REVIEW
Chlorophyll a fluorescence as a tool to monitor physiological
status of plants under abiotic stress conditions
Hazem M. Kalaji
1
•
Anjana Jajoo
2
•
Abdallah Oukarroum
3
•
Marian Brestic
4
•
Marek Zivcak
4
•
Izabela A. Samborska
1
•
Magdalena D. Cetner
1
•
Izabela Łukasik
5
•
Vasilij Goltsev
6
•
Richard J. Ladle
7,8
Received: 11 April 2015 / Revised: 29 February 2016 / Accepted: 1 March 2016 / Published online: 29 March 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Plants living under natural conditions are
exposed to many adverse factors that interfere with the
photosynthetic process, leading to declines in growth,
development, and yield. The recent development of
Chlorophyll a fluorescence (ChlF) represents a potentially
valuable new approach to study the photochemical effi-
ciency of leaves. Specifically, the analysis of fluorescence
signals provides detailed information on the status and
function of Photosystem II (PSII) reaction centers, light-
harvesting antenna complexes, and both the donor and
acceptor sides of PSII. Here, we review the results of fast
ChlF analyses of photosynthetic responses to
environmental stresses, and discuss the potential scientific
and practical applications of this innovative methodology.
The recent availability of portable devices has significantly
expanded the potential utilization of ChlF techniques,
especially for the purposes of crop phenotyping and
monitoring.
Keywords Chlorophyll fluorescence JIP-test
Photosynthesis Photosystem II Quantum efficiency
Stress detection
Abbreviations
ABS Absorption flux
Chl Chlorophyll
Communicated by AK Kononowicz.
& Hazem M. Kalaji
hazem@kalaji.pl
& Anjana Jajoo
anjanajajoo@hotmail.com
Abdallah Oukarroum
oukarroum.abdallah@uqam.ca
Marian Brestic
marian.brestic@uniag.sk
Marek Zivcak
marek.zivcak@uniag.sk
Izabela A. Samborska
izabelasam@wp.pl
Magdalena D. Cetner
magdalena.cetner@gmail.com
Izabela Łukasik
zzlukasik@gmail.com
Vasilij Goltsev
goltsev@biofac.uni-sofia.bg; goltsev@gmail.com
Richard J. Ladle
richard.ladle@ouce.ox.ac.uk
1
Department of Plant Physiology, Faculty of Agriculture and
Biology, Warsaw University of Life Sciences (WULS-
SGGW), Nowoursynowska 159, 02-776 Warsaw, Poland
2
School of Life Sciences, Devi Ahilya University,
Indore 452 017, MP, India
3
Department of Chemistry and Biochemistry, University of
Que
´
bec in Montre
´
al, Montre
´
al, QC, Canada
4
Department of Plant Physiology, Slovak University of
Agriculture, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic
5
Racławicka 106, 02-634 Warsaw, Poland
6
Department of Biophysics and Radiobiology, Faculty of
Biology, St. Kliment Ohridski University of Sofia, 8 Dr.
Tzankov Blvd., 1164 Sofia, Bulgaria
7
School of Geography and the Environment, University of
Oxford, South Parks Road, Oxford, UK
8
Institute of Biological Sciences and Health, Federal
University of Alagoas, Av. Lourival Melo Mota, s/n,
Tabuleiro do Martins, Maceio
´
, Alagoas 57072-900, Brazil
123
Acta Physiol Plant (2016) 38:102
DOI 10.1007/s11738-016-2113-y
ChlF Chlorophyll fluorescence
CS Cross section of the sample
Cyt b
6
f Cytochrome b
6
f
DF Delayed (chlorophyll) fluorescence
DFI Drought factor index
LHC (II) Light-harvesting complex (of PSII)
OEC Oxygen-evolving complex
P680* Excited PSII reaction center
P700 PSI reaction center
PAR Photosynthetically active radiation
PC Plastocyanin
PCA Principal component analysis
PF Prompt (chlorophyll) fluorescence
Pheo Pheophytin
PQ Plastoquinone
PSI, PSII Photosystem I, II
Q
A
Primary plastoquinone electron acceptor of
PSII
Q
B
Secondary plastoquinone electron acceptor
RC Reaction center
ROS Reactive oxygen species
Introduction
Over the course of the 21st century, global agriculture must
produce more food to sustain a growing human population
(Beddington et al.
2012). However, this goal is threatened
by anthropogenic climate change which has the potential to
dramatically reduce yields in affected regions (Lobell et al.
2008). Recent studies indicate that Chlorophyll fluores-
cence (ChlF) measurements may provide unique bench-
marks to improve global agricultural productivity models,
improving the reliability of crop yield projections under
climate change scenarios (Guanter et al.
2014; Malaspina
et al.
2014). More generally, ChlF is emerging as a very
powerful tool in agricultural, environmental, and ecologi-
cal studies (Gottardini et al.
2014). One of its main
advantages is that ChlF is a non-invasive tool, allowing
scientists to get information on the photosynthetic process
without destroying the tested sample.
Under natural conditions, plants are exposed to many
adverse environmental stress factors. These can disrupt the
photosynthetic apparatus, causing a decrease of plant pro-
ductivity and overall yield. Photosynthesis is particularly
sensitive to environmental constraints (see Kalaji et al.
2012), making photosynthetic measurements an important
component of plant stress studies. Howeve r, traditional
methods, even technically advanc ed ones such as the
measurements of photosynthetic rates through gas
exchange (CO
2
,H
2
O, and O
2
), are time-consuming and
provide incomplet e information on overall photosynthetic
function. In contrast, ChlF measurements represent a sim-
ple, non-destructive, inexpensive and rapid tool for ana-
lyzing light-dependent photosynthetic reac tions and for
indirectly estimating chlorophyll content within the same
sample tissue (See reviews by Govindjee
1995; Papa-
georgiou and Govindjee
2011; and by Stirbet and Govin-
djee
2011, 2012). These technical advantages of ChlF
approaches have made it a popular technique among plant
breeders (e.g., for crop phenotyping and monitoring),
biotechnologists, plant physiologists, farmers, gardeners,
foresters, ecophysiologists, and environmentalists.
Critically, from the perspective of plant stress studies,
ChlF measurements also provide indirect information
about the physiological condition of plants. Analysis of
chlorophyll fluorescence (ChlF) induction curves allows
the evaluation of the physiological condition of photosys-
tem II (PSII) and photosynthetic electron transport chain
components. It also provides information on the coopera-
tion of light-dependent photochemical reactions and light-
independent biochemical reactions. More generally, ChlF
measurements relate, directly or indirectly, to all stages of
light-dependent photosynthetic reactions, including pho-
tolysis of water, electron transport, pH gradient formation
across the thylakoid membrane, and ATP synthesis and
thus general bioenergetic condi tion of the photosynthetic
machinery (Berna
´
t et al.
2012).
Numerous ChlF techniques and applications have now
been developed, each one contributing to knowledge of
photosynthesis. In this review, we focus on results from
fast fluorescence analysis induced by continuous illumi-
nation. These studies were made possible by the develop-
ment of a reliable mathem atical model known as the JIP-
test (Strasser et al.
2004) that allowed the analysis of flu-
orescence changes that occur in less than 1 s. Such anal-
yses provide detailed information on the status and
function of PSII reaction centers, antenna, as well as on
donor and acceptor sides of PSII. The main focus of the
review is to outline the effects of stress factors on the
photochemical processes as reflected in changes in fast
ChlF kinetics and related biophysical parameters.
Analysis of polyphasic chlorophyll fluorescence
kinetics
Illumination of a dark-adapted photosynthetic sample
allows a polyphasic chlorophyll fluorescence induction
curve to be obtained (O–J–I–P-trans ient) (Fig.
1). The
curve’s trajectory provides considerable information about
the structure and function of the photosynthetic apparatus
(Kautsky and Hirsch
1931; Schreiber et al. 1994). The JIP-
test is based on the rise in polyphasic fast chlorophyll a,
102 Page 2 of 11 Acta Physiol Plant (2016) 38:102
123
and is used for investigating the correlation between light-
dependent reactions and ChlF. It is based on the theory of
‘‘energy flow’’ across thylakoid membranes (Strasser et al.
2000). This theory can be operationalized in simple alge-
braic equations, representing the balance between total
energy inflows and outflows for each of the examined light-
harvesting complexes and providing information on the
probable distribution of absorbed energy. With these
equations, it is possible to describe the energetic commu-
nication (also known as the ‘‘grouping’’ or ‘‘connectivity’’
and ‘‘overall grouping probability ’’) between the PSII
complexes (Stirbet
2013).
The name of the JIP-test (OJIP) originates from the
specific points on the induction curve formed by the
recorded ChlF signal (Fig.
1): these correspond to the
gradual reduction of Q
A
and the primary electron acceptor
of PSII. The shape of the curve depends from PSII
grouping (L-band) (Tsimilli-Michael and Strasser
2013)
and the balance between electron donation from
OEC ? P680
?
and electron accept from Q
A
-
(K-band)
(Strasser et al.
2005). The O–J part of the fluorescence rise
relates to the closure of some of the PSII reaction centers in
response to the reduction of Q
A
to a level determined by
the ratio between the trapping rate and Q
A
reoxidation rate
by Q
B
and the rest of the electron transfer chain. The J–I
part of the curve corresponds to the reduction of the sec-
ondary electron acceptor Q
B
, plastoquinone (PQ),
cytochrome (Cyt b
6
f), and PC. The increase in ChlF in the
I–P part of the induction curve is typically attributed to the
reduction of electron transporters (ferredoxin, intermediary
acceptors, and NADP) of the PSI acceptor side. Stress
conditions such as high temperature, excessive PAR,
nitrogen deficiency, or drought inhibit the oxygen-evolving
complex (OEC) and bloc k the electron transport between
the OEC and tyrosine (Guha et al.
2013). Under stressful
conditions, a peak occurs (the K-band) within the
200–300 ls range of the ChlF induction curve, indicating a
disruption of the OEC.
The JIP- test parameters characterizing the PAR energy
absorption and electron transport can be categorized into
four main groups: (1) basic measured and calculated values
[fluorescence (F
t
) and variable fluorescence (V
t
) values,
initial slope, etc.]; (2) quantum yields and probabilities; (3)
energy fluxes; and (4) vitality indices. The biophysical
parameters representing the energy fluxes are divided into
specific and phenomenological. The specific parameters are
calculated per reaction center (RC), while the phe-
nomenological parameters are calculated per sample cross
section (CS).
The vitality indices represent the products of several
independent parameters combining struct ural and func-
tional criteria. These crite ria include the density of reaction
centers, the quantum efficiency of primary photochemistry,
and conversion of excitation energy in electron transport
(Strasser et al.
2000, 2004, 2010; Zushi et al. 2012). The
vitality indices were create d as non-specific parameters to
be used mostly in practical applications, such as screening
for enhanced stress tolerance underfield conditions (Sri-
vastava et al.
1999; Strasser et al. 2004; Brestic and Zivcak
2013).
Chlorophyll fluorescence kinetics can also be used to
reveal PSII heteroge neity of photosynthetic apparatus. PSII
is naturally heterogeneous in terms of antenna and reducing
side. Antenna heterogeneity includes variations in antenna
size and in connectivity (grouping) of antenna molecules.
Based on antenna size, PSII centers have been classified as
alpha (a), beta (b ), and gamma (c) (Melis and Homann
1976). These differ from each other in life span and
number of associated chlorophylls. Reducing side hetero-
geneity is related to the ability to transfer an electron from
Q
A
. Centers which are capab le of transferring electrons
from Q
A
to Q
B
are termed Q
B
reducing centers, while those
which are unable to do so are termed Q
B
non-reducing
centers. Specific characteristics of PSII heterogeneity have
been reviewed in Jajoo (
2013). Recent studies have shown
changes in PSII heterogeneity under high temperature
(Mathur et al.
2011b), high salt (Mehta et al. 2010a), and
some pollutants like polycyclic aromatic hydrocarbons
(PAH) (Tomar and Jajoo
2013, 2014). Changes may relate
to the number of active/inactive reaction centers,
Fig. 1 A typical Chlorophyll a polyphasic fluorescence curve,
exhibited by plants (main plot). The transient is plotted on a
logarithmic time scale from 10 ls to 600 s. The same curve plotted in
regular time scale is shown in upper insertion (left). Initial part of
OJIP transient (0–30 ms) plotted on regular time scale is shown in
second insertion (bottom). The marks refer to the selected time points
used by the JIP-test for the calculation of structural and functional
parameters. The signals are the fluorescence intensity F
o
(at 30 ls);
the fluorescence intensity F
K
(at 300 ls); the fluorescence intensities
F
J
(at 2 ms) and F
I
(at 30 ms); the maximal fluorescence intensity,
F
P
= F
M
(at time denoted as t
FM
). Usually, for analysis of fluores-
cence transient, the record is limited to 1 s, creating typical OJIP-
polyphasic fluorescence rise
Acta Physiol Plant (2016) 38:102 Page 3 of 11 102
123
interconversion of active alpha centers into inactive beta
and gamma centers, and increases in the number of Q
B
non-reducing centers under various stress conditions.
Chlorophyll fluorescence kinetic parameters
in response to various abiotic stresses
In the following sections, we review the evid ence that ChlF
kinetics may serve as an useful indicator of the negative
impacts of climate change and human activities, such as
high and low temperature, drought, salinity, nutrient defi-
ciencies, and heavy metals.
Temperature effects
Climate change is likely to increase heat stress in plants,
limiting productivity and biomass production. Photosyn-
thesis is the most sensitive of plant cell processes to high
temperatures (Sharkey and Schrader
2006), which cause
changes in the reduction–oxidation properties of PSII
acceptors and reduce the efficiency of photosynthetic
electron transport in both photosystems (Mathur et al.
2014).
Heat stress affects the values of ChlF parameters
(Fig.
2a). For example, in response to high temperature
stress apples Malus x domestica Borkh reduced both the
ratio of reduced acceptors (plastoquinone) Q
A
-
to RC and
the ratio of reduced acceptors (plastoquinone) Q
B
-
to Q
A
-
.
There was also a decrease in the maximum quantum yield
of PSII and an increase in the minimal fluorescence value
(Chen et al.
2009; Brestic et al. 2013). High temperature
stress also influences the shape of the O–J–I–P curve,
decreasing F
M
and increasing F
o
. The increase in F
o
may
be due to the release of LHC II from the PSII complex,
inactivation of PSII photochemical reaction or an inhibition
of electron flow due to the reduced transfer of Q
A
to Q
B
(Mathur et al. 2011a). For example, the increase of F
o
observed in spinach and rice has been attributed to the
irreversible dissociation of LHC II from the PSII complex
and partly reversible inactivation of PSII (Yamane et al.
1997). The decrease in the fluorescence F
M
level may be
related to denaturation of chlorophyll-proteins (Yamane
et al.
1997).
The K peak (at 30 0 ls) is a well-documented symptom
of heat stress, and is thought to indicate the separation of
the OEC complex and electron transport between pheo-
phytin and primary electron acceptor Q
A
(Strasser et al.
2000; Laza
´
r 2006). In wheat, a treatment at 35 °C had no
affect on photosynthetic efficiency, while exposure to
45 °C caused irreversible damage to the OEC (Schreiber
et al.
2012). The direct cause of the ChlF curve peak (K) is
the outflow of electrons from P680 to PSII acceptors,
which over-compensates the inflow of electrons from the
donor side of PSII to P680. The K peak is also affected by
changes in the energetic relationships between photosys-
tems II. An increase in the F
K
/F
J
ratio (Srivastava and
Strasser
1995) indicates that the heat stress is inhibiting the
donation of electrons by the OEC.
The fast ChlF technique also represents a useful tool to
monitor PSII thermostability. The most efficient approach
is to estimate the critical temperature, i.e., the threshold
level above which there is a sharp increase/decrease of the
observed parameter (Brestic and Zivcak
2013). Some
genotypes can serve as donors of enhanced heat tolerance
in crop breeding programs. For example, the response of
heat-treated common bean (Phaseolus vulgaris L.) lines
and their recovery were monitored by changes in ChlF
induction and analyzed by means of the JIP-test (Stefanov
et al.
2011). PSII thermostability of 30 genotypes of Winter
wheat plants (Triticum aestivum L.) with different geo-
graphic origins were identified using the fast ChlF kinetics
(Brestic et al.
2012). ChlF has also been shown to be a
more effective than conventional methods (e.g., harvest
index, grain filling, etc.) for screening genotypes of durum
wheat (Gautum et al.
2014).
In certain latitudes, low temper atures are a major factor
limiting crop yields (Yang et al.
2009). In the northern
hemisphere, low temperatures during the winter and early
spring are usually followed by intense PAR. These condi-
tions can cause degradation of the thylakoid structure and
distortion in light-dependent photosynthetic reactions
(Suzuki et al.
2011). Cold stress also affects ChlF param-
eters (Fig.
2b). For example, a decrease was observed in
chlorophyll cont ent, OEC efficiency on the donor side of
PSII, photochemical quenching, and efficiency of open
PSII reaction centers for bitter melon plants (Momordica
charantia L.) exposed to cold stress (Yang et al.
2009).
Some plant species are known for their tolerance to low
temperatures, showing less photoinhibition of PSII. For
example, under cold stress pea plants show only small
modifications in ChlF parameters (Strauss et al.
2006; Streb
et al.
2008).
Drought stress
Drought stress effects on photosynthetic apparatus are well
known. They typically start with mostly stomat al effects at
moderate drought intensi ty, and culminate in metabolic and
structural changes caused by severe or long-lasting drought
stress (Jedmowski et al.
2013). This final changes are also
associated with enhancement of photoprotective and
antioxidant functions and pathways (Chaves et al.
2009).
PSII has high resistance to water deficit (compared to PSI)
and negative impacts therefore only occur under conditions
of extreme drough t (Lauriano et al.
2006).
102 Page 4 of 11 Acta Physiol Plant (2016) 38:102
123
ChlF measurements indicat e enhanced protection of PSII
and PSI photochemistry under drought conditions by
adjusting the energy distribution between photosystems and
by act ivating alternative electron sinks (Zivcak et al.
2013).
Drought stress may enhance the resistance of PSII to heat
stress as shown by the disappearance of the K-band from the
OJIP transient (see Fig.
2c and Oukarroum et al. 2012).
The ChlF method is potentially useful for screening
genotypes for drought tolerance (Guha et al.
2013). The
fluorescence rise during the first 2–3 ms is relat ed to pri-
mary photochemistry and it h as been suggested that stim-
ulated L- and K-bands can be used as tools for evaluating
potential to cope with (and recover from) drought stress
(Oukarroum et al.
2007). The L-band is influenced by the
excitation energy transfer between PSII units, commonly
denoted as connectivity or grouping (Strasser and Stirbet
1998).This can b e influenced composition of PSII antennae
that have been changed due to mutations (Brestic et al.
2014) or environmental conditions (Zivcak et al. 2014a).
The K-band has been associated with a dissociation of the
oxygen-evolving complex (OEC) (Guisse
´
et al.
1995). The
measurement of OLKJIP fluorescence transients and their
analysis using the JIP-test might therefore be used as
indicators for drought stress tolerance and physiological
disturbances before the appearance of visible signs of
drought stress.
The most widely used parameter from the ChlF OJIP
transient is the performance index (PI), which provides
Fig. 2 The O(K)JIP-transients of chlorophyll fluorescence in wheat
(Triticum sp. L.) plant samples exposed to different stress conditions
compared to non-stressed plants. The insertions show the changes of
amplitude of relative variable fluorescence in O–J phase (DV
OJ
), J–I
phase (DV
JI
), I–P phase (DV
IP
) and of the ratio of variable
fluorescence in time 0.3 ms (V
K
/V
J
) to variable fluorescence in time
2ms(V
J
) as an indicator of the PSII donor side limitation (K-band).
Individual graphs present comparisons of records in non-stressed
plants (control, C) of a exposed to heat stress (8 h exposed to high
temperature in moderate actinic light, the leaf temperature
was *40 °C); b exposed to long-lasting suboptimal low temperature
(10 days at 10/6 °C day/night); c exposed to severe drought stress
(12th day after withholding of irrigation, leaf relative water con-
tent *60 %); d exposed to salt (NaCl) stress; e plants cultivated at
low soil nitrogen content (low nitrogen, LN); and f exposed to lead
(Pb). Data were provided by the authors of this review
Acta Physiol Plant (2016) 38:102 Page 5 of 11 102
123