Inhibition of Photosystem II in the Green Alga Scenedesmus obliquus by Nickel

Biochem. Physiol. Pflanzen 188, 363 - 372 (1993) Gustav Fischer Verlag Jena

Inhibition of Photosystem II in the Green Alga Scenedesmus ohliquus by Nickel MOSTAFA M. EL-SHEEKH Botany Department, Faculty of Science, Tanta University , Tanta, Egypt Ke y Term lnde x: Electron transport chain , fluorescence , nickel , photosystem II, thermoluminescence ; Scenedesmus obliquus

Summary The toxic effect of Nj2+ ions on photosynthetic electron transport was investigated by monitoring Hill activity, fluorescence, oxygen evolution and thermoluminescence properties in the green algae Scenedesmus obliquus 276-1 . Nickel strongly inhibited 2,6-dichlorophenolindophenol (DCPlP) photoreduction in the broken cells of Scenedesmus, and the activity lost could not be restored by adding 1,5-diphenyl carbazide (DPC) . Oxygen evolution both measured polarographically and under flash light conditions decreased by increasing the nickel concentration. Fluorescence intensity measured at room temperature decreased upon addition of nickel chloride , both in the presence and absence of DCMU . The maximum fluorescence could not be restored by addition of artificial electron donors . Thermoluminescence studies revealed that the S2QA - charge recombination, however, was inhibited with increasing concentrations of nickel chloride. The results suggest that NiH does not block the electron flow between the primary and secondary quinone electron acceptor, but modify the QB site or interact with the non-heme iron between the QA and QB, leading to the impairment of photosystem II.

Introduction Heavy metals are known to interfere with a variety of photosynthetic functions (VAN ASSCHE and CLIJSTERS 1983; CLUSTERS and VAN ASSCHE 1985). Inhibition of electron transport by Ni2+ (TRIPATHY and MOHANTY 1980; TRIPATHY et al. 1981; MOHANTY et al. 1989) has been reported . Some of the heavy metal ions, such as NiH, Co2+ and Zn H which are required as trace elements for plant mineral nutrition, occur in some places at high concentrations, and when taken up by plants impair plant growth and development. The study of partial electron transport reactions in intact cells is difficult since the exogenous electron donors and acceptors do not easily enter the intact cells (MURTHY et al. 1990). However, measurements of fluorescence of chi a in intact algal cells provide information on the absorption and utilization of energy in photosynthesis (PAPAGEORGIOU 1975 ; FORK and MOHANTY 1986). Attempts have been made to localize the site of inhibition for Zn2+ , Co2+ and Ni2+ on Abbreviations: chi, chlorophyll ; DCMU, 3-(3,4-dichlorophenyl)-1, I-dimethylurea; DCPlP, 2,6-dichlorophenolindophenol ; DPC , 1,5-diphenyl carbazide; Fm , Fo, maximum and constant fluorescence; Fp, yield of fluorescence at Fp transient; F" variable chlorophyll fluorescence; MV, methyl viologen; QA and QB, primary and secondary quinone acceptors of PS II; PS, Photosystem II 24*

BPP 188 (1993) 6

363

the PS II driven electron transport chain. Ni2+ and Co2+ act directly on P680 (TRIPATHY et al. 1981; TRIPATHY et al. 1983). The action sites of heavy metal ions have been investigated by electron transport rate measurements (HAMPP et al. 1976; BAKER et al. 1982). Recently thermoluminescence proved to be a new useful technique in probing PS II photochemistry (DEMETER and GOVINDJEE 1989). The most important photosynthetic thermoluminescence components arise from thermally activated radiative recombination of positive and negative charges, produces in light-induced primary charge separation In PS II (V ASS and INOUE 1992). This study was initiated to investigate the effects of various concentrations of nickel ions on the photochemical activities in the intact green algae S. obliquus. The results indicate that nickel inhibited the whole chain as well as photosystern II driven electron transport on the reducing side.

Materials and Methods Organism and Growth Conditions The green algae Scenedesmus obliquus 276-1. Sammlung von Algenkulturen, Pflanzenphysiologisches Institut, Universitat G6ttingen, Germany, was cultured in a medium described by KUHL (1962). The cultures were illuminated continuously with fluorescent tubes maintaining the desired light intensity (130 watts' m- 2 ). The cultures were incubated at 27°C and aerated continuously with a mixture of 95 % air and 5 % CO 2 . Growth of the cultures was monitored by measuring the chlorophyll a and b content according to MACKINNEY (1941). For experiments, cells were harvested in their exponential growth phase by centrifugation for 5 min at 5000 x g. In all experiments, the algal cells were incubated for 30 min in dark with NiCI 2 , after being washed with the nutrient medium.

Oxygen Evolution Measurements Oxygen evolution and/or consumption and electron transport reactions were measured polarographically using a Clark-type electrode. For measuring oxygen evolution under flash light conditions, the algal cells were suspended in 50 mM phosphate buffer pH 6.5, which in addition contained 50 mM KCI. Flash induced oxygen yield was measured with a loliot-type elctrode. 100 f,ll sample aliquots with a chlorophyll concentration of 70 f,lg chl'ml- 1 were illuminated with a sequence of short flashes after 5 min adaptation in the dark on the surface of the electrode. The flash frequency was 2 Hz.

DCPIP Photoreduction Measurement The DCPIP-Hill activity was measured with the (Shimadzu UV-3000) used in split beam mode. Three ml ing 20 f,lg chlorophyll and 40 f,lM DCPIP were excited by illuminating red actinic light was 300 W· m- 2 . The DCPIP recording the absorbance change at 590 nm.

help of a spectrophotometer of the sonicated algae containred light. The intensity of the photoreduction was assayed by

Fluorescence Induction Measurement Fluorescence induction transient was measured using the intact algal cells equivalent to 15 f,lg chI· ml- 1• The algal cells were excited (white light/Kombinat VEB NARV A, TGL 10 619/10 W' m -2) and the emitted light was det~cted by an EMI 9558 B photomul364

BPP 188 (1993) 6

tiplier perpendicular to the exciting path. The fluorescence transients were recorded by a multichannel analyzer (ICA 70 KFKI).

Thermoluminescence Measurements Thermoluminescence measurements were done as described by DEMETER and V ASS (1984) . Samples containing 100 f1g chi· ml l - were quickly cooled to -40 °C and were illuminated with white light (10 W· m- 2 ) for 30 s before heating. Glow curves were then recorded between -40°C and 60°C at a rate of 20 °C . min -I .

Results and Discussion Effect of Ne+ ions on the oxygen production and PS Il-catalyzed DCP]P photoreduction

A limitation of the electron transport at the reducing side of PS II, could be responsible for a lowering of oxygen yield (WIESSNER et al. 1991). In S. obliquus, the flash induced pattern of oxygen production was drastically decreased by increasing the NiH concentration . At the concentration of 20 mM, a heavily damped pattern of flash induced oxygen production was observed (Fig. 1) and the steady state level of oxygen production was decreased by about 70 % as compared with untreated algae. These results are in agreement with the measurements of oxygen yield using a Clark-type electrode (Fig. 2). The effective concentration of NiH to inhibit the electron transport chain is relatively high. It can be assumed that NiH ions can not penetrate easily through the cell wall of Scenedesmus . Using the algal cells similar high concentrations of heavy metal

400 'in 300 :: c ::::I

- 200 --

Qj '-

"C

GI

>-

c

GI C7I

>)C

100

0

0

4

12 16 8 Flash number

20

Fig. 1. The effect of various concentrations of NiCl 2 on the flash induced oxygen yields of intact cells of Scenedesmus ob/iquus. After incubation with Ni2+, the cells were suspended in 50mM phosphate buffer, pH 6.S in addition in SOmM KCI and incubated for S min in the dark on the surface of the electrode before measuring. BPP 188 (1993) 6

365

..

~ r.. c: 0

u

~

100

0

-

~ 0

..

80


a

a. 60 :J

'l:I

c:

a 40

..c: 0

:J 0

20

>


c:


>0-

0

0

10 1S NiCl 2 (mM)

S

)(

0

20

Fig. 2. The effect of various concentrations of NiCl 2 on different partial electron transport rate reactions in Scenedesmus obliquus. Whole chain electron transport was measured without any addition (0). PS I activity (e) was measured from 40[.tM OCPIP to 2mM MV in the presence of 10 [.tM OCMU and 2mM sodium ascorbate. Control rate (100%) for whole chain = 120[.tmol O2 evolved· mg-1chl' h- 1, and for OCPIP to MV = 300 [.tmol O2 uptake' mg-1chl' h- 1. The bars indicate the standard deviation on the mean.

Table 1. Effect of various concentrations of NiCl z on the DCPlP reduction in the absence and presence of the artifical electron donor DPC (0.5 [.1M). mM NiCl 2

0.0 2.5 5.0 10 20

Hill activity (relative units) H2O

% of control

OPC

% of control

120±8 108±6 57±4 45±2 38 ±2

100 90 45 38 32

120±8 1l0±6 54±4 41 ±2 35±2

100 92 45 34 29

The chlorophyll concentration in all the experiments was the same (20[.tg· ml- 1 culture medium). Values are mean ± SE of three replicate experiments.

ions proved to be effective in several other works. POSKUTA (1991) found that Mn2+ up to 100 mM, Zn 2 + (10-40 mM), Cu 2 + (1-5 mM) and Co2+ (20-80 mM) are the effective concentrations to ihibit photosynthesis measured as oxygen evolution and dark respiration. In chloroplasts it is evident that 5 mM Ni2+ (MoHANTY et al. 1989) and (1-8 mM), (TRIPATHY et al. 1983) are the effective concentrations to inhibit electron transport chain. 366

BPP 188 (1993) 6

In PS II preparations RASHID et al. (1991) found that Zn2+ at low concentrations (1 mM) inhibited about 50 % of electron transport, while complete inhibition was only produced at approximately 40 mM ZnCh. They also obtained about 95 % inhibition of DCPIP photoreduction by addition of 20 mM ZnCh. The PS II dependent electron flow decreased progressively with increasing concentration of NiCl 2 in the broken cells of Scenedesmus (Table 1). The concentration required for approximately 70 % inhibition was 20 mM NiCI 2 . The loss of Hill activity was not restored by the addition of artifical electron donor of PS II, 1,5-diphenyl carbazide (DPC), which donate electrons to a site between the reaction center of PS II and the water splitting system (VERNON and SHAW 1969). Similar observations have been made in Nitreated thylakoids (TRIPATHY et al. 1981 and MOHANTY et al. 1989). In Tris-inactivated thylakoids, MOHANTY et al (1989), found that NH 20H restored the Hill activity, unlike in heavy metal ions-treated thylakoids. These observation suggest that Ni2+ ions inhibit at the site beyond the DPC electron donation site.

PS I catalyzed electron transport

Electron flow from the reduced donor DCPIPH 2 to methyl viologen in the presence of DCMU, sodium ascorbate, and methyl viologen is catalyzed by PS I (HAUSKA 1977). Fig. 2 shows that the PS I-catalyzed methyl viologen photoreduction and its subsequent auto-oxidation resulting in O2 uptake was not affected by Ni2+ , even at high concentration (20 mM). Contrary to this, the PS II dependent electron flow decreased progressively with increasing concentration of NiCl 2 in the broken cells of Scenedesmus. From these findings, it was concluded that nickel affects PS II activity without affecting that of PS I.

Effect of Ni2+ ions on chi a fluorescence yield

Changes in chI a fluorescence intensity are intimately associated with PS II activity and they reflect the redox states of the primary acceptor of PS II (GOEDHEER 1972; RENGER and SCHREIBER 1986) . Thus fluorescence induction was used to probe the pattern of metal ion inhibition. Fig. 3 (A and B) shows that incubation of the intact cell of Scenedesmus with Ni2+ resulted in a lowering of room temperature Fm and Fp in the presence of (Fig.3 A) and in the absence of (Fig. 3 B) DCMU, respectively. The extent of chi a fluorescence quenching increased with increasing nickel concentration. Table 2 presents a comparison of Fa, Fv and Fm as a function of NiCl 2 concentration. A gradual reduction in Fv was observed that resulted from a decrease in Fm upon Ni2+ treatment of the algal cells . In Ni2+ treated cells a decrease in FvfF m ratio was also observed. This result indicates that the primary photochemical efficiency of PS II was affected by Ni2+ treatment of Scenedesmus cells (SATOH et al. 1972; KRAUSE and WEIS 1984). Fig. 3 shows that approximately 60 % of the Fm was quenched by 20 mM Ni2+. Addition of exogenous electron donors, such as NH 2 0H and DPC to Ni2+ treated Scenedesmus cells could not restore the loss of chI a fluorescence intensitivity to the initial untreated control level (Fig. 4). BPP 188 (1993) 6

367

A

-DCMU

-

Control 2.5mM Ni Z• 5 mM Ni z• 10 mM Ni z• 20mM Niz•

"iii

·c ~

-... ~

-

>.iii

-

& B

Control

.s

+OCMU

i u

Xl...

2.SmM Ni z•

3

5 mM Ni z• 10mM Niz•

0

u..

20 mM Ni2>

o

os

to

tS

Time (sec)

2.0

Fig . 3. Fluorescence induction transients of untreated and NiH treated cells of Scenedesmus obliquue. (A) In the absence of DCMU . (B) In the presence of lO~M DCMU. The incubation with DCMU was 2 min in the dark in the measuring cuvette.

Interruption of electron flow between QA and QB , direct modification of QB, alteration of components beyond QB or inactivation of water splitting system can lead to the impairment of PS II activity (MOHANTY et al. 1989). The loss of variable fluorescence after Ni2+ treatment exclude the suggestion that Ni2+ ions inhibit between QA and QB' Since the blocking of electron flow between QA and QB by metal ions should result in a rise of fluorescence emission to the Pm level, similar to the DeMU type effect (BUTLER 1977; RENGER et al. 1984). Therefore, it can be concluded that Ni2+ ions inhibit either at water splitting side or acts as an acceptor of PS II. Effect of Ni 2 + ions on thermoluminescence emission

The thermoluminescence emission is a good diagnostic indicator of PS II photochemistry . The changes in the amplitudes and peak positions of the Band Q bands can be 368

BPP 188 (1993) 6

Table 2. Influence of NiCl2 on room temperature chlorophyll fluorescence in intact cells of Scenedesmus obliquus.

Fm

mMNiCh

Fo

Fv

Fv/Fm

9,0± 1.10 8.7±0.87 8.6±0.71 8.3 ± 0.51 8.0±0.62

15 ±2.3 12.3 ± 2.5 9.4 ± 1.6 6.7±1.7 4.0±0.7

0.63±0.04 0.58±0.04 0.52±0.04 0.44±0.03 0.33±0.02

(relative units)

24±4.66 21 ± 2.41 18 ± 2 .24 15±2.14 12± 1.40

0 2.5 5 10

20

The measuring time was 100 ms in the presence of OCMU . Values nrc mean ± SE of four repetitions .

-~ 100

c 0

v 0

~

~

80

60

>:!:::: III C CU

c

40

CU

v

c

CU

v

20

III CU &...

0 :::J

u..

0

0

5

15 10 NiCl2 (mM)

20

Fig. 4 . Effect of various concentrations of NiCl 2 on the maximum fluorescence yield of Scenedesmus obliquus. Addition of the artificial electron donors, 5 mM NH 20H or 0.5 ~M OPC had not any effect on the decay curve. The bars indicate the standard deviation of the mean.

easly followed by thermoluminescence technique, providing information about the changes in the redox states of the respective electron transport components (DEMETER and GOVINDJEE 1989). Thermoluminescence originates from the reversal of photoinduced electron flow around PS II . After change separation at P680 , the positively (S2 or S3) and negatively (QA , QB) charged moieties trapped on the donor and acceptor sides of PS II respectively, recombine to yield the thermoluminescence glow curves. The intact cells of Scenedesmus excited by saturating white light at - 60 °C for 30 s exhibited a thermoluminescence band at about 30°C (Fig. SA). This band is designated as B band and originates from the S2QB - charge recombination (RUTHERFORD et al. 1982; DEMETER and V ASS 1984). In the presence of DCMU, the B band is converted to a band at about 15°C BPP 188 (1993) 6

369

A

-OCHU

B~band

B

"iii

:!:: c

~

-40 -20

0 +20 +40 +60 -40 ~20 0 +20 +40 +60 Temperature (OC)

Fig. 5. Effect of various concentrations ofNiCl2 on the amplitude of thermoluminescence Band Q bands. (A) In the absence of DCMU. (B) 10IlM DCMU-treated cells of Scenedesmus obliquus. Control (a), 2.5mM NiCh (b), 5mM NiCl 2 (c), lOmM NiCl 2 (d) and 20mM NiCl 2 (e) DCMU was added after treatment of the algal cells with Ni2+. The. curves shown in the figures are the representatives of three independent measurements.

(Fig. 5 B). This band arises from S2QA - charge recombination and is known as Q band (RUTHERFORD et a11982; DEMETER and VASS 1984). Increasing concentrations of Ni2+ , induced marked reduction in the B band intensity (Fig. 5A) while there was no large effect on the Q band intensity (Fig. 5B). 20mM Ni2+ was found to decrease the amplitude of the B band by 52 % . At all Ni2+ concentrations tested, the S2QA - charge recombination, unlike the S2QB charge recombination was unaffected. The result suggests that the formation ofQA - is not affected by Ni2+. This indicates a preferential effect of Ni2+ on the QB acceptor because the S2 state is the common positive charge pool for both the Band Q thermoluminescence bands. I conclude that nickel ions have strong toxic effect on the photosynthetic electron transport chain. The fluorescence and thermoluminescence measurements demonstrate that the action site of Ni2+ ions is either at the level of secondary quinone acceptor QB or Ni2+ ions interact with the non-heme iron located between the QA and QB acceptors. This study reveals the effect of heavy metal ions considering its importance to the ecosystems, thereby, it could serve as a method towards a sound understanding the problem of heavy metal pollution. 370

BPP 188 (1993) 6

Acknowledgements The author would like to acknowledge the fellowship grant provided by Hungarian Academy of Sciences and UNESCO and would like to thank Dr. S. DEMETER for providing all facilities during preparing this work and for critical reading of the manuscript.

References BAKER, N. R., FERNYHOUGH, P., and MEEK, I. T.: Light dependent inhibition of photosynthetic electron transport by zinc. Physiol. Plant. 56, 217 -222 (1982). BUTLER, W. L.: Chlorophyll fluorescence as a probe for electron transfer. In: Encyclopedia of Plant Physiology, Vol. 5 (Eds. TREBST, A., and AVRON, M.) pp. 149-167. Springer-Verlag, Berlin 1977. CLIJSTERS, H., and VAN ASSCHE, F.: Inhibition of photosynthesis by heavy metals. Photosynth. Res. 7, 31-40 (1985). DEMETER, S., and GOVINDJEE: Thermoluminescence in plants. Physiol. Plant. 75, 121-130 (1989). DEMETER, S., and VAAS, I.: Charge accumulation and recombination in photosystem II studied by thermoluminescence I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence in chloroplasts. Biochim. Biophys. Acta 764, 24- 32 (1984). FORK, D. c., and MOHANTY, P.: Fluorescence and other characteristics of blue-green algae (cyanobacteria), red algae and cryptogams. In: Light Emission by Plants and Bacteria, (Eds. GOVINDJEE, AMEZ, J., and FORK, D. C.) pp. 451-496. Academic Press, London 1986. GOEDHEER, J. C.: Fluorescence in relation to photosynthesis. Annu. Rev. Plant Physiol. 23, 87-112 (1972). HAMPP, R., BEULICH, K., and ZIEGLER, H.: Effect of zinc and cadmium on photosynthetic CO 2 fixation and Hill activity of isolated spinach chloroplasts. Z. Pflanzenphysiol. 77, 336- 344 (1976). HAUSKA, G.: Artificial acceptors and donors. In: Encyclopedia of Plant Physiology, Vol. 5 (Eds. TREBST, A., and AVRON, M.) pp. 253-265. Springer-Verlag, Berlin 1977. KRAUSE, G. H., and WEIS, E.: Chlorophyll fluorescence as a tool in plant physiology. Photosynth. Res. 5, 139-154 (1984). KUHL, A.: Zur Physiologie der Speicherung kondensierter organischer Phosphate in Chlorella. In: Beitrage zur Physiologie und Morphologie der Algen. Gustav Fischer Verlag, Stuttgart 1962. MACKINNEY, G.: Absorption of light by chlorophyll solutions. J. BioI. Chem. 140, 315 - 322 (1941). MOHANTY, N., V ASS, I., and DEMETER, S.: Impairment of photosystem 2 activity at the level of secondary quinone electron acceptor in chloroplasts treated with cobalt, nickel and zinc ions. Physiol. Plant. 76, 386- 390 (1989). MURTHY, S. D. S., BUCHOV, N. G., and MOHANTY, P.: Mercury-induced alterations of chlorophyll a fluorescence kinetics in cyanobacteria: Multiple effects of mercury on electron transport. J. Photochem. Photobiol. 6, 373-380 (1990). PAPAGEORGIOU, G.: Chlorophyll fluorescence: an intrinsic probe of photosynthesis. In: Bioenergetics of Photosynthesis, (Ed. GOVINDJEE) pp. 319-371. Academic Press, New York 1975. POSKUTA, J. W.: Toxicitity of heavy metals to growth, photosynthesis and respiration of Chlorella pyrenoidosa cells grown in air or 1 % CO 2 . Photosynthetica 25, 181-188 (1991). RASHID, A., BERIER, M., PAZDERNICK, L., and CARPENTIER, R.: Interaction of Zn 2 + with the donor side of PS II. Photosynth. Res. 30, 123-130 (1991). REGNER, G., HAGEMANN, R., and VERMAAS, W. F. J.: Studies on the functional mechanism of PS II herbicides in isolated chloroplasts. Z. Naturforsch. 39c, 362-367 (1984). REGNER, G., and SCHREIBER, U.: Practical applications of fluorimetric methods to algae and higher plant research. In: Light Emission by Plants and Bacteria, (Eds. GOVINDJEE, AMEZ, A., and FORK, D. C.) pp. 587-619. Academic Press, New York 1986. BPP 188 (1993) 6

371

RUTHERFORD, A. W., CROFTS, A. R., and INOUE, Y.: Thermoluminescence as a probe of photosystem II photochemistry. The origin of flash-induced glow peaks. Biochim. Biophys. Acta 682, 557-465 (1982). SATOH, K., KATOH, S., and KATAMIYA, A.: Light and dark rate-determinating steps in electron transport reactions in spinach chloroplasts. Plant Cell Physiol. 13, 885-897 (1972). TRIPATHY, B. C., and MOHANTY, P.: Zinc inhibited electron transport of photosynthesis in isolated barley chloroplasts. Plant Physiol. 66,1174-1178 (1980). TRIPATHY, B. C., BHATIA, B., and MOHANTY, P.: Inactivation of chloroplast photosynthetic electron transport activity by Ni 2 +. Biochim. Biophys. Acta 638,217-224 (1981). TRIPATHY, B. c., BHATIA, B., and MOHANTY, P.: Cobalt ions inhibit electron transport activity of photosystem II without affecting photosystem I. Biochim. Biophys. Acta 722, 88-93 (1983). VAN ASSCHE, F., and CLUIJJSTERS, H.: Multiple effects of heavy metal toxicity on photosynthesis. In: Effects of Stress on Photosynthesis, (Eds. MARCELLE, R., CLUSTERS, H., and V ANPOUCK) pp. 371- 382. Marinus Nijhoff Publishers, The Hague 1983. V ASS, I., and INOUE, Y.: Thermoluminescence in the study of photo system II. In: The Photosystems: Structure, Function and Molecular Biology, (Ed. BARBER, 1) pp. 259-294. Elsevier Science Publishers, Amsterdam 1992. VERNON, L. P., and SHAW, E. R.: Photoreduction of 2,6-dichlorophenolindophenol by phenyl carbazide: A photosystem 2 reaction catalyzed by Tris-washed chloroplasts and subchloroplast fragments. Plant Physiol. 44, 1645-1649 (1969). WIESSNER, W., DEAK, Z. S., MENDE, D., and DEMETER, S.: Flash oxygen yield patterns of autotrophically and photoheterotrophically grown Chlamydabatrys stellata in presence and absence of lipophilic acceptors. Photosynth. Res. 29, 37-44 (1991).

Received July 2, 1992; revised farm accepted December 10, 1992 Author's address: Dr. MOSTAFA M. EL-SHEEKH, Botany Department, Faculty of Science, Tanta University, Tanta, Egypt.

372

BPP 188 (1993) 6