REVIEW
Frequently asked questions about chlorophyll fluorescence,
the sequel
Hazem M. Kalaji
1
•
Gert Schansker
2
•
Marian Brestic
3
•
Filippo Bussotti
4
•
Angeles Calatayud
5
•
Lorenzo Ferroni
6
•
Vasilij Goltsev
7
•
Lucia Guidi
8
•
Anjana Jajoo
9
•
Pengmin Li
10
•
Pasquale Losciale
11
•
Vinod K. Mishra
12
•
Amarendra N. Misra
13
•
Sergio G. Nebauer
14
•
Simonetta Pancaldi
6
•
Consuelo Penella
5
•
Martina Pollastrini
4
•
Kancherla Suresh
15
•
Eduardo Tambussi
16
•
Marcos Yanniccari
16
•
Marek Zivcak
3
•
Magdalena D. Cetner
1
•
Izabela A. Samborska
1
•
Alexandrina Stirbet
17
•
Katarina Olsovska
18
•
Kristyna Kunderlikova
18
•
Henry Shelonzek
19
•
Szymon Rusinowski
20
•
Wojciech Ba˛ba
21
Received: 26 June 2016 / Accepted: 17 October 2016 / Published online: 4 November 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Using chlorophyll (Chl) a fluorescence many
aspects of the photosynthetic apparatus can be studied, both
in vitro and, noninvasively, in vivo. Complementary tech-
niques can help to interpret changes in the Chl a fluorescence
kinetics. Kalaji et al. (Photosynth Res 122:121–158,
2014a)
addressed several questions about instruments, methods and
applications based on Chl a fluorescence. Here, additional Chl
a fluorescence-related topics are discussed again in a question
and answer format. Examples are the effect of connectivity on
photochemical quenching, the correction of F
V
/F
M
values for
PSI fluorescence, the energy partitioning concept, the inter-
pretation of the complementary area, probing the donor side
of PSII, the assignment of bands of 77 K fluorescence emis-
sion spectra to fluorescence emitters, the relationship between
prompt and delayed fluorescence, potential problems when
sampling tree canopies, the use of fluorescence parameters in
QTL studies, the use of Chl a fluorescence in biosensor
Hazem M. Kalaji and Gert Schansker have contributed equally to this
paper.
& Hazem M. Kalaji
hazem@kalaji.pl
& Gert Schansker
gert.schansker@gmail.com
Marian Brestic
marian.brestic@uniag.sk
Filippo Bussotti
filippo.bussotti@unifi.it
Angeles Calatayud
calatayud_ang@gva.es
Lorenzo Ferroni
lorenzo.ferroni@unife.it
Vasilij Goltsev
goltsev@gmail.com; goltsev@biofac.uni-sofia.bg
Lucia Guidi
lucia.guidi@unipi.it
Anjana Jajoo
anjanajajoo@hotmail.com
Pengmin Li
Lipm@nwsuaf.edu.cn
Pasquale Losciale
pasquale.losciale@crea.gov.it
Vinod K. Mishra
mishravkbhu@gmail.com
Amarendra N. Misra
misraan@yahoo.co.uk; misra.amarendra@gmail.com
Sergio G. Nebauer
sergonne@bvg.upv.es
Simonetta Pancaldi
simonetta.pancaldi@unife.it
Consuelo Penella
penella_con@gva.es
Martina Pollastrini
martina.pollastrini@unifi.it
Kancherla Suresh
sureshkancherla@rediffmail.com
Eduardo Tambussi
tambussi35@yahoo.es
Marcos Yanniccari
marcosyanniccari@conicet.gov.ar
Marek Zivcak
marek.zivcak@uniag.sk
Alexandrina Stirbet
stirbet@verizon.net
123
Photosynth Res (2017) 132:13–66
DOI 10.1007/s11120-016-0318-y
applications and the application of neural network approaches
for the analysis of fluorescence measurements. The answers
draw on knowledge from different Chl a fluorescence analysis
domains, yielding in several cases new insights.
Keywords Chl a fluorescence Delayed fluorescence
Photochemical quenching Energy partitioning Area
Abbreviations
ANN Artificial neural network
Area, Sm Complementary area above the
fluorescence rise and this area
normalized to F
V
, respectively
ATP Adenosine triphosphate
Car Carotenoid
Chl Chlorophyll
1
Chl,
3
Chl Singlet chlorophyll and tripl et
chlorophyll
Chl
D1
Accessory Chl molecule bound to the
D1 protein
CP43, CP47 Core antenna proteins of PSII of 43
and 47 kDa, respectively
CSm Cross section (in the JIP test it is
assumed that F
M
is a measure for the
cross section)
cyt Cytochrome
D1 protein One of the major PSII reaction center
proteins, the other being D2
DCMU 3-(3,4-Dichlorophenyl)-1,1-
dimethylurea
ETC Electron transport chain
ETR Electron transport rate
Fd Ferredoxin
FNR Ferredoxin NADP
?
reductase
F
O
, F
M
, F
O
0
, F
M
0
Minimum and maximum
fluorescence intensity emitted by
dark- and light-acclimated samples,
respectively
F
PSI
Chlorophyll a fluorescence emitted
by photosystem I
F
S
Steady-state chlorophyll
a fluores cence
F
V
/F
M
Maximum quantum yield of primary
photosystem II p hotochemistry
IRGA Infrared gas analyzer
JIP test Analysis framework for the
interpretation of OJIP transients
developed by Bruno and Reto
Strasser
K step Fluorescence intensity at 300 ls
Katarina Olsovska
katarina.olsovska@uniag.sk
Kristyna Kunderlikova
xkunderlikov@is.uniag.sk
Henry Shelonzek
shelonzek@gmail.com
Szymon Rusinowski
rusinowski@ietu.katowice.pl
Wojciech Ba˛ba
wojciech.baba12@gmail.com
1
Department of Plant Physiology, Faculty of Agriculture and
Biology, Warsaw University of Life Sciences – SGGW,
Nowoursynowska 159, 02-776 Warsaw, Poland
2
Wesemlinstrasse 58, 6006 Lucerne, Switzerland
3
Department of Plant Physiology, Slovak Agricultural
University, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic
4
Department of Agricultural, Food and Environmental
Sciences, University of Florence, Piazzale delle Cascine 28,
50144 Florence, Italy
5
Departamento de Horticultura, Instituto Valenciano de
Investigaciones Agrarias, Ctra. Moncada-Na
´
quera Km 4.5.,
46113 Moncada, Valencia, Spain
6
Department of Life Sciences and Biotechnology, University
of Ferrara, Corso Ercole I d’Este, 32, 44121 Ferrara, Italy
7
Department of Biophysics and Radiobiology, Faculty of
Biology, St. Kliment Ohridski University of Sofia, 8
Dr.Tzankov Blvd., 1164 Sofia, Bulgaria
8
Department of Agriculture, Food and Environment, Via del
Borghetto, 80, 56124 Pisa, Italy
9
School of Life Sciences, Devi Ahilya University, Indore,
M.P. 452 001, India
10
State Key Laboratory of Crop Stress Biology for Arid Areas,
College of Horticulture, Northwest A&F University,
Yangling 712100, Shaanxi, China
11
Consiglio per la ricerca in agricoltura e l’analisi
dell’economia agraria [Research Unit for Agriculture in Dry
Environments], 70125 Bari, Italy
12
Department of Biotechnology, Doon (P.G.) College of
Agriculture Science, Dehradun, Uttarakhand 248001, India
13
Centre for Life Sciences, Central University of Jharkhand,
Ratu-Lohardaga Road, Ranchi 835205, India
14
Departamento de Produccio
´
n vegetal, Universitat Polite
`
cnica
de Vale
`
ncia, Camino de Vera sn., 46022 Valencia, Spain
15
ICAR – Indian Institute of Oil Palm Research, Pedavegi,
West Godavari Dt., Andhra Pradesh 534 450, India
16
Institute of Plant Physiology, INFIVE (Universidad Nacional
de La Plata — Consejo Nacional de Investigaciones
Cientı
´
ficas y Te
´
cnicas), Diagonal 113 N°495, CC 327,
La Plata, Argentina
17
204 Anne Burras Lane, Newport News, VA 23606, USA
18
Department of Plant Physiology, Slovak University of
Agriculture, A. Hlinku 2, 94976 Nitra, Slovak Republic
14 Photosynth Res (2017) 132:13–66
123
kF, kN and kP Rate constants for Chl
a fluores cence, heat dissipation and
photochemistry
LED Light-emitting diode
LHC, LHCI and
LHCII
Light-harvesting complex, in
general, associated with PSI and
mainly associated with PSII,
respectively
M
o
The initial slope (first 250 ls) of the
OJIP transient times 4, normalized to
F
V
NADP
?
Nicotinamide adenine dinucleotide
phosphate, oxidized form
NO Nitric oxide
NPQ, q
N
Non-photochemical quenching
expressed as (F
M
/F
M
0
- 1) and
(1 - F
V
0
/F
V
), respectively
OEC Oxygen-evolving complex
OJIP Fluorescence rise on a dark-to-light
transition from a minimum value
O via the intermediate steps J and
I to the maximum value P, which is
F
M
if the light is saturating
P680, P700 PSII and PSI reaction center
chlorophyll dimer, respectively
PAR Photosynthetically active radiance
PCA Principal component analysis
PF, DF Prompt fluorescence and delayed
fluorescence, respectively
Pheo Pheophytin, cofactor bound to PSII
PI
abs
, PI
tot
Performance indexes of the JIP test
P
n
, I
PL
Net rate of carbon fixation and model
based calculated net rate of carbon
fixation, respectively
PPFD Photosynthetic photon flux density
PsbO, PsbP and
PsbQ
PSII extrinsic proteins
PSI, PSII Photosystems I and II, respectively
Q cycle Cyclic electron transport through cyt
b6f and the PQ pool
Q
A
, Q
B
, PQ Primary and secondary quinone
electron acceptors of PSII and free
plastoquinone, respectively
q
E
Energy quenching, fluorescence
quenching dependent on an
acidification of the lumen
q
P
, q
L
Photochemical quenching calculated
based on the puddle and lake model,
respectively
QTL Quantitative trait locus
q
Z
Non-photochemical quenching of
Chl a fluorescence related to the
xanthophyll cycle
RC Reaction center
R
Fd
Relative fluorescence decrease ratio
RLC Rapid light curve
ROS Reactive oxygen species
Rubisco Ribulose-1,5-bisphosphate
carboxylase/oxygenase
RWC Relative water content
SOM Self-organizing map
SPAD Refers to an instrument used to
estimate the leaf Chl content
S states
S0, S1, S2, S3 and
S4
Redox states of the oxygen-evolving
complex
t
F
m
Time needed to rise from O to P
Tl Leaf temperature
TL Thermoluminescence
TyrD, TyrZ Tyrosine D and Z, redox active
tyrosines in the D2 and D1 proteins
of PSII, respectively
UV Ultraviolet
V, A, Z Violaxanthin, antheraxanthin and
zeaxanthin, respectively
VDE Violaxanthin de-epoxidase
V
J
, V
I
Relative position of the J and I steps
between O and P
U
P0
Maximum quantum yield of primary
photochemistry
U
PSI
, U
PSII
PSI and PSII operating efficiency,
respectively
wE
o
JIP test parameter thought to be
related to forward electron transport,
defined as 1 - V
J
Introduction
In 2014 we published a paper in question and answer
format on a series of chlorophyll (Chl) a fluorescence-re-
lated topics (Kalaji et al.
2014a). There were, however, still
enough questions left for a sequel. In the present paper we
treat questions on the relationship between prompt fluo-
rescence (PF), measured with fluorimeters like the PAM
and the HandyPEA, and delayed fluorescence (DF), the
19
Department of Plant Anatomy and Cytology, Faculty of
Biology and Environmental Protection, University of Silesia,
ul. Jagiellon
´
ska 28, 40-032 Katowice, Poland
20
Institute for Ecology of Industrial Areas, Kossutha 6,
40-844 Katowice, Poland
21
Department of Plant Ecology, Institute of Botany,
Jagiellonian University, Lubicz 46, 31-512 Krako
´
w, Poland
Photosynth Res (2017) 132:13–66 15
123
much wea ker cousin of PF that is emitted in response to
recombination reactions within PSII; energy partitioning;
q
P
versus q
L
; the analysis of several forms of stress using
Chl a fluorescence; the JIP test parameters area and F
J
; the
consequences of fluorescence emitted by PSI for parame-
ters like F
V
/F
M
; considerations when sampling trees; the
assignment of 77 K fluorescence bands; QTL studies on
Chl a fluorescence-related traits from a Chl a fluorescence
point of view and several other topics.
Question 1: What is chlorophyll a fluorescence
and why do we study it?
Chl a fluorescence can be defined as the red to far-red light
emitted by photosynthetic tissues/organisms when illumi-
nated by light of approximately 400–700 nm (photosyn-
thetically active radiation or PAR) (McCree 1972). Within
this spectrum, blue and red light excite chlorophyll more
efficiently than green light. Although Chl a fluorescence
represents only a small fraction of the absorbed energy
[approximately 0.5–10% (Latimer et al. 1956 ; Brody and
Rabinowitch 1957; Ba rber et al. 1989; Porcar-Castell et al.
2014)], its intensity is inversely proportional to the fraction
of energy used for photosynthesis (a redox eff ect) (Duysen s
and Sweers
1963). For this reason, the Chl a fluorescence
signal can be used as a probe for photosynthetic activity. At
the same time, Chl a fluorescence is also inversely pro-
portional to changes in dissipative heat emission (a yield
effect, i.e., an increase in the yield of heat emission causes
a decrease in the yield of fluorescence emission) (e.g.,
Krause and Wei s
1991) and, therefore, Chl a fluorescence
can be used as well to monitor regulatory processes
affecting the PSII antenna (see, e.g., Question 8). Finally,
P680
?
is a strong quencher of Chl a fluorescence (Steffen
et al.
2005) and this effect allows the study of the different
redox states (S states) the oxygen-evolving complex of
PSII, due to the fact that the lifetime of P680
?
is S state
dependent. All of these things taken together could turn
Chl a fluorescence into a indecipherable signal, but thanks
to the development of specific protocols, and by using
complementary techniques, the different effects can be
separated, turning Chl a fluorescence into a powerful tool
for the study of photosynthesis: quenching anal ysis
(Bradbury and Baker
1981; Quick and Horton 1984;
Schreiber et al.
1986), JIP test (Strasser and Strasser 1995;
Strasser et al.
2004), non-photochemical quenching (NPQ)
(Demmig and Winter 1988; Horton and Hague 1988),
electron transport rate (ETR) (Genty et al.
1989; Krall and
Edwards
1990), rapid light curves (RLCs) (White and
Critchley
1999; Ralph and Gademann 2005), flash-induced
fluorescence (Robinson and Crofts
1983; de Wijn and van
Gorkom 2001; Bouges-Bocquet 1980, Ioannidis et al.
2000), dark-adaptation kinetics of OJIP transients (Bukhov
et al.
2001; Schansker et al. 2005), Chl a fluorescence and
photoacoustic spectroscopy (Buschmann and Kosca
´
nyi
1989; Snel et al. 1990; Allakhverdiev et al. 1994; Bukhov
et al.
1997), Chl a fluorescence and 820-nm
absorbance/transmission (Klughammer and Schreiber
1994; Schansker et al. 2003), Chl a fluorescence and
delayed fluorescence (Goltsev et al. 2012; Kalaji et al.
2012a), imaging (Nedbal and Whitmarsh 2004; Hideg and
Schreiber
2007; Lichtenthaler et al. 2007; Gorbe and
Calatayud 2012), the actinic light wavelength dependence
of photosynthesis (Schreiber et al.
2012) and more recently
attention has been paid to statistic aspects of the mea-
surements of parameters (e.g., Bussotti et al.
2011a). The
photosynthetic literature is huge with many topics studied
such as plant b reeding (Baker and Rosenqvist
2004; Kalaji
and Pietkiewicz 2004; Kalaji and Guo 2008), seed vigor
and seed quality assessment (Jalink et al.
1998; Dell’Aquila
et al.
2002; Konstantinova et al. 2002), fruit and veg-
etable quality determination and postharvest processing
control (Merz et al.
1996; Nedbal et al. 2000), senescence
(Adams et al. 1990a; Kotakis et al. 2014 ), climate change
effects (Ashraf and Harris
2004) and a variety of algae
(Gorbunov et al.
1999; Antal et al. 2009 ; Grouneva et al.
2009). Furthermore, Chl a fluorescence measurements have
been used for monitoring plant stresses (Guidi and Cala-
tayud
2014), such as photoinhibition (Sarvikas et al. 2010;
Matsubara et al.
2011), heat stress (Allakhverdiev et al.
2007; Ducruet et al. 2007;To
´
th et al. 2007a; Kalaji et al.
2011a; Brestic
ˇ
et al. 2012), UV stress (Vass et al. 1999; van
Rensen et al.
2007; Guidi et al. 2011), sal t stress (Kalaji
and Pietkiewicz
1993; Demetriou et al. 2007; Melgar et al.
2009; Kalaji et al. 2011b; Penella et al. 2016), drought
stress (Lu and Zhang 1998; Flexas et al. 2002;Z
ˇ
ivc
ˇ
a
´
k et al.
2013), urban tree conditions (Hermans et al. 2003; Swoc-
zyna et al.
2010a, b), environmental pollution (Bussotti
et al.
2005; Kalaji and Łoboda 2007; Romanowska-Duda
et al.
2010; Tuba et al. 2010; Bussotti et al. 2011b; Cotrozzi
et al.
2016), sulfur-deprivation/H
2
production in Chlamy-
domonas (Antal et al.
2007; Nagy et al. 2012) and water
quality (Romanowska-Duda et al.
2005; Ralph et al. 2007;
Baumann et al.
2009).
Question 2: Does Chl a fluorescence only probe
PSII?
A common misunderstanding is that variable Chl a fluo-
rescence is a specific probe for PSII. This is true for flash
experiments, in which Q
A
in all PSII RCs is reduced by a
saturating single turnover flash. However, if longer pulses
of light are given, Q
A
will become reduced and oxidized
multiple times, and under these conditions fluorescence
16 Photosynth Res (2017) 132:13–66
123
also becomes a probe for the reduction and redox state of
the PQ pool and even for the electron flow through PSI and
PSI content (Schansker et al.
2005; Ceppi et al. 2012).
Under steady-state conditions, i.e., a stable level of
photosynthesis reached after a few minutes of illumination,
the whole photosynthetic apparatus is in equilibrium and
electron flow through any of the components of the electron
transport chain (including PSII) would be indicative for the
overall photosynthetic rate (Kramer et al.
2004a; Scheibe
et al.
2005; Eichelmann et al. 2009). As a consequence,
under steady-state conditions, the electron flux calculated
on the basis of the Chl a fluorescence signal can be used as a
measure for the overall photosynthetic activity. This point
was demonstrated by Genty et al. (1989, 1990a).
Another common mistake is to interpret fluorescence
measurements in terms of single reaction centers. In the
case of photoinhibition it is, e.g., often assumed or implied
that the quant um yield of indivi dual PSII RCs changes,
whereas it is more realistic to interpret changes in the
parameter F
V
/F
M
in terms of changes in the quantum yield
of the population of PSII RCs as a whole.
The importance of looking at photosynthesis measure-
ments in stochastic terms can be illustrated by experiments
showing that at high light intensities 80% of the PSII RCs
can be inhibited before the electron transport rate becomes
affected (e.g., Heber et al.
1988).
This observation also illustrates that at high light inten-
sities PSII activity has little relevance for photo synthetic
activity, whereas at low light intensities PSII RCs become
rate limiting. This also means that the effect of a treatment on
PSII measured at a single light intensity has limited meaning.
Question 3: What is the Kautsky effect?
Kautsky and Hirsch (
1931) observed for several types of
leaves that a dark-to-light transition is characterized by an
initial fast increase of the fluorescence intensity followed
by a slow decrease to a minimum level, after which the
fluorescence intensity remains at this low intensity. The
authors assigned the stable low level of fluorescence to
steady-state photosynthesi s. They noted further that the
slow fluorescence decrease had the same time dependence
as the induction of CO
2
assimilation and concluded that the
fast fluorescence rise reflects a photochemical reaction
since it was insensitive to cyanide and temperature chan-
ges. The fluorescence changes occurring during induction
of photosynthesis have been studied intensively during the
last 50 years and, in honor of the first publication on this
phenomenon, such a fluorescence transient is called a
Kautsky transient, and the changes in the fluorescence
intensity the Kautsky effect. In Fig.
1 examples of the first
10 s of Kautsky transients measured on several angiosperm
and gymnosperm plants are shown on a logarithmic time-
scale. The fluores cence rise phase (OJIP) reflects the
reduction of the photosynthetic electron transport chain
(see Kalaji et al.
2014a for a more comprehensive discus-
sion) and its kinetics, as illustrated in Fig.
1, are quite
similar for all photosynthetic organisms. The fluorescence
decrease has kinetics that differ quite strongly between
different types of photosynthetic organisms (in Fig. 1
angiosperm vs. gymnosperm plants). The S and M steps
observed in transients of gymnosperm species lack/are
hidden in transients of angi osperm species. Using 820-nm
transmission measur ements it was shown that the initial
fluorescence kinetics beyond P depend strongly on the
activation of electron flow at the PSI acceptor side, asso-
ciated with the activation of ferredoxin-NADP
?
reductase
(FNR) (Kautsky et al.
1960; Munday and Govindj ee 1969;
Satoh
1981; Harbinson and Hedley 1993; Schansker et al.
2003, 2008; Ilı
´
k et al. 2006). Fluorescence then declines
within 3–5 min with the onset of photosynthetic CO
2
fix-
ation until it reaches a lower, steady-state fluorescence
intensity (F
S
). In fully photosynthetically active leaves this
steady-state level, especially at high light intensities, is
usually close to the F
O
level (e.g., Flexas et al. 2002).
Question 4: What is quantum yield?
In a gener al sense, the quantum yield can be defined by an
action, e.g., oxygen evolution or a stable charge separation,
divided by the number of photons that has to be absorbed
Fig. 1 Chl a fluorescence induction transients measured on angios-
perm (sugar beet, camellia and tobacco) and gymnosperm (Ginkgo
and yew) leaves. The fast induction kinetics OJIP are similar for both
types of plants with a higher F
M
/F
O
ratio in gymnosperms and the
same OJIP kinetics for all leaves/needles measured. Beyond P the
kinetics differ quite strongly between both types of plants (Schansker
et al., unpublished data)
Photosynth Res (2017) 132:13–66 17
123