Journal of Experimental Botany, Vol. 57, No. 6, pp. 1211–1223, 2006
doi:10.1093/jxb/erj104 Advance Access publication 21 March, 2006
REVIEW ARTICLE
Conservation and dissipation of light energy as
complementary processes: homoiohydric and
poikilohydric autotrophs
Ulrich Heber1,*, Otto L. Lange1 and Vladimir A. Shuvalov2
1
Julius von Sachs Institute of Biosciences, University of Würzburg, D-97082 Würzburg, Germany
Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino-na-Oke, Moscow Region, and
Laboratory of Biophysics, Belozersky Institute of Chemical and Physical Biology, Moscow State University,
Moscow 119992, Russia
2
Received 26 July 2005; Accepted 21 December 2005
Abstract
The relationship between photosynthetic energy conservation and thermal dissipation of light energy is
considered, with emphasis on organisms which tolerate full desiccation without suffering photo-oxidative
damage in strong light. As soon as water becomes
available to dry poikilohydric organisms, they resume
photosynthetic water oxidation. Only excess light is
then thermally dissipated in mosses and chlorolichens
by a mechanism depending on the protonation of a
thylakoid protein and availability of zeaxanthin. Upon
desiccation, another mechanism is activated which
requires neither protonation nor zeaxanthin although
the zeaxanthin-dependent mechanism of energy dissipation remains active, provided desiccation occurs in
the light. Increased thermal energy dissipation under
desiccation finds expression in the loss of variable,
and in the quenching of, basal chlorophyll fluorescence. Spectroscopical analysis revealed the activity
of photosystem II reaction centres in the absence of
water. Oxidized b-carotene (Car1) and reduced chlorophyll (Chl2), perhaps ChlD1 next to P680 within the
D1 subunit, accumulates reversibly under very strong
illumination. Although recombination between Car1
and Chl2 is too slow to contribute significantly to
thermal energy dissipation, a much faster reaction
such as the recombination between P6801 and the
neighbouring Chl2 is suggested to form the molecular
basis of desiccation-induced energy dissipation in
photosystem II reaction centres. Thermal dissipation
of absorbed light energy within a picosecond time
domain deactivates excited singlet chlorophyll, thereby
preventing triplet accumulation and the consequent
photo-oxidative damage by singlet oxygen.
Key words: Chlorophyll fluorescence, energy dissipation,
lichens, mosses, photoprotection, photosystem II, reaction
centre, zeaxanthin.
Introduction: photosynthesis versus
photoprotection
The central requirements for autotrophic plant life are
the availability of light, water, and CO2. Whereas light
absorption by photosynthetic pigments is proportional to
the incident light intensity, photosynthesis is not. Essentially, all absorbed light is used for photosynthetic reactions by unstressed leaves only as long as photon flux
is very low. By contrast, light is invariably far in excess
when plants are fully exposed to sunlight. CO2 then
becomes rate-limiting for photosynthesis. Light absorbed
by the photosynthetic apparatus may activate atmospheric
oxygen which, as singlet oxygen ð1 O2 Þ, is either indiscriminately oxidative (Krieger-Liszkay, 2005) or, in its univalently reduced form ðO
2 Þ, gives rise to reactive oxygen
radicals (Siefermann-Harms, 1987; Owens, 1996; Asada,
1999). The question of the nature of the mechanisms which
prevent excess excitation energy from damaging the
photosynthetic apparatus in hydrated plants by unspecific
oxidation has been and still is a subject of intensive research
(Demmig-Adams, 1990; Schreiber and Neubauer, 1990;
Krieger and Weiss, 1993; Björkman and Demmig-Adams,
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1212 Heber et al.
1995; Bruce et al., 1997; Gilmore and Govindjee, 1999;
Niyogi, 1999; Heber et al., 2001; Finazzi et al., 2004;
Holt et al., 2004, 2005; Krause and Jahns, 2004; Horton
et al., 2005; Pascal et al., 2005; and others).
Whereas the survival of the majority of higher plants
depends on the maintenance of hydration, many mosses,
lichens, and aerophilic algae tolerate full dehydration
(Green and Lange, 1995; Alpert, 2000). For metabolic
activity, they depend on moisture they take from their
environment. Even under temperate conditions, epilithic
or terrestrial lichens are hydrated for only 35–65% of
the annual time period, depending on lichen type and
habitat. Desert lichens experience less than 10% of their
life in conditions of water potential that allow for photosynthetic and/or respiratory activities (Evans and Lange,
2003). For the remainder of the time they are desiccated.
Survival under high irradiance depends on mechanisms
which permit rapid thermal dissipation of perceived radiation. The capability of poikilohydric autotrophs to cope
with strong irradiance is species-specific. Many lichens
are less easily damaged by strong light in the desiccated
state than under hydrated conditions (Demmig-Adams
et al., 1990). Tolerance of exposure has a strong adaptive
component and depends on habitat conditions or pretreatment (Gauslaa and Solhaug, 2004). Lichens from
shade habitats are damaged when they are exposed to
high light even when dehydrated (Gauslaa and Solhaug,
1999; Gauslaa et al., 2001).
In photosynthesis, only a few specialized chlorophyll
molecules are involved in converting light energy into
redox energy. A much larger number serves, together with
several carotenoids, to harvest light energy. Absorbed
excitation energy migrates to and is trapped by a limited
number of chlorophyll-containing reaction centres (RCs)
where energy conservation is initiated by charge separation within about 3 ps (Zinth and Kaiser, 1993). The RC of
photosystem II (PSII) where water is oxidized is composed
of two proteins, D1 and D2, and contains six chlorophylls,
two pheophytins, two b-carotenes, two quinones, and one
or two haems of cytochrome b559 (Fig. 1). Endergonic
oxidation of a chlorophyll dimer termed P680, or P, creates
an oxidant of a redox potential high enough to oxidize
water in several steps. The liberated electrons are transfered
first to one of the bound quinones of the RC, a plastoquinone (PQ) termed QA, then to QB and from there, through
a number of further steps involving also the reaction centre
of photosystem I, PSI, to a physiological electron acceptor
such as CO2.
As long as water is present as a donor molecule, its
oxidation reduces the oxidized primary electron donor,
P680+, which is created by charge separation in the RC of
PSII. This prevents P680+ from unspecific oxidative side
reactions which can destroy components of the photosynthetic apparatus. In the absence of effective thermal
energy dissipation, excessive excitation of light-harvesting
Stroma
D2
CP43
D1
CP47
Fe
QA
PQ
QB
Cytb559
Pheo
Pheo
Car/Car
Chlz
Chlz
Chl
Chl
P680
Tyr
Tyr
Mn
Mn
Mn e- Mn
Lumen
MSP
O2 + 4H+
2H2O
Fig. 1. Schematic representation of the PSII core complex with
chlorophyll-binding proteins CP43 and CP47, the D1 and D2 proteins
of the RC, the manganese protein of water oxidation and the protein
MSP (Vermaas, 1993: Reprinted, with permission, from the Annual
Review of Plant Physiology and Plant Molecular Biology Vol. 44 ª1993
by Annual Reviews www.annualreviews.org; pigment localization
according to Kamiya and Shen, 2003). It is widely accepted that
pheophytin (Pheo) is the primary electron acceptor, but recent evidence
attributes this role to a chlorophyll next to P680 (Shuvalov and Heber,
2003).
chlorophylls under strong irradiance leads to increased
long-lived triplet formation, not only within the pigment
bed but also within PSII RCs (Krieger-Liszkay 2005). If
triplet detoxification by carotenoids (Siefermann-Harms,
1987) cannot keep pace with triplet accumulation, highly
oxidative singlet oxygen will be created by
Chl + 3 O2 ! 1Chl + 1 O2:
Unspecific oxidation by 1 O2 will, finally, result in cell
death.
Obviously, the problem of over-excitation is exacerbated
when sun-exposed photosynthetic organisms desiccate. In
the present communication, conservation of light energy
in photosynthesis will only be considered briefly, but attention will be directed to problems of regulated thermal
energy dissipation in hydrated and desiccated photosynthetic organisms as the main means of photoprotection.
3
Conservation and dissipation of light energy
Chlorophyll fluorescence as a tool to assess the
effectiveness of thermal energy dissipation as
a sink for excess excitation energy
The energy of light absorbed by the photosynthetic
apparatus may either be used for photochemical charge
separation in the RCs, or it may be re-emitted as fluorescence, or it may be dissipated harmlessly in the form of
heat. As these processes are mutually exclusive, i.e. they
compete with one another, measurements of fluorescence
can give important information, not only on photosynthetic
electron flow but also on energy dissipation by the photosynthetic apparatus (Bradbury and Baker, 1981; Schreiber
et al., 1986; Krause and Weis, 1991; Krause and Jahns,
2004). Fluorescence is decreased, or quenched, when light
energy is used for photosynthetic electron transport and
when it is thermally dissipated. Both modes of fluorescence
quenching can be experimentally distinguished when
modulated light is used for measuring fluorescence
(Schreiber and Bilger, 1993; Krause and Jahns, 2003).
Modulated fluorescence emitted by hydrated plants is
at its minimum level (termed Fo) under strictly rate-limiting
light. Electron carriers of the photosynthetic electron
transport chain such as QA are largely oxidized under
very low light. The potential for charge separation in PSII
RCs is maximal. However, when actinic light is strong
enough to reduce QA fully, modulated fluorescence is at
its maximum level (termed Fm) because photochemical
charge separation between excited P, P*, and QA is no
longer possible. In this situation, fluorescence intensity is
determined by the state of energy dissipation. Fluorescence
is at the Fm level, when mechanisms of thermal energy
dissipation have not yet been activated. It is at the lower
F9m level when thermal energy dissipation competes effectively with fluorescence emission. Non-photochemical
fluorescence quenching NPQ = ðFm =F9m 1Þ describes the
1213
extent of energy dissipation. However, under desiccation,
F9m is often not distinguishable from F9o , which signifies
a quenched Fo level (see Figs. 4 and 5). In this case,
NPQ = ðFm =F9o 1Þ:
Thus, regulation of energy dissipation can be monitored
by following the changes in fluorescence, provided proper
precautions are taken to avoid interference by photochemical fluorescence quenching.
Use of light for photosynthesis
In rate-limiting light, almost all absorbed quanta are used
for photosynthesis by hydrated plants. As a result, the initial
slope of photosynthesis versus photon flux is practically
identical in many different plants. As the light intensity is
increased, the slope decreases. In fully sun-exposed leaves,
light is usually far in excess. Practically all absorbed light
energy needs to be converted into heat in desiccated
poikilohydric autotrophs if oxidative damage is to be
avoided. This requires highly effective mechanisms of
thermal energy dissipation. In fact, the quantum yield of
fluorescence is very low in desiccated poikilohydric plants.
Fluorescence is strongly quenched. It increases upon
hydration indicating rapid deactivation of the mechanisms
which are responsible for fluorescence quenching in the
desiccated state. Sensitive regulation of energy dissipation
ascertains the dominance of photosynthetic energy conservation after water has become available. Protons play
a key role both in photosynthesis and in the opposing
process of energy dissipation. When two molecules of
water are oxidized inside the closed thylakoid membrane
system of chloroplasts, four protons remain inside the
thylakoids. Eight more are pumped into the thylakoids as
the four electrons liberated during water oxidation are on
their path to reduce one molecule of CO2 (Fig. 2). Efflux of
TM
3 ADP, 3 Pi
AS
3 ATP
4 H+
8 H+
8 H+
4Θ
2H2O
ETC
CC
(HCHO) + H2O
4 H+
4Θ
CO2
O2
Fig. 2. Proton deposition during water oxidation within closed thylakoid membranes (TM), proton transport into the thylakoid interior during linear
electron transport and proton efflux during the synthesis of the ATP which is required for the reduction of CO2 to (HCHO). ETC, electron transport chain;
AS, ATP synthase; CC, carbon cycle; for further explanation, see text.
1214 Heber et al.
these protons lowers the acidification of the intrathylakoid
space and is sufficient, or almost sufficient (Allen, 2003a),
to power the endergonic synthesis of three molecules of
ATP which are needed for the reduction of one CO2 in
the so-called C3 pathway of carbon assimilation. Thus,
influx of protons into and efflux from the thylakoids are
largely balanced during carbon reduction. Thylakoid acidification is low because a relatively small proton gradient
satisfies the ATP requirements of carbon assimilation. The
alternative electron transport pathways of cyclic electron
transport and electron flow to oxygen support stronger
thylakoid acidification (Allen, 2003a; Heber, 2003). They
are regulated by the redox state of electron carriers. By
permitting protonation of the PsbS protein, they activate
thermal energy dissipation as will be shown below.
Thermal energy dissipation: role of
light-harvesting chlorophyll–protein
complexes
Light in excess of that used for photosynthesis is either
re-emitted as fluorescence or thermally dissipated so that
fluorescence decreases. When the main light-harvesting
complex of the photosynthetic membrane, LHCII is isolated in a detergent solution, it is strongly fluorescent. Upon aggregation, fluorescence is quenched. Fluorescence
life-times decrease accordingly from about 4 ns to values
ranging between 100 ps and 1.5 ns (Mullineaux et al.,
1993; Moya et al., 2001). Fluorescence is also quenched
in LHCII crystals (Pascal et al., 2005). The fluorescence
life-time was 890 ps compared to 4 ns for LHCII trimers.
In attempts to find a molecular basis for regulated energy
dissipation, aggregation phenomena are proposed to lead
to observed fluorescence quenching in intact organisms
(Horton et al., 1991, 2005). Still, the energy dissipation
indicated by a decrease of fluorescence life-times in isolated chlorophyll–protein complexes from 4 ns to about
100 ps or more is insufficient to drain excitation energy
sufficiently fast from open PSII RCs to afford photoprotection of PSII RCs. More effective mechanisms of
energy dissipation are observed in intact organisms.
Role of zeaxanthin in thermal energy dissipation
by hydrated plants
Although it has long been known that the concentration
of the xanthophyll zeaxanthin in photosynthetic membranes of higher plants reversibly increases in the light
at the expense of violaxanthin (Yamamoto et al., 1962;
Sapozhnikov et al., 1966), a role of zeaxanthin in thermal energy dissipation has been proposed by Barbara
Demmig at the University of Würzburg only in 1987
(Demmig-Adams, 1990). By now, an impressive amount
of documentation has become available not only on the
wide distribution of zeaxanthin-dependent thermal energy
dissipation within green plants, but also on its regulation
(Gilmore and Govindjee, 1999; Niyogi, 1999; Ruban et al.,
2002; Ma et al., 2003; Holt et al., 2004, 2005; Horton et al.,
2005). In summary, a special protein in the antenna of PSII,
the gene product of the PsbS gene, is thought to change its
conformation on protonation. Once protonated, it is capable of forming a complex with zeaxanthin which is active
as an effective sink for excitation energy. Sensitive regulation (Li et al., 2004) ensures that assimilation has precedence over thermal energy dissipation. Whereas linear
light-dependent electron transport from water to CO2 as
depicted in Fig. 2 is accompanied by balanced deposition
of protons inside thylakoids and proton efflux from the
thylakoids, additional thylakoid acidification by protoncoupled cyclic and pseudocyclic electron transport (Allen,
2003a; Heber, 2003) provides protons for protonation of
the PsbS-protein when CO2 becomes rate-limiting for
carbon reduction. Also, a low intrathylakoid pH is known
to cause the de-epoxidation of the xanthophyll violaxanthin
to zeaxanthin (Pfündel and Dilley, 1993). Availability of
zeaxanthin and protonation of the PsbS-protein make it
possible to form energy-dissipating centres which drain
excitation energy from PSII RCs.
However, activation of energy dissipation by protonation is not necessarily based on light-dependent electron
transport. It has recently been shown that CO2 which, at
a pK of 6.31, acts as a very weak protonating agent, is
capable of promoting non-photochemical quenching,
NPQ, in a moss, provided some zeaxanthin is present
(Bukhov et al., 2001a). Similar observations were made
with chlorolichens (Heber et al., 2000; Kopecky et al.,
2005). This is surprising because protonation of the
amino acids E122 and E226 of the PsbS-protein (Li
et al., 2002) is expected to require a much lower pH than
can be provided by CO2. NPQ formation increased and
the quantum efficiency of charge separation in PSII RCs
decreased with increasing CO2 concentration (Bukhov
et al., 2001a). Actinic light was not necessary. Removal
of the CO2 reversed the NPQ and increased the quantum
efficiency of charge separation in PSII RCs. Dithiothreitol,
an inhibitor of zeaxanthin formation (Yamamoto and
Kamite, 1972) inhibited NPQ formation by CO2. All this
showed that protonation governed energy dissipation. The
presence of zeaxanthin was essential, but a few molecules
of zeaxanthin per PSII RC were sufficient for effective
competition with open RCs for excitation energy (Bukhov
et al., 2001a).
Importantly, leaves which possess the zeaxanthin cycle
were very slow to respond to high concentrations of CO2
by increasing NPQ or decreasing charge separation in PSII
RCS when actinic light was absent. By contrast, responses
of the moss Rhytidiadelphus to CO2 were fast and dramatic
(Bukhov et al., 2001b). Figure 3 compares the extent
of the reversible suppression of charge separation by CO2
in the moss Rhytidiadelphus with that in a spinach leaf
Conservation and dissipation of light energy
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
10
20
30
40
50
60
Fig. 3. Transient charge separation in PSII RCs (measured as DF/Fm)
during 1 s light pulses versus CO2 concentration in air in the hydrated
moss Rhytidiadelphus squarrosus (filled circles) and in spinach leaves
(filled triangles). Actinic light was absent during the experiment, but
zeaxanthin was present (Bukhov et al., 2001b; reprinted with kind
permission of Springer Science and Business Media).
in the absence of actinic light. Apparently, regulation of
zeaxanthin-dependent energy dissipation by protonation
was different in the moss (or in lichens, Kopecky et al.,
2005) on one side and in higher plants such as spinach on
the other side. Also, protonation by CO2 suppressed Fo
fluorescence in mosses and lichens, but not in spinach or
other higher plants. Maximum rates of photosynthesis in
the different groups of photosynthetic organisms are known
to be different (Green and Lange, 1995; Lange, 2003;
Larcher, 2003). Maximum charge separation in PSII RCs
as expressed by DF/Fm is also different. It is consistently
lower in lichens and mosses than in higher plants (Jensen,
2002). Apparently, the balance between energy conservation and energy dissipation is tilted towards dissipation in
many poikilohydric autotrophs, whereas, in higher plants,
energy conservation assumes dominance over energy dissipation. It thus appears that sensitivity to excess light is
higher in the mosses and the lichens than in higher plants.
More sensitive regulation of thermal energy dissipation ensures protection against oxidative damage in these organisms.
Thermal energy dissipation in desiccated
poikilohydric autotrophs: simple consequence
of drying or the result of the activation of a
special mechanism?
After hydrated lichens or phototolerant poikilohydric
mosses have been slowly desiccated, variable fluorescence
DF=(FmFo) is lost and even Fo fluorescence is more or
1215
less dramatically decreased (see F9o in Figs 4 and 5),
depending on species and season. Whereas full quenching of
DF increases NPQ in hydrated plants from zero to less than
5 if Fo remains unchanged, NPQ may increase to values of
more than 50 during the desiccation of some lichens. The
increase in thermal energy dissipation revealed by the loss of
fluorescence yield is readily reversed by hydration.
Importantly, drying of leaves, or of poikilohydric
mosses, when these are in a photosensitive state, does not
result in much fluorescence quenching (Kopecky et al.,
2005; Heber and Shuvalov, 2005). This shows that the
large loss of fluorescence during desiccation of phototolerant poikilohydric organisms is not a simple result
of drying a photosynthetic membrane. Rather, it indicates
the activation of a specific mechanism, or of specific mechanisms, of energy dissipation.
Whereas many lichens are associated with a green
alga, others harbour a cyanobacterium as the photobiont.
Only those with a green alga possess a zeaxanthin cycle
(Demmig-Adams et al., 1990). Nevertheless, both types
of lichens exhibit strong desiccation-induced fluorescence
quenching. Figure 4 compares modulated chlorophyll
fluorescence of the desiccated chlorolichen Hypogymnia
physodes and the cyanolichen Peltigera neckeri before
and after hydration. A modulated measuring beam of a
very low average intensity (less than 0.005% of sunlight)
increased chlorophyll fluorescence to the level F9o : After
the initial fluorescence rise two short light pulses of
extremely strong actinic light (PPFD=13 000 lmol m2
s1, i.e. five times sunlight) were given while the lichens
were still dry. Although they were strong enough to excite
all chlorophylls several times, including those located
within the RCs, they caused no appreciable fluorescence
responses. Apparently, stable charge separation was suppressed in the desiccated lichens. Hydration increased
fluorescence strongly. Reversible pulse-induced fluorescence changes demonstrated that PSII RCs became rapidly
functional after hydration. After the Fo level had risen
during hydration, fluorescence was transiently quenched
below the elevated Fo level immediately after the 1 s light
pulses only in the Hypogymnia experiment of Fig. 4A,
but not in the Peltigera experiment of Fig. 4B. Transient
pulse-induced Fo quenching is typical for the presence of
active zeaxanthin-dependent energy dissipation (Katona
et al., 1992). The cyanolichen does not possess a zeaxanthin cycle.
Two different pathways of thermal energy dissipation in
poikilohydric autotrophs which possess a zeaxanthin
cycle
As long as sufficient light is available and hydration still
supports proton-coupled electron transport in poikilohydric
autotrophs which possess the zeaxanthin cycle, zeaxanthindependent energy dissipation is active while water is slowly
1216 Heber et al.
A
B
Fm
Fm
Fluorescence
Fo
Fo
10 min
Fo´
Fo´
on
off on
H2O
off
H2O
Fig. 4. Modulated chlorophyll fluorescence of the desiccated chlorolichen Hypogymnia physodes (A) and the desiccated cyanolichen Peltigera neckeri
(B) before and after hydration. A very weak modulated measuring beam (PPFD <0.1 lmol m2 s1) was turned on to elicit modulated fluorescence
(Schreiber et al., 1986). Strong 1 s light pulses (PPFD=13 000 lmol m2 s1) were given every 200 s both before and after hydration by a drop of water.
Note that pulse-induced fluorescence responses are absent while the lichens are still dry. For further explanation, see text.
A
B
50
Fm
Fluorescence
60
10 min
Fo
70
80
Fm
90
Fo´
Fo´
100
on
off
H2O
on
H2O
off
Fig. 5. Modulated chlorophyll fluorescence of the desiccated moss Rhytidium rugosum before and after hydration. In (A) the moss had been taken sundried from its habitat; in (B) it had been hydrated and kept for 2 d under dim light (PPFD=2.5 lmol m2 s1) before it was slowly dried in the dark.
Strong 1 s light pulses given every 200 s both before and after hydration failed to increase fluorescence before hydration. After hydration, they produced
less stable charge separation in (A) than in (B) indicating persisting inhibition of PSII activity in the sun-dried moss. For explanation, see text.
lost. Dissipating centres not only appear to survive desiccation but also to remain functional in the fully desiccated
state (Eickmeier et al., 1993; Deltoro et al., 1998; Kopecky
et al., 2005). Nevertheless, desiccation-induced thermal
energy dissipation does not depend on the presence of
zeaxanthin even in poikilohydric organisms which possess
the zeaxanthin cycle. This is shown in the experiment of
Fig. 5 for the sun-tolerant moss Rhytidium rugosum, but
Conservation and dissipation of light energy
can also be demonstrated in chlorolichens. The moss was
taken sun-dried from a sun-exposed habitat and used for
the experiment in Fig. 5A. A parallel sample was hydrated
and kept for 2 d in dim light in order to decrease or eliminate zeaxanthin. It was then slowly dried in darkness and
taken for the experiment in Fig. 5B. A low-intensity measuring beam increased chlorophyll fluorescence of the
desiccated mosses less in Fig. 5A than in Fig. 5B, indicating
that chlorophyll fluorescence was more strongly quenched
in the sun-dried moss than in the moss which had been
dried in darkness. Two very strong 1 s light pulses were as
ineffective in causing appreciable fluorescence responses
in the desiccated mosses as they were in the lichen experiments of Fig. 4. Apparently, stable charge separation was
suppressed, not only in the lichens but also in the desiccated
mosses.
Hydration by a drop of water changed the fluorescence
responses. A small rapid initial lowering of fluorescence
appeared to have optical reasons, but the following increase
is based on the recovery of function. Quenching was reversed by hydration. Pulse-induced fluorescence now
indicated charge separation in the PSII RCs and transient
reduction of oxidized QA both in the sun-dried and the
dark-dried moss. However, the extent of charge separation
as indicated by the size of fluorescence responses was
smaller in the sun-dried moss than in the dark-dried moss
(DF/Fm=0.14 and 0.42, respectively, in Fig. 5A and B
shortly after hydration). Appreciable transient Fo quenching following the strong light pulses occurred only in
Fig. 5A suggesting the presence of active zeaxanthindependent energy dissipation (Katona et al., 1992;
Kopecky et al., 2005). It was very small or absent in
Fig. 5B. Apparently, in the sun-dried moss, zeaxanthin
had been present. Lowered Fo fluorescence in Fig. 5A compared with Fo fluorescence in Fig. 5B suggests that it
had contributed to photoprotection during desiccation.
Nevertheless, the large reversal of quenching by hydration
in the dark-dried moss also reveals considerable desiccation-induced thermal energy dissipation which does not
depend on the presence of zeaxanthin.
Acquisition and loss of phototolerance in
poikilohydric mosses
When collected in rainy weather during the autumn or
winter seasons and then desiccated in the dark, the shadeadapted moss Rhytidiadelphus squarrosus was found to
be photosensitive. Desiccation did not result in appreciable
fluorescence quenching (Heber and Shuvalov, 2005).
Remoistening failed to increase Fo fluorescence. When
the moss was collected in the spring or summer, fluorescence decreased strongly whether or not light was present
during desiccation. Hydration reversed the fluorescence
quenching (see also Fig. 5 for Rhytidium rugosum). Labora-
1217
tory experiments with different shade-adapted mosses
species established interchangeability between the different
fluorescence responses (U Heber, VA Shuvalov, unpublished experiments). Exposing a phototolerant ‘summer’
moss in the hydrated state for prolonged periods of time
to dim light before drying it in darkness made it photosensitive, as shown by increased sensitivity to strong irradiation in the desiccated state. Damage became manifest by
the irreversible loss of fluorescence characteristics not
only after hydration but also in the desiccated state. Conversely, drying a photosensitive ‘winter’ moss under illumination made it more phototolerant, as shown by decreased
sensitivity to strong irradiation, whether or not zeaxanthin
was present.
Photosystem II reaction centres as possible
sites of desiccation-induced energy dissipation
Activation of desiccation-induced thermal energy dissipation during drying of shade-adapted mosses requires the
interaction of light and desiccation. A main factor involved
in zeaxanthin-dependent energy dissipation is protonation
of the PsbS protein. However, protonation does not appear
to be involved in desiccation-induced energy dissipation
because the protonophore nigericin did not prevent fluorescence quenching during drying. Another possibility to
explain the light requirement of the acquisition of phototolerance is redox regulation which is known to be common
in plant metabolism (Allen, 2003b). Illumination changes
the redox state of several components of the photosynthetic apparatus. In order to restrict reduction to a specific site,
the electron transport inhibitor DCMU was used. It is
known to block electron transport selectively between QA
and QB in the RC of PSII (Trebst, 1986). This restricts reduction to a component of the RC such as QA. Importantly,
desiccation of a DCMU-inhibited photosensitive moss
in the presence of light, which was sufficient to reduce
QA in the RC of PSII, but insufficient to induce zeaxanthin
synthesis, increased phototolerance in Rhytidiadelphus
squarrosus (U Heber and VA Shuvalov, unpublished
data). The experiment suggested the RC of PSII as the
site of desiccation-induced energy dissipation, but does
not rule out the possibility of a light requirement for conformational changes of chlorophyll–protein complexes
which may be involved in desiccation-induced energy
dissipation.
Spectral changes in fluorescence emission
during desiccation
Spectral changes in fluorescence excitation and emission
caused by desiccation of a chlorolichen and a cyanobacterium have been investigated by Bilger et al. (1989). The
results of low temperature fluorescence spectroscopy were
interpreted to indicate interruption of functional energy
1218 Heber et al.
transfer from the antenna of photosystem II or from accessory pigments to PSII RCs which were considered inactive.
These conclusions now appear to be in question in view
of more recently observed RC activity in dried leaves
(Shuvalov and Heber, 2003; Kopecky et al., 2005) and
desiccated mosses (Heber and Shuvalov, 2005; see also
below). Nevertheless, fluorescence spectroscopy still provides valuable information on energy dissipation. Figure 6
shows fluorescence emission spectra of two desiccated
mosses. The excitation wavelength was 440 nm. In the
lower spectrum of Fig. 6A for sun-dried Hypnum, fluorescence emission peaked at about 720 nm. The 685 nm
band of fluorescence emission shown in the upper spectrum
was largely absent. By contrast, when zeaxanthin had been
converted to violaxanthin by exposure to dim light before
drying Hypnum in the dark, a large maximum peaking
at 685 nm dominated the upper spectrum in Fig. 6A. The
720 nm emission was still present. In sun-dried Rhytidium
(Fig. 6B, lower spectrum), the 685 nm emission was less
strongly suppressed than in sun-dried Hypnum. When
10000
9000
A
Rhytidium was dried in the dark after previous exposure
to dim light to decrease zeaxanthin (upper spectrum of
Fig. 6B), the 685 nm peak was as large as in the Hypnum
experiment of Fig. 6A). The 720 nm emission was also
present. It is important to note that the spectra were taken
at room temperature where photosystem I is known to be
largely non-fluorescent. Therefore, emission at 720 nm is
thought to originate from PSII. In fact, excitation with
monochromatic far-red light up to about 740 nm was
shown to result in fluorescence emission from PSII (Heber
and Shuvalov, 2005). The observations shown in Fig. 6
suggest that, in desiccated mosses, zeaxanthin-dependent
energy dissipation drains excitation energy preferably from
centres which emit 685 nm fluorescence.
On heating desiccated mosses and lichens, fluorescence
increased reversibly. Charge separation in PSII RCs returned indicating temperature-induced reversal of thermal
energy dissipation (Heber and Shuvalov, 2005). From a
comparison of the shift of fluorescence emission towards
720 nm in desiccated mosses (Fig. 6) and lichens with the
activation energy of the reversible part of the temperaturedependent fluorescence increase, it was suggested that
energy is dissipated by dissipation centres which emit
720 nm fluorescence with a very low quantum yield.
Fluorescence (r.u.)
8000
Electron transport in PSII RCs in the
absence of water
7000
6000
5000
4000
3000
2000
1000
0
640
660
680
700
720
740
760
780
800
760
780
800
Wavelength (nm)
40000
B
Fluorescence (r.u.)
35000
30000
25000
20000
15000
10000
5000
0
640
660
680
700
720
740
Wavelength (nm)
Fig. 6. Fluorescence emission spectra of the desiccated moss Hypnum
cupressiforme (A) and Rhytidium rugosum (B). The lower spectra in (A)
and (B) are from sun-dried desiccated mosses, the higher spectra from
desiccated mosses, which had been hydrated and were then slowly dried
in the dark. For further explanation, see text.
Even very low light raised the fluorescence of dry leaves
(Kopecky et al., 2005) or desiccated photosensitive mosses
above the Fo level indicating efficient energy migration
to PSII RCs and reduction of QA. Once reduced, QA remained reduced in the dark or reoxidized only slowly. It
could be permanently kept in the fully reduced state by
low intensity background light. This was used to separate
other photoreactions of PSII from electron transport to
QA (Shuvalov and Heber, 2003). Light-dependent bleaching around 500 nm showed that b-carotene (Car), a constituent of PSII RCs, was photo-oxidized with a low
quantum yield, not only in dried spinach leaves (Fig. 7),
but also in dried spinach chloroplasts or subchloroplast
particles. Oxidation was almost proportional to light intensity up to PPFDs close to sunlight. While b-carotene
was photo-oxidized, kinetically related formation of
bands at 450 and 810 nm and bleaching at 436 and
680 nm suggested the accumulation of a reduced chlorophyll. Sieve effects, which are stronger in the blue than
in the green region of the spectrum, made quantitative
analysis of the relationship between chlorophyll reduction
and carotene oxidation difficult in dry leaves. However,
spectra obtained with subchloroplast particles suggested
a ratio Chl/Car+ close to 1. No indication was obtained
for the photoaccumulation of reduced pheophytin. Also,
redox changes around 559 nm were notably absent
suggesting that cytochrome b559 in the RC of PSII
1219
Fe
QB
QA
Pheo D1
Decrease
Absorption
Increase
Conservation and dissipation of light energy
Pheo D2 Cytb559
Car cis/Car trans
Chlz D2
Chlz D1
400
450
500
550
Chl D1
Chl D2
nm
P680
Fig. 7. Light–dark difference spectrum of a dried spinach leaf. Owing to
pronounced sieve effects particularly in the blue region of the spectrum,
the positive absorption band at 450 nm is strongly suppressed compared
with the bleaching of the carotene band at 500 nm. Bleaching at 500 nm
decreased absorption by DA=33103.
Tyr D1
Tyr D2
4 Mn
remained in the oxidized state while reversible redox reactions of b-carotene and chlorophyll were observed. On
darkening, Car+ was reduced and Chl was oxidized with
half-times below 1 s (Shuvalov and Heber, 2003).
The data were interpreted as showing that a chlorophyll
next to P680 is the primary electron acceptor in the RC
of PSII rather than pheophytin, up to now considered the
primary electron acceptor in higher plants. Charge separation in PSII resulted in the initial formation of the radical
pair P680+Chl. After Car was oxidized in the dry leaves
by P680+, reduction of Car+ by Chl completed an electron
cycle within the RC of PSII (Fig. 8). By decreasing the
concentration of P680+, both the fast recombination of
the closely spaced radical pair P680+Chl and the slower
recombination between the more widely spaced Car+ and
Chl within PSII RCs are photoprotective reactions. However, the slower recombination reaction is not expected
to contribute significantly to energy dissipation. Details
on spatial relations in the PSII RC have been published
by Kamiya and Shen (2003). Edge to edge distances
between P680 and ChlD1 are about 5 Å, between P680
and PheoD1 about 9 Å, and between ChlD1 and Car
about 23 Å (Christian Fufezan, University of Freiburg,
Germany, personal communication). The recombination P680+Chl!P680 Chl is thought to compete with
and actually eliminate, in desiccated organisms, the recombination of the pair P680+Pheo which is known to
be accompanied by fluorescence emission and by the
formation of highly reactive singlet oxygen (KriegerLiszkay, 2005).
Transmittance spectra very similar to the spectrum
shown in Fig. 7 for a dried spinach leaf were also obtained
with the poikilohydric fern Polypodium vulgare (Shuvalov
and Heber, 2003) and with the desiccated moss Rhytidia-
Fig. 8. Schematic representation of electron flow in PSII RCs. D1 and
D2 refer to the D1 and D2 proteins to which pigments are attached.
Proteins are not shown. Pigment localization is according to Kamiya and
Shen (2003). Drawn out arrows depict the presently accepted view of
linear electron transport from water to external electron acceptors
although it is likely that the anion radical of ChlD1 is an obligatory
intermediate even of this pathway. Dashed arrows show electron flow
when water is absent. Charge separation between P680 and ChlD1 and
subsequent fast recombination of the charges are emphasized by an arrow
pointing in two directions. These reactions are thought to form the basis
of RC-based thermal energy dissipation in desiccated poikilohydric
autotrophs.
delphus squarrosus, but only after the moss had been
slowly dried in the dark. Owing to strong sieve effects,
the fern and moss spectra were well resolved only in the
500 nm region. Attempts to obtain transmission spectra
from sun-dried mosses were unsuccessful because stable
charge separation in the RCs was suppressed as a consequence of increased desiccation-induced energy dissipation. Desiccated lichens do not transmit weak measuring
light. However, reflectance could be used to obtain information on photoreactions. Strong desiccation-dependent
quenching made it necessary to use extremely high photon
flux densities for analysis. In this situation, light-dependent
band formation around 800 nm was observed. Dark relaxation of the band had several phases. Since both chlorophyll cations and anions absorb in this region, no reliable
assignments could be made, although a contribution of
P700+ could be excluded by doing measurements in the
presence of a background of far-red light.
Formation of an absorption band was also observed close
to 950 nm where carotenes are known to absorb (Tracewell
et al., 2001). Absorption increased in mosses and lichens
in proportion to light intensity as carotene oxidation had
1220 Heber et al.
done in dried spinach leaves (Shuvalov and Heber, 2003)
showing that the quantum efficiency of oxidation was low.
The reaction was observed whether or not attempts had
been made to eliminate zeaxanthin. Dark relaxation was
too fast to be resolved by the available instrumentation. The
reflectance data suggest that the electron cycle within
the RC of PSII, which has been observed in dry spinach
leaves, is faster in desiccated mosses and lichens.
recently demonstrated a zeaxanthin-independent energy
dissipation pathway in hydrated leaves which is localized
in or near PSII RCs. Nothing definite is known yet about
the site of desiccation-induced energy dissipation in desiccated poikilohydric autotrophs, but there are arguments
to suggest that it also proceeds in PSII RCs where charge
separation results in the oxidation of P680 and, initially, in
the reduction of a chlorophyll (Shuvalov and Heber, 2003):
P680Chl ! P680 + Chl
Significance of Fo suppression: time scale of
thermal energy dissipation
before electrons move to pheophytin according to
As has been mentioned above, Fo fluorescence elicited
by very low PPFDs of preferably less than 0.1 lmol m2
s1 describes a situation in which essentially all PSII
RCs are open in hydrated photosynthetic organisms while
QA is oxidized. Minimum or Fo fluorescence is in equilibrium with energy capture by the reaction centres, which
is known to result in charge separation within about 3 ps
(Zinth and Kaiser, 1993). Nevertheless, protonation by
high concentrations of CO2 quenched Fo fluorescence in
hydrated mosses and lichens by activating zeaxanthindependent energy dissipation (Heber et al., 2000; Bukhov
et al., 2001a). This proves that the dissipating centres
formed as a result of protonation were capable of draining
excitation energy even from open PSII reaction centres. It
means simultaneously that energy dissipation was faster
than energy capture by PSII RCs.
Fo fluorescence is quenched not only by activating
zeaxanthin-dependent energy dissipation in hydrated
mosses and lichens, but more strongly in desiccated phototolerant mosses, chlorolichens and cyanolichens, whether
or not zeaxanthin is present, and whether or not drying
is done in near darkness to keep PSII RCs in the open state.
It was concluded that desiccation of poikilohydric autotrophs results in energy loss from dissipating centres which
is much faster than energy trapping by normally functioning PSII RCs.
Using leaves of transgenic Arabidopsis thaliana, Holt
et al. (2005) concluded from measurements of transient
absorption changes in the picosecond range, that charge
separation in an activated dissipation centre consisting of
the protonated PsbS-protein, zeaxanthin, and chlorophyll
results, within about 1 ps, in zeaxanthin oxidation and
chlorophyll reduction:
This secondary reaction appears to be suppressed by desiccation. In this situation, fast recombination of the initial
charge-separating reaction becomes very probable. It is
proposed that the reaction
+
ðPsbS-P3Zea3ChlÞ ! ðPsbS-P3Zea 3Chl Þ:
This fast charge separation initiates energy dissipation
whereas non-fluorescent recombination to (PsbS-P3Zea3
Chl) completes thermal energy degradation within about
150 ps.
Zeaxanthin-dependent energy dissipation is thought to
be localized in the antenna of PSII (Horton et al., 2005;
but see Holt et al., 2004), but Finazzi et al. (2004) have
Chl + Pheo ! Chl + Pheo
+
P680 Chl ! P680Chl
is responsible for fast energy dissipation in PSII RCs of
desiccated poikilohydric autotrophs (Fig. 8). This is in
close analogy to the recombination reaction which according to Holt et al. (2005) forms the molecular basis of
zeaxanthin-dependent energy dissipation. The simultaneous occurrence of two photoprotective pathways in desiccated chlorolichens (Kopecky et al., 2005) and mosses
and the strong suppression of Fo fluorescence in these
organisms suggest that RC-based energy dissipation operates in a picosecond time domain not very different from
that reported by Holt et al. (2005) for zeaxanthin-dependent
energy dissipation.
Concluding remarks
During the first decennium of modern photosynthesis
research, the main emphasis was on the elucidation of the
principal pathways of electron transport and of the reduction of CO2 and other physiological electron acceptors.
Later, much attention was focused on the mechanisms
involved in regulating the pathways. Always, the use of
light energy for biosynthetic purposes was in the foreground of interest although it was soon recognized that light
can also damage the photosynthetic apparatus. No mutants
devoid of carotenoids have ever been observed which can
survive illumination in an oxygen-containing atmosphere.
Also, agents interfering with carotenoid biosynthesis
are effective herbicides. Although this emphasized the
role of carotenoids in providing protection against lightsensitized oxidation, the existence of physiological mechanisms able to regulate not only photosynthesis but also
the thermal degradation of excess light energy was overlooked until fluorescence became a widely applied tool in
photosynthesis research. Then it became obvious that
changes in chlorophyll fluorescence were not only related
to photochemical light use but also gave information on
Conservation and dissipation of light energy
hitherto unknown mechanisms involved in protecting the
photosynthetic apparatus against radical damage (Krause
and Weis, 1991; Krause and Jahns, 2004). As long as water
is available and photon flux is low, light energy absorbed
by the light-harvesting pigment system of PSII is trapped
primarily by PSII RCs where charge separation takes
place, initiating energy conservation. Water is oxidized and
oxygen is evolved (Fig. 1). Zeaxanthin-dependent thermal
energy dissipation is activated in the antenna of PSII only
at higher photon flux densities by protonation of the PsbS
protein. This occurs when alternative electron transport
processes decrease the intrathylakoid pH so as to permit
protein protonation. This governs thermal energy dissipation under conditions of hydration. Once they have been
activated, zeaxanthin-containing dissipation centres remain
functional even after desiccation.
Loss of water during desiccation of poikilohydric
autotrophs activates another mechanism of thermal energy
dissipation which, like zeaxanthin-dependent energy dissipation, finds expression in reduced chlorophyll fluorescence. It is proposed to be localized in the RCs of PSII.
Loss of water during desiccation, in combination with the
reduction of an RC component such as QA, appears to
change the conformation of PSII RCs so as to transform
them from energy-conserving to energy-dissipating centres.
Figure 8 compares the established view of linear electron
transport in PSII RCs of hydrated photosynthetic organisms
with electron transport in the RCs of desiccated poikilohydric autotrophs. The latter is thought to form the basis for
desiccation-induced thermal energy dissipation. Together
with zeaxanthin-dependent energy dissipation, energy
dissipation in PSII RCs protects poikilohydric mosses and
chlorolichens from photo-oxidation when water is absent.
Both mechanisms are important for their ability to survive
full sunlight for prolonged periods of time in the desiccated
state. Cyanolichens appear to be protected primarily by the
desiccation-activated mechanism.
Acknowledgements
Our research was supported by Deutsche Forschungsgemeinschaft,
Fonds der Chemischen Industrie and Russian Fund for Basic
Research. Professor Hedrich, Würzburg, provided the working
facilities. We also acknowledge co-operation with Drs Pfündel,
Würzburg, and Bukhov and Azarkovich, Moscow. Dr Zellner,
Würzburg, kindly helped in identifying several species of mosses
and lichens.
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