Energy-Dependent Chlorophyll Fluorescence Quenching in
Chloroplasts Correlated with Quantum Yield of Photosynthesis
G. H einrich Krause and Henrik Laasch
Botanisches Institut der Universität D üsseldorf, Universitätsstraße 1, D -4000 D üsseldorf 1,
Bundesrepublik Deutschland
Z. Naturforsch.
42c, 581—584 (1987); received January 19, 1987
Chlorophyll Fluorescence Quenching, Chloroplast, Photoinhibition, Photosynthesis, Thermal
Energy Dissipation
Chlorophyll a fluorescence quenching was studied in intact, C 0 2 fixing chloroplasts isolated
from spinach. Energy-dependent quenching (qE), which is correlated with the light-induced pro­
ton gradient across the thylakoid membrane presumably reflects an increase in the rate-constant
o f thermal dissipation o f excitation energy in the photosynthetic pigm ent system . The extent o f qE
was found to be linearly related to the decrease o f quantum yield o f photosynthesis. We suggest
that this relationship indicates a dynamic property o f the membrane to adjust thermal dissipation
o f absorbed light energy to the energy requirem ent o f photosynthesis.
Introduction
W hen chloroplast pigments absorb light in excess
of the energy turnover in photosynthetic reactions
including carbon metabolism, photoinhibition of the
electron transport system may occur. Photoinhibi­
tion is characterized by partial inactivation of p hoto­
system (PS) II and to a lesser extent of PS I (see [1]).
Various protective systems are known that minimize
damage in excess light, e.g., damage by active oxy­
gen species, excited triplet states of chlorophyll (see
[2, 3]), or “overreduction” of the electron transport
chain [4, 5].
A possible way to cope with excess excitation ener­
gy in the photosynthetic apparatus before the onset
of photochemical reactions would be a regulated
non-destructive therm al deactivation of excited pig­
ments. Such a regulated “valve” for excess energy
appears to be indicated by “energy-dependent” ,
A pH -related quenching of chlorophyll a fluores­
cence, qE. The extent of this quenching was found to
be linearly related to the intrathylakoid proton con­
centration and thus to the size of the light-induced
proton gradient across the thylakoid membrane [6 ].
The quenching of fluorescence seems to be based on
an increase in the rate-constant of therm al deactiva­
tion [7, 8 ]. This is probably caused by structural
changes in the membrane due to intrathylakoid acidi­
fication and related cation exchange at the internal
thylakoid surface.
Reprint requests to Dr. H. Krause.
Verlag der Zeitschrift für Naturforschung, D -7400 Tübingen
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In a previous communication [9] it was shown that
in the presence of qE substantial protection against
photoinhibition is provided. The present study gives
evidence for a dynamic adjustm ent of thermal energy
dissipation, as indicated by qE, to the energy require­
ment of photosynthetic carbon assimilation (see also
[1 0 ]).
Materials and Methods
Intact chloroplasts were isolated from freshly har­
vested leaves of spinach (Spinacia oleracea L.) [11,
12]. Samples containing 23 fig chlorophyll (Chi) ml - 1
were illuminated in the cuvette of a Clark-type oxy­
gen electrode (light path 6 mm) with red light (L I,
see below) of different light flux densities. The reac­
tion medium contained 0.33 m sorbitol, 2 m M ED TA
(disodium salt), 1 m M MgCl2, 1 m M MnCl2, 10 m M
NaCl, 60 mM KC1, 0.5 mM KH 2 P 0 4, 40 mM (2-[4-(2hydroxyethyl)-l-piperazinyl]-ethanesulfonic
acid
(pH 7.6 with N aO H ), 2 mM K H C 0 3 and 2000 U m l " 1
catalase (EC 1.11.1.6).
Quantum yields of C 0 2-dependent 0 2 evolution
were determ ined according to Giersch and H eber [13].
A bsorption by the chloroplasts m easured with an in­
tegrating Ulbricht sphere was about 49% of incident
red light. Simultaneously with 0 2 evolution,
chlorophyll fluorescence was recorded with a pulse
amplitude m odulation fluorom eter (PAM 101, H.
Walz, Effeltrich, Germ any) [14], Non-modulated ac­
tinic light ( L 1) was obtained with glass filters Calflex
C, K65 (Balzers, Liechtenstein) and RG 645 (Schott,
Mainz, FR G ); X.max was 665 nm, half-band width
15 nm. The com ponents of fluorescence quenching,
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582
G. H. Krause and H. Laaseh • E nergy-D ependent Chlorophyll Fluorescence Q uenching
qE (A pH -dependent quenching) and qQ (photochem ­
ical quenching) [7] were determ ined in the steady
state of photosynthesis after 5 min actinic illumina­
tion at the indicated light flux densities (see Fig. 1).
Results and Discussion
Fig. 1 depicts an example of the fluorescence sig­
nals used for the determ ination of qE and qQ. It is
known from earlier studies [7, 8 , 15] that under the
conditions applied here, non-photochemical quench­
ing (qN) is approximately equal to qE. Small contribu­
tions of other mechanisms to qNhave been neglected.
Light-saturation curves of qE and qQ in the presence
of bicarbonate are depicted in Fig. 2 a. W ith increas­
ing light flux densities qE saturates at about 0.7 to
0 . 8 , whereas qQ declines to a low value, indicating
that the proportion of oxidized electron acceptor of
PS II (Q a) decreases. Fig. 2b shows the light-dependence of rates and quantum yields of C 0 2 -dependent 0 2 evolution, representing C 0 2 fixation in
the steady state. The data dem onstrate that at higher
irradiances an increased part of the absorbed light
energy is not utilized in photosynthetic carbon as­
similation. If the quantum yield is plotted versus qE,
a linear relationship is seen (Fig. 3). We suggest that
this reflects a regulation of thermal energy dissipa­
tion according to the energy requirem ent of photo­
synthesis. In low light, low qE would indicate a low
l ight
Fig. 1. Chlorophyll fluorescence recording (X > 700 nm) of
intact spinach chloroplasts excited by a weak modulated
light. Non-m odulated actinic light (L 1) causes a rise from
initial fluorescence em ission (F0) to the peak (Fp), which is
followed by slow quenching. Pulses (1 s) of saturating white
light (L 2) cause full reduction o f Q A (electron acceptor of
PS II) and reverse photochem ical quenching (qo)- W hen
chloroplasts are dark-adapted, L 2 induces maximal fluo­
rescence (F m). The difference betw een Fm and the em ission
in L 2 after prolonged illumination with L 1 indicates non­
photochem ical quenching (qN), m ost o f which consists of
energy-dependent quenching (qE)- The com ponents of
quenching are calculated as qE ~ qN = [(Fv)m—(Fv)s]/(Fv)m;
qo - [(Fv) s - F v]/(Fv)s.
0 6
0
4
0 2
100
50
Light
flux
density
(W
150
m
)
Light
flux
density
(W
m
Fig. 2a, b. Light-dependence o f energy-dependent (qE) and photochem ical (qQ) fluorescence quenching (a) and o f rate
( • ) and quantum yield (O ) of C 0 2-dependent 0 2 evolution (b) in intact chloroplasts. Data represent the steady state after
5 min illumination with L 1 at the respective light flux density. For details o f fluorescence recording see Fig. 1. The
quantum yield is defined as m oles 0 2 evolved per m ole photons absorbed.
G. H. Krause and H . Laaseh ■E nergy-D ependent Chlorophyll Fluorescence Q uenching
0.4
583
0.6
0.8
F lu o re sc e n c e quenching (q )
Fig. 3. Correlation betw een energy-dependent fluorescen­
ce quenching (qE) and quantum yield o f C 0 2-dependent 0 2
evolution (data from Fig. 2). The straight line was fitted by
linear regression; correlation coefficient, r — -0 .9 9 .
rate-constant of therm al deactivation that allows a
high quantum yield of photosynthesis. Extrapolation
of the straight line in Fig. 3 to qE = O denotes an
“optim al” quantum requirem ent of about 1 0 photons
per 0 2 molecule evolved. W hen with increasing
irradiance C 0 2 fixation approaches light-saturation
and the quantum yield declines, the rate-constant of
therm al deactivation, as indicated by qE, appears to
increase. This would avoid or minimize overenergi­
zation of the photosynthetic apparatus and thereby
protect it from photoinhibitory damage. It should be
noted that the linear relationship depicted in Fig. 3
does not extent beyond light saturation of photosyn­
thesis, because then also qE becomes light-saturated.
In this respect different responses were observed
from the more complex system of intact leaves [16].
One may argue that the quantum yield of C 0 2
fixation is solely regulated by the redox state of Q A,
as a correlation betw een quantum yield and qQ is
indeed observed (Fig. 4). A high ApH might control
reoxidation of Q A by restricting electron flow
through the plastoquinone pool. However, this is not
the case, as shown in Table I. A ddition of m oderate
concentrations of the uncoupler N H 4 C1 (5 m M ) to the
chloroplasts does not inhibit C 0 2 fixation (and thus
does not lower the quantum yield) but drastically
diminishes qE due to partial uncoupling [17]. It can be
Fig. 4. Relationship between photochem ical quenching
(qQ) and quantum yield of C 0 2-dependent 0 2 evolution
(data from Fig. 2). The quantum yield is plotted versus
1—q0 ; the latter term indicates the approximate proportion
of reduced Q A in the steady state.
Table I. Effects o f partial uncoupling with N H 4C1 (5 m M )
on fluorescence quenching in intact chloroplasts (presence
o f 2 mM K H C 0 3). Quenching was determ ined as for Fig. 1
after 5 min illumination with L 1 (light flux densities as
given).
Light (L 1)
NH4CI
C 0 2-dep.
0 2 evoln.
[^mol/mg Chi • h]
20 W m “
2
absent
present
13
18
0.58
0.03
0.34
70 W m “
2
absent
present
74
74
0.72
0.13
0.27
Conditions
Q uenching
qE
qQ
0.21
0.22
seen that under this condition Q A does not become
more oxidized (as qQ does not increase) but rather
more reduced. Thus, an increased excitation of PS II
due to diminished thermal energy dissipation ap­
pears to be the predom inant effect of partial un­
coupling.
Conclusions
The present communication shows a close linear
correlation between A pH-related quenching (qE)
and quantum yield of photosynthetic C 0 2-dependent
0 2 evolution. We view this as evidence for a regula­
584
G. H. Krause and H. Laasch ■Energy-D ependent Chlorophyll Fluorescence Q uenching
tion of therm al energy dissipation according to the
energy requirem ents of photosynthesis. Protection
against the damaging effects of excess excitation
would be the consequence of such regulation. The
effectiveness of this protective mechanism can be
judged from the data in Fig. 3. An increase of qE
from about 0.3 to 0.7 is related to an approximate
increase in quantum requirem ent from 15 to 70
(quantum requirem ent is defined here as moles
quanta absorbed per mole 0 2 evolved). We do not
suppose that the increase in the thermal dissipation
limits photosynthesis. There is strong evidence that
C 0 2 assimilation is limited by the activity of the car­
bon reduction cycle even at low, non-saturating irradiances [18]. R ather, we suggest that therm al ener­
gy dissipation is dynamically adjusted in accordance
to the proportion of excess excitation energy. This
adjustm ent would be limited, however, by the light-
saturation of the processes responsible for qE. At
irradiances below saturation, overexcitation of the
membrane and related damage should be largely
avoided. But even at light-saturation, partial protec­
tion should be provided due to the high rate-constant
of thermal dissipation, as indeed indicated by previ­
ous experiments [9, 10]. The mechanism discussed
here allows relatively fast responses (in the range of
one minute) of the photosynthetic system to chang­
ing light regimes. As recently reported [19], there
seems to be a second, long-term mechanism of pro­
tection that is also based on increased therm al dissi­
pation and would be particularly effective in light
above saturation of photosynthesis.
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Acknowledgem ents
We thank Birgit Weyers and Elke Danz for compentent assistance.