Download Molecular design of the photosystem II light

Journal of Experimental Botany, Vol. 56, No. 411,
Light Stress in Plants: Mechanisms and Interactions Special Issue, pp. 365–373, January 2005
doi:10.1093/jxb/eri023
Advance Access publication 22 November, 2004
Molecular design of the photosystem II light-harvesting
antenna: photosynthesis and photoprotection
Peter Horton* and Alexander Ruban
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank,
Sheffield S10 2TN, UK
Received 21 April 2004; Accepted 2 September 2004
Abstract
The photosystem II (PSII) light-harvesting system carries out two essential functions, the efficient collection
of light energy for photosynthesis, and the regulated
dissipation of excitation energy in excess of that which
can be used. This dual function requires structural and
functional flexibility, in which light-harvesting proteins
respond to an external signal, the thylakoid DpH, to
induce feedback control. This process, referred to as
non-photochemical quenching (NPQ) depends upon the
xanthophyll cycle and the PsbS protein. In nature, NPQ is
heterogeneous in terms of kinetics and capacity, and
this adapts photosynthetic systems to the specific
dynamic features of the light environment. The molecular features of the thylakoid membrane which may enable this flexibility and plasticity are discussed.
Key words: Light-harvesting complex, non-photochemical
quenching, photoprotection, thylakoid membrane, xanthophyll
cycle.
Introduction
During the evolution of oxygenic photosynthesis, there
have been two conflicting demands placed upon the
molecular machinery of photosystem II (PSII). On the
one hand efficient collection of sunlight is needed to deliver
excitation energy to the photosynthetic reaction centre at
a rate sufficient to drive electron transport at the highest
sustainable rate. On the other hand, excited states of
chlorophyll and the presence of molecular oxygen provide
a potentially lethal cocktail that can irreversibly damage the
proteins, lipids, and pigments of the photosynthetic membrane. To combat this, constitutive features of the photosystem, such as the presence of carotenoids to scavenge
singlet oxygen and trap triplet states of chlorophyll, are
combined with other photoprotective mechanisms which
are induced under high levels of illumination to bring about
the dissipation of excess chlorophyll excited singlet states.
In this paper those features of molecular design that
promote efficient light harvesting will be reviewed, and
then it will be shown how the dynamic nature of this system
allows for an inducible enhancement of the rate of dissipation of excited states. It will be shown how these features
of design differ in different classes of organism, and these
differences will be rationalized in terms of evolution within
particular ecological niches.
The molecular principles of light harvesting
The chlorophyll a and b molecules, bound to the pigment–
protein complexes of the light-harvesting system, are
arranged in three-dimensional space to serve as an antenna;
gathering light over a large absorption cross-section, and
‘funnelling’ the absorbed energy to the reaction centres.
Energy transfer between pigment molecules, both between
and within individual complexes is highly efficient. Equilibration of energy within the antenna is rapid, and tends
towards the red-shifted lower energy excited states of the
reaction centre core complex (van Amerongen and van
Grondelle, 2001). This highly efficient antenna depends
entirely on the specific structures of the apo-proteins of the
light-harvesting complexes: they must bind sufficient pigments to maximize absorption; they must provide for the
optimal orientation and configuration of pigments for
energy transfer; they must tune the excited state energy
levels of the pigments to promote energy transfer; and they
must permit protein–protein contacts to allow appropriate
routes of energy transfer between complexes.
There are important constraints placed upon this design.
Most notable is that the chlorophyll molecules must be
* To whom correspondence should be addressed. Fax: +44 (0)114 222 2787. E-mail: p.horton@sheffield.ac.uk
Journal of Experimental Botany, Vol. 56, No. 411, ª Society for Experimental Biology 2004; all rights reserved
366 Horton and Ruban
arranged so as to prevent the tendency for excited states
to dissipate by non-radiative decay. In the main lightharvesting complex, LHCII, the chlorophyll concentration
is approximately 0.6 M: if chlorophyll was dissolved in an
organic solvent at this concentration all absorbed energy
would be dissipated in this way, a process described as
concentration quenching (Beddard and Porter, 1976). This
occurs via close chlorophyll–chlorophyll interactions, dimers or excimers. Clearly the apoprotein provides a specific
‘solvent’, an environment that allows a high chlorophyll
concentration without the probability of quenching by such
associations. This environment arises from the formation of
hydrophobic binding pockets, liganding of the chlorophyll
Mg atoms by amino acid residues of the alpha helices and
hydrogen bonding to the porphyrin ring (Liu et al., 2004). It
is useful to regard chlorophylls as the ‘cofactors’ of the lightharvesting ‘enzymes’, and many parallels exist between
these and other proteins with essential cofactors, such as
haem proteins and flavoproteins.
PSII macro-organization
The basic structural unit of PSII is the so-called LHCII–PSII
supercomplex (Hankamer et al., 1997). This is a macromolecular dimer, consisting of the D1/D2/CP43/CP47 PSII
core complex and several light-harvesting proteins, one
copy of the minor monomeric complexes CP26 and CP29,
and one of the major trimeric complex, LHCII. In the granal
membrane of the chloroplast, the supercomplex is associated with further LHCII trimers and the monomeric CP24, to
form ‘megacomplexes’, which frequently display a semicrystalline order (Boekema et al., 2000).
The subunits of the PSII antenna are therefore associated
together with increasing levels of complexity. For example,
LHCII monomers form trimers, which are themselves
associated, on the one hand, with the monomeric complexes
and core complexes and on the other, with more LHCII
trimers, to form a 2-D organization in the thylakoid membrane. Finally, the stromal-facing surfaces of membranes are
appressed together to form the grana, with contacts between
LHCII and between LHCII and PSII cores of the adjacent
membranes. This three-dimensional macrostructure has
been referred to as the macrodomain (Garab and Mustardy,
1999). The tendency for oligomerization is displayed by
LHCII, which can in vitro self-assemble into 2-D and 3-D
structures that have a strong resemblance to thylakoid
membranes (Burke et al., 1987). Equally, oligomers of
LHCII can be prepared following mild detergent treatment
of thylakoid membranes (Ruban et al., 1999). The trimeric
state of LHCII appears to be essential for this organization of
PSII. When the main proteins that comprise LHCII are
removed by expression of antisense genes, it was found that
the amount of the normally monomeric CP26 dramatically
increased in level and it became trimeric (Ruban et al.,
2003). This change in expression of photosynthetic genes
must be a response to the LHCII deficiency, as the plant
attempts to compensate for the loss of an essential protein.
Amazingly, this response allows the wild-type oligomeric
PSII structure and the granal membrane to be maintained,
emphasizing the importance and robustness of the thylakoid
macrostructure.
Molecular accessories for regulating light
harvesting
In addition to the main protein complexes, both the PSII core
and the light-harvesting system contain a number of other
proteins, pigments, and lipids (Hankamer et al., 1997).
Whereas some of these may have essential roles in the
structure and function of the light-harvesting systems (e.g. a
specific phospholipid is required for LHCII trimerization;
Remy et al., 1982) and DGDG may enable association
between trimers (Liu et al., 2004), others appear to have
another role, that of tuning the properties of light-harvesting
proteins for regulation. Specific lipids appear to be necessary
for certain dynamic events within the 3-D organization of
LHCII (Simidjiev et al., 2000). Peripherally associated with
LHCII are the carotenoids of the xanthophyll cycle (Ruban
et al., 1999), which plays a vital role in photoprotective
energy dissipation (Demmig-Adams, 1990), observed as the
non-photochemical quenching of chlorophyll fluorescence
(NPQ). This cycle is the reversible de-epoxidation of
violaxanthin (found under light-limiting conditions) into
zeaxanthin (found under light-saturating conditions) that is
correlated with the induction of NPQ. Violaxanthin deepoxidase catalyses this reaction, and this enzyme may have
a specific docking site on LHCII (Liu et al., 2004). A group
of LHC-related proteins encoded by the members of the lhc
gene super family (Jansson, 1999), including the ELIPS, the
Lil proteins, and PsbS are also involved in stress responses
(Heddad and Adamska, 2002), with PsbS having a key role
in NPQ. Protein kinases also interact with LHCII (Bennett,
1983) and CP29 (Croce et al., 1996), phosphorylating them.
In the case of LHCII it is well-known that the redox
regulation of kinase activity provides the basis for regulating
the distribution of absorbed energy between PSII and PSI
(Horton and Black, 1980), phosphorylation modulating the
structure of the complex, and its interaction with other
photosystem proteins (Allen and Forsberg, 2001). Phosphorylation of CP29 appears to be activated under cold
stress, causing a structural change in the protein, which may
have a photoprotective role (Croce et al., 1996).
Changes in structure as the basis for regulation
of light harvesting
Whereas the higher order structure of PSII was first thought
to be important only in increasing the efficiency of light
harvesting, more recently it has been suggested that it
provides the essential dynamic properties involved in
Molecular design of the PSII light-harvesting antenna
regulation (Horton, 1999). When isolated light-harvesting
complexes self-assemble into oligomers in vitro, there is
a decrease in fluorescence yield (Mullet and Arntzen, 1980;
Ruban et al., 1992), resulting from a decrease in excitation
lifetime by non-radiative decay. Since, in vivo, the complexes also exist in an oligomeric state (Dekker et al., 1999;
Ruban et al., 1999), it could be proposed that increased
energy dissipation would result. Indeed, it has been recognized that the maximum fluorescence lifetime of chlorophyll
in vivo is always less than free chlorophyll or chlorophyll in
unaggregated LHCII (Horton and Ruban, 1994). Perhaps the
advantages of oligomeric organization outweigh the disadvantage of some waste of absorbed excitation energy.
More interesting, however, is the notion that structural
changes within the oligomeric antenna could provide
a physiological mechanism for regulating the partitioning
of energy between utilization in photosynthesis and dissipation by NPQ (Horton et al., 1991, 1996). Indeed, there
are many remarkable similarities between NPQ and in vitro
fluorescence quenching in LHCII and other antenna complexes (Horton et al., 1999). Whilst the exact mechanism of
energy dissipation in these complexes remains to be determined, it almost certainly is equivalent to the process of
‘concentration’ quenching discussed above. Hence, given
that the precise 3-D structure of the protein prevents such
quenching, it is easy to imagine how (relatively subtle)
changes in the structure could induce quenching. Examination of the structural model of LHCII reveals the presence
of several candidate quenching pairs of chlorophyll molecules (Kuhlbrandt et al., 1994; Liu et al., 2004). Although
the pair of Chl a 613/614 has been put forward as a potential
quenching site (Liu et al., 2004), it has previously been
suggested that the Chl a 610/611/612/lut1 domain was the
site of quenching (Wentworth et al., 2003). It was shown
how the differing tendencies of trimeric and monomeric
LHCII to quench could be explained if the conformation of
quenching locus was controlled by the Lut 2 domain
(Wentworth et al., 2003). This idea was consistent with
earlier observations of altered fluorescence yield of complexes when the Lut 2 site binds different xanthophylls
(Moya et al., 2001; Morosinotto et al., 2002; Formaggio
et al., 2001; Caffarri et al., 2004).
As a guiding principle, it was suggested that NPQ was
a response of the structure of the PSII light-harvesting
antenna, resulting in altered pigment interactions within
protein subunits and caused by the change in external
environment and the binding of protons (because of the
increase in transthylakoid DpH) (Horton et al., 1991; Horton
and Ruban, 1992). Of course, the notion that protein
structure and function can be altered by pH change is well
established and, extrapolating further, it is suggested that the
oligomeric nature of the light-harvesting system may allow
co-operativity in proton binding and, most importantly,
allosteric regulation by other ligands (Ruban and Horton,
1995a; Ruban et al., 2001; Horton et al., 2000).
367
A conceptual model was formulated, comprising four
conformational states of the light-harvesting system, with
different fluorescence yields, differing in de-epoxidation
and protonation state (Horton et al., 1991). The action of
zeaxanthin was explained, it is an allosteric activator of
quenching binding to a peripheral site on LHCII or another
light-harvesting antenna protein. There is considerable
experimental support for this hypothesis, although direct
participation of zeaxanthin in the quenching mechanism
cannot be excluded (Frank et al., 1994; Ma et al., 2003).
Paramount in this evidence is the startlingly different
kinetic properties of qE observed in thylakoids isolated
from leaves pretreated to have different de-epoxidation
states (Ruban et al., 2001): qE forms more rapidly, at
a lower DpH and with reduced co-operativity in the
presence of zeaxanthin, all characteristics of it being an
allosteric activator. Such properties also explain the kinetics
of NPQ formation upon illumination of dark-adapted
leaves, and the accelerated rate of formation upon a second
illumination following a brief dark interval in which DpH,
but not de-epoxidation is relaxed (Ruban and Horton,
1999). Recently it has been suggested that, rather than
being a peripheral site on an antenna protein, the internal
Lut 2 site discussed above provides the allosteric binding
site for zeaxanthin (Formaggio et al., 2001). Indeed it has
been shown that when the Lut 2 site in CP26 is occupied by
zeaxanthin, there is a decrease in fluorescence lifetime,
compared with when violaxanthin is bound (Moya et al.,
2001; Crimi et al., 2001). In this model, reversible
exchange of carotenoid binding to this site is proposed
(Bassi and Caffarri, 2000), and in vitro this has been shown
to be promoted at low pH (Morosinotto et al., 2002).
It is interesting, and rather unexpected, that there is no
evidence for any one light-harvesting complex having an
obligatory role in NPQ, at least in higher plants; wild-type
levels of NPQ are found in Arabidopsis plants expressing
antisense genes for CP29, CP26, and LHCII (Andersson
et al., 2001, 2003). Similarly, chl b less mutants, which
have only low levels of all Lhcb proteins show NPQ
(Lokstein et al., 1993). This indicates that no single Lhcb
protein can be the unique site of either energy dissipation or
zeaxanthin activation. Furthermore, it suggests that NPQ
could be ‘shared’ by all antenna proteins, including those of
the PSII core, since the above principle of control of
concentration quenching can apply to them all. It remains to
be shown what the minimum structural unit for NPQ is.
In vitro studies also show how each antenna subunit has
only a small capacity for zeaxanthin-induced quenching
(Wentworth et al., 2000; Crimi et al., 2001), but that this is
amplified when subunit interactions are enabled (Ruban
and Horton, 1992; Moya et al., 2001). Indeed, this suggests
that the intra- and inter-subunit conformational changes act
in a concerted manner, for example, a quenching locus in
LHCII or CP26 could be controlled by the Lut 2 domain,
which is in turn affected by the xanthophyll bound there,
368 Horton and Ruban
the binding of xanthophylls at the more peripheral neoxanthin (N1) and violaxanthin (V1) sites, and interactions
with neighbouring protein subunits.
The role of PsbS and zeaxanthin activation
Arabidopsis plants with mutation in the PsbS gene (npq4
mutants) show none of the rapidly reversible type of NPQ
known at qE, despite having wild-type levels of zeaxanthin
and DpH, and unaltered levels of light-harvesting and
reaction centre complexes (Li et al., 2000). It was suggested that PsbS provides the site of NPQ, by binding both
protons and zeaxanthin, and interacting with a chlorophyllcontaining complex to introduce a new quenching pathway.
This hypothesis for the role of PsbS has been subject to
intensive investigation. Evidence for proton binding has
come from the identification of binding sites for DCCD
(Dominici et al., 2002), and from the inhibition of qE by
site-directed mutagenesis of potential proton binding residues (Li et al., 2002a). The pigment-binding properties of
PsbS are unclear; purified PsbS appears to contain no
chlorophyll or carotenoid.
Finding the location of zeaxanthin binding sites has been
an important goal in understanding NPQ. It has been shown
that isolated LHCII and the minor complexes CP26 and
CP29 contain violaxanthin (Ruban et al., 1994, 1999; Bassi
and Caffarri, 2000). Only some of this is available for
de-epoxidation, shown to include that loosely bound to
the complex (Ruban et al., 1999; Caffarri et al., 2001), and
there is evidence that violaxanthin bound to the Lut 2
site on CP26 and CP24 could exchange for zeaxanthin
(Morosinotto et al., 2002). However, such studies of
zeaxanthin binding sites could be misleading, since it is
likely that only a small proportion of the zeaxanthin pool is
involved in NPQ (Gilmore, 2001). This subpopulation of
zeaxanthin has been studied using a spectroscopic approach. It is well known that qE is very strongly correlated
with an absorption change at 535 nm (Bilger et al., 1988;
Ruban et al., 1993), whilst it was considered to arise from
selective light scattering, its proximity to the carotenoid
absorption bands, suggested it may be electronic in origin.
Indeed, the DA535 spectrum could be modelled as a red
shift in 1–2 carotenoid molecules (Ruban et al., 2002).
Resonance Raman spectroscopy has been used to identify
the absorption bands from each of the xanthophylls (Ruban
et al., 2000). In leaves, induction of qE was associated with
the appearance of a zeaxanthin species selectively excited at
528 nm. This signal was absent in the npq4 mutant. These
data not only proved the electronic nature of DA535, but
also showed that it most likely arose from a small population of red-shifted zeaxanthin molecules (Ruban et al.,
2002). It is suggested that this was indicating the activation
of zeaxanthin, required to fulfil its role in qE. The Raman
signal also indicated that this species was found in a rather
specific configuration, indicating binding to a protein com-
ponent of the membrane. In order to test whether this site
could be on PsbS, zeaxanthin was reconstituted with
a purified sample of this protein; a red-shifted absorption
band was created with an identical Raman signature to
DA535 (Aspinall-O’Dea et al., 2002).
Although specific binding to another protein site is not
excluded, these data suggest that the activator role of
zeaxanthin in qE requires that it be bound to PsbS. This
binding, in vivo, requires DpH, most likely because of
proton binding to PsbS, and confers upon zeaxanthin very
new configurational and excited state features. How these
properties are related to its mechanism in qE is currently
unknown. On the one hand, these features may be needed
for zeaxanthin to quench chlorophyll excited states on
a neighbouring antenna complex directly (Ma et al., 2003).
On the other hand, protonated PsbS may ‘deliver’ zeaxanthin to its active site in the antenna: in this case it again
may be a direct quencher, or as suggested above, it may
fulfil an allosteric activating role. Finally, a protonated zea/
PsbS complex may have an allosteric role. In the latter two
cases, PsbS would be regarded as the regulatory subunit of
the light-harvesting antenna. Further work is needed to
distinguish these possibilities.
Comparative biochemistry and physiology
of NPQ
NPQ is observed in all higher plants; it has been detected in
a wide range of species in many different plant types, in
a wide range of habitats (Demmig-Adams, 1998; Johnson
et al., 1993). It has also been found in lower plants
(Campbell et al., 1998; El Bissati et al., 2000), green algae
(Niyogi et al., 1997), and diatoms (Casper-Lindley and
Björkman, 1998; Arsalane et al., 1994). It seems reasonable
to assume that the underlying molecular mechanisms are
the same in all cases. Nevertheless a comparative study of
NPQ in different species has proved to be rewarding,
different aspects of NPQ appear to be enhanced in certain
species, making them good models for further study.
Moreover, in such cases, the knowledge obtained, can give
new approaches to gaining fundamental information on the
mechanism of NPQ.
NPQ has been described as being the sum of two
different processes (Horton et al., 1996). In addition to
the rapidly relaxing qE type discussed above, there is
further NPQ that relaxes very slowly when excess light is
removed. This latter type, called qI, is usually much smaller
than qE, and is attributed to a more sustained effect of
excess light. Therefore qI has been associated with ‘photoinhibition’ rather than ‘regulation’, and was thought to arise
from quenching by photodamaged PSII reaction centres.
However, it was suggested that qI, like qE, may not be an
effect of excess light stress, but an adaptive response to it
(Horton et al., 1988, 1996; Ruban et al., 1993). Thus,
perhaps quenching by a photoinhibited reaction centre is
Molecular design of the PSII light-harvesting antenna
a useful response that provides photoprotection of the
antenna and thylakoid membrane (Chow et al., 2002).
Since strong evidence of the accumulation of damaged PSII
under photoinhibitory conditions was absent, it was further
suggested that qI occurs in the antenna, perhaps even by the
same or similar mechanism to qE (Demmig and Winter,
1988; Ruban et al., 1993; Ruban and Horton, 1995b).
In order to determine the relationship between qE and qI,
species that show particular NPQ characteristics have been
sought. The epiphytic plant, Guzmania monostachia, exhibits a large NPQ capacity (Ruban et al., 1993). Both qE
and qI are induced in excess light: successive periods of
illumination progressively ‘pump’ qE to its maximum value,
and this is activation is correlated with an increase in qI.
Thus, qI is the signature of the light ‘activation’ described
above, and is therefore attributed to the de-epoxidation of
violaxanthin. The large extent of activation and qI in this
species is also associated with a large xanthophyll cycle pool
and a high de-epoxidation state. Spectroscopy carried out on
Guzmania leaves upon the induction of qI showed the
formation of red-shifted chlorophyll emission previously
identified in aggregated LHCII. A very stable type of NPQ
found in plants acclimated to low temperature also has this
feature, the ‘cold hard band’, suggesting that evergreen
plants which can withstand freezing conditions over winter
exist in a permanently photoprotected qI state of aggregated
LHCII (Gilmore and Ball, 2000). Biochemical data on this
state suggests that factors such as LHCII phosphorylation
(Ebbert et al., 2001) and accumulation of PsbS (Öquist and
Huner, 2003) may play a role in establishing and/or stabilizing this state of the PSII antenna.
NPQ found in some algae has different features compared
with higher plants. In Chlamydomonas, NPQ is rather weak
(about 50% of a typical value in higher plants), slow to form,
and partially dependent upon zeaxanthin formation (Elrad
et al., 2002). Various NPQ-deficient mutants have been
isolated, including one with a mutation in a light-harvesting
gene, not in PsbS (Elrad and Grossman, 2004). In many
respects NPQ resembles the higher plant qI, rather than qE.
In diatoms, NPQ is similar to that found in plants, except
that it is large (sometimes about five times bigger) and it is
very slow to relax; it forms like qE but relaxes like qI
(Lavaud et al., 2002). Diatoms have a xanthophyll cycle, but
it is different from that found in plants. It is a one-step deepoxidation of diadinoxanthin to diatoxanthin, equivalent to
the second step of the plant xanthophyll cycle, antheraxanthin–zeaxanthin conversion. The light-harvesting complexes in diatoms are different from those found in higher
plants; they bind different carotenoids and chlorophylls, and
have very low sequence similarity. They are predicted
3-helix proteins. The xanthophyll cycle carotenoids account
for 20–40% of the carotenoid pool, and it is estimated that
each LHC binds 2–4 molecules of diadinoxanthin. PsbS is
absent from these organisms, although it is interesting that
some similar PsbS protein sequences are found in their LHC
369
proteins. The DA535 is absent, although a light-induced
absorbance change around 522 nm is observed, and may be
indicative of some diatoxanthin activation. The formation of
NPQ is DpH-dependent and this appears to be not only due to
stimulation of de-epoxidation at low lumen pH; collapse of
the DpH by uncouplers reverses NPQ even though the level
of diatoxanthin in unchanged. Interestingly, when NPQ has
been completely formed, it is no longer uncoupler sensitive.
Previously an attempt was made to explain both qE and
qI in terms of a 4-state model of the light-harvesting system
(Horton and Ruban 1992); qI was considered to be a state in
which de-epoxidation has taken place, but not yet protonated, whereas qE is always a protonated state that would
normally also bind zeaxanthin. The pKa of the epoxidized
and de-epoxidized states, the strength and number of
xanthophyll cycle binding sites, the xanthophyll cycle pool
size, the level of PsbS and the pH of the thylakoid lumen
would determine the amounts of qE and qI observed in
different plants. Extending this rationale to the green algae,
perhaps the DpH is not large enough, and the zeaxanthin
concentration not high enough to cause protonation of the
qE active sites in the absence of PsbS.
In diatoms, the large NPQ can be explained by either
a stronger quencher, or by a larger number of quenching
sites. There is currently insufficient data to distinguish these.
The kinetic features indicate a qI-type process, but one which
is nevertheless dependent upon protonation, and formed
rapidly. The fact that formation of diatoxanthin only requires
one de-epoxidation, could explain the rapid activation of
quenching. Since, PsbS is absent from diatoms, NPQ must
occur by direct effects on the light-harvesting complexes.
The slow relaxation could arise from extreme stability of the
protonated state of these complexes when diatoxanthin is
present, so that reversal of quenching absolutely depends
upon the rather slow kinetics of de-epoxidation. This
stability may arise from particularly strong protein interactions within this antenna that ‘locks’ the quenched state. This
blurring of the distinction between qE and qI leads to a new
suggestion about the role of the PsbS protein in plants:
perhaps it provides control over NPQ, enabling not only
induction of NPQ, but also its rapid reversibility.
Comparative ecology of NPQ
The variation in character of NPQ in different organisms
suggests that facets of this process have undergone evolutionary adaptation to particular ecological niches. In land
plants, a major requirement for the regulation of light
harvesting is a rapid, dynamic response to changes in light
levels. The activation by de-epoxidation, and triggering by
protonation, is adapted to the more rapid changes associated with leaf movements and sunflecks, against a background of diurnal and daily changes. The relatively slow
kinetics of de-epoxidation act as a molecular memory of
‘average’ light conditions, whereas protonation provides an
370 Horton and Ruban
‘on/off switch’ with a response time of just a few seconds.
This switch, apparently, requires a transition between two
states of the light-harvesting system with different excitation lifetimes, setting the upper limit on the magnitude of
NPQ. The amount of quenching depends upon the number
of units in each state. Plants acclimated, and adapted to
particular light environments, tune the dynamics of NPQ.
Plants exposed consistently to large excesses of radiation
are found to have the high NPQ capacity, driven by a high
xanthophyll cycle pool and PsbS concentration, a low
content of LHCII and perhaps an increased capacity of DpH
generation (by cyclic electron transfer). In overwintering
plants, these features appear to be accompanied by other
mechanisms, which stabilize the quenched state even in the
absence of DpH.
Diatoms are able to achieve a very high NPQ capacity.
This appears to be an adaptation to environment, with large
but relatively slow fluctuations in incident radiation, arising
from ocean currents. The requirement for a rapid off switch
is quite low in this environment. It seems likely in any case
that it is not possible to have, at the same time, a very large
NPQ, and to have it rapidly reversible.
Evolution of NPQ
The diversity of NPQ characteristics suggests a strong
evolutionary pressure upon the attainment of optimized
photoprotection. This evolutionary process can be viewed
within the context of the LHC gene super family (Green and
Pichersky, 1994; Jansson, 1999). The cyanobacterial HLIP
and plant Lil 2 genes encode single-helix proteins that
resemble the progenitors of the Lhc proteins. The Lil3 gene
then arose by duplication, to give a 2-helix protein. PsbS is
then considered as a further duplication, since it is a 4-helix
protein. ELIPS, which are 3-helix proteins, would then
have evolved from a PsbS ancestor by deletion of a C-helix
region. All of these proteins are associated with stress
tolerance (Heddad and Adamska, 2002), and probably do
not bind chlorophylls. Instead, they may have evolved as
carotenoid protein complexes. For instance, the ELIP
protein found in abundance in some algae, Cbr, may bind
carotenoids (Levy et al., 1993). Accumulation of plant
ELIPS have also been reported to correlate closely with the
amount of xanthophyll pigments (Montane et al., 1998).
Light-harvesting proteins can then be considered to have
evolved from these stress proteins, by binding chlorophylls,
and excluding many carotenoids (Fig. 1). The trimeric state
of plant LHCII is probably the most evolved complex for
light harvesting (Wentworth et al., 2003). It is interesting to
consider the LHCF protein of diatoms; this complex binds
chlorophyll, but has retained a high amount of carotenoid
binding. The level of this protein increases under conditions of high NPQ, and hence may be considered as truly
bifunctional—operating equally in both photoprotection
and light harvesting, its two modes depending upon deepoxidation. By contrast, in plants, photoprotection requires
PsbS, a protein not involved in light harvesting. However,
this protein is able to confer upon LHCII (or any PSII
antenna complex) a photoprotective function, perhaps by
providing delivery and removal of the ‘active’ zeaxanthin.
The presence of PsbS, therefore, allows the light-harvesting
complex to function as an efficient light harvester, with
a high absorption cross-section, its chlorophyll pigments not
needing to be diluted by binding high levels of carotenoid.
The extra benefit from this mode of control is rapidity as
reversal does not depend upon epoxidation of zeaxanthin.
Moreover, once the appropriate de-epoxidation state has
been reached, NPQ can be turned on and off rapidly. In
this context it is interesting to consider the effect of PsbS
concentration on NPQ. Manipulation of the amount of PsbS
in Arabidopsis shows that its stoichiometry correlates with
changes in the amplitude of the qE component of NPQ (Li
et al., 2002b). Mutants which lack PsbS altogether have only
a slowly relaxing NPQ; in the absence of PsbS the unassisted
process is revealed, one which relies upon the passive mass
action effect of the xanthophyll cycle and in which qE and qI
are indistinguishable. The evolution of qE in plants, driven
by the need to respond rapidly to changing light intensity,
resulted from the specification of an ancestral carotenoid–
protein complex to a new role reversibly binding xanthophyll cycle carotenoids and protons, and interacting with the
PSII light-harvesting antenna proteins.
Conclusions
The principles that have guided the molecular design of the
PSII light harvesting have been elucidated. Modifications to
this design are found in different organisms’ adaptations to
Fig. 1. Evolutionary and functional relationships between proteins involved in light harvesting and photoprotection. LHCII is seen as the end-product of
a process that maximizes light harvesting through high amounts of chlorophyll binding. These may have evolved from stress-responsive single-helix
proteins such as HLIP and ELIPS, which bind only carotenoids. PsbS provides an essential function in plant photoprotection, with only minimal pigment
binding. On the other hand, LHCF binds both chlorophyll and carotenoids in high amounts, functioning in both light harvesting and photoprotection.
Molecular design of the PSII light-harvesting antenna
specific environments. An essential feature is the structural
flexibility of the light-harvesting proteins, which allow
control over the transfer of excitation energy to particular
quenching species, associates of chlorophyll or chlorophyll
and xanthophyll. The recent elucidation of the threedimensional structure of LHCII has revealed the possible
molecular mechanisms by which this may occur (Liu et al.,
2004). High resolution electron microscopy and image
analysis of wild-type and genetically altered plants are
showing how these structures are organized within the
intact membrane (Yakushevska et al., 2003; Ruban et al.,
2003) and new spectroscopic approaches to the investigation of intact systems are beginning to reveal details of how
such mechanisms operate (Robert et al., 2004).
References
Allen JF, Forsberg J. 2001. Molecular recognition in thylakoid
structure and function. Trends in Plant Science 6, 317–326.
Andersson J, Ruban AV, Walters R, Howard C, Wentworth M,
Horton P, Jansson S. 2003. Absence of Lhcb1 and Lhcb2 proteins of
the light-harvesting complex of photosystem II: effects on photosynthesis, grana stacking and fitness. The Plant Journal 35, 350–361.
Andersson J, Walters RG, Horton P, Jansson S. 2001. Antisense
inhibition of the photosynthetic antenna proteins CP29 and CP26:
implications for the mechanism of protective energy dissipation.
The Plant Cell 13, 11930–1204.
Arsalane W, Rousseau B, Duval JC. 1994 Influence of the pool size
of the xanthophyll cycle on the effects of light stress in a diatom:
competition between photoprotection and photoinhibition. Photochemistry and Photobiology 60, 237–243.
Aspinall-O’Dea M, Wentworth M, Pascal A, Robert B, Ruban
AV, Horton P. 2002. The PsbS subunit of photosystem II binds
zeaxanthin and activates it for non-photochemical fluorescence
quenching. Proceedings of the National Academy of Sciences,
USA 99, 16331–16335.
Bassi R, Caffarri S. 2000. Lhc proteins and the regulation of
photosynthetic light harvesting function by xanthophylls. Photosynthesis Research 64, 243–256.
Beddard GS, Porter G. 1976. Concentration quenching in chlorophyll. Nature 260, 366–367.
Bennett J. 1983. Regulation of photosynthesis by reversible phosphorylation of light-harvesting a/b protein. Biochemical Journal
212, 1–13.
Bilger W, Heber U, Schreiber U. 1988. Kinetic relationship between energy-dependent fluorescence quenching, light-scattering,
chlorophyll luminescence and proton pumping in intact leaves.
Zeitschrift für Naturforschung 43, 877–887.
Boekema EJ, van Breemen JFL, van Roon H, Dekker JP. 2000.
Arrangement of photosystem II supercomplexes in crystalline
macrodomains within the thylakoid membrane of green plant
chloroplasts. Journal of Molecular Biology 301, 1123–1133.
Burke JJ, Ditto CL, Arntzen CJ. 1987. Involvement of the lightharvesting complex in cation regulation of excitation energy
distribution in chloroplasts. Archives of Biochemistry and Biophysics 187, 252–263.
Caffarri S, Croce R, Breton J, Bassi R. 2001. The major antenna
complex of photosystem II has a xanthophyll binding site not
involved in light harvesting. Journal of Biological Chemistry 276,
35924–35933.
Caffarri S, Croce R, Cattivelli L, Bassi R. 2004. A look within
LHCII: differential analysis of the Lhcb1–3 complexes building
371
the major trimeric antenna complex of higher plant photosynthesis.
Biochemistry 43, 9467–9476.
Campbell D, Hurry V, Clarke AK, Gustafsson P, Oquist G. 1998.
Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation. Microbiology and Molecular Biology
Reviews 62, 667–677.
Casper-Lindley C, Bjorkman O. 1998. Fluorescence quenching in
four unicellular algae with different light-harvesting and xanthophyll cycle pigments. Photosynthesis Research 56, 277–289.
Chow WS, Lee HY, Park YI, Park YM, Hong YN, Anderson JM.
2002. The role of inactive photosystem-II-mediated quenching in
a last-ditch community defence against high light stress in vivo.
Philosophical Transactions of the Royal Society of London, Series
B 357, 1441–1449.
Crimi M, Dorra D, Bosinger CS, Giuffra E, Holzwarth AR, Bassi
R. 2001. Time-resolved fluorescence analysis of the recombinant
photosystem II antenna complex CP29: effects of zeaxanthin, pH and
phosphorylation. European Journal of Biochemistry 268, 260–267.
Croce R, Breton J, Bassi R. 1996. Conformational changes induced
by phosphorylation in the CP29 subunit of photosystem II.
Biochemistry 35, 11142–11148.
Dekker JP, van Roon H, Boekema EJ. 1999. Heptameric association of light-harvesting complex II trimers in partially sollubilized
photosystem II membranes. FEBS Letters 449, 211–214.
Demmig-Adams B. 1990. Carotenoids and photoprotection: a role
for the xanthophyll zeaxanthin. Biochimica et Biophysica Acta
1020, 1–24.
Demmig-Adams B. 1998. Survey of thermal energy dissipation and
pigment composition in sun and shade leaves. Plant and Cell
Physiology 39, 474–482.
Demmig B, Winter K. 1988. Characterization of 3 components of nonphotochemical fluorescence quenching and their response to photoinhibition. Australian Journal of Plant Physiology 15, 163–177.
Dominici P, Caffarri S, Armenante F, Ceoldo S, Crimi M, Bassi
R. 2001. Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant. Journal of
Biological Chemistry 277, 22750–22758.
Ebbert V, Demmig-Adams B, Adams WW, Mueh KE, Staehelin
LA. 2001. Correlation between persistent forms of zeaxanthindependent energy dissipation and thylakoid protein phosphorylation. Photosynthesis Research 67, 63–78.
El Bissati K, Delphin E, Murata N, Etienne AL, Kirilovsky D.
2000. Photosystem II fluorescence quenching in the cyanobacterium Synechocystis PCC 6803: involvement of two different
mechanisms. Biochimica et Biophysica Acta 1457, 229–242.
Elrad D, Grossman AR. 2004. A genome’s-eye view of the lightharvesting polypeptides of Chlamydomonas reinhardtii. Current
Genetics 45, 61–75.
Elrad D, Niyogi KK, Grossman AR. 2002. A major light-harvesting
polypeptide of photosystem II functions in thermal dissipation. The
Plant Cell 14, 1801–1816.
Formaggio E, Cinque G, Bassi R. 2001 Functional architecture of
the major light-harvesting complex from higher plants. Journal of
Molecular Biology 314, 1157–1166.
Frank HA, Cua A, Chynwat V, Young A, Gosztola D, Wasielewski MR. 1994. Photophysics of the carotenoids associated with the
xanthophyll cycle in photosynthesis. Photosynthesis Research 41,
389–395.
Garab G, Mustardy L. 1999. Role of LHCII-containing macrodomains in the structure, function and dynamics of grana.
Australian Journal of Plant Physiology 26, 649–658.
Gilmore AM. 2001. Xanthophyll cycle-dependent nonphotochemical quenching in photosystem II: mechanistic insights gained from
Arabidopsis thaliana L. mutants that lack violaxanthin deepoxidase activity and/or lutein. Photosynthesis Research 67, 89–101.
372 Horton and Ruban
Gilmore AM, Ball MC. 2000. Protection and storage of chlorophyll
in overwintering evergreens. Proceedings of the National Academy of Sciences, USA 97, 11098–11101.
Green BR, Pichersky E. 1994. Hypothesis for the evolution of
3-helix chl a/b and chl a/c light-harvesting antenna proteins from
2-helix and 4-helix ancestors. Photosynthesis Research 32, 149–162.
Hankamer B, Barber J, Boekema EJ. 1997. Structure and
membrane organization of photosystem II in green plants. Annual
Reviews in Plant Physiology and Plant Molecular Biology 48,
641–671
Heddad M, Adamska I. 2002. The evolution of light stress proteins
in photosynthetic organisms. Comparative and Functional
Genomics 3, 504–510.
Horton P. 1999. Are grana necessary for regulation of light harvesting? Australian Journal of Plant Physiology 26, 659–669.
Horton P, Black MT. 1980. Activation of adenosine 59 triphosphateinduced quenching of chlorophyll fluorescence by reduced plastoquinone. FEBS Letters 119, 141–144.
Horton P, Ruban AV. 1992. Regulation of photosystem II. Photosynthesis Research 34, 375–385.
Horton P, Ruban AV. 1994. The role of LHCII in energy quenching.
In: Baker NR, Bowyer JR, eds. Photoinhibition of photosynthesis—
from molecular mechanisms to the field. Oxford: Bios Scientific
Publishers, 111–128.
Horton P, Oxborough K, Rees D, Scholes JD. 1988. Regulation of
the photochemical efficiency of photosystem II; consequenes for
the light response of field photosynthesis. Plant Physiology and
Biochemistry 26, 453–460.
Horton P, Ruban AV, Rees D, Pascal A, Noctor GD, Young A.
1991. Control of the light-harvesting function of chloroplast
membranes by the proton concentration in the thylakoid lumen:
aggregation states of the LHCII complex and the role of zeaxanthin. FEBS Letters 292, 1–4.
Horton P, Ruban AV, Walters RG. 1996. Regulation of light
harvesting in green plants. Annual Reviews of Plant Physiology
and Plant Molecular Biology 47, 655–684.
Horton P, Ruban AV, Wentworth M. 2000. Allosteric regulation of
the light-harvesting system of photosystem II. Philosophical
Transactions of The Royal Society London B 355, 1361–1370.
Horton P, Ruban AV, Young AJ. 1999. Regulation of the structure
and function of the light-harvesting complexes of photosystem II
by the xanthophyll cycle. In: Frank HA, Young AJ, Cogdell JC,
eds. The photochemistry of carotenoids: applications in biology.
Kluwer Academic Publishers, 271–291.
Jansson S. 1999. A guide to the Lhc genes and their relatives in
Arabidopsis. Trends in Plant Science 4, 236–240.
Johnson GN, Young AJ, Scholes JD, Horton P. 1993. The
dissipation of excess excitation-energy in British plant-species.
Plant, Cell and Environment 16, 673–679.
Kuhlbrandt W, Wang DN, Fujiyoshi Y. 1994. Atomic model of
plant light-harvesting complex by electron crystallography. Nature
367, 211–214.
Lavaud J, Rousseau B, van Gorkom HJ, Etienne A-L. 2002.
Influence of the diadinoxanthin pool size on the photoprotection in
marine planktonic diatom Phaeodactylum tricornutum. Plant
Physiology 129, 1398–1406.
Levy H, Tal T, Shaish A, Zamir A. 1993. Cbr, an algal homolog of
plant early light-inducible proteins, is a putative zeaxanthin binding
protein. Journal of Biological Chemistry 268, 20892–20896.
Li XP, Bjorkman O, Shih C, Grossman AR, Rosenquist M,
Jansson S, Niyogi KK. 2000. A pigment-binding protein essential
for regulation of photosynthetic light harvesting. Nature 403,
391–395.
Li XP, Muller-Moule P, Gilmore AM, Niyogi KK. 2002b. PsbSdependent enhancement of feedback de-excitation protects photo-
system II from photoinhibition. Proceedings of the National
Academy of Sciences, USA 99, 15222–15227.
Li XP, Phippard A, Pasari J, Niyogi KK. 2002a. Structure–
function analysis of photosystem II subunit S (PsbS) in vivo.
Functional Plant Biology 29, 1131–1139.
Liu Z, Yan H, Wang K, Kuang T, Zhang J, Gui L, An X, Chang
W. 2004. Crystal structure of spinach major light-harvesting
complex at 2.72 Å resolution. Nature 428, 287–292.
Lokstein H, Hartel H, Hoffmann P, Renger G. 1993. Comparison
of chlorophyll fluorescence quenching in leaves of wild type with
a chlorophyll b-less mutant of barley. Journal of Photochemistry
and Photobiology 19, 217–225.
Ma YZ, Holt NE, Li XP, Niyogi KK, Fleming GR. 2003. Evidence
for direct carotenoid involvement in the regulation of photosynthetic light harvesting. Proceedings of the National Academy of
Sciences, USA 100, 4377–4382.
Montane MH, Tardy F, Kloppstech K, Havaux M. 1998. Differential control of xanthophylls and light-induced stress proteins, as
opposed to light-harvesting chlorophyll a/b proteins, during
photosynthetic acclimation of barley leaves to light irradiance.
Plant Physiology 118, 227–235.
Morosinotto T, Baronio R, Bassi R. 2002. Dynamics of chormophore binding to Lhc proteins in vivo and in vitro during operation
of the xanthophyll cycle. Journal of Biological Chemistry 277,
36913–36920.
Moya I, Silvestri M, Vallon O, Cinque G, Bassi R. 2001. Timeresolved fluorescence analysis of the photosystem II antenna
proteins in detergent micelles and liposomes. Biochemistry 40,
12552–12561.
Mullet JE, Arntzen CJ. 1980. Simulation of grana stacking in
a model system. Mediation by a purified light-harvesting pigment–
protein complex from chloroplasts. Biochimica et Biophysica Acta
589, 100–117.
Niyogi KK, Bjorkman O, Grossman AR. 1997. Chlamydomonas
xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching. The Plant Cell 9, 1369–1380.
Oquist G, Huner NPA. 2003. Photosynthesis of overwintering
evergreen plants. Annual Review of Plant Biology 54, 329–355.
Remy R, Tremolieres, A, Duval JC, Ambardbretteville F,
Dubacq JP. 1982. Study of the supramolecular organization of
light-harvesting chlorophyll protein (LHCP): conversion of the
oligomeric form into the monomeric one by phospholipase-a2 and
reconstitution with liposomes. FEBS Letters 137, 271–275.
Robert B, Pascal AA, Horton P, Ruban AV. 2004. Insights into the
molecular dynamics of the plant light-harvesting proteins in vivo.
Trends in Plant Science 9, 385–390.
Ruban AV, Horton P. 1992. Mechanism of DpH-dependent dissipation of absorbed excitation energy by photosynthetic membranes. I. Spectroscopic analysis of isolated light-harvesting
complexes. Biochimica et Biophysica Acta 1102, 30–38.
Ruban AV, Horton P. 1995a. Regulation of nonphotochemical
quenching of chlorophyll fluorescence in plants. Australian Journal of Plant Physiology 22, 221–230.
Ruban AV, Horton P. 1995b. An investigation of the sustained
component of nonphotochemical quenching of chlorophyll fluorescence in isolated chloroplasts and leaves of spinach. Plant
Physiology 108, 721–726.
Ruban AV, Horton P. 1999. The xanthophyll cycle modulates the
kinetics of nonphotochemical energy dissipation in isolated lightharvesting complexes, intact chloroplasts and leaves. Plant Physiology 119, 531–542.
Ruban AV, Lee PJ, Wentworth M, Young, AJ, Horton P. 1999.
Determination of the stoichiometry and strength of binding of
xanthophylls to the photosystem II light-harvesting complexes.
Journal of Biological Chemistry 274, 10458–10465.
Molecular design of the PSII light-harvesting antenna
Ruban AV, Pascal A, Robert B. 2000. Xanthophylls of the major
photosynthetic light-harvesting complex of plants: identification,
conformation and dynamics. FEBS Letters 477, 181–185.
Ruban AV, Pascal AA, Robert B, Horton P. 2002. Activation of
zeaxanthin is an obligatory event in the regulation of photosynthetic
light harvesting. Journal of Biological Chemistry 277, 7785–7789.
Ruban AV, Rees D, Pascal AA, Horton P. 1992. Mechanism of
DpH-dependent dissipation of absorbed excitation energy by
photosynthesis II. The relationships between LHCII aggregation
in vitro and qE in isolated thylakoids. Biochimica et Biophysica
Acta 1102, 39–44.
Ruban AV, Wentworth M, Yakushevska, AE, Andersson J, Lee
PJ, Keegstra W, Dekker JP, Boekema EJ, Jansson S, Horton,
P. 2003. Plants lacking the main light-harvesting complex retain
photosystem II macro-organization. Nature 421, 648–652.
Ruban AV, Wentworth M, Horton P. 2001. Kinetic analysis of nonphotochemical quenching of chlorophyll fluorescence. I. Isolated
chloroplasts. Biochemistry 40, 9896–9901.
Ruban AV, Young A, Pascal A, Horton P. 1994. The effects of
illumination on the xanthophyll composition of the photosystem II
light-harvesting complexes of spinach thylakoid membranes. Plant
Physiology 104, 227–234.
373
Ruban AV, Young A, Horton P. 1993. Induction of nonphotochemical energy dissipation and absorbance changes in leaves; evidence
for changes in the state of the light-harvesting system of photosystem II in vivo. Plant Physiology 102, 741–750.
Simidjiev I, Stoylova S, Amenitsch H, Javorfi T, Mustardy L,
Laggner P, Holzenburg A, Garab G. 2000. Proceedings of the
National Academy of Sciences, USA 97, 1473–1476.
van Amerongen H, van Grondelle R. 2001. Understanding the
energy transfer function of LHCII, the major light-harvesting
complex of green plants Journal of Physical Chemistry B 105,
604–617.
Wentworth M, Ruban AV, Horton P. 2000. Chlorophyll fluorescence quenching in isolated light-harvesting complexes induced by
zeaxanthin. FEBS Letters 471, 71–74.
Wentworth M, Ruban AV, Horton P. 2003. Thermodynamic
investigation into the mechanism of the chlorophyll fluorescence
quenching in isolated photosystem II light-harvesting complexes.
Journal of Biological Chemistry 278, 21845–21850.
Yakushevska AE, Keegstra W, Boekema EJ, Dekker JP, Jansson
S, Ruban AV, Horton P. 2003. The structure of photosystem II in
Arabidopsis: localization of the CP26 and CP29 antenna complexes. Biochemistry 42, 608–613.