Quantitative data on laser flash-induced variable fluorescence in the 100 ns to 1 ms time range (Belyaeva et al. in Photosynth Res 98:105–119, 2008) confirming those of others (Steffen et al. in Biochemistry 40:173–180, 2001, Biochemistry 44:3123–3132, 2005; Belyaeva et al. in Biophysics 51(6):976–990, 2006), need a substantial correction with respect to magnitude of the normalized variable fluorescence associated with single turnover-induced charge separation in RCs of PS II. Their data are conclusive with the involvement of donor side quenching, the release of which occurs with a rate constant in the range of tens of ms-1, and presumed to be associated with reduction of YZ+ by the OE

Vredenberg, W.J.

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Kinetic models of photosystem II should accommodate the effect of donor side quenching on variable chlorophyll a fluorescence in the microseconds time

Wim J. Vredenberg

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LETTER TO THE EDITOR
Kinetic models of photosystem II should accommodate the effect
of donor side quenching on variable chlorophyll a fluorescence
in the microseconds time range
Wim J. Vredenberg
Received: 6 January 2009 / Accepted: 13 July 2009 / Published online: 28 July 2009
 The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract Quantitative data on laser flash-induced variable
fluorescence in the 100 ns to 1 ms time range (Belyaeva et al.
in Photosynth Res 98:105–119, 2008) confirming those of
others (Steffen et al. in Biochemistry 40:173–180, 2001,
Biochemistry 44:3123–3132, 2005; Belyaeva et al. in Bio-
physics 51(6):976–990, 2006), need a substantial correction
with respect to magnitude of the normalized variable fluo-
rescence associated with single turnover-induced charge
separation in RCs of PS II. Their data are conclusive with
the involvement of donor side quenching, the release of
which occurs with a rate constant in the range of tens of ms-1,
and presumed to be associated with reduction of Yþz by
the OEC.
Keywords Chlorophyll a fluorescence  PS II modeling 
Donor side quenching
Measurement of chlorophyll (Chl) fluorescence with high
time resolution is a non-invasive method, which offers a
valuable tool for quantitative analyses of the dynamics and
time patterns of the primary processes in photosystem II
(PS II) of photosynthesis. In a recent paper ‘‘PS II model-
based simulations of single turnover flash-induced tran-
sients of fluorescence yield monitored within the time
domain of 100 ns–10 s on dark-adapted Chlorella pyre-
noidosa cells’’ (Belyaeva et al. 2008). Natalia Belyaeva
et al. from Andrew Rubin’s and Gernot Renger’s groups
have shown impressive results of a quantitative analysis of
the chlorophyll fluorescence transients in a time domain
that covers eight decades.
Their paper raises, however, a problem with respect to the
magnitude of the variable fluorescence FSTFv (=F
STF
m - Fo)
that is associated with a single turnover of PS II which
comprises charge separation and stabilization in its reaction
center (RC). Fo is the initial dark fluorescence level and
minimal due to full photochemical quenching of fluores-
cence emission in antennas of so-called open RCs; FSTFm is
the maximal fluorescence of so-called semi-closed RCs
which all have made one turnover and an electron trapped at
the secondary acceptor QA and the positive charge at the
donor side beyond the primary donor P680. The single
turnover-induced formation of QA
-(QA - reduction) has
caused an increase in fluorescence emission due to the
release of photochemical quenching by QA. Usually time
responses of fluorescence emission F(t) in the light are
plotted relative to Fo.
F(t)/Fo data in Chlorella (Belyaeva et al. 2008, see
Figs. 2, 3) show, in agreement with those reported by
Ronald Steffen et al. for other species, that the maximum of
the normalized variable fluorescence nFSTFv (=[F
STF
m - Fo]/
Fo) upon a saturating 10 ns laser flash is reached in the
time range between 10 and 100 ls with 0.8 \ nFSTFv \ 1.
Values of nFSTFv in this range are at variance with and 50%
below nFSTFv * 2 reported for a variety of organisms and
routinely measured with flashes of 30 ls duration in a
Dual-Modulation Kinetic Fluorometer (PSI, Brno, Cz).
These 30 ls-flashes can be considered as STFs under the
conditions used. Moreover, it has been reported that double
(TTF) and multiple excitations with these STFs causes a
relatively small and transient increase in nFSTFv ascribed to
quenching release associated with electron trapping in
reduced QB-nonreducing (semi-open) RCs (Vredenberg
et al. 2007).
W. J. Vredenberg (&)
Department of Plant Physiology, Wageningen University
and Research, Droevendaalsesteeg 1, 6708 PB Wageningen,
The Netherlands
e-mail: wim.vredenberg@wur.nl
123
Photosynth Res (2009) 102:99–101
DOI 10.1007/s11120-009-9477-4
If one would accept nFSTFv = 1 from Belyaeva’s model
and experiments, it would mean that the release of photo-
chemical quenching (QA reduction) has to be supplemented
with an approximate threefold higher release of fluores-
cence quenching from other origin, in order to accommo-
date nFSTFv * 4 in multi-turnover light pulses (MTF-
excitation). The latter value corresponds with FMTFv /
FMTFm * 0.8, which is a common and ‘proper’ value for
healthy preparations. It is difficult to imagine that the
candidates for this formidable quenching job that are
mentioned in their paper can do it.
In addition, the kinetic pattern of the decay in the 100 ls
to 10 s time range suggests that, according to size and
pattern of the decay in the time range above 20 ms, re-
oxidation of QA
- in*50% of RCs occurs in a time above
20 ms. One would expect such high fraction of RCs with
low turnover rate of PS II only in preparations with
attenuated photosynthetic efficiency. However, the decay
patterns presented in Figs. 2 and 3 of the referred paper are
also at variance with those reported by other research
groups. These routinely show that the fraction with slow
decay in the time range above 10 ms is 10–30% of the total
RCs and has been attributed to that of QB-nonreducing RCs
(Vredenberg et al. 2006).
Size and kinetic pattern of the F(t)/Fo response are
determined by the rate constants of the release of fluores-
cence quenching by the (dark) oxidized primary acceptor
pair pheophytin (Phe) and QA and by (photo-) oxidized
intermediates in the PS II donor side electron transfer
pathway (Vredenberg 2008). Specifically it has to be con-
sidered that the kinetics of laser-induced fluorescence
changes in the 1–200 ls time range are determined (i) by
the rate constant(s) of the fluorescence increase due to
release of donor side quenching (DSQ) and (ii) by that of
the fluorescence decrease due the recovery of fluorescence
quenching associated with the re-oxidation of QA
- at the
acceptor side.
Briefly, a non-quenching condition (or state) of RCs
with QA
- and life time (1/kAB) in the range between 150 and
500 ls is formed with rate constant (ke) of the order of
106 ms-1 (Belyaeva et al. 2008; Vredenberg 2008). The
rate of quenching release is substantially attenuated with
respect to ke and is determined by the rate constant of DSQ-
release, which we might call kdsq. It follows that the nor-
malized fluorescence response F(t)/Fo in this simplified
concept with 100% QB-reducing RCs can be approximated
by the relation
FðtÞ
Fo
¼ 1 þ nFSTFv ð1  ekdsqtÞekABt ð1Þ
in which, nFSTFv is the normalized variable fluorescence
associated with STF excitation (see for an extensive deri-
vation and explanation Vredenberg and Prasil 2009).
For QB-nonreducing RCs kAB in Eq. 1 is replaced by
k-nqb where k-nqb  kAB is the approximate average rate
constant of the slow re-appearance of quenching associated
with recovery of these RCs. For a heterogeneous system
with a b-fraction (S0) of QB-nonreducing RCs, Eq. 1 can be
rewritten with
FDSQðtÞ ¼ FðtÞ
Fo
¼ 1 þ nFSTFv ½ð1  ekdsqtÞfð1  bÞekABt
þ beknqbtg: ð1aÞ
Ample evidence has been given that nFSTFv * 2 in leaves
and thylakoids. This value, with according to definition
nFSTFv (=F
MTF
m /Fo - 1) * 2nF
STF
v * 4, corresponds with
FMTFv /F
STF
v * 0.8, which is the ‘proper’ value for healthy
preparations. Under conditions at which kAB  0.1 ms-1
which is true for QB-nonreducing RCs or in the presence of
DCMU, the graph of Eqs. 1 and 1a will show an exponential
rise with reaction time 1/kdsq toward a maximum with F(t)/
Fo = 1 ? nF
STF
v * 3. This level will also be reached under
conditions at which kdsq  kAB. In this context, it is
noteworthy that in the papers of Belyaeva (2006, 2008)
and of Steffen et al. (2001, 2005), the maximum F(t)/Fo
values are around 1.9. The significantly reduced level of
maximal variable fluorescence after laser flash excitation
could be due to (i) either a poor quality of the preparations or
(ii) to the rate constant kdsq of DSQ release when this is less
than 2 orders of magnitude smaller than that of QA
-
re-oxidation (kAB). A closer analysis, using Eqs. 1 and 1a,
will point to evidence for the second interpretation.
Figure 1, with experimental data (closed black dia-
monds) reproduced from Steffen (Steffen et al. 2005, see
Fig. 2 therein), and of similar shape as that reported by
Belyaeva et al. (2006, 2008) will serve a further explana-
tion and illustration. The best fit (solid red line) for
FDSQ(t) = F(t)/Fo shows (i) a rise from 1 (at 100 ns) to
*1.9 reached at t * 20 ls, and (ii) the well documented
biphasic decay with fast (F) phase in the 0.02–1 ms time
range towards an intermediate plateau level Fpl at
FDSQ(t) * 1.3 followed by the slow (S) phase far into the
tens of seconds time range. We have assumed the following
parameter values which are in the range commonly found
in thylakoids and intact leaves: normalized variable fluo-
rescence in STF, nFSTFv = 1.8, rate constants (in ms
-1) for
DSQ release (kdsq), QA
- re-oxidation (kAB), and quenching
recovery in double reduced QB-nonreducing RCs (k-nqb) 35,
10, and 0.025, respectively, and fraction of QB-nonreduc-
ing RCs b (=Fpl/nFSTFv
)*18%. After substitution in Eq. 1a
one obtains the simulated time responses of FDSQ(t). The
rough simulation, illustrated in Fig. 1 and based on a
simplified reaction scheme, shows a reasonable corre-
spondence of the simulation with experimental curve
(Steffen et al. 2005, Fig. 2), and a substantial attenuation of
100 Photosynth Res (2009) 102:99–101
123
the maximum in the F(t)/Fo curve with respect to
nFSTFv = 1.8. The attenuation decreases with a decrease in
kAB, i.e. with attenuation of electron transport at the
acceptor side of PS II. This indicates that the low F(t)/Fo
maximum in the experimental curve is due to the inter-
ference of the rate constants DSQ release and of QA
- re-
oxidation (recovery of quenching). Similarly one can show
that the F(t)/Fo response changes (blue solid curve) when
the rate constant of the release of DSQ is assumed to be 50-
fold higher with kdsq* 15 ls
-1, which would mean the
ignorance of DSQ release in a time domain above *10 ls.
In summary, the quantitative data on laser flash-induced
variable fluorescence from the 100 ns to 1 ms time range
(Belyaeva et al. 2008) confirming those of others (Steffen
et al. 2001, 2005; Belyaeva et al. 2006), need a substantial
correction with respect to magnitude of the normalized
variable fluorescence associated with single turnover-
induced charge separation in RCs of PS II. Their data are
conclusive with the involvement of donor side quenching,
the release of which occurs with a rate constant in the range
of tens of ms-1, and presumed to be associated with
reduction of Yþz by the OEC.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
References
Belyaeva NE, Paschenko VZ, Renger G, Riznichenko GYu, Rubin
AB (2006) Application of photosystem II model for analysis of
fluorescence induction curves in the 100 ns to 10 s time domain
after excitation with a saturating light pulse. Biophysics
(translated from Biofizika) 51(6):976–990
Belyaeva NE, Schmitt F-J, Steffen R, Paschenko VZ, Riznichenko
GYu, Chemeris YuK, Renger G, Rubin AB (2008) PS II model-
based simulations of single turnover flash-induced transients of
fluorescence yield monitored within the time domain of 100 ns–
10 s on dark-adapted Chlorella pyrenoidosa cells. Photosynth
Res 98:105–119
Steffen R, Christen G, Renger G (2001) Time-resolved monitoring of
flash-induced changes of fluorescence quantum yield and decay
of delayed light emission in oxygen-evolving photosynthetic
organisms. Biochemistry 40:173–180
Steffen R, Eckert H-J, Kelly AA, Do¨rmann PG, Renger G (2005)
Investigations on the reaction pattern of photosystem II in leaves
from Arabidopsis thaliana by time-resolved fluorometric anal-
ysis. Biochemistry 44:3123–3132
Vredenberg WJ (2008) Algorithm for analysis of OJDIP fluoresecnce
induction curves in terms of photo- and electrochemical events
in photosystems of plant cells. Derivation and application.
J Photochem Photobiol B 91:58–65
Vredenberg WJ, Prasil O (2009) Modeling of chlorophyll a fluores-
cence kinetics in plant cells. Derivation of a descriptive
algorithm. In: Laisk A, Nedbal L, Govindjee (eds) Photosyn-
thesis in silico. Understanding complexity from molecules to
ecosystems. Springer, Dordrecht, The Netherlands, pp 125–149
Vredenberg WJ, Kasalicky V, Durchan M, Prasil O (2006) The
chlorophyll a fluorescence induction pattern in chloroplasts upon
repetitive single turnover excitations: accumulation and function
of QB-nonreducing centers. Biochim Biophys Acta 1757:173–
181
Vredenberg WJ, Durchan M, Prasil O (2007) On the chlorophyll
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synth Res 93:183–192
1
1.4
1.8
2.2
2.6
0.0001 0.01 1 100 10000
time[ms]
F/
Fo
FDSQ(t),Eq.1a
0.01
0.6
4
10
~60
3.103
Fpl
Fig. 1 Relative chlorophyll a fluorescence change (closed black
diamonds) F(t)/Fo of 1 h dark-adapted Arabidopsis thaliana leaf in
100 ns to 10 s time range (logarithmic) upon saturating laser flash
(6.2 9 1015 photons cm-2/flash), reproduced from Fig. 2 in Steffen
et al. (2005). Bold red curve is the simulated response FDSQ(t) using a
modification of Eq. 1a. The modification accounts for a S0 (b):S1:S2
heterogeneity of 0.2:0.4:0.4 with corresponding rate constants of
donor side quenching kdsq = 300, 60, and 7 ms
-1, kAB*9 ms
-1 and
a biphasic decay of QB-nonreducing RCs with rate constants
k-nqb*25 and 0.5 s
-1 and nFv = 1.8. Note that F
pl is from (reduced)
QB-nonreducing RCs at the fractional size b * 0.3/1.8*18%. The
red dashed curves (closed triangles, diamonds and squares) are
simulations with variable rate constant of quenching recovery (kAB)
due to QA
- reoxidation. Parameter values of variable quenching-
regeneration (kAB) are indicated at the right-hand side of the
respective curves. The blue-colored dashed curve shows the FDSQ(t)
response when, at constant kAB (*10 ms
-1), kdsq is increased 50-fold
(for instance when donor side quenching (DSQ) is ignored). The
dashed curves illustrate the effect of interference between kdsq and
kAB on the maximum of F(t)/Fo with an increasing disproportion
between nFSTFv and the maximum of F
DSQ(t) with the increase in rate
(kAB) of quenching recovery
Photosynth Res (2009) 102:99–101 101
123


Kinetic models of photosystem II should accommodate the effect of donor side quenching on variable chlorophyll a fluorescence in the microseconds time range

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Wageningen Yield 

Application of photosystem II model for analysis of fluorescence induction curves in the 100 ns to 10 s time domain after excitation with a saturating light pulse. Biophysics (translated from

Modeling of chlorophyll a fluorescence kinetics in plant cells. Derivation of a descriptive algorithm. In: Laisk A, Nedbal L, Govindjee (eds) Photosynthesis in silico. Understanding complexity from molecules to ecosystems.

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Kinetic models of photosystem II should accommodate the effect of donor side quenching on variable chlorophyll a fluorescence in the microseconds time

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