The polyphasic chlorophyll a fluorescence rise measured under high intensity of exciting light

Abstract

Chlorophyll a fluorescence rise caused by illumination of photosynthetic samples by high intensity of exciting light, the O–J–I–P (O–I 1 –I 2 –P) transient, is reviewed here. First, basic information about chlorophyll a fluorescence is given, followed by a description of instrumental setups , nomenclature of the transient, and samples used for the measurements. The review mainly focuses on the explanation of particular steps of the transient based on experimental and theoretical results, published since a last review on chlorophyll a fluorescence induction [Lazár D (1999) Biochimica et Biophysica Acta 1412, 1–28]. In addition to 'old' concepts (e.g. changes in redox states of electron acceptors of photosystem II (PSII), effect of the donor side of PSII, fluorescence quenching by oxidised plastoquinone pool), 'new' approaches (e.g. electric voltage across thylakoid membranes, electron transport through the inactive branch in PSII, recombinations between PSII electron acceptors and donors, electron transport reactions after PSII, light gradient within the sample) are reviewed. The K-step, usually detected after a high-temperature stress, and other steps appearing in the transient (the H and G steps) are also discussed. Finally, some applications of the transient are also mentioned.

Full text

CSIRO PUBLISHING
www.publish.csiro.au/journals/fpb Functional Plant Biology, 2006, 33, 9–30
Review:
The polyphasic chlorophyll a fluorescence rise measured under high
intensity of exciting light
Du
ˇ
san Laz
´
ar
Palack
´
y University, Faculty of Science, Department of Experimental Physics, Laboratory of Biophysics,
t
ˇ
r. Svobody 26, 771 46 Olomouc, Czech Republic. Email: lazard@seznam.cz
Abstract. Chlorophyll a fluorescence rise caused by illumination of photosynthetic samples by high intensity of
exciting light, the O–J–I–P (O–I
1
–I
2
–P) transient, is reviewed here. First, basic information about chlorophyll a
fluorescence is given, followed by a description of instrumental set-ups, nomenclature of the transient, and samples
used for the measurements. The review mainly focuses on the explanation of particular steps of the transient based on
experimental and theoretical results, published since a last review on chlorophyll a fluorescence induction [Laz
´
ar D
(1999) Biochimica et Biophysica Acta 1412, 1–28]. In addition to ‘old’ concepts (e.g. changes in redox states of
electron acceptors of photosystem II (PSII), effect of the donor side of PSII, fluorescence quenching by oxidised
plastoquinone pool), ‘new’ approaches (e.g. electric voltage across thylakoid membranes, electron transport through
the inactive branch in PSII, recombinations between PSII electron acceptors and donors, electron transport reactions
after PSII, light gradient within the sample) are reviewed. The K-step, usually detected after a high-temperature
stress, and other steps appearing in the transient (the H and G steps) are also discussed. Finally, some applications
of the transient are also mentioned.
Keywords: fluorescence induction, G step, H step, K step, model, O–J–I–P (O–I
1
–I
2
–P) transient, theory.
I Chlorophyll a fluorescence and the F
0
and F
M
levels
The quantum yield of chlorophyll (Chl) a fluorescence
in a solution (where excitation energy transfer and
photochemistry do not occur) is ∼20–35% (Latimer et al.
1956; Weber and Teale 1957) and this fluorescence has a
lifetime of ∼6–20 ns (M
¨
uller et al. 1969; Avarmaa et al. 1977;
Pfarrherr et al. 1991; Brody 2002). In contrast, the quantum
yield of Chl a fluorescence from the photosynthetic apparatus
is only ∼2–8% (from open to closed reaction centres of
photosystem II, RCII; Latimer et al. 1956; Trissl et al. 1993)
with an average lifetime of ∼300 ps (for open RCII; Keuper
and Sauer 1989; Marder and Raskin 1993; Briantais et al.
1996; Gilmore et al. 1996) and ∼1.6 ns (for closed RCII;
Keuper and Sauer 1989; Marder and Raskin 1993; Brody
2002). Under physiological conditions, fluorescence signal
during fluorescence rise (FLR) is assumed to originate
mainly from photosystem II (PSII) [reviewed by Govindjee
et al. (1986); Krause and Weis (1991); Dau (1994)].
Contribution of photosystem I (PSI) to the overall
fluorescence signal during FLR at room temperature is
∼15–20% (Strasser and Butler 1977; Wong and Govindjee
1979; Stahl et al. 1989; Roelofs et al. 1992; Trissl et al. 1993)
Abbreviations used: See Table 1 for a complete list of abbreviations used.
and its fluorescence is assumed to have a constant level;
fluorescence from PSI contributes only to minimal
fluorescence, F
0
, [however, see also Ikegami (1976) and
Byrdin et al. (2000); section II.4.2.10]. But at emission
wavelengths greater than 700 nm the contribution of the PSI
fluorescence to F
0
can be up to 30–55% (Pf
¨
undel 1998;
Gilmore et al. 2000; Franck et al. 2002). Nevertheless, in a
first approximation, which is well accepted in photosynthesis
research, FLR is understood to originate from PSII.
Minimal fluorescence, F
0
, is defined as the fluorescence
when all RCIIs are open, i.e. when the first quinone electron
acceptor of PSII, Q
A
, is oxidised [see also Vredenberg
(2000); Strasser and Stirbet (2001), and section II.4.2.3 for
alternative approaches]. As Butler (1977, 1978) postulated
that every fluorescence signal comes from Chls of light
harvesting antennae (LHA), F
0
is the fluorescence signal
coming from excited Chls of LHA before the excitations
reach RCII (Mathis and Paillotin 1981). Owens (1996)
suggested that F
0
level is a consequence of the transfer
equilibrium: an equilibrium between the formation of the
excited states among all the light harvesting pigments and
P680 and utilisation of the excited states for reversible
© CSIRO 2006 10.1071/FP05095 1445-4408/06/010009

10 Functional Plant Biology D. Laz
´
ar
Table 1. List of abbreviations used in the text
Abbreviation Definition
A
515
,A
520
Absorbance signal measured at 515 or 520 nm, respectively
ADRY Accelerator of the deactivation reactions of the enzyme ‘Y’ in OEC
Chl Chlorophyll
CP43, CP47 A 43-kDa, 47-kDa Chl containing inner LHAs of PSII
cyt Cytochrome
DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-bezoquinone
DCMU 3-(3

,4

-dichlorophenyl)-1,1-dimethylurea; diuron
DF Delayed fluorescence
F
0
Minimal Chl a fluorescence
FI Chl a fluorescence induction (both the FLR and fluorescence decay)
F
I
Chl a fluorescence signal at the I step in FLR measured under high intensity of exciting light
F
J
Chl a fluorescence signal at the J step in FLR measured under high intensity of exciting light
F
K
Chl a fluorescence signal at the K step in FLR measured under high intensity of exciting light
FLR Chl a fluorescence rise
F
M
Maximal Chl a fluorescence
FNR Ferredoxin-NADP
+
oxidoreductase
FP Fluorescence parameter
F
V
Variable fluorescence (= F
M
− F
0
)
F
V
/ F
M
Maximal quantum yield of PSII photochemistry
HQ 1,4-benzenediol; hydroquinone
LED Light-emitting diode
LHA Chl containing light harvesting antenna(e)
LHCII Chl containing light harvesting complex of PSII
MCA Metabolic control analysis
MV Methylviologen
NADP Nicotinamide adenine dinucleotide phosphate
NH
2
OH Hydroxylamine
O,K,J(≡ I
1
≡ i),I(≡ I
2
), H, G, P Particular steps in FLR measured under high intensity of exciting light
OEC Oxygen-evolving complex
P680 Primary electron donor in PSII
A
3
P680* Triplet excited state of P680
P700 Primary electron donor in PSI
A
PAM Pulse amplitude modulation
PBQ 1,4-benzenedione; p-benzoquinone
PC Plastocyanin
PEA Plant efficiency analyser
Pheo Primary electron acceptor in PSII, pheophytin, localised in D1 protein
Pheo2 Pheophytin localised in D2 protein
PQ Plastoquinone
PSI Photosystem I
PSII Photosystem II
Q
2
A component of PSII
Q
A
The first quinone electron acceptor in PSII
Q
B
The second quinone electron acceptor in PSII
RCII Reaction centre of PSII
RRP Reversible radical pair
Rubisco Ribulose-1,5-bisphosphate carboxylase / oxygenase
S
i
(i = 0, 1, 2, 3) S-states of OEC
T
820
Transmission signal measured at 820 nm
TMPD N,N,N

,N

-tetramethyl-p-phenylenediamine
Tris Tris(hydroxymethyl)aminomethane
TSTM Three-state trapping model
V
I
Relative variable fluorescence at the I step
V
J
Relative variable fluorescence at the J step
Y
Z
Tyrosine 161 of D1 protein
A
What is denoted as P680 and P700 is generally thought to be the primary electron donors in PSII and PSI, respectively, but recent results
indicate that the primary electron donors in both PSII and PSI are probably accessory chlorophylls and not P680 and P700, respectively.
For more details see van Mourik (2004), Novoderezhkin et al. (2005), and Groot et al. (2005) for PSII and M
¨
uller et al. (2003)
and Holzwarth et al. (2005a, b) for PSI.

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 11
primary photochemistry (i.e. charge separation and charge
recombination; Laible et al. 1994; see also Laz
´
ar 2003).
Maximal fluorescence F
M
is defined as the fluorescence
when all the RCIIs are closed, i.e. when all Q
A
is
reduced (Vredenberg 2000; Strasser and Stirbet 2001, and
section II.4.2.3 for alternative approaches). Even if charge
separation (i.e. formation of P680
+
Pheo
−
from P680*Pheo)
can occur in the closed RSII, its rate constant is ∼3–6 times
smaller [see Laz
´
ar (2003) for a summary of the rate constants
according to different authors] than in the case of open RCII.
Further, in the case of the closed RCII, the rate constant of
the charge recombination (i.e. formation of P680*Pheo from
P680
+
Pheo
−
) is higher than in the case of open RCII. The
changes in the rate constants of the charge separation and
recombination lead to increased accumulation (lifetime) of
the excited states in the closed RCII when compared with
open RCII; this leads to higher fluorescence emission in the
closed RCII than in the open RCII.
II The polyphasic chlorophyll a fluorescence rise:
O–J–I–P (O–I
1
–I
2
–P) transient
The O–J–I–P (O–I
1
–I
2
–P) FLR is reviewed in this section.
A summary of the instrumental set-ups for the measurement
of the FLR is given first, followed by a description of the
nomenclature used for the FLR and of the samples used
for the measurements. Finally, particular steps of the FLR
are explained in detail. Additional information can be found
in related reviews (Govindjee et al. 1986; Krause and Weis
1991; Dau 1994; Govindjee 1995; Joshi and Mohanty 1995;
Mohammed et al. 1995; Schreiber et al. 1995; Laz
´
ar 1999;
Roh
´
a
ˇ
cek and Bart
´
ak 1999; Samson et al. 1999; Chaerle and
Van Der Straeten 2000, 2001; Maxwell and Johnson 2000;
Roh
´
a
ˇ
cek 2002; Sayed 2003; Baker and Rosenqvist 2004;
Oxborough 2004a). Readers are also referred to a recently
published book ‘Chlorophyll a fluorescence: a signature
of photosynthesis’ (Papageorgiou and Govindjee 2004), in
which the state of the art, related to the FLR, is reviewed
extensively (e.g. Govindjee 2004; Schreiber 2004; Strasser
et al. 2004; Vredenberg 2004).
II.1 Instrumental set-ups
Three conditions must be guaranteed by an experimental
set-up to measure and distinguish the polyphasic
FLR: a high (saturating) intensity of exciting light
(∼3000–10000 µmol photons m
−2
s
−1
), a fast beginning
of illumination of a sample, and a fast time resolution of
detected fluorescence signal. The first condition is realised
by high intensity light sources (halogen and xenon lamps,
light emitting diodes, lasers) and the second condition
by using a fast enough shutter or shutter-less set-up.
A fast enough analogue / digital converter must be used
to ensure a satisfactory time resolution of the detected
fluorescence signal.
The first results on the measurements of the FLR under
high intensity of excited light were published by Morin
(1964) and Delosme (1967). Both these investigators used
laboratory made set-up where a fast enough shutter was
realised by using a gun whose ‘22 long rifle’ bullet blew apart
a metal plate that was between the sample and the illuminating
xenon lamp, serving as a light source. More user-friendly
instruments and commercial fluorometers have recently
replaced such a ‘dangerous’ set-up. Ruth (1990, 1991)
used a laboratory-made fluorometer with a helium–neon
laser as a light source and acousto-optic modulator (Bragg
cell) as a shutter, ensuring a fast enough beginning of the
illumination, to measure the FLR. However, the laser used by
Ruth did not provide a sufficiently high intensity of exciting
light. Another laboratory-made fluorometer was used for the
measurement of the FLR by Posp
´
ı
ˇ
sil and Dau (2000, 2002).
In this fluorometer, light emitting diodes (LED) were used
as a light source, which also ensures a fast beginning of
the illumination.
Ulrich Schreiber and co-workers (Schreiber 1986;
Schreiber et al. 1986; Schreiber and Neubauer 1987;
Neubauer and Schreiber 1987) published the first
measurements of the FLR under high intensity of exciting
light, using a commercial fluorometer (Pulse Amplitude
Modulation, PAM 101–103; Walz, Germany) where a
halogen lamp serves as a light source and a mechanical
shutter (full shutter opening within 800 µs) ensures a fast
beginning of the illumination. In 1991 and 1992 the first
FLR measurements with another commercial fluorometer,
the PEA (Plant Efficiency Analyser; Hansatech, England)
fluorometer were published by Strasser and Govindjee
(1991, 1992). There is no shutter in the PEA fluorometer
and a fast enough beginning of the illumination and the
illumination itself is achieved by LEDs. The same set-up for
the measurement of the FLR is also used by the Double-
Modulation Fluorometer (Photon Systems Instruments,
Brno, Czech Republic). This fluorometer even enables
measurements of the FLR with time resolution of 100 ns
during extremely strong (∼200 000 µmol photons m
−2
s
−1
)
short (up to ∼50–100 µs) saturating light flash, the so-
called flash fluorescence induction (Nedbal et al. 1999;
Kobl
´
ı
ˇ
zek et al. 2001).
The fluorometers most often used for the measurements
of the FLR, the PAM and PEA fluorometers, however,
use different approaches for the detection of fluorescence
signal. In the PEA fluorometer, continuous illumination of a
sample is used to induce photosynthetic electron transport
(i.e. as actinic light), but also serves to assess the ‘state’
of a sample via its fluorescence signal (i.e. as measuring
light). In the PAM 101–103 fluorometer, pulse-modulated
measuring light and continuous actinic light are separated.
Hence, photosynthetic electron transport (and closure of
RCIIs) is induced by continuous illumination, but the ‘state’
of the sample is probed by short (1 µs) measuring light

12 Functional Plant Biology D. Laz
´
ar
flashes placed 10 µs apart (during the FLR measurement).
By measuring the fluorescence signal during each individual
measuring flash as well as a few microseconds thereafter
and then subtracting the two signals, the fluorescence
excited by the actinic light is eliminated. Therefore, as
intensity of the measuring light is constant, PAM fluorometers
measure fluorescence yield, irrespective of the fluorescence
intensity excited by actinic illumination. Fluorescence yield
may vary by about a factor of five (between all RCIIs
being open or closed), whereas there is no limit for
fluorescence intensity, as it is proportional to intensity of
excitation light.
The different approaches used by PAM and PEA
fluorometers for the measurements are reflected in the
way the F
0
level is measured or calculated. In the PEA
fluorometer, the F
0
level is not measured but calculated
from experimental data; FLR data points in the range of
80–120 µs are fitted by a linear function and then extrapolated
to time zero, whose fluorescence signal is considered as the
F
0
level. The F
0
level determined in this way is within
10% of the fluorescence signal measured at 40–50 µsof
the FLR (e.g. Strasser et al. 1995; Su
ˇ
sila et al. 2004).
Therefore, the FLRs measured by PEA fluorometer are
usually presented starting from 40–50 µs (the O level).
Using different Chl concentrations (acetone extracts, where
excitation energy transfer and photochemistry do not occur)
and sample thicknesses, Su
ˇ
sila et al. (2004) pointed out that
the fluorescence signal detected by the PEA fluorometer, is
distorted by the fluorometer up to ∼50 µs but starting from
this time to the end of the measurement period, the detected
0.00
0.05 0.10
0.15
0.20
1
2
3
4
B
M
I
2
I
1
O
Time (s)
1
2
3
4
5
b
a
O
P
I
J
O
–1
0
–2
–3
–4
A
Log time (s)
1
2
3
4
–4
–3
–2
–1
Log time (s)
Fluorescence yield (r. u.)
Fluorescence intensity (relative units)
Fluorescence yield (relative units)
Fig. 1. FLRs measured with dark-adapted barley leaves (part A; curve a, no treatment; curve b, leaf incubated in 32 mM DCMU solution for
5 h; data from Laz
´
ar et al. 1998) and tobacco leaf (part B, no treatment; data from Laz
´
ar 1999) by PEA and PAM fluorometer, respectively, under
high intensity of exciting light [3400 µmol photons m
−2
s
−1
of red light (A) and 9000 µmol photons m
−2
s
−1
of white light (B)]. The same curve
as in main part B is presented on a logarithmic time-axis in the inset of part B.TheO,J(I
1
),I(I
2
), and P (M) steps are labelled.
fluorescence signal is constant. In contrast, the PAM 101–103
fluorometer measures the F
0
level by the measuring flashes
before the onset of actinic illumination. Although the energy
of the individual measuring flashes is very high, their short
duration and repetition with enough time between two flashes
(625 µs for the F
0
measurement) ensure that integral energy
of measuring flashes is very small and does not cause any
significant photochemical electron transport in the sample.
However, it is necessary to establish that ‘true’ F
0
is being
measured by checking it at decreased light intensity of the
measuring flashes.
II.2 Nomenclature
All steps in the FLR can be clearly revealed only when
logarithmic presentation of time axis is used. In the following
text, the term ‘step’ is used for a hump or a wave or a peak
that appears visually at a given time in the FLR. A ‘transient’
or a ‘phase’ is used in the following text as a part of the
FLR that starts at a step and is completed in another step.
The J (I
1
) and I (I
2
) steps usually appear at 2–3 ms and
30–50 ms, respectively (see Fig. 1A, curve a). The O step
stands for F
0
and the P step represents F
M
usually reached
at ∼200–500 ms. The J and I notation of particular steps
in the FLR is based on the original work by Strasser and
Govindjee (1991, 1992). However, the two steps appearing
between the F
0
and F
M
levels were denoted earlier as I
1
and
I
2
by U. Schreiber and co-workers (Schreiber 1986; Schreiber
et al. 1986; Schreiber and Neubauer 1987; Neubauer and
Schreiber 1987; see Fig. 1B). Strasser et al. (1995) established
equivalencies between the J and I
1
steps and the I and I
2
steps

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 13
since (i) the steps appear at the same time in the FLR (compare
curve a in Fig. 1A and the inset in Fig. 1B) and (ii) the relative
heights of the J and I
1
steps on the one hand and of the I
and I
2
steps on the other hand have the same light intensity
dependencies. Delosme (1967) denoted by the small letter ‘i’
the only step he measured between F
0
and F
M
at ∼2 ms; it
seems to be equivalent to the J (≡ I
1
) step. As the FLR is
now often measured by PEA, the O–J–I–P notation is used
in the text.
As the initial O–J transient reflects primary photochemical
reactions (see below), it was called the photochemical phase
of the FLR (Delosme 1967; Neubauer and Schreiber 1987;
Strasser et al. 1995). The main feature of this phase is that
the initial slope and relative height of the phase strongly
depends on the intensity of the exciting light (see e.g. Strasser
et al. 1995; Tomek et al. 2001). In contrast, subsequent
J–I–P transient cannot be speeded up by further increase
in the intensity of exciting light (see e.g. Strasser et al.
1995; Tomek et al. 2001) and it was called the thermal
phase of the FLR (Delosme 1967; Neubauer and Schreiber
1987) because it depends on the temperature of measurement
(within physiological range).
Under certain conditions, additional steps, the K, G, and
H steps, can appear in the FLR. These steps are described in
detail later under separate sections.
II.3 Samples
Chlorophyll a fluorescence rise can be measured with
any photosynthetic organism, but it has been measured
mostly with whole leaves, mosses, algae, cyanobacteria,
chloroplasts, and thylakoid membranes. FLRs from these
samples measured at room temperature are usually
characterised by typical O–J–I–P transients, as mentioned
above (but see also section III.2). While a detailed explanation
of the O–J–I–P transient measured with these ‘usual’ samples
is given in the following section, a short description of
measurements of the FLR with ‘unusual’ samples and the
results are briefly summarised now.
When the FLR is measured with PSII membranes, the
I step is not present (Posp
´
ı
ˇ
sil and Dau 2000, 2002; Heredia
and De Las Rivas 2003). Exploration of the FLR measured
with PSII membranes led to new suggestions for the origin
of the particular steps in the FLR (see sections II.4.2.2
and II.4.2.12). Fluorescence quenching by the oxidised
plastoquinone (PQ) pool (see section II.4.2.4 for more
details) is more pronounced in PSII membranes than in
more structurally organised samples (thylakoid membranes,
chloroplasts, leaves) (Kurreck and Renger 1998; Kurreck
et al. 2000; Posp
´
ı
ˇ
sil and Dau 2000, 2002). J. Kurreck and
co-workers (Kurreck and Renger 1998; Kurreck et al. 2000)
explained this finding by greater affinity of PQ molecules
for LHA to form a quenching complex in the case of
PSII membranes than in the case of the more structurally
organised samples.
The FLR was also measured with aggregates of light
harvesting complex of PSII (LHCII) and with trimeric PSI
to explore the generation of fluorescence quenchers from the
triplet states of chlorophyll (Barzda et al. 2000) and quantum
yield of fluorescence in PSI with initially reduced or oxidised
primary electron donor in PSI, P700 (Byrdin et al. 2000; see
also section II.4.2.10), respectively.
II.4 Explanation
In this section possible explanations of the particular steps in
the O–J–I–P FLR, suggested in the literature, are summarised,
separately for the photochemical and thermal phases of the
FLR. It seems that each of these explanations separately
cannot explain particular steps of the FLR and probably all
of the processes described below occur simultaneously and
affect the steps of the FLR to some extent.
II.4.1 The photochemical phase
II.4.1.1 Accumulation of reduced Q
A
with Q
B
being oxidised
Duysens and Sweers (1963) had already proposed
the existence of a quencher ‘Q’ that was removed as
Chl a fluorescence rose. According to the suggestion by
Delosme (1967), the photochemical phase (O–J transient)
‘corresponds to the destruction of a quencher Q which is the
primary reactant of the photoreaction II in photosynthesis’.
Using present notations, Q should be Q
A
. In agreement
with this suggestion, a working hypothesis was introduced
by Strasser and Govindjee (1992) proposing that the J step
reflects light-driven accumulation of Q
A
−
with Q
B
, the
second quinone electron acceptor in PSII, being oxidised,
that is, J ≈ Q
A
−
Q
B
state.
A main experimental proof that the O–J FLR represents
accumulation of only reduced Q
A
comes from measurements
with 3-(3

,4

-dichlorophenyl)-1,1-dimethylurea (DCMU), an
inhibitor of electron transport between Q
A
and PQ molecules
of the PQ pool [Oettmeier and Soll 1983; Trebst and Draber
1986; Trebst 1987; Shigematsu et al. 1989; DCMU inhibits
this reaction by binding to the Q
B
pocket of PSII (Velthuys
1981)]. When a sample is treated with DCMU, the FLR
measured at high intensity of exciting light is characterised
by a steep fluorescence increase, reaching maximal saturation
level at approximately the position of the J step measured in
the sample without DCMU (Strasser et al. 1995; Laz
´
ar et al.
1998, 2001; Tomek et al. 2001; compare curves a and b in
Fig. 1A). Theoretical simulations of the FLR either with or
without DCMU also confirmed the suggestion that it is mainly
Q
A
that accumulates in the reduced state in the position of the
J step (Stirbet and Strasser 1995a, b, 2001; Stirbet et al. 1995,
1998; Laz
´
ar et al. 1997b, 2005b; Laz
´
ar and Posp
´
ı
ˇ
sil 1999;
Strasser and Stirbet 2001; Tomek et al. 2001; Lebedeva et al.
2002; Laz
´
ar 2003; Zhu et al. 2005). It is important to realise
that when DCMU is not present, the Q
A
may not be fully

14 Functional Plant Biology D. Laz
´
ar
reduced in the J step of the FLR because electrons from Q
A
−
are continuously transferred to Q
B
and further on towards
PSI. Therefore, when DCMU is not present, the J step may
represent only a partial and not full accumulation of Q
A
−
.
II.4.1.2 The donor side of PSII
Using different treatments (Tris, high temperature, ADRY-
reagents, NH
2
OH, pH) that inhibit donor side of PSII, the
photochemical phase of the FLR was shown to be partially
controlled by the donor side of PSII (see e.g. Schreiber and
Neubauer 1987; Bukhov et al. 2004). The conclusion that
the donor side of PSII can affect the photochemical phase
of the FLR was also made on the basis of measurement of
the FLR under low intensity of exciting light (Hsu 1993;
Lavergne and Leci 1993) and using the flash fluorescence
induction measurements (Kobl
´
ı
ˇ
zek et al. 2001). However, the
effect of the donor side of PSII on photochemical phase of
the FLR found by Hsu (1993) and Lavergne and Leci (1993)
was determined on the basis of assumption of a different
fluorescence quenching in different redox states (the S-states)
of oxygen evolving complex (OEC) and is therefore related to
the ‘donor side’ fluorescence quenching and not to the rates
of the S-state transitions of OEC as such.
Also theoretical simulations of the FLR revealed an effect
of the donor side on the photochemical phase of the FLR
(Stirbet et al. 1998; Laz
´
ar and Posp
´
ı
ˇ
sil 1999; Laz
´
ar 2003).
As inhibition of the donor side of PSII results in appearance
of the K step in the FLR, the effect of the donor side of PSII
on the FLR is discussed more extensively in section III.1.
II.4.1.3 Excitation energy transfer among PSIIs
Theoretical calculations and simulations of the FLR
indicated that excitation energy transfer among PSIIs also
affect the photochemical phase of the FLR (Lavergne and
Leci 1993; Stirbet et al. 1998; Laz
´
ar 2003; Zhu et al. 2005).
II.4.1.4 Electric voltage across thylakoid membranes
Using simultaneous recordings of the FLR and absorbance
changes at 515 nm (A
515
, reflecting electric voltage across
thylakoid membranes), Schreiber and Neubauer (1990)
found that A
515
has a maximum (there is a maximum in
light-induced electric voltage across thylakoid membranes)
approximately at the position of the J step. Schreiber and
Neubauer (1990) suggested that the electric voltage favours
formation of P680 triplet excited state (
3
P680*). Formation
of
3
P680* as such may lead to lower fluorescence emission
but energy of
3
P680* can be further quenched either in RCII
or in LHA; this may lead to fluorescence quenching at the
J step. Similarly, using different frequencies of sinusoidal
modulation of light source intensity, Dau et al. (1991)
found that a time constant of fluorescence signal equals a
time constant of A
520
, showing that the fluorescence signal
increases when electric voltage across thylakoid membrane
increases. Subsequent analysis of the measured data revealed
that formation of electric voltage across thylakoid membrane
is responsible for 7% of the photochemical phase of FLR
(Dau et al. 1991).
II.4.1.5 Electron transport through the inactive
branch in PSII
Schreiber (2002, 2004) has discussed a hypothetical
mechanism to be responsible for the fluorescence quenching
at the J step. He proposed that in PSII with Q
A
−
, an electron
transport through the inactive branch in PSII can occur,
that is, an electron may be transferred from P680 to Pheo
localised in D2 protein of PSII (Pheo2) followed by an
electron transport from the reduced Pheo2 to Q
B
or Q
B
−
.
The proposed P680 → Pheo2 → Q
B
(Q
B
−
) electron transport
is assumed to be highly inefficient due to high yield of
back non-radiative recombination. The proposed electron
transport as such and the assumed high yield of back non-
radiative recombination cause a fluorescence quenching at
the J step, which results in fluorescence at the J step being
smaller than at the P step of the FLR. Although it has already
been shown that Q
B
can be reduced via the inactive branch in
mutants of anoxygenic photosynthetic bacteria Rhodobacter
capsulatus and Rhodobacter sphaeroides (Kirmaier et al.
2003; Breton et al. 2004; Wakeham et al. 2004; Frolov et al.
2005; Paddock et al. 2005), a consideration of the inactive
branch to affect the J step of the FLR as described above was
only hypothetical.
As Q
B
is involved in the mechanism mentioned above,
it was also called the Q
B
-quenching mechanism (Schreiber
2002). However, further mechanisms in which Q
B
is involved
can be also called Q
B
-quenching mechanisms as Schreiber
(2004) has discussed in more details. According to this view,
the results of O Pr
´
a
ˇ
sil, ZS Kolber, and PG Falkowski, as
described in section II.4.2.1, which, however, have not been
accepted by others, may be considered as a manifestation of
aQ
B
-quenching mechanism. Further research is necessary to
test this hypothesis.
II.4.1.6 Recombination between PSII electron
acceptors and donors
Goltsev and co-workers (Goltsev and Yordanov 1997;
Goltsev et al. 2003, 2005; Zaharieva and Goltsev 2003)
used a phosphoroscope method and theoretical simulations
of delayed fluorescence (DF) and the FLR to study effects of
recombinations between PSII electron acceptors and donors
leading to DF detected in the range of 350 µs–5.5 ms (for
review on DF see Tyystj
¨
arvi and Vass 2004). The authors
found that the time at which the first peak (denoted as I
1
)
in the DF intensity appears, corresponds with the time of
a maximal rate of the FLR during the O–I phase measured
under low intensity of exciting light (Goltsev and Yordanov
1997; Goltsev et al. 2003; Zaharieva and Goltsev 2003). The
origin of the I
1
in the DF is probably in recombination of Q
A
−

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 15
with Y
Z
+
when Q
B
is singly reduced (Goltsev and Yordanov
1997; Zaharieva and Goltsev 2003; Goltsev et al. 2005) and
the lifetime of Y
Z
+
Q
A
−
Q
B
−
is determined by the rate of
electron transport from Q
A
−
to Q
B
−
(Goltsev et al. 2005).
II.4.1.7 Effects of other processes
Laz
´
ar (2003) made a detailed analysis of the FLR by means
of theoretical simulations of the FLR. For these simulations,
Laz
´
ar used a model that was obtained by combination of three
existing models for the description of energy and electron
transport steps in PSII: (i) the reversible radical pair (RRP)
model (Breton 1983; van Grondelle 1985; Schatz et al. 1988;
Leibl et al. 1989; Roelofs et al. 1992) describing energy
utilisation leading to primary photochemistry, i.e. charge
separation, recombination, and stabilisation (see Dau 1994
for a review); (ii) the model of Kok et al. (1970) describing the
function of the donor side of PSII, i.e. that the Mn cluster of
OEC undergoes a cycle through its four S-states; and (iii) the
two-electron gate model (Bouges-Bocquet 1973; Velthuys
and Amesz 1974; Crofts and Wraight 1983) describing the
function of the acceptor side of PSII, i.e. that Q
B
, unlike Q
A
,
is a two-electron acceptor. Therefore, this model (almost)
completely included reactions on both the donor and the
acceptor side of PSII.
By changing values of particular rate constants or initial
concentrations of states in the model, theoretical simulations
by Laz
´
ar (2003) indicated that the photochemical phase of
the FLR is also affected by non-photochemical fluorescence
quenching by P680
+
and by oxidised PQ pool, by charge
recombination between P680
+
and Q
A
−
, by initial state
of OEC, and by rate of electron transport from Y
Z
to
P680
+
(for further details, see Laz
´
ar 2003). Theoretical
simulations by other authors also produced similar results
(Stirbet et al. 1998; Zhu et al. 2005).
II.4.1.8 The Q
B
-reducing / non-reducing heterogeneity
of PSII
All previous explanations were made assuming PSII
to be homogeneous. However, it is well documented in
the literature that heterogeneity of PSII exists (reviewed
by Lavergne and Briantais 1996). One type of the PSII
heterogeneity is with respect to the ability of PSII to reduce
Q
B
. In this way PSIIs can be divided into two parts:
(i) PSIIs that can reduce Q
B
, the Q
B
-reducing PSII, and
(ii) PSIIs that cannot reduce Q
B
, the Q
B
-non-reducing PSII
(Graan and Ort 1984, 1986; Whitmarsh and Ort 1984; Melis
1985; McCauley and Melis 1987; Chylla and Whitmarsh
1989; Lavergne and Leci 1993). With respect to this type
of PSII heterogeneity, the photochemical phase was found
to reflect accumulation of Q
A
−
of the Q
B
-reducing but also
of the Q
B
-non-reducing PSIIs (Hsu 1992a, b; Laz
´
ar et al.
1997b; Strasser and Stirbet 1997; Tomek et al. 2001, 2003;
Laz
´
ar 2003).
II.4.2 The thermal phase
II.4.2.1 Accumulation of reduced Q
B
in addition
to reduced Q
A
According to the suggestion by Delosme (1967),
the thermal phase (J–I–P transient) ‘corresponds to the
destruction of a quencher R’. Using present notations,
R should be PQ, either bound to PSII as Q
B
or free in
thylakoid membranes. In agreement with this suggestion,
as in the case of the photochemical phase of the FLR (see
section II.4.1.1), Strasser and Govindjee (1992) suggested a
working hypothesis proposing that the I and P steps reflect
light-driven accumulation of Q
B
−
and Q
B
2−
, respectively, in
addition to the accumulation of Q
A
−
, that is, I ≈ Q
A
−
Q
B
−
state and P ≈ Q
A
−
Q
B
2−
state.
Although an accumulation of particular redox forms of
PSII at the time of the appearance of the I and P steps
in the FLR was confirmed only by theoretical simulations
(Stirbet and Strasser 1995a, b, 2001; Stirbet et al. 1995, 1998;
Laz
´
ar et al. 1997b; Strasser and Stirbet 2001; Tomek et al.
2001; Lebedeva et al. 2002; Laz
´
ar 2003; Zhu et al. 2005),
assignment of the I and P steps to the redox forms seems
to be reasonable, at least as the first approximation. Even if
several different models were used for the simulations, their
results are, in general, the same as for the accumulation of
particular redox forms.
In agreement with the above-mentioned interpretation of
the FLR are the unpublished results of O Pr
´
a
ˇ
sil, ZS Kolber,
and PG Falkowski, obtained by site-directed mutants of the
green alga Chlamydomonas reinhardtii, with substitution of
Ala251 in the Q
B
-pocket of the D1 protein, which affects
the affinity of the PQ molecules for D1. These unpublished
data may suggest that the thermal phase of the FLR is related
to occupancy of the Q
B
-site by the PQ molecule and by a
capacity of Q
B
to deoxidise Q
A
−
(see also Samson et al.
1999). In agreement with this hypothesis, Yaakoubd et al.
(2002) found, by measuring F
M
induced by a single turnover
flash or by continuous illumination with samples treated with
DCMU and exogenous PQs, that oxidised Q
B
is responsible
for ∼56% of the thermal phase of the FLR.
II.4.2.2 Protonation of Q
B
2−
Consistent with the explanation given in the previous
section are the results of Heredia and De Las Rivas
(2003), using PSII membranes treated with 1,4-
benzenediol (hydroquinone, HQ) and with 1,4-benzenedione
(p-benzoquinone, PBQ). Heredia and De Las Rivas found
that the addition of reduced and protonated quinones (HQ,
PBQH
2
) resulted in fluorescence quenching of the very
last part of the thermal phase of the FLR. As they used
PSII membranes for the measurements, which do not
show the I step in FLR (see section II.3), they labelled the
fluorescence level that is unquenched by the quinones as the
‘H’ level. However, this H fluorescence level seems to be

16 Functional Plant Biology D. Laz
´
ar
different from the H step (peak) described in section III.2
and from a hump H described in section II.4.2.12. Heredia
and De Las Rivas interpreted the J–H transient to represent
the reduction of Q
B
to both Q
B
−
and Q
B
2−
and the
H–P transient to represent the protonation of Q
B
2−
.An
effect of protonation of Q
B
2−
was also considered in
theoretical simulations of the FLR (Laz
´
ar et al. 1997b;
Stirbet et al. 1998).
II.4.2.3 The donor side of PSII
An inhibition of the donor side of PSII suppresses the
thermal phase of the FLR (Schreiber and Neubauer 1987;
Posp
´
ı
ˇ
sil and Dau 2000; Bukhov et al. 2004). A partial
inhibition of OEC, caused either by various treatments that
deplete components on the donor side of PSII, or high
temperature treatment of PSII membranes (which lack the
I step; see section II.3), led to a correlation between a rate
constant of the J–P transient of the FLR (see section II.3)
and a steady-state rate of oxygen evolution (Posp
´
ı
ˇ
sil and
Dau 2000). Since a partial inhibition of the acceptor side of
PSII, caused by addition of subsaturating concentrations of
DCMU, resulted in a correlation between the rate constant
and the steady-state rate of oxygen evolution too, the
rate constant of the J–P transient may be considered an
indicator of the extent of electron flow from water to PQ
molecules generally (Posp
´
ı
ˇ
sil and Dau 2000). An effect of
the inhibition of OEC on the thermal phase of the FLR
was also revealed from theoretical simulation of the FLR
(Laz
´
ar 2003).
Participation of the donor side of PSII in the J–I phase
of the FLR was discussed by Vredenberg et al. (2005) on
the basis of a theoretical analysis of the three-state trapping
model (TSTM) formulated by Vredenberg (2000) [see also
Strasser and Stirbet (2001); Vredenberg (2004)]. In the
TSTM, the open (PheoQ
A
), semi-open(closed) (PheoQ
A
−
),
and closed (Pheo
−
Q
A
−
) states of RCIIs are defined. A
smaller rate constant of P680
+
reduction by Y
Z
in the
higher S-states of OEC [summarised by Laz
´
ar (2003)]
results in a lower efficiency of closing of the semi-open
RCIIs that leads to a slow FLR during the J–I phase
(Vredenberg et al. 2005).
II.4.2.4 Fluorescence quenching by the oxidised
PQ pool
As inhibition of OEC leads to a lack of electrons for
the reduction of the PQ pool, the pool remains oxidised,
and acts as a fluorescence quencher (Vernotte et al. 1979;
Hsu and Lee 1995; Kramer et al. 1995; Kurreck and Renger
1998; Kurreck et al. 2000; Haldimann and Tsimilli-Michael
2005). This fluorescence quenching by the oxidised PQ pool
is suggested to lead to the suppression of the thermal phase
(Posp
´
ı
ˇ
sil and Dau 2000; Bukhov et al. 2004). Also using
the pump and probe method and flash fluorescence induction
measurements, the thermal phase was suggested to reflect
the removal of the fluorescence quenching by the oxidised
PQ pool (Samson and Bruce 1996; Kobl
´
ı
ˇ
zek et al. 2001).
However, measurement of F
M
, induced by single turnover
flash or by continuous illumination, in samples treated with
DCMU and by exogenous PQs, revealed that fluorescence
quenching by the oxidised PQ pool is responsible for only
25% of the thermal phase of the FLR (Yaakoubd et al. 2002).
Therefore a question arises as to which part of the FLR reflects
the fluorescence quenching by the oxidised PQ pool. As
DCMU and chemicals that accept electrons from the acceptor
side of PSI (both keep the PQ pool oxidised) suppress the I–P
phase of the FLR, it is the I–P phase that reflects fluorescence
quenching by the oxidised PQ pool (Neubauer and Schreiber
1987; Schreiber et al. 1989). An effect of the fluorescence
quenching by oxidised PQ pool on the FLR generally was
also considered in theoretical simulations of the FLR (Stirbet
et al. 1998; Laz
´
ar 2003; Zhu et al. 2005).
T
´
oth et al. (2005) have recently found that if intact leaves
are treated with DCMU ‘carefully’, the F
M
level is the same
in the DCMU-treated leaves as in the controls (i.e. untreated
leaves). This fact implies that the F
M
level is not sensitive
to the redox state of the PQ pool and also that removal
of the fluorescence quenching by the oxidised PQ pool, as
discussed above, is not responsible for the thermal phase
of the FLR, at least in intact leaves. As discussed by T
´
oth
et al. (2005), the F
M
level is lowered in leaves treated with
DCMU by a ‘not-so-careful’ procedure because the treatment
probably causes a damage of PSII enabling a better contact of
oxidised PQ molecules from the pool with excited chlorophyll
molecules in LHA. Therefore, T
´
oth et al. (2005) suggested
that extent of the PQ pool quenching expressed as lowering
of the F
M
level could provide a tool to access the intactness
of the PSII protein complex and quality of the isolation
procedure used.
II.4.2.5 The Q
2
component
Samson and Bruce (1996) suggested that the thermal
phase of the FLR could also reflect a reduction of a
component, labelled as Q
2
. The term Q
2
was first used
by Joliot and Joliot (1977, 1979) as a putative electron
acceptor, which needs more than one flash to be reduced
in the presence of DCMU; it was part of an alternative
electron pathway of very low quantum efficiency. However,
instead of defining the putative electron acceptor, Lavergne
and Rappaport (1998) suggested that measured data can be
explained by charge recombination between P680
+
and Q
A
−
.
Nevertheless, consideration of the charge recombination had
almost no effect on the simulated thermal phase of the FLR
(Laz
´
ar 2003).
II.4.2.6 Cytochrome b
559
Laz
´
ar et al. (2005b) experimentally showed that an
increase of the amount of initially reduced cytochrome (cyt)
b
559
in DCMU-treated thylakoid membranes resulted in an
increase of the F
M
level. Theoretical simulations, where cyt
b
559
was assumed to accept electrons from Pheo
−
and donate

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 17
electrons to P680
+
(in the presence of DCMU), further
supported this experimental finding. Therefore, with respect
to ‘definition’ of the Q
2
component, as mentioned above,
such a cyt b
559
could be identified as the Q
2
component.
The reduced Pheo from work of Laz
´
ar et al. (2005b) need
not be the Pheo localised in the D1 protein of PSII but it
can be Pheo2 localised in the D2 protein of PSII, which
is a part of the ‘inactive’ electron transport branch in
PSII (see section II.4.1.5) and is closer to cyt b
559
than
Pheo in D1 as known from the structure of PSII (Zouni
et al. 2001; Kamiya and Shen 2003; Ferreira et al. 2004).
Further, Schreiber and Neubauer (1987) had suggested that
if fluorescence quenching by P680
+
(Butler 1972; Mauzerall
1972; Sonneveld et al. 1979; Deprez et al. 1983; Shinkarev
and Govindjee 1993; Bruce et al. 1997) causes quenching of
fluorescence signal at the J step, the reduction of P680
+
by
an alternate electron donor, e.g. by cyt b
559
or a carotenoid,
could result in fluorescence increase during the thermal phase
of the FLR.
II.4.2.7 Fluorescence quenching in light
harvesting antennae
On the basis of measurements of fluorescence decays
after an application of single or multiple turnover flash, and
fitting of the experimental data by a model, Vasil’ev and
Bruce (1998) suggested that removal of the fluorescence
quenching in LHA of PSII is responsible for the
thermal phase of the FLR. Moise and Moya (2004a, b)
reached a similar conclusion on the basis of their phase
and modulation fluorometry measurements: variable and
transitory fluorescence quenching occurs during the thermal
phase, and the quenching results from a conformational
change in a LHA of PSII and the LHA of PSII, where the
conformational changes occur is CP47.
II.4.2.8 Changes in yield of delayed
(recombination) fluorescence
Schreiber and Krieger (1996) suggested another
interpretation for the thermal phase of the FLR. These
authors assumed that fluorescence signal during the FLR
consists of both the prompt fluorescence and DF, the former
originating directly from the excited states and the latter
indirectly after the formation of the excited states, via
radiative charge recombination between P680
+
and Pheo
−
(nanosecond range; for review on DF see Tyystj
¨
arvi and Vass
2004). Schreiber and Krieger (1996) further suggested that a
gradual removal of non-radiative loss of the charge-separated
state occurs in PSII in the P680
+
Pheo
−
Q
A
−
state resulting
in an increase of the yield of DF that leads to an increase
of fluorescence signal during the thermal phase of the FLR.
The origin of changes in the DF was discussed by the authors
to be connected to changes in positive charges stored at the
donor side of PSII.
Goltsev and co-workers (Goltsev and Yordanov 1997;
Zaharieva and Goltsev 2003; Goltsev et al. 2003, 2005)
studied an effect of recombinations between PSII electron
acceptors and donors by a phosphoroscope method
and theoretical simulations of the DF and FLR (see
section II.4.1.6). The authors found that the time at which
the second peak (denoted as I
2
) in the DF intensity appears,
corresponds with the time of a maximal rate of the FLR
during the I–P phase measured under low intensity of
exciting light (Goltsev and Yordanov 1997; Goltsev et al.
2003; Zaharieva and Goltsev 2003). The origin of the I
2
in the DF is probably in recombination of Q
A
−
with Y
Z
+
when Q
B
is doubly reduced (Goltsev and Yordanov 1997;
Zaharieva and Goltsev 2003; Goltsev et al. 2005) and the
lifetime of Y
Z
+
Q
A
−
Q
B
2−
is determined by the sum of
the rate constants of electron transport from the S-state
transitions of OEC to Y
Z
+
and of exchange between the
reduced and oxidised PQ molecules in the Q
B
-site of PSII
(Goltsev et al. 2005).
II.4.2.9 Electron transport reactions after PSII
When the FLR is measured, in the green alga Chlorella,
under low intensity of excitation light, a dip D usually
appears after the I step (Munday and Govindjee 1969a).
It was suggested that appearance of the dip is related to
function of PSI (Munday and Govindjee 1969b; Schreiber
et al. 1971; Satoh and Katoh 1981; Hansen et al. 1991).
However, using theoretical modelling of the FLR measured
under low intensity of excitation light, Baake and Strasser
(1990) and Baake and Schl
¨
oder (1992) showed that inclusion
of electron transport reactions occurring after the PQ pool
[cyt b
6
/ f complex, plastocyanin (PC), PSI] in a model
does not improve a fit of the model to a part of the FLR
around the position of the dip D of the experimental FLR
curves measured with leaves. Also other authors assumed
the electron transport reactions occurring after the PQ pool
in theoretical simulations of the FLR (Goltsev and Yordanov
1997; Lebedeva et al. 2002).
New information about the electron transport chain
after the PQ pool has been obtained by simultaneous
measurement of the FLR and transmission changes at 820 nm
(T
820
), which should reflect changes in redox state of
PC and P700. Schreiber et al. (1989), R. J. Strasser and
co-workers (Strasser et al. 2001; Schansker et al. 2003,
2005) showed that T
820
has a minimum exactly at the
position of the I step in the O–J–I–P FLR measured with
leaves. On the basis of 2,5-dibromo-3-methyl-6-isopropyl-
p-bezoquinone (DBMIB) and methylviologen (MV) action
on the FLR and T
820
signal, Schansker et al. (2005)
suggested that a transient limitation at the acceptor side of
PSI leading to a traffic jam of electrons transiently formed
in the electron transport chain are responsible for the I–P
transient of the FLR (cf. Munday and Govindjee 1969b).
It is likely that this limitation on the PSI side can be
first caused by inactive ferredoxin-NADP
+
oxidoreductase

18 Functional Plant Biology D. Laz
´
ar
(FNR) and consequently by the inactive Calvin–Benson cycle
(Schansker et al. 2003).
II.4.2.10 Fluorescence coming from PSI
Observations of direct fluorescence from PSI has been
recently reviewed by Itoh and Sugiura (2004). Here, I present
a discussion of the effect of PSI reactions on OJIP FLR.
Applying saturating light pulses of varying lengths and
measuring the T
820
signal with leaves treated with MV
and using far-red background illumination (to avoid any
limitation at the acceptor side of PSI), Schreiber et al. (1989)
found that the electron transport chain is reduced within
∼50 ms; that is at a time when the I step appears in the
FLR. On the basis of this result and of measured changes
in the T
820
signal, as mentioned above, the I–P transient in
the FLR was suggested to reflect a reduction of P700 and
of PSI acceptor side caused by a limitation at the acceptor
side of PSI (Munday and Govindjee 1969b; Schreiber et al.
1989). Further, Ikegami (1976) found that when P700 is
reduced by dithionite in P700-enriched particles, the particles
upon illumination (by blue light) showed an increase in
fluorescence signal (detected at 694 nm) at room temperature.
This result together with the suggestion mentioned above
led Ulrich Schreiber and co-workers (Schreiber et al. 1989;
Schreiber 2002) to suggest that the I–P transient of the FLR
could reflect an increase in fluorescence signal coming from
PSI. More recently, Byrdin et al. (2000) also found that
the trimeric PSIs with initially reduced PSI by ascorbate
(i.e. P700 being initially present) show a FLR (excited by
He–Ne laser at 633 nm and detected at wavelengths above
665 nm) at room temperature with variable fluorescence
F
V
(= F
M
− F
0
) equal to ∼12 ± 5% of F
M
.However,to
prove that the I–P transient of the FLR reflects exclusively
fluorescence signal coming from PSI, direct comparison
of the FLRs measured at different emission wavelengths
is necessary.
II.4.2.11 Light gradient within a sample
As any photosynthetic sample used for measurements
of the FLR curves represents layers of pigments, a light
gradient within the sample, in addition to other optical
effects, is created along the light path. The gradient then
results in the excitation of particular sub-layers of the sample
by light of different intensities and the detected ‘overall’
fluorescence signal is a sum of the signals coming from
the sub-layers. Assuming this simple rationale and using
different sample preparations, Hsu and Leu (2003) suggested
that the I step in the FLR originates in the abaxial layer(s)
of the sample (when it is illuminated from the adaxial
side). As it is the photochemical phase of the FLR that
is very sensitive to the intensity of exciting light (see
section II.2), the I step in the ‘overall’ FLR should in fact
reflect the photochemical phase of the FLR coming from
the abaxial layer(s) of the sample (Hsu and Leu 2003).
Franck et al. (2005) also suggested a participation of
fluorescence signals coming from particular layers of the
sample to overall fluorescence signal of the FLR on the basis
of measurements of fluorescence emission spectra at room
temperature during the FLR with different samples and using
theoretical simulations of the spectra. On the basis of the
theoretical simulations, Franck et al. (2005) also concluded
that as the time elapses from the onset of excitation, the
measured fluorescence signal during the FLR originates from
deeper layers.
Even though an effect of the light gradient within
the sample on the ‘overall’ FLR was explored by both
experimental measurements and theoretical simulations
(Su
ˇ
sila et al. 2004), it was not proven that the light gradient
is responsible for the appearance of the I step in the FLR.
However, if the FLR is used as an analytical tool to access
information about photosynthetic function, care must be
exercised in interpreting the data (Hsu and Leu 2003; Su
ˇ
sila
et al. 2004). Su
ˇ
sila et al. (2004) surmised that the light
gradient within the sample can significantly affect the results
of the JIP test (see section V.1) mainly in the case when the
concentration of Chls is changed when a plant is stressed.
II.4.2.12 Electric voltage across
the thylakoid membrane
When the FLR is measured with PSII membranes the
I step is missing in the FLR, in contrast to more intact
samples where the I step is present (see section II.3).
However, PSII membranes do not form closed compartments,
which would enable the formation of electric voltage across
the membrane. These facts, together with the existing
coincidence in the times of the I step appearance in the
FLR (at 30–50 ms) and the formation of a peak in light
induced electric voltage across the thylakoid membrane (at
20–50 ms but with different samples, for reviews see
Bulychev and Vredenberg 1999; Vredenberg 2004), led
Posp
´
ı
ˇ
sil and Dau (2002) to suggest that the I step reflects
light induced changes in electric voltage across thylakoid
membranes. Application of valinomycin with potassium
ions, led to a ‘short-circuit’ with respect to the membrane
voltage and the I step disappeared from the FLR measured
with thylakoid membranes. Similarly the appearance of a
hump, labelled as H (note: this hump is different from
the H fluorescence level described in section II.4.2.2.2 and
from the H peak described in section III.2), in the FLR
but measured under low intensity of exciting light with
Bryopsis chloroplasts was related to changes in light-induced
membrane voltage (Satoh and Katoh 1981).
However, appearance of the I step in FLR and a peak in
light-induced electric voltage across thylakoid membranes in
the same time as assumed by Pospíšil and Dau (2002) is in
contrast to results of Schreiber and Neubauer (1990), who
found that the light-induced electric voltage across thylakoid
membranes has a maximum at the J step and not at the

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 19
I step of the FLR (see section II.4.1.4). Further, Schreiber
and Neubauer (1990) suggested that increase of fluorescence
signal during the thermal phase of the FLR reflects a lowering
of fluorescence quenching driven by a relaxation of the
membrane voltage, where the fluorescence quenching occurs
by a mechanism as described in section II.4.1.4.
In addition to the participation of
3
P680* as described
in section II.4.1.4, there are two other mechanisms related
to changes in electric voltage across thylakoid membranes
that affect changes in fluorescence emission directly and
indirectly. In the direct mechanism, formation of the electric
voltage causes a decrease in Gibbs free-energy difference,
G
0
, between the excited states in RCII and the charge
separated state (P680
+
Pheo
−
): the decrease in G
0
leads to a
decrease in the rate constant of primary charge separation and
increase in the rate constant of primary charge recombination,
both resulting in an increased accumulation of excited
states leading to increased fluorescence emission (Dau et al.
1991; Dau and Sauer 1991, 1992). In connection to the
indirect mechanism, Graan and Ort (1983) found that a
marked stimulation of the electron transport rate occurred
when chloroplasts were treated with valinomycin, and they
explained this effect by an increased rate of plastoquinol
oxidation. Therefore, when valinomycin is not present a
light-induced electric voltage across the thylakoid membrane
is formed, causing a decrease in the rate of plastoquinol
oxidation that leads to an increased accumulation of reduced
Q
B
and Q
A
that results in an increased fluorescence
emission. Similarly, Goltsev and Yordanov (1997) assumed
a decrease in the rate constant of plastoquinol oxidation
caused by an intra-thylakoid space acidification in theoretical
simulations of the FLR. It is evident from the above text
that both the ‘direct’ and the ‘indirect’ mechanisms involve
changes in the rate constants of electron transport steps.
Therefore it is clear that when a light-induced electric field
is present, rate constants of all the electron transport steps
will be changed and will somehow contribute to changes
in fluorescence emission. Such an approach was used by
Lebedeva et al. (2002), who assumed a dependence of
the rate constants of all the electron transport steps in
thylakoid membranes on the light-induced electric voltage
across thylakoid membranes in theoretical simulations
of the FLR.
An effect of electrogenic events across thylakoid
membranes on the FLR has been extensively studied by
WFJ Vredenberg and AA Bulychev (Bulychev and Niyazova
1989; Vredenberg 2000; Bulychev and Vredenberg 2001;
Vredenberg and Bulychev 2002, 2003). Bulychev and
Vredenberg (2001) found that electrogenic events generated
in PSI, when PSII photochemical activity was absent
(caused by NH
2
OH treatments), affect the PSII. Thus,
Vredenberg and Bulychev (2002) suggested the so-called the
photoelectrochemical control of fluorescence yield: when the
photochemical activity of PSII is absent at the I step of
the FLR (assuming that all the electron acceptors in PSII
are already reduced), the subsequent I–P transient is due to
changes in fluorescence yield caused by electric events across
the thylakoid membrane (see also Vredenberg 2004).
An effect of light induced changes in pH or the
electric voltage across thylakoid membrane was also included
in models, which simulated FLR curves for different
intensities of exciting light (Goltsev and Yordanov 1997;
Lebedeva et al. 2002).
II.4.2.13 The heterogeneity in the rate of PQ
pool reduction
All the explanations, thus far discussed in this review,
were made assuming PSII to be homogeneous. As already
mentioned above (see section II.4.1.8), PSII is heterogeneous
in many aspects. One aspect of the PSII heterogeneity is in
the rate of reduction of particular PQ pools (Joliot et al.
1992; Kirchhoff et al. 2000). In addition to a fast-reduced
PQ pool localised mainly in thylakoid grana, there also
exists a slowly reduced PQ pool localised mainly in stroma-
exposed thylakoid membranes. To incorporate this type of
heterogeneity into explanation of the FLR, the J–I and
I–P transients of the thermal phase were suggested to reflect
a reduction of the fast and slow PQ pool, respectively
(Strasser et al. 1995; Barth
´
elemy et al. 1997; Schreiber 2002).
Further, Bukhov et al. (2003) found that addition of a weak
concentration of N,N,N

,N

-tetramethyl-p-phenylenediamine
(TMPD) leads to a clearer appearance of the I step in the FLR
measured with thylakoid membranes. Since Bukhov et al.
(2003) used TMPD as an electron acceptor from the reduced
PQ pool (see also Joly et al. 2005), the resolution of the I step
resulted from a decreased reduction of both fast and slowly
reducing PQ pool. Similarly, theoretical simulations of the
FLR revealed that assuming a slowly reducing PQ pool in a
model leads to an appearance of a step in the thermal phase
of the FLR (Laz
´
ar 2003).
II.4.3 A summary
The results presented in the previous sections suggest that
many events may affect the final shape of measured FLR.
Some of the conclusions were based on precise experiments
and their detailed analyses but some of them were only
from hypotheses. It is not yet possible to say definitively
which mechanisms really (or only) contribute to the final
shape of the FLR and which do not. It is highly likely
that all the mechanisms mentioned contribute to the FLR to
some extent.
One way to quantify the extent by which a particular
mechanism contributes to a measured quantity is to form a
mathematical model describing all the involved mechanisms
and evaluate the extent to which a mechanism contributes
to the measured quantity by means of exact mathematical
formulae. Such an approach is common in mathematical
modelling of metabolic pathways and is known as metabolic

20 Functional Plant Biology D. Laz
´
ar
control analysis [MCA; for reviews on MCA, see Fell
(1992); Visser and Heijnen (2002)]. In MCA, in addition
to other coefficients, the control coefficient C, which exactly
quantifies the extent to which a particular model parameter
(concentration or activity or rate constant) controls a selected
variable of the model (concentration or flux), is calculated.
MCA has already been used for exploration of several plant
metabolic pathways, e.g. the Calvin–Benson cycle (Poolman
et al. 2000) and the malate valve (Fridlyand et al. 1998),
but it has also been used for exploration of fluorescence
data related to photosynthetic function; a simple MCA-like
method was used for the analysis of changes in oscillations in
steady-state fluorescence signal caused by high temperature
treatment (Laz
´
ar et al. 2005c) and MCA was used for
the analysis of changes in F
M
of the DCMU-FLRs due
to changes in initial redox state of cyt b
559
(Laz
´
ar et al.
2005b; see section II.4.2.6). Therefore, it is a challenge for
future work to construct a mathematical model of the FLR,
which would include all suggested mechanisms and evaluate
contributions of particular mechanisms to FLR by means
of MCA.
III The K, H, and G steps in the fluorescence rise
III.1 The K step
When samples are treated with high temperature, a new
step at ∼300–400 µs, denoted as K, appears in FLR (Guiss
´
e
et al. 1995a, b; Srivastava et al. 1997; Laz
´
ar and Il
´
ık 1997;
Laz
´
ar et al. 1997a; Strasser 1997; see Fig. 2, curve b). Reto
G
H
d
b
c
a
O
P
I
P
P
J
J
K
K
O
–1 0
–2
–3
–4
1
2
3
4
5
Log time (s)
Fluorescence intensity (relative units)
Fig. 2. Chlorophyll a fluorescence rise measured with dark-adapted
pea leaves (curve a, no treatment; curve b, leaf incubated at 47
◦
C
in water for 5 min), potato leaf (curve c, leaf incubated at 44
◦
Cin
water for 13 min), and with lichen Umbilicaria hirsuta (curve d, no
treatment) by PEA fluorometer under high intensity of exciting light
[3400 µmol photons m
−2
s
−1
of red light; data from Laz
´
ar 1999 and
courtesy of P. Il
´
ık and M. Bart
´
ak (curve d)]. The O, K, J, I, H, G, and P
steps are labelled.
Strasser and his co-workers (Guiss
´
e et al. 1995a; Strasser
1997) suggested that the appearance of the K step reflects
an inhibition of OEC, probably together with an inhibition
of the acceptor side of PSII (Laz
´
ar et al. 1999). More
generally, Strasser (1997) suggested that the K step arises
when the rate of electron flow from P680 to the acceptor
side of PSII exceeds the rate of electron flow from the
donor side of PSII to P680. Srivastava et al. (1997) and
Laz
´
ar and Posp
´
ı
ˇ
sil (1999) found that the appearance of
the K step reflects changes in the energetic connectivity
between PSIIs.
As the K step reflects an accumulation of reduced Q
A
(Strasser 1997), the O–K rise is also the photochemical
phase of FLR. However, the photochemical phase of the
FLR measured at room temperature (the O–J transient) does
not simply move to shorter times (the O–K transient) when
measured with high temperature treated samples because
both the K and J steps can be measured together under
certain conditions (Guiss
´
e et al. 1995a, b; Srivastava et al.
1997; see Fig. 2, curve c). This fact suggests that the K
and J steps might reflect two different phenomena (Guiss
´
e
et al. 1995a, b; Srivastava et al. 1997). Therefore, the source
of the K step is expected to be present even in unstressed
samples but for dynamic reasons it does not appear as a
clear step in the FLR (Guiss
´
e et al. 1995a, b; Srivastava
et al. 1997; Strasser 1997). The same conclusion can also
be drawn from positions of peak accumulations of excited
states simulated on the basis of a theoretical model of the FLR
(Laz
´
ar 2003).
III.2 The H and G steps
The O–J–I–P FLR is not a typical property of all
photosynthetic organisms under ‘standard’ conditions. The
P step, measured at high intensity of excitation light at
room temperature, was found to be ‘split’ into two steps in
the FLR in foraminifers (Tsimilli-Michael et al. 1998a, b),
zooxanthellae (Hill et al. 2004), lichens (Bukhov et al. 2004),
and in lichens and lichenised algae (Il
´
ık et al. 2006; see Fig. 2,
curve d). To continue with the previous O–K–J–I notation,
the two steps were labelled as H and G (Tsimilli-Michael
et al. 1998a, b).
On the basis of simultaneous measurements of FLR and
T
820
signal with different samples treated with different
chemicals, Il
´
ık et al. (2006) found that fluorescence decrease
from the H step to a dip between H and G steps is caused
by a removal of limitation on the acceptor side of PSI,
probably caused by light-induced activation of FNR (see
section II.4.2.9) or cyclic electron flow around PSI or Mehler
reaction, resulting in a transient reoxidation of reduced PQ
pool (and Q
A
−
). Following fluorescence increase from the
dip between H and G steps to the G step is then caused by
subsequent PQ pool (and Q
A
) re-reduction. The PQ pool re-
reduction is not associated with cyclic electron flow around
PSI and is probably caused by inability of the cyt b
6
/ f
complex to rapidly reoxidise the PQ pool (Il
´
ık et al. 2006).

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 21
In light of the discussion above, the H step mentioned
here seems to be different from the H fluorescence level and
from the hump H described in sections II.4.2.2 and II.4.2.12,
respectively. Thus, a different nomenclature is ultimately
needed to describe this H step.
IV Statistical properties of parameters determined
from the fluorescence rise
IV.1 Presentation of parameters determined
from the fluorescence rise
Statistical evaluation of FLR data is not routine in the
literature and when it is used, it is only to fulfill standard
requirements for the data presentation. The values of F
0
, F
M
,
F
V
/ F
M
parameters [and also other fluorescence parameters
(FPs) determined from FLR] are very often presented by
means of the mean and standard deviation (or standard error)
in the literature (see e.g. Bj
¨
orkman and Demmig 1987).
But to present any parameter by means of the mean and
standard deviation (or standard error), the distribution of the
parameter’s data should be Gaussian. However, the FPs (F
0
,
F
J
, F
I
, F
M
, F
V
, V
J
, V
I
, F
V
/ F
M
, etc.) generally do not have
Gaussian distribution (Laz
´
ar and Nau
ˇ
s 1998; Laz
´
ar et al.
2003, 2005a, 2006). Therefore, presentation of data by means
of the mean and standard deviation (or standard error) is
not appropriate and it masks the real data distribution of the
FPs. The use of median, quartiles, and maximal and minimal
values better describes a real situation of data distribution
(Laz
´
ar and Nau
ˇ
s 1998). Further, because the FPs of the
FLR generally do not have Gaussian distributions, the non-
parametric test should be used for statistical comparisons of
the FPs rather than the parametric tests, which are based on
the assumption of Gaussian distribution of data.
IV. 2 Changes in statistical distributions of fluorescence
parameters caused by stress
Generally, statistical distribution (histogram) of a FP,
measured under non-stressed conditions, is not Gaussian
but skewed to a side (Laz
´
ar and Nau
ˇ
s 1998; Laz
´
ar et al.
2003, 2005a, 2006). When plant material suffers from stress,
changes in statistical distributions of the parameters occur
and these changes depend on the extent of the stress.
For example, the F
M
level, measured with barley leaves
upon high temperature treatment, is skewed to the left for
measurements at room temperature (i.e. most of the F
M
values are high, on the right in the histogram, but there are
also some smaller F
M
values, on the left in the histogram),
then symmetrical (44
◦
C), then skewed to the right (51
◦
C),
and finally symmetrical again (65
◦
C) (Laz
´
ar et al. 2005a).
The observed changes in distributions of F
M
can be well
explained in the sense of changes in functional heterogeneity
of PSIIs, assuming that a stress leads to a malfunction of
PSII and causes a decrease in the F
M
level (Laz
´
ar et al.
2005a). However, complete changes in the distributions as
described above can be observed only when plant material
suffers progressively from weak to severe stress (Laz
´
ar 2005):
when a stress is not severe, not all stages of change in the
distribution can be observed, as was the case for measurement
with pumpkin leaves that suffered from senescence and
fungal infection (Laz
´
ar and Nau
ˇ
s 1998) or with wheat
leaves that suffered from senescence and other stresses
(Laz
´
ar et al. 2003).
Changes in distributions of FPs, determined from FLR,
caused by stress, as described above, are also accompanied
by changes in variances of measured data and the changes
in variances are different for different FPs (Laz
´
ar et al.
2003, 2005a). The different variances of the FPs can be
indicative of different processes described by the FPs (Laz
´
ar
et al. 2005a). The difference in variances of the FPs can
be used for an evaluation of mutual independence of two
FPs and, thus, also of mutual independence of processes
described by the FPs (Laz
´
ar et al. 2003, 2005a). For
example, it was found, for the case of a high temperature
stress, that for almost all combinations of two basic FPs
(F
0
, F
K
, F
J
, F
I
, F
M
), there is an increase in mutual
independence of the FPs in the temperature range 44–51
◦
C
and therefore also an increase in mutual independence of
processes characterised by the FPs in this temperature range
(Laz
´
ar et al. 2005a).
Laz
´
ar et al. (2006) showed that a detailed analysis of
changes in statistical distributions of FPs could be useful
for early detection of plant stress. To obtain a large amount
of experimental data for a given FP (necessary for detailed
analysis), they measured FI with leaf segments under a
low intensity of exciting light with an imaging fluorometer.
Selected FP of control (no stress) and stressed samples
(stress mainly by a dehydration of the segments in this
work) were compared by classical statistical comparison
(Mann-Whitney test; compares values of the medians) and
by statistical comparison of shapes of distributions of the
FPs (two-sample Smirnov test). The authors found that
examples exist in which statistically significant difference
is not revealed by the classical statistical comparison (for
given critical level) but statistically significant difference is
revealed by comparisons of distributions (for the same critical
level). It implies that the shape of statistical distribution of
a FP is more sensitive to stress than the median of the FP
and that the comparison of changes in shapes of statistical
distributions of FPs is therefore more suitable for early
detection of plant stress than a classical statistical comparison
(Laz
´
ar et al. 2006).
V Applications of the fluorescence rise
V.1 The JIP test for the so-called ‘vitality’ screening
With the main goals to measure several hundreds of samples
per hour and to provide a manual that non-specialists
can execute the measurements and obtain results in a
standard form, a test, known as the JIP test, was formulated
by Strasser and Strasser (1995) for ‘vitality’ screening

22 Functional Plant Biology D. Laz
´
ar
upon changing environmental conditions. The test was
later produced as a free software Biolyzer (Laboratory
of Bioenergetics, University of Geneva, Switzerland;
http://www.unige.ch/sciences/biologie/bioen/jipsoftware.
html; verified 12 September 2005). The basis of the test is
measurement of the FLRs with PEA fluorometer followed
by an analysis of measured curves. In this JIP test, basic
parameters evaluated from the FLR curves, such as F
0
, F
M
,
fluorescence signals at 100 and 300 µs and at the J, I and
P steps, are used for subsequent calculation of different
parameters that are somehow related to energy and electron
fluxes in PSII and therefore to photosynthetic function
generally (see Strasser et al. 2004).
The JIP test seems to be very popular now; when a
search term ‘jip and test and fluorescence’ was used in
the Web of Science search engine, 31 articles were listed
(search performed on 7 July 2005) published during a
period from 1997 to 2005. For example, choosing only
from ten of the most recently published articles, the
JIP test was used for study of effects of ambient v.
reduced UV-B radiation on Salix arctica plants (Albert
et al. 2005), comparison of ozone foliar symptoms in
woody plant species (Bussotti et al. 2005), quantification
of the photosynthetic performance of phosphorus-deficient
Sorghum plants (Ripley et al. 2004), assessment of stress
conditions in Quercus ilex L. leaves (Bussotti 2004),
description of effect of salinity stress on PSII in Ulva
lectuca (Xia et al. 2004), exploration of low temperature
tolerance of tobacco plants (Parvanova et al. 2004),
exploration of ozone action on Mediterranean evergreen
broadleaves and on woody plant leaves (Paoletti et al.
2004; Gravano et al. 2004), phenotyping of dark- and light-
adapted barley plants (Oukarroum and Strasser 2004), and
detection of draught and salinity tolerance chickpea varieties
(Epitalawage et al. 2003).
Although the JIP test is very often used now, the results
should be taken with care, especially in the case when the
concentration of Chls is changed during stress (Su
ˇ
sila et al.
2004; see section II.4.2.11).
V.2 Remote sensing and pattern recognition
in precise agriculture
There is a rapidly growing interest for plant identification in
agriculture practice with the aim of recognising cultivated
plants from weeds. Then, using a sprayer mounted on a
tractor, herbicides and other agrochemicals could be applied
selectively and precisely. This approach would result in
precision farming or precision agriculture, the terms that are
used very often now.
Precision agriculture requires a tool that enables remote
sensing of the plants and simultaneous recognition. One
way to determine the plant species is to measure FLRs,
which are characteristic for given species and can be used
as fingerprints. Application of sophisticated mathematical
procedures (the genetic algorithms and neural networks
classifiers) then would enable recognition of particular
species on the basis of their ‘fingerprints’. Fluorescence
signal can be also measured by a CCD camera from
a large area (i.e. measurement of fluorescence imaging;
for reviews see Chaerle and Van Der Straeten 2000,
2001; Nedbal and Whitmarsh 2004; Oxborough 2004a, b),
enabling fast and effective remote sensing (for a review
see Moya and Cerovic 2004). Therefore, it seems that
chlorophyll fluorescence signal is the tool needed for
precision agriculture.
The first results of the pattern recognition using
fluorescence techniques came from measurements of
fluorescence induction (FI) by a PAM fluorometer (i.e. with
low time resolution, but with both FLR and fluorescence
decay being measured). A special design of illumination
routine, given to dark-adapted samples, consisted of
application of red light of moderate intensity, far-red light,
high intensity white light, and dark intervals (Tyystj
¨
arvi et al.
1999). However, this type of data collection required a long
time, which conflicts with the need for rapid data capture
during tractor movement in field conditions. Therefore,
FLRs measured with leaves without dark adaptation and
using a PEA fluorometer were also used for subsequent
analysis by the same research group (Ker
¨
anen et al. 2003).
Importantly, the analysis based on the PEA data had
about the same or, in some cases, even better accuracy of
correct identification as the analysis based on the PAM data
(Ker
¨
anen et al. 2003).
When FLR or FI is used for pattern recognition, whole
measured curves are not used but, instead, only some
‘features’ of the curves are analysed. Currently, the features
used are slopes and y-axis intercepts of regression lines
to experimental FLR or FI data in selected time intervals.
The best selection of the time intervals is therefore very
important to obtain features that are species-specific and
therefore usage of which would result in best pattern
recognition. This problem was addressed by Codrea et al.
(2003, 2004), who used an optimiser (genetic algorithm)
to tune the endpoints of the time intervals to improve the
pattern recognition.
V. 3 In silico photosynthesis to understand photosynthetic
regulations and limitations – future prospects
A summary of possible origins of the steps in the FLR, given
in sections II.4 and III, clearly shows that there exist several
regulatory mechanisms that can fine tune photosynthetic
performance, even during the very first second of illumination
of photosynthesising organisms. What is important is that not
only the function of PSII can be reflected in the FLR but also
a function of the electron transport chain within thylakoid
membranes and a means of subsequent electron usage by
FNR and the Calvin–Benson cycle. Therefore, in principle,
all the photosynthetic reactions, involved in different

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 23
levels of organisation, should be somehow reflected in the
FLR or FI.
Prolonged light illumination and consideration of higher
levels of organisation lead to very large numbers of
different regulatory mechanisms involved in different levels
of organisation. One way to understand the complexity of
operating machinery is to use mathematical modelling of all
the events, i.e. to form in silico photosynthesis. Mathematical
modelling is therefore an important tool for the better
understanding of the explored processes. For recent reviews
on mathematical modelling of plant metabolic pathways, see
Giersch (2000) and Morgan and Rhodes (2002).
At present there is no global model of photosynthesis
(but see http://www.e-photosynthesis.org for a developing
project; verified 12 September 2005) that would satisfactorily
describe all the related phenomena measured by respective
quantities. However, several detailed models describing
particular photosynthetic events and regulations have been
suggested. For example, in addition to exploration of the
O–J–I–P FLR, models describing electron transport reactions
within thylakoid membrane were also used for theoretical
exploration of the electron transport chain as such (Berry and
Rumberg 2001), flash and continual light induced oxygen
evolution (Ka
ˇ
na et al. 2002), dependence of fluorescence
signal on linearly increasing temperature of the sample
(Kou
ˇ
ril et al. 2004), and forced fluorescence oscillations
(Nedbal et al. 2005). It is therefore a challenge for future work
to construct a mathematical model of whole photosynthesis.
Such model would be used, for example, in revealing the
regulatory mechanisms that are not directly accessible or
understandable from the available experimental data and
for finding the limitations of photosynthesis under different
experimental conditions.
When limitations of photosynthesis under different
conditions are found, they can be used in an effort
to increase photosynthetic efficiency and crop yield
by means of specific mutations. However, engineering
mutants, which lead to increased efficiencies and yields,
remains a long-term goal. Sinclair et al. (2004) showed,
by means of theoretical calculations, that assumed
50% increase in mRNA production responsible for the
synthesis of small and large subunits of ribulose-1,5-
bisphosphate carboxylase / oxygenase (Rubisco), the key
enzyme in Calvin–Benson cycle, would lead only to
37% increase in Rubisco content, 33% increase in
light-saturated leaf photosynthetic rate, 30% increase
in photosynthetic rate of isolated plant, 18% increase in
accumulated crop mass, 6% increase in grain yield with
additional nitrogen supply, but possibly a 6% decrease in
grain yield without additional nitrogen supply. Therefore it
seems that there may not be much room to increase efficiency
and yield of natural photosynthesis. Thus, efforts should be
focused on the construction of an artificial photosynthesis to
serve our increasing consumption demands.
VI Conclusions
This review has summarised the current understanding of
the FLR measured under high intensity of exciting light, the
O–J–I–P transient. The review also included a discussion
of other steps, K, H, and G, which appear in the FLR
under certain conditions. Some specific applications of the
FLR were also mentioned. Although as the current text
implies, many interpretations have been suggested for the
particular steps of the FLR, yet this phenomenon is not
fully understood. Further research is necessary to fully
understand the relationship of the O–J–I–P transient to
photosynthetic reactions.
Acknowledgments
This work was financially supported by the Ministry of
Education of the Czech Republic by a grant number
MSM 6198959215. This review was also a part of the
Habilitation Thesis of the author (Laz
´
ar 2005). I thank
Professors Govindjee, Ulrich Schreiber, and Reto J. Strasser
for their valuable comments that have improved this
presentation. In addition, Govindjee has also edited parts of
the manuscript.
References
Albert KR, Mikkelsen TN, Ro-Poulsen H (2005) Effects of ambient
versus reduced UV-B radiation on high arctic Salix arctica assessed
by measurements and calculations of chlorophyll a fluorescence
parameters from fluorescence transients. Physiologia Plantarum
124, 208–226. doi: 10.1111/j.1399-3054.2005.00502.x
Avarmaa R, Soovik T, Tamkivi A, Tonissoo B (1977)
Fluorescence lifetimes of chlorophyll-a and some related
compounds at low temperatures. Studia Biophysica Berlin 65,
213–218.
Baake E, Strasser RJ (1990) A differential equation model for
the description of the fast fluorescence rise (O–I–D–P-transient)
in leaves. In ‘Current research in photosynthesis. Vol. II’.
(Ed. M Baltscheffsky) pp. 567–570. (Kluwer Academic Publishers:
Dordrecht)
Baake E, Schl
¨
oder JP (1992) Modelling the fast fluorescence rise of
photosynthesis. Bulletin of Mathematical Biology 54, 999–1021.
doi: 10.1016/S0092-8240(05)80092-8
Baker NR, Rosenqvist E (2004) Applications of chlorophyll
fluorescence can improve crop production strategies: an examination
of future possibilities. Journal of Experimental Botany 55,
1607–1621. doi: 10.1093/jxb/erh196
Barth
´
elemy X, Popovic R, Franck F (1997) Studies on the O–J–I–P
transient of chlorophyll fluorescence in relation to photosystem II
assembly and heterogeneity in plastids of greening barley. Journal
of Photochemistry and Photobiology. B, Biology 39, 213–218.
doi: 10.1016/S1011-1344(97)00012-2
Barzda V, Vengris M, Valkunas L, van Grondelle R, van Amerongen H
(2000) Generation of fluorescence quenchers from the triplet states
of chlorophylls in the major light-harvesting complex II from green
plants. Biophysical Journal 39, 10468–10477.
Berry S, Rumberg B (2001) Kinetic modeling of the photosynthetic
electron transport chain. Bioelectrochemistry 53, 35–53.
doi: 10.1016/S0302-4598(00)00108-2

24 Functional Plant Biology D. Laz
´
ar
Bj
¨
orkman O, Demmig B (1987) Photon yield of O
2
evolution of
chlorophyll fluorescence characteristics at 77 K among vascular
plants of diverse origins. Planta 170, 489–504. doi: 10.1007/
BF00402983
Bouges-Bocquet B (1973) Electron transfer between the two
photosystems in spinach chloroplasts. Biochimica et Biophysica
Acta 314, 250–256.
Breton J (1983) The emission of chlorophyll in vivo. Antenna
fluorescence or ultrafast luminescence from reaction centre
pigments. FEBS Letters 159, 1–5. doi: 10.1016/0014-5793
(83)80405-0
Breton J, Wakeham MC, Fyfe PK, Jones MR, Nabedryk E
(2004) Characterization of the bonding interactions of Q
B
upon
photoreduction via A-branch or B-branch electron transfer in mutant
reaction centers from Rhodobacter sphaeroides. Biochimica et
Biophysica Acta 1656, 127–138.
Briantais J-M, Dacosta J, Goulas Y, Ducruet J-M, Moya I
(1996) Heat stress induces in leaves an increase of the
minimum level of chlorophyll fluorescence, F
0
: a time-resolved
analysis. Photosynthesis Research 48, 189–196. doi: 10.1007/
BF00041008
Brody SS (2002) Fluorescence lifetime, yield, energy transfer and
spectrum in photosynthesis, 1950–1960. Photosynthesis Research
73, 127–132. doi: 10.1023/A:1020405921105
Bruce D, Samson G, Carpenter C (1997) The origins of
nonphotochemical quenching of chlorophyll fluorescence in
photosynthesis. Direct quenching by P680
+
in photosystem II
enriched membranes at low pH. Biochemistry 36, 749–755.
doi: 10.1021/bi962216c
Bukhov NG, Govindachary S, Egorova EA, Joly D, Carpentier R (2003)
N,N,N

,N

-tetramethyl-p-phenylenediamine initiates the appearance
of a well-resolved I peak in the kinetics of chlorophyll fluorescence
rise in isolated thylakoids. Biochimica et Biophysica Acta 1607,
91–96.
Bukhov NG, Egorova EA, Govindachary S, Carpentier R (2004)
Changes in polyphasic chlorophyll a fluorescence induction curve
upon inhibition of donor or acceptor side of photosystem II
in isolated thylakoids. Biochimica et Biophysica Acta 1657,
121–130.
Bulychev AA, Niyazova MM (1989) Modelling of potential-depending
changes of chlorophyll fluorescence in the photosystem 2. Biofizika
34, 63–67.
Bulychev AA, Vredenberg WJ (1999) Light-triggered electrical
events in the thylakoid membrane of plant chloroplasts.
Physiologia Plantarum 105, 577–584. doi: 10.1034/j.1399-3054.
1999.105325.x
Bulychev AA, Vredenberg WJ (2001) Modulation of photosystem II
chlorophyll fluorescence by electrogenic events generated by
photosystem I. Bioelectrochemistry 54, 157–168. doi: 10.1016/
S1567-5394(01)00124-4
Bussotti F (2004) Assessment of stress conditions in Quercus ilex L.
leaves by O–J–I–P chlorophyll alpha fluorescence analysis. Plant
Biosystems 138, 101–109. doi: 10.1080/11263500412331283708
Bussotti F, Agati G, Desotgiu R, Matteini P, Tani C (2005)
Ozone foliar symptoms in woody plant species assessed with
ultrastructural and fluorescence analysis. New Phytologist 166,
941–955. doi: 10.1111/j.1469-8137.2005.01385.x
Butler WL (1972) On the primary nature of fluorescence yield
changes associated with photosynthesis. Proceedings of the National
Academy of Sciences USA 69, 3420–3422.
Butler WL (1977) Chlorophyll fluorescence: a probe for electron
transfer and energy transfer. In ‘Encyclopedia of plant physiology.
Photosynthesis I, volume 5’. (Eds VA Trebs, M Avron) pp. 149–167.
(Springer-Verlag: Berlin)
Butler WL (1978) Energy distribution in the photochemical apparatus
of photosynthesis. Annual Review of Plant Physiology and Plant
Molecular Biology 29, 345–378.
Byrdin M, Rimke I, Schlodder E, Stehlik D, Roelofs TA (2000) Decay
kinetics and quantum yields of fluorescence in photosystem I from
Synechococcus elongatus with P700 in the reduced and oxidized
state: are the kinetics of excited state decay trap-limited or transfer-
limited? Biophysical Journal 79, 992–1007.
Chaerle L, Van Der Straeten D (2000) Imaging techniques and the
early detection of plant stress. Trends in Plant Science 5, 495–501.
doi: 10.1016/S1360-1385(00)01781-7
Chaerle L, Van Der Straeten D (2001) Seeing is believing: imaging
techniques to monitor plant health. Biochimica et Biophysica Acta
1519, 153–166.
Chylla RA, Whitmarsh J (1989) Inactive photosystem II complexes in
leaves. Plant Physiology 90, 765–772.
Codrea CM, Aittokallio T, Ker
¨
anen M, Tyystj
¨
arvi E, Nevalainen OS
(2003) Feature learning with a genetic algorithm for fluorescence
fingerprinting of plant species. Pattern Recognition Letters 24,
2663–2673. doi: 10.1016/S0167-8655(03)00109-0
Codrea CM, Aittokallio T, Ker
¨
anen M, Tyystj
¨
arvi E, Nevalainen OS
(2004) Genetic feature learning algorithm for fluorescence
fingerprinting of plants. Lecture Notes in Computer Science 2936,
371–384.
Crofts AR, Wraight CA (1983) The electrochemical domain
of photosynthesis. Biochimica et Biophysica Acta 726,
149–185.
Dau H (1994) Molecular mechanism and quantitative models of variable
photosystem II fluorescence. Photochemistry and Photobiology 60,
1–23.
Dau H, Sauer K (1991) Electric field effect on chlorophyll fluorescence
and its relation to photosystem II charge separation reactions studied
by a salt-jump technique. Biochimica et Biophysica Acta 1098,
49–60.
Dau H, Sauer K (1992) Electric field effect on the picosecond
fluorescence of photosystem II and its relation to the energetics and
kinetics of primary charge separation. Biochimica et Biophysica Acta
1102, 91–106.
Dau H, Windecker R, Hansen U-P (1991) Effect of light-induced
changes in thylakoid voltage on chlorophyll fluorescence of
Aegopodium podagraria leaves. Biochimica et Biophysica Acta
1057, 337–345.
Delosme R (1967)
´
Etude de l’induction de fluorescence des algues vertes
et des chloroplastes au d
´
ebut d’une illumination intense. Biochimica
et Biophysica Acta 143, 108–128.
Deprez J, Dobek A, Geacintov NE, Paillotin G, Breton J (1983) Probing
fluorescence induction in chloroplast on a nanosecond time scale
utilizing picosecond laser pulse pairs. Biochimica et Biophysica Acta
725, 444–454.
Duysens LNM, Sweers HE (1963) Mechanism of the two photochemical
reactions in algae as studied by means of fluorescence. In ‘Studies
on microalgae and photosynthetic bacteria’. (Ed. Japanese Society
of Plant Physiologists) pp. 353–372. (University of Tokyo Press:
Tokyo, Japan)
Epitalawage N, Eggenberg P, Strasser RJ (2003) Use of fast chlorophyll
a fluorescence technique in detecting drought and salinity tolerant
chickpea (Cicer arietinum L.) varieties. Archives des Sciences
Gen
´
eve 56, 79–93.
Fell DA (1992) Metabolic control analysis: a survey of its theoretical
and experimental development. Biochemical Journal 286,
313–330.
Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004)
Architecture of the photosynthetic oxygen-evolving center. Science
303, 1831–1838. doi: 10.1126/science.1093087

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 25
Franck F, Juneau P, Popovic R (2002) Resolution of the photosystem I
and photosystem II contributions to chlorophyll fluorescence of
intact leaves at room temperature. Biochimica et Biophysica Acta
1556, 239–246.
Franck F, Dewez D, Popovic R (2005) Changes in the room-temperature
emission spectrum of chlorophyll during fast and slow phases of the
Kautsky effect in intact leaves. Photochemistry and Photobiology
81, 431–436. doi: 10.1562/2004-03-01-RA-094.1
Fridlyand LE, Backhausen JE, Scheibe R (1998) Flux control of the
malate valve in leaf cells. Archives of Biochemistry and Biophysics
349, 290–298. doi: 10.1006/abbi.1997.0482
Frolov D, Wakeham MC, Andrizhiyevskaya EG, Jones MR,
van Grondelle R (2005) Investigation of B-branch electron transfer
by femtosecond time resolved spectroscopy in a Rhodobacter
sphaeroides reaction centre that lacks the Q
A
ubiquinone.
Biochimica et Biophysica Acta 1707, 189–198.
Giersch C (2000) Mathematical modelling of metabolism. Current
Opinion in Plant Biology 3, 249–253.
Gilmore AM, Hazlett TL, Debrunner PG, Govindjee (1996)
Photosystem II chlorophyll a fluorescence lifetimes and intensity are
independent of the antenna size differences between barley wild-type
and chlorina mutants: photochemical quenching and xanthophyll
cycle-dependent nonphotochemical quenching of fluorescence.
Photosynthesis Research 48, 171–187. doi: 10.1007/BF00041007
Gilmore A, Itoh SS, Govindjee (2000) Global spectral-kinetic analysis
of room temperature chlorophyll a fluorescence from light
harvesting antenna mutants of barley. Philosophical Transactions
of Royal Society of London Series B-Biological Sciences 335,
1–14.
Goltsev V, Yordanov I (1997) Mathematical model of prompt
and delayed chlorophyll fluorescence induction kinetics.
Photosynthetica 33, 571–586.
Goltsev V, Zaharieva I, Lambrev P, Yordanov I, Strasser R (2003)
Simultaneous analysis of prompt and delayed chlorophyll a
fluorescence in leaves during the induction period of dark to
light adaptation. Journal of Theoretical Biology 225, 171–183.
doi: 10.1016/S0022-5193(03)00236-4
Goltsev V, Chernev P, Zaharieva I, Lambrev P, Strasser RJ (2005)
Kinetics of delayed chlorophyll a fluorescence registered in
milliseconds time range. Photosynthesis Research 84, 209–215.
doi: 10.1007/s11120-004-6432-2
Govindjee (1995) Sixty-three years since Kautsky: chlorophyll a
fluorescence. Australian Journal of Plant Physiology 22, 131–160.
Govindjee (2004) Chlorophyll a fluorescence: a bit of basics
and history. In ‘Chlorophyll a fluorescence: a signature of
photosynthesis’. (Eds GC Papageorgiou, Govindjee) pp. 1–42.
(Springer: Dordrecht)
Govindjee, Amesz J, Fork DC (Eds) (1986) ‘Light emission by plants
and bacteria.’ (Academic Press: Orlando, FL)
Graan T, Ort DR (1983) Initial events in the regulation of electron
transfer in chloroplasts. The role of the membrane potential. Journal
of Biological Chemistry 258, 2831–2836.
Graan T, Ort DR (1984) Quantitation of the rapid electron donors
to P700, the functional plastoquinone pool, and the ratio of
the photosystems in spinach chloroplasts. Journal of Biological
Chemistry 259, 14003–14010.
Graan T, Ort DR (1986) Detection of oxygen-evolving photosystem II
centers inactive in plastoquinone reduction. Biochimica et
Biophysica Acta 852, 320–330.
Groot ML, Pawlowicz NP, van Wilderen JLGW, Breton J,
van Stokkum IHM, van Grondelle R (2005) Initial electron donor
and acceptor in isolated photosystem II reaction centers identified
with femtosecond mid-IR spectroscopy. Proceedings of the National
Academy of Sciences USA 102, 13087–13092.
Gravano E, Bussotti F, Strasser RJ, Schaub M, Novak K, Skelly J,
Tani C (2004) Ozone symptoms in leaves of woody plants in
open-top chambers: ultrastructural and physiological characteristics.
Physiologia Plantarum 121, 620–633. doi: 10.1111/j.1399-3054.
2004.00363.x
Guiss
´
e B, Srivastava A, Strasser RJ (1995a) The polyphasic rise of
the chlorophyll a fluorescence (O–K–J–I–P) in heat-stressed leaves.
Archives des Sciences Gen
´
eve 48, 147–160.
Guiss
´
e B, Srivastava A, Strasser RJ (1995b) Effects of high temperature
and water stress on the polyphasic chlorophyll a fluorescence
transient of potato leaves. In ‘Photosynthesis: from light to
biosphere. Vol. IV’. (Ed. P Mathis), pp. 913–916. (Kluwer Academic
Publishers: Dordrecht)
Haldimann P, Tsimilli-Michael M (2005) Non-photochemical
quenching of chlorophyll a fluorescence by oxidised
plastoquinone: new evidences based on modulation of the
redox state of the endogenous plastoquinone pool in broken
spinach chloroplasts. Biochimica et Biophysica Acta 1706,
239–249.
Hansen U-P, Dau H, Br
¨
uning B, Fritsch T, Moldaenke C (1991) Linear
analysis applied to the comparative study of the I–D–P phase of
chlorophyll fluorescence as induced by actinic PS-II light, PS-I
light and changes in CO
2
-concentration. Photosynthesis Research
28, 119–130. doi: 10.1007/BF00054125
Heredia P, De Las Rivas J (2003) Fluorescence induction of
photosystem II membranes shows the steps till reduction and
protonation of the quinone pool. Journal of Plant Physiology 160,
1499–1506. doi: 10.1078/0176-1617-01011
Hill R, Larkum AWD, Frankart C, K
¨
uhl M, Ralph PJ (2004)
Loss of functional photosystem II reaction centres in
zooxanthellae of coral exposed to bleaching conditions: using
fluorescence rise kinetics. Photosynthesis Research 82, 59–72.
doi: 10.1023/B:PRES.0000040444.41179.09
Holzwarth AR, M
¨
uller MG, Niklas J, Lubitz W (2005a) Charge
recombination fluorescence in photosystem I reaction centers from
Chlamydomonas reinhardtii. Journal of Physical Chemistry B 109,
5903–5911.
Holzwarth AR, M
¨
uller MG, Niklas J, Lubitz W (2005b) Ultrafast
transient absorption studies on photosystem I reaction centers from
Chlamydomonas reinhardtii. 2. Mutations near the P700 reaction
center chlorophylls provide insight into the nature of the primary
electron donor. Biophysical Journal, (in press).
Hsu B-D (1992a) The active photosystem II centers can make
a significant contribution to the initial fluorescence rise from
F
0
to F
i
. Plant Science 81, 169–174. doi: 10.1016/0168-9452
(92)90039-O
Hsu B-D (1992b) A theoretical study on the fluorescence induction
curve of spinach thylakoids in the absence of DCMU. Biochimica et
Biophysica Acta 1140, 30–36.
Hsu B-D (1993) Evidence for the contribution of the S-state
transitions of oxygen evolution to the initial phase of fluorescence
induction. Photosynthesis Research 36, 81–88. doi: 10.1007/
BF00016272
Hsu B-D, Lee J-Y (1995) Fluorescence quenching by plastoquinone in
an oxygen evolving photosystem-II-enriched preparation. Journal
of Photochemistry and Photobiology. B, Biology 30, 57–61.
doi: 10.1016/1011-1344(95)07146-S
Hsu B-D, Leu K-L (2003) A possible origin of the middle phase
of polyphasic chlorophyll fluorescence transient. Functional Plant
Biology 30, 571–576. doi: 10.1071/FP03012
Ikegami I (1976) Fluorescence changes related in the primary
photochemical reaction in the P-700-enriched particles isolated
from spinach chloroplasts. Biochimica et Biophysica Acta 449,
245–258.

26 Functional Plant Biology D. Laz
´
ar
Il
´
ık P, Schansker G, Kotabov
´
aE,V
´
aczi P, Strasser RJ, Bart
´
ak M (2006)
A dip in the chlorophyll fluorescence induction at 0.2–2 s
in Trebouxia-possessing lichens reflects a fast reoxidation of
photosystem I. A comparison with higher plants. Biochimica et
Biophysica Acta, (in press).
Itoh S, Sugiura K (2004) Fluorescence of photosystem I. In
‘Chlorophyll a fluorescence: a signature of photosynthesis’.
(Eds GC Papageorgiou, Govindjee) pp. 231–250. (Springer:
Dordrecht)
Joliot P, Joliot A (1977) Evidence for a double hit process in
photosystem II based on fluorescence studies. Biochimica et
Biophysica Acta 462, 559–574.
Joliot P, Joliot A (1979) Comparative study of the fluorescence yield and
of the C550 absorption change at room temperature. Biochimica et
Biophysica Acta 546, 93–105.
Joliot P, Lavergne J, B
´
eal D (1992) Plastoquinone compartmentation in
chloroplasts. I. Evidence for domains with different rates of photo-
reduction. Biochimica et Biophysica Acta 1101, 1–12.
Joly D, Bigras C, Harnois J, Govindachary S, Carpentier R (2005)
Kinetic analyses of the OJIP chlorophyll fluorescence rise in
thylakoid membranes. Photosynthesis Research 84, 107–112.
doi: 10.1007/s11120-004-7763-8
Joshi MJ, Mohanty P (1995) Probing photosynthetic performance
by chlorophyll-a-fluorescence — analysis and interpretation of
fluorescence parameters. Journal of Scientific and Industrial
Research 54, 155–174.
Kamiya N, Shen J-R (2003) Crystal structure of oxygen-evolving
photosystem II from Thermosynechococcus vulcanus at 3.8-
˚
A
resolution. Proceedings of the National Academy of Sciences USA
100, 98–103. doi: 10.1073/pnas.0135651100
Ka
ˇ
na R, Laz
´
ar D, Pr
´
a
ˇ
sil O, Nau
ˇ
s J (2002) Experimental and theoretical
studies on the excess capacity of photosystem II. Photosynthesis
Research 72, 271–284. doi: 10.1023/A:1019894720789
Ker
¨
anen M, Aro E-M, Tyystj
¨
arvi E, Nevalainen OS (2003) Automatic
plant identification with chlorophyll fluorescence fingerprinting.
Precision Agriculture 4, 53–67. doi: 10.1023/A:1021863005378
Keuper HJK, Sauer K (1989) Effect of photosystem-II reaction
center closure on nanosecond fluorescence relaxation kinetics.
Photosynthesis Research 20, 85–103. doi: 10.1007/BF00028623
Kirchhoff H, Horstmann S, Weis E (2000) Control of the
photosynthetic electron transport by PQ diffusion microdomains in
thylakoids of higher plants. Biochimica et Biophysica Acta 1459,
148–168.
Kirmaier C, Laible PD, Hanson DK, Holten D (2003) B-side charge
separation in bacterial photosynthetic reaction centers: nanosecond
time scale electron transfer from H
B
−
to Q
B
. Biochemistry 42,
2016–2024. doi: 10.1021/bi0269730
Kobl
´
ı
ˇ
zek M, Kaftan D, Nedbal L (2001) On the relationship between
the non-photochemical quenching of the chlorophyll fluorescence
and the photosystem II light harvesting efficiency. A repetitive flash
fluorescence induction study. Photosynthesis Research 68, 141–152.
doi: 10.1023/A:1011830015167
Kok B, Forbush B, McGloin M (1970) Cooperation of charges in
photosynthetic O
2
evolution — I. A linear four step mechanism.
Photochemistry and Photobiology 11, 457–475.
Kou
ˇ
ril R, Laz
´
ar D, Il
´
ık P, Skotnica J, Krch
ˇ
n
´
ak P, Nau
ˇ
s J (2004)
High-temperature induced chorophyll fluorescence rise in plants
at 40–50
◦
C: experimental and theoretical approach. Photosynthesis
Research 81, 49–66. doi: 10.1023/B:PRES.0000028391.70533.eb
Kramer DM, DiMarco G, Loreto F (1995). Contribution of
plastoquinone quenching to saturation pulse-induced rise of
chlorophyll fluorescence in leaves. In ‘Photosynthesis: from light to
biosphere. Vol. I’. (Ed. P Mathis) pp. 147–150. (Kluwer Academic
Publishers: Dordrecht)
Krause GH, Weis E (1991) Chlorophyll fluorescence and
photosynthesis: the basis. Annual Reviews in Plant Physiology and
Plant Molecular Biology 42, 313–349. doi: 10.1146/annurev.pp.42.
060191.001525
Kurreck J, Renger G (1998) Investigation of the plastoquinone pool size
and fluorescence quenching in photosystem II (PS II) membrane
fragments. In ‘Photosynthesis: mechanisms and effects. Vol. 2’.
(Ed. G Garab) pp. 1157–1160. (Kluwer Academic Publishers:
Dordrecht)
Kurreck J, Sch
¨
odel R, Renger G (2000) Investigation of the
plastoquinone pool size and fluorescence quenching in
thylakoid membranes and photosystem II (PS II) membrane
fragments. Photosynthesis Research 63, 171–182. doi: 10.1023/
A:1006303510458
Laible PD, Zipfel W, Owens TG (1994) Excited state dynamics
in chlorophyll-based antennae: the role of transfer equilibrium.
Biophysical Journal 66, 844–860.
Latimer P, Bannister TT, Rabinowitch E (1956) Quantum
yields of fluorescence of plant pigments. Science 124,
585–586.
Lavergne J, Briantais J-M (1996) Photosystem II heterogeneity. In
‘Oxygenic photosynthesis: the light reactions’. (Eds DR Ort,
CF Yocum) pp. 265–287. (Kluwer Academic Publishers:
Dordrecht)
Lavergne J, Leci E (1993) Properties of inactive photosystem II centers.
Photosynthesis Research 35, 323–343. doi: 10.1007/BF00016563
Lavergne J, Rappaport F (1998) Stabilization of charge separation
and photochemical misses in photosystem II. Biochemistry 37,
7899–7906. doi: 10.1021/bi9801210
Laz
´
ar D (1999) Chlorophyll a fluorescence induction. Biochimica et
Biophysica Acta 1412, 1–28.
Laz
´
ar D (2003) Chlorophyll a fluorescence rise induced by high
light illumination of dark-adapted plant tissue studied by means
of a model of photosystem II and considering photosystem II
heterogeneity. Journal of Theoretical Biology 220, 469–503.
doi: 10.1006/jtbi.2003.3140
Laz
´
ar D (2005) The O–K–J–I–P chlorophyll a fluorescence transient:
theory and experiments. Habilitation Thesis, Palack
´
y University
Olomouc, Czech Republic.
Laz
´
ar D, Il
´
ık P (1997) High-temperature induced chlorophyll
fluorescence changes in barley leaves. Comparison of the critical
temperatures determined from fluorescence induction and from
fluorescence temperature curve. Plant Science 124, 159–164.
doi: 10.1016/S0168-9452(97)04602-5
Laz
´
ar D, Nau
ˇ
s J (1998) Statistical properties of chlorophyll
fluorescence induction parameters. Photosynthetica 35, 121–127.
doi: 10.1023/A:1006886202444
Laz
´
ar D, Posp
´
ı
ˇ
sil P (1999) Mathematical simulation of chlorophyll a
fluorescence rise measured with 3-(3

,4

-dichlorophenyl)-1,
1-dimethylurea-treated barley leaves at room and high
temperatures. European Biophysics Journal 28, 468–477.
doi: 10.1007/s002490050229
Laz
´
ar D, Broke
ˇ
sM,Nau
ˇ
sJ,Dvo
ˇ
r
´
ak L (1998) Mathematical
modelling of 3-(3

,4

-dichlorophenyl)-1,1-dimethyl urea action
in plant leaves. Journal of Theoretical Biology 191, 79–86.
doi: 10.1006/jtbi.1997.0566
Laz
´
ar D, Il
´
ık P, Nau
ˇ
s J (1997a) An appearance of K-peak in
fluorescence induction depends on the acclimation of barley leaves
to higher temperature. Journal of Luminescence 72–74, 595–598.
doi: 10.1016/S0022-2313(96)00293-1
Laz
´
ar D, Nau
ˇ
s J, Matou
ˇ
skov
´
aM,Fla
ˇ
sarov
´
a M (1997b) Mathematical
modelling of changes in chlorophyll fluorescence induction caused
by herbicides. Pesticide Biochemistry and Physiology 57, 200–210.
doi: 10.1006/pest.1997.2277

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 27
Laz
´
ar D, Posp
´
ı
ˇ
sil P, Nau
ˇ
s J (1999) Decrease of fluorescence intensity
after the K step in chlorophyll a fluorescence induction is suppressed
by electron acceptors and donors to photosystem II. Photosynthetica
37, 255–265. doi: 10.1023/A:1007112222952
Laz
´
ar D, Tomek P, Il
´
ık P, Nau
ˇ
s J (2001) Determination of the
antenna heterogeneity of photosystem II by direct simultaneous
fitting of several fluorescence rise curves measured with DCMU
at different light intensities. Photosynthesis Research 68, 247–257.
doi: 10.1023/A:1012973402023
Laz
´
ar D, Nau
ˇ
s J, Hlav
´
a
ˇ
ckov
´
aV,
ˇ
Spundov
´
a M, Mieslerov
´
aB
(2003) Statistical changes in chlorophyll fluorescence rise within
adaxial leaf area caused by worsening of photosystem II
function. In ‘Proceedings of international conference on advances
in statistical inferential methods: theory and applications’.
(Ed. V Voinov) pp. 339–346. (KIMEP: Almaty, Kazakhstan
Republic)
Laz
´
ar D, Su
ˇ
sila P, Nau
ˇ
s J (2005a) Statistical properties of parameters
evaluated from the O–J–I–P chlorophyll a fluorescence transients
and their changes upon high temperature stress. In ‘Fundamental
aspects to global perspectives. [CD-ROM]’. (Eds A van der Est,
D Bruce) (Allen Press: Montreal)
Laz
´
ar D, Il
´
ık P, Kruk J, Strzałka K, Nau
ˇ
s J (2005b) A theoretical
study on effect of the initial redox state of cytochrome b
559
on
maximal chlorophyll fluorescence level (F
M
). Implications for
photoinhibition of photosystem II. Journal of Theoretical Biology
233, 287–300. doi: 10.1016/j.jtbi.2004.10.015
Laz
´
ar D, Ka
ˇ
na R, Klinkovsk
´
yT,Nau
ˇ
s J (2005c) Experimental
and theoretical study on high temperature induced changes
in chlorophyll a fluorescence oscillations from barley leaves
upon 2% CO
2
. Photosynthetica 43, 13–27. doi: 10.1007/
s11099-005-3027-x
Laz
´
ar D, Su
ˇ
sila P, Nau
ˇ
s J (2006) Early detection of plant stress from
changes in distributions of chlorophyll a fluorescence parameters
measured with fluorescence imaging. Journal of Fluorescence,
(in press).
Lebedeva GV, Belyaeva NE, Demin OV, Riznichenko GY, Rubin AB
(2002) Kinetic model of primary photosynthetic processes
in chloroplasts. Description of the fast phase of chlorophyll
fluorescence induction under different light intensities. Biophysics
47, 968–980.
Leibl W, Breton J, Deprez J, Trissl H-W (1989) Photoelectric
study on the kinetics of trapping and charge stabilization in
oriented PS II membranes. Photosynthesis Research 22, 257–275.
doi: 10.1007/BF00048304
M
¨
uller A, Lumry R, Walker MS (1969) Light-intensity dependence
of in vivo fluorescence lifetime of chlorophyll. Photochemistry and
Photobiology 9, 113–126.
Marder JB, Raskin VI (1993) The assembly of chlorophyll into pigment–
protein complexes. Photosynthetica 28, 243–248.
Mathis P, Paillotin G (1981) Primary processes of photosynthesis. In
‘The biochemistry of plants. Vol. 8’. (Eds MD Hatch, NK Boardman)
pp. 97–161. (Academic Press: New York)
Mauzerall D (1972) Light-induced fluorescence changes in Chlorella,
and the primary photoreactions the production of oxygen.
Proceedings of the National Academy of Sciences USA 69,
1358–1362.
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence —
a practical guide. Journal of Experimental Botany 51, 659–668.
doi: 10.1093/jexbot/51.345.659
McCauley S, Melis A (1987) Quantitation of photosystem II activity
in spinach chloroplasts. Effect of artificial quinone acceptors.
Photochemistry and Photobiology 46, 543–550.
Melis A (1985) Functional properties of photosystem II
β
in spinach
chloroplasts. Biochimica et Biophysica Acta 808, 334–342.
Mohammed GH, Binder WD, Gillies SL (1995) Chlorophyll
fluorescence—areviewofitspractical forestry applications
and instrumentation. Scandinavian Journal of Forest Research 10,
383–410.
Moise N, Moya I (2004a) Correlation between lifetime heterogeneity
and kinetics heterogeneity during chlorophyll fluorescence induction
in leaves: 1. Mono-frequency phase and modulation analysis reveals
a conformational change of a PSII pigment complex during the IP
thermal phase. Biochimica et Biophysica Acta 1657, 33–46.
Moise N, Moya I (2004b) Correlation between lifetime heterogeneity
and kinetics heterogeneity during chlorophyll fluorescence induction
in leaves: 2. Multi-frequency phase and modulation analysis
evidences a loosely connected PSII pigment–protein complex.
Biochimica et Biophysica Acta 1657, 47–60.
Morgan JA, Rhodes D (2002) Mathematical modelling of
plant metabolic pathways. Metabolic Engineering 4, 80–89.
doi: 10.1006/mben.2001.0211
Morin P (1964)
´
Etudes des cin
´
etiques de fluorescence de la chlorophylle
in vivo, dans lea premiers instants qui suivent le d
´
ebut de
l’illumination. Journal of Chemical Physics 61, 674–680.
van Mourik F, Groot M-L, van Grondelle R, Dekker JP,
van Stokkum IHM (2004) Global and target analysis of fluorescence
measurements on photosystem 2 reaction centers upon red
excitation. Physical Chemistry Chemical Physics 6, 4820–4824.
Moya I, Cerovic ZG (2004) Remote sensing of chlorophyll fluorescence:
instrumentation and analysis. In ‘Chlorophyll a fluorescence:
a signature of photosynthesis’. (Eds GC Papageorgiou, Govindjee)
pp. 429–445. (Springer: Dordrecht)
M
¨
uller MG, Niklas J, Lubitz W, Holzwarth AR (2003) Ultrafast
transient absorption studies on photosystem I reaction centers
from Chlamydomonas reinhardtii. 1. A new interpretation of the
energy trapping and early electron transfer steps in photosystem I.
Biophysical Journal 85, 3899–3922.
Munday JC Jr, Govindjee (1969a) Light-induced changes in the
fluorescence yield of chlorophyll a in vivo. III. The dip and the peak
in the fluorescence transient of Chlorella pyrenoidosa. Biophysical
Journal 9, 1–21.
Munday JC Jr, Govindjee (1969b) Light-induced changes in the
fluorescence yield of chlorophyll a in vivo. IV. The effect
of preillumination on the fluorescence transient of Chlorella
pyrenoidosa. Biophysical Journal 9, 22–35.
Nedbal L, Whitmarsh J (2004) Chlorophyll a fluorescence imaging
of leaves and fruits. In ‘Chlorophyll a fluorescence: a signature of
photosynthesis’. (Eds GC Papageorgiou, Govindjee) pp. 389–407.
(Springer: Dordrecht)
Nedbal L, Trt
´
ılek M, Kaftan D (1999) Flash fluorescence induction:
a novel method to study regulation of photosystem II. Journal
of Photochemistry and Photobiology. B, Biology 48, 154–157.
doi: 10.1016/S1011-1344(99)00032-9
Nedbal L, B
ˇ
rezina V,
ˇ
Cerven
´
yJ,Trt
´
ılek M (2005) Photosynthesis in
dynamic light: systems biology of unconventional chlorophyll
fluorescence transients in Synechocystis sp. PCC 6803.
Photosynthesis Research 84, 99–106. doi: 10.1007/s11120-
004-6428-y
Neubauer C, Schreiber U (1987) The polyphasic rice of chlorophyll
fluorescence upon onset of strong continuous illumination:
I. Saturation characteristics and partial control by the
photosystem II acceptor side. Zeitschrift f
¨
ur Naturforschung 42c,
1246–1254.
Novoderezhkin VI, Andrizhiyevskaya EG, Dekker JP, van Grondelle R
(2005) Pathways and timescales of primary charge separation in
the photosystem II reaction center as revealed by a simultaneous fit
of time-resolved fluorescence and transient absorption. Biophysical
Journal 89, 1464–1481.

28 Functional Plant Biology D. Laz
´
ar
Oettmeier W, Soll HJ (1983) Competition between plastoquinone
and 3-(3,4-dichlorphenyl)-1,1-dimethylurea at the acceptor side of
photosystem II. Biochimica et Biophysica Acta 724, 287–297.
Oukarroum A, Strasser RJ (2004) Phenotyping of dark and light adapted
barley plants by the fast chlorophyll a fluorescence rise OJIP. South
African Journal of Botany 70, 277–283.
Owens TG (1996) Processing of excitation energy by antenna pigments.
In ‘Photosynthesis and the environment’. (Ed. NR Baker) pp. 1–23.
(Kluwer Academic Publishers: Dordrecht)
Oxborough K (2004a) Imaging of chlorophyll a fluorescence:
theoretical and practical aspects of an emerging technique for the
monitoring of photosynthetic performance. Journal of Experimental
Botany 55, 1195–1205. doi: 10.1093/jxb/erh145
Oxborough K (2004b) Using chlorophyll a fluorescence imaging
to monitor photosynthetic performance. In ‘Chlorophyll a
fluorescence: a signature of photosynthesis’. (Eds GC Papageorgiou,
Govindjee) pp. 409–428. (Springer: Dordrecht)
Paddock ML, Chang C, Xu Q, Abresch EC, Axelrod HL, Feher G,
Okamura MY (2005) Quinone (Q
B
) reduction by B-branch electron
transfer in mutant bacterial reaction centers from Rhodobacter
sphaeroides: quantum efficiency and X-ray structure. Biochemistry
44, 6920–6928. doi: 10.1021/bi047559m
Paoletti E, Bussotti F, Della Rocca G, Lorenzini G, Nali C, Strasser RJ
(2004) Fluorescence transient in ozonated Mediterranean shrubs.
Phyton-Annales REI Botanicae 44, 121–131.
Papageorgiou GC, Govindjee (Eds) (2004) ‘Chlorophyll a
fluorescence: a signature of photosynthesis.’ (Springer: Dordrecht,
The Netherlands)
Parvanova D, Popova A, Zaharieva I, Lambrev P, Konstantinova T,
Taneva S, Atanassov A, Goltsev V, Djilianov D (2004)
Low temperature tolerance of tobacco plants transformed
to accumulate proline, fructans, or glycine betaine. Variable
chlorophyll fluorescence evidence. Photosynthetica 42, 179–185.
doi: 10.1023/B:PHOT.0000040588.31318.0f
Pfarrherr A, Tencher K, Leupold D, Hoffmann P (1991) Chlorophyll b
in solution: fluorescence lifetimes, absorption and emission spectra
as criteria of purity. Journal of Photochemistry and Photobiology. B,
Biology 9, 35–41. doi: 10.1016/1011-1344(91)80002-Y
Pf
¨
undel E (1998) Estimating the contribution of photosystem I to
total leaf chlorophyll fluorescence. Photosynthesis Research 56,
185–195. doi: 10.1023/A:1006032804606
Poolman MG, Fell DA, Thomas S (2000) Modelling photosynthesis
and its control. Journal of Experimental Botany 51, 319–328.
doi: 10.1093/jexbot/51.suppl
1.319
Posp
´
ı
ˇ
sil P, Dau H (2000) Chlorophyll fluorescence transients of
photosystem II membrane particles as a tool for studying
photosynthetic oxygen evolution. Photosynthesis Research 65,
41–52. doi: 10.1023/A:1006469809812
Posp
´
ı
ˇ
sil P, Dau H (2002) Valinomycin sensitivity proves that light-
induced thylakoid voltages result in millisecond phase of chlorophyll
fluorescence transients. Biochimica et Biophysica Acta 1554,
94–100.
Ripley BS, Redfern SP, Dames J (2004) Ozone foliar symptoms
in woody plant species assessed with ultrastructural and
fluorescence analysis. South African Journal of Science 100,
615–618.
Roelofs TA, Lee C-H, Holzwarth AR (1992) Global target analysis of
picosecond chlorophyll fluorescence kinetics from pea chloroplasts.
A new approach to the characterization of the primary processes
in photosystem II α- and β-units. Biophysical Journal 61,
1147–1163.
Roh
´
a
ˇ
cek K (2002) Chlorophyll fluorescence parameters: the definitions,
photosynthetic meaning, and mutual relationships. Photosynthetica
40, 13–29.
Roh
´
a
ˇ
cek K, Bart
´
ak M (1999) Technique of the modulated chlorophyll
fluorescence: basic concepts, useful parameters, and some
applications. Photosynthetica 37, 339–363.
Ruth B (1990) A device for determination of the microsecond
component of the in vivo chlorophyll fluorescence induction
kinetics. Measurement Science & Technology 1, 517–521.
doi: 10.1088/0957-0233/1/6/010
Ruth B (1991) Measurement of the chlorophyll fluorescence induction
kinetics with 10 µs time resolution and its application in the
forest decline research. Radiation and Environmental Biophysics 30,
321–332. doi: 10.1007/BF01210516
Samson G, Bruce D (1996) Origins of the low yield of chlorophyll a
fluorescence induced by single turnover flash in spinach thylakoids.
Biochimica et Biophysica Acta 1276, 147–153.
Samson G, Pr
´
a
ˇ
sil O, Yaakoubd B (1999) Photochemical and thermal
phases of chlorophyll a fluorescence. Photosynthetica 37, 163–182.
doi: 10.1023/A:1007095619317
Satoh K, Katoh S (1981) Fluorescence induction in chloroplasts isolated
from the green alga, Bryopsis maxima IV. The I–D dip. Plant & Cell
Physiology 22, 11–21.
Sayed OH (2003) Chlorophyll fluorescence as a tool in cereal
crop research. Photosynthetica 41, 321–330. doi: 10.1023/
B:PHOT.0000015454.36367.e2
Schansker G, Srivastava A, Govindjee, Strasser RJ (2003)
Characterization of the 820-nm transmission signal paralleling the
chlorophyll a fluorescence rise (OJIP) in pea leaves. Functional
Plant Biology 30, 785–796.
Schansker G, T
´
oth SZ, Strasser RJ (2005) Methylviologen and
dibromorhymoquinone treatments of pea leaves reveal the role of
photosystem I in the Chl a fluorescence rise OJIP. Biochimica et
Biophysica Acta 1706, 250–261.
Schatz GH, Brock H, Holzwarth AR (1988) Kinetic and energetic model
for the primary processes in photosystem II. Biophysical Journal 54,
397–405.
Schreiber U (1986) Detection of rapid induction kinetics
with a new type of high-frequency modulated chlorophyll
fluorometer. Photosynthesis Research 9, 261–272. doi: 10.1007/
BF00029749
Schreiber U (2002) Assessment of maximal fluorescence yield:
donor-side dependent quenching and QB-quenching. In
‘Plant spectrofluorometry: applications and basic research’.
(Eds O van Kooten, JFH Snel) pp. 23–47. (Rozenberg Publishers:
Amsterdam)
Schreiber U (2004) Pulse-amplitude-modulation (PAM) fluorometry
and saturation pulse method: an overview. In ‘Chlorophyll a
fluorescence: a signature of photosynthesis’. (Eds GC Papageorgiou,
Govindjee) pp. 279–319. (Springer: Dordrecht)
Schreiber U, Neubauer C (1987) The polyphasic rice of chlorophyll
fluorescence upon onset of strong continuous illumination:
II. Partial control by the photosystem II donor side and
possible ways of interpretation. Zeitschrift f
¨
ur Naturforschung 42c,
1255–1264.
Schreiber U, Neubauer C (1990) O
2
-dependent electron flow, membrane
energization and the mechanism of non-photochemical quenching
of chlorophyll fluorescence. Photosynthesis Research 25, 279–293.
doi: 10.1007/BF00033169
Schreiber U, Krieger A (1996) Two fundamentally different types
of variable chlorophyll fluorescence in vivo. FEBS Letters 397,
131–135. doi: 10.1016/S0014-5793(96)01176-3
Schreiber U, Bauer R, Franck UF (1971) Chlorophyll fluorescence
induction in green plants at oxygen deficiency. In ‘Proceedings
of the IInd international congress on photosynthesis’.
(Eds G Forti, M Avron, A Melandri) pp. 169–179. (Junk:
The Hague)

The polyphasic chlorophyll a fluorescence rise Functional Plant Biology 29
Schreiber U, Schliwa U, Bilger W (1986) Continuous recording of
photochemical and nonphotochemical chlorophyll fluorescence
quenching with a new type of modulation fluorometer.
Photosynthesis Research 10, 51–62. doi: 10.1007/BF00024185
Schreiber U, Neubauer C, Klughammer C (1989) Devices and
methods for room-temperature fluorescence analysis. Philosophical
Transactions of the Royal Society of London. Series B, Biological
Sciences 323, 241–251.
Schreiber U, Hormann H, Neubauer C, Klughammer C (1995)
Assessment of photosystem II photochemical quantum yield by
chlorophyll fluorescence quenching analysis. Australian Journal of
Plant Physiology 22, 209–220.
Shigematsu Y, Satoh F, Yamada Y (1989) A binding model
for phenylurea herbicides based on analysis of a Thr 264
mutation in D-1 protein of tobacco. Pesticide Biochemistry
and Physiology 35, 33–41. doi: 10.1016/0048-3575(89)
90100-4
Shinkarev VP, Govindjee (1993) Insight into the relationship of
chlorophyll a fluorescence yield to the concentration of its natural
quenchers in oxygenic photosynthesis. Proceedings of the National
Academy of Sciences USA 90, 7466–7469.
Sinclair TR, Purcell LC, Sneller CH (2004) Crop transformation and
the challenge to increase yield potential. Trends in Plant Science 9,
70–75. doi: 10.1016/j.tplants.2003.12.008
Sonneveld A, Rademaker H, Duysens LNM (1979) Chlorophyll a
fluorescence as a monitor of nanosecond reduction of the
photooxidized primary donor P-680
+
of photosystem II. Biochimica
et Biophysica Acta 548, 536–551.
Srivastava A, Guiss
´
e B, Greppin H, Strasser RJ (1997) Regulation
of antenna structure and electron transport in photosystem II of
Pisum sativum under elevated temperature probed by the fast
polyphasic chlorophyll a fluorescence transient: OKJIP. Biochimica
et Biophysica Acta 1320, 95–106.
Stahl U, Tusov VB, Paschenko VZ, Voigh J (1989) Spectroscopic
investigations of fluorescence behaviour, role and function of
the long-wavelength pigments of photosystem I. Biochimica et
Biophysica Acta 973, 198–204.
Stirbet A, Govindjee, Strasser BJ, Strasser RJ (1995) Numerical
simulation of chlorophyll a fluorescence induction in plants. In
‘Photosynthesis: from light to biosphere. Vol. II’. (Ed. P Mathis)
pp. 919–922. (Kluwer Academic Publishers: Dordrecht)
Stirbet A, Govindjee, Strasser BJ, Strasser RJ (1998) Chlorophyll a
fluorescence induction in higher plants: modelling and numerical
simulation. Journal of Theoretical Biology 193, 131–151.
doi: 10.1006/jtbi.1998.0692
Stirbet A, Strasser RJ (1995a) Numerical simulation of the
fluorescence induction in plants. Archives des Sciences Gen
´
eve 48,
41–60.
Stirbet A, Strasser RJ (1995b) Numerical simulation of the in vivo
fluorescence in plants. In ‘First international symposium on
mathematical modelling and simulation in agriculture and bio-
industries. Vol. 1’. pp. I.B.2-1–I.B.2-6. (Free University of Brussels:
Brussels)
Stirbet A, Strasser RJ (2001) The possible role of pheophytine in the fast
fluorescence rise OKJIP. In ‘Proceedings of the 12th international
congress on photosynthesis. [CD-ROM]’. pp. S11–027. (CSIRO
Publishing: Melbourne)
Strasser BJ (1997) Donor side capacity of photosystem II probed by
chlorophyll a fluorescence transients. Photosynthesis Research 52,
147–155. doi: 10.1023/A:1005896029778
Strasser RJ, Butler WL (1977) Yield of energy transfer and spectral
distribution of excitation energy in photochemical apparatus
of flashed bean leaves. Biochimica et Biophysica Acta 462,
295–306.
Strasser RJ, Govindjee (1991) The F
0
and the O–J–I–P fluorescence rise
in higher plants and algae. In ‘Regulation of chloroplast biogenesis’.
(Ed. JH Argyroudi-Akoyunoglou) pp. 423–426. (Plenum Press:
New York)
Strasser RJ, Govindjee (1992) On the O–J–I–P fluorescence transient in
leaves and D1 mutants of Chlamydomonas reinhardtii. In ‘Research
in photosynthesis. Vol. 2’. (Ed. M Murata) pp. 29–32. (Kluwer
Academic Publishers: Dordrecht)
Strasser RJ, Strasser BJ (1995) Measuring fast fluorescence transients
to address environmental questions: the JIP test. In ‘Photosynthesis:
from light to biosphere. Vol. V’. (Ed. P Mathis) pp. 977–980. (Kluwer
Academic Publishers: Dordrecht)
Strasser RJ, Stirbet A (1997) Influence of photosystem II heterogeneity
on the simulated Chl a fluorescence induction transients.
In ‘Proceeding of the second international symposium on
mathematical modelling simulation in agriculture bio-industries’.
(Ed. I Farkas) pp. 21–26. (G
¨
od
¨
oll
¨
o University of Agricultural
Sciences: Budapest)
Strasser RJ, Stirbet AD (2001) Estimation of the energetic connectivity
of PS II centres in plants using the fluorescence rise O–J–I–P. Fitting
of experimental data to three different PS II models. Mathematics
and Computers in Simulation 56, 451–461. doi: 10.1016/S0378-
4754(01)00314-7
Strasser RJ, Srivastava A, Govindjee (1995) Polyphasic chlorophyll a
fluorescence transient in plants and cyanobacteria. Photochemistry
and Photobiology 61, 32–42.
Strasser RJ, Schansker G, Srivastava A, Govindjee (2001) Simultaneous
measurement of photosystem I and photosystem II probed by
modulated transmission at 820 nm and by chlorophyll a fluorescence
in the sub ms to second time range. In ‘Proceedings of
the 12th international congress on photosynthesis. [CD-ROM]’.
pp. S14–003. (CSIRO Publishing: Melbourne)
Strasser RJ, Tsimilli-Michael M, Srivastava A (2004) Analysis of
chlorophyll a fluorescence transient. In ‘Chlorophyll a fluorescence:
a signature of photosynthesis’. (Eds GC Papageorgiou, Govindjee)
pp. 321–362. (Springer: Dordrecht)
Su
ˇ
sila P, Laz
´
ar D, Il
´
ık P, Tomek P, Nau
ˇ
s J (2004) The gradient of exciting
radiation within a sample affects relative heights of steps in the
fast chlorophyll a fluorescence rise. Photosynthetica 42, 161–172.
doi: 10.1023/B:PHOT.0000040586.39903.db
Tomek P, Laz
´
ar D, Il
´
ık P, Nau
ˇ
s J (2001) On the intermediate steps
between the O and P steps in chlorophyll a fluorescence rise
measured at different intensities of exciting light. Australian Journal
of Plant Physiology 28, 1151–1160.
Tomek P, Il
´
ık P, Laz
´
ar D,
ˇ
Stroch M, Nau
ˇ
s J (2003) On the
determination of Q
B
-non-reducing photosystem II centers from
chlorophyll a fluorescence induction. Plant Science 164, 665–670.
doi: 10.1016/S0168-9452(03)00029-3
T
´
oth SZ, Schansker G, Strasser RJ (2005) In intact leaves, the
maximum fluorescence level (F
M
) is independent of the redox state
of the plastoquinone pool: a DCMU-inhibition study. Biochimica et
Biophysica Acta 1708, 275–282.
Trebst A (1987) The three-dimensional structure of the herbicide
binding niche on the reaction centre polypeptides of photosystem II.
Zeitschrift f
¨
ur Naturforschung 42c, 742–750.
Trebst A, Draber W (1986) Inhibitors of photosystem II and
the topology of the herbicide and Q
B
binding polypeptide in
the thylakoid membrane. Photosynthesis Research 10, 381–392.
doi: 10.1007/BF00118304
Trissl H-W, Gao Y, Wulf K (1993) Theoretical fluorescence
induction curves derived from coupled differential equations
describing the primary photochemistry of photosystem II by
an exciton-radical pair equilibrium. Biophysical Journal 64,
974–988.

30 Functional Plant Biology D. Laz
´
ar
Tsimilli-Michael M, P
ˆ
echeux M, Strasser RJ (1998a) Biomonitoring of
coral reef and temperate foraminifers by the Chl a fluorescence rise
O–J–I–P of their symbionts. In ‘Photosynthesis: mechanisms and
effects. Vol. 5’. (Ed. G Garab) pp. 4113–4116. (Kluwer Academic
Publishers: Dordrecht)
Tsimilli-Michael M, P
ˆ
echeux M, Strasser RJ (1998b) Vitality and
stress adaptation of the symbionts of coral reef and temperature
foraminifers probed in hospite by the fluorescence kinetics OJIP.
Archives des Sciences Gen
´
eve 51, 205–240.
Tyystj
¨
arvi E, Vass I (2004) Light emission as a probe of charge
separation and recombination in the photosynthetic apparatus:
relation of prompt fluorescence to delayed light emission and
thermoluminescence. In ‘Chlorophyll a fluorescence: a signature of
photosynthesis’. (Eds GC Papageorgiou, Govindjee), pp. 363–388.
(Springer: Dordrecht)
Tyystj
¨
arvi E, Koski A, Ker
¨
anen M, Nevalainen O (1999) The Kautsky
curve is a built-in barcode. Biophysical Journal 77, 1159–1167.
van Grondelle R (1985) Excitation energy transfer, trapping and
annihilation in photosynthetic systems. Biochimica et Biophysica
Acta 811, 147–195.
Vasil’ev S, Bruce D (1998) Nonphotochemical quenching of excitation
energy in photosystem II. A picosecond time-resolved study of the
low yield of chlorophyll a fluorescence induced by single-turnover
flash in isolated spinach thylakoids. Biochemistry 37, 11046–11054.
doi: 10.1021/bi9806854
Velthuys BR (1981) Electron dependent competition between
plastoquinone and inhibitors for binding to Photosystem II. FEBS
Letters 126, 272–276. doi: 10.1016/0014-5793(81)80259-1
Velthuys BR, Amesz J (1974) Charge accumulation at the reducing side
of system 2 of photosynthesis. Biochimica et Biophysica Acta 333,
85–94.
Vernotte C, Etienne A-L, Briantais L-M (1979) Quenching of the
system II chlorophyll fluorescence by the plastoquinone pool.
Biochimica et Biophysica Acta 545, 519–527.
Visser D, Heijnen J (2002) The mathematics of metabolic
control analysis revisited. Metabolic Engineering 4, 114–123.
doi: 10.1006/mben.2001.0216
Vredenberg WJ (2000) A three-state model for energy trapping and
chlorophyll fluorescence in photosystem II incorporating radical pair
recombination. Biophysical Journal 79, 26–38.
Vredenberg WJ (2004) System analysis and photoelectrochemical
control of chlorophyll fluorescence in terms of trapping models of
photosystem II: a challenging view. In ‘Chlorophyll a fluorescence:
a signature of photosynthesis’. (Eds GC Papageorgiou, Govindjee)
pp. 133–172. (Springer: Dordrecht)
Vredenberg WJ, Bulychev A (2002) Photo-electrochemical
control of photosystem II chlorophyll fluorescence in vivo.
Bioelectrochemistry 57, 123–128. doi: 10.1016/S1567-5394
(02)00062-2
Vredenberg WJ, Bulychev A (2003) Photoelectric effects on chlorophyll
fluorescence of photosystem II in vivo. Kinetics in the absence
and presence of valinomycin. Bioelectrochemistry 60, 87–95.
doi: 10.1016/S1567-5394(03)00053-7
Vredenberg WJ, van Rensen JJS, Rodrigues GC (2005) On
the sub-maximal yield and photo-electric stimulation of
chlorophyll a fluorescence in single turnover excitations in
plant cells. Bioelectrochemistry (Amsterdam, Netherlands) 68,
83–90.
Wakeham MC, Breton J, Nabedryk E, Jones MR (2004) Formation of
a semiquinone at the Q
B
site by A- or B-branch electron transfer in
the reaction center from Rhodobacter sphaeroides. Biochemistry 43,
4755–4763. doi: 10.1021/bi035726x
Weber G, Teale FWJ (1957) Determination of the absolute quantum
yield of fluorescent solutions. Transactions of the Faraday Society
53, 646–655. doi: 10.1039/tf9575300646
Whitmarsh J, Ort DR (1984) Stoichiometries of electron
transport complexes in spinach chloroplasts. Archives of
Biochemistry and Biophysics 231, 378–389. doi: 10.1016/0003-
9861(84)90401-6
Wong D, Govindjee (1979) Antagonistic effects of mono- and divalent
cations on polarization of chlorophyll fluorescence in thylakoids and
changes in excitation energy transfer. FEBS Letters 97, 373–377.
doi: 10.1016/0014-5793(79)80124-6
Xia JR, Li YJ, Zou DH (2004) Effects of salinity stress on
PSII in Ulva lactuca as probed by chlorophyll fluorescence
measurements. Aquatic Botany 80, 129–137. doi: 10.1016/
j.aquabot.2004.07.006
Yaakoubd B, Andersen R, Desjardins Y, Samson G (2002) Contribution
of the free oxidized and Q
B
-bound plastoquinone molecules to
the thermal phase of chlorophyll-a fluorescence. Photosynthesis
Research 74, 251–257. doi: 10.1023/A:1021291321066
Zaharieva I, Goltsev V (2003) Advances on photosystem II
investigation by measurement of delayed chlorophyll
fluorescence by a phosphoroscopic method. Photochemistry
and Photobiology 77, 292–298. doi: 10.1562/0031-8655(2003)
077<0292:AOPIIB>2.0.CO;2
Zhu X-G, Govindjee, Baker NR, deSturler E, Ort DR, Long SP
(2005) Chlorophyll a fluorescence induction kinetics in leaves
predicted from a model describing each discrete step of excitation
energy and electron transfer associated with photosystem II. Planta,
(in press).
Zouni A, Witt HT, Kern J, Fromme P, Krauß N, Saenger W, Orth P
(2001) Crystal structure of photosystem II from Synechococcus
elongatus at 3.8
˚
A resolution. Nature 409, 739–743. doi: 10.1038/
35055589
Manuscript received 19 April 2005, accepted 18 August 2005
http://www.publish.csiro.au/journals/fpb