Is PsbS the site of non-photochemical quenching in
Krishna K. Niyogi*, Xiao-Ping Li, Vanessa Rosenberg and Hou-Sung Jung
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA
Received 29 July 2004; Accepted 22 October 2004
The PsbS protein of photosystem II functions in the
regulation of photosynthetic light harvesting. Along
with a low thylakoid lumen pH and the presence of de-
epoxidized xanthophylls, PsbS is necessary for photo-
protective thermal dissipation (qE) of excess absorbed
light energy in plants, measured as non-photochemical
quenching of chlorophyll fluorescence. What is known
about PsbS in relation to the hypothesis that this
protein is the site of qE is reviewed here.
Key words: Arabidopsis, chlorophyll fluorescence, light har-
vesting, mutant, non-photochemical quenching, photosynthesis,
Light is necessary for photosynthesis in plants, but the
supply of light in natural environments is not constant.
Incident light can vary rapidly due to passing clouds or
sunflecks, as well as on a daily or seasonal basis. With
increasing light intensity, photosynthetic utilization of
absorbed light energy reaches saturation, while light ab-
sorptioncontinues toincrease.Thiscanresultin amismatch
betweenexcitation ofphotosyntheticpigments anda plant’s
ability to use the excitation energy for photosynthesis.
Under such excess light conditions, how do plants manage
to balance the input and utilization of light energy in
One of the ways in which this balancing act is accom-
plished is through the regulation of photosynthetic light
harvesting. On a time scale of seconds to minutes, non-
photochemical quenching (NPQ) processes in photosystem
II (PSII) can be induced or disengaged in response to
changes in light intensity. The term NPQ reflects the way
in which these processes are routinely assayed through
measurements of chlorophyll fluorescence (Maxwell and
Johnson, 2000; Mu ¨ller et al., 2001). Under most circum-
stances, the major component of NPQ is due to a regulatory
mechanism, called qE, which results in the thermal dissipa-
tion of excess absorbed light energy in the light-harvesting
antenna of PSII. qE is induced by a low thylakoid lumen pH
(i.e. a high DpH) that is generated by photosynthetic
electron transport in excess light, so it can be considered
as a type of feedback regulation of the light-dependent
reactions of photosynthesis (Fig. 1). Because qE involves
the de-excitation of singlet excited chlorophyll, it is also
sometimes referred to as feedback de-excitation (Ku ¨lheim
et al., 2002).
The low thylakoid lumen pH that induces qE has two
roles (Fig. 2). One role is the pH-dependent activation of a
lumen-localized violaxanthin de-epoxidase (VDE) enzyme
that catalyses the conversion of violaxanthin to zeaxanthin
via the intermediate antheraxanthin (Demmig-Adams and
Adams, 1996). Zeaxanthin and/or antheraxanthin (xantho-
phylls with a de-epoxidized 3-hydroxy b-ring end group)
are necessary for qE in plants (Demmig-Adams, 1990;
Demmig-Adams et al., 1990; Gilmore and Yamamoto,
1993; Niyogi et al., 1998). In limiting light, zeaxanthin
epoxidase (ZE) converts zeaxanthin back to violaxanthin.
Together, these light intensity-dependent interconversions
The second role of low thylakoid lumen pH is in driving
qE (Horton and Ruban, 1992). It has been hypothesized
that protonation activates a binding site for zeaxanthin in
one of the proteins (Gilmore, 1997), and as a result an ab-
* To whom correspondence should be addressed. Fax: +1 510 642 4995. E-mail: firstname.lastname@example.org
Abbreviations: DCCD, N,N9-dicyclohexylcarbodiimide; LHC, light-harvesting complex; NPQ, non-photochemical quenching of chlorophyll fluorescence; PSII,
photosystem II; qE, pH- and xanthophyll-dependent component of NPQ; QTLs, quantitative trait loci; VDE, violaxanthin de-epoxidase; ZE, zeaxanthin
Journal of Experimental Botany ª Society for Experimental Biology 2004; all rights reserved
Journal of Experimental Botany, Page 1 of 8
Light Stress in Plants: Mechanisms and Interactions Special Issue
Advance Access published December 20, 2004
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etal., 2002). Thisalteration ofthe properties of one ora few
zeaxanthin molecules per PSII might allow zeaxanthin to
facilitate directly the de-excitation of singlet excited chloro-
phyll via energy or electron transfer (Ma et al., 2003;
Holt et al., 2004).
qE is a plant trait of major ecophysiological significance
(Demmig-Adams et al., 1999). Plants that experience
excess light stress in their environment (i.e. sun plants)
generally have higher qE capacities and larger xanthophyll
cycle pool sizes (violaxanthin+antheraxanthin+zeaxanthin)
than plants, growing in shaded environments (Thayer and
Bjo ¨rkman, 1990; Demmig-Adams and Adams, 1992,
1994; Johnson et al., 1993; Demmig-Adams, 1998), and
the maximum extent of qE is considered to be an important
sive light. Furthermore, mutants that lack qE (see below)
are less resistant to light stress (Graßes et al., 2002; Li et al.,
2002b) and have decreased ecological fitness in fluctuating
light environments (Ku ¨lheim et al., 2002).
The importance of qE as a photosynthetic regulatory
process has stimulated tremendous interest in understand-
ing its ecophysiology, genetics, biochemistry, and biophys-
ical mechanism. This paper reviews the role that a specific
PSII protein, PsbS, plays in qE.
A genetic approach revealed a role for PsbS
By isolating and characterizing Arabidopsis thaliana
mutants that lack qE, it was shown that qE requires PsbS,
in addition to a low lumen pH and the presence of de-
epoxidized xanthophylls like zeaxanthin (Li et al., 2000).
qE-deficient mutants were isolated by video imaging of
chlorophyll fluorescence quenching during exposure of
mutagenized Arabidopsis seedlings to excess light (Niyogi
et al., 1998; Shikanai et al., 1999; Peterson and Havir,
2000). The npq1 and npq2 (aba1) mutants were defective
in VDE and ZE, respectively (Niyogi et al., 1998), whereas
npq4 mutants lacked qE and DA535 but had a normal
xanthophyll cycle (Li et al., 2000; Peterson and Havir,
2000). The npq4-1 mutation was mapped to chromosome 1
and ultimately shown to affect the gene encoding PsbS (Li
et al., 2000). The npq4-1 mutant had a complete deletion of
the psbS gene, so the PsbS protein was missing. Other PSII
proteins, however, were present at wild-type levels in the
mutant, and light harvesting and photosynthesis appeared
to be normal (Li et al., 2000). Characterization of several
independently isolated alleles of npq4 with various lesions
in the psbS gene (Peterson and Havir, 2001; Graßes et al.,
2002; Li et al., 2002c) confirmed that PsbS is necessary for
qE in Arabidopsis.
A working hypothesis for the role of PsbS
The PsbS protein had been identified previously as an
integral component of PSII in other plants through bio-
chemical approaches (Ljungberg et al., 1984; Ghanotakis
Fig. 1. Diagram depicting feedback regulation of photosynthetic light
harvesting (qE) by one of the products of the light reactions of
photosynthesis (DpH). The DpH is used to drive ATP synthesis, and
NADPH and ATP are used in CO2 fixation and other assimilatory
reactions. qE down-regulates photosynthetic light harvesting by de-
exciting singlet excited chlorophyll in the PSII antenna and thereby
dissipating excess absorbed light energy as heat.
Fig. 2. Schematic model for qE in plants. (Left) In limiting light, the steady-state thylakoid lumen pH is greater than 6 (Kramer et al., 1999).
Violaxanthin (Viola) is bound mainly to the V1 site in LHCII and the L2 site in other LHC proteins (such as CP29 and CP26) (Caffarri et al., 2001;
Morosinotto et al., 2002). For simplicity, other pigments (chlorophylls and other carotenoids) are not shown, and only one Viola and one LHC protein
are shown per PSII. The various components are not drawn to scale. (Middle) In excess light, the thylakoid lumen pH drops below 6, driving protonation
of carboxylate side chains in VDE and PsbS. Protonation of VDE activates the enzyme and allows for its association with the membrane (Hager and
Holocher, 1994), where it converts multiple Viola molecules to zeaxanthin (Zea). Protonation of glutamate residues E122 and E226 in PsbS activates
symmetrical binding sites for xanthophylls with a de-epoxidized b-ring endgroup (i.e. zeaxanthin). (Right) Zea binding to protonated sites in PsbS results
in the qE state in which singlet chlorophyll de-excitation is facilitated. Other Zea molecules bind to sites in LHCII and other LHC proteins.
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et al., 1987), but its function was uncertain. Cloning and
sequencing of the spinach psbS gene (Kim et al., 1992;
Wedel et al., 1992) had revealed similarity to chlorophyll-
and xanthophyll-binding proteins that are members of
the light-harvesting complex (LHC) protein superfamily
(Green and Pichersky, 1994; Jansson, 1999), and pigment
binding by isolated PsbS had been reported (Funk et al.,
1994, 1995b). This information, coupled with the npq4
mutant characterization, led to the hypothesis that PsbS
might be the site of qE in PSII (Li et al., 2000). Based on
this working hypothesis, several predictions could be made.
If PsbS is the site of qE, then (i) other PSII antenna
proteins should not be required for qE, except perhaps for
the pigment–protein complex from which excitation energy
is transferred to the quenching site in PsbS. (ii) PsbS should
bind both zeaxanthin and chlorophyll, the pigments that are
necessary for qE. (iii) Protonation of PsbS at low thylakoid
lumen pH should be necessary for qE. (iv) More quenching
sites would mean more quenching, so qE capacity should
depend to some extent on the amount of PsbS. In the
following sections, the experiments that have been per-
formed to test these predictions will be considered.
Where is PsbS?
PsbS was discovered more than 20 years ago as a 22 kDa
protein in isolated PSII preparations (Berthold et al., 1981),
but its exact location within PSII is still unknown. In order
for PsbS to function as the site of qE, the presumed
pigments bound to PsbS would have to be coupled excita-
tionally to one or more chlorophylls in the PSII light-
harvesting antenna system. Early biochemical studies
showed that PsbS could be co-immunoprecipitated with
the (non-pigmented) 33 kDa and 23 kDa subunits of the
oxygen-evolving complex in PSII (Ljungberg et al., 1984),
suggesting a close association with the PSII reaction centre
core. However, PsbS is still present in etiolated plants
(Funk et al., 1995a) and in mutants that lack PSII
(Dominici et al., 2002), indicating that it might be a more
peripheral subunit of PSII. Selective extraction of PsbS was
shown to disrupt interaction between the peripheral LHCII
and the reaction centre, consistent with a location at the
interface between these subcomplexes (Kim et al., 1994). A
similar conclusion was reached in some studies of PSII
supercomplexes, in which PsbS was found to be more
closely associated with the reaction centre than with an
LHCII fraction (Dominici et al., 2002; Thidholm et al.,
2002). Homodimers of PsbS have been described recently,
and a dimer-to-monomer transition seems to be triggered by
low pH or high light (Bergantino et al., 2003). The dimeric
form of PsbS was shown to cofractionate more with the
PSII reaction centre, whereas the monomer was also
associated with an LHC fraction, and LHC proteins could
be co-immunoprecipitated with PsbS (Bergantino et al.,
2003). Investigation of qE in chlorophyll b-less mutants
and antisense plants that lack various LHC proteins
(Lokstein et al., 1993; Andersson et al., 2001, 2003),
however, have shown that no single LHC protein seems to
be necessary for qE (unlike the situation in algae such as
Chlamydomonas), which casts some doubt on the func-
tional significance of an association between PsbS and
a specific LHC protein. Considering all of these results, it
seems likely that PsbS is located somewhere between the
PSII reaction centre core and the peripheral LHCII (Fig. 2),
with the functional association possibly occurring between
PsbS and the PSII core antenna.
Electron microscopic studies of plant PSII–LHCII super-
complexes have revealed the positions of most peripheral
antenna and core subunits of PSII (Hankamer et al., 2001;
Yakushevska et al., 2003), but this approach has not thus
far been successful in revealing the specific location of
PsbS. It turns out that PsbS was not present in these super-
complexes (Nield et al., 2000), because it was removed by
the b-dodecylmaltoside detergent that was used to solubi-
lize the supercomplexes (Harrer et al., 1998; Nield et al.,
2000). It was recently found that extraction of PSII particles
with a-dodecylmaltoside results in the retention of PsbS in
supercomplexes (Dominici et al., 2002), so there is hope
that a home for PsbS will be found soon.
Does PsbS actually bind pigments?
Although the initial biochemical studies of PsbS did not
provide any hint of pigment binding (Ljungberg et al.,
1986; Bowlby and Yocum, 1993), the finding that PsbS is
a member of the LHC protein superfamily suggested this as
a strong possibility (Kim et al., 1992; Wedel et al., 1992).
However, PsbS is considered to be a distant relative of the
well-known chlorophyll- and xanthophyll-binding mem-
bers of this superfamily (Green and Durnford, 1996;
Jansson, 1999), and it differs from all others in having
four transmembrane domains instead of the usual three
(Kim et al., 1994) (Fig. 3). Inspection of the predicted
amino acid sequence of PsbS shows little conservation of
the residues that provide binding sites for chlorophylls in
the LHC proteins that are known to function in light
harvesting (Ku ¨hlbrandt et al., 1994; Bassi et al., 1999;
Croce et al., 1999; Liu et al., 2004). The only ligands that
appear to be conserved in LHCII and PsbS are the two
charge-compensated glutamates (Funk et al., 1995b) (Fig.
3) that also have a critical role in the proper folding and
stability of LHCII (Bassi et al., 1999; Croce et al., 1999).
Furthermore, unlike most other LHC proteins, PsbS is
stable in the absence of chlorophyll in vivo (Funk et al.,
Efforts to isolate PsbS with bound pigments have met
with mixed success. Funk et al. (Funk et al., 1994, 1995b)
were the first to publish evidence supporting pigment
binding by isolated PsbS, and they called the protein
CP22 (for chlorophyll-binding protein of 22 kDa). More
PsbS and non-photochemical quenching 3 of 8
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recently, Dominici et al. (2002) were unable to demonstrate
a stable association of pigments with isolated PsbS, and
their attempts to reconstitute recombinant PsbS with pig-
ments in vitro were unsuccessful. Aspinall-O’Dea et al.
(2002) also found no pigment binding that could withstand
purification of PsbS, but they were able to provide evidence
for an interaction between PsbS and zeaxanthin in vitro.
This interaction resulted in a change in the absorption
spectrum of zeaxanthin that reconstituted the DA535that is
associated with qE in leaves and thylakoids (Aspinall-
O’Dea etal., 2002).Overexpression ofPsbS in tobacco was
shown to increase the extent of violaxanthin de-epoxidation
under relatively low light conditions in vivo, and it was
suggested that this result could be explained by zeaxanthin
binding to PsbS, which would sequester zeaxanthin and
prevent feedback inhibition of de-epoxidation by VDE
(Hieber et al., 2004). Ultrafast transient absorption studies
of qE in isolated thylakoids revealed the PsbS-dependent
presence of singlet excited zeaxanthin following the exci-
tation of chlorophyll (Ma et al., 2003). Assuming that the
excited zeaxanthin is bound to PsbS, this result implies that
there must be chlorophyll in very close proximity to the
zeaxanthin, either bound to PsbS itself or on the periphery
of a closely associated protein.
The bottom line from the work to date seems to be that, if
PsbS does indeed bind pigments in vivo, then the nature of
this binding interaction must differ substantially from that
in other LHC proteins. There is now some evidence for
zeaxanthin binding by PsbS (Aspinall-O’Dea et al., 2002),
which is consistent with the hypothesis that PsbS is the site
of qE, but it is also consistent with other, more complicated
hypotheses in which zeaxanthin has an indirect, allosteric
role in qE (Horton et al., 2000; Aspinall-O’Dea et al.,
2002). Unfortunately, the results showing a lack of chloro-
phyll binding, because they are negative results, neither
support nor rule out the hypothesis.
PsbS as a sensor of lumen pH
It was suggested early on that one or more carboxylate side
chains in PSII proteins might bind protons at low lumen pH
and thereby trigger qE (Horton and Ruban, 1992). This idea
was supported by the inhibition of qE by N,N9-dicyclo-
hexylcarbodiimide (DCCD) (Ruban et al., 1992), which
binds to proton-active residues in hydrophobic environ-
ments. DCCD was shown to bind to LHC proteins, such as
CP29 and CP26 (Walters et al., 1996; Pesaresi et al., 1997),
which had been suspected to be involved in qE (Horton and
Ruban, 1992; Bassi et al., 1993; Jahns and Schweig, 1995),
but antisense experiments showed that CP29 and CP26 are
unlikely to be sites of qE (Andersson et al., 2001).
After the involvement of PsbS in qE was discovered,
it was shown that DCCD binds to PsbS as well (Dominici
et al., 2002), and sequence analysis showed that PsbS has
eight conserved acidic amino acid residues (glutamate and
aspartate) located at or near the lumen side of the protein
Fig. 3. Topological model of Arabidopsis PsbS. Triangles and horizontal arrows denote positions of two highly conserved, charge-compensated
glutamates that serve as ligands to bound chlorophylls in LHCII (Ku ¨hlbrandt et al., 1994). Squares denote the positions of eight acidic amino acid
residues (seven glutamates and one aspartate in Arabidopsis) located at or near the lumen side of the protein that are conserved in all known PsbS
sequences. The two glutamates that are necessary for qE and DCCD binding are numbered and marked by vertical arrows. Numbering is relative to the
predicted initiator methionine of the PsbS precursor protein (prior to import into chloroplasts). Modified from Li et al. (2002c).
4 of 8 Niyogi et al.
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that are candidate proton- and/or DCCD-binding sites
four symmetrical pairs, and they are conserved in all known
PsbS sequences, including the recently identified sequences
from the green algae C. reinhardtii and Volvox carteri
(Anwaruzzaman et al., 2004). Although many npq4 point
mutant alleles had been isolated following chemical muta-
genesis in Arabidopsis, none of these mutations affected a
genesis approach was used to test the role of the lumenal
acidic amino acid residues in PsbS (Li et al., 2002c).
Each of seven glutamates and one aspartate in Arabidopsis
PsbS was changed (both individually and as symmetrical
pairs) by mutagenesis in vitro to glutamine or asparagine,
respectively. The site-directed mutants were transformed
into the npq4-1 mutant that lacks the wild-type psbS gene,
and the function of each mutant was tested in vivo. One
pair of glutamates (E122 and E226; Fig. 3) was shown to be
necessary for qE, DA535, and DCCD binding, strongly
suggesting that protonation of these residues in excess light
is necessary for qE and that PsbS serves as a sensor of
lumen pH (Li et al., 2004).
PsbS and qE capacity
If PsbS is the site of qE, then the level of qE should be
related to the amount of PsbS per PSII. If there are more
quenching sites, then there should be more quenching (up
to a limit, of course). Molecular and genetic analysis of the
npq4-1 mutant showed that there is indeed a psbS gene
dosage effect on qE. Heterozygous npq4-1/NPQ4 plants
have half the number of psbS genes as the wild type, and
they have a correspondingly lower level of psbS mRNA,
PsbS protein, and qE (Li et al., 2002a). Increasing PsbS
expression in transgenic plants confers a higher qE capacity
(Li et al., 2002b; Hieber et al., 2004), with saturation
occurring in Arabidopsis at ;5 times the wild-type level of
PsbS on a per PSII basis (Fig. 4). This saturation indicates
that there is a maximum number of functional binding sites
for PsbS per PSII. Thus, the PsbS protein level can be
a determinant of qE capacity, and PsbS expression seems to
limit qE in wild-type Arabidopsis and tobacco (Li et al.,
2002b; Hieber et al., 2004). Alterations in PsbS level have
been reported as explanations for lower qE in LHCII-
deficient plants (Andersson et al., 2003) and higher qE in
PsaD-deficient plants (Haldrup et al., 2003).
Does the stoichiometry of PsbS vary naturally in plants?
The stoichiometry of PsbS has been reported to be two
copies of PsbS per PSII in wild-type spinach thylakoids
(Funk et al., 1995b), but the Arabidopsis mutants and
transgenics show that the stoichiometry can vary widely,
from zero (in the npq4-1 mutant) to many times the wild-
type value (Fig. 4). The stoichiometry in low-light-grown,
wild-type Arabidopsis plants has not yet been determined.
It seems plausible that variations in PsbS expression might
explain at least some cases of environmental (i.e. sun versus
shade) and species-dependent variation in qE capacity
(Johnson et al., 1993; Demmig-Adams and Adams, 1994;
To investigate this hypothesis, the genetic basis for
natural variation in qE capacity in Arabidopsis accessions
(often referred to as ‘ecotypes’) has started to be examined.
A survey of the qE capacity was conducted in more than 50
accessions, and it was found that there is substantial intra-
species variation for qE in Arabidopsis (Fig. 5). However,
selected high and low qE accessions appeared to have the
Fig. 4. Relationship between PsbS protein level and qE. The npq4-1
mutant was transformed with the wild-type Arabidopsis psbS gene under
the control of its own promoter (Li et al., 2000). T1transformants were
selected on agar medium containing gentamycin, and the level of NPQ in
the transformants was initially assessed using chlorophyll fluorescence
video imaging. T1 seedlings exhibiting a range of NPQ values were
transferred to soil and grown to maturity. NPQ was then measured using
a commercial fluorometer (FMS2; Hansatech, King’s Lynn, UK) after
illumination at 1200 lmol photons m?2s?1for 10 min. qE of each plant
was calculated as (NPQ in T1plant)–(NPQ in npq4-1). The PsbS protein
level in each T1plant was determined by immunoblotting and normalized
to the level of the PSII reaction centre protein D1 (Li et al., 2002b). The
qE values of 39 independent T1plants are shown with the open circles.
Fig. 5. Natural variation of qE in 38 Arabidopsis accessions. Accessions
were grown under identical low light conditions (100 lmol photons m?2
s?1), and NPQ induction was measured during illumination with high
light (1500 lmol photons m?2s?1) for 10 min followed by a relaxation
period of 5 min in darkness.
PsbS and non-photochemical quenching5 of 8
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same level of PsbS expression (H-S Jung and KK Niyogi,
unpublished results). Analysis of F2plants resulting from
a cross between a high qE accession (Sf-2) and a low qE
accession (Col-0) showed continuous variation of the qE
phenotype, indicating that qE capacity in these accessions
is a quantitative genetic trait that is controlled by multiple
genes. Mapping of quantitative trait loci (QTLs) uncovered
two major QTLs that are responsible for much of the
variation in qE, but neither of these QTLs mapped to the
position of the psbS gene, indicating that the naturally
occurring variation in qE between these two accessions is
not attributable to PsbS (H-S Jung and KK Niyogi, un-
published results). The extent to which this conclusion can
be generalized to other Arabidopsis accessions and to other
species remains to be determined.
PsbS in algae
Is PsbS a key player in qE in other photosynthetic organ-
isms? Besides angiosperms like Arabidopsis and tobacco,
genes encoding PsbS have been identified in a moss (Phys-
V. carteri) (Anwaruzzaman et al., 2004), but the function of
these genes in qE has not yet been tested. C. reinhardtii is
an interesting case, because qE-deficient mutants have been
isolated (Niyogi et al., 1997), but so far no mutants have
been shown to affect PsbS. In fact, the most extensively
studied C. reinhardtii mutant, npq5, turned out to be
defective in an LHCII gene called Lhcbm1 (Elrad et al.,
2002). This finding raises intriguing questions about the
possible relationship between LHCII and PsbS in this alga,
and this can be resolved by studying PsbS-deficient mutants
generated through reverse genetics.
all have PsbS. For example, diatoms exhibit robust qE that
depends on a low thylakoid lumen pH and the presence of
a de-epoxidized xanthophyll (diatoxanthin instead of zeax-
anthin and antheraxanthin), but the first completely se-
quenced genome of a diatom, Thalassiosira pseudonana
members of the LHC protein superfamily, so it is possible
that another member of the family performs the function of
lenge will be to identify the proteins that play the role that
PsbS has in plants. It is likely that investigation of qE in
algae will provide interesting insights into the evolution of
function in the LHC protein superfamily.
The discovery that PsbS is necessary for qE in Arabidopsis
was an important breakthrough in the study of qE (Li et al.,
2000), but it certainly did not mean that the problem of
understanding qE was solved. On the contrary, the mech-
anism of qE still remains one of the last major unresolved
mysteries in photosynthesis.
A simple hypothesis has been proposed that PsbS is the
site of qE in plants (Li et al., 2000). Several experimental
tests of the hypothesis have now been conducted, and at
present the hypothesis remains viable, although more com-
plicated scenarios are also consistent with the available
data. Previously hypothesized sites of qE, such as LHCII,
CP29, and CP26, look less promising in the light of recent
antisense experiments (Andersson et al., 2001, 2003). On
the other hand, the amount of the PsbS protein in thylakoids
has been shown to be a determinant of qE capacity (Fig. 4)
(Li etal., 2002b; Hieberet al., 2004),and two lumen-facing
glutamate residues in PsbS (Fig. 3) have been identified as
proton-binding sites that are probably involved in sensing
lumen pH and turning qE on and off (Li et al., 2002c,
2004). Evidence for zeaxanthin binding by PsbS in vitro
has been reported (Aspinall-O’Dea et al., 2002), and a
follow-up of these results is eagerly anticipated. Ultrafast
PsbS-dependent excitation of zeaxanthin following laser
excitation of chlorophyll has been demonstrated (Ma et al.,
2003). This places strict constraints on the distance between
the nearest chlorophyll and the excited zeaxanthin, which is
assumed to reside in PsbS, but chlorophyll binding to PsbS
remains to be unequivocally demonstrated. It is possible
that the coupled chlorophyll might be located on the periph-
ery of PsbS, perhaps at the interface between PsbS and
PSII, which might explain the difficulty in isolating PsbS
with bound chlorophyll.
The next major breakthrough in understanding the role of
PsbS in qE will probably depend on biochemical reconsti-
tution of qE in a much simpler system than isolated thy-
lakoid membranes, the simplest system to date. Indeed,
a holy grail of qE research is the isolation of a complex
containing PsbS, zeaxanthin, and chlorophyll that exhibits
pH- and zeaxanthin-dependent de-excitation of singlet
excited chlorophyll (qE). In conjunction with methodolog-
ical advances in spectroscopy and structural biology, it will
then be possible to obtain a full picture of the mechanism
This work was supported by the Director, Office of Science, Office
of Basic Energy Sciences, Chemical Sciences Division, of the US
Department of Energy under contract No. DE-AC03-76SF00098.
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