Photosynthesis Research 27: 41-55, 1991.

(~) 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

A theoretical and experimental analysis of the qt, and qN coefficients of

chlorophyll fluorescence quenching and their relation to photochemical
and nonphotochemical events

Michel Havaux 1'2, Reto J. Strasser I & Hubert Greppin 2

IUniversitd de GenOve, Laboratoire de Biodnerg~tique, Station de Botanique, CH-1254 Lullier-Gen~ve

(address for correspondence); 2Laboratoire de Physiologie v~g~tale, 3 place de l'Universit~, CH-1211
GenOve 4, Switzerland

Received 5 March 1990; accepted 13 September 1990

Key words: energy dissipation, quantum yield, photochemistry, PS II

Abstract

The initial (Fo), maximal (FM) and steady-state (Fs) levels of chlorophyll fluorescence emitted by intact
pea leaves exposed to various light intensities and environmental conditions, were measured with a
modulated fluorescence technique and were analysed in the context of a theory for the energy fluxes
within the photochemical apparatus of photosynthesis. The theoretically derived expressions of the
fluorescence signals contain only three terms, X = J2p2F/(1 -- G), Y = T/(1 - G) and V, where V is the
relative variable fluorescence, J2 is the light absorption flux in PS II, P2r is the probability of
fluorescence from PS II, G and T are, respectively, the probabilities for energy transfer between PS II
units and for energy cycling between the reaction center and the chlorophyll pool: F 0 = X, F M = X/
( 1 - Y) and F s = X(1 + (YV/(1 - Y))). It is demonstrated that the amplitudes of the previously defined
coefficients of chlorophyll fluorescence quenching, qp and qr~, reflect, not just photochemical (qr) or
nonphotochemical (qN) events as implied in the definitions, but both photochemical and nonphoto-
chemical processes of PS II deactivation. The coefficient qp is a measure of the ratio between the actual
macroscopic quantum yield of photochemistry in PS II (thp) in a given light state and its maximal value
measured when all PSII traps are open (4~pp°n) in that state, with v-pa~°Pen=(F~-Fo)/FM and
~bp = 1 - (Fs/FM). When the partial connection between PS II units is taken into consideration, 1 - qp is
nonlinearily related to the fraction of closed reaction centers and is dependent on the rate constants of
all (photochemical as well as nonphotochemical) exciton-consuming processes in PS II. On the other
hand, 1 - qN equals the (normalized) ratio of the rate constant of photochemistry (k2b) to the combined
rate constant (kN) of all the nonphotochemical deactivation processes excluding the rate constant k22 of
energy transfer between PS II units. It is demonstrated that additional (qualitative) information on the
individual rate constants, kN-k22 and k2b, is provided by the fluorescence ratios 1/F M and (1/Fo) - (1/
FM), respectively. Although, in theory, .~p'hnpe~is determined by the value of both k2b and kN-k22 ,
experimental results presented in this paper show that, under various environmental conditions, rh
v.p°pen is
modulated largely through changes in kN, confirming the idea that PS II quantum efficiency is
dynamically regulated in vivo by nonphotochemical energy dissipation.

Abbreviations: Chl - chlorophyll; Fo, F M and F s - initial, maximal and steady-state levels of modulated
Chl fluorescence emitted by light-adapted leaves; PS I and I I - photosystem I and II; qp and qr~-
(previously defined) photochemical and nonphotochemical components of Chl fluorescence quenching
42
Introduction
Under continuous illumination, photosynthetic
organisms attain a state characterized by a rela-
tively low chlorophyll (Chl) fluorescence emis-
sion (see Fig. 1). This quenching of in vivo Chl
fluorescence has been ascribed to two kinds of
processes (for review, see Krause and Weis 1984,
Briantais et al. 1986). Firstly, Chl fluorescence is
controlled by the redox state of the primary
electron acceptor QA of photosystem (PS) II,
with oxidized QA being a quencher and reduced
QA a nonquencher of fluorescence. Secondly,
light energy absorbed by PS II-chlorophylls can
be dissipated by a variety of mechanisms which
are nonphotochemical in nature, such as thermal
deexcitation, which is possibly modulated by the
light-induced proton gradient across the
thylakoid membrane, and the transfer of energy
to the weakly fluorescent PSI by 'spillover'. In
addition, the antenna size of the photosystems
(and therefore the level of light absorption) is
regulated by the state-transition phenomenon in-
volving (de)phosphorylation of the light harvest-
ing complexes and their subsequent migration in
the lipid matrix of the thylakoid membranes.
This diversity of mechanisms controlling Chl
fluorescence emission in vivo makes the meas-
ured fluorescence signal rich in information but
also very complex and difficult to interpret. The
recent development of selective modulation
fluorometers (see technical review by Bolhfir-
Nordenkampf et al. 1989) has, however, opened
new possibilities for quantitative interpretation
of Chl fluorescence. By combining modulated
and nonmodulated lights, modulated fluorimetry
allows the measurement, under any steady-state
condition, of the actual modulated fluorescence
level (Fs) and the fluorescence level correspond-
ing to a situation where all PS II reaction centers
are either open or closed in the same energized
state (F 0 and FM, respectively, see Fig. 1).
Schreiber and co-workers (Schreiber et al. 1986,
Bilger and Schreiber 1986) have introduced the
use of the so-called photochemical (qv) and non-
photochemical (qN) fluorescence quenching fac-
tors calculated from the F s, F M and F 0 levels: qv
compares the actual modulated fluorescence
level with the initial (F0) and maximal (FM)
levels in a given light state ( q p = ( F u - F s ) /
(FM-F0)) whereas qN compares the F 0 and F M
levels before and after illumination
(1 - qN = [(FM-F0)/F0]/[(FM-Fo)/F0]dark). It
was assumed that those empirical parameters
allow the two types of fluorescence quenching
mechanisms (photochemical/nonphotochemical)
to be quantitated separately: the 'photochemical
fluorescence quenching qp' would specifically re-
flect the changes in fluorescence intensity linked
to the oxidation/reduction of QA and the 'non-
photochemical fluorescence quenching qN' would
monitor exclusively nonphotochemical deactiva-
tion of PS II. This approach has recently been
used in a number of fluorimetric studies of the
photosynthetic electron transport and its regula-
tion in vivo as well as in studies on the effects of
environmental stresses on them (examples of
applications can be found in Lichtenthaler 1988
or Baltscheffsky 1990).
In this paper, we present a detailed theoretical
analysis of the qp and qN fluorescence parame-
ters (as defined by Bilger and Schreiber 1986).
This theoretical treatment of the modulated fluo-
rescence signals is substantiated by experimental
data obtained with pea leaves. It is demonstrated
that the value of the qp and qN coefficients
depend on the rate constant of all the exciton-
consuming processes in PS II and, consequently,
both parameters can be regarded as indicator of
photochemical and nonphotochemical fluores-
cence quenching processes. Thus, the nomencla-
ture qp for 'photochemical fluorescence quench-
ing' and qN for 'nonphotochemical fluorescence
quenching' is not suitable. Nevertheless, qp and
qN are useful empirical parameters, in particular
when used in combination with other fluores-
cence ratios which provide additional informa-
tion of the rate constants of the various dissipa-
tive pathways. For instance, 1 - qN equals the
ratio of the rate constant of photochemistry (k2b)
to the combined rate constant (kN) of nonphoto-
chemical deexcitation processes (which include
fluorescence, heat and spillover), normalized to
the dark state. Using the equations and relations
given in this paper, it is thus possible to obtain
the general picture of energy dissipation in PS II
in a given light-adapted state. Experimental data
showed that, under various environmental condi-
tions, k N plays a dominant r61e as compared to
k2bin the light-induced modification of the qN

coefficient and the PSII photochemical ef-
ficiency.
Materials and methods
Experiments were performed on intact leaves of
pea (Pisum sativum L.) grown in a glasshouse
under controlled temperature, light and air
humidity conditions. Rapid water stress (leading
to a leaf water potential of around -17 bars) was
induced as previously described (Havaux et al.
1988): detached pea leaflets were placed on filter
paper in the dark and dehydrated in air for 4 h at
room temperature (---22°C) and at a relative air
humidity of around 55%. Mild heat stress was
induced by transferring for 10 min pea leaflets
into distilled water maintained at a constant
temperature of 36°C.
In vivo 685-nm Chl fluorescence emission from
leaves exposed to air with 0.03% CO 2 was meas-
ured at room temperature using an Hansatech
modulated fluorescence instrument connected to
a micro-processor power supply (Electro-
Automatik, model EA-3040). A typical example
of modulated fluorescence curve obtained with
this fluorometer is shown in Fig. 1. Chl fluores-
cence was excited with a modulated yellow light
(peak wavelength, 585 nm; modulation frequen-
cy, 870 Hz) provided by an array of yellow light-
emitting diodes combined with an Ealing Beck
35-5404 filter. Fluorescence emission was de-
tected with a photodiode through a 685-nm inter-
ference filter. The intensity of the modulated
light was sufficiently low not to induce any vari-
able fluorescence, so that the fluorescence level
recorded was close to the basic fluorescence level
F 0. The presence of inactive PS II (capable of
active water oxidation but ineffective in electron
flow to plastoquinone) might however introduce
some inaccuracy in the determination of the F 0
level using the modulated fluorescence tech-
nique. Indeed, inactive PS II centers are believed
to be responsible for the O - I rise in the
Kautsky-type fluorescence induction curves (Cao
and Govindjee 1990). Possibly, in very low light
conditions, fluorescence emission from inactive
centers could slightly increase the monitored F 0
to a level close to the I level. As inactive centers
are converted to active centers in light (Guen-
43
DARK
z
5 rain
I I
L
I
z
0
3
u_
M®UATE0 UGHT
Fig. I. Typical curve of modulated Chl fluorescence emitted
by an attached pea leaflet. The basic fluorescence level F 0
was induced by a very weak beam of modulated yellow light.
The transitory closure of the PS II reaction centers by a short
pulse of intense blue light (Ls) allowed the maximal level of
Chl fluorescence F M to be measured. Photosynthesis was
induced by an actinic blue light LA, the intensity of which
was adjusted using neutral density filters. After prolonged
illumination of the leaves with LA, Cht fluorescence reached
a steady-state level F s. LVR, 2-s pulse of far-red light. F0d~'k
dark
and F M , initial and maximal fluorescence levels measured in
leaves previously adapted to darkness.
ther et al. 1990), F 0 measured in leaves adapted
to strong actinic light (i.e., with few inactive
PS II centers) could be closer to the 'true' F 0
level than the level measured in dark-adapted
leaves - a phenomenon which could partially ac-
count for the apparent F0-quenching observed in
high light (cf. below). Short pulses (1 s) of super-
saturating blue light L s (500 W m -2) were used
to measure the maximal level F M of modulated
Chl fluorescence in dark- or light-adapted leaves.
Photosynthesis was induced by a non-modulated
(actinic) beam of blue light LA, the intensity of
which was adjusted from 0 to 500 W m -2 using
neutral density filters. After prolonged illumina-
tion (ca. 15 min) of the leaves with LA, the
fluorescence yield reached a low steady-state
level F s. The basic fluorescence yield F 0 of those
light-adapted leaves was determined by simulta-
neously turning off L A and applying a 2-s pulse
of far-red light LFR. We assumed that the meas-
urement of F 0 was fast enough to avoid relaxa-
tion of the quenching processes established
under light conditions, allowing to measure the
true dark level of the light-adapted leaves. This
44
assumption was supported by the finding (not
shown here) that the application of an intense
light pulse L s on the F 0 level, just after L A was
switched off, gave a fluorescence peak with the
same height as that of the F M level. The non-
modulated lights LFR, L A or L s were supplied by
Schott KL1500 light sources combined with
adequate filters (320-620-nm broad-band filter
for L A and L s and 730-nm interference filter for
LFR) and were delivered via fiber optics to the
leaf placed in the Hansatech leaf-clip. The fluo-
rescence signals were displayed on a poten-
tiometric chart recorder or stored on diskettes.
All light intensities were measured with a YSI-
Kettering 65A radiometer.
Results
Theoretical analysis of the measured Chl
fluorescence levels
Our theoretical approach is based on the analysis
of the energy fluxes within the photochemical
apparatus of photosynthesis as outlined by Stras-
ser (1978) and Sironval et al. (1984). In the
present work, the two photosystems will be con-
sidered as an assemblage of a reaction center
(denoted a and b for PS I and PS II, respectively)
and a pool of Chls (denoted by the suffix 1 or 2
for PSI or PS II). Figure 2 shows a schematic
model of the photosystems and the energy fluxes
entering and leaving PS II. Deactivation pro-
cesses in the Chl pool 2 include fluorescence,
heat loss and transfer of excitation energy to the
reaction center (trapping), to PS I-Chls (PS II-
PS I transfer called 'spillover') and to other PS II
units (PS II-PS II transfer called 'grouping').
The energetic behavior of this simplified sys-
tem can be mathematically described using basic
equations for the energy input and output of the
Chl pool 2. The total energy influx (E2) is
E2 = J2 + Ei2 (1)
where J2 is the light absorption flux in the Chl
pool 2 (mol photon s- 1 ) and El2 is the sum of all
energy transfer fluxes from surrounding pigment
systems (i.e., from the reaction c e n t e r (Eb2) and
from the Chl pool of other PS II units (E22)).
ABSORPTION
J2
E2F FLUORESEENEE
SPILLOVER V _ E2H HEAT
I I I I
PSI PS II
Fig. 2. Schematic model of the grouped PS II units. In this
model, PS II is seen as an assemblage of a reaction center (b)
and a large pool of Chls (2) constituted by the core antennae
and the light harvesting Chl complexes. The energy fluxes
entering this pigment system 2 (white arrows) are the light
absorption J2 and the energy migration from the reaction
center (Eb2) and the surrounding Chl pools 2 (E22). Energy
outfluxes (black arrows) are E2b (trapping), E2F (Chl fluores-
cence emission), E2n (thermal deactivation), E2~ (energy
transfer to PSI by spillover) and E22 (energy exchange
between PS II units called 'grouping'). PSI is represented
here by its reaction center (a) and its pool of Chls (1).
The energy outfluxes (E2j) are
Ezi = E2P2i (2)
where P2i is the probability of pigment deexcita-
tion by fluorescence (PEF), heat emission (P2H)
or energy transfer to the reaction center (P2b)
and to PS I by spillover (P21). The probabilities
P2j a r e related to the rate c o n s t a n t s kEi by the
following equation (Sironval et al. 1984):
P2i = k2i/ZJ k2j (3)
The measured fluorescence signal F 2 is propor-
tional to E2F which is equal to the product E2P2F.
By definition, a reaction center is in the open
configuration when all trapped energy is used for
photochemistry. For closed reaction centers,
photochemistry cannot be accomplished but the
 45

energy trapped by the reaction center Chl may (10) becomes:

be transferred back to the Chl pool 2 (thus
increasing the flux through the PS II dissipative K2F
(10A)
pathways) with a probability Pb2 (Butler 1978). FM = J 2 Ek2i-k22- k2b
In the case where all reaction centers are in the i
open configuration,
On the other hand, Eqs. (7) and (8) yields
Eopen = J2 4- n !~ °pen (4)
2 - 1-,22~2 T
Fv = F M - F ° = P 2 F J 2 ( 1 - G ) ( 1 - G - T )
since Pb2 = 0 in open centers. Consequently, (11)
Fopen Eopen (5)
2 = P2F 2 = PZFJ2/( 1 -- P22) Fv/F 0 = T/(1 - G - T) (12)

In the context of our experimental protocol pre- Fv/F M = T/(1 - G) (13)

sented in Fig. 1, ~2r~°Penis equal to the fluores-
cence level F 0 (or ~0l~'darkfor a sample previously
adapted to darkness) measured with the weak
modulated light alone. If all reaction centers are
closed (cl) (e.g., by illumination of the (dark- or
light-adapted) sample with an intense light pulse
Ls, as in Fig. 1), the measured signal F M or
Fdark el cl el cl
M = F2 = P2F(J2 + E22 + Eb2) with Eb2 =
cl cl cl cl
E2bPb2 = E2 P2bPbz and E22 = P22Ez. Therefore
F~l = FM ----P2FJ2/( 1 -- P= -- P2bPb2) (6)
Denoting P22 and P2bPb2, respectively, by G
(grouping term) and T (trapping product for
energy cycling between the reaction center and
the Chl pool), we have the following system:
When the leaves were exposed for a prolonged
time to the actinic light L A (Fig. 1), Chl fluores-
cence reached a steady-state level F s which can
be mathematically expressed as the sum of the
fluorescence of PS II with open reaction center
--2(0 -----F0(1 - V ) ) and the fluorescence of PS II
l:?°pen
with closed center (F~l(,) = FMV) (Strasser 1981):
F s = F o ( 1 - V ) + FMV= F o + (F M - F o ) V
(14)
where F 0 and F M are the basic and maximal
fluorescence levels measured in the steady-state
and V is the relative variable fluorescence,

F o = p:FJ2/(1 - G) (7) V = (F s - F o ) / ( F M - F o ) (15)

F M = p2FJ2/(1 - G - T) (8) Using Eqs. (7) and (8), Eq. (14) can be re-
written
which can also be expressed in terms of rate
constants (cf. Eq. (3)): J2P2F ( T )
F s - i----G 1+ 1 - G - T V (16)
K2F
(9) It can be demonstrated (see appendix) that V is
Fo = J2 E k2i -- k22 an hyperbolic function of the fraction of closed
i reaction centers B:
K2F
B
FM = J2 E k2i - k22 - k2bPb2 (10) V= Fv (17)
I+•G(1-B)
As in Kitajima and Butler (1975), we shall as-
sume that kb2 is much higher than the rate Equations (7), (8) and (16) indicate that the
constant kbH of thermal energy dissipation in the theoretical expression of the experimental signals
reaction center so that, for closed centers, F0, F~ and F s contains only three terms with
Pb2 = kb2/(kb2 + kbu) ~ 1. Consequently, Eq. physical meaning: X = J2P2F/(1 -- G), Y = T/
46

( 1 - G) and V. As illustrated in Fig. 3, F o = X, intensity affected differently the F0, F M and F s

Era = X/(1 - Y) and F s = X(1 + (YV/(1 - Y))).
The F0, Fra and F s Chl fluorescence levels
described above were measured in intact (at-
tached) pea leaves adapted to various actinic
light intensities L A ranging from zero to satura-
tion (ca. 400 W m-2). As shown in Fig. 4, light
P2F
~o: J2"I_-W-
~-
PZF I 1 being much more pronounced than the latter
FM= Jz' 1--'~- " l 1 YT'"
PZF ",.I- G.,': • iV::)
F: J '-izT'0 ÷ 1- ....... "
-, . . . .
Fig. 3. The experimental signals F 0, F M and F (actual Chl
fluorescence level, =F s when measured at the steady-state)
can be expressed using three terms having a physical mean-
ing: J2P2F/(1 -- G), T/(1 - G) and V where P~F is the prob-
ability for energy dissipation by fluorescence, G is tile group-
ing term (in our model, 13 = P22, probability for energy
transfer between PS II units), T is the trapping term (i.e., the
product of the probabilities of energy transfer between the
reaction center and the Chl pool = P2bPb2), V is the relative
variable fluorescence (which is nonlinearily related to the
fraction of closed P$ II centers). See text for details.
levels. The steady-state modulated Chl fluores-
cence F s was observed to be virtually constant
and independent of the actinic light intensity,
providing an interesting example of biological
homeostasis. A detailed phenomenological de-
scription of this phenomenon and its sensitivity
to variations of the environmental conditions will
be presented in a separate paper. In contrast to
this constancy of the F s level, increases in light
irradiance resulted in a drastic quenching of the
maximal fluorescence F M and to a noticeable
decrease in F0-fluorescence, the former change
one. A roughly linear relationship was observed
between F 0 and the logarithm of the light intensi-
ty whereas the light dependence of Fra was
.......
biphasic, with a first linear part at low light
intensities and a sharp decrease at intensities
higher than around 2 0 W m -2. Equation (10A)
indicates that such a decrease in Fra amplitude
can be due to processes affecting one or several
of the following parameters: J2, k2F and the sum
k2F + k2H + k21. The current hypothesis ascribes
the light-induced quenching of F M to the build-
up of the transthylakoid pH gradient, which is
believed to influence k2x (Krause et al. 1982,
1988), and the state 1-state 2 transitions which
regulate the light distribution between PS I and

.-// I I I i

• • Q

,.e
Q',
Fm
F--
@@
Z

@", @

....... ° .... Q.... I~l.~...l~... i"1 o rl .O.O. O

C~
0 ......... " ....
1.1-
I I I I I I I I I
I=0 2 ~ 6 8 10
Log21
Fig. 4. Initial (F0), maximal (FM) and steady-state (Fs) levels of modulated Chl fluorescence in intact pea leaves illuminated for
about 15 rain with various intensities (I in W m -2) of the actinic blue light LA. F~ was obtained after illuminating the sample with
an intense light pulse whereas F o was obtained by switching off the LA light. Each set of experimental points (Fo, FM, Fs)
corresponds to a different leaf. Please note that the x-axis is logarithmic. Log to the base 2 (log~) is used because the various fight
intensities examined differed by a factor of around 2.
 47

P S I I via changes in J2 a n d / o r k21 (Fork and
Satoh 1988). In addition to those processes,
light-induced alteration of the F 0 level can also
be caused by a modification of the rate constant
of photochemistry k2b (cf. Eq. (9)).
under steady-state conditions depends of the
proportion of open reaction centers and is equal
to:
~bp = (1 - B ) E 2 P e n p 2 b / J 2 (23)

Quantum yields Remembering that B is the fraction of closed

reaction centers, J z = F 0 ( 1 - G ) / P 2 v (cf. Eq.
The quantum yields for photochemistry ~be and (7)), r~°Pe"
-2 = F0(1 - V ) * and 1/(1 - G) = Fv/
non-photochemical energy dissipation ~bN can be (FMT) (cf. Eq. (13)), one obtains:
estimated from the F0, F M and F s levels. By
definition, the maximal quantum yield for photo- (~p = Fv/FM(1 - V ) = ~b~Pe"(1 - V )
chemistry in PS II (~b~,p~n) is the maximal rate of = 1 - (Fs/FM) (24)
photochemistry cr~°Pe"~
' , ~ 2 b ] divided by the light ab-
sorption in PS II (J2): It is interesting to note that this expression of ~be
])open = l~,open/| pop .... (18) is in agreement with a recent work of Genty et
P ~2b "~2 = P2btZ2 /J2 al. (1989) who observed a close correlation be-
tween the fluorescence parameter (F M - F s ) / F M
Thus, according to Eq. (5), (= 1 - (Fs/FM)) and the quantum yield of non-
~b °p~n = p 2 b / ( 1 - - G ) (19) cyclic electron transport estimated from CO 2
P fixation measurements.
The quantum yield for nonphotochemical
Consequently energy dissipation in PS II with open reaction
p b2"4"
d'°pen = T/(1 - G) (20) center is
p

,hope" = (Ezv + E, n + E2: + E 2 2 ) / J ,

Under the assumption that Pb2 = 1 in closed "~N . .
centers and Pb2 = 0 in open centers and as a = (E 2 -E2b)/J 2
consequence of Eq. (13), = (E2/J2) _ (Ezb/J2)

(~open
p = T/(1 - G) = F v / F M (21) = (E,/J2)
_ _ ",rp
.open (25)

When PS II unit-unit transfer is zero (G = 0),
Eq. (20) reads
(~open
P = T = P2b (21A)
Equation (21A) belongs to a highly simplified
model and corresponds to the expression of the
quantum yield of photochemistry originally pro-
posed by Kitajima and Butler (1975). When T
and G are replaced by their expressions in terms
of rate constants, we have
Due to the conformation of the system allowing
energy recycling between PS II units (G) and
also between the reaction center and the Chl
pool (T), one photon absorbed can provoke
more than one excitation event: E J J 2 > 1 (see
Eq. (4)) and thus ,~op~.
"¢"N + ,~op~.
W'p > 1. It is only
when there is no grouping (i.e., E 2 = J2) that
a,°P~" = 1 - , va,°Pe"
v-N p (26)
A trivial consequence of Eqs. (21) and (26) is

t~open
p = k 2 b / ( k N + k2 b _ k22) (22) rh°pen
V-N = 1 -- d~°pen
v-v = F 0 / F M (27)

with k~ being the sum of all nonphotochemical

rate constants (k N = k2F -~- k21 --~ k2H 31- k22)" *F 0 is the Chl fluorescence level when all reaction centers are
open whereas F~p~" is the fluorescence emitted by PS II with
On the other hand, the actual quantum yield their reaction center present in the open configuration in the
~be in light-adapted leaves photosynthesizing steady-state: F ~ P " " = ( I - B ) E ~ P ' " p 2 F .
48

ON = 1 -- 0p = Fs/FM (28) Fs - F° = V (29A)

The effects of the L A intensity on the quantum
yields 0p, ¢h
"~aopen ,0N, 0~ pe" in the steady-state are
shown in Fig. 5. It can be seen that low light
intensities (below about 20 W m -z) had very lit-
tie effect on 0P and no effect on ,.~ .~popen At .
intensities higher than 20 W m -2, an increase in
irradiance resulted in a strong decrease in both
0p and v'p'C°pen.At the highest intensity tested, 0p
was zero (photochemistry was light-saturated)
and ,q-p
"h°Pcn fell to approx. 40% of the maximal
value recorded in low light. When P22 = 0, the
light dependences of ON and ,,~open "~N are the re-
versed image of the plot of 0p and 0ppCn VS. L A
intensity.
Chl fluorescence quenching factors, qe and qN
As already mentioned in the Introduction, mod-
ulated Chl fluorescence data such as those pre-
sented in Fig. 4 are usually analyzed using the
now familiar qp and qN fluorescence quenching
factors. Under conditions of F0-quenching, the
coefficients were defined by Bilger and Schreiber
(1986) as follows:
1 - qp = FM_ F0
(Fv/F0)
1 - qN -- (Fv/F0)dar k (30)
The early definition of 1 - qN (Schreiber et al.
1986) did not take into account the light-induced
quenching of the F o level; F o was considered to
be constant and therefore 1 - qN = Fv/(Fv)dark"
By definition, all four expressions V, 1 - V , qp
and 1 - qp are fractions or probabilities ranging
from zero to unity. This is not the case for the
term 1 - qN which is the ratio of two values
characterizing two different physiological states
and can range theoretically from zero to infinity.
Then, the expression 1 - qN is somewhat mis-
leading, giving the impression that it is a prob-
ability like qp or 1 - q p .
Equation (29) can be rewritten in the follow-
ing way:
1 - (Fs/FM)
qp = Fv/F M
or, according to Eqs. (21) and (24),

F M - US Op

qp - FM -- F 0
(at steady state) (29) q P - 0popen (31)

1 -II [ I I I I I I I

c~" ~N
--
0 0 "" "" ~pen
N
w
)- ",0 • , •
0..~
o .,{o

Z ., .-'i, " "4,

;D
0
.o~R~~ .=.-= (~ ~,..e

q, ~p
I I I I I I I I O-Z.-b
2 4 6 8 10
Log21
Fig. 5. Maximal and actual quantum yields of photochemistry in PS II (~pcn and ~bp) and minimal and actual quantum yields for
energy dissipation via nonphotochemical processes (~b~pen and ~bN)in pea leaves adapted to various intensities (I in W m -2) of the
actinic blue light L^. Calculated from the data presented in Fig. 3. Quantum yields for nonphotochemical energy dissipation were
calculated assuming P22 = 0.
 49

Thus, the qp parameter in a given steady-state is photochemistry (k2b) as well as on the rate con-
an indicator of the changes in the actual quan- stants of non-photochemical processes (kN).
tum yield of photochemistry in PS II relative to On the other hand, Eq. (30) can be rewritten
the maximal quantum yield in that state (if all as follows
reaction centers were open). As shown in Figs. 5
and 6, 6p was very close to vp ,h open
(qv ~ 1) in a ( 1 -_ (Fo/FM) ~/ ( 1 - (Fo/FM)
relatively large range of light intensities from 0 1 - qN = F0/F M //\ -Fo--7-~M dark
to around 2 0 W m -2. Above this, the actual
quantum yield strongly differed from the maxi-
(34)
mal quantum yield (i.e., qp decreased)- an ef-
fect attributable (in part) to the progressive After proper substitution of Eq. (9), Eq. (10A)
(steady-state) closure of the reaction centers. and Eq. (22) into Eq. (34), we obtain
Comparison of Eqs. (15) and (29) shows that
k2b ~/( k2b
qp = 1 - V and, consequently, when energy ex-
1 - qN = ( k N _-k~2 ] / \ ~-N~-"k22/dark (35)
changes between PS II units are taken into con-
sideration (G ~ 0), qp depends not only on the
fraction of closed reaction centers B but also on The above relationship indicates that, like qp,
F v G / F 0 (cf. Eq. 17). This latter ratio can be the 'nonphotochemical' fluorescence quenching
rewritten as follows qN is a function of both the rate constants of
photochemical and non-photochemical events.
TG However, in contrast to what we observed for qp
1 -T-G
(32) (Eq. (33)), the rate constant of grouping (k22)
has no influence on the qN values (indeed, k N -
If we express T and G in terms of rate constants, k22 = k2F + kEH + k21 ). When expressed in terms
we obtain: of quantum yields ( ~ b 2 i = E 2 i / J 2 = ( k 2 i P * ) / J 2
where P* is the concentration of excited pig-
Fv k22k2b ments, Strasser 1978), Eq. (35) becomes:
F--~ G = (k N + kEb)(k N _ k22) (33)
1 -- qN = ,hope, ,hopen ] / ;6pen --open /
Thus, the above relationship clearly indicates "WN -- '+'22 --- ~N -- q)22 /dark
that the so-called photochemical fluorescence
quenching qp depends on the rate constant of (36)
-# I I I I I I I I I
0 0
1 . . . . . . . • , •
-oO "', o
', 1 -qN / "
0 ",0 0

, ID
0.5
o".o, :'e
", O*
~.,0

1- • b..o,..o
qP :~"'"" " -o.. o
o! ....
•
I''"
2
•
-
...11~-~"
I
¢
I I
6
I I I
8
-.
I
10
Log 2 I
Fig. 6. Dependence on the actinic light intensity (I in W m -2) of the fluorescence quenching parameters, 1 - qp and 1 - qN, in pea
leaves. Calculated from the data of Fig. 3.
50
Thus, the parameter 1 - q N is a measure of the
relative values of the quantum yield for photo-
chemistry and the quantum yield of a part of the
nonphotochemical energy dissipation ($~pe~_
~b2°pe~
2 ) by open P S I I centers in a given light
condition compared to their value in the dark-
adapted state. An adequate name describing this
physical meaning has still to be proposed.
The light dependence of 1 - qN (Fig. 6) shows
that the ratio k2b/(k N - k22 ) remained practically
unchanged in leaves adapted to low light inten-
sities (below around 20 W m-2) and was strongly
reduced at high light intensities. It is however
impossible to tell, from the qN value, whether
high lights caused an increase in kN, a decrease
in k2b or a change in both rate constants. This
problem can be partially solved by examining
raw fluorescence data such as t / F M and (1/
F0) - (1/FM). Indeed,
1/F M = (k N - k22)/(J2k2F )
(l/F0) - (1/FM) = k2J(J2k2F )
It is unlikely that the product J2k2F differed
much from one light-adapted state to the other.
Indeed, the measured signals are the modulated
Chl fluorescence elicited by the pulsed light
whose intensity was maintained constant. Al-
though J2 can change with the light energy redis-
tribution associated with the state 1-state 2 tran-
sitions, it is known that, at the most, 5-10% of
t,o
t,
~3
k.$
2
.... ~......©...~ .... .O.-.~-O~.°~we..ar..
~! , I
2 6
Fig. 7. Dependence on the actinic light intensity (I in W m-2) of the Chl fluorescenceparameters, 1/FMand 1/F0 - 1/Fu, in pea
leaves. Data are calculated from Fig. 3 and normalized to the values determined in dark-adapted leaves.
the light input of PS II is involved in this process
(Malkin et al. 1986). In addition, state-transi-
tion-related quenching of Chl fluorescence has
been shown to be saturated at low light inten-
sities (Horton and Hague 1988). On the other
hand, Chl fluorescence has a small rate constant
which is believed to be constant over a wide
range of physiological conditions. As no changes
in the shape of the Chl fluorescence emission
spectra were observed in pea leaves under the
light conditions used here (data not shown), the
pigment molecules and their close environment
were presumably not affected and it is therefore
very unlikely that the rate constant of radiative
deexcitation of those pigments was changed. In
fact, the assumption that k2F = constant has been
admitted even in cases where marked conforma-
tional changes can be measured such as in ex-
periments with chloroplasts placed in low or high
salt media (Butler and Kitajima 1975). Thus, in
(37) the first approximation, the fluorescence parame-
ters 1/F M and ( 1 / F o ) - ( 1 / F M ) can be used as
(38) q u a l i t a t i v e indicators of the individual rate con-
stants k N - k22 and k~b, allowing to monitor the
general trends in the energy dissipation adjust-
ments in PSII. In other terms, 1/F M
(k N - k22 ) and [(1/Fo) - (1/FM) ] ~ k2b.
Figure 7 shows that an increase in irradiance
brought about an increase in 1/F M and a con-
comitant decrease in (l/F0) - (1/FM). However,
the changes in the latter parameter were less
marked than the changes in the 1/F M amplitude,
I ! I !
O ,"
] 0"" 0
0 •
0,"
°'0
0," • 0
Oo.-" 1 1
• Fo F~
° °" ~" ~-o.-.9
a t I I I I I
8 10
L0g2 I
 51

suggesting that light affected the qN-quenching tial reduction of 1 - qn. This effect was particu-
predominantly through a change in the rate con- larly marked in heated leaves and was observed
stant of nonphotochemical energy dissipation over the whole range of intensities (from 0 to
k n -k22. The fact that the two fluorescence saturation). The extent of the water stress-
ratios changed in an opposite manner is an addi- induced increase in qn was more limited and was
tional argument in favor of the assumption that observed at moderate and high light intensities
changes in J2k2F are negligible since, otherwise, only. The analysis of the F M and F 0 amplitudes
1/F M and ( l / F 0 ) - (1/Fra) would have simulta- (Fig. 8B) suggested that the changes in qn could
neously increased or decreased. have different causes in heated and dehydrated
leaves. Indeed, thermally stressed leaves ex-
Stress effects hibited a concomitant increase in 1/F M and de-
crease in ( l / F 0 ) - (1/FM) whereas rapid water
Figure 8A shows that environmental stresses, stress affected 1/F m only. Changes in J2 under
such as mild heat stress (36°C for 10 min) and stress conditions appeared negligible since the
leaf dehydration (for 4 h), resulted in a substan- values of the 1/F M and (l/F0) - (1/Fro) fluores-

// , , I I I I I I I

"~-°o A
"Z~ 0-.

,~ O-q
,p
z
'P
0.5 'A ',,

0"'-.
".n O,
-. [] ". .,

.... u Ip.%_ o . . . . o ]a

I I I I I I
4 6 o 10
Log 2 1

41 , I I I I I I I I

I B

•. "-: " "m

...•-IF k "
- " II
..-'* • 4"
z
~3 -'n

~- 2 ~¢
3 .- --- 1 1
. O. _ _ 0_.0~-.8. -e t" •

" "0 O - O..

[ --d
..
°-
I I I 1 I I I I I
2 6 10 6 8
Log2 I
Fig. 8. Light intensity dependence of A) the Chl fluorescence quenching factor 1 - qs and B) the fluorescence parameters, 1/F M
(closed symbols) and 1/F 0 - 1/FM (open symbols), in control pea leaves (O, O) and in leaves subjected to mild heat stress (E], U;
36°C for 10 min) or rapid leaf dehydration (for 4 h; A, A). Data are normalized to the values determined in dark-adapted
samples. Units of I: W m -2.
52
cence ratios measured after dark-adaptation
were observed to be very similar in stressed and
unstressed leaves (data not shown).
Discussion
Our theoretical study shows that the nomencla-
ture of 'photochemical fluorescence quenching'
for qp and 'nonphotochemical fluorescence quen-
ching' for qN is not suitable since the amplitude
of those two fluorescence parameters depends
simultaneously on the value of the rate constant
of photochemistry (k2b) and those of nonphoto-
chemical processes of pigment deexcitation (i.e.,
fluorescence and heat emissions, energy ex-
changes by spillover and, in the case of qp,
grouping). Then, qp and qN can be considered as
indicators of both photochemical and nonphoto-
chemical events.
In fact, the parameter 1 - qp is nothing else
than the variable fluorescence function
V = (F s - F0)/(F M - F0), the properties of which
have been previously analyzed in numerous
studies (see, for example, Joliot and Joliot 1964,
Malkin and Kok 1966, Lavorel and Etienne
1977, Butler and Strasser 1977, Strasser 1980).
Using a different model (in which the F 0 level is
a 'dead' fluorescence unrelated to the photo-
chemical apparatus), Joliot and Joliot (1964) re-
lated, for the first time, the fraction of closed
centers and the probability of intersystem energy
transfer as: V = (1 - p22)B/(1 - p22B). Equation
(17) is a more general form of the expression of
V given by Butler and Strasser (1977) for a
partial matrix model of the photochemical ap-
paratus. A common equation of V for various
theoretical models where G indicates the overall
grouping probability can be found in Strasser
(1981). In all those cases, V is nonlinearily re-
lated to the fraction of closed reaction centers
(B). It follows that qp can be used to directly
monitor the closure of the PS II traps only under
very particular conditions when G = 0 (and
hence V= B). Examples of such pigment systems
without inter-system energy transfer is provided
by leaves lacking the light harvesting Chl-protein
complexes, such as those of Chl-b-less mutants
and 'flashed' leaves during the greening process
(Strasser and Butler 1977), or to some extent by
chloroplasts in low salt media (Butler and Kita-
jima 1975). However, even in those particular
cases, qp cannot be considered as a pure in-
dicator of photochemical events since B is a
function of both the total light input and all the
rate constants of energy distribution in the sys-
tem PS I + PS II (Strasser 1985).
In 'normal' leaves, there are various lines of
evidence supporting the idea of energy ex-
changes between PS II units (Joliot and Joliot
1964, Williams 1977, Velthuys 1987) and, conse-
quently, the condition G = 0 is likely not to be
fulfilled. Thus, in actual fact, V = 1 - qp is not
proportional to B and, at the best, qp provides a
qualitative estimate of the proportion of open
centers. Another consequence of Eqs. (17) and
(33) is that the light-induced decrease in the
average macroscopic quantum yield (l~p) of
photochemistry through PSII relative to the
maximal quantum yield Ih°pen ,,/,,p in the same ener-
gized state (Figs. 5 and 6) is attributable at the
same time to the increased fraction of closed
centers and the modification of the photochemi-
cal and nonphotochemical energy dissipation
rate constants. In fact, ,rh°pen ~p itself is determined
by the particular constellation of the various rate
constants of energy dissipation established in a
given light environment (see Eq. (22)). In pea
leaves exposed to intense, photosynthetically
saturating, light, •,~p
-h o p e n
was observed to drop to a
low value corresponding to around 40% of its
maximal value measured after dark adaptation.
As shown in Fig. 7, this drastic reduction of the
photochemical efficiency of open PS II reaction
centers in the steady state resulted from a de-
crease in the rate constant of photochemistry
and, for a very large part, from a considerable
rise in the combined rate constant ( k ~ - k22) of
all the other, nonphotochemical, exciton-
consuming processes (except grouping). This ob-
servation is a confirmation of the idea, suggested
in recent studies (Weis and Berry 1987, Krause
et al. 1988, Genty et al. 1989, Horton 1990), that
the efficiency of PS II photochemistry in vivo is
dynamically regulated by nonphotochemical
events. In the present work, the photosynthetic
activity was modulated by several means, namely
changes in the incident light, high temperature
treatment and leaf dehydration. In all the situa-
tions tested, changes in k N were responsible for a

very large part for the changes in v.p'~°Pe",suggest-
ing that the modulation of the photochemical
yield of PS II via adjustments of the k N value
occurs under various physiological conditions. A
possible function of this down-regulation of
~b°pe"
P by nonphotochemical energy dissipation
could be to adjust the rate of photochemistry to
match that of carbon metabolism and hence to
avoid over-excitation of the PS II reaction cen-
ters. Light-induced increase in kN can then be
seen as a protective mechanism which nondes-
tructively diverts excess excitation energy from
the PS II reaction centers sensitive to photoinhi-
bition (Krause 1988). This idea is corroborated
by the finding that, after exposure of leaves to
environmental constraints (water stress and heat,
cf. Fig. 8) which are known to markedly reduce
the light level at which photoinhibitory damage
appear (Ludlow 1987, Ludlow and Powles 1988),
the kN-increase became apparent at much lower
light intensities than in control leaves.
Our data do not provide information on the
changes in the individual rate constants k2F, k2H
and k21 which are responsible for the light-
induced rise in k N - k22. However, recent photo-
acoustic studies have shown a spectacular en-
hancement of the heat emission signals in leaves
exposed to bright light (Buschmann 1987,
Havaux 1989), suggesting that the kN-changes
could possibly reflect a modification of k2H. The
molecular bases of the photoregulation of ther-
mal energy dissipation in leaves are still un-
known. It is believed that increased heat emis-
sion is caused by structural alteration in
thylakoid membranes due to intrathylakoid
acidification and related cation exchange at the
internal thylakoid surface (Krause et al. 1982,
1988), although this hypothesis is still waiting for
experimental support (Havaux 1990).
Equation (35) indicates that 1 - qN is the (nor-
malized) ratio k2b/(k N -k22 ). In this sense, it is
a useful empirical parameter which compares
photochemical vs. nonphotochemical deactiva-
tion of PS II. As shown in Fig. 6, adaptation to
saturating light induced a decrease in the above
ratio to less than 10% of its value in the dark.
When used in combination with other Chl fluo-
rescence parameters, such as the ratios 1/FM and
( l / F 0 ) - ( 1 / F M ) , qN allows us to examine the
general trends in the light-induced changes in the
53
photochemical and nonphotochemical pathways
of pigment deexcitation in PS II. It should how-
ever be kept in mind that even if the above
fluorescence parameters remain constant,
changes in the grouping-type nonphotochemical
energy dissipation (indicated by kaz ) can still
occur. Incidentally, another interesting fluores-
cence parameter could be the Fo/F M ratio which
is the probability of nonphotochemical energy
dissipation PN =kN/(kN +kzb) when the PSII
cooperativity is neglected (k2a = 0).
It is interesting to note that the general picture
of energy dissipation in PS II requires the knowl-
edge of the 'extreme' (F0, FM) fluorescence
levels only. The additional information provided
by the steady-state level of Chl fluorescence (Fs)
is related to B which is merely an indicator of the
energy flow level in the PS II centers. It is de-
termined by the balance between the excitation
E2b of the PS II traps, which depends on the rate
constants of all the deexcitation processes in
PS II (kN, kab), and the reactions downstream
involved in the reopening of the closed centers.
In fact, the intensity of steady-state modulated
fluorescence was almost constant and indepen-
dent of the L A intensity, at least in control,
unstressed leaves. Whether the maintenance of a
constant F s level has a precise physiological func-
tion remains an open question.
Concluding remarks
The present theoretical study indicates that the
empirical terms qe and qN do not allow the
Qn-related photochemical quenching of in vivo
Chl fluorescence and the nonphotochemical com-
ponent of fluorescence quenching to be sepa-
rated and quantified. It is indeed demonstrated
that the amplitudes of both qp and qr~ are de-
termined at the s a m e time by the value of the rate
constant of photochemistry and that of non-
photochemical deactivation of PS II. Thus, the
nomenclature 'photochemical fluorescence quen-
ching qe' and 'nonphochemical fluorescence
quenching qN' is not adequate and highly mis-
leading. Theoretical data presented in this paper
suggest a more rigorous approach for estimating
the two types of fluorescence quenching via the
analysis of the relative values of photochemical
54
and nonphotochemical rate constants of energy
dissipation in PS II. For example, the Chl fluo-
rescence ratio Fv/F 0 provides a direct measure of
the rate constant ratio k2b/(kN-k22 ) whereas
changes in k2b and kN-k22 can be monitored on a
qualitative basis by the Chl fluorescence ratios
1/F M and (1/Fo) - (1/Fu). It should be stressed
that all equations and relations given above are
valid only if they refer to the same energetic
state; this study is dealing with steady states in
which Fo, F s and F~a have their relations with
specific, but state-dependent, rate constants of
energy dissipation. In addition, it should also be
kept in mind that some of the equations (those
where the trapping product T = P2bPb2 is in-
volved) were derived from our model under the
simplifying assumption that the main distinctive
feature of open and closed reaction centers is
their (in)ability of recycling all the transferred
energy back to the Chl pool (i.e., Pb2 = 1 in
closed centers and 0 in open centers). The exten-
tion of those equations to a situation where Pb2 is
allowed to vary and take any value between 0
and 1 will be examined in future work.
Acknowledgments
We wish to thank Dr S. Drenkard and Dr Gov-
indjee for critical reading of the manuscript and
helpful comments.
Appendix
Relationship between V, the relative variable Chl fluores-
cence, and B, the fraction of closed PS II reaction centers.
At a macroscopic level,
F 2= P2rE2
and
E 2 = (1 - B)E2 pen + BE~l
If we consider the microstate (i.e., one PS II unit),
E~pen = J2 + (1 - B)E2Penp22 + BE2~P22
E2CJ = J2 + (1
- B)E2openP22 + BE2~lP22 + E2elP2bPb2
= E2 pe~ + E~T
After proper substitution of Eqs. (1A), (2A), (3A) and (4A)
into Eq. (15), we obtain
V = E2 - E2P~n B (5A)
cl open Fv
E2 - E 2 1 + ~ G(1-B)
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