The action of oxygen on chlorophyll fluorescence quenching and absorption spectra in pea thylakoid...

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Journal of Photochemistry and Photobiology B: Biology 77 (2004) 79–92

The action of oxygen on chlorophyll fluorescence quenchingand absorption spectra in pea thylakoid membranes under the

steady-state conditions

Maciej Garstka *, Patrycja Nejman 1, Małgorzata Rosiak

Department of Metabolic Regulation, Institute of Biochemistry, Faculty of Biology, Warsaw University, Miecznikowa 1, PL-02-096 Warszawa, Poland

Received 19 December 2003; received in revised form 21 August 2004; accepted 24 August 2004

Available online 22 October 2004

Abstract

The effect of oxygen concentration on both absorption and chlorophyll fluorescence spectra was investigated in isolated pea thyl-

akoids at weak actinic light under the steady-state conditions. Upon the rise of oxygen concentration from anaerobiosis up to 412 lMa gradual absorbance increase around both 437 and 670 nm was observed, suggesting the disaggregation of LHCII and destacking of

thylakoids. Simultaneously, an increase in oxygen concentration resulted in a decline in the Chl fluorescence at 680 nm to about 60%

of the initial value. The plot of normalized Chl fluorescence quenching, F(�O2)/F(+O2), showed discontinuity above 275 lM O2,

revealing two phases of quenching, at both lower and higher oxygen concentrations. The inhibition of photosystem II by DCMU

or atrazine as well as that of cyt b6f by myxothiazol attenuated the oxygen-induced quenching events observed above 275 lM O2,

but did not modify the first phase of oxygen action. These data imply that the oxygen mediated Chl fluorescence quenching is par-

tially independent on non-cyclic electron flow. The second phase of oxygen-induced decline in Chl fluorescence is diminished in thyl-

akoids with poisoned PSII and cyt b6f activities and treated with rotenone or N-ethylmaleimide to inhibit NAD(P)H-plastoquinone

dehydrogenase. The data suggest that under weak light and high oxygen concentration the Chl fluorescence quenching results from

interactions between oxygen and PSI, cyt b6f and Ndh. On the contrary, inhibition of non-cyclic electron flow by antimycin A or

uncoupling of thylakoids by carbonyl cyanide m-chlorophenyl hydrazone did not modify the steady-state oxygen effect on Chl flu-

orescence quenching. The addition of NADH protected thylakoids against oxygen-induced Chl fluorescence quenching, whereas in

the presence of exogenic duroquinone the decrease in Chl fluorescence to one half of the initial level did not result from the oxygen

effect, probably due to oxygen action as a weak electron acceptor from PQ pool and an insufficient non-photochemical quencher. The

data indicate that mechanism of oxygen-induced Chl fluorescence quenching depends significantly on oxygen concentration and is

related to both structural rearrangement of thylakoids and the direct oxygen reduction by photosynthetic complexes.

� 2004 Elsevier B.V. All rights reserved.

Keywords: Absorption changes; NAD(P)H-plastoquinone dehydrogenase; Oxygen; Pea thylakoids; Steady-state chlorophyll fluorescence quenching;

Plastoquinone pool; PSII inhibition

1. Introduction

Changes in absorption and chlorophyll a (Chl) fluo-

rescence reflect changes in properties and structure of

1011-1344/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jphotobiol.2004.09.001

* Corresponding author. Tel.: +48 22 5543215; fax: +48 22 5543221.

E-mail address: [email protected] (M. Garstka).1 Present address: Department of Pneumology, Medical University

of Warsaw, PL-02-097.

thylakoid membranes. In isolated chloroplasts Chl fluo-

rescence yield depends on: (i) equilibrium between redox

state of the first PSII quinone acceptor (QA) and plasto-

quinone (PQ) pool [1,2], (ii) the extent of thylakoid

membranes stacking [3,4], (iii) redistribution of excita-

tion energy between PS I and PSII [5], (iv) singlet–singletexcitation transfer to carotenoids [6], (v) LHCII aggre-

gation induced by the acidification of thylakoid lumen

mailto:[email protected]

80 M. Garstka et al. / Journal of Photochemistry and Photobiology B: Biology 77 (2004) 79–92

and violaxanthin deepoxidation [7]. In addition, other

quenchers like membrane energization [7] and effect of

donor side of PSII (cf. [2]) have to be taken into consid-

eration. Moreover, the light-induced LHCII aggregation

may occur upon detaching of these complexes from PSII

[8], suggesting that fluorescence quenching takes placeindependently at both PSII and LHCII. It seems likely

that in thylakoid membranes the change in Chl fluores-

cence yield is the common event of stacking, photosys-

tems segregation, transthylakoid pH gradient and

LHCII aggregation [4]. In fact, these phenomena are

closely related to each other, e.g. dissagregation of

LHCII-containing microdomains is associated with

destacking of thylakoid membranes [4,9].The reduction/oxidation ratio of PQ pool effect on

chlorophyll fluorescence depends on: (i) non-cyclic elec-

tron flow from PSII to stromal acceptors as well as

molecular oxygen, (ii) cyclic electron transport around

PSI and (iii) chlororespiration, including dark reduction

of PQ by endogenous reductants and its oxidation by

molecular oxygen [10,11]. The existence of two different

pathways of cyclic electron flow was suggested [12]: (i)an antimycin-sensitive one, requiring probably ferre-

doxin and hypothetical ferredoxin-plastoquinone reduc-

tase (FQR) [13,14], and (ii) a route including the

chloroplast NAD(P)H-plastoquinone dehydrogenase

(Ndh) activity [12,14].

The direct oxygen reduction by PSI in intact chloro-

plasts, the Mehler reaction [15], is closely associated

with superoxide dismutase and ascorbate peroxidaseactivities, which detoxify superoxide and hydrogen

peroxide in sequential reactions producing

monodehydroascorbate (MDA) andwater [15,16]. Ferre-

doxin-dependent reduction of MDA by monodehydro-

ascorbate reductase completes these reactions in

water–water cycle, which is thought to involve 30% of to-

tal non-cyclic electron flow [16]. However, this reaction is

likely favored at extreme electron supply, i.e. at highlight, due to competition for other stromal electron

acceptors, e.g. NADP+ or oxaloacetate [17].

Reduction of oxygen in chloroplasts can be performed

not only at PSI acceptor side but probably also at PQ oxi-

dase [10,11]. It was suggested that oxidation of plastoqu-

inol under both dark and light conditions occurs via

chlororespiration pathway [10,18,19]. However, as chlo-

rorespiration complexes involved in PQ reduction havebeen recognized [11,13,14,20], the nature of enzyme par-

ticipating in PQ oxidation remains under debate

[10,18,21]. This plastid terminal oxidase (PTOX) activity

seems to be linked to both carotene desaturation and

chlororespiration [22,23]. Contribution of cyt b559[21,24] andperoxidase [18] to dark plastoquinol oxidation

has been suggested. Moreover, the non-enzymatic mech-

anism of PQ oxidation has been recently proposed [25].The relationship between a light-induced shrinkage

of chloroplasts, oxygen concentration and light quality

had been observed many years ago [26,27]. Recently,

following oxygen treatment of isolated pea chloroplasts

a decline in excitation of chlorophyll fluorescence

accompanied by an increase in absorbance around

435, 470 and 674 nm was observed [28]. Moreover,

the kinetics of light-induced chlorophyll a fluorescencequenching in aggregates of LHCII was sensitive to the

oxygen presence [29], while the violaxanthin photo-

isomerization at the weak light closely associated with

structural rearrangement of LHCII was detected exclu-

sively in the presence of oxygen [30].

The oxygen concentration in chloroplast suspension

is proportional to O2 partial pressure in atmosphere

and amounts 256–275 lM O2 [31,32], while in thylakoidlipid phase it is 20% higher than in water phase and

amounts to about 300 lMO2 [25,33]. Furthermore, oxy-

gen concentration, depending on photosynthetic activity

and environmental conditions, may control the distribu-

tion of electrons between the cyclic and noncyclic elec-

tron pathways [15,17,31]. Both participation of oxygen

in electron transport [15,17] and structural changes of

thylakoid membranes [15,28,30] have been proved, butthe relationships between these phenomena are far from

the complete understanding. Hence, the aim of the pre-

sent investigation was to study these two events by

measuring of both absorbance and chlorophyll fluores-

cence in isolated thylakoids at various oxygen concen-

trations increasing from anaerobiosis up to 412.5 lMO2 at weak light conditions. The experiments were per-

formed under the steady-state conditions, i.e. when thefinal equilibrium between oxygen concentration and

both PSII-driven reduction of PQ pool and the level of

reduced centers of photosynthetic complexes is estab-

lished. Hence, the fluorescence and absorbance changes

reflect the oxygen effect on membrane components un-

der weak light conditions which limit the rate of photo-

synthesis [15]. In view of the data presented one might

conclude that the oxygen-induced Chl fluorescencequenching is partially independent on non-cyclic elec-

tron flow and may be related to structural rearrange-

ment of thylakoid membranes. The contributions of

PSI, cyt b6f and Ndh to oxygen-induced quenching phe-

nomena under the steady-state conditions at weak light

are discussed.

2. Materials and methods

2.1. Plant material and thylakoids preparation

The pea (Pisum sativum L. cv. Baron) plants were

grown in perlite-containing pots in the controlled envi-

ronment at 24 �C/20 �C day/night under 16 h photope-

riod with irriadiance of 80 lmol m�2 s�1. Plants werefertilized with diluted Knop�s solution. Leaves of 14

days old seedlings were homogenized in 330 mM

M. Garstka et al. / Journal of Photochemistry and Photobiology B: Biology 77 (2004) 79–92 81

sorbitol, 20 mM tricine–NaOH (pH 7.5), 15 mM NaCl,

4 mM MgCl2 and 40 mM ascorbate. The homogenate

was filtered and centrifuged at 2000g for 5 min to obtain

chloroplasts. Thylakoids were prepared from chloro-

plasts by osmotic shock in the medium containing 20

mM tricine–NaOH (pH 7.0), 15 mM NaCl and 4 mMMgCl2 and centrifugation at 6000g for 10 min. The thyl-

akoids were resuspended in the medium of 330 mM

sorbitol, 20 mM Hepes–NaOH (pH 7.0), 15 mM NaCl

and 4 mM MgCl2. Following the second centrifugation

at 6000g for 10 min the pellet was resuspended in the

above buffer and kept on ice in dark. Thylakoids were

always freshly prepared before experiments. Chloro-

phyll concentration was quantified after extraction with80% acetone [34].

2.2. Preparation of samples

Samples for spectroscopic assays were prepared in

buffer used for resuspending of thylakoids, but pH was

adjusted to 7.5. All procedures were performed at

25 �C in 4 ml closed cuvettes filled completely with reac-tion mixture. The thylakoids suspension and reagents

were added using Hamilton syringe through 0.5 mm

hole made in teflon plug. Anaerobic conditions were

achieved by the addition of 1 mM glucose, glucose oxi-

dase (18.4 U/ml) and catalase (3600 U/ml) to the nitro-

gen washed-medium 5 min before the thylakoids were

included. The increased concentrations of O2 were

achieved by sequential addition of appropriate amounts(2–8 ll) of 1% H2O2 under dark conditions. A relation-

ship between amount of H2O2 and O2 concentration was

estimated with Clark electrode. In control conditions no

artificial electron acceptors were added. Inhibitors and

electron acceptors were dissolved either in ethanol (anti-

mycin, atrazine, DCMU, DBMIB, myxothiazol, rote-

none) or in water (MV, NEM). No effect of ethanol

on absorbance and fluorescence values was observed atused ethanol concentration (up to 1%). DQ was reduced

to DQH2 following the treatment with NaHB4 immedi-

ately before use [35]. The inhibitors affected significantly

neither glucose oxidase and catalase activities nor the

oxygen concentration.

2.3. Determination of absorbance and fluorescence emis-

sion spectra under the steady-state conditions

Absorption spectra were measured with the use of

D-600 Beckman spectrophotometer whereas fluores-

cence excitation spectra were determined with the Shim-

adzu RF-5301PC spectrofluorometer. The scans were

recorded for each 0.1 and 0.2 nm and the mean accumu-

lation times were 300 and 70 s for absorbance and fluo-

rescence spectra, respectively. Fluorescence emissionspectra were recorded with the excitation and emission

slit set to 3 and 10 nm, respectively. Samples were illumi-

nated with probing light of spectrofluorometer at 435

nm (36 lmol m�2 s�2). Changes of fluorescence were

measured from 630 to 760 nm with magnetical stirring

to prevent thylakoids settling. For absorbance and fluo-

rescence measurements the thylakoids suspensions were

diluted to concentration of 6 lg Chl ml�1.Thylakoids were incubated for 2–8 min with indi-

cated oxygen concentration in darkness, i.e. under con-

ditions sufficient to establish the constant oxygen

concentration. Samples were scanned thrice for each

measurement, i.e. under conditions necessary to attain

the invariable absorbance or fluorescence level. Time

of illumination at relative weak light was sufficient to in-

duce a reversible Chl fluorescence quenching [8,30], butdid not cause the Chl degradation. No significant

changes in oxygen concentration during period of thyl-

akoid illumination were observed in agreement with pre-

vious observations [25,31]. The changes in oxygen

concentration related to both O2 evolution in PSII and

reduction by PSI or plastoquinone pool were negligible

[25]. The first and the second absorbance scans were car-

ried out to eliminate light-dependent changes in theabsorbance amplitude. The third scan, which did not

show significant changes in comparison with the second

scan, was used to estimate the oxygen action. In order to

evaluate fluorescence measurements the second and the

third scans were used to obtain an average maximum

emission spectrum of thylakoids. The differences in the

absorbance or fluorescence between the first scan and

subsequent one did not exceed 1.5% and 1% (at 437and 680 nm), respectively. This procedure made us able

to obtain a steady-state equilibrium between each oxy-

gen concentration and redox state of membrane compo-

nents at weak light illumination.

2.4. Analysis of absorbance and fluorescence changes

The spectrum for anaerobic conditions was taken as areference one in calculations of the difference spectra.

The maxima of fluorescence emission spectra at 680 nm

for anaerobiosis and appropriate oxygen concentration

were utilized for the calculation of the oxygen-induced

quenching curves. Ratio of fluorescence in the absence

and presence of oxygen, F(�O2)/F(+O2), as a function

of the O2 concentration was presented as a normalized

fluorescence quenching, according to the principle ofquenching analysis [2]. Linear segments of the plot were

calculated as the best fitted simple or pair of regression

lines. For samples treated with exogenous chemicals

the F(�O2) values were determined under anaerobic con-

ditions in the presence of appropriate agent.

Inhibitors were added to cuvettes under anaerobic

conditions following the first fluorescence measurement.

Oxygen treatment affected the spectral properties of glu-cose oxidase/catalase in the UV absorbance region,

while fluorescence emission spectra were free of


significant interferences. The corresponding difference

absorbance enzyme spectra were subtracted from the

thylakoid absorbance ones.

3. Results and discussion

3.1. Effect of oxygen concentration increase on thylakoid

absorbance spectra

As the increase of absorbance after transfer of chlo-

roplasts from anaerobic (6 nM O2) to air-saturation

conditions (�250 lM O2) was recently established [28],

in the present study a gradual elevation of thylakoidabsorption induced by increasing oxygen concentrations

from anaerobiosis to 412.5 lM O2 concentration was

investigated (Fig. 1(a)). Difference spectra of the scans

Fig. 1. Effect of oxygen concentration on difference absorbance spectra (a) an

in isolated pea thylakoids. (a) shows absorbance difference spectra for thylako

were normalized at 540 nm. Equilibrium with desidered oxygen concentratio

shows the absorbance difference spectrum recorded following restoration

concentration. Spectrum was normalized at 400 nm. The spectra are represe

(with anaerobic spectrum taken as the reference one)

yielded positive broad band at 437 with shoulders at

470 and 480 nm and positive peak at 670 nm, while a

small negative band was detected at around 700 nm.

The Chl a difference absorbance bands were shifted to

blue region in comparison with those at 439 and 681nm revealed in the anaerobic spectrum. However, ampli-

tudes of the Soret band transitions at 437 nm rised with

the oxygen concentration increase (Fig. 1(a)). The ab-

sence of negative bands around 436 and 680 nm, charac-

teristic for Chl a irreversible bleaching under strong

illumination at aerobic conditions in both PSII and

PSI reaction centers [36,37], suggests that in the presence

of oxygen (cf. Fig. 1(a)), the absorbance changes are notassociated with degradation of photosynthetic com-

plexes under the steady-state conditions. The small oxy-

gen-induced negative bands around 700 nm recorded for

(a)

(b)

d difference spectrum following restoration of anaerobic conditions (b)

ids sequentially treated with increasing oxygen concentrations. Spectra

n was reached in dark and was followed by scanning of the sample. (b)

of anaerobic conditions versus spectrum recorded at 250 lM O2

ntative for scans obtained for five separate experiments.


increased O2 concentration may be related to oxidation

of PSI reaction center [37]. However, this effect is fully

reversible following oxygen removal (Fig. 1(b)).

Oxygen-induced absorption changes might be effec-

tively reversed upon the decrease in O2 concentrations

from 250 lM to 15 nM following nitrogen and glucose(10 mM) treatment. The amplitude of difference spec-

trum of thylakoids (the second anaerobic–aerobic)

(Fig. 1(b)) was reversed in comparison with that for oxy-

gen-treated thylakoids in the Soret region of scans (Fig.

1(a)). However, significant red shifts at 442 nm were ob-

served. In the reversed spectrum both a wide positive

absorbance band between 520 and 640 nm and a sharp

negative one at 681 nm have appeared, whereas respec-tive symmetrical bands were not detected in the oxygen-

induced spectrum (Fig. 1(a)).

Stacking of thylakoids [4,28] and aggregation of iso-

lated LHCII [40] cause a decrease of absorbance in the

Soret region due to an increase in light scattering and

the flattening effect [41]. These scattering changes have

been related to formation of multiaggregates of LHCII

[40,41] and changes in intrinsic thylakoid membranestructure [4]. On the other hand, a weak light-induced

increase in absorption values at 400–520 and 690 nm

is accompanied by a simultaneous decrease in light scat-

tering associated with disaggregation of LHCII com-

plexes [30]. In this case the light-driven isomerization

of violaxanthin under aerobic conditions induces proba-

bly the conformational reorganization of LHCII [30].

An oxygen-induced increase of absorption (cf. Fig.1(a)) is possibly related to this event. The destacking

of thylakoids, also visible as an absorbance increase

[4,28] (cf. Fig. 1(a)), may occur in the aftermath of

light-induced and oxygen-dependent reorganization of

LHCII at trimeric level [9,30]. However, in contrast to

isolated LHCII, in thylakoids the participation of other

membrane components should be also considered. In

pea chloroplasts changes in the excitation spectrum fol-lowing oxygen-treatment are very similar to those in-

duced by Mg2+-depletion [28], suggesting that similarly

to the action of ions, oxygen may induce thylakoids

destacking resulting from a change in the surface mem-

branes potential [3,4,28]. Since oxygen is required for

LHCII kinase activation [42], the influence of oxygen

on LHCII phosphorylation and subsequent thylakoids

destacking is possible in intact chloroplasts.As shown in Fig. 1 following increase or decrease of

oxygen concentration the Chl a absorption bands of pea

thylakoids were reversibly shifted to blue or red, respec-

tively. Furthermore, after oxygen removal the reversion

of bands at 442 and 488 nm to the initial amplitude was

observed (cf. Fig. 1). Similarly, both the red shift of

bands at 680 and 490 nm and the decrease of absorbance

in the Soret region of scan have been observed after theaggregations of LHCII [41]. On the other hand, the

destacking of thylakoid membranes is associated with

the blue shift of bands at 681 and 439 nm [4,28]. Hence,

the red and blue shifts observed at various oxygen con-

centrations suggest changes in both degree of membrane

stacking and aggregation of LHCII complexes. How-

ever, the formed bands (positive at 540–620 and negative

at 681 nm) in the reversed spectrum (cf. Fig. 1(b)) sug-gest that restoration of anaerobic conditions caused

additional changes in thylakoid membranes in compari-

son with those induced by oxygen treatment (cf. Fig.

1(a)). Both increase in steady-state transmittance in the

range from 548 to 575 nm [4] and decrease in absorbance

around 520 nm [28] were observed under cation-depend-

ent moderate thylakoid destacking. Moreover, changes

in absorbance region between 505 and 550 nm are alsoknown to be associated with (i) formation of trans-

thylakoid pH gradient [15,26,39], (ii) electrochromic

shifts of light-harvesting pigments reflecting the trans-

thylakoid electric field [38] and (iii) aggregation of

LHCII [7,39,40]. Since in our experiments the relatively

weak light (36 lmol m�2 s�2) was used, it seems possible

that positive absorbance band between 520 and 640 nm

in reversed spectrum is associated with increasing stack-ing of thylakoid membranes. On the other hand, the

presence of negative band at 680 nm may be related to

positive charge accumulation on thylakoid membranes

[38], suggesting that restoration of anaerobic conditions

also caused changes in membrane surface potential [28].

The results indicate that at weak light and at wide range

of O2 concentrations (from 0.08 to 412.5 lM O2), the

oxygen action is related to substantial, partly reversiblerearrangement of thylakoid membranes, probably

resulting of both disaggregation of LHCII and destack-

ing of thylakoids.

3.2. Relationship between oxygen concentration and Chl

fluorescence quenching

As shown in Fig. 2(a), with increasing oxygen con-centration the maximum fluorescence at 680 nm has de-

creased, while the quenching curve exhibited two phases.

At oxygen concentration equal to the 275 and 412 lMO2, the fluorescence decreased by 15% and 40%, respec-

tively. It should be noted that under the atmospheric

pressure the oxygen concentration at air saturated buffer

in 25 �C corresponds to 256 lM. Above this oxygen con-

centration the normalized fluorescence quenching,F(�O2)/F(+O2), showed the break point and the linear

phase of plot exhibiting slope 6-fold higher than the lin-

ear phase below this point (Fig. 2(b)). A similar decrease

in fluorescence at 680 nm was observed with excitation

at 455, 470 and 490 nm (data not shown).

Two phase dependence of Chl fluorescence on oxygen

concentration (cf. Fig. 2(a)) suggests diverse mecha-

nisms of quenching at low and high oxygen. In the ab-sence of exogenous electron acceptors during

illumination of thylakoids with a weak light, changes

(a) (b)

(c) (d)

Fig. 2. The dependence of chlorophyll fluorescence emission change on the oxygen concentrations in control (a, b) and PSII inhibited (c, d)

thylakoids. (a) and (c) show fluorescence changes measured under anaerobic conditions and at increasing oxygen concentrations for control sample

(solid line, open circles) and following the addition of 30 lM DCMU (dotted line, solid circles) or 30 lM atrazine (dashed line, solid triangles). The

fluorescence quenching (a, c) at each oxygen concentration was calculated upon subtraction of anaerobic value at 680 nm from each aerobic one. The

initial anaerobic fluorescence level of fluorescence in samples before agents addition amounted 338, 365 and 328 arbitrary units (a.u.) for control,

DCMU and atrazine-treated thylakoids, respectively. Under anaerobic conditions a decrease in fluorescence upon DCMU or atrazine addition was

marked on the abscissa axis (a, c). The data are means ± SD for 6, 10 and 5 separate experiments for control, DCMU and atrazine-treated samples,

respectively. The set at two panels has the same arbitrary scale. (b) and (d) show ratios of the fluorescence values measured in the absence and

presence of oxygen, F(�O2)/F(+O2), as a function of oxygen concentration, for control (b) and DCMU- or atrazine-treated thylakoids (d). The

fluorescence value in presence of inhibitors under anaerobic conditions was taken as a initial fluorescence level F(�O2). Control curves are added to

(c) and (d).


in Chl fluorescence intensity are dependent on the redox

state of QA and equilibrium between QA and PQ pool

[1,2,43]. The subsequent dark incubation under aerobicconditions caused the reoxidation of PQH2 by molecular

oxygen [2], probably by both enzymatic [23,24] and

autocatalytic processes [25]. However, the degree of

PQ reduction is dependent on light conditions and thyl-

akoid structure. In chloroplasts QA is reduced by 10%

when the PQ pool is reduced by 50% and achieves

50% of reduction when PQ is reduced by 90% [1]. In de-

stacked thylakoids nearly all PQ becomes reduced by thesaturating light, while in stacked membranes about 50%

of PQ remains oxidized due to microdomain structure of

thylakoids and restricted PQ diffusion [4]. On the other

hand, the PQ pool contributed only to 25% of the high

light-induced non-photochemical quenching phenomena

[44]. In contrary, under the weak light the maximum flu-

orescence level was achieved during few minutes of illu-

mination [43]. According to this finding [1,4,43] it islikely that under the weak light applied only the part

of PQ pool became reduced (cf. Fig. 2(a)). An efficient

oxygen uptake associated with PQ oxidation was

observed at relatively high light [25], suggesting that at

weak light this reaction can be limited. Moreover, under

the illumination of samples no significant changes in

oxygen concentration were observed (not showed).

These data suggest that at weak light oxidation of PQpool is not the only cause responsible for oxygen-

dependent Chl fluorescence quenching.

In Mehler reaction the Km for oxygen achieves 75–

100 lM in leaves [16], but in the ferredoxin supplied

thylakoids it amounts to 60 lM (cf. [17], for review).


The affinity of ferredoxin-free PSI preparation to oxy-

gen is high (Km is equal to 3–10 lM) (cf. [17], for re-

view), so under these conditions (cf. Fig. 2(a)) oxygen

reduction is not limited in isolated thylakoids. However,

the break point in normalized fluorescence quenching

(cf. Fig. 2(b)) is considerably above the cited Km values,suggesting that under the weak light the oxygen-induced

decrease in Chl fluorescence does not derive solely from

oxygen reduction by PSI.

It has been proposed that in chloroplasts the oxygen-

dependent decrease in Chl excitation [28] as well as exci-

tation and emission in LHCII aggregates [30] are related

to structural reorganization of thylakoid membranes and

LHCII trimer, respectively. These effects were observedfollowing the increase in oxygen concentration from 4.7

nM to 272 lMO2. Thylakoid membranes have a high

oxygen permeability [33], but the apoprotein of LHCII

may act as a barrier against O2 transport, protecting

Chl and xanthophylls associated with these complexes

[45]. On the contrary, in isolated LHCII the light-induced

decrease in fluorescence is more pronounced at increased

oxygen concentration, indicating that oxygen presenceenables light-induced isomerization of violoxanthin

followed by structural reorganization of LHCII trimer

[30]. Hence, the weak light-induced dissagregation of

aggregated LHCII [30] facilitates oxygen penetration to

complexes resulting in a chlorophyll fluorescence

quenching.

3.3. Effect of PSII inhibitors on oxygen-induced Chl

fluorescence quenching

To evaluate the contribution of PSII and PQ pool to

oxygen-induced fluorescence quenching we have per-

formed experiments in the presence of atrazine and

DCMU, i.e. under conditions when inhibitors block

Q�A oxidation by QB and prevent reduction of the PQ

pool [2,3]. The addition of inhibitors in the dark underanaerobic conditions followed by the period of continu-

ous illumination caused a decrease in maximum fluores-

cence level by about 5% and 8% for atrazine and

DCMU, respectively (Fig. 2(c)). DCMU quenching

action has been ascribed to a non-photochemical

quenching by oxidized PQ pool and might result from

the binding of plastoquinone molecule to the site other

than QB [2].As shown in Fig. 2(c), at saturated concentrations (30

lM) both atrazine and DCMU diminished the overall

oxygen action by about 30 and 50%, respectively. In

view of the normalized fluorescence quenching (Fig.

2(d)) it is clear that the second phase of steady-state

quenching, emerging above 275 lM oxygen concentra-

tion, is affected by atrazine/DCMU treatment. In thyl-

akoids with inhibited PSII (Fig. 2(d)), oxygenconcentration above 275 lM poorly influenced the fluo-

rescence quenching in contrast to quenching processes

observed in control thylakoids, in which electron dona-

tion to PQ pool and PSI was not prohibited (cf. Fig.

2(b)). These data imply the contribution of factors lo-

cated outside PSII to the second phase of Chl fluores-

cence quenching.

The PSII reaction center poisoned with atrazine/DCMU exists in Z+ P680 Phe Q�

A state and cannot acts

as fluorescence quencher [3]. In this case the Chl fluores-

cence yield may be regulated by structural changes in

thylakoid membranes related to the stacking phenome-

non [3] or Chl quenching in LHCII aggregates [7,8].

The hypothetical DCMU-insensitive oxidation of PSII

endogenous plastoquinone different from QA [2] and/

or PSII oxidation by PQ pool at low potential cyt b559[21] may contribute to oxygen-induced Chl fluorescence

quenching phenomenon. Moreover, at high oxygen con-

centration the difference between DCMU and atrazine

action (cf. Fig. 2(d)) may be related to alternative elec-

tron leakage from PSII [2,21]. The small oxygen uptake

observed in chloroplasts, thylakoids and isolated PSII

particles treated with DCMU [19,25] excludes participa-

tion of QB site in oxygen reduction and suggests that QA

is probably the site of electron donation to oxygen inside

PSII complex. The oxygen may react directly with both

QA producing superoxide [46] and non-haem iron cou-

pled with QA and QB co-factors [47]. Hence, in atra-

zine/DCMU-treated thylakoids oxygen-induced Chl a

fluorescence quenching at low oxygen concentration is

probably due to oxidation of P680 by alternative path-

ways [2,47] and/or structural rearrangements of thylak-oids (cf. Fig. 1(a)) [3,4,28,30].

3.4. Chl fluorescence quenching in view of competition

between exogenous quinones and oxygen

The addition of DQ in the presence of DCMU under

anaerobic condition caused decrease in fluorescence to

about 45% of its initial value. Moreover, the fluores-cence values were practically insensitive to increasing

oxygen concentrations (Fig. 3(a)). Upon the addition

of DQ alone, a decrease in Chl fluorescence under anaer-

obic conditions was higher (Fig. 3(a)) than overall oxy-

gen-induced fluorescence quenching (cf. Fig. 2(a)). In

presence or absence of DCMU the DQ action was sim-

ilar, indicating that the effect of 30 lM duroquinone on

fluorescence yield is independent on QA oxidation state.As shown in Fig. 3(c) DBMIB did not affect oxygen-

induced quenching up to 125 lM O2 (Figs. 3(c) and (d)),

indicating that inhibition of electron flow from PSII to

cyt b6f is negligible compared to oxygen action on Chl

fluorescence quenching. However, above 125 lM O2

concentration a sudden decline of Chl fluorescence was

observed. It should be noted that this effect is twice more

pronounced than the overall oxygen action in the ab-sence of electron acceptor (cf. Fig. 2(a)), and leads to

complete quenching of the Chl fluorescence.

(a) (b)

(c) (d)

Fig. 3. Fluorescence quenching by oxygen in the presence of exogenous quinones. (a) shows fluorescence decrease following the addition of 30 lMDCMU and 50 lM DQH2 (dotted/dashed line, solid triangles), 30 lM DCMU and 50 lM DQ (dashed line, solid inverted triangles) and 50 lM DQ

alone (dropped line, solid squares). The action of 30 lM DBMIB is shown in panel C (dashed line, solid triangles). Before the addition of particular

components initial anaerobic fluorescence level amounted 402, 355, 375 and 368 for DQH2 + DCMU, DQ + DCMU, DQ and DBMIB-treated

thylakoids, respectively. The data are means ± SD for three and four separate experiments with respect to DBMIB and other compounds,

respectively. The arbitrary scale for (a) is the same as in Fig. 2, while the scale for (b) is twice diminished. (b) and (d) show changes in the normalized

fluorescence, F(�O2)/F(+O2), for appropriate samples from (a) and (c), respectively. The control curves from Fig. 2(a) and (b) (solid line, open

circles) are added to figures. The other details are described in the legend to Fig. 2.


Exougenously added quinones, duroquinone (DQ)

and dibromo-3-methyl-6-isopropyl-p-benzoqinone

(DBMIB), are able to act as a Chl a fluorescence quenc-hers. During the dark–light–dark transition the behavior

of these artificial quinones is similar to that ascribed to

endogenous plastoquinones [48], which can be reduced

under light and oxidized under dark conditions [1,2].

DBMIB is considered to act only as PSII electron accep-

tor while DQ shuttles electrons from PSII to PSI being

reduced by PSII and oxidized by the cyt b6f [35,48].

The turnover of oxidized and reduced form of DQ underillumination is possible when the oxygen acts as a termi-

nal electron acceptor [48]. On the other hand, DBMIB

at low concentration (<5 lM) is widely used as inhibitor

of quinol-oxidation site (QO) in the cyt b6f complex

[49,50].

Under aerobic conditions and weak light the plast-oquinol pool cannot be filled up with electrons due to

the photochemical activity of PSI [48] and/or oxygen

reduction inside the PQ pool [25], so DQ can compete

with oxygen for electron donated by PSII [48]. However,

changes in normalized fluorescence quenching (cf. Fig.

3(b)) showed that DQ effects in both the presence and ab-

sence of DCMU and various oxygen concentrations were

similar to those observed under anaerobic conditions.Thus, under steady-state conditions DQ-quenching effect

is related to quinone capability as a direct quencher of

excited Chl (cf. [48], for review). On the other hand,


above 350 lMO2, the oxygen effects on Chl fluorescence

were poorly visible in the presence of DQ, indicating that

DQ prevents the oxygen-induced quenching action due

to higher affinity for excited Chl molecules in comparison

with that for oxygen. Exogenous duroquinol (DQH2) is

an artificial electron donor to PQ pool [35,43] and cantransfer electrons to PSI under illumination [48]. How-

ever, as shown in Fig. 3(b) the effect of simultaneously

added DCMU and DQH2 on oxygen-induced fluores-

cence quenching was not significantly different from that

of DCMU alone (cf. Fig. 1(b)). Thus, it is likely that

reduction of membrane components by DQH2 is rather

of little importance for steady-state oxygen effect in

poisoned thylakoids.A significant effect of DBMIB on Chl fluorescence

may be associated with diaphorase activity of FNR,

which transfers electrons from PQ pool to DBMIB

being subsequently reoxidised by O2 with Km amounted

to 16 lM [49]. Since the role of DBMIB in quenching

phenomena is determined by its photoreduction [48],

the reoxidation of DBMIB by oxygen may prevent the

exhaustion of the quinone and increase the DBMIB effi-cacy on Chl fluorescence quenching. On the other hand,

DBMIB did not affect the fluorescence under anaerobic

conditions (cf. Fig. 3(c)), suggesting that a direct reduc-

tion of DBMIB at QB site of PSII can be ruled out [51].

Thus, it is likely that oxygen above 125 lM plays a sec-

ondary role in Chl fluorescence quenching, essentially

increasing the DBMIB action. Below 125 lM oxygen,

the Chl fluorescence quenching does not result fromcompetition between DBMIB and oxygen.

3.5. Oxygen effect on uncoupled thylakoids

As shown in Fig. 4(a) in a presence of 5 lM CCCP, a

lipid soluble proton carrier, Chl fluorescence was de-

creased by about 20% under anaerobic conditions, indi-

cating the CCCP as efficient Chl fluorescence quencher.Sequential addition of oxygen to uncoupled thylakoids

augmented the decline of the fluorescence quenching

by 40% (Fig. 4(a)) resulting in a decrease in the overall

fluorescence to 40% of the initial level. However, the

plot of oxygen-induced normalized fluorescence quench-

ing with the breakpoint above 275 lM O2 (Fig. 4(b)),

was not significantly different from the control one (cf.

Fig. 2(a)).Both the electron flow toward oxygen and cyclic elec-

tron transport around PSI, which can occur in isolated

thylakoids in the absence of artificial acceptors, increase

the transmembrane proton gradient [15,17]. The DpHplays a regulatory role in photosynthetic electron trans-

port at the level of PQ oxidation or the cyt b6f complex

[15,17]. FCCP, acting as a lipid soluble uncoupler, may

increase the proton availability within membranes, andenhance the dismutation of superoxide generated during

O2 reduction [47]. Moreover, the stimulation of oxygen

uptake by the methylamine and gramicidin D in the ab-

sence of artificial acceptors was observed [25]. The data

presented (cf. Fig. 4(a)) do not exclude the contribution

of oxygen to DpH formation [15,17], but obviously sug-

gest that under the weak light conditions uncoupling of

thylakoids did not influence the oxygen-induced fluores-cence quenching.

3.6. Attenuation of oxygen action in the presence of

NADH

As shown in Fig. 4(a), during the steady-state condi-

tions NADH did not influence the fluorescence level in

pea thylakoids under anaerobic conditions, while it effi-ciently attenuated the oxygen effect on Chl fluorescence

and almost completely prevented the quenching below

275 lM O2 (Fig. 4(b)). Moreover, at oxygen concentra-

tions increased up to 412.5 lM O2 overall NADH utili-

zation (measured at 340 nm) did not exceed 10%. These

data are in agreement with no NADH oxidation in the

absence of exogenous acceptors in thylakoids under aer-

obic conditions [52].The NAD(P)H-dependent reduction of PQ pool

may be catalyzed by Ndh [11,20] and/or rotenone-in-

sensitive Ndh [43], whereas a direct participation of

FNR in PQ reduction is not clearly established

[49,52]. The addition of NADH to chloroplasts evokes

a fluorescence rise and reduces the PQ pool due to

chloroplastic dehydrogenase(s) activities [43]. This phe-

nomenom is more pronounced under anaerobic condi-tions suggesting that in the presence of oxygen a

significant part of electrons entering PQ pool is used

for O2 reduction [43]. Thus, the attenuating effect of

NADH on oxygen-induced Chl a fluorescence quench-

ing in isolated thylakoid membranes probably requires

the Ndh activity [43,52]. The successful prevention of

oxygen-induced Chl fluorescence quenching by NADH

under the weak light is understood in view of theobservation that in isolated intact chloroplasts oxygen

is a weak electron acceptor [17].

3.7. Effect of cytochrome inhibitors on oxygen-induced

Chl fluorescence quenching

As shown in Fig. 4(c), under anaerobic condition

both 30 lM antimycin A and 30 lM myxothiazol de-creased the Chl fluorescence level by 8% and 20%,

respectively. Antimycin A did not influence the oxy-

gen-induced Chl fluorescence decline, whereas the

myxothiazol diminished the overall oxygen action by

30% (Fig. 4(d)). Antimycin A is an efficient inhibitor

of cyclic electron flow at FQR site [13,14]. Both antimy-

cin A and myxothiazol inhibit the cyt bc pathway in the

mitochondrial respiration [14,50], while myxothiazol isgenerally considered as inefficient inhibitor of the cyt

b6f complex [50]. Recently, it has been reported that

(a) (b)

(c) (d)

Fig. 4. Effect of electron donor, uncoupler and cytochrome inhibitors on the oxygen-induced chlorophyll fluorescence quenching. Data in (a) present

the action of 200 lM NADH (dashed/dropped line, solid circles) and 5 lM CCCP (dotted line, solid triangles). They are means ± SD for four

separate experiments. The effects of 30 lM antimycin (dashed line, solid circles) and 30 lMmyxothiazol (dotted line, solid triangles) are shown in (c).

The initial anaerobic fluorescence level amounted 328, 363, 395 and 318 for NADH, CCCP, antimycin and myxothiazol-treated thylakoids,

respectively. The data are means ± SD for four separate experiments. The arbitrary scale is the same as in Fig. 2. (b) and (d) show changes in the

normalized fluorescence, F(�O2)/F(+O2), for appropriate samples from (a) and (c), respectively. The control curves from Figs. 2(a) and (b) (solid

line, open circles) are added figures. The other details are described in the legend to Fig. 2.


the inhibition capacity of cyt b6f inhibitors (stigmatellin,

DBMIB, DNP-INT and myxothiazol) depends on light

conditions [42]. In the dark the Qo site in cyt b6f com-

plex might more closely resemble the cyt bc complex

making the myxothiazol binding possible [42]. Since in

the present study inhibitors and oxygen were added inthe dark, and the illumination period needed for fluores-

cence measurent was shorter than the dark period, the

inhibition of electron transfer from PQ pool to cyt b6f

by myxothiazol should be considered.

Data obtained with antimycin A-treated thylakoids

(cf. Fig. 4(c)) suggest that cyclic electron flow does

not play a significant role in establishing the steady-

state equilibrium at various oxygen concentrations. In

view of the normalized fluorescence quenching (cf.

Fig. 4(d)) it is clear that the myxothiazol effect resem-

bles that caused by atrazine and DCMU (cf. Fig. 2(d)).

These data indicate that the second phase of steady-

state quenching, emerging above 275 lM O2 concentra-tion (cf. Figs. 2(d) and 4(b) and (d)), is attenuated by

PSII or cyt b6f inhibition. Hovewer, the effect of anti-

mycin A and myxothiazol cannot be simply explained

by inhibition of electron flow at the cyt b6f level. Since

antimycin A is also known as an inhibitor of LHCII

aggregation [39], and the myxothiazol is probably an

inhibitor of FNR diaphorase activity [53], other, less


specific, sites of inhibition may be responsible for these

phenomena.

3.8. Effect of inhibitors of chlororespiration pathways on

oxygen-induced Chl fluorescence quenching

As shown in Fig. 5(a), N-ethylmaleimide (NEM), an

inhibitor of sulfhydryl groups [56] and a potent inhibitor

of FNR and chloroplastic dehydrogenases [43] caused a

decrease in Chl fluorescence quenching at rather low (60

lM) concentration compared to that reported for inhi-

bition of NAD(P)H oxidation by FNR (3mM) or Ndh

(0.5mM) [43]. NEM did not influence the first phase of

Chl fluorescence quenching, whereas above 275 lM O2

the effect of oxygen was strongly diminished (Fig.

5(b)). The addition of 60 lM NEM in the presence of

30 lM DCMU under the steady-state conditions (Fig.

5A) attenuated the oxygen action in the same way as

NEM or DCMU alone (cf. Fig. 2(c)), indicating that

these inhibitors did not influence the Chl fluorescence

quenching evoked by oxygen concentration increased

up to 275 lM (cf. Fig. 2(b)). In illuminated thylakoids

(a) (b

(c) (d

Fig. 5. Influence of Ndh inhibitors on the oxygen-induced chlorophyll fluores

line, solid triangles) or 30 lM DCMU and 60 lM NEM (dashed line, solid

solid triangles). The initial anaerobic fluorescence level was 374, 383, 381 for

The data are means ± SD for four separate experiments. The arbitrary scale

fluorescence, F(�O2)/F(+O2), for appropriate samples from (a) and (c), res

circles) are added to figures. The other details are described in the legend to

NEM at milimolar concentrations is very effective elec-

tron acceptor from PQ pool [56]. However, at applied

low NEM concentration the acceptor properties of this

inhibitor are visible probably only under anaerobic con-

ditions, when NEM decreases the fluorescence level by

6% (Fig. 5(a)).As shown in Fig. 5(c) in the presence of 60 lM rote-

none, the inhibitor of mitochondrial complex I and chlo-

roplastic Ndh [43], oxygen caused the overall Chl

fluorescence decrease only by 15% in comparison with

40% estimated in control samples (cf Fig. 2(a)). Moreo-

ver, above 275 lM oxygen concentration the Chl fluo-

rescence quenching was completely diminished by

rotenone (Fig. 5(d)), suggesting the importance of Ndhcomplex in oxygen-induced quenching under the steady-

state conditions.

The effect of oxygen on the relationship between Chl

fluorescence and PQ pool oxidation has been studied

using the propyl gallate and SHAM, inhibitors of alter-

native oxidase [10,23]. Although the plastid terminal

oxidase (PTOX) is probably insensitive to SHAM and

sensitive to propyl gallate [10,23], these two inhibitors

)

)

cence quenching. Data in (a) present the action of 60 lMNEM (dotted

circles). The action of 60 lM rotenone is presented in (c) (dashed line,

NEN, NEM + DCMU and rotenone-treated thylakoids, respectively.

is the same as in Fig. 2. (b) and (d) show changes in the normalized

pectively. The control curves from Figs. 2(a) and (b) (solid line, open

Fig. 2.


did not affect the overall oxygen action (data not

shown). The PTOX displays the relatively low affinity

for oxygen [23], which may explain the insignificant ef-

fect of inhibitors on oxygen-induced Chl fluorescence

quenching under steady-state conditions.

Recently, it has been proposed that the oxidationof PQ pool by molecular oxygen may be performed

by: (i) autocatalytic reactions inside thylakoid mem-

branes [25], (ii) oxidation at low potential site of cyt

b559 [21] and (iii) reaction catalyzed by PTOX

[10,23]. Reduction of oxygen by reactions sequence

involving Ndh complex, peroxidase, superoxide dismu-

tase and Mehler reaction was proposed by Casano

et al. [18] in view of in vitro experiments with purifiedenzymes. Molecular interactions between FNR and

Ndh [54] as well as FNR and cyt b6f [55] suggest

the complex relationship between PQ pool and mem-

brane complexes. Hence, these findings and presented

results (cf. Figs. 4 and 5) imply participation of cyt

b6f, PSI and Ndh complex in Chl fluorescence quench-

ing at weak light and oxygen concentration above

275 lM.

4. Conclusions

The results presented in this investigation indicate

that:

(i) The structural changes in thylakoids membrane arecorrelated to oxygen concentration. At weak light

oxygen induces both disaggregation of LHCII and

destacking of thylakoids (cf. Fig. 1), probably due

to oxygen-dependent and light-driven structural

rearrangement of LHCII complexes [8,30]. The

oxygen-induced thylakoids reorganization does

not seem to be the only component responsible

for Chl fluorescence decrease.(ii) The Chl fluorescence quenching is related to oxygen

concentration and reveals two phases with the

break point above 275 lM oxygen concentration

(cf. Figs. 2(a) and (b) and 4(b) and (d)).

(iii) The oxygen-induced decrease in Chl fluorescence

appearing at low oxygen concentration does not

relate to both redox state of QA (cf. Figs. 2(c) and

(d)) and electron flow from PSII to cyt b6f (cf. Figs.4(c) and (d)).

(iv) At high oxygen concentration (above 275 lM O2)

Chl fluorescence decrease seems to be associated

with oxygen reduction by PSI, cyt b6f and/or Ndh

complex (cf. Figs. 4(c) and (d) and 5).

(v) Uncoupling of thylakoids by CCCP does not result

in modification of the steady-state oxygen effect on

Chl fluorescence quenching (cf. Figs. 4(a) and (b)),while oxygen action may be modulated by electron

donors and acceptors, NADH (cf. Fig. 4(a)), DQ

(cf. Fig. 3(a)) and DBMIB (cf. Fig. 3(b)), exhibiting

a higher affinity to membrane complexes than

oxygen.

5. Abbreviations

CCCP carbonyl cyanide m-chlorophenyl hydra-

zone

Chl chlorophyll

DBMIB 2,5-dibromomethyl-6-isopropyl-p-benzo-

quinone

DCMU 3-(3,4-dichlorphenyl)1,1-dimethylurea

DNP-INT dinitrophenylether of 2-iodoo-4-nitrothy-mol

DQ duroquinone

FNR ferredoxin-NADP+-reductase

FQR ferredoxin-plastoquinone reductase

LHCII light harvesting chlorophyll a/b protein

complex II

Ndh NAD(P)H-plastoquinone dehydrogenase

NEM N-ethylmaleimidePQ plastoquinone

PSI, PSII photosystem I, II

SHAM salicylhydroxamic acid

Acknowledgements

This work was partly supported by the grants of Pol-

ish Ministry of Scientific Research and Information

Technology (No. 1455/15 and No. 3 P04C 109 23).

The authors are grateful to Professor J. Bryła for a crit-

ical reading of manuscript. We thank to Dr. H.Grubek-

Jaworska for making available the absorption spectro-

photometer. The assistance of Mrs. A. Dro _zak in

experiments with oxidase inhibitors is acknowledged.

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The action of oxygen on chlorophyll fluorescence quenching and absorption spectra in pea thylakoid...

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