REVIEW

Chlorophyll a fluorescence: beyond the limits of the QA model

Gert Schansker • Szilvia Z. Toth • Alfred R. Holzwarth •

Gy}oz}o Garab

Received: 27 September 2012 / Accepted: 18 February 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Chlorophyll a fluorescence is a non-invasive

tool widely used in photosynthesis research. According to

the dominant interpretation, based on the model proposed

by Duysens and Sweers (1963, Special Issue of Plant and

Cell Physiology, pp 353–372), the fluorescence changes

reflect primarily changes in the redox state of QA, the

primary quinone electron acceptor of photosystem II

(PSII). While it is clearly successful in monitoring the

photochemical activity of PSII, a number of important

observations cannot be explained within the framework of

this simple model. Alternative interpretations have been

proposed but were not supported satisfactorily by experi-

mental data. In this review we concentrate on the processes

determining the fluorescence rise on a dark-to-light tran-

sition and critically analyze the experimental data and the

existing models. Recent experiments have provided addi-

tional evidence for the involvement of a second process

influencing the fluorescence rise once QA is reduced. These

observations are best explained by a light-induced con-

formational change, the focal point of our review. We also

want to emphasize that—based on the presently available

experimental findings—conclusions on a/ß-centers, PSII

connectivity, and the assignment of FV/FM to the maximum

PSII quantum yield may require critical re-evaluations. At

the same time, it has to be emphasized that for a deeper

understanding of the underlying physical mecha-

nism(s) systematic studies on light-induced changes in the

structure and reaction kinetics of the PSII reaction center

are required.

Keywords Chl a fluorescence �Fluorescence yield changes � Light-induced conformational

changes � OJIP transient � Thermal phase

Abbreviations

Chl Chlorophyll

CP43, CP47 Core antenna proteins of photosystem II

of 43 and 47 kDa, respectively

DCMU 3-(30,40-Dichlorophenyl)-1,1-

dimethylurea

ETC Electron transport chain

FCCP Carbonylcyanide-p-

trifluoromethoxyphenylhydrazone

F0 Minimum chl a fluorescence yield in the

dark-adapted state

FM Maximum chl a fluorescence yield in the

dark-adapted state

FV Variable fluorescence defined as FM-F0

LED Light emitting diode

P680 Reaction center pigments of photosystem II

PFD Photon flux density

Pheo Pheophytin

PMS Phenazine methosulfate

PQ Plastoquinone

PSII and PSI Photosystem II and I, respectively

QA and QB Primary and secondary quinone electron

acceptors of photosystem II, respectively

RC Reaction center

S-states S0, S1,

S2, S3, and S4

Different redox states of the oxygen-

evolving complex

G. Schansker (&) � S. Z. Toth � G. Garab (&)

Institute of Plant Biology, Biological Research Center Szeged,

Hungarian Academy of Sciences, Szeged 6701, Hungary

e-mail: gert.schansker@gmail.com

G. Garab

e-mail: garab.gyozo@brc.mta.hu

A. R. Holzwarth

Max-Planck-Institute for Chemical Energy Conversion, 45470

Mulheim an der Ruhr, Germany

123

Photosynth Res

DOI 10.1007/s11120-013-9806-5

SSTF Saturating single turnover flash

TMPD N,N,N’,N’-tetramethyl-

p-phenylenediamine dihydrochloride

TRIS Tris(hydroxymethyl)aminomethane

TyrZ, TyrD Tyrosine donors to P680 localized on the

PSII reaction center D1 and D2 proteins,

respectively

Introduction

Measurements of chlorophyll a fluorescence transients are

used as a non-invasive tool to probe the activity of the

photosynthetic apparatus. It is undoubtedly the most widely

applied technique in photosynthesis research (reviewed

e.g., in Papageorgiou and Govindjee 2004). Upon exposing

a dark-adapted leaf or algal cells to a strong saturating light

pulse (3,000–12,000 lmol photons m-2 s-1, 200–1,000 ms),

the fluorescence increases from a minimum level F0 to the

maximum level FM, which is reached in about 200 ms. If

presented at a high time resolution a polyphasic OJIP

transient is observed (e.g., Strasser et al. 2004; see Fig. 1).

On a logarithmic time scale (Strasser and Govindjee 1991)

three rise phases can be identified: OJ (0–3 ms), JI

(3–30 ms), and IP (30–200 ms) (Neubauer and Schreiber

1987; Strasser and Govindjee 1991). When using the phase

amplitude modulation (PAM) technique, the fluorescence

induced by a saturating pulse is generally presented as a

spike (e.g., Schreiber 2004).

According to the most widely accepted interpretation,

hereafter called the QA model, which was proposed in 1963

by Duysens and Sweers and reviewed recently by Stirbet

and Govindjee (2012), the fluorescence rise from F0 to FM

is a reflection of the reduction of QA. This forms the basis

of nearly all theoretical treatments of fluorescence mea-

surements (Butler and Kitajima 1975; Paillotin 1976;

Strasser 1978; Trissl and Lavergne 1994; Kramer et al.

2004) including the quenching analysis (Schreiber 2004)

and the JIP test, a mathematical framework for the analysis

of OJIP transients (Strasser and Strasser 1995; Strasser

et al. 2004) and is the main explanation model in most

reviews (e.g., Maxwell and Johnson 2000; Baker 2008).

The QA model has been clearly successful in monitoring

the photochemical activity of photosynthetic samples.

However, it has also been recognized that a number of

important observations, related to the time and temperature

dependencies of the fluorescence rise, cannot be explained

within the framework of this simple model (e.g., Samson

et al. 1999; Schansker et al. 2011).

The QA model is, implicitly or explicitly, based on two

assumptions: (1) the fluorescence rise between F0 and FM is

a reflection of the progressive reduction of QA, i.e., the

transition from open to closed PSII reaction centers, and

(2) no changes in the fluorescence yield of open or closed

centers during the transition between the two states F0 and

FM occur. Assumption 2 means that the rate constants for

fluorescence emission and heat dissipation of neither open

nor closed centers change during the transition from F0 to

FM. With the introduction of the quenching analysis, a

method to separate photochemical (electron transport

related) and non-photochemical (fluorescence yield change

related) quenching of the fluorescence intensity, this

assumption was formulated more explicitly (van Kooten

and Snel 1990).

The vast literature related to fluorescence changes on a

dark-to-light transition has recently been thoroughly

reviewed by Stirbet and Govindjee (2012). According to

that review, the QA model, i.e., the model of Duysens and

Sweers (1963) is ‘‘the most credible theory.’’ The authors

suggest that the whole rise kinetics, in particular including

the IP phase, originates essentially from a gradual photo-

reduction of QA only. At the same time, however, Stirbet

and Govindjee (2012) also point out that the complex

fluorescence rise kinetics can only be understood by

modifying the Duysens and Sweers theory. They state that

during the fluorescence rise ‘‘… the presence of several

quenchers and modifiers that influence the fluorescence

yield during the O–J–I–P transient …’’ are also to be taken

into account. This actually implies that neither of the two

basic assumptions of the Duysens and Sweers model is

Fig. 1 Example of a fluorescence transients induced by red light of

15,000 lmol photons m-2 s-1 applied to dark-adapted, untreated, and

DCMU-treated pea leaves. Both the steps and the assignment of the

different rise phases as photochemical phase and thermal phaseaccording to Delosme (1967) are indicated. At this light intensity the

photochemical phase represents almost 70 % of FV

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accepted. The implied ‘‘modifiers’’ in some cases form the

basis for alternative models presented in the literature;

some of these alternative models will be discussed below.

Most ‘‘modifiers,’’ e.g., P680?, TyrZ?, and TyrD, carot-

enoid triplets, or the potentially functioning alternative

reaction pathways, such as cyclic PSII and PSI electron

transfers, do affect (or may affect) part of the initial kinetic

phases of the fluorescence rise. However, none of them

offer an explanation for the crucial slow fluorescence rise

phase, the IP phase.

Experimental observations which are difficult

to reconcile with the QA model

A saturating single turnover flash (SSTF) induces at best

60–65 % of FM

The fluorescence intensity induced by a single SSTF in

intact spinach chloroplasts does not surpass the fluores-

cence intensity of the J step (Schreiber 1986) which is

approx. 60–65 % of FM. On the other hand, it has been

shown that an SSTF can reduce all or nearly all QA at room

temperature, as shown by the fact that it induces a nearly

maximum DA320nm signal (Joliot and Joliot 1981), which is

a direct measure for reduced QA, and a nearly maximum S2

multiline signal, arising from the manganese cluster of the

water splitting complex (Brudvig et al. 1983). Also, the

analysis of period-4 oscillations in the flash-oxygen yield

indicates, based on the miss factor, that the fraction of

unreduced QA on each single turnover flash is less than

7–8 % (Shinkarev (2005) re-analyzing the data of Forbush

et al. (1971)).

FM is reached only after many excitations and multiple

PSII turnovers

A photon flux density (PFD) of 3,000 lmol photons

m-2 s-1 white or red light means that each PSII becomes

excited once every *200 ls (cf. Neubauer and Schreiber

1987; Lazar and Pospısil 1999). This equates *1,000

excitations per 200 ms (the time needed to reach FM). It

has also been shown that to reach FM, the photosynthetic

electron transport chain (ETC) has to become reduced

(Schansker et al. 2005, 2006, 2008); in particular, it has

been shown by Joliot and Joliot (2002) that in leaves, under

their experimental conditions, about 20 electrons had to be

transported, i.e., 20 stable charge separations had to take

place, to reach FM. In addition, it has been demonstrated

that electron flow through PSI (Kautsky et al. 1960;

Munday and Govindjee 1969; Schreiber et al. 1989;

Harbinson and Hedley 1993; Schansker et al. 2005) in

combination with inactive FNR (Kautsky et al. 1960;

Munday and Govindjee 1969; Satoh 1981; Carillo et al.

1981; Schansker et al. 2005, 2006, 2008) affects the fluo-

rescence rise in leaves and intact chloroplasts.

J step (2–3 ms) also requires multiple charge separations

It follows (see point 2) that during the time needed to reach

the J step C10 excitations of each PSII occur. This multiple

excitation also induces multiple charge separations, which

is demonstrated by at least two observations: (i) In heat-

treated leaves as well as in TRIS-washed thylakoids, where

only one electron is available for donation to P680? in this

time range (Toth et al. 2007a), a fluorescence peak

(K) appears after *300 ls of illumination, which is fol-

lowed by a fluorescence decline due to forward electron

transport to QB. (ii) Experiments with SSTFs indicate that a

pair of electrons needs about 2 ms to reach the PQ-pool

(Petrouleas and Crofts 2005).

Kinetics in the presence of DCMU

In isolated thylakoid membranes in the presence of DCMU

(3-(3,4-dichlorophenyl)-1,1-dimethylurea), which blocks

the electron transport between QA and QB, an SSTF

induces about 70–75 % of FM (Joliot and Joliot 1979;

Samson and Bruce 1996). Joliot and Joliot (1979) observed

for spinach thylakoid membranes that while DA550, indi-

cating the local field due to the reduction of QA, reached

almost its highest value already after one saturating flash in

the presence of DCMU; the fluorescence yield was only

about 70–75 % of the maximum variable fluorescence.

Further flashes, usually 3–5, are needed to reach the

maximum fluorescence intensity. In leaves in continuous

strong light FM is reached after about 10 ms (Toth et al.

2005a), i.e., also only after multiple excitations.

The sigmoidicity of the fluorescence rise in the presence

of DCMU

Traditionally, the sigmoidicity of the fluorescence rise in the

presence DCMU is explained in terms of the connectivity

between PSII antennae (Joliot and Joliot 1964; Paillotin

1976; Strasser 1978). However, according to Vredenberg

(2004, 2008) this feature is difficult to explain within a QA

model. The kinetic analysis of Vredenberg (2008) suggests

that the sigmoidicity is related to the double excitation nature

of the fluorescence rise in the presence of DCMU.

Alternative interpretations assuming

that the fluorescence yield is determined by both QA

and (an) additional process(es)

Morin (1964) and Delosme (1967)—using a special setup,

with a shutter opening time of 30 and 5 ls, respectively,

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which also allowed the use of high light intensities—were

the first to observe an intermediate step in the fluorescence

rise (without inhibitors). Their recorded fluorescence rise

had, therefore, more complex kinetics than those published

by Duysens and Sweers (1963). It should be noted here that

such measurements could only be done routinely 25 years

later with the introduction of fast LED-based systems.

Delosme (1967) proposed that only the first 2 ms of the

fluorescence rise was due to the reduction of QA. The

nature of the remaining part, called the ‘‘thermal phase’’

due to its temperature dependence, was not identified.

Since then different models and interpretations have been

proposed. They all agree on the crucial involvement of QA-

and at least one further component but differ from each

other concerning the nature of this additional process. They

also differ from each other whether or not they accept one

or the other of the two basic assumptions of the QA model.

(It is to be noted here that the term ‘‘thermal phase’’ as

introduced by Delosme in 1967 is somewhat misleading. It

has been thoroughly documented that this ‘‘thermal

phase,’’ i.e., the slow rise of the fluorescence induction is

also a light-induced reaction. We adhere to this terminol-

ogy throughout this review, but in order to avoid any

misunderstanding we italicize it).

Redox reactions

Most of the authors who studied the thermal phase, starting

with Delosme (1967), proposed the involvement of a light-

induced redox reaction. In these models, Assumption 2, but

not 1, was considered valid. Joliot and Joliot (1979, 1981)

proposed a redox factor called Q2, involved in a non-

electrogenic reaction; the identity of which, however,

remained elusive. Recently, Boussac et al. (2011) found

that in Thermosynechococcus elongatus QB– could extract

an electron from the non-heme iron leading to oxidized

non-heme iron in a fraction of PsbA3-PSII (a stress

inducible cyanobacterial gene product) but not in PsbA1-

PSII. They proposed that Q2 might be identical with the

non-heme iron. This interpretation, however, has not been

confirmed by studies monitoring the redox state of the non-

heme iron and of the chl a fluorescence in green leaves.

Vredenberg (2000) suggested that the redox component

was pheophytin (Pheo), and that both QA- and Pheo- are

required for the full closure of the PSII reaction center

(RC) and for reaching FM. However, accumulation of

Pheo- was not demonstrated (for extensive literature on

Pheo- and chl a fluorescence quenching see Stirbet and

Govindjee 2012). Schreiber (2002) proposed a reduction of

the inactive branch of PSII but, again, experimental evi-

dence was not presented. We also stress that low-temperature

fluorescence induction data suggest that the process associated

with the thermal phase cannot be a redox factor on the

acceptor side of PSII (Schansker et al. 2011).

Delosme (1971) noted that the process reflected by the

thermal phase must be closely linked to the reduction of QB

and the PQ-pool. Later it was suggested that the occupancy

state of the QB-site (the redox state and/or the presence of a

PQ molecule bound to the QB-site) determines the thermal

phase (Vasil’ev and Bruce 1998; Samson et al. 1999;

Yaakoubd et al. 2002). Amesz and Fork (1967) showed that

oxidized artificial quinones can quench fluorescence and

subsequently it was shown that oxidized PQ is also a fluo-

rescence quencher in isolated thylakoid membranes (Joliot

1974; Vernotte et al. 1979) and especially in PSII membranes

(Kurreck et al. 2000). However, in intact leaves PQ-pool

quenching does not occur (Toth et al. 2005a). Further, the

observation that FM can only be reached following the

reduction of the ETC (Schansker et al. 2005, 2006, 2008)

could in principle be explained by assuming that the thermal

phase is determined by the QB-site occupancy state (Vasil’ev

and Bruce 1998; Samson et al. 1999; Yaakoubd et al. 2002),

since it depends on the PQ-pool redox state, which will, to

some extent, be sensitive to the electron flow through PSI.

However, there are three problems with the QB-site occu-

pancy hypothesis: (i) there is no clear link between the

QB-site occupancy state and processes that could lead to an

increase in the fluorescence yield; (ii) it cannot explain

measurements made on DCMU-inhibited samples, where

the QB-site occupancy state does not play a role; and (iii) the

thermal phase can relax and be re-induced without changes

in the QB-site occupancy state (Schansker et al. 2011).

a/ß-centers

The concept of PSII a/ß heterogeneity was introduced to

explain the biphasicity of the fluorescence rise in the

presence of DCMU (Melis and Homann 1975, 1976).

According to this model, which is based on the validity of

both Assumptions 1 and 2, but operates with two QA

molecules in two different types of RCs, the two rise

phases originate from two different populations of PSII

with different antenna sizes. The sigmoidal fluorescence

rise was proposed to arise from a-centers located in the

grana; they were thought to possess large light-harvesting

antennae interconnected via energy migration. In contrast,

the ß-centers, which were proposed to be located in the

stroma lamellae and to exhibit an exponential fluorescence

rise, explained by their small antenna sizes exhibiting no

connectivity (Melis and Homann 1975). In later work

Melis (1985) proposed overlap between ß-centers and

QB-non-reducing centers (Lavergne 1982). However, the

a/ß-center model does not provide a satisfactory explanation

for the complexity of the fluorescence rise under different

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conditions: (i) the model does not explain the observation

that the slow fluorescence rise in the presence of DCMU

exhibits a strong temperature dependence (Schansker et al.

2011)—which cannot be explained either by antenna or RC

heterogeneity; (ii) the model cannot account for the

observations of France et al. (1992) that biphasicity arises

only after double (or multiple) excitations with (a) short

dark interval(s) between the excitations. This does not

mean that there is no (antenna size) heterogeneity of

PSII—for which there exists extensive biochemical evi-

dence (e.g., Danielsson et al. 2006)—it only suggests that

antenna size heterogeneity of PSII does not necessarily

induce a distinguishable kinetic phase in the fluorescence

rise. Instead, antenna heterogeneities may exert diffuse

effects on the OJ rise.

Vredenberg (2004) noted that the interpretation of the

a/ß-concept is model dependent, having a different meaning

within his three-state model. As shown by Vredenberg

(2008), the sigmoidicity of the fast fluorescence rise phase

in the presence of DCMU—an important feature of the a/ß-

concept—can mathematically be described by two kineti-

cally overlapping exponential functions if the two rate

constants differ by less than one order of magnitude.

Hence, the wealth of experimental observations obtained

and interpreted within the framework of a/ß heterogeneity

require a critical re-evaluation.

The transmembrane uniform electric field

The role of the light-induced transmembrane uniform

electric field, induced by a primary charge separation and

consecutive ‘‘delocalization’’ of the field (Junge 1977), as a

co-determinant of the fluorescence rise kinetics has been

considered in several studies (Pospısil and Dau 2002;

Bulychev and Vredenberg 2001; Vredenberg and Bulychev

2003; Rubin and Riznichenko 2009). Electrochemical

effects on the fluorescence yield have been shown under

special conditions like a salt jump or redox titrations (e.g.,

Dau and Sauer 1991; Krieger and Rutherford 1997).

According to this interpretation, Assumption 2 is not valid.

Especially in the model of Vredenberg the electric field is

an important driving force for the fluorescence rise,

determining the IP phase. Vredenberg (2000) showed that

following a pre-flash the F0 of the OJIP transient measured

shortly afterward was increased by 18 % and showed a

relaxation time in darkness of 105 s. Vredenberg linked

this to changes in the lateral resistance of the chloroplast

compartments showing similar relaxation kinetics. How-

ever, as shown by Schansker and Strasser (2005), QB-

induced by a 10-s FR light pulse also causes an increase of

F0 and relaxation time of 100 s. This effect—also induced

by an STF—is due to the equilibrium between QA and QB-,

where the electron at pH 7 spends 5 % of its time on QA

and 95 % of its time on QB (Diner 1977) and the equilib-

rium shifts further to QA as the stromal pH increases

(Robinson and Crofts 1984). Bulychev and Vredenberg

(2001) worked with samples with a destroyed PSII donor

side. They used N,N,N’,N’-tetramethyl-phenylenediamine

dihydrochloride (TMPD) and phenazine methosulfate

(PMS) as electron donors, assuming that these compounds

only donated electrons to PSI and showed that under these

conditions TMPD and PMS stimulate the fluorescence rise.

The authors interpreted this as an influence of the PSI-

generated electrical field on PSII fluorescence. However,

both ascorbate (midpoint potential of 58 mV (Loach

1976)) and TMPD (midpoint potential of 240 mV (Prince

et al. 1981)) can donate electrons to the donor side of Mn-

depleted PSII (see Mano et al. (2004) and Toth et al. (2009)

for ascorbate and Tamura et al. (1990) for TMPD), making

it likely that PMS with a midpoint potential of 85 mV

(Prince et al. 1981) can do this as well. In addition, PMS is

also a fluorescence quencher (Wientjes and Croce 2012).

Vredenberg et al. (2006) observed that a train of 40 STFs

leads to a gradual increase of both the initial and maximum

flash-induced fluorescence intensity. In the presence of

FCCP (carbonylcyanide-p-trifluoromethoxy-phenylhy-

drazone) this increase is no longer observed. The authors

assumed that FCCP acting as a protonophoric uncoupler

abolishes the alkalinization of the stroma and that, there-

fore, the observed flash-induced fluorescence increase was

related to the alkalinization of the stroma and an accom-

panying shift of the equilibrium between QA and QB-

toward QA (Diner 1977; Robinson and Crofts 1984).

However, FCCP is also an ADRY reagent (McCauley et al.

1987) and as shown by de Wijn and van Gorkom (2001),

under flash conditions FCCP mediates electron transport

between the PQ-pool and the S2 and S3 states of the Mn-

cluster of PSII—possibly via cyt b559, which has been

shown to undergo autooxidation and fast, flash-induced

reduction in the presence of ADRY reagents (Barabas et al.

1993)—keeping the PQ-pool oxidized. This interpretation

is also much more in line with the work of Schansker et al.

(2005) and Toth et al. (2007b) on the relationship between

the PQ-pool redox state and the kinetic properties of OJIP

transients. The inactive centers that Vredenberg et al.

(2006) describe in their paper are in this context substrate

(PQ)-limited PSII reaction centers. Vredenberg and Bulychev

(2003) observed that valinomycin (VMC) infiltrated in

leaves suppresses the IP phase of the fluorescence rise.

VMC can dissipate the electric field and the authors

interpreted this result to indicate that the electric field

drives the IP phase. This interpretation is, however, in

disagreement with the measurements of Schreiber and

Neubauer (1990), who observed that the electric field

decreases during the IP phase. In addition, the effect of the

oxygen concentration on the fluorescence decline beyond

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the P-step in the presence of VMC observed by Sokolove

and Marsho (1979) can also be interpreted as a secondary

effect of VMC on the acceptor side of PSI. This is con-

firmed by the observation that the suppression of the IP

phase in the presence of VMC is accompanied by an

inhibition of the 820-nm transmission increase normally

observed during the IP phase (G Schansker and SZ Toth,

unpublished data). That VMC may have multiple effects on

the ETC is also suggested by the observation that it has an

effect on the JI rise in PSII and thylakoid membranes

(Pospısil and Dau 2002). Vredenberg (2011) and Vreden-

berg et al. (2012) proposed that CET is the electric field

creating driving force during the IP phase. However, there

is consensus in the literature that under high light condi-

tions, under which OJIP transients are measured, the

PQ-pool is largely reduced at the I step, defined as the

fluorescence intensity reached after 20–30 ms of illumi-

nation by e.g., 3,000 lmol photons m-2 s-1 (Lazar 2003,

Zhu et al. 2005, Toth et al. 2007b), and as a consequence

the redox poise necessary for CET (reviewed by Heber

2002) is missing. In addition, it is in that case difficult to

explain why the FM in the presence of DCMU, which has

the same value as in the absence of DCMU (Toth et al.

2005a), can be reached within 10 ms.

In summary, the transmembrane uniform electric field

may very well have a small effect on the fluorescence rise;

however, unambiguous experimental data supporting such

a role, for the moment, are missing. We also want to point

out that the local field within PSII is two-to-three orders of

magnitude more intense than the uniform transmembrane

electric field (Zimanyi and Garab 1989); local fields may

play a significant role in the fluorescence rise, an effect

which will be discussed below.

Conformational changes

A fluorescence yield change induced by a light-driven

conformational change within the RC is another possible

determinant of the fluorescence rise; in that case both

Assumptions 1 and 2 of the QA model must be considered

to be invalid. Moise and Moya (2004a, b) were the first to

consider conformational changes as an explanation for the

thermal phase in detail. Using multi-frequency phase and

modulation fluorometry, they determined the fluorescence

lifetimes and fluorescence yields along the fluorescence

rise (induced by a light intensity of 70 lmol photons

m-2 s-1). A non-linear relationship between lifetime and

yield was observed, which was proposed to reflect a con-

formational change within PSII, in particular, in CP47

(Moise and Moya 2004a, b). These conformational changes

were observed both in the physiological temperature range

and at cryogenic temperatures, and both in the absence and

presence of DCMU, but did no longer occur below

-50 �C. Based on a study of the temperature dependences

and induction/relaxation kinetics of fluorescence transients

in a wide temperature interval Schansker et al. (2011) came

to the same conclusion as Moise and Moya (2004a, b): the

process associated with the thermal phase is related to a

conformational change. However, these authors were still

able to detect dark relaxation of the fluorescence intensity

attributable to conformational changes at temperatures as

low as -60 �C. The difference between the two sets of

low-temperature data, criticized by Stirbet and Govindjee

(2012), can readily be explained by the different light

intensities used in the two studies and the fact that charge

stabilization (Brudvig et al. 1983; Schlodder 2008) and

probably also the conformational changes (e.g., via protein

flexibility) become hindered at low temperatures. Schansker

et al. (2011) used not only much stronger light, 3,500 lmol

red photons m-2 s-1, but also gradually increased the pulse

length when decreasing the temperature, from 80 ms at

-20 �C to 300 ms at -60 �C. Further, Schansker et al.

(2011) showed that at cryogenic temperatures the two

fluorescence rise components exhibited substantial activa-

tion energies, and only one process occurred in a temper-

ature independent, nearly activationless manner (as

expected for the primary charge separation within PSII (cf.

Dekker and van Grondelle 2000)). Also, the fast relaxation

of the rise at physiological temperatures could not be

attributed to known redox changes. These observations are

consistent with the involvement of conformational changes

in the fluorescence rise as proposed by Moise and Moya

(2004a, b).

Nature, mechanism, and significance

of the conformational changes

In this section we provide an overview on additional

experimental evidences demonstrating the occurrence of

conformational changes and we will discuss their signifi-

cance as far as the interpretation of the fluorescence rise

parameters is concerned. First, however, we have to

address the problem related to the thermal phase in the

presence of DCMU—mainly because its occurrence is

doubted by some researchers. We also comment on pub-

lished model calculations, which show that the pure QA

model—despite many efforts—cannot offer an appropriate

explanation for the slow fluorescence rise kinetics.

The occurrence of the thermal phase in the presence

of DCMU

When considering the role of conformational changes on

the fluorescence yield of closed centers, we implicitly

assume that the thermal phase is also expressed in the

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presence of PSII inhibitors such as DCMU, and the phys-

ical mechanism(s) underlying the quantitative fluorescence

rise are the same both in the presence or absence of

DCMU, and that the difference is confined to the kinetics,

due to the absence of electron acceptors beyond QA. We

must note, however, that this view is not shared by all

researchers. Stirbet and Govindjee (2012), considering the

arguments in favor or disfavor of either side, suggest that

the thermal phase is absent in the presence of DCMU. In

their view, the ‘‘theories supporting [the idea of its pres-

ence] do not have sufficient experimental support.’’

Concerning the occurrence of thermal phase in the

presence of DCMU, first, we would like to point out that, as

discussed above, the widely accepted model on the fluo-

rescence rise in the presence of DCMU, i.e., explaining the

fluorescence rise in terms of a/ß heterogeneity, is difficult

to reconcile with the very different temperature depen-

dences of the two fluorescence rise phases in the presence

of DCMU. We would also like to stress that, to our

knowledge, there is no experimental evidence supporting

the (in our view much stronger) assumption that the basic

processes and effects related to the fluorescence induction

within PSII in the presence of DCMU are principally dif-

ferent from those in its absence. In fact, the experimental

observation that neither the F0 nor the FM intensities

depend on the presence of DCMU in intact systems (Toth

et al. 2005a) argues against such differences due to the

presence of DCMU in the same way as it argues against

PQ-pool quenching of PSII fluorescence in intact systems.

Also, at low temperature (below -20 �C) fluorescence

properties appear to be identical in the presence and

absence of DCMU. Minor differences, due to differences

e.g., in the recombination kinetics, might be induced by the

shift in the redox potential of QA in the presence of DCMU

(Krieger-Liszkay and Rutherford 1998). Also, the induc-

tion of a further oxidation of non-heme iron upon the

addition of DCMU, in a fraction of PsbA3 PSII, but not of

PsbA1 PSII, as shown by Boussac et al. (2011), to some

extent might pose the possibility of differences between the

nature of fluorescence inductions in untreated and DCMU-

treated samples. On the other hand, Samson and Bruce

(1996) studying the effect of a pre-oxidized non-heme iron

in the presence of DCMU on the flash-induced fluorescence

intensity concluded that the non-heme iron did not signif-

icantly contribute to the low fluorescence yield induced by

an SSTF. It thus seems more likely that a partial oxidation

of the non-heme iron, if it occurs in higher plant leaves

under physiological conditions, will affect only the shape

of the fluorescence rise to some extent but not the final

fluorescence level(s).

Hence, we believe that there exists no convincing evi-

dence that DCMU suppresses the thermal phase. There is,

however, an apparent discrepancy, which requires an

explanation: an SSTF induces a larger FV in the presence of

DCMU than in its absence, *85 versus 65 % in leaves

(e.g., Schansker et al. 2011), and 75 versus 50 % in isolated

thylakoid membranes (Joliot and Joliot 1979; Samson and

Bruce 1996; Vredenberg 2000). (This difference between

leaves and isolated thylakoid membranes can be explained

by the occurrence of PQ-pool quenching in isolated thy-

lakoid membranes (cf. Samson and Bruce 1996) but not in

leaves (Toth et al. 2005a) (see above). Alternatively, a

structural re-organization of the entire thylakoid membrane

and/or changes in the extent of stacking of the thylakoid

membranes during isolation could as well have an effect on

the ratio between the photochemical and thermal phase).

This apparent discrepancy between the data in the presence

and absence of DCMU can be resolved, however, by

analyzing the dark relaxation kinetics instead of the

induction kinetics (Schansker et al. 2011). In that case

*30 % of FV is associated with the thermal/slow rise

phase—both in the presence and absence of DCMU.

Schansker et al. (2011) argued that an explanation for the

observation that only *15 % of FV is observed for the

slow rise phase in the presence of DCMU is a kinetic

overlap between the photochemical phase and the thermal

phase.

Taken together, the above-mentioned data and argu-

ments strongly suggest that the thermal phase is present

both in the absence and presence of DCMU, and thus there

is no need to assume that the physical background of the

phenomena is substantially different in the presence and

absence of DCMU.

Simulations

Stirbet and Govindjee (2012) in their argumentation

emphasized the role of simulations as tools to characterize/

explain the OJIP transient but did not discuss the literature

in detail. Researchers simulating OJIP transients based on

the QA model are faced with simulated fluorescence tran-

sients that rise much faster than the experimentally

observed transients. Processes introduced in the models to

slow the fluorescence rise down mentioned by Lazar and

Schansker (2009) are an electron acceptor X that accepts

electrons from QB at a high rate, PQ-pool quenching, slow

and fast reducing PQ-pools, and extremely slow PSII RCs.

The two models that consist of the most comprehensive

description of the ETC are the models of Lazar (2003,

2009) and Zhu et al. (2005). These are actually two com-

ponent models in the sense that QA as well as the oxidized

PQ-pool are considered to be fluorescence quenchers. In

both cases the maximum fluorescence level is simulated

after *20 ms of illumination. This indicates clearly that

OJIP transients measured at high light intensities PQ-pool

quenching, if it did exist, would not provide an explanation

Photosynth Res

123

for a theoretical OJIP transient reproducing the correct

experimental time course. As demonstrated by Lazar

(2009) the problem is not so much the kinetic model, since

the PSI reactions can be modeled with a correct time

dependence, but the fact that this does not translate into the

right fluorescence amplitudes of the fluorescence rise

phases. In the model of Vredenberg, which is kinetically

simpler, the kinetic phase assigned by Lazar (2003, 2009)

and Zhu et al. (2005) to PQ-pool quenching is attributed to

the release of Pheo quenching (Vredenberg 2000) [or to

active RCs that turn during illumination into inactive RCs

that can then release Pheo quenching (Vredenberg et al.

2006)]. On the basis of QA and Pheo only the OJI rise can

be simulated, whereas the IP phase is simulated assuming

an effect of the electric field on the fluorescence yield

(Vredenberg and Bulychev 2002, 2003; Vredenberg et al.

2007).

The fact that on the basis of realistic, experimentally

proven parameters (see I.2.4 for a discussion of the electric

field), no satisfactory simulation has been obtained so far,

suggests that (i) either of the basic assumptions are not

valid and/or (ii) additional factors must be taken into

consideration. A simulation of the OJIP transient using a

conformational change model as proposed by Schansker

et al. (2011) has not been tried yet; it definitely will require

a deeper understanding of the nature of the changes and the

underlying physical mechanism(s).

The nature and mechanism of the conformational

changes

The temperature dependence of the fast and slow rise

phases in the presence of DCMU yields important infor-

mation on the character of these phases. Schansker et al.

(2011) showed that the fast rise phase was nearly temper-

ature independent, whereas the slow rise phase had a strong

temperature dependence, strongly suggesting that the fast

and the slow rise phases represent two fundamentally dif-

ferent processes. As a consequence, the kinetic overlap

observed at room temperature disappears, leading to a

disappearance of the sigmoidicity of the fluorescence rise

(Schansker et al. 2011). We do stress, however, that

(i) these observations are unrelated to sigmoidicity that

may occur in the absence of DCMU and (ii) this does not

necessarily imply that connectivity between PSII antennae

does not exist. In fact, recent quenching analyses based on

fluorescence lifetime measurements show that in isolated

thylakoid membranes at least four PSII supercomplexes

appear to form connected domains (Lambrev et al. 2011).

Concerning the nature of the conformational change,

another feature of the fluorescence rise in the presence of

DCMU is important. The initial part of the fluorescence

rise is sigmoidal. This is traditionally interpreted as a

consequence of energy transfer between PSII antennae

(Joliot and Joliot 1964). However, Vredenberg (2004,

2008) showed that sigmodicity can also be observed if

kinetic overlap between two consecutive processes with

exponential kinetics occurs. As noted above, a precondition

for this sigmoidicity is that the two rate constants differ by

less than one order of magnitude. Schansker et al. (2011)

proposed that the overlapping process originated from a

light-induced conformational change. Both France et al.

(1992) and Steffen et al. (2001) observed that the fluores-

cence rise induced by a single excitation is exponential.

This suggests that a single excitation induces only a single

process and not both a stable charge separation and the

additional process in parallel: a conformational change

according to the interpretation of Schansker et al. (2011).

Vredenberg (2004) also interpreted the observation of

Steffen et al. (2001) in sequential terms. In addition, France

et al. (1992) using a pump-probe approach observed that

short light pulses of 2 ls or less induced an exponential

fluorescence rise, irrespective of the intensity of the pulse

(the number of excitations per pulse). Sigmoidicity could

only be achieved with two short flashes spaced by a dark

interval of some tens of microseconds or by a sufficiently

long (C50 ls) light pulse. This confirms the involvement

of two sequential reactions (first the reduction of QA and

only then the induction of the conformational change), of

which the second can only occur following a short dark

interval. These results were contested by Hemelrijk and

van Gorkom (1992); however, the latter authors used

continuous measuring light instead of a pump-probe

approach. Thus the comparability of the two sets of

experiments is not straightforward.

The experiments discussed above demonstrate that there

exist two processes involved in the fluorescence rise that

differ in their properties and of which only one can be

assigned to the reduction of QA. Presently, it is unclear why

two excitations of a PSII RC occurring within 2 ls fail to

induce the conformational change(s). A possible explana-

tion is that the stabilization of the charge separated state, or

just the stabilization of the QA- state requires 2–50 ls. As

argued by Schansker et al. (2011) and discussed in more

detail in the following section, the redox state of QA seems

to be important for the stability of the conformational

changes and the amplitude of the associated fluorescence

change.

The stability of QA- determines the stability

of the conformational changes in PSII

Although experimental observations inconsistent with the

pure QA model already exist for many years, alternative

interpretations never gained wider popularity because a

credible mechanism for such an alternative interpretation

Photosynth Res

123

was missing. The QB-site occupancy theory (Vasil’ev and

Bruce 1998; Samson et al. 1999; Yaakoubd et al. 2002)

provides a good starting point for an argument that allows

us to identify the factor that determines the stability of the

conformational changes and how the light-induced con-

formational changes can follow the reduction of QA.

As the ETC becomes reduced, fewer oxidized PQ mole-

cules will be bound to the QB-sites and the re-oxidation rate

of the QA molecules will decrease, i.e., QA will become more

stably reduced. At a light intensity of 15,000 lmol photons

m-2 s-1, the excitation rate (*1 excitation per PSII per

40 ls) is much higher than the forward electron transfer from

QA to QB (halftime of 100–200 ls for the first and

400–600 ls for the second electron (Petrouleas and Crofts

2005). After 2 ms of illumination [99 % of QA may be

reduced whereas the fluorescence intensity is still 30–40 %

below FM (cf. Schansker et al. 2006). However, the lifetime

of QA- in each PSII RC does not depend on the light intensity

but on the rate of forward electron transport, which in turn

depends on the redox state of the ETC. In other words, if the

induction and stability of the conformational changes would

depend on the stability of QA- in each PSII RC the accom-

panying fluorescence yield changes would be sensitive to the

reduction of the ETC (Schansker et al. 2011). It is possible

that as the electron transport chain becomes gradually

reduced there is a continuous induction and relaxation of the

proposed conformational changes and that the fluorescence

yield change associated with these conformational changes

also makes a small contribution to the OJ rise. The rela-

tionship between ETC redox state and thermal phase can also

explain the dependence of the area above the OJIP transient

on the number of electrons needed to reduce the ETC

(Strasser and Strasser 1995; Joliot and Joliot 2002; Toth et al.

2007b).

The above concept linking the stability of QA-, in broad

terms, to the light-induced conformational changes

(Schansker et al. 2011), offers an explanation for the cor-

relation of the JIP phase with the ETC redox state. In order

to explain the observation that the thermal phase and the

slow fluorescence rise in the presence of DCMU, as

stressed above, can only be induced by light following the

reduction of QA, further assumptions must be made. We

must assume that stabilization of the charge separated state

or that of QA- occurs with some delay relative to the

reduction step. It can be speculated that electrostatic cou-

pling between the donor and acceptor sides can account for

such an effect. The existence of such an effect can be

inferred from data showing that treatments affecting the

donor side of PSII like Ca2? depletion can exert an effect

on the redox potential of QA (e.g., Krieger and Rutherford

1997). Because of the presence of strong local fields, the

role of ionic movements should also be considered in these

processes (see below).

Experimental evidence for the occurrence

of conformational changes in PSII and purple bacteria

Several independent observations suggest the occurrence of

conformational changes in PSII, and also in purple bacte-

rial RCs. It has been shown that external electric fields,

applied during illumination and cooling, can induce chan-

ges in the thermoluminescence and thermodepolarization

currents of isolated thylakoid membranes, suggesting

transitions between conformational states of PSII RCs

(Knox et al. 1984). Delosme et al. (1994) studying spinach

PSII membranes and Chlamydomonas reinhardtii core

particles reported that charge separations induce volume

changes (contractions) in PSII. This was confirmed for

manganese-depleted Synechocystis PCC6803 core parti-

cles (Hou et al. 2001), showing that the conformational

changes do not depend on the activity of the oxygen-

evolving complex. In addition, a secondary reaction lead-

ing to an expansion of the sample (5–10 ls of illumination)

was observed; an expansion missing in bacterial RCs and

PSI (Hou et al. 2001).

There exists a vast literature demonstrating light-

induced conformational changes in purple bacterial RCs

(e.g., Abgaryan et al. 1998; McMahon et al. 1998;

Goushcha et al. 2000; Barabash et al. 2002). All these

observations strongly suggest a close association between

conformational transitions and the functioning of PSII-type

RCs. The light-induced conformational changes in bacte-

rial RCs are often rather short-lived. Nagy et al. (2008)

ascribed fluorescence changes in Rhodobacter sphaeroides

with a lifetime of 30–40 ls to a conformational change. In

purple bacteria a change in the protein environment of the

bacteriochlorophyll dimer P has been proposed to occur in

response to these conformational changes (Deshmukh et al.

2011a, b). Of particular interest to the present discussion of

the thermal phase in the PSII fluorescence induction are,

however, studies on bacterial RCs which demonstrate that

(i) the effects of light-induced conformational changes on

the fluorescence parameters (and their relaxation) depend

critically on the lifetimes of the internal electric fields

related to the type and lifetime of the various radical pair

species as intermediates of the charge separation/recom-

bination process (Muller et al. 1995, Holzwarth and Muller

1996), (ii) that the light-induced conformational changes

can be accumulated by subsequent multi-excitations

(Goushcha et al. 1997a; Abgaryan et al. 1998), and (iii) are

principally following a highly non-linear system behavior

(Christophorov et al. 2000, 2003; Goushcha et al. 1997b,

1999, 2000). It should be noted that the term ‘‘light-

induced conformational change’’ used here explicitly

includes not only changes in the conformational backbone

of the protein surrounding the cofactors, but also—and

very likely—different protonation states of amino acid side

Photosynth Res

123

chains which can be brought about by small conforma-

tional changes of the backbone, by a strong electric field

and modified redox states of cofactors. Quite generally, the

implied ‘‘conformational changes’’ are caused by the light-

induced stimulation of charge species (either radical pairs,

charged states, or explicit proton movements) that move or

migrate through the RC protein complex.

Potential processes connecting conformational changes

to a fluorescence yield change causing the additional

fluorescence

When searching for the nature and mechanism of the

conformational changes in PSII responsible for the addi-

tional fluorescence rise component, there are several key

observations which must be taken into consideration.

From the data discussed above, it seems clear that the

conformational changes are primarily induced after the

reduction of QA (Schansker et al. 2011); also, it has been

shown that the additional fluorescence rise, which is

proposed to be associated with the conformational chan-

ges, can only be induced by an additional flash following

the first SSTF with a lag phase, in the 2–50 ls range,

rather than by increasing the intensity of the second flash

fired with a shorter dark interval (France et al. 1992); the

analysis of the fluorescence rise kinetics with long flashes

also indicates that the activationless process attributable

to a charge separation occurs first and it is followed by

the slower rise phase(s) assigned to conformational

changes (Schansker et al. 2011). These findings are in line

with the notion discussed above, that the extent of con-

formational changes induced in the RC complex is con-

trolled primarily by the strength and lifetime of electric

fields and charges moving through or being localized

within the RC. For the present discussion the QA- state is

the most long-lived charged state in the RC. Using the

same arguments, similar effects as caused by internal

electric fields, but perhaps of smaller magnitude, can then

be also caused by external electric fields across the

membrane in which the RC is located.

The lag phase and the above sequence of events may

give us a hint concerning the process(es) occurring after the

reduction of QA. One can speculate that this might be

correlated with (subsequent) charge redistributions in the

aqueous (electrolyte) phases following the generation of

vectorial charge separation in the membrane (Fowler and

Kok 1974; Junge 1977). Indeed, Osvath et al. (1994), by

investigating the light gradient electric signal induced by

short laser flashes on magnetically aligned thylakoid

membranes, have shown that the ionic equilibration in the

lateral direction occurs with a time constant of about 20 ls.

This may bring about conformational changes in the

vicinity of the local charges.

Considering closed RCs (QA reduced), an additional

light reaction and the mechanism responsible for the light-

induced conformational changes, the most likely hypothe-

sis is that they can be induced by the multiple, transient

formation of a P?Pheo- charge separations, which also

occur in closed RCs (Szczepaniak et al. 2008, 2009;

Martinez-Junza et al. 2008) and which can transiently

induce a very strong electrical field, that persists longer

than in open RCs. Such conformational changes do not

necessarily relax fully and can be accumulated by multiple

turnovers. It cannot be ruled out either that protein con-

formational changes are induced or driven locally by the

ultrafast local heat packages produced via (nonradiative)

dissipative processes due to recombination of the charges

in the RC and/or due to dissipation of the excited triplet

states of chl molecules in the core; these, in turn, can

induce elementary structural changes (Cseh et al. 2000;

Gulbinas et al. 2006). The generation of local electric field

and/or thermal transients may facilitate the conformational

adjustment of the reaction center proteins to the local field

due to the presence of QA- coupled to D?, where D is an

electron donor molecule.

Conformational changes may readily explain (light-

induced) changes in the fluorescence yield by affecting the

redox potential of for example the P680/P680? couple.

A similar process has been proposed in photosynthetic

bacteria (Deshmukh et al. 2011a, b). Most likely, a variety

of molecular localizations and conformationally induced

changes of internal rates or redox potentials in the RCs add

up to provide the overall effects of light-induced confor-

mational changes on the observed fluorescence yields. The

picture is complex and at present the various effects are not

studied in detail. Nevertheless, the principal effects are

sufficiently clear to conclude that light-induced confor-

mational changes do have significant effects on fluores-

cence yields in RCs. It is important to note that these

effects are intimately related to the functioning of RCs.

Suffice it to note that it is very clear now that energetic

relaxations of the radical pair energies do occur after

charge separation in RCs (both in open as well as closed

RCs) without which photosynthetic primary processes

would not be efficient and stable charge separation would

not occur over sufficiently long time scales to allow the

subsequent catalytic processes for energy storage (Holzwarth

2008a, b).

The data presented in Schansker et al. (2011) are also

compatible with the suggestion that the fluorescence yield

increase following the reduction of QA reflects a molecular

switch between a photosynthetic and a dissipative state as

earlier suggested by Raszewski and Renger (2008) for the

entire FV. Experimental support for a change in the prop-

erties of PSII RCs following the induction of the confor-

mational changes was obtained by Martinez-Junza et al.

Photosynth Res

123

(2008) and Braslavsky and Holzwarth (2012). Again, it is

quite conceivable that this dissipative state helps—inter

alia—to protect PSII RCs in vivo from damage due to over-

excitation in situations where PSII RCs are closed. The

induction of this putative dissipative state takes less than

10 ms in the presence of stably reduced QA and the

reversal to the energy conversion state takes less than

100 ms once the light is turned off and may even be faster

following the oxidation of QA (Schansker et al. 2011). The

kinetic analysis of these authors also indicates that in a

small fraction of PSII reaction centers QA- becomes re-

oxidized by recombination with the donor side within the

first 100 ms of darkness (relaxation time of 0.4–0.6 s

(Schansker et al. 2005, 2011)) but this does not affect the

analysis and interpretation of the data. The idea is illus-

trated in Fig. 2.

Interpretation of the FV/FM value

The FV/FM ((FM-F0)/FM) is undoubtedly the most widely

used parameter in photosynthesis research. Kitajima and

Butler (1975) using a theoretical treatment of energy fluxes

in PSII based on the concept of Duysens and Sweers (1963)

showed that FV/FM is an expression of the maximum

quantum yield of primary photochemistry of PSII. How-

ever, this analysis is only correct if the redox state of QA is

the only determinant of the fluorescence yield. Within the

context of the conformational change concept, as well as

with the other alternative models, this derivation is not

valid. Another argument that has been put forward against

the FV/FM has to do with the FM value. Neubauer and

Schreiber (1987) suggested that the IP phase was due to a

release of PQ-pool quenching. Schreiber et al. (1995)

considered on this basis the use of the fluorescence

intensity at the I step for the calculation of the FV/FM.

Falkowski and co-workers (e.g., Prasil et al. 1996) argued

that the FM induced by an SSTF was the correct FM value

for the calculation of the FV/FM and that the rest was the

release of PQ-pool quenching. However, as discussed

above, PQ-pool quenching is not observed in leaves.

Recently, Vredenberg and Prasil (Vredenberg et al. 2012)

argued that the IP phase was due to a CET-induced fluo-

rescence yield change and, therefore, in their view, the FM

was problematic for this reason (however, see I.2.4 for a

discussion on the role of CET in the fluorescence rise).

It may be noted here that in the case of photoinhibition,

where there is a linear correlation between the FV/FM value

and the severity of the photoinhibition treatment, it has to

be assumed that the maximum quantum yield of each PSII

RC can change gradually. Although redox titrations of QA

show that potential and variable fluorescence correlate

(e.g., Krieger-Liszkay and Rutherford 1998), this has not

been shown to occur.

The meaning of the FV/FM value can also be defined in a

more phenomenological way. Measurements on protoplasts

of Valerianella locusta (Van Wijk and Krause 1991) and

pea leaves (Schansker and Van Rensen 1999) showed that

the decline of the FV/FM value induced by photoinhibition

paralleled the loss of the oxygen-evolving capacity of PSII,

making it a good parameter to follow the inactivation of

PSII RCs. In the case of heat stress a decline in the FV/FM

value was shown to be due to the increasing inability of the

remaining PSII RCs to reduce the ETC before FNR is

activated (Toth et al. 2005b). Other studies have demon-

strated that linear correlations can be obtained between the

parameter FV’/FM’ (determined in the light-adapted state)

and the quantum yield of CO2 fixation (e.g., Harbinson

et al. 1990; Oberhuber et al. 1993).

Fig. 2 Schematic

representation of the relaxation

and regeneration of the thermalphase. A first strong pulse of

light generates a full OJIP

transient in *200 ms; in

darkness the thermal phaserelaxes within 100 ms and can

be regenerated within 3 ms by a

second light pulse. For the

measurement pea leaves were

used. The chl a fluorescence was

induced by two 5,000 lmol

photons m-2 s-1 pulses of

700 ms and 2 s, respectively,

spaced 80 ms apart. The figure

is modified from Fig. 7 of

Schansker et al. (2011)

Photosynth Res

123

Flash-oxygen measurements may be more appropriate to

determine the PSII quantum yield of primary photochem-

istry. As pointed out above, Shinkarev (2005) calculated an

average miss factor of 7–8 % for the flash-oxygen yield,

implying that the PSII quantum yield of primary photo-

chemistry is[0.92/0.93. In other words, the PSII quantum

yield of primary photochemistry is considerably higher

than normally thought and probably also considerably

more invariable than FV/FM.

Conclusions

Although the QA model, proposed by Duysens and Sweers

nearly four decades ago, has proven to be very successful

and, in broad terms, provided a good model as far as the

photochemical activity of PSII is concerned, it is also

becoming more and more evident that (an) additional

process(es) has/have to be taken into account to understand

the complexity of the fluorescence rise kinetics, most

particularly the so-called thermal phase, the slowly rising

kinetic components of the fluorescence transient.

In this paper we have given an overview of the most

important observations that are difficult to understand

within the framework of the pure QA model and have

critically reviewed alternative explanations, paying partic-

ular attention to the role of light-induced conformational

changes. They appear to offer the most consistent and

convincing explanation for the wide body of presently

available experimental data both in the absence and pres-

ence of DCMU and both at physiological and cryogenic

temperatures. Although many open questions remain,

especially with regard to the precise molecular nature and

mechanism of these changes, the fact that conformational

changes have been observed in PSII reaction centers in

independent experiments, and also in purple bacterial RCs,

adds strong support to this proposal. The observation that

there exist important differences between the electron

transport rates and intermediates of open and closed RCs

points toward the crucial role of conformational changes in

controlling a molecular switch between photosynthetic and

dissipative states.

Amending the pure QA model by the QA plus confor-

mational change model has important consequences for the

interpretation of the parameter FV/FM. While it evidently

remains a sensitive parameter related to the photochemical

activity of PSII, its exact quantitative meaning will have to

be determined again. Further, since the sigmoidal and

biphasic rise kinetics of the chl a fluorescence (in the case

of DCMU blocked RCs) depend on the induction of this

second process, i.e., a superimposition of a fluorescence

yield change driven by light-induced conformational

changes on the reduction kinetics of QA, conclusions

regarding the connectivity of PSII units and on PSII het-

erogeneity, in terms of e.g., a/ß centers, will have to be re-

evaluated critically.

Acknowledgments The authors thank Drs. Petar Lambrev, Laszlo

Kovacs, and Fabrice Rappaport for useful discussions. This work was

supported by the Hungarian Research Foundation (OTKA, grant no.

MB08B82403, PD72718 and CNK80345 to G.S., S.Z.T. and G.G.,

respectively), and by NIH-A*STAR grant (TET-10-1-2011-0279) to

GG. S.Z.T. acknowledges financial support by the Bolyai Janos

Research Foundation of the Hungarian Academy of Sciences. This

work was also supported by the Marie Curie Initial Training Network

‘‘HARVEST’’ sponsored by the 7th Framework Program of the

European Union (grant number 238017 to G.G. and A.R.H.).

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