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Photochemistry andphotobiology. 1969. Vol. 10, pp. 119-123. Pergamon Press. PrintedinGreat Britain EFFECT OF DESASPIDIN AND DCMU ON PHOTOKINESIS OF BLUE-GREEN ALGAE* W. NULTSCH Department of Botany, University of MarburglL., Germany (Received21 March 1969) Abstract-The effect of desaspidin and DCMU (3-(3.4-dichlorophenyl)-l ,I-dimethylurea) on the speed of movement in light and dark of the blue-green alga Phormidium uncinatum has been investigated. Desaspidin, which uncouples oxidative phosphorylation and predominantly cyclic photosynthetic phosphorylations, inhibits movement in the dark and light as well, but dark movement is more sensitive. Movement in the light is more sensitive under anaerobic conditions than in air. The inhibitory effect of desaspidin is markedly increased by DCMU, which inhibits non-cyclic electron transport and coupled phosphorylation in air as well as under argon. There is no evidence for any photodestruction of desaspidin in air, provided that no ferricyanide is present in the medium. These findings are interpreted to confirm the concept that photokinesis (i.e. a light induced acceleration of movement in microorganisms) is the result of an increased ATP production by photosynthetic phosphorylation and that both cyclic and non-cyclic photophosphorylations supply energy for the movement in blue-green algae. INTRODUCTION EARLIER investigations [ 1-31 have demonstrated that in blue-green algae of the genus Phormidium and in the purple bacterium Rhodospirillum rubrum photokinesis (i.e. an acceleration of movement by light) is the result of an increased ATP supply by photosynthetic phosphorylation, whereas movement in the dark is supported by ATP produced by oxidative phosphorylation and, to a very small extent, by anaerobic- phosphorylation coupled with glycolysis. Consequently, in these organisms the speed of the movement in light and dark may be used to measure the activities of phosphoryla- tion processes within the living cell. In this connection the question arose whether cyclic or non-cyclic phosphorylations or both contribute to the photokinetic effect. Based on the results of experiments with photosynthetic inhibitors [41, uncouplers [ 11 and redox systems [3], it has been concluded that photokinesis may be caused by both cyclic and non-cyclic photophosphorylation. However, because the ATP produced by non-cyclic phosphorylation is to a large extent consumed by C0,-fixation under normal conditions, cyclic phosphorylation may be the main energy source of photokinesis. In order to verify these conclusions the inhibitory effect of desaspidin and DCMU on the movement of the blue-green alga Phormidium uncinatum in light and dark has been investigated. While DCMU is a well known inhibitor of non-cyclic electron transport and coupled phosphorylation [5], desaspidin is reported by Baltscheffski and Kiewiet[61 and Arnon and co-workers[7,8] to be a specific uncoupler of cyclic phosphorylation at lower concentrations. At high concentrations desaspidin inhibits non-cyclic phosphorylation, too. Gromet-Elhanan and Avron [9] have re-examined the desaspidin effect. They interpreted their results to indicate that desaspidin is a non- *A preliminary account of this work was presented at the Fifth International Congress on Photobiology, 26-3 1 August, 1968, Hanover, New Hampshire (Abstract Gf-5). 119

EFFECT OF DESASPIDIN AND DCMU ON PHOTOKINESIS OF BLUE-GREEN ALGAE

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Photochemistry andphotobiology. 1969. Vol. 10, pp. 119-123. Pergamon Press. PrintedinGreat Britain

EFFECT OF DESASPIDIN AND DCMU ON PHOTOKINESIS OF BLUE-GREEN ALGAE*

W. NULTSCH Department of Botany, University of MarburglL., Germany

(Received21 March 1969)

Abstract-The effect of desaspidin and DCMU (3-(3.4-dichlorophenyl)-l ,I-dimethylurea) on the speed of movement in light and dark of the blue-green alga Phormidium uncinatum has been investigated. Desaspidin, which uncouples oxidative phosphorylation and predominantly cyclic photosynthetic phosphorylations, inhibits movement in the dark and light as well, but dark movement is more sensitive. Movement in the light is more sensitive under anaerobic conditions than in air. The inhibitory effect of desaspidin is markedly increased by DCMU, which inhibits non-cyclic electron transport and coupled phosphorylation in air as well as under argon. There is no evidence for any photodestruction of desaspidin in air, provided that no ferricyanide is present in the medium. These findings are interpreted to confirm the concept that photokinesis (i.e. a light induced acceleration of movement in microorganisms) is the result of an increased ATP production by photosynthetic phosphorylation and that both cyclic and non-cyclic photophosphorylations supply energy for the movement in blue-green algae.

I N T R O D U C T I O N

EARLIER investigations [ 1-31 have demonstrated that in blue-green algae of the genus Phormidium and in the purple bacterium Rhodospirillum rubrum photokinesis (i.e. an acceleration of movement by light) is the result of an increased ATP supply by photosynthetic phosphorylation, whereas movement in the dark is supported by ATP produced by oxidative phosphorylation and, to a very small extent, by anaerobic- phosphorylation coupled with glycolysis. Consequently, in these organisms the speed of the movement in light and dark may be used to measure the activities of phosphoryla- tion processes within the living cell.

In this connection the question arose whether cyclic or non-cyclic phosphorylations or both contribute to the photokinetic effect. Based on the results of experiments with photosynthetic inhibitors [41, uncouplers [ 11 and redox systems [3], it has been concluded that photokinesis may be caused by both cyclic and non-cyclic photophosphorylation. However, because the ATP produced by non-cyclic phosphorylation is to a large extent consumed by C0,-fixation under normal conditions, cyclic phosphorylation may be the main energy source of photokinesis.

In order to verify these conclusions the inhibitory effect of desaspidin and DCMU on the movement of the blue-green alga Phormidium uncinatum in light and dark has been investigated. While DCMU is a well known inhibitor of non-cyclic electron transport and coupled phosphorylation [ 5 ] , desaspidin is reported by Baltscheffski and Kiewiet[61 and Arnon and co-workers[7,8] to be a specific uncoupler of cyclic phosphorylation at lower concentrations. At high concentrations desaspidin inhibits non-cyclic phosphorylation, too. Gromet-Elhanan and Avron [9] have re-examined the desaspidin effect. They interpreted their results to indicate that desaspidin is a non-

*A preliminary account of this work was presented at the Fifth International Congress on Photobiology, 26-3 1 August, 1968, Hanover, New Hampshire (Abstract Gf-5).

119

I20 W. NULTSCH

specific uncoupler of all types of photophosphorylation, and that its uncoupling effect is quickly destroyed by photooxidation under oxidizing conditions. This interpretation, however, has been questioned by Tsujimoto, McSwain and Arnon[ 101, who gave new evidence that desaspidin at lower concentrations distinguishes between photo- phosphorylation coupled with the photooxidation of water and all other types of photophosphorylation.

MATERIAL A N D METHODS

In our experiments a strain of the blue-green alga Phormidium uncinatum has been used, which contains abundant amounts of phycoerythrin but only small amounts of phycocyanin. The culture technique, the experimental conditions and the methods of evaluation of the photokinetic effect have been described elsewhere[ 1 I , 123. The photokinetic experiments have been carried out in white light (1000 Ix) or in far-red light at 7 14 nm using an interference filter (Schott & Gen., Mainz).

RESULTS

As shown in Fig. 1, increasing amounts of desaspidin increasingly inhibit the move- ment of Phormidium uncinatum in both light and dark. The dark movement is more sen- sitive to desaspidin and is completely arrested at about 5 X M. This is almost the same value as reported by Urbach and Simonis[l3] to inhibit oxidative phosphoryla- tion in the green alga Ankistrodesmus braunii.

On the other hand, photokinesis is more sensitive to desaspidin under argon atmosphere than in air. Under argon it is completely arrested by M desaspidin, while in air it is inhibited only to an extent of 50 per cent of the control by the same concentration. Similar results have been obtained by Urbach and Simonis [ 131 in their photophosphorylation experiments in Ankistrodesmus braunii. Under argon (i.e. in absence of CO,) the cyclic type of photophosphorylation predominates. This would

Fig. 1 . Effect of desaspidin on the speed of movement of Phormidium uncinatum in dark and light, in air and under argon atmosphere. Abscissa: molarity of des-

aspidin; ordinate: speed of movement as per cent of the controls.

Effect of desaspidin and DCMU 121

explain the higher sensitivity of photokinesis to desaspidin under argon than in air. However, with respect to the conclusions of Gromet-Elhanan and Avron [9], the stronger effect of desaspidin in argon could also be due to the photodestruction of desaspidin in air.

M DCMU decreases the speed of the movement in light to about 50 to 60 per cent of the control, and that less than 50per cent of the DCMU resistant movement in light is due to oxidative phosphorylation, which is obviously inhibited in light. As anaerobic phosphorylation in glycolysis does not play an important role [4], the remaining oxygen independent and DCMU resistant part of the movement must be due mainly to cyclic photophos- phorylation. Consequently, it should be sensitive to lower desaspidin concentrations. As shown in Fig. 2, concentrations of 2 X lop6 and 5 X low6 M of desaspidin, which are ineffective if applied alone, reveal a distinct inhibitory effect if combined with 5 X M DCMU. At M desaspidin the movement is completely arrested in presence

In earlier papers[4, 141 it has been shown that 5 x

Fig. 2. Effect of desaspidin and DCMU on the speed of movement of Phormidiurn uncinaturn in the light, in air and argon. Abscissa: molarity of desaspidin; ordinate: speed of

movement as per cent of the controls.

of DCMU, while it is inhibited to only about 50 per cent by desaspidin alone. In cor- responding experiments carried out under argon atmosphere movement in the light is more strongly inhibited by DCMU than in air, which is consistent with the findings of Urbach and Simonis[l3] in their phosphorylation experiments. This may be due to oxidative phosphorylation, which is only partially impaired by higher DCMU concen- trations in air, but is completely inhibited under anaerobic conditions. Another possible explanation for the different effect of DCMU under aerobic and anaerobic conditions has been given by Urbach and Simonis[l3], who suggest that the pseudocyclic type of phosphorylation, which depends on oxygen, may also exist in vivo.

As a result of the stronger effect of DCMU under argon the combination of desaspidin and DCMU is also more effective under argon than in air (Fig. 2). As the

I22 W. NULTSCH

photodestruction of desaspidin should be prevented by argon in this case, too, one should expect that these dose response curves also reveal different shapes, as it is the case if desaspidin is applied alone. However, the curves turned out to be parallel. This favours the assumption that the stronger effect of desaspidin in argon is the result of the inhibition of non-cyclic phosphorylation in C0,-free atmosphere and is not due to the photodestruction of desaspidin in air.

Experiments with far-red light of 7 14 nm confirm the results of the aforementioned experiments. The results are summarized in Table 1. Under argon atmosphere, the photokinetic effect of far-red light (which excites only photosystem 1) is more strongly inhibited by 2 X M desaspidin than is the photokinetic effect of white light (which excites both photosystems I and 11). On the other hand, 5 X M DCMU are less effective in far-red than in white light. Since cyclic photophosphorylation is driven by photosystem I, the specific effect of desaspidin on cyclic phosphorylation seems to be proved. These findings are consistent with the results of investigations on the effect of desaspidin and DCMU on photophosphorylation in intact cells of Ankistrodesmus braunii, reported by Urbach and Simonis [ 131.

Table 1 . Effect of desaspidin (2 X M) and DCMU ( 5 X M) on photokinesis in white light and at 7141-1113 (6000erg

X cm-* x sec-I) under argon

Photokinetic effect (%) Treatment White light 714 nm

Control 100 100 Desaspidin 82 56 DCMU 52 76

In order to demonstrate that the uncoupling effect of desaspidin depends on the oxidation-reduction state of the system investigated, Gromet-Elhanan and Avron [9] have compared the effect of desaspidin on photophosphorylation reactions in presence of ferricyanide and ferrocyanide. They found that desaspidin is the less effec- tive the more oxidizing the conditions (i.e. the more ferricyanide is present). The decrease of the effectiveness of desaspidin by ferricyanide is prevented by the addition of considerable amounts of ferrocyanide. Based on these results Gromet-Elhanan and Avron [9] have concluded that the uncoupling effect of desaspidin is quickly destroyed by photooxidation.

Therefore, in corresponding experiments the influence of ferricyanide and ferro- cyanide on the inhibitory effect of desaspidin on photokinesis has been investigated. The results of these experiments are presented in Table 2. According to the experiments of Gromet-Elhanan and Avron[9] it has been found, that the inhibitory effect of desaspidin was completely canceled by 5 x lop4 M ferricyanide (which is almost ineffec- tive if applied alone), while ferrocyanide, (which slightly stimulates photokinesis if applied alone), does not impair the inhibitory effect of desaspidin significantly even at very high concentrations.

DISCUSSION Hind[ 151 has shown that desaspidin is photodestroyed if illuminated in the presence

of ferricyanide at pH 8, and that the oxidation product fails to uncouple photophos-

Effect of desaspidin and DCMU 123

Table 2. Effect of ferricyanide and ferrocyanide on the inhibition of photokinesis by desaspidin in white light

( I 000 Ix) and air

Photokinetic effect (%) Treatment Control M Desaspidin

None 100 51 Ferricyanide 5 X M 93 96 Ferrocyanide 5 X M 109 60

2 x 10-2M 109 55

phorylation. This may be the explanation for the loss of activity of desaspidin in all experiments in which ferricyanide is present, e.g. in the photophosphorylation experi- ments of Gromet-Elhanan and Avron[9] as well as in our photokinetic experiments.

However, neither the results of our experiments on the effect of desaspidin upon photokinesis nor the investigations of Urbach and Simonis[l3] on the effect of des- aspidin upon 32P incorporation gave any evidence that desaspidin is photodestroyed within the living cell in the absence of ferricyanide or other oxidizing agents.

Therefore, we may conclude that both cyclic and non-cyclic photophosphorylations occur in vivo, and that desaspidin at lower concentrations predominantly inhibits the cyclic one, as was postulated by Arnon and co-workers[7,8, 101. Moreover, the results of our experiments give further evidence that both cyclic and non-cyclic photophos- phorylations supply energy for movement, and that the light induced acceleration of movement, called photokinesis, is due to the increased production of ATP by photosyn- thetic phosphorylation processes in blue-green algae.

Acknowledgements-This investigation has been supported by the Deutsche Forschungsgemeinschaft. I am indebted to Miss W. Hauber for valuable technical assistance.

R E F E R E N C E S 1 . W. Nultsch,Z. Pjfanzenphysiol. 56, 1 (1967). 2. W. Nultsch,Arch. Mikrobiol. 63,295 (1968). 3. W. Nultsch and G. Throm, Nature 218,697 (1968). 4. W. Nultsch and Jeeji-Bai, Z. Pjfanzenphysiol. 54,84 ( 1 966). 5. L. N. M. Duysens, Prog. Biophys. 14,l (1964). 6. H. Baltscheffsky and D. Y. de Kiewiet,Acta Chem. Scand. 18,2406 (1964). 7. D. J. Arnon, H. Y. Tsujimoto and B. D. McSwain, Nature 207, 1367 (1965). 8. Z. Gromet-Elhanan and D. I. Arnon, Plant Physiol. 40, 1060 (1965). 9. Z. Gromet-Elhanan and M. Avron, Plant Physiol. 41, 123 1 (1966).

10. H. Y. Tsujimoto, B. D. McSwain and D. J. Arnon, Plant Physiol. 41, 1376 (1966). 1 1 . W. Nultsch, Planta 56,632 (1961). 12. W. Nultsch,Planta 57,613 (1962). 13. W. Urbach and W. Simonis,Z. Naturf. 22b, 537 (1967). 14. W. Nultsch, Photochem. Photobiol. 4,613 (1965). 15. G . Hind, Nature 210,703 (1966).