REGULAR PAPER

Characterization of photosystem II heterogeneity in responseto high salt stress in wheat leaves (Triticum aestivum)

Pooja Mehta • Suleyman I. Allakhverdiev •

Anjana Jajoo

Received: 5 May 2010 / Accepted: 2 August 2010 / Published online: 21 August 2010

� Springer Science+Business Media B.V. 2010

Abstract The effect of high salt stress on PS II hetero-

geneity was investigated in wheat (Triticum aestivum)

leaves. On the basis of antenna size, PS II has been clas-

sified into three forms, i.e., a, b, and c centers while on the

basis of electron transport properties of the reducing side

of the reaction centers, two distinct forms of PS II have

been suggested, i.e., QB reducing centers and QB non-

reducing centers. The chlorophyll a (Chl a) fluorescence

transients, which can quantify PS II behavior, were

recorded using PEA to derive OJIP in vivo with high time

resolution and further analyzed according to JIP test. Our

results showed that with an increase in the salt concen-

tration during growth, the number of QB non-reducing

centers increased. In antenna size heterogeneity the num-

ber of b and c centers increased while the number of acenters decreased. A change in the energetic connectivity

between the PS II units was also observed. Recovery

studies showed that antenna heterogeneity was completely

recovered from damage at 0.5 M NaCl concentration and

partially recovered at 1 M NaCl concentration while

reducing side heterogeneity showed no recovery at all after

0.5 M onwards.

Keywords Photosystem II � Heterogeneity � High salt

stress � Antenna size

Abbreviations

ABS Absorption

Chl Chlorophyll

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

ETo Electron transport flux beyond QA

FR Fluorescence rise

FI Fluorescence induction curve

RCs Reaction centers

TRo Energy trapping flux by PS II centers

Introduction

PS II is a multisubunit integral membrane protein complex

used by higher plants and cyanobacteria to catalyze water

oxidation and plastoquinone reduction (Boekema et al.

2000). The reaction center of PS II consist of D1–D2

proteins, which contain accessory Chl on D1 branch as

primary electron donor (Groot et al. 2005; Holzwarth et al.

2006), the secondary electron donor P680, Yz, and YD

(tyrosine), the intermediatory electron acceptor Pheophytin

(Pheo) and two plastoquinone electron acceptor QA and QB

along with an associated non-heme iron. PS II is a light-

dependent water-plastoquinone oxidoreductase enzyme

that uses light energy to oxidize water and is mainly

located in the appressed grana stacks (Carpentier et al.

2005; Allakhverdiev et al. 2008).

PS II has been found to be more heterogeneous than

other components such as PS I and Cyt b6f in various

aspects and differs in its structure and function both. This

P. Mehta � A. Jajoo (&)

School of Life Science, Devi Ahilya University, Indore 452017,

MP, India

e-mail: anjanajajoo@hotmail.com

S. I. Allakhverdiev

Institute of Plant Physiology, Russian Academy of Sciences,

Botanicheskaya Street 35, Moscow 127276, Russia

S. I. Allakhverdiev

Institute of Basic Biological Problems, Puschino, Moscow

Region 142290, Russia

123

Photosynth Res (2010) 105:249–255

DOI 10.1007/s11120-010-9588-y

diverse nature of PS II is known as PS II heterogeneity. The

fluorescence rise measured with DCMU treated plant

material cannot be described by a single exponential

increase (Doschek and Kok 1972). The biphasic fluores-

cence induction kinetics observed upon illumination of

higher plant chloroplast suspended in the presence of her-

bicides (DCMU) was used for a description of one type of

PS II heterogeneity, the PS II antenna heterogeneity (Melis

and Homann 1975, 1976). Two main aspects of PS II

heterogeneity have been studied widely: antenna hetero-

geneity and reducing side heterogeneity. Antenna hetero-

geneity is related to the antenna size as well as to the

energetic connectivity between PS II units. By using

kinetic analysis of fluorescence induction curve of DCMU

poisoned chloroplast, PS II has been resolved into three

components of different antenna sizes, i.e., a, a-s, and b(Hsu et al. 1989) and lately renamed to PS II a, PS II b, and

PS II c (Strasser 1981; Strasser and Greppin 1981; Melis

1985; Hsu and Lee 1991), respectively, while Sinclair and

Spence (1990) demonstrated four types of PS II, i.e., a, b,

U, and d. PS II a are mainly located in the grana region of

thylakoid membranes, have a large light harvesting antenna

and show the possibility of excited states transfer between

PS II units which displays a sigmoidal fluorescence rise

when measured with DCMU. On the other hand PS II b are

mainly located in stromal region of thylakoid membranes,

have 2.5 times smaller light harvesting antenna and show

impossibility of the excited states transfer between PS IIs

displays in the form of an exponential fluorescence rise

when measured with DCMU. Smaller antenna size in PS II

b fraction has been ascribed to the absence of peripheral

LHC II in PS II. The intrinsic trapping and fluorescence

property of a and b centers are considered to be similar

(Melis 1991). The antenna size of PS II c is the smallest

amongst all. PS II a and PS II b both are photochemically

competent and are able to transfer electron from QA to QB

(Thielen and Van Gorkom 1981; Lavergne 1982; Melis

1985; Graan and Ort 1986; Ghirardi and Melis 1988;

Greene et al. 1988; Guenther et al. 1988).

According to the concept of connectivity (also called

grouping) closed PS II reaction centers (RC) may transfer

their excitation energy to open neighboring PS II units that

results in sigmoidal fluorescence rise instead of exponential

rise (Lavergne and Trissl 1995; Joliot and Joliot 1964; Joliot

et al. 1973; Strasser et al. 2004). It was suggested that the

three populations of PS II units (a, b, and c) are different in

their connectivity properties, i.e., the a-centers are supposed

to be grouped, whereas the two others are not, and the trap-

ping efficiency of the c-centers is thought to be lowest.

Based on the electron transport properties on the

reducing side of the reaction centers, PS II in green plants

occurs in two distinct forms: centers with efficient electron

transport from QA to QB are known as QB reducing type,

while centers that are photochemically competent but

unable to transfer electron from QA to QB are known as QB

non-reducing type (Graan and Ort 1986). In such centers

QA- can only be reoxidized by a back reaction with the

donor side of PS II. QB reducing centers are active centers

while QB non-reducing centers are inactive centers. Using

fluorescence transients, the measurement of reducing side

heterogeneity has been done in two ways: (i) by measuring

height of the plateau (Fpl) in the presence of DCMU under

low light intensity (Tomek et al. 2001), (ii) by double hit

method where two fluorescence transients were induced by

two subsequent pulses (Strasser et al. 2004).

Various stress condition like high temperature, high salt,

etc., adversely affect photosynthetic efficiency, particularly

at the donor side and acceptor side of Photosystem II

(Mathur et al. 2010; Mehta et al. 2010). Extent and nature

of PS II heterogeneity may vary under different physio-

logical conditions, i.e., high salt stress, temperature stress,

etc. High salt stress is one of the major environmental

factors that limit the plant productivity. Salt stress leads to

reduction in the growth of the plant, which is associated

with a decrease in the rates of photosynthesis. Many crop

species are sensitive to high concentration of salt with

negative impact on the crop production. There are many

studies about the effects of salt stress on fluorescence

induction kinetics (Mehta et al. 2010), however, investi-

gation of change in PS II heterogeneity in response to high

salt stress has not yet been studied. In the present work we

have determined the effect of high salt stress on antenna

and reducing side heterogeneity of PS II in wheat. Since

most of the changes obtained in the heterogeneity of PS II

were found to be reversible, we have proposed that alter-

ation in the antenna size and reducing side heterogeneity of

PS II may be a temporary adaptive mechanism exhibited by

PS II to tolerate stress condition in the initial stages.

Materials and methods

Plant material: wheat (Triticum aestivum)

Lok-1 cultivar of wheat was used for experiments. Wheat

seeds were allowed to germinate and then transferred to

petriplates containing Knop solution with a photosynthet-

ically active photon flux density (PPFD) of 300 lmol m-2 s-1, 20�C and a photoperiod of 16/8 h light/dark.

High salt treatment

High salt stress in the form of NaCl concentrations, i.e.,

0.1, 0.2, 0.3, 0.4, 0.5, and 1 M NaCl was given to the

seedlings gradually. For gradual stress, treatment of 0.1 M

NaCl was given in steps to the seedlings after every 48 h

250 Photosynth Res (2010) 105:249–255

123

from day one when it was transferred on nutrient medium

(Knop solution) till when the concentration reached to 1 M.

Measurement of fluorescence induction kinetics

The chlorophyll a (Chl a) fluorescence induction kinetics

was measured at room temperature using a Plant Efficiency

Analyzer (PEA, Hansatech, King’s Lynn, Norfolk, Eng-

land). The fluorescence measurements were performed two

inches away from the tip and base, i.e., in the middle

portion on the abaxial surface of the leaves. Thirty mea-

surements were taken for each treatment. Excitation light

of 650 nm (peak wavelength) from array of three light—

emitting diodes is focused on the surface of the leaf to

provide a homogenous illumination. Light intensity

reaching the leaf was 3000 l mol m-2 s-1 which was

sufficient to generate maximal fluorescence for all the

treatments. The fluorescence signal is received by the

sensor head during recording and is digitized in the control

unit using a fast digital converter. Energy pipeline model

was deduced using the Biolyzer HP 3 software (the chlo-

rophyll fluorescence analyzing program by Bioenergetics

Laboratory, University of Geneva, Switzerland).

Determination of reducing side heterogeneities

The double hit method was followed for the calculation of

QB reducing and QB non-reducing centers (Strasser and

Tsimilli-Michael 1998; Strasser et al. 2004). In this method

two fluorescence transients were induced by two sub-

sequent pulses (each of 1 s duration). The first pulse

(denoted as 1st hit) was conducted after a dark period long

enough to ensure the reopening of all reaction centers,

followed by a second pulse (2nd hit). The duration of the

dark interval between two hits is 500 ms (Fig. 1).

Fv ¼ Fm� Fo, Fv� ¼ Fm� � Fo�

where Fv: variable fluorescence of 1st hit, Fm: maximal

fluorescence of 1st hit, Fv*: variable fluorescence of 2nd hit,

Fm*: maximal fluorescence of 2nd hit, Fo: minimal fluo-

rescence of 1st hit, Fo* minimal fluorescence of 2nd hit.

QB non-reducing centers were calculated by the fol-

lowing equation.

Vo Boð Þ ¼ Fv=Fmð Þ � Fv�=Fm�ð Þ½ �= Fv=Fmð Þ

where Bo = relative amount of QB non-reducing PS II

centers (Strasser and Tsimilli-Michael 1998).

The relative amounts of QB non-reducing center have

been shown to vary with light intensity as suggested in

Tomek et al. (2001), however, in this work, we have used

high light intensity (3000 l mol m-2 s-1) for carrying out

experiments in vivo. The advantage of the high light

intensity is that the Fm can be clearly distinguished (Toth

and Strasser 2005). Similar light intensity has been used for

control and salt stressed plants and so it was possible to

measure relative changes in QB non-reducing centers.

Determination of antenna heterogeneity

For calculation of antenna heterogeneity, DCMU poisoning

method (Strasser 1981; Hsu et al. 1989) was used. The

method is as follows: wheat plants were put in darkness for

*1 h before the DCMU treatment, and then pairs of leaves

were placed into small trays (without detaching them from

the plant) filled with 100 ml DCMU solution overnight and

in complete darkness (the DCMU concentration was

200 lM, and the solution contained 1% ethanol, which was

used to dissolve the DCMU). Following the treatment,

leaves were removed from the DCMU solution (still not

detached and in darkness), wiped and left in the air for

*1 h to avoid possible effects of anaerobiosis. DCMU

keeps QA in its reduced state during the measurements and

does not reflect the photochemical reactions (Lazar et al.

2001).

Determination of a, b, and c centers:

Alpha (a), beta (b), and gamma (c) centers were calculated

from the complementary area (CA) growth curve (Melis

1985). The CA is an area between the curve of fluorescence

induction and line determining the level of the maximal

fluorescence intensity Fm (Lazar 1999). Kinetics of

Fig. 1 A representative fluorescence induction curve (log time scale)

obtained from double hit method (as described in ‘‘Materials and

methods’’ section). Fluorescence measurement was induced by two

subsequent pulses (each of 1 s). The first pulse (denoted as 1st hit)

was conducted after a dark period long enough to ensure the

reopening of all reaction centers, followed by a second pulse (2nd

hit). The duration of the dark interval between two hits was 500 ms.

The graphs have time axis in logarithmic scale

Photosynth Res (2010) 105:249–255 251

123

complementary area of the dark adapted sample was fitted

with three exponentials phases (corresponding to a, b, and

c). Based on the lifetimes (s) of each of the fraction, their

contribution to the total amplitude (A) of the kinetics of

complementary area was calculated and has been indicated

as percentage of a, b, and c centers (Strasser 1981; Hsu

et al. 1989). It has been suggested that in the presence of

DCMU, the kinetics of QA- accumulation may be obtained

by integrating the kinetics of complementary area. The

complementary area is fitted by exponentials only and thus

considers measurements of only energetically separated PS

II units under salt stress.

Results and discussion

Polyphasic chlorophyll a fluorescence transient was mea-

sured to evaluate the effects of high salt stress on the

photochemical efficiency of PS II. The OJIP transient

represents the successive reduction of electron transport

pool of PS II (Govindjee 1995). The intensity of fluores-

cence in the OJIP transient decreased with increase in NaCl

concentration, as shown in Fig. 2. An increase in salt

concentration causes a significant decrease in the minimal

fluorescence (Fo), variable fluorescence (Fv), and maximal

fluorescence (Fm). A decrease in the fluorescence yield of

leaves can be attributed to an inhibition of electron flow at

oxidizing site of PS II (Lu and Vonshak 2002). The

decrease in Fm and fluorescence at J, I, P may be due to

two reasons, first by inhibition of electron transport at the

donor side of the PS II which results in the accumulation of

P680? (Govindjee 1995; Neubauer and Schreiber 1987) and

second due to a decrease in the pool size of QA-.

These chlorophyll a fluorescence transient curves mea-

sured in the absence (Fig. 2a) and presence of DCMU

(Fig. 2b) were further used to evaluate the effect of high

salt stress on reducing side and antenna size heterogeneity

of Photosystem II.

Effect of high salt stress on reducing side heterogeneity

To study reducing side heterogeneity of PS II, relative

amount of QB reducing and QB non-reducing centers were

measured from the fluorescence rise (FR) curve as descri-

bed in material and methods. The amount of QB non-

reducing centers was increased in salt stressed leaves. In

control leaves, the QB non-reducing centers were found to

be 14% (Table 1) which became 32% in 0.5 M salt treat-

ment. To study recovery process, the salt stressed leaves

were kept in distilled water for 24 h and then fluorescence

was measured. Leaves treated with 0.5 M NaCl showed no

recovery, the number of QB non-reducing centers remained

almost the same as in the 0.5 M NaCl treated leaves

(Table 1). This result suggests that the damage at the

reducing side of PS II was permanent.

Effects of high salt stress on antenna heterogeneity

The kinetics of complementary area of DCMU treated

fluorescence induction curve was calculated by the equation

B ¼R

Fm� Ftð Þdt� �

, where B is the double normalized

(between 0 and 1) kinetics of complementary area (Strasser

Fig. 2 The OJIP Chl a fluorescence transient curve (log time scale)

in wheat leaves exposed to various concentration of NaCl for 1 h

dark. The graphs have time axis in logarithmic scale

Table 1 Relative changes in QB non-reducing and QB reducing

centers in response to high salt stress in wheat leaves

NaCl

concentration (M)

% of QB

non-reducing centers

% of QB

reducing centers

Control 14 ± 1 86 ± 4

0.1 15 ± 2 85 ± 3

0.2 21 ± 1 79 ± 2

0.3 23 ± 2 77 ± 3

0.4 25 ± 1 75 ± 2

0.5 32 ± 2 68 ± 3

Recovery 32 ± 2 68 ± 3

Experiment was repeated 5 times

252 Photosynth Res (2010) 105:249–255

123

et al. 2000; Murata et al. 1966; Malkin and Kok 1966) and the

B kinetics of the first light pulse were fitted with three

exponentials that correspond to three different types of PS II

centers namely PS II a, b, and c. They were further analyzed

and later assigned as PS II a, b, and c centers on the basis of

their life times. As is evident from Fig. 3, the lifetime of the

fastest a component was 0.37 ms and contributed 70% of the

total amplitude. The beta component was about 3.8-fold

slower (life time *1.44 ms) and it was responsible for 26%

of the total amplitude. The gamma component was very slow

with lifetime of 9.14 ms and small, being only 4% of the total

amplitude in control leaves. The changes in percentage of a,

b, and c components with different salt concentrations are

shown in Fig. 3. With increase in salt concentration the

percentage of a centers decreased while that of b and ccenters increased. The number of c centers has increased

more as compared to b centers with increase in the salt

concentration. The relative ratio of a:b:c centers in control

leaves was 70:26:4 while it became 38:36:26 in salt stressed

leaves (1 M NaCl). Recovery for antenna heterogeneity was

also studied. It was observed that in antenna size heteroge-

neity the recovery of 0.5 M NaCl treatment was equal to

control while in case of 1 M NaCl treatment recovery has

been occurred but not exactly equal to the control. After

recovery in 0.5 and 1 M NaCl stressed plant, the relative

proportions of a:b:c centers became 70:26:4 and 66:29:5.

These results indicate that the damage caused due to high salt

stress in antenna size heterogeneity was temporary and

largely reversible, suggesting that the a, b, and c centers were

interconvertible.

It has been shown that the relative variable fluorescence

[(Ft - Fo)/(Fm - Fo)] is not only directly proportional to

the number of closed RCs, but it is linearly related to the

rate at which centers close (Bennoun and Li 1973). This

may be explained by the connectivity of PS II units. PS IIbwere characterized by an exponential rise, of the time

course of complementary area (CA) whereas PS IIa showed

a non-exponential (sigmoidal) rise (Melis and Homann

1976). The exponential shape of this rise for PS IIb was

suggested to reflect mutual energetic separation of these PS

II. On the other hand, the non-exponential fluorescence rise

of PS IIa is generally believed to reflect energetic con-

nectivity between these PS IIs as originally suggested by

Joliot and Joliot (1964). The fluorescence rise (FR) curves

measured with DCMU and high salt-treated wheat leaves

are shown in Fig. 4. The curves are presented by means of

relative variable fluorescence (rFvt), which is defined as

(Ft - Fo)/(Fm - Fo), where Fo, Fm, and F(t) are the

minimal and maximal measured fluorescence intensity at

time t, respectively (Lazar et al. 2001). With increase in

NaCl concentration sigmoidicity of the curve decreased

indicating that high salt stress reduces the connectivity

between the PS II units. These results also suggest that the

alpha component (sigmoidal phase) of chlorophyll a fluo-

rescence induction curve decreased and beta component

(exponential phase) increased. A loss in connectivity also

Fig. 3 Complementary area

curves (linear time scale)

showing percentage of alpha,

beta, and gamma centers in

control and salt-treated wheat

leaves

Photosynth Res (2010) 105:249–255 253

123

indicates that the fraction of closed RCs, i.e., QB non-

reducing centers has also increased (Strasser and Tsimilli-

Michael 1998).

The antenna and reducing side heterogeneity were

studied by energy pipeline models of the photosynthetic

apparatus (Kruger et al. 1997; Strasser 1987; Strasser et al.

2000) and specific energy fluxes were calculated. ABS/RC

demonstrates average antenna size and expresses the total

absorption of PS II antenna chlorophylls divided by the

number of active (in the sense of QA reducing) reaction

centers. Therefore, the antenna of inactivated reaction

centers are mathematically added to the antenna of the

active reaction centers. TRo/RC refers only to the active

(QA to QA-) centers (Force et al. 2003). As a result of high

salt stress, the flux ratios ABS/RC, TRo/RC, and DIo/RC

increased (Table 2). The ratio of ABS/RC increased due to

inactivation of some active RCs. TRo/RC represents the

maximal rate by which an exciton is trapped by the RC

resulting in the reduction of QA. An increase in this ratio

indicates that all the QA has been reduced but it is not able

to oxidize back due to stress, i.e., the reoxidation of QA- is

inhibited so that QA cannot transfer electrons efficiently to

QB. DIo/RC represents the ratio of the total dissipation of

untrapped excitation energy from all RCs with respect to

the number of active RCs. Dissipation may occurs as heat,

fluorescence and energy transfer to other systems. It is

influenced by the ratios of active/inactive RCs. The ratio of

total dissipation to the amount of active RCs increased

(DIo/RC) due to the high dissipation of the active RCs.

These ratios conclusively describe that the number of

inactive centers have increased due to high salt stress in

wheat leaves.

Conclusion

From these results we conclude that increase in salt con-

centration caused an alteration in both the types of PS II

heterogeneity. An increase in salt concentration caused an

increase in the relative amounts of QB non-reducing centers

as well as a change in the relative amounts of a, b, and ccenters. Change in response to salt stress led to the con-

version of the active a centers into inactive b and c centers.

It was observed that the change in reducing side hetero-

geneity could not be recovered while that of antenna size

was recovered. Changes in heterogeneity of PS II seem to

be an adaptive mechanism of plants to face harsh envi-

ronmental conditions. The structure and function of PS II is

manipulated temporarily under high salt stress in the form

of change in heterogeneity.

Acknowledgments PM thanks Council of Scientific and Industrial

Research (CSIR), India for the Senior Research Fellowship [09/301/

(0019)09/EMR-I]. Project (INT/ILTP/B-6.27) to AJ by Department of

Science and Technology (DST), New Delhi, India is thankfully

acknowledged. This work was financially supported, in part, by grants

from the Russian Foundation for Basic Research (08-04-00241; 09-

04-91219-CT). We are also thankful to Prof. Reto J. Strasser for

gifting Biolyzer HP 3 Software.

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