Photosynthesis Research 25: 241-248, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

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Comparison of effects of air pollutants (802, 03, NO2) on intact leaves by measurements of chlorophyll fluorescence and PToo absorbance changes Wolfgang Schmidt, Christian Neubauer, J6rg Kolbowski, Ulrich Schreiber & Wolfgang Urbach Lehrstuhl Botanik I, Universitiit Wiirzburg, Mittl. Dallenbergweg 64, 8700 Wiirzburg, FRG Received 14 May 1990; accepted in revised form 14 May 1990

Key words: leaves

Air pollutants, Calvin cycle activity, electron transport, photosystems I and II, spinach


The immediate effects of short exposures to high concentrations of different air pollutants (20 min SO2, 2 h 03, and 4 h NO 2, 5 ppm each) on chlorophyll fluorescence and PT00absorbance changes at 830 nm of intact spinach leaves were investigated. Three different types of fluorescence measurements were used: Fluorescence rise kinetics in saturating light, fast fluorescence induction kinetics (Kautsky-effect), and slow induction kinetics with repetitive application of saturation pulses (saturation pulse method). The results show that the various air pollutants caused rather different damage in the photosynthetic apparatus of the leaves: 1. SO 2 : The main effect is due to the acidifying action, weakening the PS II donor side (suppression of I1-I2-P phase in fluorescence) and inhibiting Calvin cycle activation (no relaxation of membrane energization). 2. 03: Ozone has apparently no specific point of attack due to its high reactivity. It obviously reacts with all cell membranes, but primarily with the plasma membrane which it first passes on the way into the leaf. 3. NO2: NO 2 produces H N O 3 and H N O 2, when dissolved in the leaf water. The nitrite reductase, however, is highly effective, so that (in the light) nearly all nitrite is reduced. By the reduction of nitrite to ammonia, O H - is produced preventing net acidification. Obviously, the electron transport rates, which are possible with nitrite as acceptor are very high, being comparable to those observed with the well-known Hill reagent methylviologen, as revealed by PT00 measurements in saturating light. Such high reactivities with NO 2 must prevent assimilatory electron flow.

Abbreviations: Cyt. b / f - Cytochrome b/f; F R - Far-red light; M V - Methylviologen; p p m - Parts per million; P Q - Plastoquinone; PS I I / I - P h o t o s y s t e m II/I; Q A - Ubiquinone A, the primary electron acceptor of PS II Introduction

Measurements of chlorophyll fluorescence parameters are well suited as indicators for the state of the photosynthetic apparatus, not only indicating changes in overall photosynthetic performance, but also allowing to localize sites of

primary damage. During recent years, fluorescence measurements have proven to provide a useful analytical tool in investigations of the effects of natural environmental stress factors such as heat (Bilger et al. 1984, Bilger et al. 1987), water stress (Schreiber and Bilger 1987), and low temperatures (Smillie et al. 1987,

242 Hetherington and Oquist 1988), as well as of the effects of air pollutants as SO 2 (Shimazaki et al. 1984, Schmidt et al. 1988) or 0 3 (Schreiber et al. 1978). As was first demonstrated by Harbinson and Woodward (1987) and also by Weis et al. (1987), PT00 measurements can be readily performed with intact leaves via the absorption changes of the PT00÷ cation radical around 820nm. Very recently, it became possible to obtain additional information on events concerning photosystem (PS) I in intact leaves by measuring P700 absorbance changes with the PAM Chlorophyll Fluorometer using a new emitter-detector-unit (Schreiber et al. 1988). We have carried out a comparative study of the effects of air pollutants on intact leaves by measurements of various fluorescence parameters and of P700 absorbance changes to investigate, in which ways these pollutants affect photosynthesis.

Materials and methods

The experiments were carried out with intact leaves of Spinacia oleracea; for fumigation, detached leaves were used. The leaf-stalks were cut again under water to maintain transpiration. The leaves were kept in small flasks filled with water and sealed with terostate to prevent solution of gases in the water. The fumigation experiments were carried out with gas-concentrations of 5 ppm of SO 2, 0 3 or NO2, respectively, for a period of 20 min (SO2), 2 h (03) or 4 h (NO2). If other fumigation periods were used, this is noted in the figure legends. SO 2 and NO 2 were taken from gas-bottles (Messer-Griesheim GmbH, Duisburg, FRG) containing 5 ppm of SO 2 or NO 2, respectively, in synthetic air. 0 3 was produced by an ozone generator (Fischer M 500, Fischer, Miinchen, FRG) and measured with potassium iodide in neutral solution according to Byers and Saltzman (1956). The leaves were fumigated within cuvettes with a volume of 0 . 4 x 1 0 - 3 m -3 at room temperature (about 20°C) and a light intensity of about 100 W m -2. Control experiments were carried out under identical conditions using ambient air. After fumigation, the leaves were dark-adap-

ted for I h before measurements of the following responses: 1. Fluorescence rise kinetics in saturating light (O-I1-I2-P), 2. Rapid fluorescence induction kinetics (Kantsky-effect, O - I - P ) , 3. Slow induction kinetics with repetitive application of saturation pulses, 4. P700 absorbance changes at 830 nm. All measurements were carried out using a modulation fluorometer (PAM Chlorophyll Fluorometer, H. Walz, Effeltrich, FRG). For further information concerning the different types of measurements see Results and Discussion.

Results and discussion

1. Fluorescence rise kinetics in saturating light ( 0 - I 1-I2-P ) Figure 1 shows fluorescence rise kinetics at saturating light intensity (2000 W m -2) of intact leaves, which were pretreated by fumigation with SO 2, 0 3 or NO 2 in the light (at about 100 W m-2). The characteristic kinetics (see control) are changed markedly by SO 2 and 0 3. With SO 2, F 0 and 11 are somewhat increased; the 11-12 and the I2-P phases are strongly inhibited. This behaviour is indicative for damage caused e.g., by acidification at PS II involving donor and acceptor side (Neubauer and Schreiber 1987). Fumigation with ozone causes a small general lowering of all rise components. Remarkably, after treatment with nitrogen oxides there is no effect in spite of a 24 h lasting fumigation. It is known that in the light, nitrite can be reduced via the nitrite reductase (dependent on the nitrate supply, see Kaplan et al. 1979), which is present in the chloroplasts using reduced ferredoxin as electron donor. Therefore, the fumigation experiment with NO 2 was repeated under dark conditions. In Fig. 2 the fluorescence rise in saturating light (2000 W m -z) of leaves fumigated with air (control) or with NO 2 for 4.5, 6 and 7 h in the dark with all other experimental conditions being identical is shown. With increasing fumigation times, both the 11-12 and the I2-P rises become suppressed. The dark-level fluoresence as well as









P "

1 2 ~

contrOlox % soe



100 ms


Fig. I.

Fluorescence rise kinetics at saturating light (white light, 2 0 0 0 W m 2) of intact spinach leaves fumigated with air (control), SO 2, 0 3 or NO 2 in the light.


control 4 . 5 h NOx





6 h NoX 7 h NOx



100 m s

Fig. 2.

Fluorescence rise kinetics at saturating light (white light, 2000 W m -2) of intact spinach leaves fumigated with air (control) or with NO 2 for 4.5, 6 and 7 h in the dark.

the 11 level are not affected. These effects are reminiscent to the effects of electron acceptors, known to selectively suppress the I2-P rise at saturating light intensities ( N e u b a u e r and Schreiber 1987). These results emphasize the importance of the illumination state in fumigation experiments with N O 2. In all following experiments, NO2-fumigation was performed in darkness.

2. Rapid fluorescence (Kautsky-effect, O - l - P )



In Fig. 3, fast fluorescence induction transients (Kautsky-effect) of leaves with different pretreatments are depicted. After recording the

dark-level fluorescence F 0 (corresponding to one relative unit of fluorescence yield), actinic light (red light) with an intensity of 2 0 W m -2 was switched on (indicated by the arrows) and the resulting trace was recorded, until the P-level was reached. The control curve shows the typical O - I - P transients. With fumigation of a leaf with SO 2, the I-level rises, the I-P-rise is slowed down, and the decline from P is retarded. This result confirms the conclusion of Fig. 1 that SO 2 causes damage at the level of PS II. Ozone also increases the I-level and retards the decline from the P-level, but not as drastically as SO 2. Treating the leaves with nitrogen oxides affects the Kautsky-curves in a very different man-










0 i

5 sec


Fig. 3. Rapid fluorescence induction curves of leaves pretreated with air (control), SO2, 03 and NO2. ner: Upon onset of light, fluorescence rises to a very high I-level, from where it does not further increase. As concluded from Fig. 2, dark-fumigation with NO 2 appears to create a large pool of electron acceptors. In addition, the rise in the I-level signals damage at the PS II acceptor side.

3. Slow induction kinetics with repetitive application of saturation pulses The typical behaviour of a control leaf is depicted in Fig. 4 (see control trace): After recording of the dark-level fluorescence, a single saturating light pulse (flash symbol) is given to monitor maximal fluorescence. Two minutes later, when fluorescence yield has returned to F0, actinic illumination and saturation pulses are started (arrow). After an initial rise, fluorescence decays with complex kinetics to a steady state level. Initially there is a fast reduction of the electron transport chain, which is followed by a slower reoxidation. The peaks induced by the saturation pulses display two types of changes: First, there is a decrease indicating energization of the thylakoid membrane, and second, there is an increase to a steady state value, as membrane energization relaxes part of the energy is consumed in form of ATP in the (meanwhile light activated) Calvin cycle. This normal pattern is strongly modified by fumigation. SO 2 decreases the maximal fluorescence intensity, slows down the reoxidation of QA, and inhibits Calvin cycle activity, thus preventing relaxation of energization. With 0 3, the

maximal fluorescence also becomes somewhat suppressed, whereas the reoxidation of QA appears almost not affected (but see Fig. 3). The saturation pulses show no longer the relaxation of membrane energization. The slow fluorescence induction curve after NO2-fumigation looks rather similar to that after fumigation with SO2: Again maximal fluorescence yield is suppressed and relaxation of membrane energization is prevented. However, the kinetic information from Figs. 1 and 3, together with additional information presented below, shows clearly that the mechanisms of SO 2 and NO 2 action are fundamentally different. 4. /°700 absorbance changes at 830 nm PT00 acts as the reaction center of PS I, which can be driven by far-red light (700-730nm). PT00 absorbance changes reflect the dynamic interaction of the two photosystems, mediated by the electron carriers PQ, Cyt b / f and plastocyanin (Schreiber et al. 1988). Measurements were carried out as follows (see Fig. 5): In the beginning, with a dark-adapted control leaf, PT00 is reduced. When illuminated with continuous far-red light (FR, closed arrow up), PT00 becomes oxidized. For information on the rate of oxidation, 5 s light o n - 5 s light off cycles were applied. When PT00 reached a constant oxidation level, continuous FR was switched on; then strong white background light was applied (open arrows), resulting in re-reduction of PT00 by electrons from PS II.




t~ cj


, 5rain



03 ql


Fig. 4. Slow fluorescence induction kinetics with repetitive application of saturation pulses of leaves pretreated with air (control), SO2, 0 3 and NO 2.


fi g



T P700 Ox,


'° 1 5



t 30 sec

Fig. 5. Measurement of P700 absorbance changes of a dark adapted control leaf. t FR on; ~ FR off. ~ White light on; ~ White light off.

Figure 6 shows that after fumigation with 802, both the rate and the extent of oxidation are decreased; in particular, the first phase of the biphasic oxidation kinetics of the control is slowed down. O z o n e (Fig. 7) causes only minor changes in PT00 oxidation and reduction kinetics. The main effect of N O 2 (Fig. 8) is on the response upon onset of white light. P700 is not mostly reduced as usual, but a rather high oxidation level is kept even with white light. This means that the electrons coming from PS II are withdrawn f r o m P S I by N O 2. The following e x p e r i m e n t was conceived to d e m o n s t r a t e the high efficiency with which N O 2 acts as an electron acceptor in intact leaves, by comparison with the potent Hill-reagent methyl-




PTO0 ox.

~ PToo ox. NO2

SO2 Io






8 8

8 o





f 30 sec

30 sec


Fig. 6. Measurement of P70o absorbance changes of a dark adapted leaf pretreated with SO 2. ~ FR on; t FR off. White light on; ~ White light off

Fig. 8. Measurement of PT0o absorbance changes of a dark adapted leaf after pretreatment with NO 2. f FR on; [ FR off. ~ White light on; ~ White light off.

viologen (MV). Leaves were infiltrated with different concentrations of MV and illuminated with FR to oxidize P700; then a 200 ms pulse of saturating white light was given (see Fig. 9). First, in all cases there is rapid PT00 oxidation followed after a delay of approximately 60 ms by re-reduction. The higher the MV-concentration, the lower this re-reduction becomes. At saturating MV-concentration there is even an additional P700 oxidation instead of re-reduction. Upon termination of the saturating light pulse in all cases there is rapid reduction of PT00, which reflects dark-relaxation of the redox-gradient between

the reduced PQ-pool and the oxidised PSI donor-side, which was built up in the saturating light pulse due to the rate limiting step between P Q H 2 and the cyt b/f complex. In close analogy to the behaviour of MVinfiltrated leaves, also leaves fumigated with increasing doses of NO 2 (Fig. 10) show a decreasing extent of PToore-reduction during the saturating light pulse. This demonstrates that the electron acceptor (presumably NO2) accumulating in intact leaves is capable of supporting high rates of electron transport. f

PTO0 ox.




o~ 8


5 8

8 8 .,

Comparison of effects of air pollutants (SO2, O 3, NO 2) on intact leaves by measurements of chlorophyll fluorescence and P700 absorbance changes.

The immediate effects of short exposures to high concentrations of different air pollutants (20 min SO2, 2 h O3, and 4 h NO2, 5 ppm each) on chlorophy...
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