REGULAR PAPER

Elucidating the site of action of oxalate in photosynthetic electrontransport chain in spinach thylakoid membranes

Anjana Jajoo Æ Archna Sahay Æ Pooja Singh Æ Sonal Mathur ÆSergei K. Zharmukhamedov Æ Vyacheslav V. Klimov ÆSuleyman I. Allakhverdiev Æ Sudhakar Bharti

Received: 12 March 2008 / Accepted: 19 May 2008

� Springer Science+Business Media B.V. 2008

Abstract The effects of oxalate on PS II and PS I photo-

chemistry were studied. The results suggested that in

chloride-deficient thylakoid membranes, oxalate inhibited

activity of PS II as well as PS I. To our knowledge, this is the

only anion so far known which inhibits both the photosys-

tems. Measurements of fluorescence induction kinetics, YZ•

decay, and S2 state multiline EPR signal suggested that

oxalate inhibited PS II at the donor side most likely on the

oxygen evolving complex. Measurements of re-reduction of

P700+ signal in isolated PS I particles in oxalate-treated

samples suggested a binding site of oxalate on the donor, as

well as the acceptor side of PS I.

Keywords Electron transport chain � EPR �Fluorescence � Oxalate anion � Photosystem II �Photosystem I

Abbreviations

DCPIP 2,6-Dichlorophenolindophenol

DAD(red) Reduced 3,6-diaminodurene

Fo Initial fluorescence, where all QA are oxidized

Fv Variable fluorescence

MV Methyl viologen

TMPD(red) Reduced N,N,N1,

N1-tetramethyl-p-phenylenediamine

Introduction

Transformation of light energy into chemical energy in the

course of oxygenic photosynthesis takes place with the

contribution of sequentially functioning photosystem II (PS

II) and photosystem I (PS I), the thylakoid membrane

complexes containing reaction center and the core antenna.

PS II is a light-dependent water-plastoquinone oxidore-

ductase enzyme that uses light energy to oxidize water and

is mainly located in the appressed grana stacks. Various

crystallographic investigations of cyanobacterial photo-

system II have provided high resolution structures from 3.8

to 3.0 A (Kamiya and Shen 2003; Ferreira et al. 2004; Loll

et al. 2005) that explain the general arrangement of the

protein matrix and cofactors. The structural and functional

aspects of PS II are interrelated. PS I functions as a light-

driven plastocyanin-Fd oxidoreductase (Chitnis et al.

1995). The structure of the PS I core complex from the

thermophilic cyanobacterium Thermosynechococcus elon-

gates is known at 2.5 A resolution (Jordan et al. 2001).

A large number of organic and inorganic anions affect

various reactions associated with PS II and PS I. Certain

organic anions like glyoxylate, oxalate, and glycolate com-

pete with anions like formate (HCO2-) and bicarbonate

(HCO3-) for binding to the PS II (Petrouleas et al. 1994). It is

well established that bicarbonate is required for the func-

tional activity at the acceptor side of PS II, providing efficient

re-oxidation of the QA. The non-heme Fe between QA and QB

has been shown to play an essential role in bicarbonate

binding (Govindjee and Van Rensen 1993). On the other

hand, bicarbonate requirement for the donor side of PS II has

been clearly demonstrated (Klimov et al. 1995, 1997; All-

akhverdiev et al. 1997). Formate slows down the electron

transfer from QA- to QB and competes with HCO3

- for the

binding to the non-heme iron at the acceptor side of PS II

A. Jajoo (&) � A. Sahay � P. Singh � S. Mathur � S. Bharti

School of Life Sciences, Devi Ahilya University, Vigyan

Bhavan, Khandwa Road, Indore 452017, M.P., India

e-mail: anjanajajoo@hotmail.com

A. Jajoo � S. K. Zharmukhamedov � V. V. Klimov �S. I. Allakhverdiev � S. Bharti

Institute of Basic Biological Problems, Russian Academy

of Sciences, Pushchino, Moscow Region 142290, Russia

123

Photosynth Res

DOI 10.1007/s11120-008-9314-1

(Petrouleas and Diner 1990). Several studies have shown that

HCO2- induces inhibition of water oxidation reactions, as

well as electron transfer on the PS II acceptor side (Stemler

and Lavergne 1997). Interaction of HCO2- with chloride

(Cl-) on the donor and acceptor sides of PS II has been

reported (Feyziev et al. 2000; Jajoo et al. 2005a).

Inorganic anion Cl- has been shown to be an indis-

pensable cofactor involved in the photosynthetic oxygen

evolution (Lindberg and Andreasson 1996). Chloride is

required for high oxygen evolution activity in PS II and is

responsible for the normal electron paramagnetic reso-

nance (EPR) properties of the S2 state (Wincencjusz et al.

1999). Activating Cl- can be replaced by several mono-

valent anions including bromide (Br-), nitrate (NO3-),

iodide (I-) and nitrite (NO2-) with varying efficiency. In

Cl--depleted PS II membranes, substitution of Cl- by Br-,

I-, NO2-, and fluoride (F-) suggests a possible binding site

of these anions at the PS II donor side as well as at the

acceptor side (Jajoo et al. 2005b).

Different functions have been proposed for oxalate in

plants, including Ca2+ regulation, ion balance (e.g., Na+

and K+), plant protection, tissue support and heavy metal

detoxification (Nakata 2003). More attention and interest

has been focused on the roles of oxalate in plant abiotic

stress resistance, such as heavy metal toxicity and phos-

phorus deficiency (Dong et al. 2004). In view of its

function as a strong chelator of manganese and other cat-

ions (Schlosser and Hofer 2002), it is necessary to examine

the role of oxalate in photosynthetic reactions also. In the

photosynthetic electron transport chain among many

anions so far studied only oxalate was found to inhibit both

the photosystems (Jajoo and Bharti 1993a). It was, there-

fore, of interest to determine the specific effect and site of

action of oxalate in the photosynthetic electron transport

chain. In this study using oxalate-treated thylakoids, we

present data on: (i) electron transfer rates through PS II; (ii)

chl a fluorescence induction kinetics at room temperature;

(iii) decay kinetics of YZ• by time-resolved EPR at 253 K;

(iv) S2 state multiline EPR spectra; (v) partial reactions

mediated by PS I in the absence and presence of HgCl2;

(vi) re-reduction of P700+ in purified PSI particles. Based

on our results, we report for the first time that oxalate

inhibited PS II at the donor side on the oxygen evolving

complex (OEC) and inhibited PS I by binding on the donor

as well as the acceptor side of PS I.

Materials and methods

Isolation and storage

PS II membranes and thylakoid membranes were prepared

from fresh market spinach following method as described

in Kuwabara and Murata (1982). The membranes were

stored at 77 K with 50% glycerol added until use. Prior to

the experiment, the membranes were thawed slowly at 0�C

and washed with suspension medium containing 0.1 M

sucrose and 50 mM HEPES–NaOH buffer (pH 7.6) in

order to remove glycerol and centrifuged at 4,500g for

10 min. For preparation of ion-deficient membranes (-ion)

which did not contain any exogenous Cl-, the pellet was

suspended in an isolation medium containing 0.33 M

sucrose, 50 mM HEPES–NaOH buffer (pH 7.6). For

preparation of ion-sufficient membranes (+ion) isolation

medium consisted of 0.33 M sucrose, 50 mM HEPES–

NaOH buffer (pH 7.6), 10 mM NaCl and 1 mM MgCl2. All

steps were performed at 0–4�C. The thylakoid membranes

were stored in dark on ice. The chlorophyll content was

measured according to the method as described in Porra

et al. (1989). It is to be emphasized that we did not use

chloride-depleted membranes that require harsher treat-

ments, e.g., high pH, treatment with sulfate to remove

endogenous Cl-.

Purified PS I particles were prepared following the

method of Shuvalov et al. (1976).

Measurements of rates of electron transfer through PS

II and PS I

The activity of PS II was measured spectrophotometrically

as photo-reduction of DCPIP (H2O ? DCPIP reaction).

White light was employed for illuminating the reaction

mixture for 30 s and its absorbance was measured at

605 nm. The PS II activity was expressed in terms of lmol

DCPIP reduced mg chl-1 h-1.

The activity of PS I was monitored polarographically by

YS I model Clark-type oxygen electrode (Yellow Springs,

USA) with a water-jacketed reaction vessel thermostated at

25�C connected to a graphic recorder. The activity of PS I

was measured with either DCPIPH2, TMPD(red) or DAD(red)

as electron donor and methyl viologen (MV) as terminal

auto-oxidizable electron acceptor. The reaction mixture

was constantly stirred with the help of a magnetic stirrer.

Light intensity (150 W m-2) was employed for illuminat-

ing the reaction mixture. The PS I activity was expressed in

terms of lmol oxygen consumed mg chl-1 h-1.

Fluorescence measurements

Fluorescence induction kinetics of chl a was monitored

using a Photosynthetic Efficiency Analyzer (PEA, Hansa-

tech, UK). Re-reduction of P700+ in purified PS I particles

were measured in a 1 cm cuvette using a homemade

phosphoroscopic set-up as described in Klimov et al.

(1982).

Photosynth Res

123

EPR measurements

In order to measure rate of decay of YZ• , time-resolved EPR

measurements were performed using Varian X-band

spectrometer and homemade nitrogen gas-flow cryostat

with a temperature controller. Samples were directly illu-

minated using HOYA-SCHOTT MegaLight-100 to excite

YZ• signals. To measure S2 state multiline EPR signal, CW-

EPR measurements were performed using a Bruker-300E

X-band spectrometer, and an ST4102 standard cavity. An

Oxford-900 continuous gas-flow cryostat and ITC-4 tem-

perature controller were used to regulate the sample

temperature at 6.0 K. Samples (pH 6.8) were illuminated

with 500 mW tungsten-halogen lamp through an 8 cm

thick water filter to induce the formation of the S2 state

multiline signal at 200 K in an ethanol/solid CO2 bath. The

illuminated samples were quickly cooled to 200 K and then

stored at 77 K. The EPR spectra of S2 state multiline sig-

nals were recorded at 6 K. Chlorophyll concentration used

in all EPR measurements was 3–4 mg/ml.

Results and discussion

Effects of oxalate on PS II

The effects of oxalate (as sodium salt) on PS II photo-

chemistry were monitored in broken thylakoid membranes

by measuring H2O ? DCPIP reaction, both in ion-sufficient

(+ion) and in ion-deficient (-ion) thylakoid membranes

(Fig. 1). Hill activity in case of +ion membranes, and -ion

membranes was found to be 149 and 84, respectively. As

expected, activity of control thylakoid membranes in -ion

membranes was significantly less than the activity of control

thylakoid membranes in +ion membranes. This is because of

the fact that Cl- is required for the optimum activity of PS II.

Effects of oxalate on PS II activity were different in ion-

sufficient and ion-deficient samples. Oxalate (5 mM) could

cause only 7% decrease in PS II rates in +ion samples as

compared to 46% inhibition in -ion samples. Presence of

Cl- probably masked the inhibitory effect of oxalate. In

other words presence of Cl- did not allow oxalate to bind to

the sites on PS II as has been reported in case of other

inhibitory anions like NO2-, HCO2

- (Jajoo and Bharti

1993a, b). Thus for further study, -ion membranes were

used to observe the effects of oxalate independent of Cl-.

In order to investigate the site of action of oxalate on PS II,

DPC ? DCPIP reaction was measured in heat-shocked

thylakoid membranes in the absence and presence of oxalate.

Within the photosynthetic membranes, the donor side of PS

II, i.e., OEC, is thought to be most susceptible to the heat-

induced damage (Yamane et al. 1998; Bukhov and Mohanty

1999). Heat-induced decline in PS II photochemical

efficiency can be restored by the addition of exogenous

electron donor like hydroxylamine or diphenylcarbazide

(DPC). In case of oxygen evolving complex inactivation,

DPC reduces P680+ as a result of electron donation through

YZox (Ghirardi et al. 1996). Rates obtained in heat-treated

thylakoid membranes were 3–5% of control values sug-

gesting that the OEC was almost completely inactivated in

heat-treated thylakoid membranes. Effects of anion were

observed equally well in heat-shocked thylakoids that are

known to have lost the ability to evolve oxygen and so the

action of anions was suggested to be on the acceptor side of

PS II (Jajoo and Bharti 1993b). However in case of oxalate,

inhibition of PS II activity was not observed in

DPC ? DCPIP reaction (Table 1). This suggested that

oxalate probably inhibits PS II at a site before DPC donation

site. Binding of oxalate (100 mM) on the acceptor side of PS

II at non-heme iron was suggested (Petrouleas et al. 1994).

However, we could not observe an acceptor side effect of

oxalate in our samples at oxalate concentration upto 20 mM.

Fluorescence induction kinetics at room temperature

Chl a fluorescence induction kinetics was measured in +ion

and -ion thylakoid membranes. Fluorescence induction

curves are characterized by Fo which is minimum fluores-

cence level having QA in the maximally oxidized form, and

0 5 10 15 2040

60

80

100

120

140

160

µ m

ol D

CP

IP r

educ

ed m

gChl

-1 h

-1

Oxalate Conc.(mM)

Fig. 1 Change in the PS II electron transport rates (H2O ? DCPIP

reaction) in the presence of oxalate in (d) ion-sufficient; (o) ion-

deficient thylakoid membranes. The reaction mixture for ion-

sufficient thylakoid membranes contained 0.33 M sucrose, 50 mM

HEPES–NaOH buffer (pH 7.6), 1 mM NaCl, 1 mM MgCl2 and

thylakoid suspension equivalent to 10 lg of chl/ml. The reaction

mixture for PS II activity measurement in ion-deficient thylakoid

membranes contained 0.33 M sucrose, 50 mM HEPES–NaOH buffer

(pH 7.6), and thylakoid suspension equivalent to 10 lg of chl/ml. The

thylakoids were incubated for 10 min in the dark. All experiments

were performed three times in triplicate

Photosynth Res

123

Fm, maximum fluorescence level with QA in the maximally

reduced form (QA-). The difference in fluorescence at Fo and

Fm defines the variable fluorescence, Fv. The yield of fluo-

rescence is regulated by the redox state of QA, which is a

quencher of fluorescence in the oxidized form (Shinkarev

and Govindjee 1993). The ratio Fv/Fm relates to the quantum

yield of PS II primary photochemistry, i.e., reduction of QA.

The fluorescence induction curves with different con-

centrations of oxalate in -ion thylakoid membranes are

shown (Fig. 2) and Fv/Fm, Fv/Fo ratios were calculated

from these traces (shown in inset). In +ion thylakoid

membranes, the Fv/Fm ratio was 0.753 (data not shown)

while in -ion thylakoid membranes this ratio was 0.545,

indicating towards decreased PS II photochemistry in -ion

samples, as was also evident from decreased Hill activity

(Fig. 1). Concentration dependent decrease in the Fv/Fm

ratios was observed in the presence of oxalate. About 34%

decrease in the Fv/Fm ratio was found with 5 mM oxalate.

Decrease in Fv/Fm ratio indicates less quantum efficiency

of the reaction center. This suggested that the inhibition

caused by oxalate was on the donor side of PS II and hence

less electrons reached to QA resulting in less accumulation

of QA-. Oxalate (5 mM) decreased the Fv/Fo ratio by about

43%. A decrease in Fv/Fo ratio is an indicator of structural

alterations in PS II (Havaux and Lannoye 1985). The Fv/Fo

ratio also reflects the efficiency of electron donation to PS

II reaction center (Skorzynska and Baszynski 2000) and the

rate of photosynthetic quantum conversion at PS II reaction

center (Babani and Lichtenthaler 1996). Thus a decrease in

the Fv/Fm ratio and Fv/Fo ratio in the oxalate-treated

samples supported the contention that oxalate affected

mainly the donor side of PS II.

Time-resolved EPR measurements

Light-induced yield and decay kinetics of tyrosine radical

anion (YZ• ) was measured to further explore the role of

oxalate on the donor side of PS II. Electron transfer

between the OEC and P680 is mediated by a tyrosine

residue, YZ• , which is a tyrosine-161 of the D1 polypeptide.

A second redox active tyrosine YD• has been identified as

tyrosine-161 of the D2 polypeptide (Debus et al. 1988). YZ•

and YD• EPR signals can be distinguished on the basis of

their decay rates as YD• is typically present as a dark stable

neutral radical and takes several minutes to decay at room

temperature. On the contrary, YZ• decays very fast in oxy-

gen evolving PS II, in a time range faster than 1 ms at room

temperature (Hoganson and Babcock 1988).

The light-induced EPR signal of tyrosine radical

includes the YD• as well as the YZ

• component. The EPR

spectrum of YZ• is normally measured as the light minus

dark difference spectrum after a relatively short dark time,

and hence the spectrum due to YD• , which is relatively

stable in dark, can be subtracted. Figure 3 shows the EPR

signal of tyrosine recorded in dark (a) and after irradiation

(b). The difference spectrum (c) shows the YZ• spectrum

recorded in oxalate-treated thylakoid membranes. The

arrow indicates the position of magnetic field fixed for the

kinetic experiments. At room temperature the decay of YZ•

is very fast and difficult to observe and hence we measured

the EPR signal of YZ• at 253 K. In normal oxygen evolving

PS II membranes (untreated), the decay of YZ• is too fast to

Table 1 Change in the PS II electron transport rates (DPC ? DCPIP

reaction) in heat-treated thylakoid membranes

Treatment PS II activity (lmol DCPIP

reduced mg chl-1 h-1)

Normal thylakoid membranes 84 ± 2

Heat-treated (HT) thylakoids 4 ± 0.1

HT + DPC 70 ± 1 (100)

HT + DPC + 5 mM oxalate 68 ± 1 (97)

HT + DPC + 10 mM oxalate 67 ± 1 (96)

HT + DPC + 20 mM oxalate 67 ± 1 (96)

The reaction mixture for PS II activity measurement contained

0.33 M sucrose, 50 mM HEPES–NaOH buffer (pH 7.6), and thyla-

koid suspension equivalent to 10 lg of chl/ml. The thylakoids were

incubated for 10 min in the dark. Heat treatment was performed as

follows: thylakoids containing 1 mg chl ml-1 were heated at 45�C for

5 min in the dark with continuous gentle shaking. Heat-treated thy-

lakoids were then immediately cooled in an ice bath and kept in dark

until further use. Diphenylcarbazide (DPC, 2 mM) was used as an

electron donor for measuring the DPC ? DCPIP reaction. All

experiments were performed three times in triplicates. Values in

parenthesis show the normalized values

25

30

35

40

45

50

55

Flu

ores

cenc

e In

tens

ity, r

el. u

n.

Time (sec)

0.350.400.450.500.550.600.650.700.750.80

Flu

ores

cenc

e In

tens

ity

Oxalate Conc.(mM)

Fv/FmFv/F0

50 10 15 20

1E-5 1E-4 1E-3 0.01 0.1 1

Fig. 2 Room temperature chl a fluorescence induction curves in -

ion oxalate-treated thylakoid membranes. (j) control; (d) 5 mM

oxalate; (m) 10 mM oxalate. The reaction mixture contained 0.33 M

sucrose, 50 mM HEPES–NaOH buffer (pH 7.6), and thylakoid

suspension equivalent to 10 lg of chl/ml. The experiment was

performed three times in triplicates. The inset shows the changes in

Fv/Fm (D) and Fv/Fo (m)

Photosynth Res

123

be measured even at 253 K. However, as shown in Fig. 4,

oxalate treatment led to an increase in the light-induced

intensity of YZ• and slowed down the rate of biphasic decay

of YZ• thus increasing the life time of YZ

• decay. The t1/2 of

decay of YZ• in 5 mM oxalate-treated PS II was found to be

1.10 s (for fast phase) and 227 s (for slow phase), while in

10 mM oxalate-treated PS II it was 1.39 s (for fast phase)

and 230 s (for slow phase).

In the case of active, oxygen evolving PS II, YZ is not

only rapidly oxidized by P680+, but the oxidized YZ• is in

turn, rapidly reduced by water molecules through the

turnover of the S-states. Only when the electron transfer

pathways from the OEC is blocked and PS II looses its

oxygen evolution ability, the reduction of YZ• become much

slower. If electron transfer is affected between Mn cluster

and YZ• , decay of YZ

• would be controlled by QA-. Due to

electron vacancy in QB, recombination between QA and YZ•

would be slowed and intensity of YZ• would be high (Jajoo

et al. 2006). Increase in the light-induced amplitude of YZ•

and decrease in the rate of decay of YZ• further suggested a

possible binding of oxalate on the OEC. This was further

confirmed by measurements of S2 state multiline signal in

oxalate-treated PS II membranes.

S2 state multiline (ML) EPR signal

S2 state is characterized by a S2 state multiline (ML) signal

and a g = 4.1 signal. The spectral characteristics of the ML

signal reflects the strength with which the Mn ions of the Mn

complex are coupled with one another (Miller and Brudwig

1991). In +ion PS II membranes, a large ML signal between

2500 and 4000 G and a g = 4.1 signal is observed between

1200 and 2200 G (Fig. 5). In -ion PS II membranes, about

35% decrease in the amplitude of ML signal intensity was

observed. Oxalate induced further decrease in the amplitude

of the ML signal in -ion PS II membranes confirming that

oxalate binds at the donor side of PS II.

sign

al in

tens

ity, r

el.u

n.

3340332033003280

(c)

(b)

(a)

Magnetic Field (G)

Fig. 3 EPR spectrum for tyrosine radical anions recorded at 253 K in

oxalate-treated thylakoid membranes. (a) Spectrum recorded in dark

(b) after illumination (c) difference spectrum of illuminated minus

dark spectra. The arrowhead indicates the position of the magnetic

field fixed for the kinetic experiments. Experimental conditions:

microwave frequency 9.31 GHz, microwave power 2 mW, modula-

tion amplitude 5 G

sign

al in

tens

ity, r

el.u

n.

10 mM Ox

5 mM Ox

control

642Time [min]

Fig. 4 EPR traces of the decay kinetics of YZ• in oxalate-treated

thylakoid membranes at 253 K. Experimental conditions: microwave

frequency 9.31 GHz, microwave power 2 mW, modulation amplitude

5 G. The magnetic field was fixed at 3300 G for the kinetic

experiments. (a) control; (b) 5 mM oxalate; (c) 10 mM oxalatesi

gnal

inte

nsity

, rel

.un.

10 mM Ox

5 mM Ox

-ion

+ion

Magnetic field (Gauss)5000400030002000

g = 4.1

Fig. 5 S2 state EPR spectra in oxalate-treated PS II membranes. (a)

+ion sample; (b) -ion sample; (c) 5 mM oxalate; (d) 10 mM oxalate.

The EPR spectra are presented as light minus dark difference spectra.

Experimental settings: temperature 6 K, microwave power 2 mW,

microwave frequency 9.417, modulation amplitude 16 G. Chlorophyll

concentration used was 3–4 mg/ml

Photosynth Res

123

Effects of oxalate on PS I

A concentration response of oxalate on PS I activity was

measured. PS I rates were inhibited significantly in the

presence of oxalate (Table 2). There was about 20% inhi-

bition in PS I activity with 5 mM oxalate. To precisely

locate the site of action of oxalate on PS I, PS I mediated

partial electron transfer chain reactions (DCPIPH2 ? MV,

TMPD(red) ? MV, and DAD(red) ? MV) were measured

(Table 3), where the addition of 5 mM oxalate caused

17%, 40% and 36% inhibition, respectively. DAD(red) and

TMPD(red) are known to donate electrons near cytochrome

f in the electron transport chain. DCPIPH2 donate electrons

at two sites, one directly to P700 and the other via plas-

tocyanin (Jajoo and Bharti 1993a, b).

In order to pinpoint the site of action of oxalate on PS I,

we carried out the above mentioned partial reactions in the

presence of mercuric chloride (HgCl2). Mercury specifi-

cally inhibits PS I rates by binding to plastocyanin as well

at the acceptor side (FA/FB). In the presence of HgCl2,

62%, 78%, and 84% inhibition in DCPIPH2 ? MV,

TMPD(red) ? MV, and DAD(red) ? MV reactions were

observed, respectively. On addition of oxalate and HgCl2together, predominant effects of oxalate were observed

(Table 3). In Table 3, we observe that the donor systems

give different maximum rates, whereas in the presence of

oxalate (5 mM), all donor systems give about the same

rates. This suggests that oxalate imposes the same rate

limiting step regardless of the donor. It implies that oxalate

works on the acceptor or reducing side of PS I. Oxalate

could form insoluble salt with HgCl2 thus preventing

inhibition caused by HgCl2. However, this possibility is

ruled out as according to standard chemical data both

mercury chloride (Ksp = 1.4 9 10-18) and mercury oxa-

late (Ksp = 1.7 9 10-13) have very low solubility. Steady

state measurements of photo-induced absorbance changes

at 700 nm related to photoaccumulation of the oxidized

primary donor of PS I were carried out using purified PSI

particles. If we assume a monophasic decay in all cases and

take the half-time value on X-axis, then as shown in Fig. 6,

rate of re-reduction of P700+ is a little slower in oxalate-

treated (5 mM) PS I particles as compared to control. The

back reaction of dark reduction of preliminary primary

electron donor of PS, chlorophyll P700, is characterized by

a half-life of 11.0 ± 0.5 s in the absence of any other

additions, while it was 15.0 ± 0.5 s and 25.7 ± 0.5 s in

the presence of 5 mM and 10 mM oxalate respectively.

Rate of reduction of preliminary primary electron donor of

PS I, chlorophyll P700, was 0.91 9 10-3 mol mg-1 h-1

in the absence of other additions, while it was 0.67 9

10-3 mol mg-1 h-1 and 0.39 910-3 mol mg-1 h-1 in the

presence of 5 mM and 10 mM oxalate, respectively.

Comparison of this data with Table 3 suggests that the

main effect of 5 mM oxalate is on the reducing side of PS

I, but higher concentration of oxalate (10 mM) may also

start to affect the oxidizing side as well.

This study reports for the first time that: (i) unlike all

other anions reported so far, only oxalate inhibits both PS I

and PS II, (ii) oxalate shows its inhibitory effects on PS I

and PS II only when exogenous chloride is not present in

the medium, (iii) the site of action of oxalate is on the

Table 2 Changes in the PS I mediated electron transport rate

(DCPIPH2 ? MV reaction) in the presence of oxalate

Treatment PS I activity (lmol oxygen

consumed mg chl-1 h-1)

Control 409 ± 5 (100)

1 mM oxalate 384 ± 6 (94)

5 mM oxalate 339 ± 5 (83)

10 mM oxalate 339 ± 7 (83)

20 mM oxalate 331 ± 5 (81)

The reaction mixture for PS I measurement contained 0.1 M Sucrose,

20 mM HEPES–NaOH buffer (pH 7.6), 3 mM ascorbate, 5 lM

DCMU, 0.1 mM DCPIP, 0.1 mM methyl viologen, 5 lM sodium

azide and thylakoid suspension equivalent to 20 lg chl/ml. All

experiments were performed three times in triplicates. Values in

parenthesis show the normalized values

Table 3 Changes in various partial reactions mediated by PS I in the presence of oxalate and HgCl2

Treatment PS I activity (lmol oxygen consumed mg chl-1 h-1)

DCPIPH2 ? MV TMPD(red) ? MV DAD(red) ? MV

Control 409 ± 5 (100) 617 ± 16 (100) 502 ± 8 (100)

5 mM oxalate 339 ± 9 (83) 376 ± 4 (60) 322 ± 12 (64)

10 nmol HgCl2 155 ± 8 (38) 136 ± 4 (22) 75 ± 6 (16)

5 mM oxalate + 10 nmol HgCl2 327 ± 9 (78) 364 ± 3 (59) 274 ± 12 (61)

The reaction mixture for PS I measurement contained 0.1 M Sucrose, 20 mM HEPES–NaOH buffer (pH 7.6), 3 mM ascorbate, 5 lM DCMU,

0.1 mM methyl viologen, 5 lM sodium azide and thylakoid suspension equivalent to 20 lg chl/ml. DCPIPH2, TMPD(red) and DAD(red) were

used as electron donors. Concentrations of DCPIPH2, DAD and TMPD were 0.1 mM, 0.05 mM and 0.05 mM, respectively. Values in paren-

thesis show normalized values

Photosynth Res

123

donor side of PS II, (iv) Oxalate inhibits PS I by binding to

donor as well as the acceptor side of PS I.

Acknowledgment This work was supported by the Indo-Russian

Joint project INT/ILTP/B-6.27 and by grants from the Russian

Foundation for Basic Research.

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