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REVI EW
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Frequently asked questions about in vivo chlorophyll uorescence:
3
practical issues
4 Hazem M. Kalaji

Gert Schansker

Richard J. Ladle

Vasilij Goltsev

5 Karolina Bosa

Suleyman I. Allakhverdiev

Marian Brestic

Filippo Bussotti

6 Angeles Calatayud

Piotr Dabrowski

Nabil I. Elsheery

Lorenzo Ferroni

7 Lucia Guidi

Sander W. Hogewoning

Anjana Jajoo

Amarendra N. Misra

8 Sergio G. Nebauer

Simonetta Pancaldi

Consuelo Penella

DorothyBelle Poli

9 Martina Pollastrini

Zdzislawa B. Romanowska-Duda

Beata Rutkowska

10 Joao Serodio

Kancherla Suresh

Wiesaw Szulc

Eduardo Tambussi

11 Marcos Yanniccari

Marek Zivcak
12 Received: 9 February 2014 / Accepted: 2 June 2014
13 The Author(s) 2014. This article is published with open access at Springerlink.com
14 Abstract The aim of this educational review is to provide
15 practical information on the hardware, methodology, and
16 the hands on application of chlorophyll (Chl) a uores-
17 cence technology. We present the paper in a question and
18 answer format like frequently asked questions. Although
19 nearly all information on the application of Chl a uores-
20 cence can be found in the literature, it is not always easily
21 accessible. This paper is primarily aimed at scientists who
22 have some experience with the application of Chl a uo-
23 rescence but are still in the process of discovering what it
24 all means and how it can be used. Topics discussed are
25 (among other things) the kind of information that can be
26 obtained using different uorescence techniques, the
27 interpretation of Chl a uorescence signals, specic
28 applications of these techniques, and practical advice on
29 different subjects, such as on the length of dark adaptation
30 before measurement of the Chl a uorescence transient.
The authors dedicate this paper to Professor Govindjee on the
Occasion of his 80th Birthday
H. M. Kalaji (&)
Department of Plant Physiology, Faculty of Agriculture and
Biology, Warsaw University of Life Sciences SGGW,
Nowoursynowska 159, 02-776 Warsaw, Poland
e-mail: hazem@kalaji.pl
G. Schansker (&)
Avenue des Amazones 2, 1226 Chene-Bougeries, Switzerland
e-mail: gert.schansker@gmail.com
R. J. Ladle
Institute of Biological and Health Sciences, Federal University
of Alagoas, Praca Afranio Jorge, s/n, Prado, Maceio, AL, Brazil
e-mail: richard.ladle@ouce.ox.ac.uk
V. Goltsev
Department of Biophysics and Radiobiology, Faculty of
Biology, St. Kliment Ohridski University of Soa, 8 Dr.
Tzankov Blvd., 1164 Soa, Bulgaria
e-mail: goltsev@gmail.com; goltsev@biofac.uni-soa.bg
K. Bosa
Department of Pomology, Faculty of Horticulture,
Biotechnology and Landscape Architecture, Warsaw University
of Life Sciences SGGW, Nowoursynowska 159,
02-776 Warsaw, Poland
e-mail: karolinabosa@gmail.com
S. I. Allakhverdiev
Institute of Plant Physiology, Russian Academy of Sciences,
Botanicheskaya Street 35, Moscow 127276, Russia
e-mail: suleyman.allakhverdiev@gmail.com
S. I. Allakhverdiev
Institute of Basic Biological Problems, Russian Academy of
Sciences, Pushchino, Moscow Region 142290, Russia
M. Brestic M. Zivcak
Department of Plant Physiology, Slovak Agricultural University,
Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic
e-mail: marian.brestic@uniag.sk
M. Zivcak
e-mail: marek.zivcak@uniag.sk
F. Bussotti M. Pollastrini
Department of Agricultural, Food and Environmental Sciences,
University of Florence, Piazzale delle Cascine 28,
50144 Florence, Italy
e-mail: lippo.bussotti@uni.it
M. Pollastrini
e-mail: martina.pollastrini@uni.it
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Photosynth Res
DOI 10.1007/s11120-014-0024-6
Journal : Large 11120 Dispatch : 5-7-2014 Pages : 38
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31 The paper also provides the physiological background for
32 some of the applied procedures. It also serves as a source of
33 reference for experienced scientists. 34
35 Keywords Chlorophyll a uorescence Fluorescence
36 imaging Complementary techniques Frequently asked
37 questions Plant stress monitoring Photosynthesis
38 Abbreviations
39 A
n
Net CO
2
assimilation rate
40 ATP synthase Enzyme responsible for the synthesis of
ATPfromADPand inorganic phosphate
41 Car Carotenoid
42 Chl Chlorophyll
43 Chlz Accessory chlorophyll in the
photosystem II reaction center
44 CP43, CP47 Core antenna proteins of PSII of 43
and 47 kDa
45 Cyt b
6
/f Cytochrome b
6
/f complex
46 D1 protein One of the major PSII reaction center
proteins, the other being D2
47 DBMIB 2,5-Dibromo-3-methyl-6-isopropyl-p-
benzoquinone
48 DCMU 3-(3,4-Dichlorophenyl)-1,1-
dimethylurea
49 DF Delayed uorescence
50 ETC Electron transport chain
51 ETR Electron transport rate
52 F
O
Minimum Chl a uorescence yield in
the dark-adapted state
53 F
M
Maximum Chl a uorescence yield in
the dark-adapted state
54 F
t
Fluorescence intensity at time t
55 F
V
Maximum variable uorescence,
dened as F
M
- F
O
56 F
V
/F
M
A quantity related to the maximum
quantum yield of PSII photochemistry
57 F
O
/F
M
A parameter related to changes in heat
dissipation in the photosystem II
antenna
58 F
O
0
, F
V
0
, F
M
0
, F
S
Minimum, variable, maximum and
steady state uorescence intensity in
the light-adapted state
59 F
q
0
F
M
0
F
0
[with F
0
= Fs in the steady
state]
60 F
q
0
/F
M
0
Photosystem II operating efciency
61 Fd Ferredoxin
62 FER Fluorescence excitation ratio
63 FNR Ferredoxin-NADP
?
-reductase
64 I
1
Fluorescence intensity at 23 ms
65 IRGA Infra red gas analyzer
66 LED Light-emitting diode
67 LHCII Light harvesting complex II
68 NADP
?
Nicotinamide adenine dinucleotide
phosphate, oxidized form
69 NPQ Non-photochemical quenching,
expressed as (F
M
/F
M
0
- 1)
70 OJIP transient Chl a uorescence rise
induced during a dark-to-strong light
transition, where O is equivalent to F
O
,
P is for peak, equivalent to F
M
(when
measured at saturating light) and J and
I are inections between O and P
A. Calatayud C. Penella
Departamento de Horticultura, Instituto Valenciano de
Investigaciones Agrarias, Ctra. Moncada-Naquera Km 4.5,
Moncada, 46113 Valencia, Spain
e-mail: calatayud_ang@gva.es
C. Penella
e-mail: penella_con@gva.es
P. Dabrowski
Department of Environmental Improvement, Faculty of Civil
and Environmental Engineering, Warsaw University of Life
Sciences SGGW, Nowoursynowska 159, 02-776 Warsaw,
Poland
e-mail: piotr.andrzej.dabrowski@gmail.com
N. I. Elsheery
Agricultural Botany Department, Faculty of Agriculture, Tanta
University, Tanta, Egypt
e-mail: nshery@yahoo.com
L. Ferroni S. Pancaldi
Department of Life Sciences and Biotechnologies, University of
Ferrara, Corso Ercole I dEste 32, 44121 Ferrara, Italy
e-mail: frrlnz@unife.it
S. Pancaldi
e-mail: pcs@unife.it
L. Guidi
Department of Agriculture, Food and Environment, Via del
Borghetto, 80, 56124 Pisa, Italy
e-mail: guidilu@agr.unipi.it
S. W. Hogewoning
Plant Lighting BV, Veilingweg 46, 3981 PC Bunnik, The
Netherlands
e-mail: info@plantlighting.nl
A. Jajoo
School of Life Sciences, Devi Ahilya University,
Indore 452 001, M.P, India
e-mail: anjanajajoo@hotmail.com
A. N. Misra
Centre for Life Sciences, Central University of Jharkhand, Ratu-
Lohardaga Road, Ranchi 835205, India
e-mail: misraan@yahoo.co.uk; misra.amarendra@gmail.com
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71 O (F
O
), K (F
K
),
72 J (F
J
), I (F
I
),
73 P (F
P
)
Fluorescence intensities at
20, 300 ls, 23 ms, *30 and
*200 ms, respectively
74 P680 Photosystem II reaction center
chlorophyll dimer
75 P700 Photosystem I reaction center
chlorophyll dimer
76 PAM Pulse amplitude modulation
77 PFD Photon ux density
78 PEA Photosynthesis efciency analyser
79 PIabs A JIP test parameter also called
performance index
80 PQ Plastoquinone
81 PSI, PSII Photosystem I, Photosystem II
82 Q
A
Primary quinone electron
acceptors of PSII
83 Q
B
Secondary quinone electron
acceptor of PSII
84 qE, qT, qI Non-photochemical quenching
components dened by their
relaxation times in darkness, where E
stands for energy-dependent changes,
T for state changes, and I for
photoinhibition
85 qN Non-photochemical quenching,
expressed as (1 - F
V
0
/F
V
)
86 qP Photochemical quenching
87 RLC Rapid light curve
88 ROS Reactive oxygen species
89 Rubisco Ribulose-1,5-bisphosphate
carboxylase/oxygenase
90 S-states S0
91 S1, S2, S3
92 and S4
Different redox states of the oxygen
evolving complex
93 Sm Normalized area above the OJIP transient
94 STF Single turnover ash
95 TL Thermoluminescence
96 XC Xanthophyll cycle
97 UV Ultraviolet
98 DV
IP
Relative amplitude of the IP phase of
Chl a uorescence induction
Uco
2
Quantum yield of CO
2
xation
U
PSII
PSII operating efciency 99
100 Introduction
101 The measurement of chlorophyll (Chl) a uorescence is one
102 of the most widely used methods to probe photosynthesis
103 (see Papageorgiou and Govindjee 2004 for reviews on
104 application of Chl a uorescence to different aspects of
105 photosynthesis; also see Govindjee (2004) for an overview
106 of important publications on Chl a uorescence). Any
107 researcher who tries to nd his or her way in the uorescence
108 literature will initially be overwhelmed by the number of
109 published articles and by all the conicting ideas. Such a
110 researcher will also quickly discover that it is not easy to nd
111 an answer for many simple and basic questions. We plan to
112 ll this gap in this educational review focusing mainly on
113 plants, green algae, and diatoms.
114 The Chl a uorescence signal is very rich in its content;
115 it is very sensitive to changes in photosynthesis and can be
116 recorded with great precision. Many processes affect the
117 uorescence yield and/or intensity, and using a variety of
118 light protocols (ashes, pulses, continuous light, etc.),
119 different processes can be studied. However, most authors
120 have used only a limited set of experimental protocols
121 based on methods that have been developed over time.
S. G. Nebauer
Departamento de Produccion vegetal, Universitat Polite`cnica de
Vale`ncia, C de Vera sn, 46022 Valencia, Spain
e-mail: sergonne@bvg.upv.es
D. Poli
Department of Biology, Roanoke College, 221 College Lane,
Salem, VA 24153, USA
e-mail: poli@roanoke.edu
Z. B. Romanowska-Duda
Department of Ecophysiology and Plant Development,
University of Lodz, Banacha 12/16, Lodz 90-237, Poland
e-mail: romano@biol.uni.lodz.pl
B. Rutkowska W. Szulc
Agricultural Chemistry Department, Faculty of Agriculture and
Biology, Warsaw University of Life Sciences SGGW,
Nowoursynowska 159, 02-776 Warsaw, Poland
e-mail: beata_rutkowska@sggw.pl
W. Szulc
e-mail: wieslaw_szulc@sggw.pl
J. Serodio
Departamento de Biologia, CESAM Centro de Estudos do
Ambiente e do Mar, Universidade de Aveiro, Campus de
Santiago, 3810-193 Aveiro, Portugal
e-mail: jserodio@ua.pt
K. Suresh
Directorate of Oil Palm Research, West Godavari Dt.,
Pedavegi 534 450, Andhra Pradesh, India
e-mail: sureshkancherla@rediffmail.com
E. Tambussi M. Yanniccari
Institute of Plant Physiology, INFIVE (Universidad Nacional de
La Plata Consejo Nacional de Investigaciones Cient cas y
Tecnicas), Diagonal 113 N495, 327 La Plata, Argentina
e-mail: tambussi35@yahoo.es
M. Yanniccari
e-mail: marcosyanniccari@conicet.gov.ar
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122 With the available commercial equipment, it is very
123 easy to make a uorescence measurement, but as the lit-
124 erature shows, the interpretation of such measurements is
125 still very contentious. There is not even agreement on the
126 processes that determine the uorescence rise from F
O
to
127 F
M
, i.e., the variable uorescence (F
V
). The dominant
128 interpretation assumes that the variable uorescence is
129 determined by the redox state of Q
A
, the rst quinone
130 acceptor of PSII, as originally proposed by Duysens and
131 Sweers (1963) and recently defended by Stirbet and Gov-
132 indjee (2012). Delosme (1967) on the other hand argued
133 that Q
A
was not enough and that there was another
134 important process explaining part of F
V
. This position has
135 recently been supported and extended by Schansker et al.
136 (2011, 2014); see Question 21 for a broader discussion of
137 this point.
138 Another attractive feature of Chl a uorescence is its
139 non-invasive character, which allows the measurement on
140 leaves and even on canopies of trees during long periods of
141 time. A range of instruments has been developed focusing
142 on different aspects of photosynthesis and on different
143 properties of Chl a uorescence. An overview will be given
144 here of the available types of instruments, and we will
145 discuss also what kind of information can be obtained with
146 these instruments.
147 It is important to understand that a uorescence value by
148 itself has no meaning. A well-dened reference state for
149 the photosynthetic sample measured is needed to allow an
150 appropriate interpretation of the data. Processes that relax
151 following illumination will be discussed here as well as the
152 time needed to reach the dark-adapted state, which is an
153 important reference state.
154 A widely read introductory paper on the use of Chl
155 a uorescence is by Maxwell and Johnson (2000), and two
156 more recent papers treating the application of Chl a uo-
157 rescence techniques are by Logan et al. (2007) and Mur-
158 chie and Lawson (2013). These papers focus on the
159 analysis of what is called the steady state: the stable pho-
160 tosynthetic activity after 510 min of illumination at a
161 chosen light intensity. Here, our focus is broader, consid-
162 ering a wider range of uorescence techniques. We make
163 the point that interpretation of uorescence data can be
164 improved making use, at the same time, of different classes
165 of uorescence techniques, as well as by the use of com-
166 plementary techniques such as gas exchange and 820 nm
167 transmission/absorption measurements. We also emphasize
168 that there are still controversies with respect to the inter-
169 pretation of Chl a uorescence data.
170 The educational review is meant to be a starting point
171 for researchers interested in further exploiting Chl a uo-
172 rescence measurements to understand photosynthetic sys-
173 tems. Some questions arise are trivial, e.g.,
174 Question 1: should the instrument be called uorimeter
175 or uorometer?
176 Both versions are allowed, the former being British-Eng-
177 lish and the latter American-English. Answers to other
178 questions may make the difference between a successful
179 and a failed experiment.
180 Question 2. Which types of instruments are available
181 for uorescence measurements?
182 For a rough classication of uorescence instruments used
183 to probe electron transfer reactions involving photosystem
184 II (PSII) and/or photosystem I (PSI), three major classes
185 can be distinguished (see Fig. 1 for an illustration of this
186 classication and see Question 33 for a discussion of fast
187 repetition rate (FRR) measurements and equipment).
188 [1] Instruments based on short light ashes (few ls or
189 less). With such instruments, information on the
190 electron transfer reactions within PSII can be
191 obtained: re-oxidation kinetics of Q
A
-
via forward
192 electron transfer to Q
B
or recombination with the
193 donor side of PSII (see Fig. 2).
194 [2] Instruments based on a saturating pulse (few hundred
195 ms strong light). With such instruments, information
196 on the photosynthetic electron transport chain (ETC)
197 can be obtained: reduction kinetics of the ETC, PSII
198 antenna size, relative content of ETC components
199 like PSI (see Fig. 3).
200 [3] Instruments designed to study the steady state
201 (relatively stable photosynthetic activity after
202 510 min of illumination). With such instruments,
203 light-induced regulatory mechanisms, interaction
204 between ETC, CalvinBenson cycle, stomatal open-
205 ing, and photorespiration (the process initiated when
206 the enzyme Rubisco reacts with O
2
instead of CO
2
)
207 are studied (see Fig. 4).
208 Flash uorescence measurements
209 Figure 2 shows an example of a typical ash uorescence
210 experiment. These measurements are based on the concept
211 of a single turnover ash (STF). An STF has to meet two
212 requirements: (1) The intensity of a STF must be high
213 enough to excite the antennae of all PSII reaction centers
214 (RCs) followed by a charge separation in all PSII RCs
215 leading to a reduction of essentially all Q
A
; (2) A STF must
216 be short enough to induce only one charge separation in
217 each PSII RC. In practice, this situation is never completely
218 reached, and either misses or double hits are induced in a
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219 small fraction of PSII RCs (see e.g., Kok et al. 1970;
220 Shinkarev 2005). The re-oxidation of Q
A
-
can then be fol-
221 lowed: in active RCs, most electrons will be transferred to
222 Q
B
and following a second ash to Q
B
-
(see Fig. 2). The
223 rst reaction has a half-time of 100200 ls, and the second
224 reaction has a half-time of 400600 ls (reviewed by Pet-
225 rouleas and Crofts 2005). If no PQ is bound to the Q
B
-site,
226 the electron on Q
A
-
has to wait, till a PQ molecule binds to
227 the Q
B
-site, and this process can take a few ms (Crofts and
228 Wraight 1983). In the case of inactive PSII centers, forward
229 electron transfer cannot take place, and re-oxidation of
230 Q
A
- occurs via a recombination reaction with the donor
231 side of PSII (Lavergne 1982a; Chylla et al. 1987; Lavergne
232 and Leci 1993; Schansker and Strasser 2005). These
233 instruments can also be used to study the S-states (oxida-
234 tion states S0, S1, S2, S3 and S4) of the oxygen evolving
235 complex of PSII. A series of STFs induces period-4
236 oscillations in the F
O
-level as a function of the S-states (see
237 Delosme 1972; Delrieu 1998; Ioannidis et al. 2000 for
238 examples of such measurements).
239 To probe the oxidation of reduced Q
A
following a sat-
240 urating ash, there are two possible approaches:
241 (1) The easiest method makes use of low-intensity
242 modulated light, which excites only a small fraction
243 of the PSII RCs per unit of time. Figure 2 shows an
244 example of such a measurement. For control samples,
245 in which re-oxidation of Q
A
-
via forward electron
246 transport can occur, this approach works well. How-
247 ever, when the sample is inhibited, e.g., by an electron
Fig. 1 The processes that can be studied analyzing the uorescence
decay following a single turnover ash, the analysis of OJIP
transients, or the quenching analysis. With the analysis of the
uorescence decay kinetics (STF analysis, purple line), it is possible
to obtain information on electron transport reactions inside PSII and
via the occupancy state of the Q
B
-site on the PQ-pool redox state;
OJIP transients (green line) can be used to obtain information on the
redox state of the photosynthetic ETC, on the stoichiometry of the
components of the ETC and on the relative PSII antenna size; the
quenching analysis (rosa line) gives information on dynamic
processes, electron ow, under steady state conditions, is sensitive
to short-term regulatory processes in the antenna (see text) and to
CalvinBenson cycle activity, changes in photorespiration and
stomatal opening (modied from Kalaji and Loboda 2010)
Fig. 2 Example of the uorescence decay kinetics following a single
turnover xenon ash to a suspension of PSII-enriched membranes
isolated from spinach. Several pre-ashes had been given to induce a
partial reduction of the PQ-pool (G. Schansker, unpublished data)
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248 transfer inhibitor such as DCMU (3-(3,4-dichloro-
249 phenyl)-1,1-dimethylurea), which displaces Q
B
from
250 its binding site (Velthuys 1981; Lavergne 1982b), the
251 low-intensity modulated light leads to the accumula-
252 tion of a considerable population of Q
A
-
complicating
253 the analysis of the experiment, because re-oxidation
254 of Q
A
-
by recombination with the donor side is much
255 slower than forward electron transport to Q
B
.
256 (2) The second method uses a combination of a STF
257 followed by a probe ash that probes the redox state of
258 Q
A
at the time of the probe ash (this is called a pump
259 probe experiment) (Mauzerall 1972; Robinson and
260 Crofts 1983). The intensity of the probe ash is much
261 lower than that of the STF. In this case, the experiment
262 is repeated many times and each time at a variable time
263 t after the STF, a probe ash is given to probe the redox
264 state of Q
A
. In this way, the re-oxidation kinetics are
265 constructed point by point. The actinic light problem,
266 described above for DCMU inhibited samples, does
267 not exist in this case. On the other hand, identical
268 samples do not exist, and therefore, the biological
269 variability between samples will lead to experimental
270 noise and the need for repetitions to obtain smooth
271 kinetics. To make different phases in the re-oxidation
272 kinetics visible, the use of a logarithmic time scale has
273 been introduced (see e.g., Cser and Vass 2007).
274 Commercial equipment to make this type of measure-
275 ments is the superhead uorometers (Photon Systems
276 Instruments, Brno, Czech Republic), which can also
277 be used to measure OJIP transients and saturating
278 pulse protocols (see below).
279 Complementary techniques for ash uorescence mea-
280 surements are thermoluminescence (TL) (reviewed by Vass
281 and Govindjee 1996; Misra et al. 2001a, b; Ducruet and
282 Vass 2009) and delayed uorescence (DF) (recently
283 reviewed by Goltsev et al. 2009) measurements that pro-
284 vide specic information on recombination reactions
285 within PSII RCs.
Fig. 3 OJIP transients (double normalized between O and P)
measured on a bean leaf (Phaseolus vulgaris) shown on a linear
timescale (a) and a logarithmic timescale (b). A measurement on dark
adapted (closed symbols) which has an oxidized PQ-pool and a low
J-step and a measurement made 5 s later (open symbols) where Q
A
had become re-oxidized in part of the PSII RCs due to recombination
(O level considerably below P), the PQ-pool is still almost completely
reduced (J level near P), and the acceptor side of PSI is almost
completely re-oxidized (I level close to that of the dark-adapted state)
(G. Schansker, unpublished data)
Fig. 4 Slow Chlorophyll a uorescence kinetics (in arbitrary units)
using a PAM-2100 uorometer. The dark-adapted leaf is illuminated
with weak modulated measuring light to give the zero uorescence
level F
0
. Application of a saturation pulse (SP) allows measurement
of the maximum uorescence level in the dark F
M
. Photosynthesis is
then activated by an actinic light source (in this case 250 lmol
photons m
-2
s
-1
). SPs during the light phase were triggered spaced
1 min apart (indicated by arrows) to determine the maximum
uorescence intensity in the light (F
M
0
), and for each SP, q
P
, U
PSII,
and NPQ parameters were calculated, and these are indicated in the
gure (Penella et al. unpublished data)
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286 Flash uorescence measurements are frequently used to
287 study PSII mutants (e.g., Etienne et al. 1990; Nixon et al.
288 1991; Cser and Vass 2007) and can also be used in the case
289 of treatments that affect the function of PSII [e.g., stresses
290 like heat stress (Yamasaki et al. 2002)] or to probe the PQ
291 redox state (Dannehl et al. 1996).
292 Saturating pulse or OJIP measurements
293 Upon a dark-to-light transition, the uorescence intensity
294 of a leaf or other photosynthetic samples increases from a
295 low value (F
O
or O) via two intermediate steps (F
J
or J and
296 F
I
or I) in 200300 ms to a maximum value (F
M
or P)
297 during the application of a saturating pulse of light (see
298 Fig. 3a, b; Strasser and Govindjee 1991; Strasser et al.
299 1995). The different uorescence rise phases (OJ, JI and
300 IP) can be related to different steps of the reduction of the
301 ETC: OJ parallels the reduction of the acceptor side of PSII
302 (Q
A
? Q
B
); JI parallels the reduction of the PQ-pool and
303 IP parallels the reduction of the electron transport acceptors
304 in and around PSI (Schansker et al. 2005). This means that
305 OJIP transients give information on the state of the ETC.
306 Although complex simulations of OJIP transients use a
307 kinetic model based on the gradual reduction of the ETC
308 (see e.g., Lazar 2003; Zhu et al. 2005), it has been shown
309 that the transients can also be approximated assuming that
310 the transients consist of three kinetic components (Boisvert
311 et al. 2006; Vredenberg 2008; Joly and Carpentier 2009)
312 indicating that the rate limitations (exchange of PQ at the
313 Q
B
-site of PSII and re-oxidation of PQH
2
by cyt b
6
/f) quite
314 effectively separate the three rise phases kinetically. The
315 kinetics of the OJIP transient are, e.g., sensitive to the PQ
316 redox state (Toth et al. 2007a) and PSI content (Oukarroum
317 et al. 2009; Ceppi et al. 2012). During the isolation of
318 thylakoid membranes, the properties of the ETC are
319 modied, and this is reected by changes in the uores-
320 cence kinetics. Attempts have been made (see e.g., Bukhov
321 et al. 2003) to make the uorescence induction kinetics of
322 thylakoid membranes look more like those of leaves.
323 Using a pulse-probe approach, a rst pulse reduces the
324 ETC and a second probe pulse given at time t after the rst
325 pulse probes the redox state of the ETC. The analysis of the
326 regeneration kinetics of the OJIP transient gives informa-
327 tion on the rate of re-oxidation of Q
A
-
by recombination
328 with the donor side of PSII, the re-oxidation of the PQ-pool
329 due to plastoquinol oxidase activity (see Question 17), and
330 the rate of re-oxidation of the acceptor side of PSI in
331 darkness (Schansker et al. 2005).
332 Complementary techniques for OJIP measurements are
333 820 nm absorbance/transmission measurements that probe
334 the redox state of PSI (plastocyanin, P700 and ferredoxin)
335 and DF measurements that give information on the
336 occurrence of recombination reactions in PSII as a function
337 of the redox state of the ETC. The interpretation of these
338 measurements can also improved by determining the chl a/
339 b ratio and the chl content of the leaves/cells. OJIP mea-
340 surements have been used widely to study the effects of
341 stress (see Questions 19, 24, 2628).
342 Steady state measurements
343 The steady state refers to the relatively stable photosyn-
344 thetic activity that is obtained when leaves or other pho-
345 tosynthetic samples are illuminated at a chosen light
346 intensity during approximately 510 min (or more). The
347 Chl a uorescence intensity in the steady state is affected
348 both by the redox state of the ETC (and Q
A
in particular)
349 and by changes in the uorescence yield, i.e., a change in
350 the probability that absorbed light is emitted as Chl
351 a uorescence. These yield changes not only can be due to
352 the formation of the transthylakoid DpH (Krause et al.
353 1983) and xanthophyll cycle (XC) related changes (Bilger
354 and Bjorkman 1991), antenna size changesfor example,
355 due to state transitions, which are especially obvious for
356 algae such as Chlamydomonas reinhardtii (see e.g., Iwai
357 et al. 2008)or photoinhibition (see e.g., Bjorkman and
358 Demmig 1987; Van Wijk and Krause 1991; Tyystjarvi and
359 Aro 1996) but are also due to the activation of ferredoxin
360 NADP
?-
reductase (FNR) on the acceptor side of PSI
361 (Schansker et al. 2006, 2008). In the 1980s, an analysis was
362 developed, called the quenching analysis (see Question 15
363 for a more detailed discussion of the quenching analysis)
364 that can distinguish between redox changes (photochemical
365 quenching) and uorescence yield changes. A uorescence
366 yield change occurs when the rate constant for either
367 uorescence or heat emission changes. If this leads to a
368 smaller F
M
value (and in many cases smaller F
O
value),
369 this is called non-photochemical quenching. Figure 4 gives
370 an example of such a protocol. Just as in the case of the
371 ash uorescence measurements (see above), the uores-
372 cence intensity is probed using low-intensity modulated
373 light. The steady state is induced using continuous actinic
374 light of a chosen intensity, and in addition every 100 or
375 200 s (this can be variable time interval), a saturating pulse
376 (comparable to an OJIP transient) is given to reduce the
377 ETC and all Q
A
. On turning off the actinic light, relaxation
378 of the induced non-photochemical quenching can be fol-
379 lowed using saturating light pulses to probe changes in the
380 F
M
level. In general, three relaxation phases are observed
381 (Demmig and Winter 1988; Horton and Hague 1988): the
382 qE which relaxes within 100200 s as a consequence of the
383 dissipation of the transmembrane DpH, the qT, whose
384 relaxation is complete within 15 min and the qI which
385 covers all processes that need more than 15 min to recover.
386 As will be discussed later in detail (see Question 15) the qT
387 and qI are less well dened. It is worth mentioning here
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388 that by measuring Chl a uorescence induced by the sat-
389 urating pulses with a higher time resolution (i.e., measuring
390 OJIPs), it is possible to obtain more information on the
391 character of the qT and qI phases (Schansker et al. 2006).
392 The relaxation of the different non-photochemical
393 quenching phases can be treated as the sum of three
394 exponentials (see e.g., Walters and Horton 1991; Rohacek
395 2010; and Question 15).
396 Obtaining the maximum F
M
0
value is not a trivial issue.
397 Markgraf and Berry (1990) and Earl and Ennahli (2004)
398 observed that in the steady state, high light intensities are
399 needed to induce the maximum F
M
0
value. Earl and Ennahli
400 (2004) observed that more than 7,500 lmol photons
401 m
-2
s
-1
(the maximum intensity of their light source) were
402 needed to reach the maximum F
M
0
value of their maize
403 leaves and that at higher actinic light intensities, more light
404 was needed to saturate F
M
0
. Schansker et al. (2006) observed
405 the same actinic light intensity dependence measuring both
406 uorescence and 820 nm transmission and suggested that
407 the ferridoxin/thioredoxin system that is thought to contin-
408 uously adjust the activity of several CalvinBenson cycle
409 enzymes (see Question 6), is responsible for the actinic light
410 intensity dependence. Earl and Ennahli (2004) proposed an
411 extrapolation method based on the measurement of F
M
0
at
412 two light intensities to obtain the true F
M
0
value. Loriaux
413 et al. (2013) studied the same light intensity dependence of
414 F
M
0
and proposed the use of a single multiphase ash lasting
415 approximately 1 s to determine the maximum F
M
0
value.
416 This ash consists of two high light intensity phases sepa-
417 rated by a short interval at a lower light intensity during
418 which the uorescence intensity decreases. The second high
419 light intensity phase of this protocol has a higher light
420 intensity than the rst phase (see also Harbinson 2013 for a
421 commentary on this paper).
422 Complementary techniques for this type of uorescence
423 measurement are gas exchange measurements (to probe Cal-
424 vinBenson cycle activity, stomatal opening, CO
2
conduc-
425 tance) and 820 nm absorbance/transmission measurements.
426 77 K uorescence spectra
427 Low temperature (77 K) uorescence measurements repre-
428 sent another technique to obtain information on the photo-
429 systems. At room temperature, variable uorescence is
430 emitted nearly exclusively by PSII. Byrdin et al. (2000)
431 detected only a small difference in the quenching efcien-
432 cies of P700 and P700
?
at room temperature. This is sup-
433 ported by the observation that inhibiting PSII by DCMU
434 (Toth et al. 2005a) or cyt b
6
/f by DBMIB (Schansker et al.
435 2005) does not affect F
M
despite a big difference in the redox
436 state of P700 in the absence and presence of inhibitors.
437 However, variable uorescence emitted by PSI can be
438 induced on lowering the temperature to 77 K. Although
439 measurements of light-induced uorescence changes can be
440 made at 77 K, in most cases, the uorescence emission
441 spectrum (600800 nm) is measured. This type of mea-
442 surement is used to obtain information on the PSII and PSI
443 antennae. This type of measurement is used to obtain
444 information on the PSII and PSI antennae. A common
445 application of 77 K measurements is the detection of the
446 occurrence of state transitions (e.g., Bellaore et al. 2005;
447 Papageorgiou and Govindjee 2011; Drop et al. 2014), where
448 changes in the relative amplitudes of the PSII and PSI bands
449 are indicators for this process. Figure 5 gives an example of
450 a measured 77 K spectrum. Emission bands at 685 and
451 695 nm are related to the antenna of PSII, and peaks around
452 730 nm are related to the antenna of PSI (Govindjee 1995;
453 S
punda et al. 1997; Srivastava et al. 1999).

454 Complementary techniques are ultrafast femto- or
455 picosecond absorbance or uorescence measurements that
456 give information on energy transfer within the antenna
457 (e.g., Gilmore et al. 1998; Richter et al. 1999) but which
458 are beyond the scope of this educational review.
459 Fast uorescence techniques (ns, ps, fs time range)
460 As noted in the previous paragraph, fast uorescence (and
461 absorption) techniques, which probe energy transfer
462 between chlorophylls or between carotenoids and chloro-
463 phylls in the photosynthetic antennae and the charge sep-
464 aration processes in the RCs of PSII and PSI will not be
Fig. 5 77 K uorescence emission spectra of leaves of plants grown
hydroponically on a complete medium (black line) and on medium
containing only traces of sulfate (green line). Sulfate deciency led to
extensive chlorosis and in addition to a rather specic loss of PSI.
This reduced the long wavelength bands around 730 nm and
increased the 685 and 695 bands due to a decreased re-absorption
by PSI reaction centers of Chl a uorescence emitted by PSII
(Schansker and Ceppi, unpublished data)
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465 discussed in this paper. See e.g., Holzwarth (1996, 2008)
466 and Berera et al. (2009) for introductory reviews on the
467 application of these methods.
468 Question 3. What is the effect of wavelengths at which
469 the uorescence is measured on the character
470 of the uorescence signal?
471 Most commercial instruments measure Chl a uorescence
472 at wavelengths longer than 700 nm. At room temperature,
473 at wavelengths longer than 700 nm, PSI becomes an
474 important source of uorescence emission. As shown by
475 Genty et al. (1990) and Pfundel (1998) in C3 plants, about
476 30 % of the F
O
emission is due to PSI uorescence, and in
477 C4 plants, this percentage is even higher (Pfundel 1998).
478 This causes, e.g., a systematic underestimation of the F
V
0
/
479 F
M
0
value, which is used as a measure of the maximum
480 quantum yield of PSII. Detecting Chl a uorescence
481 emission at wavelengths below 700 nm can considerably
482 reduce this problem. However, in measuring equipment
483 such as photosynthetic efciency analyser (PEA) and
484 HandyPEA instruments (Hansatech Instruments Ltd, UK)
485 which use red LEDs with an emission peak around 650 nm,
486 this would have led to an overlap between the actinic
487 wavelengths and the detecting wavelengths. With the
488 introduction of (strong) LEDs emitting at shorter wave-
489 lengths, e.g., in the blue (see e.g., Nedbal et al. 1999), it is
490 now technically possible to avoid this overlap and to detect
491 uorescence below 700 nm. Interference of PSI uores-
492 cence at wavelengths longer than 700 nm should be taken
493 into account especially when measuring uorescence
494 parameters in the light-adapted state. Non-photochemical
495 quenching induced in the light quenches the variable
496 uorescence (F
M
F
O
) to a larger extent than F
O
uores-
497 cence. This makes the underestimation of the true F
V
0
/F
M
0
498 value light intensity dependent as well, since a higher light
499 intensity induces more non-photochemical quenching.
500 Question 4. Which part of the leaf is probed
501 and analyzed by a uorescence measurement?
502 The leaf is optically complex. In a dorsiventral leaf, the
503 palisade parenchyma cells have been shown to act as light
504 guides, keeping the light more or less focused (Vogelmann
505 and Martin 1993; Vogelmann et al. 1996). The lobed cells of
506 the spongy mesophyll and the spaces that surround these
507 cells, on the other hand, disperse the light (Vogelmann and
508 Martin 1993). At the same time, there is a strong light gra-
509 dient within the leaf (Vogelmann 1989, 1993). This means
510 that the light intensity decreases rapidly as light penetrates
511 into the leaf. As a consequence, illuminating and probing
512 Chl a uorescence emission on the adaxial surface of the
513 leaf, chloroplasts located deep in the leaf will be excited by a
514 much lower photon ux density than those located close to
515 the adaxial side of the leaf (Terashima and Saeki 1985;
516 Fukshansky and Martinez von Remisowsky 1992). At the
517 same time, the spectral distribution of the light changes as
518 well: as light penetrates the mesophyll, the relative contri-
519 bution of green and far-red (FR) light progressively
520 increases, because the absorption of these wavelengths by
521 the leaf is less efcient (Sun et al. 1998; Rappaport et al.
522 2007). The chloroplasts located deeper in the leaf, i.e., those
523 of the spongy tissue, acclimate to these lower, FR-enriched
524 light intensities by increasing the antenna size of PSII,
525 reducing the number of RCs, and decreasing the PSI/PSII
526 ratio (Terashima et al. 1986; Evans 1999; Fey et al. 2005;
527 Pantaleoni et al. 2009). Since the emitted uorescence is a
528 linear function of the light intensity (Vogelmann and Evans
529 2002; cf. Schansker et al. 2006), chloroplasts located deeper
530 in the leaf will contribute to a lesser extent to the detected
531 uorescence signal. In practice, uorescence measurements
532 will probe mainly chloroplasts in the palisade parenchyma
533 cells (Vogelmann and Evans 2002). The assumption that not
534 all chloroplasts are assayed is supported by the observation
535 that a vefold decrease in the chlorophyll content of the leaf
536 does not affect the detected F
O
and F
M
values (Dinc et al.
537 2012). In fact, since the total amount of uorescence emitted
538 by the leaf does not change, it suggests that the light beam
539 probes deeper in the leaf as more chlorophyll is lost. The
540 optical properties of the leaf also mean that measurements
541 made on the abaxial (bottom) side of the leaf have charac-
542 teristics that differ considerably from those made on the
543 adaxial (top) side of the leaf (Schreiber et al. 1977). Oxygen
544 and CO
2
assimilation measurements on the other hand assay
545 the whole leaf, and this may lead to deviations when com-
546 paring, for example, measurements of the oxygen evolving
547 activity with uorescence measurements (Bjorkman and
548 Demmig 1987; Tyystjarvi and Aro 1996).
549 Given the gradient of photosynthetic properties that
550 exists within the leaf (Terashima et al. 1986; Evans 1999),
551 the photosynthetic response of a leaf depends on the
552 wavelength composition of the exciting light. Deeper
553 penetrating green light probes more low light acclimated
554 chloroplasts located in the lower cell layers than blue light
555 that is strongly absorbed by the leaf and mainly probes
556 chloroplasts close to the adaxial side of the leaf.
557 Question 5. How to dark-adapt leaves?
558 For the interpretation of Chl a uorescence measurements,
559 it is important that the state of the photosynthetic apparatus
560 at the beginning of the measurement is well dened. The
561 dark-adapted state of the leaf is a well-dened state of the
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562 photosynthetic apparatus and, therefore, for most experi-
563 ments, photosynthetic samples are rst dark adapted.
564 There are four main methods to achieve dark adaptation
565 in leaves:
566 1. In the case of an intact plant, a leaf can be put into a
567 leaf clip shielding it from ambient light. However, if
568 the ambient light intensity is high, and the leaf is not
569 entirely at, there is a chance that some stray light
570 reaches the shielded area.
571 2. Detached leaves can be kept for a while between wet
572 lter paper in darkness and subsequently measured in
573 the laboratory. Detachment of leaves has consequences
574 for the physiological state of the leaf: it causes, for
575 example, a closure of the stomata (Raschke 1970). See
576 Potvin (1985) and Weng et al. (2011) for a comparison
577 of the properties of attached and detached leaves and
578 Kato et al. (2002) for a discussion of the differences
579 between leaves and leaf disks.
580 3. Under laboratory conditions, measurements can be
581 made in the dark or in a dimly lit room under
582 conditions that induce very little photosynthetic activ-
583 ity. Traditionally, low-intensity green light has been
584 used as a kind of safe light (see Sun et al. 1998 for a
585 discussion of this point) although we note that leaves
586 can still absorb and use most of the green light for
587 photosynthesis (cf. Sun et al. 1998; Vogelmann and
588 Evans 2002; Rappaport et al. 2007).
589 Loss of time for dark adaptation can be avoided when
590 the measurements are made directly in the eld at night (no
591 need for leaf clips). In this case, the leaves are allowed to
592 dark adapt for many hours, and the results of such mea-
593 surements differ from measurements on leaves following a
594 relatively short dark-adaptation period during the day.
595 Question 6. What is a good dark-adaptation time?
596 Dark adaptation of samples that will be used for Chl a uo-
597 rescence measurements, is often associated with the re-
598 oxidation of Q
A
-
. However, dark adaptation is a considerably
599 more complicated process, and there are more factors that
600 can affect a subsequent uorescence measurement.
601 In dark-adapted leaves, several enzymes are inactivated
602 to prevent wasteful reactions. Examples of such enzymes
603 include Rubisco (e.g., Streusand and Portis 1987); four other
604 thioredoxin-dependent enzymes: D-fructose1,6-bisphos-
605 phatase, phosphoribulokinase, and seduheptulose-1,7-bis-
606 phosphatase (Buchanan 1984; Scheibe 1990) and ATP
607 synthase (Stumpp et al. 1999); and FNR (Carillo et al. 1981;
608 Satoh 1981). These enzymes are active in the light, and
609 during a light-to-dark transition, they gradually become
610 inactive again. The half-time of inactivation of Rubisco
611 under in vivo conditions is 24 min (Stitt et al. 1987; Laisk
612 and Oja 1998). Inactivation of ATP synthase and the three
613 other CalvinBenson cycle enzymes is under control of the
614 thioredoxin system (Scheibe 1990), and their inactivation
615 depends on the re-oxidation of stromal components such as
616 ferredoxin and NADPH. FNR inactivation varies depending
617 on the species: pea leaves need *15 min for full inactiva-
618 tion (Schansker et al. 2006), whereas in a Pinus species, an
619 hour is needed (Schansker et al. 2008). Once inactivated, all
620 of these enzymes must rst be activated again before steady
621 state photosynthesis is induced, and this affects the uo-
622 rescence induction kinetics (see Papageorgiou et al. 2007;
623 Papageorgiou and Govindjee 2011 for an in-depth discus-
624 sion of the uorescence kinetics beyond P or F
M
in a variety
625 of photosynthetic organisms). In addition, active FNR (i.e.,
626 an activated acceptor side of PSI) has an effect on the IP
627 phase of the OJIP transients and on the amplitude of the F
M
628 that can be reached by a strong pulse of light (Schansker
629 et al. 2008). In most uorescence studies, many are not
630 interested in the processes mentioned above, and in that
631 case, it is best to make the dark-adaptation time long enough
632 to allow at least FNR to become inactive again (a marker for
633 this is a regeneration of the uorescence IP phase and in
634 addition a regeneration of 820 nm re-reduction phase par-
635 alleling the IP phase, see Schansker et al. 2006, 2008).
636 As mentioned in Question 2 Sect. 3, several regulatory
637 and stress-related processes that affect the uorescence
638 yield (quench F
M
) are induced in the light. Following a
639 light-to-dark transition, i.e., on turning off the light, these
640 processes are reversed. State transitions (the transfer of a
641 part of the antenna system among PSII and PSI) and XC
642 related processes may take a considerable amount of time
643 to reverse (Fork and Satoh 1986; Ruban and Horton 1999)
644 and the recovery of a plant from photoinhibition takes
645 hours (Havaux 1989; Long et al. 1994).
646 An answer to the question as to what a good dark-
647 adaptation time is, depends on the information we want to
648 obtain. If the aim is the study of the regulatory and pho-
649 toinhibition-related processes, a dark-adaptation time of
650 15 min that allows FNR (at least in plants like pea) to
651 become inactive again would be sufcient. If someone is
652 interested in long term adaptation responses of a leaf or
653 other photosynthetic organism to a treatment, much longer
654 dark-adaption times that allow also the regulatory pro-
655 cesses and processes like photoinhibition to recover may be
656 considered (see also the next question).
657 Question 7. How to obtain the best reference F
O
and F
M
658 values for the quenching analysis?
659 In eld experiments, predawn measurements are often used
660 to obtain reference F
O
and F
M
values for measurements
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661 made during the day (Logan et al. 1999; Maxwell and
662 Johnson 2000; Demmig-Adams et al. 2006). Under these
663 conditions, NPQ is assumed to be completely relaxed and
664 therefore zero, and the photoinhibition induced during the
665 previous day is expected to have been reversed (Flexas
666 et al. 1998; Logan et al. 1999; Demmig-Adams et al. 2006).
667 However, in some cases, chronic photoinhibition occurs,
668 which can be easily detected by lowered predawn F
V
/F
M
669 values (Osmond and Grace 1995; for a review see Dem-
670 mig-Adams et al. 2012). We note that the absence of light
671 during recovery experiments may prevent a full repair of
672 photoinhibitory (Greer et al. 1986) and heat stress damage
673 (Toth et al. 2005b). Light is needed for the synthesis of
674 ATP, which is needed for the synthesis of the D1 protein
675 (Kuroda et al. 1992). Edhofer et al. (1998) have reported
676 that light is needed for translation elongation of the D1
677 protein; these are processes that are part of the PSII repair
678 cycle following damage to PSII (recently reviewed by
679 Nixon et al. 2010). Low-intensity actinic light generates the
680 ATP needed for the PSII repair cycle, and at the same time,
681 it does not induce additional photoinhibition and is thereby
682 more effective than a complete dark recovery (see e.g.,
683 Elsheery et al. 2007).
684 Question 8. What can go wrong during a uorescence
685 measurement on leaves? Technical issues
686 To dark-adapt leaves in the eld, leaf clips have been
687 developed. They cover the area of the leaf to be measured.
688 The measuring head of, for example, a HandyPEA can be
689 connected to a leaf clip, after which the clip can be opened,
690 and the measurement made. Since such measurements are
691 normally evaluated afterward, it should be kept in mind
692 that unopened or partially opened leaf clips are a common
693 reason for transients showing no or little uorescence rise.
694 A smooth leaf can also lead to problems, since the clip may
695 shift while attaching the measuring head, and in that case, a
696 non-dark-adapted part of the leaf will be measured. If the
697 leaf is not at, some stray light may enter the leaf clip via
698 the spaces left between the leaf clip and the leaf surface.
699 Especially on a bright day, this may prevent a full dark
700 adaptation of the covered leaf area. The same problems can
701 occur with pulse amplitude modulated (PAM) type
702 instruments developed for eld applications, which use leaf
703 clips to allow dark adaptation.
704 When working with a PAM instrument, the measuring
705 light intensity must be chosen in such a way that the F
M
706 stays within the measuring window. If the measured signal
707 is too strong, then the highest values will be cut off. For
708 example, as a rule of thumb the uorescence intensity
709 induced by the measuring light (associated with F
O
) should
710 be approximately 10 % of the total scale. In any case,
711 absolute values and their limits depend on the manufac-
712 turer, and its instructions should be carefully read before
713 starting any measurements. Further, the distance between
714 the leaf and the ber optics has to be adjusted; it is usually
715 set between 1 and 1.5 cm. Background uorescence signals
716 from the environment must be suppressed by zeroing the
717 signal in the absence of a leaf sample.
718 Using direct uorescence equipment like the Handy-
719 PEA, there is also a risk that the emitted uorescence
720 intensity causes an overload of the detector. It is therefore
721 important to check if, at a given gain and excitation light
722 intensity, the measured uorescence kinetics remain below
723 the maximum measurable uorescence intensity. If the
724 emitted uorescence intensity is too strong, then the top
725 part of the transient will be cut off, and in that case, the
726 gain has to be reduced.
727 Question 9. Why was it so difcult to determine the F
O
728 before ~1985?
729 It may be hard to imagine nowadays, but the determination
730 of a correct F
O
value was a major problem for researchers
731 using Chl a uorescence up to the mid-1980s (see Kalaji
732 et al. 2012a, b for a historical overview of instrument
733 development). The shutters used at the time had a full
734 opening time of anywhere between 0.8 ms (e.g., Neubauer
735 and Schreiber 1987) and 2 ms. At high light intensities, the
736 J-step is reached after *0.82 ms of illumination. To
737 minimize the effect of the shutter opening time, in many
738 studies, low-intensity light was used to slow down the
739 uorescence induction kinetics. In the 1980s, two funda-
740 mentally different solutions for the shutter problem were
741 introduced in the form of modulated systems (Schreiber
742 et al. 1986) and PEA-type instruments (Strasser and Gov-
743 indjee 1991). These two measuring concepts are explained
744 and compared in Questions 10 and 11.
745 Question 10. What is the principle of modulated
746 uorescence measurements?
747 Modulated systems pulse amplitude modulated uorome-
748 ters, (PAM) use a trick to separate the effect of the actinic
749 light that drives photosynthesis and the low-intensity
750 measuring light that is used to probe the state of the pho-
751 tosynthetic system on the measured uorescence intensity
752 (see also Question 2 Sect. 3). A so-called login amplier
753 only registers the uorescence changes induced by the
754 modulated measuring light and ignores the uorescence
755 changes induced by the continuous actinic light. This way
756 the low-intensity measuring light can be used to measure
757 both the F
O
(induced by the measuring light itself) and F
M
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758 (induced by a strong light pulse) values (Schreiber et al.
759 1986). The effective light intensity of modulated light
760 depends on the pulse frequency. In the case of a modern
761 PAM instrument, the modulated measuring light consists of
762 13 ls ashes of red or white light, and ash frequencies
763 between 100 and 20,000 Hz can be chosen. At the lowest
764 frequency, the effective photosynthetic photon ux density
765 is \0.2 lmol photons m
-2
s
-1
; an intensity that is 200
766 times higher when the highest frequency is chosen. The
767 choice of a low frequency gives not only a very small
768 actinic effect (= measuring light-induced F
V
) but also a
769 relatively poor signal-to-noise ratio. A high frequency not
770 only is considerably more actinic but gives also a much
771 better signal-to-noise ratio. The actinic effect of the mea-
772 suring light becomes especially visible (and problematic) if
773 PSII electron transfer inhibitors such as DCMU are being
774 used (see Question 2 Sect. 1). Compared to PEA-type
775 instruments an advantage of the modulated uorimeters is
776 that the measured uorescence yield is independent of the
777 intensity of both the actinic light and light of the saturating
778 pulse (Schreiber et al. 1986). In the case of PEA-type
779 instruments, the measured uorescence intensity is a linear
780 function of the actinic light intensity used, and as a con-
781 sequence, the measured uorescence intensities must be
782 normalized rst (e.g., divided by the light intensity) before
783 measurements made at different light intensities can be
784 compared (see e.g., Schansker et al. 2006).
785 Question 11. What is the principle of direct uorescence
786 measurements?
787 In the so-called direct uorescence instrumentsi.e.,
788 instruments in which the actinic light that drives photosyn-
789 thesis is also used as measuring lightthe F
O
problem is
790 solved by using strong light emitting diodes (LEDs): light
791 sources that can be switched on/off very quickly (Strasser
792 and Govindjee 1991). In modern equipment, a stable light
793 intensity emitted by the LEDs is reached in less than 10 ls.
794 Initially, only red (650 nm) LEDs were available for this
795 type of measurement but now other orange (discussed by
796 Oxborough 2004), green (Rappaport et al. 2007), and blue
797 (Nedbal et al. 1999) or a mix of LEDs of different colors
798 (Schreiber 1998) are also available. In the original PEA
799 instrument, the response time of the LEDs was still in the
800 order of the 4050 ls (e.g., Strasser et al. 1995) necessitat-
801 ing the use of extrapolation to estimate the F
O
value; in the
802 current instruments, a response time of 1020 ls is good
803 enough for an accurate determination of the F
O
value for
804 light intensities below *10,000 lmol photons m
-2
s
-1
(cf.
805 Schansker et al. 2006). The absence of a measuring light
806 source means that between pulses, there is true darkness. As
807 a consequence, the F
O
can be determined more accurately
808 than in the case of a modulated system (see Schansker and
809 Strasser 2005 for a discussion on the effects of very lowlight
810 intensities on the F
O
value). The absence of measuring light
811 is particularly advantageous when the samples to be ana-
812 lyzed have been inhibited with electron transfer inhibitor
813 such as DCMU. Another important difference between PEA
814 instruments and modulated PAM instruments is the data
815 sampling strategy. In PEA instruments, the data sampling is
816 non-linear. In HandyPEA instruments, during the rst
817 300 ls of illumination one measuring point is collected
818 every 10 ls; between 300 ls and 3 ms one point per 100 ls,
819 between 3 and 30 ms one point per ms, and between 30 and
820 300 ms one point per 10 ms. In this way, an OJIP transient
821 measured at a high time resolution is dened by approxi-
822 mately 120 measuring points. In the case of a PAM instru-
823 ment, a measurement with the same initial time resolution
824 would yield at least 20,000 measuring points (for 200 ms).
825 This makes the HandyPEA les much easier to handle when
826 analyzing them using spreadsheet programs like Microsoft
827 Excel.
828 Question 12. Why use a logarithmic timescale
829 to visualize uorescence transient measurements?
830 As described above, PEA instruments allow a shutter-less
831 measurement of OJIP transients. However, PEA instru-
832 ments make use of a second innovation and that is the use
833 of a logarithmic timescale to visualize the measurements of
834 the OJIP uorescence rise (Strasser and Govindjee 1991).
835 Bannister and Rice (1968) had already used this idea more
836 than 20 years earlier, but at that time, it was not picked up
837 by others. The logarithmic timescale was later exploited by
838 researchers measuring uorescence relaxation following a
839 STF, as well (see Question 2 Sect. 1; e.g., Cser and Vass
840 2007). The logarithmic time scale distorts the time
841 dependence somewhat but, at the same time, allows the
842 visualization of considerably more kinetic features than is
843 possible on a linear time scale. This additional kinetic
844 detail makes it much easier to detect changes in the uo-
845 rescence kinetics. Fluorescence measurements shown on a
846 linear timescale are always dominated by the slower
847 changes (see Fig. 3a). A logarithmic timescale turns
848 exponential rise phases into sigmoidal rise phases, and we
849 must keep in mind that the sigmoidicity of the uorescence
850 rise cannot be derived on the basis of uorescence tran-
851 sients visualized on a logarithmic timescale.
852 Question 13. Direct or modulated uorescence?
853 It is possible to measure OJIP transients using a modulated
854 system (Schreiber 1986; Neubauer and Schreiber 1987;
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855 Schreiber and Neubauer 1987), and at the same time, it is
856 possible to make a quenching analysis (see Questions 2.3
857 and 15) using a PEA-type instrument (Schansker et al.
858 2006). However, modulated instruments are much better
859 suited for a quenching analysis, and PEA-type instruments
860 are the instruments of choice for a study of the OJIP
861 kinetics. Thus, we recommend that both must be used to
862 get a complete picture.
863 Question 14. What kind of additional information can
864 be obtained using uorescence imaging?
865 All the instruments, discussed thus far, integrate the signal
866 of the measured area. Fluorescence imaging permits the
867 study of spatial heterogeneities in the uorescence emis-
868 sion intensity within cells, leaves, or whole plants; heter-
869 ogeneities caused by a range of internal plant factors
870 (Gorbe and Calatayud 2012). It can also be used to average
871 and analyze the uorescence signal from much larger leaf
872 areas than classical methods would allow, and at the same
873 time, it allows the simultaneous measurement/screening of
874 many samples/mutants in, for example, a microwell plate
875 or of colonies grown on a Petri dish (see e.g., Niyogi et al.
876 1997; Serodio et al. 2012) or all the leaves of an rosette of
877 Arabidopsis. There are several commercial imaging
878 instruments on the market. It is a technique whose devel-
879 opment has kept pace with improvements in LED tech-
880 nology. For reliable imaging measurements, it is critical
881 that the whole sample area be illuminated homogeneously.
882 Several introductory texts and reviews have been published
883 on uorescence imaging (e.g., Buschmann et al. 2001;
884 Oxborough 2004; Lenk et al. 2007; Scholes and Rolfe
885 2009). Since it was not possible to image F
O
0
with the
886 imaging systems available in the late 1990s, Oxborough
887 and Baker (1997) derived an equation to estimate it:
F
O
0

F
O
F
V
F
M
?
F
O
F
M
0
:
889 889 This equation allows the calculation of the parameters
890 qP [=(F
M
0
- F
S
)/(F
M
0
- F
O
0
)] and F
V
0
/F
M
0
. The challenge
891 using uorescence imaging is to process all the data col-
892 lected in a scientically meaningful way. Meyer and Genty
893 (1998) analyzed their data making frequency distributions
894 of parameters of interest; we recommend that this method
895 is considered for future experiments.
896 Imaging can be used, e.g., to assess the dynamics and
897 heterogeneous behavior of stomatal opening/closure over a
898 leaf, a phenomenon also called stomatal patchiness. A
899 palette of false colors is used to cover the range of uo-
900 rescence intensities (normalized between 0 and 1),
901 assigning a color to each pixel of the image (Gorbe and
902 Calatayud 2012). Based on the image, different areas of the
903 leaf can be chosen, the associated uorescence data aver-
904 aged, uorescence parameters can be calculated, and sub-
905 sequently, the photosynthetic properties of the chosen area
906 can be studied.
907 Using uorescence imaging, it is easy to detect photo-
908 synthetic heterogeneities in a leaf (Meyer and Genty 1998)
909 and to follow how any stress affects the leaf in spatial
910 terms. In a popular early experiment, the imaging tech-
911 nique was used to show the gradual inltration of PSII
912 inhibiting herbicides in the leaf (e.g., Daley et al. 1989;
913 Lichtenthaler et al. 1997; Chaerle et al. 2003) or the effect
914 of reactive oxygen species (ROS)-inducing herbicides
915 (e.g., Hideg and Schreiber 2007). Spatial heterogeneities
916 that have been studied using uorescence imaging include
917 heterogeneities occurring during the following processes:
918 induction of photosynthesis (Genty and Meyer 1995; Daley
919 et al. 1989), the onset of senescence (Wingler et al. 2008),
920 chilling (Hogewoning and Harbinson 2004), the response
921 to drought (Woo et al. 2007), nutrient stress (Landi et al.
922 2013), ozone stress (Gielen et al. 2006; Guidi et al. 2007),
923 wounding (Quilliam et al. 2006), and during infection with
924 viruses (Balachandran et al. 1994) or fungi (Guidi et al.
925 2007). Several studies, using imaging to study Chl a uo-
926 rescence parameters under various conditions (high/low
927 ambient CO
2
concentration, high/low light intensity, etc.),
928 have yielded information on the relationship between leaf
929 structure and organization on the one hand and the
930 response to stress conditions on the other (Baker 2008;
931 Rohacek et al. 2008; Guidi and DeglInnocenti 2011;
932 Gorbe and Calatayud 2012).
933 Serodio et al. (2013) have introduced, a new application
934 of uorescence-imaging systems, which allows the rapid
935 generation of light-response curves (see Question 18)
936 simultaneously illuminating replicates of samples using
937 spatially separated beams of actinic light of different
938 intensities.
939 Question 15. What kind of information can be obtained
940 using the quenching analysis (see Question 2)?
941 In leaves exposed to a certain irradiance, the uorescence
942 intensity is affected by changes both in the redox state of
943 the ETC (particularly the redox state of Q
A
) and in the
944 uorescence yield due to light-induced changes in the
945 properties of the PSII antenna. A method called the
946 quenching analysis was developed to separate these two
947 types of process. In most cases, the quenching analysis is
948 used to describe the steady state, i.e., the stable photo-
949 synthetic activity, which is usually reached after approxi-
950 mately 510 min of illumination at a chosen actinic light
951 intensity.
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952 A protocol was developed (Schreiber et al. 1986; Fig. 4)
953 based among others on the work of Bradbury and Baker
954 (1981) in which the measurements are initiated by switching
955 on the measuring light to determine the F
O
value of a dark-
956 adapted sample. A saturating light pulse is then applied to
957 determine the F
M
. The measurement is continued switching
958 on an actinic light source to induce photosynthesis, until the
959 uorescence emission stabilizes at a level called F
S
. The F
M
0
960 is then determined by applying another strong pulse of light
961 followed some time later (e.g., 10 s) by turning off the
962 actinic light. Turning off, the actinic light will cause a quick,
963 partial, re-oxidation of the photosynthetic ETC. Within the
964 rst 100 ms of darkness, the PQ-pool will be largely re-
965 oxidized by forward electron transport toward PC
?
and
966 P700
?
, and a value close to F
O
0
can be measured. The F
O
0
967 level subsequently increases again due to non-photochemi-
968 cal reduction of the PQ-pool by NADPH and possibly Fd
red
969 (Mano et al. 1995; Gotoh et al. 2010; Guidi and DeglIn-
970 nocenti 2012). This so-called F
O
0
rise can be almost
971 completely suppressed by a short pulse of FR light (e.g., of
972 1 s duration) following the turning off of the actinic light.
973 The increase of the uorescence intensity from F
S
to F
M
0
is
974 related to a change in the redox state of the ETC, whereas the
975 difference between F
M
0
and the dark-adapted F
M
is then a
976 measure of the uorescence yield change, which in the case
977 of qE is associated with increased heat dissipation. In
978 quenching analysis terminology, this approach splits the
979 uorescence changes into a photochemical quenching
980 (redox related) and a non-photochemical quenching (uo-
981 rescence yield related) part. On turning off the actinic light,
982 the relaxation of the non-photochemical quenching, i.e., the
983 increase of F
M
0
to F
M
, can be followed and several con-
984 tributing processes can be resolved (Walters and Horton
985 1991; Rohacek 2010). Schreiber et al. (1986) introduced the
986 parameter qN = 1 - F
V
0
/F
V
to quantify changes in the non-
987 photochemical quenching. The parameter qN can range
988 between 0 and 1, and for its calculation, the F
O
0
value is
989 needed. In 1990, Bilger and Bjorkman (1990) introduced the
990 parameter NPQ = F
M
/F
M
0
- 1 which has as advantages
991 over the parameter qN that its range is not restricted (see
992 Question 21), and in addition, it is not necessary to know the
993 F
O
0
value. However, Holzwarth et al. (2013) evaluating the
994 parameter NPQ, concluded that in this treatment of the
995 uorescence data, the relationship between the quenching
996 parameter and the underlying processes becomes distorted,
997 especially when the time dependence of NPQ is considered.
998 For the analysis of the relaxation kinetics of the parameter
999 qNsemi-logarithmic plots of Log(qN) versus time are made.
1000 This linearizes the slowest component. Using linear
1001 regression, the decay half-time and amplitude of this com-
1002 ponent can be determined. This component (an exponential
1003 function) can then be subtracted fromthe original data, and a
1004 new semi-logarithmic plot can be made of the remaining
1005 qN. The procedure can then be repeated (e.g., Walters and
1006 Horton 1991; for a discussion of the theoretical basis of the
1007 resolution method, see Rohacek 2010).
1008 The least controversial of these kinetic processes is the
1009 process relaxing during the rst 100200 s of darkness, with
1010 a relaxation half-time of *30 s. In quenching analysis
1011 terms, this is called the qE or high-energy quenching; it
1012 depends on a low lumen pH and is affected by the XC
1013 (reviewed by Horton et al. 1996; Muller et al. 2001; Gilmore
1014 2004; Krause and Jahns 2004; Ballottari et al. 2012). How-
1015 ever, the exact mechanism of the induction of the qE and the
1016 exact components involved in this process are still a hotly
1017 debated issue (e.g., Caffari et al. 2011; Johnson et al. 2011;
1018 Miloslavina et al. 2011). Aset of mutants has been generated
1019 playing an important role in the study of the qE, in which
1020 different components and processes related to qE have been
1021 modied (Niyogi et al. 1998). The second process, the qT,
1022 with a half-time of 510 min has been assigned to state II to
1023 state I transitions (transfer of LHCII units from PSI to PSII)
1024 based on the observation that it was already induced at low
1025 light intensities (Demmig and Winter 1988) and on its
1026 possible sensitivity to the phosphatase inhibitor NaF (Horton
1027 and Hague 1988). Schansker et al. (2006) studying the
1028 kinetics of the saturating pulses showed that the main uo-
1029 rescence change occurring in this time interval in pea leaves
1030 is the regeneration of the IP phase suggesting that the qT
1031 reects the inactivation of the acceptor side of PSI (the
1032 inactivation of FNR). Other processes that have been asso-
1033 ciated with the qT are some slowly relaxing component(s) of
1034 qE (Lokstein et al. 1993; Joliot and Finazzi 2010) and light-
1035 dependent movements of chloroplasts (Cazzaniga et al.
1036 2013). In practice, there are several arguments making it
1037 doubtful that the qT is a reliable measure for state transi-
1038 tions. The slowest relaxation phase, the qI, which may last
1039 several hours can consist of several processes: photoinhi-
1040 bition of PSII and XC related changes (reviewed by Krause
1041 and Jahns 2004) and possibly also state II to state I transi-
1042 tions (Schansker et al. 2006) if a change in the JI amplitude is
1043 related to state transitions as suggested by Schreiber et al.
1044 (1995) for cyanobacteria. It should be noted that the rate with
1045 which these processes reverse in darkness is not necessarily
1046 the same in all photosynthetic organisms. For example, the
1047 regeneration of the IP phase parallels the qT phase in pea
1048 leaves (Schansker et al. 2006), and it is complete within
1049 15 min, whereas the same process in needles of Pinus
1050 halepensis takes 1 h (Schansker et al. 2008).
1051 Question 16. Why is far-red light used to determine
1052 the F
O
and F
O
0
values?
1053 For leaves, it is reasonable to assume that under most
1054 conditions, nearly all PSII RCs are in the open state (Q
A
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1055 oxidized) following dark adaptation. However, the
1056 assumption is not true for heat-stressed leaves (Ducruet
1057 1999; Toth et al. 2007b) and leaves that show a high rate of
1058 chlororespiration. Chlororespiration refers to the non-pho-
1059 tochemical reduction of the plastoquinone pool by reducing
1060 equivalents derived from Fd
red
or NADPH in the stroma
1061 (Bennoun 2002). Feild et al. (1998) showed a high chlo-
1062 rorespiratory in light acclimated sunower leaves follow-
1063 ing a light-to-dark transition leading to considerably higher
1064 F
O
0
values. This F
O
0
increase is due to a population of
1065 reduced Q
A
associated with a more reduced PQ pool. There
1066 is redox interaction between the PQ-pool and Q
A
leading to
1067 a redox-equilibrium (Diner 1977); for pea leaves, it was
1068 shown that a completely reduced PQ-pool (induced by
1069 anaerobiosis) is in equilibrium with reduced Q
A
in 20 % of
1070 the PSII RCs (Toth et al. 2007a).
1071 To assure maximum oxidation of the PQ pool, the leaf
1072 can be pre-illuminated with FR light. For this purpose, FR
1073 light in the 720735 nm range is normally used. FR light
1074 preferentially excites PSI and thereby causes an oxidation
1075 of the PQ pool. We note that FR light can induce charge
1076 separations in PSII (Pettai et al. 2005; Schansker and
1077 Strasser 2005). Pettai et al. (2005) demonstrated that FR
1078 light at 740 nm still induces a low level of oxygen evolu-
1079 tion even though the activity is three times less than that
1080 induced by FR light at 720 nm. In practice, FR light
1081 induces about 2.5 % of F
V
associated with Q
B
-
in 50 % of
1082 the RCs (Schansker and Strasser 2005). However, this
1083 observation is only of importance for direct uorescence
1084 measurements, since the effects induced by FR light are
1085 also induced by the measuring beam of a modulated uo-
1086 rescence instrument.
1087 A short FR pulse (*1 s, at *720735 nm) given to a
1088 light-adapted leaf has two main effects: (i) it re-oxidizes
1089 the PQ-pool within 100 ms and (ii) it suppresses the tran-
1090 sient F
O
0
increase, which is normally observed following a
1091 light-to-dark transition (Mano et al. 1995; Gotoh et al.
1092 2010; Guidi and DeglInnocenti 2012). It is related to non-
1093 photochemical reduction of the PQ-pool by NADPH or
1094 Fd
red
; this process is mediated by an enzyme complex
1095 called NADPH dehydrogenase (NDH) (Burrows et al.
1096 1998). The induction of the qE component of non-photo-
1097 chemical quenching leads to a quenching of the F
M
level
1098 and in many plant species to a quenching of the F
O
0
level as
1099 well (Bilger and Schreiber 1986; Bilger and Bjorkman
1100 1991; Noctor et al. 1991). This qE quenching relaxes
1101 quickly in darkness. To determine the associated F
O
0
1102 quenching accurately, the F
O
0
level must be determined
1103 immediately after turning off the actinic light. The non-
1104 photochemical reduction of the PQ-pool affects the F
O
0
1105 level as well, and this may complicate an accurate deter-
1106 mination of the extent of F
O
0
quenching. Since the non-
1107 photochemical reduction of the PQ-pool is a rather slow
1108 process peaking approx. 40 s after turning off the light
1109 (Burrows et al. 1998), and the maximum re-oxidation of
1110 the PQ-pool following lights off takes less than 100 ms
1111 (Ceppi 2010), the F
O
0
level can be determined quite
1112 accurately before the transient non-photochemical reduc-
1113 tion of the PQ-pool sets in. However, using *1 s of FR is
1114 the most straightforward approach to obtain an oxidized
1115 PQ pool.
1116 Question 17. How can the NPQ index be calculated
1117 when NPQ is formed in the dark?
1118 As noted in Question 16, a process called chlororespiration
1119 has been identied in higher plants (Bennoun 1982, 2002;
1120 Rumeau et al. 2007). Cyanobacteria, which are thought to
1121 be the ancestors of the chloroplast, lack mitochondria;
1122 instead they have a respiratory chain that shares the PQ-
1123 pool with the photosynthetic ETC (Vermaas 2001; Sch-
1124 metterer and Pils 2004; Hart et al. 2005). It allows the
1125 creation of a pH gradient over the thylakoid membrane in
1126 the dark, and this gradient is utilized to synthesize ATP. In
1127 the dark, the respiratory activity in cyanobacteria is con-
1128 siderably higher than in higher plants. In fact, chlorore-
1129 spiration in higher plants is seen as a rudiment of the
1130 original respiratory chain. Also in green algae, the respi-
1131 ratory chain is still quite active (see Beardall et al. 2003 for
1132 a discussion of this topic). Another group of organisms that
1133 have been shown to have a high chlororespiratory activity
1134 are some microalgae, including diatoms (e.g., Caron et al.
1135 1987). As a consequence, there is no complete relaxation of
1136 qE in the dark. XC activity in dark grown diatoms occurs
1137 as a result of the acidication of the thylakoid lumen due to
1138 this chlororespiratory activity (Jakob et al. 1999).
1139 One effect of this high chlororespiratory activity in
1140 diatoms is that the F
M
level of dark-adapted diatoms is
1141 lower than the F
M
0
observed under low actinic light (Cruz
1142 et al. 2010). This means that it is not possible to apply the
1143 commonly used NPQ equation:
NPQ
F
M
F
M
0
1; 1
1145 1145 since the calculated value would be negative [F
M
\F
M
0
].
1146 A practical solution for this problem is the determination of
1147 the light-response curve (see Question 18) and to replace
1148 F
M
by the maximum F
M
0
level measured (F
M
0
max
; Serodio
1149 et al. 2006) in Eq (1):
1150 So,
NPQ
F
0
Mmax
F
M
0
1: 2
1152 1152 In this way, NPQ values will always be positive and
1153 approach a minimum value close to zero under conditions
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1154 closely corresponding to a state with a very small trans-
1155 thylakoid proton gradient.
1156 Question 18. Can the time that is needed for a complete
1157 quenching analysis be shortened?
1158 To characterize the properties of parameters such as qP,
1159 U
PSII
[= (F
M
0
- F
S
0
)/F
M
0
] and NPQ, it is common practice
1160 to determine the light intensity dependence of these
1161 parameters (see e.g., Bilger and Bjorkman 1991; Gray et al.
1162 1996; Verhoeven et al. 1997). The classical approach is to
1163 illuminate the leaf at each light intensity, until steady state
1164 is reached (see Questions 2.3 and 10). This process can be
1165 quite time-consuming, especially if the uorescence
1166 quenching analysis is performed for eld experiments.
1167 To reduce the time needed for this type of measurement, a
1168 faster procedure was developed and called rapid light curves
1169 (RLCs) (White and Critchley 1999; Ralph and Gademann
1170 2005). RLCs can be used to study the physiological exi-
1171 bility of the photochemistry in response to rapid changes in
1172 irradiation (Guarini and Moritz 2009). Such changes occur
1173 frequently in natural environments. An RLC is a plot of the
1174 electron transport rate (ETR: U
PSII
9 PFD 9 0.5 9 leaf
1175 absorptivity coefcient) as a function of the actinic light
1176 intensity, which is applied for xed short-time periods (e.g.,
1177 10 s or 1 min). Here, PFDstands for photon ux density, and
1178 here, it is assumed that the PSI:PSII ratio is 1:1. However,
1179 this is only a rough approximation and the real ratio will
1180 differ between samples (see Question 26). For this type of
1181 analysis, two criteria are important: (1) the samples must be
1182 dark adapted, and (2) photosynthesis must be induced
1183 [activation of the CalvinBenson cycle enzymes that
1184 become inactive during incubation in darkness (see Ques-
1185 tion 6)] before the measurement sequence is started (White
1186 and Critchley 1999). Dark adaptation of the samples allows
1187 the determination of the reference F
O
and F
M
values needed
1188 for the calculation of qN and/or NPQ. If light-adapted
1189 samples are used for the experiments, for which reference
1190 F
O
and F
M
values are missing, then the effective quantum
1191 yield (U
PSII
) and ETRcan still be calculated, but not the non-
1192 photochemical quenching parameters, nor qP. In other
1193 words, the best protocol consists of a dark acclimation of the
1194 sample, a weak modulated beam and a saturating pulse to
1195 determine the reference F
O
and F
M
, respectively, and then a
1196 pre-illumination with a moderate light intensity (approx.
1197 50 % of the ambient light intensity applied for several
1198 minutes is appropriate for this purpose) after which the RLC
1199 protocol is applied (see Lichtenthaler et al. 2005).
1200 Examples of RLCs (Fig. 6a) illustrate the importance of
1201 the duration of light intervals. In addition to differences in
1202 the values determined for individual light intensities, there
1203 is also a difference in the shape of the curves (Fig. 6b). Pre-
1204 illumination at moderate light intensities ensures faster
1205 induction. Thus, in pre-illuminated samples, a 30-s interval
1206 is sufcient to obtain appropriate values and shapes of the
1207 curves that are comparable to those measured with 2-min
1208 intervals (Fig. 6c).
1209 RLCs have frequently been used in studies dealing with
1210 plant stress (reviewed in Brestic and Zivcak 2013). The
Fig. 6 Rapid light curves. a Example of RLCs (PAR vs. ETR) for
which the duration of light intervals (20, 30, 60, 120 s) had been
varied. Closed symbols represent the values measured after 30 min
dark acclimation (without pre-illumination), and open symbols
represent values measured following 30 min of dark acclimation
and 5 min of pre-illumination at a moderate light intensity (100 lmol
photons m
-2
s
-1
). b The ETR/ETRmax ratio (ETRmax represents the
maximum value for each curve) of measurements with light intervals
of 120 and 20 s. c ETR values of experiments without pre-
illumination (NO PI) and with 5 min of pre-illumination (5 min PI,
350 lmol photons m
-2
s
-1
). Measurements were made on Citrus
leaves using a Dual-PAM uorometer (Walz, Germany) (Brestic and
Zivcak, unpublished data)
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1211 value of the RLC approach increases if a second technique,
1212 e.g., 820 nm or gas exchange measurements, is applied
1213 simultaneously, or if uorescence-imaging measurements
1214 are also made.
1215 Question 19. What is the JIP test?
1216 The idea that the uorescence rise OJIP contains a lot of
1217 information on the photosynthetic system is already quite
1218 old. OJIP transients have been compared to a bar code for
1219 photosynthesis (Tyystjarvi et al. 1999) and extensive
1220 attempts to simulate OJIP transients have been made (see
1221 Lazar and Schansker (2009) for a review of these efforts). In
1222 1991, Strasser and Govindjee published an article on the
1223 recording of the full uorescence rise kinetics OJIP between
1224 40 ls and 1 s using a PEA instrument (see Strasser et al.
1225 1995 for details). Four years later, Strasser and Strasser
1226 (1995) proposed a method to analyze these OJIP transients
1227 that was centered on the J-step [observed after 23 ms of
1228 strong illumination and equivalent to the I
1
step of Schreiber
1229 (1986)], which they called the JIP test (see Fig. 7).
1230 The theoretical basis of the JIP test has been described
1231 in detail by Strasser et al. (2004). In the JIP test, OJIP
1232 transients are used to make a ux analysis, i.e., an analysis
1233 of the fate of photons absorbed by the PSII antennae
1234 (trapping, forward electron transport beyond Q
A
and dis-
1235 sipation as heat). In the JIP test, the J-step is taken as the
1236 border between single and multiple turnovers. If we dene
1237 multiple turnovers here as events related to electron
1238 transport beyond PSII, then this claim still remains valid.
1239 The JIP test depends strongly on the assumption that the
1240 F
O
-to-F
M
rise reects the reduction of Q
A
. The concept is
1241 internally consistent but the theoretical foundation of the
1242 interpretation of the parameters disappears the moment that
1243 this assumption turns out to be wrong (see Schansker et al.
1244 2011, 2014 for a discussion of this point). An alternative
1245 approach to the interpretation of the OJIP transients is a
1246 classical physiological characterization of the various fea-
1247 tures of the uorescence rise.
1248 In the JIP test, it is assumed that the relative position of
1249 the J-step between F
O
and F
M
(i.e., V
J
, giving rise to the
1250 JIP-parameter 1 - V
J
or W
O
) gives information on photo-
1251 synthetic electron transport beyond Q
A
(e.g., Strasser et al.
1252 1995, 2004). A physiological characterization of this fea-
1253 ture, on the other hand, suggests that the parameter V
J
1254 depends on the redox state of the PQ-pool in darkness
1255 (Toth et al. 2007a) and, under certain stress conditions,
1256 may also be affected by other factors, possibly the extent of
1257 stacking of the thylakoid membranes. In this case, electron
1258 transport beyond Q
A
means a slowdown of the re-oxidation
1259 of Q
A
-
as the PQ-pool becomes more reduced, and fewer
1260 PQ molecules are bound to the Q
B
-site. Changes in W
O
1261 may certainly point to stress.
1262 In the JIP test, the parameters F
O
and F
M
were suggested
1263 to be a measure for the absorption ux (i.e., the number of
1264 photons absorbed per unit of time) per cross section
1265 (Strasser et al. 1995, 2004). With respect to this interpre-
1266 tation, it may be noted that a characterization of the
1267 changes in the F
O
and F
M
levels as a function of the Chl
1268 content of leaves showed that they are nearly insensitive to
1269 changes in the leaf chlorophyll content as long as the
1270 antenna sizes of the RCs remain unaffected (Dinc et al.
1271 2012). However, we note that this observation probably
1272 does not apply to dilute algal and thylakoid suspensions.
1273 Malkin (1966) and Murata et al. (1966) showed that the
1274 complementary area between the uorescence transient and
1275 F
M
in the presence of DCMU is proportional to population
1276 of reduced Q
A
molecules. In the JIP test, this principle is
1277 extended to the situation in the absence if DCMU, where
1278 the area between the uorescence transient and F
J
is
1279 assumed to equate one charge separation in all RCs, i.e.,
1280 one electron transported, to which the total area above the
1281 OJIP transient can be normalized (see e.g., Strasser et al.
1282 2004). Schansker et al. (2011, 2014) support and explain
1283 the relationship between the area above the OJIP transients
1284 (see Fig. 7) and the number of electrons that must be
1285 transported through the ETC before F
M
is reached.
1286 In the JIP test, it is assumed that the slope taken between
1287 F
O
and F
150 ls
is sensitive to a phenomenon called con-
1288 nectivity, i.e., the energy transfer between the antennae of
1289 several PSII RCs, whereas the slope taken between F
O
and
1290 F
300 ls
is insensitive to connectivity (Strasser and Stirbet
1291 2001; and see Stirbet 2013 for a more in-depth discussion
1292 of connectivity in the absence of PSII inhibitors like
1293 DCMU).
Fig. 7 Time points and parameters used in the JIP test. On the left
hand side, the unnormalized F scale associated with the complemen-
tary Area and on the right hand side, the V scale double normalized
between O and P associated with the normalized area Sm (Goltsev,
unpublished data)
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1294 The performance index [PI(ABS)] was introduced as an
1295 attempt to catch three different aspects of the photosyn-
1296 thetic activity of PSII in a single parameter (see Clark et al.
1297 2000 for an early application of this parameter). PI(ABS) is
1298 the product of a parameter sensitive to the effective
1299 antenna size, a parameter based on the primary quantum
1300 yield of PSII and a parameter sensitive to changes in the
1301 relative position of F
J
. It is dened as:
PI(ABS)
F
V
F
M
V
J
4F
270 ls
F
O
F
M
F
O
F
V
F
M
1
F
V
F
M
1 V
J
V
J
1303 1303 with V
J
= (F
J
- F
O
)/F
M
- F
O
). It is another JIP test
1304 parameter that has been shown to correlate with other stress
1305 parameters under a series of conditions (e.g., Clark et al.
1306 2000; Misra et al. 2001a, b; Oukarroum et al. 2006).
1307 Physiological studies have further shown that the IP phase
1308 of the uorescence rise is related to electron transport
1309 through PSI (Kautsky et al. 1960; Munday and Govindjee
1310 1969; Schansker et al. 2005) and that the (relative)
1311 amplitude of the IP phase is linked to the PSI content of the
1312 leaf (Oukarroum et al. 2009; Ceppi et al. 2012). The JIP
1313 test approach remains a good and fast way to screen a large
1314 number of samples (Kalaji et al. 2011a, b). However, once
1315 parameters that correlate with certain features of a stress
1316 have been identied, it should not be blindly assumed that
1317 the interpretation of these parameters as given by the JIP
1318 test is correct (see also Stirbet and Govindjee 2011 for a
1319 discussion of this topic). In addition, it should be kept in
1320 mind that the JIP test depends strongly on normalizations
1321 which are very sensitive to the correctness of the deter-
1322 mined F
O
and F
M
values. For example, in the case of heat
1323 stress, it is not easy to determine the F
O
and F
M
values
1324 correctly (see Toth et al. 2007b).
1325 Question 20. What kind of values may one expect
1326 for particular uorescence parameters?
1327 The F
V
/F
M
values of plant species average approximately
1328 0.830.84 in C3 plants under optimal conditions (Bjorkman
1329 and Demmig 1987; Pfundel 1998) and 0.78 in C4 plants
1330 (Pfundel 1998). Somewhat higher values have been
1331 described in certain broadleaved species. Lower values, on
1332 the other hand, are common in algae and lichens (see Trissl
1333 and Wilhelm 1993 for a discussion of these values). Stress
1334 conditions (e.g., photoinhibition) can signicantly reduce
1335 these values (e.g., Bjorkman and Demmig 1987; Van Wijk
1336 and Krause 1991; Tyystjarvi and Aro 1996).
1337 Photochemical quenching qP, non-photochemical
1338 quenching dened as qN [= 1 - (F
M
0
- F
O
0
)/(F
M
- F
O
)],
1339 and the PSII operating efciency in the light (U
PSII
) can
1340 vary between 0 and 1 (see Question 14 for denitions of qP
1341 and U
PSII
). The theoretical range for the values of the non-
1342 photochemical quenching parameter NPQ [= F
M
/F
M
0
- 1]
1343 is from zero to innity, but in most cases, it gives values
1344 between 0 and approximately 10. However, NPQ values
1345 higher than 10 have been reported in bryophytes from sun-
1346 exposed habitats (Marschall and Proctor 2004; see Bus-
1347 chmann 1999 for a discussion and comparison of qN and
1348 NPQ). High U
PSII
values indicate that a large proportion of
1349 the light absorbed by the chlorophylls of the PSII antenna
1350 is converted into photochemical energy. At its upper limit,
1351 U
PSII
could reach a value of 1, which would mean that all
1352 absorbed energy is used for stable charge separations in
1353 PSIIs. From a practical point of view, this cannot be the
1354 case, due to the fundamental inefciency of PSII (triplet
1355 formation, a small probability of uorescence, and heat
1356 emission on each transfer of excitation energy between
1357 chlorophylls), and the contribution of uorescence emitted
1358 by PSI has also an effect on the calculation (see Question
1359 3). Therefore, U
PSII
can vary between zero and the F
V
/F
M
1360 value, which in C3 plants is about 0.830.85, C4 plants
1361 around 0.78 and in algae often below 0.7 (Pfundel 1998;
1362 Trissl and Wilhelm 1993). qP values near zero indicate that
1363 most of the PSII RCs are closed, and their Q
A
is in the
1364 reduced state. Values near 1 indicate that Q
A
is in the
1365 oxidized state, and almost all of the PSII centers are open
1366 for photochemistry. The non-photochemical quenching
1367 coefcients qN and NPQ are assumed to be zero in the
1368 dark-adapted state, because then F
V
0
= F
V
and F
M
0
= F
M
.
1369 However, in some cases, positive values of these coef-
1370 cients can also occur in darkness (see Question 17).
1371 In higher plants, the induction kinetics of non-photo-
1372 chemical quenching triggered by high light usually have a
1373 typical time dependence, increasing during the rst minute
1374 of illumination due to initiation of electron transport and
1375 DpH formation preceding the activation of ATP synthase
1376 (e.g., Nilkens et al. 2010) and decrease again once the
1377 CalvinBenson cycle is activated. This quenching is sen-
1378 sitive to the balance between the electron transport rate and
1379 its associated proton transfer toward the thylakoid lumen
1380 on the one hand and the rate of ATP synthesis and the
1381 associated release of protons from the thylakoid lumen on
1382 the other hand. This form of quenching (corresponding to
1383 qE quenching, see Question 15) relaxes quickly as soon as
1384 electron transport stops, e.g., as soon as the light is turned
1385 off (see e.g., Nilkens et al. 2010). Other processes con-
1386 tributing to NPQ have slower induction kinetics (see
1387 Questions 2.3 and 15) whose induction (e.g., photoinhibi-
1388 tion) depends as well on light intensity. Higher non-pho-
1389 tochemical quenching values related to higher values of qE
1390 under steady state conditions suggest a stronger imbalance
1391 between photosynthetic electron transport and the utiliza-
1392 tion of NADPH (reected by lower qP values) (see e.g.,
1393 Walters and Horton 1993). Under continuous and/or
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1394 extreme stress, non-photochemical quenching can attain
1395 low values. This may in part be due to a loss of RCs.
1396 Photoinhibited PSII RCs lose their variable uorescence,
1397 and as a consequence, this variable uorescence can then
1398 no longer be quenched, which means less NPQ (Schansker
1399 and Van Rensen 1999). Low values may also be caused by
1400 decreased rates of linear electron transport generating a
1401 smaller transthylakoid proton gradient or to an increased
1402 permeability of the membrane due to lipid peroxidation
1403 caused by oxygen radicals, which will also reduce the build
1404 up of a DpH over the membrane.
1405 Deviations from the NPQ induction kinetics have been
1406 described in some green algae, where the NPQ induction
1407 capacity varies strongly depending on the species (see e.g.,
1408 Bonente et al. 2008). For example, in Ulva laetevirens,
1409 NPQ was induced with an early peak within the rst minute
1410 of exposure to high light, followed by a decrease and a
1411 subsequent rise (Bonente et al. 2008).
1412 Question 21. Which assumptions are made
1413 when interpreting uorescence transient
1414 measurements?
1415 Both the quenching analysis and the JIP test (see Questions
1416 15 and 19 for a discussion) are based on assumptions that
1417 were commonly made in the 1990s (e.g., van Kooten and
1418 Snel 1990 for the quenching analysis, Strasser 1996 for the
1419 JIP test and see also Stirbet and Govindjee 2011 for a list of
1420 assumptions). The most important assumption is that the
1421 uorescence increase from F
O
to F
M
reects mainly the
1422 reduction of Q
A
. This idea was rst put forward by Duy-
1423 sens and Sweers (1963). However, this assumption was
1424 challenged almost from the beginning (see e.g., Delosme
1425 1967). Delosme (1967) proposed the existence of two
1426 processes determining the uorescence rise. His suggestion
1427 that the redox state of the PQ-pool could play a role
1428 (Delosme 1971) led to the idea that the Q
B
-site occupancy
1429 state was the second factor (see Samson et al. 1999); an
1430 idea that was extended further by Schansker et al. (2011)
1431 who suggested that the Q
B
-site occupancy state controlled
1432 the re-oxidation rate of Q
A
-
and who proposed on the basis
1433 of this idea that in the presence of Q
A
-
further excitations
1434 could induce conformational changes in the PSII RCs
1435 which would then cause an increase of the uorescence
1436 yield. Considering the occupancy state idea, Schreiber
1437 (2002) proposed that the thermal phase might be explained
1438 by a reduction of the inactive branch of PSII. Vredenberg
1439 and co-workers (Vredenberg 2000; Vredenberg et al. 2006)
1440 developed another interpretation model, in which, in
1441 addition to Q
A
-
, the IP phase is determined by the electric
1442 eld, and JI rise reects an inactivation of PSII RCs
1443 (associated with proton transport over the membrane) in
1444 which Pheo
-
can accumulate. These alternative interpre-
1445 tations were challenged by Stirbet and Govindjee (2012).
1446 The rst assumption that the F
O
-to-F
M
rise is a reection of
1447 the reduction of Q
A
implies that it should always be pos-
1448 sible to reach F
M,
since all Q
A
can be reduced if the light
1449 intensity is high enough (i.e., when the excitation rate is
1450 much higher than re-oxidation rate of Q
A
-
by forward
1451 electron transport and/or the exchange of PQH
2
for PQ at
1452 the Q
B
-site). However, Schreiber (1986), Samson and
1453 Bruce (1996) and Schansker et al. (2006, 2008) showed in
1454 several ways that this is not the case.
1455 A second, related, assumption is that there are no
1456 changes in non-photochemical quenching during a satu-
1457 rating pulse. Finally, a third assumption is that the
1458 parameters F
V
/F
M
and UPSII are measures of the PSII
1459 quantum yield and that UPSII can be used to calculate the
1460 photosynthetic electron transport rate. For UPSII, this
1461 assumption has been partially veried experimentally,
1462 showing under several conditions a linear correlation
1463 between the calculated photosynthetic electron transport
1464 rate and the CO
2
assimilation rate (Genty et al. 1989; Krall
1465 and Edwards 1992 and see Questions 29 and 30). We note
1466 that the meaning of the parameter F
V
/F
M
has not been
1467 derived experimentally but is based on an analysis of so-
1468 called competitive rate equations (uorescence emission
1469 competes with other processes like heat emission and
1470 photosynthesis) for the F
O
and F
M
states (Kitajima and
1471 Butler 1975; Kramer et al. 2004). This analysis is correct as
1472 long as the uorescence rise between F
O
and F
M
is
1473 determined by the reduction of Q
A
only (see Schansker
1474 et al. 2014 for a discussion of this point).
1475 Question 22. Are there naturally occurring uorescence
1476 quenchers other than Q
A
?
1477 Another uorescence quencher that has been described
1478 extensively is P680
?
(Butler 1972; Zankel 1973; Shinkarev
1479 and Govindjee 1993; Steffen et al. 2005). The short life-
1480 time of P680
?
keeps the population of this quencher low
1481 under most conditions. Simulation work has shown that
1482 under high light conditions, the highest concentration
1483 should occur around the J-step (Lazar 2003), which was
1484 supported by experimental observations (Schansker et al.
1485 2011). However, P680
?
quenching does not affect the F
O
1486 and F
M
levels. Oxidized PQ molecules can also quench
1487 uorescence, but only in isolated thylakoids and in PSII-
1488 enriched membranes (Vernotte et al. 1979; Kurreck et al.
1489 2000; Toth et al. 2005a) and not in leaves (Toth et al.
1490 2005a). Other quenchers such as Car
?
and Chl
?
have been
1491 proposed and shown to play a role at temperatures below
1492 100 K (Schweitzer and Brudvig 1997) in the case of Chl
Z
?
;
1493 an accessory chlorophyll molecule in the RC of PSII or to
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1494 have a very short lifetime at room temperature (Steffen
1495 et al. 2001) in the case of Car
?
. Neither of these quenchers
1496 seems to play a role in the uorescence measurements
1497 discussed in this paper.
1498 Question 23. What is the difference
1499 between uorescence emission spectra recorded at 77 K
1500 and those recorded at room temperature?
1501 In Question 2 Sect. 4, measurements of 77 K uorescence
1502 emission spectra were introduced as a method to study PSII
1503 and PSI antennae. The recording of uorescence emission
1504 spectra is much easier at room temperature. In this case,
1505 one dominant peak at *684 nm is recorded, which is
1506 attributed principally to uorescence emission by the PSII-
1507 core complex (including the core antennae CP47 and
1508 CP43) and further a shoulder at 710740 nm corresponding
1509 to several uorescence emission sourcesparticularly PSI-
1510 LHCI and several minor PSII bands (Fig. 8) (Franck et al.
1511 2005; Krausz et al. 2005; Pancaldi et al. 2002). When the
1512 temperature is lowered, the 684 nm band is replaced by
1513 two bands, peaking at 685 and 695 nm, respectively; bands
1514 that in rst instance were shown to be associated with the
1515 PSII core (Gasanov et al. 1979; Rijgersberg et al. 1979).
1516 The 695 nm band is due to uorescence emission from
1517 CP47, whereas the 685 nm has been associated with uo-
1518 rescence emission by CP43 [(Nakatani et al. 1984; for
1519 spectroscopic analyses of CP47 and CP43: see Alfonso
1520 et al. 1994 (for both); van Dorssen et al. 1987 (CP47);
1521 Groot et al. 1999 (CP43)]. Srivastava et al. (1999) showed
1522 with an experiment on greening of peas how the 695 nm
Fig. 8 Examples of applications of room temperature (RT) uores-
cence emission spectra. a, b RT spectra of two developmental stages
of chloroplasts of the fruit of Arum italicum. In its early stage of
development (ivory stage), the fruit contains a rudimentary thylakoid
system in amyloplasts which upon maturation are converted to
chloroplasts (green stage; see Bonora et al. 2000). A difference
spectrum (normalized green stagenormalized ivory stage) b shows
that a distinctive trait of the amyloplast-to-chloroplast transition is the
gain in emission at around 691 nm, roughly corresponding to a PSII-
core contribution. An in-depth analysis of spectra in this system
showed that the F695/F680 uorescence ratio undergoes changes
parallel to F
V
/F
M
, assembly of LHCII-PSII supercomplexes, and
carbon xation (Ferroni et al. 2013). c, d RT spectra to improve the
description of chloroplast responses to stress. In the example, spectra
were recorded from leaves of the aquatic plant Trapa natans, which
were treated or not with manganese. In this species, acclimation to
manganese includes an accumulation of LHCII in the leaf chloro-
plasts (Baldisserotto et al. 2013). Increased RT emission at long
wavelength, as shown in the difference spectrum (d), points to the
occurrence in vivo of uncoupled aggregates of LHCII which
contribute uorescence at around 700 nm (Ferroni and Pancaldi,
unpublished data)
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1523 band increases in intensity as the PSII antenna size
1524 increases. In other words, despite CP47 being the source of
1525 the 695 nm emission, it is sensitive to the number of LHCII
1526 subunits bound to PSII. The relationship between the
1527 antenna size of PSII and the amplitude of the 695 nm band
1528 is further strengthened by the observation that chloroplast
1529 samples frozen in the presence of a DpH show a quenching
1530 of the 695 nm band (Krause et al. 1983). Based on a
1531 comparative study of photosynthetic mutants of Chla-
1532 mydomonas reinhardtii, a relationship between LHCII-PSII
1533 association and emission intensity at *695 nm has also
1534 been proposed at room temperature (Ferroni et al. 2011).
1535 To detect uorescence emitted by LHCII itself as an
1536 individual peak at 680 nm, it is necessary to freeze the
1537 sample further to 4 K (see Govindjee 1995). However, a
1538 more or less distinct shoulder at 680 nm is often reported
1539 also at 77 K and attributed to the free LHCII trimers not
1540 linked with PSII in a stable association (Hemelrijk et al.
1541 1992; Siffel and Braunova 1999; van der Weij-de Wit et al.
1542 2007; Pantaleoni et al. 2009; Ferroni et al. 2013). At room
1543 temperature, the emission region around 680 nm, never
1544 visible as an individual peak in the spectrum, was also
1545 assigned to a contribution by free LHCII (Ferroni et al.
1546 2011). Strasser and Butler (1976) showed that the strong
1547 band at 730 nm at 77 K is in part caused by energy transfer
1548 from PSII to PSI. Weis (1985) demonstrated that the
1549 absorption of PSII uorescence emission by PSI can be
1550 reduced considerably using leaf powder instead of whole
1551 leaf fragments. When using liquid samples, such as mic-
1552 roalgae suspensions or isolated thylakoids, the PSI re-
1553 absorption of emitted light can be reduced by an adequate
1554 dilution of the sample. The re-absorption phenomenon also
1555 affects room temperature spectra, resulting in a relative
1556 increase in the emission at 710740 nm and in a red shift of
1557 PSII emission (Franck et al. 2002).
1558 Room temperature uorescence emission spectra are not
1559 frequently used for photosynthesis studies, because the
1560 spectral components are not as well characterized as the
1561 77 K spectra are (Franck et al. 2002; Ferroni et al. 2011).
1562 However, methods have been developed to resolve at room
1563 temperature the contribution of PSII and PSI to Chl
1564 a uorescence under F
O
, F
M
, and steady state conditions
1565 (F
t
) (Franck et al. 2002, 2005). Figure 8 gives examples of
1566 two such applications. Room temperature uorescence
1567 spectra have also been used to evaluate the response of
1568 photosynthetic organisms (microalgae and in higher plants)
1569 to some environmental stresses (Romanowska-Duda et al.
1570 2005, 2010; Ferroni et al. 2007; Baldisserotto et al. 2010,
1571 2012; Burling et al. 2011; Hunsche et al. 2011). Finally,
1572 such spectra have been used as well to characterize
1573 developmental aspects of the photosynthetic membrane
1574 (Pancaldi et al. 2002; Baldisserotto et al. 2005; Ferroni
1575 et al. 2009, 2013) and, as discussed in Question 25, to
1576 estimate leaf chlorophyll content.
1577 Question 24. Are the uorescence rise kinetics sensitive
1578 to the chlorophyll content of the leaf?
1579 For dilute solutions of chlorophyll molecules, the measured
1580 uorescence intensity is proportional to the quantum yield
1581 of uorescence multiplied by the number of photons
1582 absorbed and the chlorophyll concentration (Lakowicz
1583 2009). On this basis, one would expect that the uores-
1584 cence intensity emitted by a leaf depends on the chloro-
1585 phyll content of that leaf. However, as described under
1586 Question 4, the leaf is complex in optical terms, and it is
1587 difcult to predict if this physical law is really critical in
1588 determining the relationship between the chlorophyll con-
1589 tent of the leaf and the uorescence emission. Several
1590 experimental studies have addressed this question. Hsu and
1591 Leu (2003) showed that two leaves placed on top of each
1592 other emitted more Chl a uorescence than a single leaf.
1593 However, this is a quite articial construct, and it can easily
1594 be shown that the outcome of the experiment strongly
1595 depends on the way the leaves were oriented (e.g., both
1596 adaxial sides up, or adaxial side up for the top leaf and the
1597 abaxial side for the bottom leaf) (Ceppi and Schansker,
1598 unpublished observations, 2008). Susila et al. (2004)
1599 attempted to show an effect of chlorophyll content using
1600 thylakoid suspensions differing in their chlorophyll con-
1601 tent. Thylakoid suspensions are homogeneous in their
1602 properties, whereas under natural conditions, a change in
1603 the chlorophyll content will be accompanied by an adap-
1604 tation (change in antenna sizes and/or changes in PSI:PSII
1605 ratio) of the individual chloroplasts inside the leaf to their
1606 new light environment (see Question 4). To address the
1607 effect of changes in the chlorophyll content of a leaf on the
1608 measured uorescence properties, it is important to nd a
1609 natural system in which the leaves can acclimate to the
1610 effects of the changing chlorophyll content. Sugar beet
1611 plants grown hydroponically in the absence of magnesium
1612 or low sulfate concentrations show a gradual loss of
1613 chlorophyll; the activity of the remaining ETCs remains
1614 largely unaffected, and there were no overall changes in the
1615 antenna size (effect on Chl a/b ratio was small). Under
1616 these conditions, an up to vefold decrease in the chloro-
1617 phyll content left the F
O
and F
M
values unchanged and had
1618 only a marginal effect on the uorescence rise kinetics
1619 (Dinc et al. 2012). On the other hand, changes in the PSII
1620 antenna size did have an effect on the F
M
-intensity (Dinc
1621 et al. 2012). In conclusion, there is little indication that a
1622 stress-induced Chl loss in leaves would complicate the
1623 interpretation of Chl a uorescence measurements.
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1624 Question 25. Can the leaf chlorophyll content be
1625 measured using uorescence?
1626 Chlorophyll uorescence emission spectra can be used to
1627 determine the chlorophyll content of green plants (Buschmann
1628 2007). The ratio between chlorophyll uorescence at 735 nm
1629 and that at 700 nm (F735/F700) is linearly proportional to
1630 chlorophyll content (Gitelson et al. 1999). Conversely, as dis-
1631 cussed in Question 24, the F
M
and F
O
values are not related to
1632 the chlorophyll content in leaves (Dinc et al. 2012). It may also
1633 be noted that there are simple chlorophyll meters on the market
1634 (CL-01, Hansatech Instruments, UK; SPAD meter, Minolta,
1635 Japan; CCM-200, Opti-Sciences, USA) that can be used to
1636 follow changes in the leaf chlorophyll content (see e.g., Cassol
1637 et al. 2008; Dinc et al. 2012). These measurements can then be
1638 calibrated against measurements of the chlorophyll extracted
1639 from leaf areas measured before with the chlorophyll meter
1640 (see e.g., Dinc et al. 2012). Chl measurements on dark-adapted
1641 leaves seem to give more reproducible results than measure-
1642 ments made on light-adapted leaves (Ceppi and Schansker,
1643 unpublished data, 2008). If the chlorophyll meter is used over
1644 the day on the same leaf, the readings change (Mishra,
1645 unpublished data, 2010), e.g., due to chloroplast movements,
1646 which change the absorbance properties of the leaf (see Wada
1647 2013 for a reviewon chloroplast movements). Chloroplasts are
1648 known to re-arrange themselves inside the cell in response to
1649 the ambient blue light intensity, adapting the absorbance
1650 properties of the leaf to the circumstances (Sakai et al. 2001;
1651 Kasahara et al. 2002). This does not only affect chlorophyll
1652 meter measurements, but also normal uorescence measure-
1653 ments (Brugnoli and Bjorkman 1992).
1654 In practice, values measured using a Chl meter are often
1655 used as indicators for relative Chl changes. In that case, we
1656 assume that the measured values are a linear function of the
1657 leaf chlorophyll content between zero and the value mea-
1658 sured on control leaves. However, in that case, it is important
1659 to test the validity of this assumption for each plant species
1660 and for each stress studied (Mishra, unpublished data, 2013).
1661 Question 26. Is it possible to compare different leaves?
1662 It is easy to take randomly two leaves from two plants of the
1663 same species and to make a uorescence measurement. But
1664 is it truly possible to compare these two measurements? It is
1665 likely that a difference in maximum uorescence amplitude
1666 will be observed. Especially, when studying OJIP transients,
1667 the kinetics are often more interesting than the absolute
1668 amplitude, and in that case, the difference in the uorescence
1669 amplitude is eliminated by double normalization between
1670 F
O
and F
M
. Arithmetically, this is done in the following
1671 way: (F
t
- F
O
)/(F
M
- F
O
). The effect of this calculation is
1672 to rescale each uorescence value in a range going from 0
1673 (corresponding to F
O
) to 1 (corresponding to F
M
). For a
1674 comparison of the kinetics of the individual rise phases of
1675 the OJIP transient, the same approach can be used. To
1676 compare the kinetics of the OJ-rise, the measured transient
1677 can be double normalized between O and J [i.e., (F
t
- F
O
)/
1678 (F
J
- F
O
)]. In terms of nomenclature, double normaliza-
1679 tions turn F values into so-called V values, like V
J
, which is
1680 the double normalized F
J
value (see Strasser et al. 2004).
1681 An important source of variability between leaves is the
1682 development of stress symptoms. A common stress-related
1683 effect is chlorosis, and it has been argued that a change in the
1684 chlorophyll content of the leaf has an impact on the uores-
1685 cence kinetics and thereby invalidates the analysis (Hsu and
1686 Leu 2003; Susila et al. 2004) but as discussed in Question 24,
1687 this is not the case as long as chloroplasts can adapt to their
1688 new light environment. In addition, if the development of the
1689 stress effects is followed over time, the gradually changing
1690 uorescence properties will help the interpretation of the data.
1691 A comparison of leaf uorescence measurements on
1692 stressed and unstressed plants in the eld is hampered by the
1693 fact that such leaves are often acclimated to completely dif-
1694 ferent light environments. It is important to realize that growth
1695 light intensity affects the stoichiometries and composition of
1696 many components of the photosynthetic membrane like the
1697 PSII to PSI ratio, the LHCII to PSII ratio, and the amount of
1698 PSII-LHCII supercomplexes (e.g., Leong and Anderson
1699 1984a, b; Walters and Horton 1994; Dietzel et al. 2008;
1700 Wientjes et al. 2013). Therefore, it is of fundamental impor-
1701 tance that the light environment (full sunlight, shade, deep
1702 shade) of leaves/plants to be compared has been adequately
1703 analyzed before the effect of a certain stress is addressed by
1704 uorimetric techniques. Several papers illustrate this, e.g.,
1705 stressed and unstressed plants were compared by van Heerden
1706 et al. (2007), whereas Zubek et al. (2009) compared leaves of
1707 plants with and without mycorrhiza, both ascribing the
1708 observed difference in the initial slope of the measured OJIP
1709 transients toaneffect onthe oxygenevolvingcomplexof PSII.
1710 An alternative and more likely explanationa difference in
1711 the effective antenna size between the samples due to differ-
1712 ences in the growth light conditionswas not considered.
1713 In summary, comparing leaves that develop under sim-
1714 ilar light conditions is relatively easy; however, comparing
1715 leaves that were growing under different light regimes is
1716 fraught with complications and should be avoided.
1717 Question 27. Can measurements made with different
1718 instruments during a large-scale eld survey be
1719 compared in absolute terms?
1720 It is important to be aware that the use of different
1721 instruments, even from the same company and the same
1722 type, may yield different results in absolute terms. The
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1723 light source used for saturating pulses of modulated
1724 instruments may age over time reducing its light intensity.
1725 The strength of the red LEDs of HandyPEAs often differs
1726 between instruments. When comparing measurements
1727 made with different types of instruments, differences may
1728 also be due to the specic geometry of the measuring cell
1729 or to the use of light sources emitting at different wave-
1730 lengths. It is possible to reduce these differences by
1731 determining light intensity dependence of the parameters of
1732 interest and using these data to change settings in order to
1733 obtain comparable results. Differences in wavelengths of
1734 the exciting light may be impossible to correct for. Green
1735 light for example has been shown to probe deeper in the
1736 leaves than red light; blue light is even more efciently
1737 absorbed than red light (Terashima et al. 2009).
1738 An example of the phenomenon, described above, is a study
1739 in which the same leaves were measured with different
1740 HandyPEA instruments (Bussotti et al. 2011a) calibrated with
1741 identical settings (lamp intensity = 3,000 lmol photons
1742 m
-2
s
-1
, time = 1 s, gain = 1). Both original and normalized
1743 transient curves were compared. Original curves differed
1744 consistently (both the extreme values of F
O
and F
M
showed a
1745 large range of variability), but the differences decreased con-
1746 sistently after normalization (double normalization betweenF
O
1747 and F
M
see Question 26 for a denition). The parameter F
O
/
1748 F
M
(parameter which is sensitive to changes in heat dissipation
1749 in the PSII antenna), as well as the normalized steps of OJIP
1750 transientsJ and I (uorescence intensities at 23 and 30 ms,
1751 respectively)showed very little variability when comparing
1752 the measurements of the different instruments with a coef-
1753 cient of variation (CV = SD/Mean) ranging from 3 to 5 %.
1754 The parameter PIabs, which consists of the product of a
1755 parameter sensitive to the effective antenna size, a parameter
1756 based on the maximumquantumyield of PSII, and a parameter
1757 sensitive to changes in the relative position of F
J
(see Question
1758 19) showed a very high variability among instruments (PIabs
1759 showed a CV = 30 %; Bussotti et al. 2011a). The high
1760 intrinsic variability of PIabs between instruments is due to the
1761 fact that this parameter is sensitive to the initial slope of the
1762 uorescence rise and the relative position of the J-step, two
1763 factors that are both relatively sensitive to the light intensity of
1764 the beam. This high intrinsic variability makes the PIabs less
1765 useful for large, multi-instrument surveys.
1766 In conclusion, in the case of small-scale experiments, it
1767 is always preferable to use the same instrument for all the
1768 measurements of an experiment.
1769 Question 28. How should a sampling campaign be
1770 organized for an ecosystem?
1771 Large-scale surveys should be carried out using a robust
1772 sampling design. Criteria and examples of such designs can
1773 be found in many statistical manuals and textbooks (see
1774 Elzinga et al. 2001). Here, we discuss some specic issues
1775 related to the assessment of uorescence parameters.
1776 Two problems widely discussed in the context of forest
1777 health monitoring (Luyssaert et al. 2002) and other eco-
1778 systems (Tuba et al. 2010) are intercalibration and har-
1779 monization. Here, intercalibration refers to procedures
1780 aimed at reducing the differences between instruments
1781 discussed in Question 27, and harmonization refers to
1782 the sampling strategy. The main issues are the variability of
1783 the leaf responses within the crown/canopy and the eco-
1784 logical scale of the investigation (assessment of the
1785 response of the whole tree/plant, or of a target population
1786 of leaves).
1787 A complete representation of a plant should take into
1788 account the different levels, age, and position of leaves.
1789 This would be the approach of choice but would require a
1790 large number of samples, and this would be difcult to
1791 realize in large-scale sampling. Thus, normally only one or
1792 a few leaf positions (e.g., sun leaves in the upper part of the
1793 crown, south exposed leaves, ag leaves, or fully devel-
1794 oped leaves) are considered, depending on the purpose of
1795 the survey.
1796 The number of leaves to be sampled depends on the
1797 internal variability of the parameters of interest. The fol-
1798 lowing formula can be used for this calculation:
n Z
2
a
s
2
= B
2
1800 1800 where n is the sample size; Z
a
is the standard normal
1801 coefcient (= 1.96 for a 95 % condence level); s is the
1802 SD; B is the desired precision level expressed as percent of
1803 the mean value (Elzinga et al. 2001; Gottardini et al. 2014).
1804 A recent study of boreal forests (Pollastrini et al. 2014)
1805 found that, in the higher external part of a crown of Betula
1806 pendula, the CV among different leaves was very low for
1807 F
V
/F
M
(1.6 %), and increased for the parameters related to
1808 the step J (1 - V
J
, CV = 7 %) and the step I
1809 (DV
IP
= 1 - V
I
, CV = 14 %). We mention here that this
1810 type of studies demonstrated that the IP phase, linked to the
1811 PSI content (Oukarroum et al. 2009; Ceppi et al. 2012), is
1812 quite sensitive to different types of stress; e.g., it decreased
1813 in response to ozone (Bussotti et al. 2011b) and nitrogen
1814 deprivation (Nikiforou and Manetas 2011), while it
1815 increased in response to high light conditions (Desotgiu
1816 et al. 2012).
1817 In order to sample as many leaves as possible during a
1818 single day, sampling must be performed during the whole
1819 day and cannot be limited to specic hours. As a conse-
1820 quence, leaves are sampled under different conditions of
1821 short-term light acclimation and different extents of pho-
1822 toinhibition. To reduce the associated variability, it is
1823 necessary to allow the regulatory mechanisms induced by
1824 the ambient light to relax and to allow the leaves to recover
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1825 from photoinhibition, which means a sufcient period of at
1826 least 45 h of dark acclimation at a constant temperature
1827 must be made before measurement. In addition, to avoid
1828 the onset of leaf senescence or the induction of other stress
1829 factors that can change the physiological state of the leaf
1830 during sampling and dark acclimation of the leaves, all
1831 eldwork must be performed as fast as possible. Managing
1832 a large number of samples in a short time, e.g., 1,000
1833 samples in one day, requires fast instruments/experimental
1834 protocols. OJIP transients need less than 1 s of illumina-
1835 tion, and their analysis is best suited for this kind of
1836 application.
1837 Question 29. What additional information can be
1838 obtained from simultaneous measurements of CO
2
1839 exchange and chlorophyll uorescence?
1840 Modern Infra red gas analyzers (IRGAs; such as the
1841 CIRAS-3, PP Systems and LI-COR 6400) allow gas
1842 exchange and uorescence to be measured simultaneously.
1843 This combination can provide information about effects on
1844 the photosynthetic ETC, CalvinBenson cycle activity, and
1845 diffusional limitations at the same time. Additionally, it is
1846 possible to determine chlorophyll uorescence parameters
1847 under particular conditions (e.g., increasing CO
2
concen-
1848 trations or low O
2
concentrations) to determine the maxi-
1849 mum electron transport rate. In this way, effects of a certain
1850 treatment can be more precisely assigned to a particular
1851 process in the whole photosynthetic apparatus than the use
1852 of these techniques individually would allow (see e.g.,
1853 Laisk and Loreto 1996; Laisk et al. 2005).
1854 Three potential applications for simultaneous measure-
1855 ments have been proposed in the literature:
1856 (i) Analysis of alternative sinks of electrons (e.g.,
1857 Flexas et al. 1998; Bota et al. 2004). Discrepancies
1858 between the electron transport rate (ETR) and the
1859 net CO
2
assimilation rate (A
n
) are an indicator of
1860 the existence of alternative electron sinks. For
1861 example, an increased ETR/A
n
ratio indicates the
1862 existence of other electron sinks (e.g., Mehler
1863 reaction, photorespiration, nitrate reduction) in
1864 competition with CO
2
assimilation (e.g., Bota
1865 et al. 2004). An important cause for an increase in
1866 ETR/A
n
is photorespiration (e.g., Galmes et al.
1867 2007). Comparing measurements made at 2 % O
2
1868 (suppression of photorespiration) with measure-
1869 ments made at 21 % O
2
(ambient) allows a
1870 quantication of this process (Rosenqvist and
1871 van Kooten 2003).
1872 (ii) Calculation of CO
2
diffusion resistance/conduc-
1873 tance in the mesophyll, which in bifacial leaves is
1874 formed by the palisade and spongiform tissues
1875 (von Caemmerer 2000). Mesophyll conductance is
1876 an important variable controlling CO
2
diffusion to
1877 the carboxylation site of Rubisco. Several meth-
1878 ods have been proposed to estimate mesophyll
1879 conductance in leaves (for a detailed description
1880 of these methods, see e.g., Warren 2006; Flexas et.
1881 al. 2008). One of these methods is based on IRGA
1882 measurements (measurements of CO
2
assimila-
1883 tion, A
n
/C
i
curves) and the electron transport rate
1884 from chlorophyll uorescence (e.g., Flexas et al.
1885 2006)a detailed description of this method is
1886 available elsewhere (Loreto et al. 1992; Evans and
1887 Loreto 2000; Flexas et al. 2008).
1888 (iii) Sink limitations in photosynthesis (Rosenqvist and
1889 van Kooten 2003). In a variation of point
1890 (i) above, simultaneous IRGA and chlorophyll
1891 uorescence measurements made at low (2 % O
2
,
1892 which suppresses photorespiration in C3 plants),
1893 and ambient (21 % O
2
) oxygen concentrations can
1894 be used to estimate changes in sourcesink
1895 relationships in leaves (Rosenqvist and van Koo-
1896 ten 2003). Under non-sink restrictions and 2 %
1897 oxygen, the CO
2
assimilation rate (A
n
) should
1898 increase, and the ETR should remain the same. By
1899 contrast, if the leaf is sink-limited, lowering the
1900 oxygen concentration to 2 % will not affect A
n
,
1901 whereas the ETR will decrease (down-regulation
1902 by nal product).
1903 Question 30. Can the wavelength dependence
1904 of the quantum yield for CO
2
xation be predicted
1905 by measuring chlorophyll uorescence?
1906 Emerson and Lewis (1943) observed that the quantum
1907 yield for O
2
evolution is wavelength dependent and that it
1908 dropped off quickly at wavelengths longer than 700 nm.
1909 Similar wavelength dependence is observed for Uco
2
1910 (McCree 1972; Inada 1976; Hogewoning et al. 2012).
1911 Typically, photosynthetic rates are higher when a leaf is
1912 illuminated with light in the red region (600680 nm),
1913 compared with an equal number of photons in the blue or
1914 the green regions of the light spectrum. Beyond 700 nm
1915 (i.e., the FR region), Uco
2
declines rapidly to nearly zero at
1916 about 730 nm.
1917 Genty et al. (1989) demonstrated that the PSII operating
1918 efciency (i.e., F
q
0
/F
M
0
or U
PSII
) correlates linearly with
1919 Uco
2
if the photosynthetic steady state is induced by white
1920 light of different intensities, while photorespiratory activity
1921 is low. This is always the case in C4 plants and in C3
1922 plants; this occurs when the O
2
concentration is low
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1923 (12 %) (see also Question 29; Genty et al. 1989; Krall and
1924 Edwards 1992). In contrast to the relationship between
1925 Uco
2
and light intensity, Chl a uorescence measurements
1926 are unsuitable for the estimation of the relationship
1927 between Uco
2
and the wavelength of irradiance used. To
1928 understand why, it is important to consider the factors that
1929 may affect the wavelength dependence of both Uco
2
and
1930 U
PSII
.
1931 First, different wavelengths are not reected and trans-
1932 mitted to the same extent by leaves. Hence, the fraction of
1933 light absorbed by a leaf is wavelength dependent (e.g.,
1934 Vogelmann and Han 2000; see also Question 4). This also
1935 explains why most leaves are green and not, for example,
1936 blackrelatively more green light is reected and trans-
1937 mitted than red and blue light, and therefore, the fraction of
1938 red and blue light absorbed by a leaf is higher than the
1939 fraction of green light that is absorbed (Terashima et al.
1940 2009). A lower fraction of incident light reaching the
1941 photosystems will directly result in a loss of Uco
2
on an
1942 incident light basis. However, at low light intensities in the
1943 linear part of the light-response curve, there are no limi-
1944 tations for the electron ow on the acceptor side of PSII.
1945 Therefore, within a range of low light intensities (typically
1946 between PPFD of 0 and 50 lmol photons m
-2
s
-1
, or an
1947 even narrower range for shade-leaves), U
PSII
does not
1948 necessarily change as a result of small changes in the light
1949 intensity. Beyond this range of low light intensities, U
PSII
1950 decreases when the light intensity increases, due to limi-
1951 tations for the electron ow on the acceptor side of PSII
1952 (see Question 2 Sect. 1 for electron transfer rates on the
1953 acceptor side of PSII). Thus, wavelength-dependent dif-
1954 ferences in the fraction of incident light reaching the
1955 photosystems are reected by differences in Uco
2
, but at
1956 low light intensities not necessarily by differences in U
PSII
.
1957 Second, carotenoids differ in the efciency (3590 %)
1958 with which they transfer excitation energy to chlorophylls,
1959 whereas the chlorophyll to chlorophyll energy transfer
1960 efciency in antenna complexes is nearly 100 % (Croce
1961 et al. 2001; de Weerd et al. 2003a, b; Caffarri et al. 2007).
1962 The transfer efciency of carotenoids depends on their
1963 chemical structure and position within the photosynthetic
1964 apparatus. Carotenoids have absorption maxima in the blue
1965 and green regions, and therefore, blue light is used less
1966 efciently by the photosystems than e.g., red light.
1967 Wavelength-dependent differences in the fraction of light
1968 absorbed by carotenoids affect the fraction of absorbed
1969 light reaching the RCs of the photosystems. This leads to
1970 the same argument as in the previous paragraph, i.e., this
1971 effect decreases Uco
2
but at low light intensities does not
1972 necessarily decrease U
PSII
.
1973 Third, leaves contain non-photosynthetic pigments such
1974 as avonoids and free carotenoids. These pigments pre-
1975 dominantly absorb light in the UV region but also in the
1976 blue and green part of the spectrum. These non-photosyn-
1977 thetic pigments are not connected to the photosystems and
1978 do not transfer the absorbed energy to the photosynthetic
1979 apparatus (see Question 31 for a discussion of these com-
1980 pounds and their detection). The absorption of light by non-
1981 photosynthetic pigments will reduce the fraction of the
1982 incident light reaching the photosystems especially in the
1983 blue and to a smaller extent in the green. Again this will
1984 affect Uco
2
at these wavelengths but at low light intensities
1985 not necessarily U
PSII
.
1986 Finally, the pigment composition and absorbance prop-
1987 erties of PSI and PSII differ, and therefore, the balance of
1988 excitation between the two photosystems is wavelength
1989 dependent for a given state of the photosynthetic apparatus
1990 (e.g., Evans 1986; Chow et al. 1990a, b; Melis 1991;
1991 Walters and Horton 1995; Hogewoning et al. 2012). In
1992 practice, when light within a narrow-band wavelength
1993 range is used to illuminate a white-light acclimated leaf,
1994 one of the two photosystems is often excited more strongly
1995 than the other. Any imbalance in excitation between the
1996 two photosystems results in a loss of Uco
2
. This wave-
1997 length dependence is especially clear in the FR region. FR
1998 light still quite efciently excites PSI but is very inef-
1999 ciently absorbed by PSII (see Question 16). This is called
2000 the red drop and, as noted above, this leads to a rapid
2001 decline of UO
2
and consequently of Uco
2
as well at
2002 wavelengths longer than 685 nm. Obviously, when PSI is
2003 excited strongly by FR light, but PSII is excited only very
2004 weakly, electron ow from PSII to PSI is not restricted, and
2005 therefore, U
PSII
will be high. However, due to the inef-
2006 cient absorption of the FR photons by PSII, linear electron
2007 ow is low, and therefore, Uco
2
is low for FR light. On the
2008 other hand, if PSII is excited more strongly than PSI, the
2009 consequent loss of U
PSII
is reected by a proportional loss
2010 of Uco
2
. Wavelengths in the range around 480 nm (blue)
2011 result in the strongest preferential excitation of PSII and
2012 therefore the strongest loss of both Uco
2
and U
PSII
(Ho-
2013 gewoning et al. 2012). However, U
PSII
is also an unreliable
2014 measure of Uco
2
for these blue wavelengths, due to the
2015 absorption by carotenoids and non-photosynthetic pig-
2016 ments (see above).
2017 In summary, U
PSII
calculated from chlorophyll a uo-
2018 rescence measurements is an unsuitable parameter for
2019 estimating the wavelength dependence of Uco
2
. Wave-
2020 length-dependent changes in (1) the absorbed light fraction,
2021 (2) the light fraction absorbed by photosynthetic carote-
2022 noids, and (3) the light fraction absorbed by non-photo-
2023 synthetic pigments, directly affect the fraction of photons
2024 reaching the photosystems and therefore Uco
2
. However, at
2025 low light intensities, changes in the fraction of photons
2026 reaching the photosystems may not affect U
PSII
. Further-
2027 more, (4) some wavelengths preferentially excite PSI,
2028 resulting in high U
PSII
values but low Uco
2
values. As a
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2029 consequence, for a reliable measurement of the wavelength
2030 dependence of Uco
2
, gas exchange measurements remain
2031 the gold standard.
2032 Question 31. Can anthocyanins and avonols be
2033 detected by chlorophyll uorescence?
2034 In vivo non-destructive determination of anthocyanins and
2035 avonols in green parts of plants can be made using the
2036 uorescence excitation ratio method (FER) (Bilger et al.
2037 1997; Agati et al. 2011). The FER method is based on the
2038 measurement of chlorophyll uorescence induced by dif-
2039 ferent excitation wavelengths. The extent of absorbance of
2040 light by the epidermal polyphenols can be derived on the
2041 basis of the ratio of chlorophyll uorescence emission
2042 intensities induced by a standard red beam and a UVVIS
2043 beam (wavelengths strongly absorbed by epidermal poly-
2044 phenols). The role of different anthocyanins and avonols
2045 can be distinguished by choosing appropriate wavelengths
2046 based on the specic absorbance spectra of the different
2047 anthocyanins and avonols.
2048 The chlorophyll uorescence excitation technique was
2049 originally developed to assess UV-absorbing compounds in
2050 the leaf epidermis (Bilger et al. 1997). Ounis et al. (2001)
2051 extended the method developing remote sensing equipment
2052 (dual excitation FLIDAR) to study polyphenols not only in
2053 leaves but also in canopies of trees. This method has also
2054 been used for the determination of the presence of avo-
2055 noids, including anthocyanins, in the skins of fruits like
2056 grapes (Kolb at al. 2003), apples (Hagen et al. 2006), and
2057 olives (Agati et al. 2005). Betemps et al. (2011) showed
2058 that in fruits, the anthocyanins and other avonoids local-
2059 ized in the outer skin layers reduce the chlorophyll uo-
2060 rescence signal in proportion to the concentration of these
2061 polyphenols.
2062 Pfundel et al. (2007) investigated two different types of
2063 commercial portable UV uorometers for in vivo screening
2064 of anthocyanins and carotenoids in leaves. The UV-A-
2065 PAM uorometer (Walz, Germany) makes use of a blue
2066 reference beam, whereas the Dualex uorometer (FORCE-
2067 A, France) makes use of a red reference beam. For mea-
2068 surements on green leaves, the two instruments gave sim-
2069 ilar results, whereas the anthocyanins common in fruits
2070 absorbed part of the blue light of the UV-A-PAM reference
2071 beam which led, for fruits, to higher estimates for epider-
2072 mal UV transmittance compared to that by the Dualex
2073 uorometer. Pfundel et al. (2007) also noted that the
2074 absence of Chl b (e.g., in the barley chlorina f2 mutant)
2075 affected the determination of the polyphenols. Ben Ghoz-
2076 len et al. (2010) developed and described an improved
2077 instrument, which they called the Multiplex (FORCE-A,
2078 France). It contains four light-emitting diodes (LEDs): UV-
2079 A (370 nm), blue (460 nm), green (515 nm), and red
2080 (637 nm) and three diodes to detect uorescence emission
2081 at 590, 685, and 735 nm. The three diodes allow correc-
2082 tions for differences in the chlorophyll content of the
2083 sample. The red LED provides the reference beam, because
2084 it corresponds to a wavelength not absorbed by anthocya-
2085 nins or avonols. The uorescence induced at this wave-
2086 length is compared with the uorescence intensity induced
2087 by the excitation wavelength specic for the polyphenol of
2088 interest (e.g., green 515 nm light for anthocyanins or
2089 370 nm UV-A light for avonols). Ben Ghozlen et al.
2090 (2010) derived formulas to correlate these ratios with the
2091 actual polyphenol content of the sample.
2092 In summary, a uorescence-based method and accom-
2093 panying equipment have been developed to determine the
2094 anthocyanin and avonol content of leaves and fruits. In
2095 the case of fruits, the choice of the color (blue or red) of the
2096 reference beam inuences the results, something that does
2097 not affect leaf measurements.
2098 Question 32. Can Chl a uorescence be used
2099 as an indicator for a specic stress in plants?
2100 To use Chl a uorescence as a tool to identify a specic
2101 stress, the effects of that stress on the photosynthetic
2102 apparatus must be understood (Kalaji et al. 2012a, b). If
2103 heat stress destroys the donor side of part of the PSII RCs,
2104 it reduces the electron donation capacity of all PSII RCs
2105 together and, as a consequence, causes a slow down of the
2106 JI rise as measured by a PEA-type instrument (Srivastava
2107 et al. 1997 and see also Schreiber and Neubauer 1987). It
2108 also changes the recombination properties of the affected
2109 PSII RCs when measuring DF (C
ajanek et al. 1998). In

2110 extreme cases, when all or nearly all PSII donor sides have
2111 been destroyed, the uorescence rise levels off after
2112 *300 ls of illumination (i.e., one charge separation) and
2113 then declines; this uorescence pattern is called the K-peak
2114 (Guisse et al. 1995; Srivastava et al. 1997; Lazar et al.
2115 1997). UV radiation may also destroy the donor side of
2116 PSII (e.g., Ohnishi et al. 2005; Hakala et al. 2005), but, at
2117 the same time, may have additional affects on the PSII RC
2118 (e.g., Vass et al. 1996) and, thereby, on the uorescence
2119 kinetics. For both drought stress and sulfate deciency, it
2120 was shown that they affect PSI (Oukarroum et al. 2009;
2121 Ceppi et al. 2012). Again, a combination of experimental
2122 phenomena is needed to distinguish these stress conditions.
2123 Another complication is that the PSII to PSI ratio that
2124 affects the parameter DV
IP
is regulated by the growth light
2125 intensity and quality as well (Leong and Anderson 1984b;
2126 Lee and Whitmarsh 1989; Chow et al. 1990a, b). Finally,
2127 there are considerable kinetic differences between the OJIP
2128 transients obtained from different plant species (Kirova
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2129 et al. 2009). This means that good references are needed to
2130 determine if something is a stress effect, taking into
2131 account the normal plasticity of the OJIP transients. The
2132 available physiological studies often concentrate on the
2133 effects of severe stress under laboratory conditions. In the
2134 eld, milder stress effects are often observed, which pos-
2135 sibly have to be distinguished from other sources of vari-
2136 ability, so that additional research efforts will be needed to
2137 obtain reliable ngerprints for a particular stress. An
2138 example of the type of research needed is a study by Kalaji
2139 (2011) who characterized the effects of 16 abiotic stresses
2140 on the uorescence properties of two Syrian landraces (cvs.
2141 Arabi Abiad and Arabi Aswad) of barley (see also Kalaji
2142 and Guo 2008).
2143 Another approach is to make mathematical analyses of
2144 sets of OJIP transients in combination with DF and 820 nm
2145 transmission transients. Goltsev et al. (2012) trained an
2146 articial neural network to estimate the relative water
2147 content (RWC) of leaves; they obtained a correlation value
2148 of R
2
= 0.98 between the estimated RWC value and the
2149 gravimetrically determined RWC value of the analyzed
2150 leaves.
2151 In France, commercial software was developed that
2152 compares measured OJIP transients with a database of
2153 uorescence transients measured on plants of dozens of
2154 genotypes of agricultural and horticultural crops suffering
2155 from deciencies of the following elements: N, Fe, Mn,
2156 Mg, P, S, Ca, and B. This approach has similarities with
2157 the one discussed above, but it is more ambitious in its
2158 scope. This software is at the moment very popular
2159 among farmers, especially in Poland, Ukraine, and Rus-
2160 sia, where it is promoted by producers of fertilizer. Kalaji
2161 et al. (unpublished data, 2013) did many experiments to
2162 test the software and suggested analysis, comparing the
2163 uorescence analysis with the chemical analysis of sev-
2164 eral plant species grown under different conditions of
2165 nutrient deciency. These studies suggested that this
2166 method needs further improvements to achieve a general
2167 validity.
2168 For the moment, it is not possible to identify specic
2169 stresses using Chl a uorescence. As noted above, different
2170 stresses may have similar effects on the photosynthetic
2171 system. In addition, in the eld, plants are often subjected
2172 to several stresses at the same time, e.g., a combination of
2173 drought, high light, and heat stress. In the laboratory, it is
2174 possible to induce clear symptoms, whereas in the eld, a
2175 combination of a less severe stress and acclimation may
2176 cause less specic symptoms. In other words, the compli-
2177 cated relationship between uorescence kinetics, stress,
2178 and natural variation is not yet sufciently well understood
2179 to use uorescence measurements as ngerprints for spe-
2180 cic stresses under natural conditions.
2181 Question 33. Is Chl a uorescence a useful tool
2182 for the monitoring of aquatic ecosystems?
2183 The use of Chl a uorescence measurements for the study
2184 of aquatic environments is a topic by itself, and here only a
2185 few points are made. This topic was reviewed in depth in a
2186 recent book edited by Suggett et al. (2011).
2187 The estimation of biomass production in aquatic envi-
2188 ronments is one of the research topics in which uores-
2189 cence techniques have played a major role and for which
2190 special equipment was developed. Falkowski and Kolber
2191 (1990) developed a submersible pump-probe instrument
2192 (see Question 2 Sect. 1 for the principle) to study biomass
2193 productivity proles along the water column in the ocean.
2194 Further, Kolber et al. (1998) discussed a new uorescence
2195 approach, which they called the FRR approach which was
2196 originally developed for aquatic studies. Instead of con-
2197 tinuous light, subsaturating excitation ashes (of which the
2198 spacing can be varied) are used to induce photosynthesis.
2199 With these ashlets, the authors could create STFs as well
2200 as multiple turnover pulses and, at the same time, study the
2201 dark relaxation kinetics of uorescence. One of the
2202 parameters that could be determined was the effective PSII
2203 antenna cross section. Using a Xenon-PAM (Walz, Ger-
2204 many), Geel et al. (1997) studied several classes of aquatic
2205 organisms in order to derive the oxygen evolution activity
2206 of these organisms on the basis of uorescence measure-
2207 ments. Kromkamp and Forster (2003) have reviewed such
2208 studies.
2209 Another important difference between measurements on
2210 plants and measurements in an aquatic environment is that
2211 aquatic samples often consist of a mixture of photosyn-
2212 thetic organisms. To cope with this problem, several
2213 instruments were developed that make use of differences in
2214 the pigment composition of different classes of photosyn-
2215 thetic organisms. Schreiber (1998) has described an
2216 instrument built by Kolbowski and Schreiber called the
2217 PHYTO-PAM Phytoplankton analyzer (Walz, Germany).
2218 The instrument does not use a monochromatic modulated
2219 beam but excites the samples alternately with weak 10 ls
2220 light pulses of 470, 535, 620, and 650 nm (inducing F
O
) to
2221 distinguish between cyanobacteria, green algae, and dia-
2222 toms. Deconvolution of the algal composition was possible
2223 using reference spectra derived from pure cultures of par-
2224 ticular classes of organisms. In addition, the instrument
2225 allowed the estimation of the activity of these classes of
2226 organisms using saturating light pulses (see Questions 2.3,
2227 10, and 15).
2228 Beutler et al. (2002) built a submergible instrument
2229 called bbe Fluoroprobe
TM
(Moldaenke, Germany) that
2230 made use of ve excitation wavelengths (450, 525, 570,
2231 590, and 610 nm) with which particular accessory
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2232 pigments can be relatively specically excited allowing the
2233 detection of peridinin containing dinoagellates and Pyr-
2234 rophyta, chlorophyll b containing green algae, fucoxanthin
2235 containing diatoms, and zeaxanthin as well as phycobili-
2236 protein containing cyanobacteria or cryptophycaea. Refer-
2237 ence spectra were used to determine the chlorophyll
2238 content associated with each class. Rolland et al. (2010)
2239 using this equipment for a monitoring study of the Marne
2240 reservoir summarize its application in monitoring studies
2241 up till that time and note that it can be used down to 100 m,
2242 and that it has a short response time.
2243 Further, Schreiber et al. (2012) have developed a new
2244 Multi-Color-PAM (Walz, Germany) instrument that com-
2245 bines multi-spectral excitation (400, 440, 480, 540, 590,
2246 and 625 nm) with the possibility to measure fast uores-
2247 cence kinetics as well as the absorption cross section of
2248 PSII antennae.
2249 Photosynthetic aquatic organisms (including aquatic
2250 plants such as Spirodela) in combination with uorescence
2251 measurements can also be used to monitor the presence of
2252 pesticides, heavy metals, and natural compounds that affect
2253 the photosynthetic apparatus. Snel et al. (1998) using a
2254 modulated PAM uorometer and monitoring ETR followed
2255 the effect of low concentrations of linuron in microcosm
2256 experiments. Another example of the application of a PAM
2257 uorometer was published by Perreault et al. (2010) who
2258 evaluated the effect of copper oxide nanoparticles on
2259 Lemna gibba using among other things the quenching
2260 analysis. Srivastava et al. (1998) using a PEA instrument
2261 showed that the cyanobacterial toxin scherellin A caused
2262 an increase of F
J
; this indicates that scherellin A affects
2263 the acceptor side of PSII like DCMU does. Bueno et al.
2264 (2004) showed an effect of lindane on the cyanobacterium
2265 Anabaena; they observed that this pesticide initially
2266 affects the amplitude of the JIP phase and after longer
2267 incubation times (1224 h) causes a general suppression
2268 of the uorescence intensity. In other studies, the effects
2269 of heavy metals like cadmium (Romanowska-Duda et al.
2270 2005) or chromate (Susplugas et al. 2000) on Spirodela
2271 oligorrhiza have been studied. Finally, Chl a uorescence
2272 is also a useful tool for the study of hydrogen production
2273 in e.g., Chlamydomonas reinhardtii (see e.g., Antal et al.
2274 2006)
2275 Concluding remarks
2276 For anyone who is beginning to use Chl a uorescence, the
2277 overwhelming number of studies that already has been
2278 carried out may make it difcult to quickly discover what is
2279 already known and which experiments will add something
2280 new to the literature. Even so, it is important to formulate
2281 rst some questions that are worth answering. Two points
2282 are worth keeping in mind. In the rst place, the ash,
2283 pulse, and steady state communities live often in
2284 parallel universes; as a consequence, there are still many
2285 opportunities for a more integrated use of these techniques.
2286 In the second place, the currently available uorescence
2287 devices can do much more than the few standard protocols
2288 that are most frequently used.
2289 As this educational review suggests, there are many
2290 aspects of uorescence that can be studied with different
2291 devices best adapted for the study of these different
2292 aspects. Flash experiments can be used to study the elec-
2293 tron transfer reactions within PSII, direct uorescence
2294 measurements are best for the measurement of the OJIP
2295 transients, which follow the reduction of the photosynthetic
2296 electron chain, and modulated measurements are best for
2297 steady state photosynthesis and the study of light-induced
2298 regulatory mechanisms affecting the antenna of PSII. The
2299 power of uorescence techniques can be increased con-
2300 siderably by simultaneously measuring other parameters,
2301 such as 820 nm transmittance changes (probing PSI) or
2302 CO
2
assimilation.
2303 There are only a few basic principles that determine the
2304 yield of uorescence. However, due to the fact that it is
2305 sensitive to many processes that differ between photosyn-
2306 thetic organisms, light acclimation states, intactness of
2307 samples, and stress conditions, a myriad of responses has
2308 been documented in the literature. The uorescence liter-
2309 ature may often be confusing and contradictory, but it
2310 contains a wealth of data and observations that we all need
2311 to understand. Only in that way, the wealth of information
2312 generated by past uorescence research can be maximally
2313 exploited.
2314 The contributing authors are available to be contacted
2315 by researchers for further discussions on the application of
2316 Chl a uorescence through the following website: https://
2317 groups.google.com/forum/?hl=en#!forum/chlorophyll
2318 uorescence where they will provide regular feedback.
2319 Acknowledgments The authors thank Govindjee (University of
2320 Illinois at Urbana-Champaign, USA) for his support, assistance, and
2321 helpful comments during the preparation of the manuscript. The
2322 authors are also grateful to Dr Giles Johnson (University of Man-
2323 chester, UK), Peter Hooda (Kingston University, UK), Dr. Mahendra
2324 Rai (SGB Amravati University, India), Dr. Szilvia Z. Toth (Biological
2325 Research Centre Szeged, Hungary), and Dr. Gerald E. Edwards
2326 (Washington State University, USA) for their valuable comments and
2327 suggestions to improve the quality of this paper. M.H. Kalaji
2328 acknowledges Prof Helmut Lichtenthaler, Dr Ulrich Schreiber, Dr
2329 Alexandra Stirbet, and Dr Dusan Lazar for their encouragement.
2330 Open Access This article is distributed under the terms of the
2331 Creative Commons Attribution License which permits any use, dis-
2332 tribution, and reproduction in any medium, provided the original
2333 author(s) and the source are credited.
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punda et al. 1997; Srivastava et al. 1999).

ajanek et al. 1998). In

troch M, Lachetova I, Kalina J, Spunda V (1998)

tude de linduction de uorescence des algues

iffel P, Braunova Z (1999) Release and aggregation of the light-

ajanek M, Il k P, Kalina J, Naus J (1997) Appearance of

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