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EnvironmentalandExpedmenlalBotany, Vol. 35, No. 4, pp. 435 439, I995 Elsevier Science Ltd

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I N H I B I T I O N OF PS II A C T I V I T Y BY C O P P E R AND ITS E F F E C T O N

S P E C T R A L P R O P E R T I E S O N I N T A C T CELLS IN ANACYSTISNIDUL4NS

ANU GUPTA* and GAURI S. SINGHAL School of Life Sciences,Jawaharlal Nehru University New Delhi 110 067, India

(Received 20 July 1993; accepted in final revised form 14 July 1995)

Gupta A. and Singhal G. S. Inhibition of PS II activi~ by copper and its effect on spectral properties on intact cells in Anacystis nidulans. Environmental and Experimental Botany 35, 435 439, 1995. Photosystem II (PS II) activity (pBQ-supported) in Anacystis nidulans was inhibited by copper con- centrations of 5, 10 and 15 /~M; the F,./Fm ratio also decreased. The decrease in Fm was due to a decrease in F,., thus showing that at all the concentrations tested copper affects PS II photochemistry. Higher concentrations of copper (10 and 15 #M) completely quenched the variable fluorescence of the cells. Fluorescence spectra of intact cells showed a shift in chlorophyll peak indicating that 15 /*M copper inhibits PS II activity by causing structural alterations in the Chl~roteins of the PS II complex. The absorption and fluorescence spectra also indicated that 5, 10 and 15/~M of copper cause bleaching of phycocyanin, thereby affecting the light harvesting complex as well.

Key words: Anacystis, chlorophyll fluorescence, copper, phycocyanin.

INTRODUCTION

Copper is an essential micronutrient for plant cells and is a cofactor ofpolyphenol oxidase, some super- oxide dismutases and plastocyanin, i7~ At con- centrations higher than 1 #M, copper is toxic to algae and higher plants. Copper inhibits photo- synthesis, ostensibly on the oxidizing side of PS II, (1'4'12'13) the c type cytochrome, (~4) the reducing side of PS II close to P680, (7i the reducing side of PS I (1) and ferredoxin. (13) Although the sensitivity of the PS II photosystem to copper is well known, there is presently no general agreement regarding the site of action. The purpose of this study is to identify

the sites of action of copper on PS II complex of Anacystis nidulans. The decrease in efficiency of PS II complex in the presence of copper could be due to its effect on the light-harvesting complex. This may lead to a decrease in transfer of energy to PS II. To study these possibilities, we chose Anacystis nidulans which offers a good model system due to its fast growth, easy handling and a distinct light- harvesting complex (phycobilisomes) whose spectral properties are different from those of the PS II complex, thus making it easier to interpret whether copper is effecting the PS II or light-harvesting complex.

Earlier studies have reported that concentrations

*For acceptance/revision comments on manuscript, not for reprint requests: 448, Basant Avenue, Amritsar, 1430 001, India.

Abbreviations: DPC = Diphenyl carbazide, F,,, = Maximum fluorescence, Fo = Initial fluorescence, F v = Variable fluorescence, MV = Methyl viologen, pBQ= Para-benzoquinone, PS II = Photosystem II.

435

436 A. GUPTA and G. S. SINGHAL

higher than 1 #M are toxic for algae and higher plants (5'6/ but these experiments were performed with either isolated chloroplasts or algae in lag phase. As the cells in log phase can tolerate higher concentrations, they were exposed to 5, 10 and 15 #M of copper. The choice of mid-log phase is close to the situation naturally occurring in the environ- ment, as the water bodies where these algae grow exponentially are being exposed to industrial efflu- ents.

M A T E R I A L S A N D M E T H O D S

Culture condititons Axenic cultures of the unicellular cyanobacteria

Anacystis nidulans (ARM 336) were obtained from the National Facility for Blue-green Algae, Indian Agriculture Research Institute, New Delhi (110012) and the culture was grown autotrophically in BG II medium (is/ pH 8.0 and autoclaved at 15 lbs for 20 min. The medium was inoculated under sterile conditions and the cultures were continuously aer- ated with aquarium air pumps (model Tropi-666 India) at 25 _+ 2°C and illuminated with cool flu- orescent bulb light intensity of 20 W/ m 2. The expo- nentially growing cultures were regularly trans- ferred to fresh medium and routinely checked for bacterial contamination. Cells were subjected to different concentrations of copper chloride (CuC12; 5, 10 or 15 #M) in mid-log phase.

Measurement of PS II activity Anacystis cells were grown in the presence of 5,

10 or 15 #M of copper, harvested after 2, 4 and 6 days of treatment and then centrifuged at 6000 g for 10 min. The cells (equivalent to a chlorophyll content of 25 #g) were placed into a cuvette con- taining 2 ml of reaction buffer (25 mM HEPES N a O H pH 7.5, 25 mM NaC1) and 2 #M pBQ. The PS II activity was measured at 25°C with an oxygen electrode (CBID, Hansatech, U.K.). Saturating actinic light was provided by an LS-2 tungsten hal- ogen light source (100 W bulb).

ChlorophyU fluorescence measurements Fluorescence induction transient curves were rec-

orded in a stirred glass cuvette at room temperature for 6 s on a transient recorder (TRI, Hansatech, U.K.) after maintaining the samples in complete darkness for 1 hr. The samples were excited with

120 [ - o----o C

~a= 100 ~ 5gMCu

o ~ 10~tMCu

80 e - - e 15~xMCu

60 Z o

40

20 -

0 2 4 6

Number of days Fig. 1. Effect of copper (5, 10 and 15 #M) on pBQ- supported PS II activity in intact cells ofAnacystis nidulans

over a 6-day assay period.

blue actinic light using a Corning CS 4-96 glass filter between the sample and lamp. Emission kin- etics were recorded through an interference filter (Wratten 89 B).

Spectra of cells Absorption spectra of cells grown in the presence

of 5, 10 or 15 #M copper were recorded with a UV 160A spectrophotometer (Shimadzu, Japan) and fluorescence spectra were recorded at room tem- perature (Perkin-Elmer LS-5, U.K.) with a 10 nm excitation slit and a 5 nm emission slit. Cells were exposed to 440 nm light to excite Chl a and to 545 nm light to excite phycobilisomes specifically. Cells equivalent to 5 #gm1-1 of Chl a were used for recording the spectra. The chlorophyll con- centrations of cells were determined according to Mackinney's formula. (a/

R E S U L T S A N D D I S C U S S I O N

The Anacystis cultures showed a decrease in PS II activity with an increase in copper concentration and increase in exposure time (Fig. 1). At 5 #M Cu, PS II activity was inhibited by 25% whereas at higher concentrations, there was a gradual decrease in PS II activity with increase in incubation time; total inhibition occurred after 6 days of treatment. PS II activity was inhibited by 70% with incubation of cells in 15 pM of copper for 2 days; prolonged

INHIBITION OF PS II ACTIVITY BY COPPER 437

1.2 m

o----0 c

1.0 - ~ 5gMCu 10p.MCu

0.8 _ ~a~ o o ~ o 15~tMCu

~--. 0.6

o4 f 2 4 6

Number of days Fig. 2. F,./F~, ratios of Anacystis cells treated with various

concentrations of(5, 10 and 15/2M) copper.

incubation completely inhibited the activity (Fig. 1). The effect of copper on pBQ-supported PS II activity indicated that copper affects PS II photo- chemistry.

The Fv/F,~ ratio of copper-treated cells showed a decrease with increasing concentrations of copper (Fig. 2). Copper treatment reduced the Fro; this decline was due to a decrease of the variable flu- orescence. There was no effect on F~,, i.e. the initial fluorescence of the cells. The decrease in Fv/F,n and a decrease in F, indicated a decrease in photo- chemical efficiency of the PS II complex showing that copper affects photosystem II which could be due to inefficient energy transfer from light-har- vesting complex to the reaction centre or due to inability of reaction centre to accept light due to structural alterations in PS II complex, i2'3'9!

To check for these possibilities, the absorption spectra of cells were recorded (Fig. 3). Absorption spectra showed a decrease in absorbance of phy- cocyanin as well as chlorophyll with increase in copper concentrations, but the peak at 620 nm was the more affected. At 5/~M copper, there was decrease in phycocyanin absorbance, but at 15/tM copper the peak at 620 nm completely disappeared along with a considerable reduction in chlorophyll absorbance thereby changing the PC/Chl a ratio from 1.09 to 0.85. (Fig. 3)

Since, copper affected the absorbance of phy- cocyanin (620 nm) and chlorophyll (680 nm), the fluorescence emission spectra of intact cells were

0.6

0.4

d~ < 0.2

~ C - - - 51aMCu

440 ~ ~ 101xMCu - - ' - - 15gMCu

0 I I I I 400 500 600 700 800

Wavelength (nm)

Fig. 3. Absorption spectra of cells treated with different concentrations (5, 10 and 15/~M) of copper for 2 days.

60 m

~ C - - - 5~tMCu

684 10gMCu

50 -- ~ - ~ 15gMCu / ' ~ 1

e-

40 --

e~

20

/ 1 1 6 4 5 / ; / \\

0 I I 600 650 700

Wavelength (nm) Fig. 4. Fluorescence spectra (room temperature) of cells treated with different concentrations (0, 5, 10 and 15/tM) of copper for 2 days. Cells were excited at 440 nm with a 5 nm excitation slit and a 10 nm emission slit. Cells

equivalent to 5/~g Chl a were taken.

recorded to elucidate the nature of alterations induced in these pigments (Fig. 4). On excitation at 440 nm, the cells showed two emission peaks. Emission at 650 nm emanated from phyco- bilisomes, at 685 nm from chlorophyll. Cells exposed to 5 #M of copper showed a decrease in

438 A. GUPTA and G. S. SINGHAL

e~

8 0

7 0 -

6 0 - -

5 0 - -

4 0 - -

30

20

10

0 u._ 600

6 5 0

- - C

-- - - 5 g M C u

- - - - 1 0 l x M C u

- - ' - - 1 5 l x M C u

I I

I 6 4 7

' / \ / \ '

' 6 4 5 \

6 5 0 7 0 0

Wavelength (nm)

Fig. 5. Fluorescence spectra of cells treated with different concentrations of copper. Cells were excited at 545 nm to excite the phycobilisomes with a 5 nm excitation slit and a 10 nrn emission slit. Cells equivalent to 5 pg Chl a

w e r e used.

absorbance of phycocyanin at 650 nm and a peak shift of 3 nm. At 15/tM, the peak emanating from phycocyanin disappeared. There was a large decrease in fluorescence at 685 nm at all copper concentrations, but a blue shift of 4 nm was observed only at the higher concentrations, i.e. 10 and 15/~M copper (Fig. 4).

The cells were excited at 545 nm to study the changes in phycocyanin fluorescence (Fig. 5). The spectra of normal cells showed a prominent peak at 650 nm emanating from PC and a shoulder at 685 nm. At 15 /~M copper, the peak at 650 nm decreased by 90% and the peak shifted from 650 nm to 645 nm.

The absorption and fluorescence spectra clearly

show that copper affects the light harvesting complex, i.e. phycobilisomes, as well as the PS II reaction centre. As copper caused bleaching ofphy- cocyanin in in vitro conditions also (unpublished data), this shows that copper causes structural alter- ations in the phycobilisomes leading to a decrease in energy transfer to PS II. Complete quenching of Fv further shows that PS II reaction centre itself is incapable of accepting the light energy which occurs due to change in structure of PS II reaction centre. This is evident from shift in peak at 685 nm as well. The results of our study further show that copper induced decrease in photosynthesis is due to a change in the pigment protein interactions of PS II and phycocyanin.

Acknowledgement~Anu Gupta would like to thank Council of Scientific and Industrial Research, New Delhi, India for financial assistance.

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INHIBITION OF PS II ACTIVITY BY COPPER 439

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