. 6, Germany

ABSTRACT

The characteristic variable fluorescence of green plants (Kautsky-effect) was used to investigate the effects of air pollutants on metabolic reactions in chloroplasts. This new technique in the field of environmental research, in conjunction with 02measurements and pigment analysis, gave an indication of effects-mechanisms .for particular heavy metals, such as copper, manganese, and zinc, as well as for SO 2. An example of the routine use of the Kautsky-effect in the testing of settled dusts is described.

INTRODUCTION

Heavy metal dusts are found in phytotoxic concentrations near certain industrial works, such as foundries and smelters, and are receiving increasing attention in the realm of environmental protection (Garber, 1970; Lee et al., 1972). In this respect, copper has to be mentioned as an active and widespread component (Dunn & Bloxam, 1933; Harrison et al., 1971). Observations on the effects of heavy metals range from sedimentation of the dusts on the plant organs to, at times, strong accumulation in the soil (Delas, 1963). Symptomatology of plant injury is not always specific for one element but, in fact, depends on combined effects of heavy metals and SO2. A knowledge of the mode of action of the single damaging agents can be a great help in the diagnosis and identification of the particular emission source, as well as in the establishment of eventual protective measures. To this end, the following results on the mechanism of injury from heavy metals and SO 2 are presented. Results of these investigations are based mainly on the measurement of the Kautsky-effect, a new method in the field of environmental research, which is 181 Environ. Pollut. (6) (1974)--© Applied Science Publishers Ltd, England, 1974 Printed in Great Britain

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UWE ARNDT

presented here for discussion. As an example of the application of this method, the determination of the relative phytotoxicity of settled dusts in the Ruhr area is shown.

MATERIALS AND METHODS

Plants of the alga Chlorella pyrenoidosa (algae collection G6ttingen, Stock No. 21 I-8b) were grown in cuvettes under a 28°C temperature regime, in an air stream enriched with 2 ~ CO2, and a light-dark rhythm of 14:10 h. Nutrient solutions were prepared according to Soeder et al. (1967). After 48 h the synchronised organisms were removed and counted in Thoma chambers. They were then removed from the nutrient medium and placed in distilled water at a concentration of 25 × 106 cells/ml water. Sensitivity of the cells is dependent on pH, therefore a pH of 6.5-7 was maintained throughout the experiments (Steemann-Nielsen & Kamp-Nielsen, 1970). Four to five hours after the formation of autospores the algal suspensions for the experiments were mixed with various salts at different concentrations and exposed to the air for 90 min in the cuvettes. For the SO2 experiments, a fumigation occurred at this time. The cells were then held in darkness for 15 min, temperature and ventilation were constant. Changes in the Kautsky-effect served as the effect criterion. Tile measuring apparatus is shown in Fig. I. The incident light intensity was 80 W/m 2. Evaluation was carried out visually, as well as through a comparision of the areas under the control and experimental curves. Measurement of oxygen development was carried out, using standard techniques, in the Warburg apparatus after the algae were exposed to the heavy metals (cJ~ Greenfield, 1942). The influence of CO2 gas exchange was eliminated with bicarbonate buffer. For the experiments on the comparison of toxicity of dusts, 750 ltg dust/ml algal-suspension was weighed out. The dust was taken from each of the 35 sampling sites of the 1st Monitoring Programme of the province of Northrhine-Westphalia and was prepared according to Herpertz (1969). Samples were mixed for the months January-February, April-May and OctoberNovember 1964. Working times and methods were as described above. A standard solution of 5 x 10 -5 molar CuCI 2 was tested at the same time. This was used to compare sensitivity of the plants in the various experiments. Standard deviation of the technique was determined for the control of each experiment, that is, without consideration of possible differences in sensitivity, and gave a value for S of +9.1 ~ . Chemical analyses of the dusts were carried out with atomic absorption on the Leitz Unicam instrument. With only a few exceptions, the values for cadmium were below the threshold limit of the instrument.

183

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Monitoring of photosynthesis in plants can yield information on the effects of air pollutants on vegetation. Effects of these pollutants on chloroplast metabolism can be studied without destroying the plant itself because gas exchange is maintained with the external atmosphere through metabolic processes which can be divided into a light and dark reaction. Investigations on the connection between air pollutants and CO2 gas exchange had already been carried out at the turn of the century (Wieler, 1905) and external monitoring of the dark reaction was achieved. The light-dependent part of photosynthesis and its sensitivity to air pollutants has not been so thoroughly investigated. Two methods for external monitoring of the light reaction are possible; the measurement of oxygen" production and the observation of fluorescence (Fig. 2). The gas (02) is freed through the splitting of water molecules in the light, while fluorescence occurs through the return of excited electrons to their initial level during photosynthesis, whereby red quanta are emitted. Dark-adapted plants, suddenly illuminated, show the induction phenomenon called the Kautsky-effect or variable fluorescence. This effect'can be traced to discharge-interferences in the redox-chain of the light reaction (Kautsky & Hirsch, 1931, 1934; Wassink & Katz, 1939; Kautsky & Franck, 1948). It is

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expressed by characteristic fluorescence curves, dependent on species and physiological stage of the plant (Fig. 3, control curve). Position and form of the curve are dependent on the activity state of the light reaction, as well as the ions present in the chloroplast. In general, it can be said that an increase in fluorescence indicates a poorer use of available light energy, whereas a drop in the Kautsky-cffect signals an acceleration of the electron flow; that is, an increase in activity state (Franck et a/., 1969). For several reasons, the variable fluorescence is particularly suited to the study of effects from air pollutants. Changes in the photosynthetic light reaction can be shown and compared with this relatively simple method without damage to the test plant itself. Supplementing the results through O2 gas exchange measurements and other methods can give evidence for the mechanism of the effects (Arndt, 1972).

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Fig. 3. Fluorescence intensity dependent on CuCI2 concentration. Of all the elements found in heavy-metal dusts, such as cadmium, zinc, mercury, and lead, copper and manganese cause particularly characteristic changes in the Kautsky-effect. After the addition of different amounts of copper chloride to the Chlorella-suspensions a concentration-dependent droP in fluorescence can be seen (Fig. 3). The effect is dependent on the physiological condition of the algae and is especially obvious when the algae are removed from their nutrient medium and allowed to grow in a solution containing the damaging agent (see Steemann-

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Nielsen et al., 1969). Under constant experimental conditions, effects on the light reaction can be demonstrated even with concentrations of 10-6 molar Cu ++ The drop in fluorescence that occurred in the experiments with Cu contradicts the above description since a rise in the activity state in the light reaction would not be expected with the addition of copper ions. To check this, oxygen measurements were carried out in the Warburg apparatus. These measurements showed that oxygen development, that is, the electron-producing system of the light reaction, is negatively influenced (Table I).

TABLE 1 EFFECTS OF HEAVY METALSON THE PHOTOSYNTHETICOXYGEN PRODUCTION, IN ~o OF CONTROL FOR 25 X l06 SYNCHRONISEDALGAL CELLS/ME SUSPENSION.STANDARD DEVIATION ~8"8

Concentration in mol/l Element CuCI2 (CH 3COO) 2Zn MnC12 CuCI2 + ( C H 3 C O O ) z Z n

1×10 0 22.8

3

5x10 12 25.5 103.1

4

lxl0 14 90-3 107.4

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5×10 69.1 106.9 101.5 66'8

5

1×10-5

5×10-6

89.3 99-8 90.2

96.8

Retardation in the splitting of water molecules, as shown in earlier experiments (Greenfield, 1942), can lead to a scarcity of electrons in photosystem II and, therefore, to a drop in fluorescence. It is obvious, therefore, that the action of copper in photosystem I| must be a blockage in the splitting of water (Habermann, 1969), especially since manganese shows a reactivating effect. This hypothesis, however, cannot satisfy all cases since, with stronger copper concentrations, changes in the pigment content of higher and lower plants occur. These changes can also be traced directly to an influence on photosystem II. Becker (1957), working with live grapevine sprayed with a copper sulphate/calcium mixture, demonstrated a copper-pigment combination which exhibited a blue-green colour. In the investigations of Gross et al. (1970) a displacement to the blue range of the absorption spectrum occurred after the addition of copper salts to Chlorellasuspensions. These results were confirmed in the experiments reported here (Fig. 4). After extraction and chromatographic purification, a blue-green pigment was obtained which, on the basis of its absorption spectrum, must be called a copperporphyrin due to its relationship to chlorophyll (Fig. 5). It is presumed that the central magnesium atom of chlorophyll is displaced by copper, a strong chelating agent. Spectral analysis of the purified pigment shows a, strong Cu-band but no Mg-band. This exchange process has been observed quite often in vitro (Strell & Zuther, 1958). A portion of the chlorophyll is removed from the pigment system of the chloroplast in the formation of this copper complex. It cannot be used in photosynthesis as light energy is not sufficient to stimulate the

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electrons. The isolated substance does not fluoresce (see Haurowitz, 1935). These processes necessarily lead to a drop in the Kautsky-effect and to lower oxygen production. Manganese showed another mode of action. Different concentrations of this element increased fluorescence induction but had little influence on oxygen production (Tables I and 2). Manganese probably works as a catalyst in the splitting of water molecules during the photosynthetic light reaction (Cheniae & Martin, 1970) but can also act as an electron donator (Ben-Hayyim & Avron, 1970). This latter property appears to play a role in the increase in fluorescence as these additional electrons cannot be taken up by the redox-chain and must fall back to its original energy level. With additional experiments it was determined that, to a certain extent, manganese weakens the effects of copper (Table 2 and Fig. 6). TABLE 2 EFFECTS OF HEAVY METALS ON THE KAUTSKY-EFFECT IN ~o OF AREA UNDER CONTROL CURVE FOR 25 X 106 SYNCHRONISEDALGAL CELLS/ML SUSPENSION

Element

Concentration in mol/I Ix10--3 lx10-4

5 x 1 0 -5 l × 1 0 - 5 5 x 1 0 - 6 1 x 1 0 - 6 5 x 1 0 - 7

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" 92

4 5x10 101

98 99 100

Ix10-7 99

101

5

It could not be conclusively determined if the formation of copper-porphyrin is retarded by manganese or if, in the photosynthetic light reaction, an enzyme is present whose activity as a copper complex is blocked but which is active as a manganese complex, as proposed by Habermann (1969). In both cases, the oxidation side of photosystem II has to be considered as the site of highest copper sensitivity (Cedeno-Maldano et al., 1972). Using the Kautsky-effect to test the action of zinc, little change in fluorescence was shown for concentrations under 10 -4 molar. This indicates that, for the metabolic processes considered here, zinc is less toxic than copper (Table 2). The combined effects of zinc and copper on oxygen production are hardly stronger than the effects from the single elements (Table 1). Because the combined action of zinc and copper causes a decrease in fluorescence in chloroplasts (Table 2), it is doubtful if an uptake-antagonism exists, such as is supposed for higher plants (Bowen, 1969). As localised air pollutants, heavy-metal-containing dusts often occur along with SO2 which is widespread in higher and lower concentrations. Therefore, the

189

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investigation of the mode of action for this gas is of particular interest. It has been known for some time that SO2 restricts the CO2-uptake of plants; that is, photosynthesis is influenced (Wieler, 1905; Thomas & Hill, 1937). The investigations of Ziegler (1972) have shown that the retardation of CO2-uptake can be traced to a blockage of a key enzyme of the Calvin cycle. This enzyme, called carboxydismutase or ribulose l'5-diphosphate carboxylase, takes part directly in the first steps of CO2 reduction in the dark reaction of photosynthesis. It is highly probable that the light reaction is also influenced by SO2. Since experiments to answer this question have only been carried out with inorganic sulphites and sulphates, one must consider that the gas would be active in the oxidised form SO 3 (Ziegler, 1972). Higher concentrations of sulphate (2 x 10 -2 molar) caused a blockage in the formation of adenosine triphosphate (ATP, see Fig. 2) (Asada et al., 1968). However, as shown on broken chloroplasts with artificial electron receptors, there was no negative influence on the system for splitting of water molecules or on electron transport (Baldry et al., 1968). Hall & Telfer (1969) demonstrated that sulphates and sulphites replace phosphate during energy conversions and hinder ATP-production, causing an acceleration in electron flow. With the method used in the investigations reported here for the measurement of fluorescence, it was possible to demonstrate the activity of SO2, although this gas exhibits two modes of action in the chloroplast. The first, namely the

190

UWE ARNDT

r e t a r d a t i o n o f carboxydismutase, leads to a build up o f p r o d u c t s f r o m the light reaction which should cause an increase in the Kautsky-effect. On the o t h er hand, the described acceleration in electron flow m u s t lead to a reduction in fluorescence. These expected results were, in fact, achieved with specific experiments (Table 2). A l t h o u g h the changes o f the Kautsky-effect were n o t great, higher c o n c e n t r a t i o n s o f SO3 lead to a reduction, whereas lower c o n c e n t r a t i o n s lead to a stimulation o f the fluorescence.

RELATIVE TOXICITY OF DUSTS W i t h the Kautsky-effect, in c o n n e c t i o n with other criteria, it is possible to study the effect o f air pollutants on m e t a b o l i c processes in chloroplasts. W i t h certain

TABLE 3 A N A L Y S E S O F T H E S O L U B L E F R A C T I O N O F D U S T S A M P L E S IN P P M O F T H E T O T A L

Sampling site 2550/5696 2550/5698 2551/5701 2551/5702 2552/5696 2552/5697 2552/5698 2552/5699 2552/5700 2552/5701 2552/5702 2552/5703 2552/5704 2553/5697 2553/5698 2553/5699 2553/5700 2553/5701 2554/5697 2554/5698 2554/5699 2554/5700 2554/5701 2554/5702 2555/5697 2555/5698 2555/5699 2555/5700 2555/5701 2555/5702 2556/5699 2556/5701 2556/5702 2556/5703 2556/5704

Manganese 729 891 1095 II11 53 1041 1232 1416 1761 1480 1073 20 340 971 1102 1293 456 1398 1339 1395 1517 1090 1713 1472 1485 1268 1176 1128 1377 1369 1441 1362 2150 1809 1535

Zinc

Copper

Cadmium

821 6008 6861 4769 67 242 10099 14164 7570 5600 2630 27 91 2834 4055 6034 2433 3647 5512 5465 5108 4829 5324 3252 5200 4131 5752 4205 3713 3571 5856 4131 5300 407 2697

traces 1008 1168 949 traces 242 911 1445 2711 1040 69 traces traces 243 394 1293 304 456 630 727 898 374 787 368 571 376 490 410 449 268 405 281 450 traces 166

traces traces traces traces traces traces 30 34 traces traces traces traces traces traces traces traces traces traces traces traces 31 traces traces traces traces traces traces traces traces traces traces traces traces traces traces

THE KAUTSKY-EFFECT

191

Fig. 7. Effects of settled dusts of the western Ruhr area on the photosynthetic light reaction. Area changes under the Kautsky-curve in ~o of control (black sections). Standard deviation ± 9.06 ~ .

192

UWE ARNDT

assumptions, it is also possible to use this method in the routine testing of relative and absolute toxicity of air and water pollutants. Because the Kautsky-effect is concerned with a very sensitive metabolic reaction, one of these assumptions is the standardisation of the test plants. Algae, with their large numbers of individuals, can serve as homogeneous raw material which, with identical numbers of cells/ml suspension and the same test procedure, always yield identical Kautsky-curves. The reaction of these organisms is, in general, reproducible for the same type and amount of pollutant. Therefore, a basis is available from which the effects of dust, gaseous, and liquid pollutants from different sampling sites can be compared with one another. As an example, relative toxicity of dusts from a monitoring network in the Western Ruhr area was tested. The dust samples were taken from the 35 sampling sites of the 1st Monitoring Programme of the province of Northrhine-Westphalia. The dust was collected in Bergerhoff dust-fall jars and suspended in water. The water was then evaporated and the dust weighed. Because of chemical conversion, certain changes in toxicity occurred which, however, played no role in this example. In the dust samples used, amounts of manganese, zinc, copper and cadmium were determined because it was known from the previous investigations that these would have strong effects on photosynthesis (Table 3). The changes in the Kautskycurve, or the change in the area under the curves--the criterion used in evaluation --does not appear to be solely dependent on the concentrations of these elements, although the antagonistic workings of copper and manganese were considered (Fig. 7). It is more likely that other substances such as organic compounds had a compensating or stimulating effect. This was particularly noticeable at sampling sites 2552/5700 and 2552/5701 (Table 3).

CONCLUSIONS

The work done so far with the Kautsky-effect shows that there are advantages in the use of this technique when compared with other methods but that there are also certain difficulties when it is used routinely. Since the method is very sensitive and can be employed without destroying the test plant, monitoring of fluorescence is an easy-to-use and informative tool in the investigation of effects-mechanisms in chloroplasts. Certain problems are revealed, however, when more extensive information is needed as, for example, in comparative measurements. Questions about selection, standardisation, and sensitivity of the test plants then arise; problems which previously have not been thoroughly investigated. This is also true for uptake and eventual antagonism of the pollutants which would be clarified by extensive analytical work.

THE KAUTSKY-EFFECT

193

ACKNOWLEDGEMENTS I a m g r a t e f u l to M r C. J. B r a n d t , M . S c . , f o r the E n g l i s h t r a n s l a t i o n o f this text.

REFERENCES

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RICHTEr, G. (1971). Stoffwechselphysiologie der Pflanzen. 2nd Ed. Stuttgart, Georg Thieme Verlag. SOEDER, C. J., SCHULZE, G. & TmELE, D. (1967). Einfluss verschiedener Kulturbedingungen auf das Wachstum in Synchronkulturen von Chlorella fusca Sh. et Kr. Arch. Hydrobiol., 33, 127-71. STEEMANN-NIELSEN, E., KAMP-NIELSEN, L. ~. WIuM-ANDERSEN, S. (1969). The effect of deleterious concentrations of copper on the photosynthesis of Chlorella pyrenoidosa. Physiologia PI., 22, 1121-33. StEEMANN-NIELSEN, E. & KAMP-NIELSEN, L. (1970). Influence of deleterious concentrations of copper on the growth of Chlorella pyrenoidosa, Physiologia PI., 23, 828-40. STRELL, M. & ZUIHER, F. (1958). Reaktionen in der Chlorophyllreihe. Ver/inderungen von Phorbiden und Chlorinen durch komplexbildende Metalle. Justus Liebigs. Annln Chem., 612, 264-71. THOMAS, M. D. & HXLL, G. R. (1937). Relation of sulfur dioxide in the atmosphere to photosynthesis and respiration of Alfalfa. PI. Physiol., Lancaster, 12, 309-83. WASSINK, E. C. & K a t z , E. (1939). The initial changes of chlorophyll-fluorescence in Chlorella. E,zymologia, 6, 145-72. WtELER, A. (1905). Untersuchungen iiber Einwirkungen schwefliger Sdure auf die Pflanzen. Gebr. Borntr/iger, Berlin. ZIEGLER, |MGARD (1972). The effect of SO3 on the activity of ribulose-l,5-disphosphate caboxylase in isolated spinach chloroplasts. Planta, 103, 155-63.