JXB Advance Access originally published online on December 20, 2004
Journal of Experimental Botany 2005 56(411):375-382; doi:10.1093/jxb/eri056
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RESEARCH PAPER |
Is PsbS the site of non-photochemical quenching in photosynthesis?
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA
* To whom correspondence should be addressed. Fax: +1 510 642 4995. E-mail: niyogi{at}nature.berkeley.edu
Received 29 July 2004; Accepted 22 October 2004
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Abstract |
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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 photoprotective 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 harvesting, mutant, non-photochemical quenching, photosynthesis, PsbS, zeaxanthin
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Introduction |
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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 absorption continues to increase. This can result in a mismatch between excitation of photosynthetic pigments and a 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 photosynthesis?
One of the ways in which this balancing act is accomplished 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
; Müller et al., 2001). Under most circumstances, the major component of NPQ is due to a regulatory mechanism, called qE, which results in the thermal dissipation 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 
pH) 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 (Külheim et al., 2002).
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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 (xanthophylls with a de-epoxidized 3-hydroxy ß-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 are known as the xanthophyll cycle (Yamamoto et al., 1999). The second role of low thylakoid lumen pH is in driving protonation of one or more PSII proteins that are involved in 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 absorbance change (A535) is detectable in leaves and isolated thylakoids, which might be due to a change in the absorption spectrum of zeaxanthin (Aspinall-O'Dea et al., 2002; Ruban et al., 2002). This alteration of the properties of one or a few zeaxanthin molecules per PSII might allow zeaxanthin to facilitate directly the de-excitation of singlet excited chlorophyll via energy or electron transfer (Ma et al., 2003; Holt et al., 2004).
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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 Björkman, 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 ecophysiological factor for the adaptation of plants to excessive 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 (Külheim et al., 2002). The importance of qE as a photosynthetic regulatory process has stimulated tremendous interest in understanding its ecophysiology, genetics, biochemistry, and biophysical mechanism. This paper reviews the role that a specific PSII protein, PsbS, plays in qE.
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A genetic approach revealed a role for PsbS |
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 TopAbstractIntroduction A genetic approach revealed... A working hypothesis for...Where is PsbS?Does PsbS actually bind...PsbS as a sensor...PsbS and qE capacityPsbS in algaeConclusionReferences |
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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 A535 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 |
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The PsbS protein had been identified previously as an integral component of PSII in other plants through biochemical approaches (Ljungberg et al., 1984
; Ghanotakis 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 performed to test these predictions will be considered.
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Where is PsbS? |
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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 excitationally 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 functional 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 supercomplexes 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 supercomplexes (Nield et al., 2000), because it was removed by the ß-dodecylmaltoside detergent that was used to solubilize the supercomplexes (Harrer et al., 1998; Nield et al., 2000). It was recently found that extraction of PSII particles with 
-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.
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Does PsbS actually bind pigments? |
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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 members 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 (Kühlbrandt 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., 1995a).
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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 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 pigments 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 A535 that is associated with qE in leaves and thylakoids (Aspinall-O'Dea et al., 2002). Overexpression of PsbS 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 excitation 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 chlorophyll binding, because they are negative results, neither support nor rule out the hypothesis.
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PsbS as a sensor of lumen pH |
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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,N'-dicyclohexylcarbodiimide (DCCD) (Ruban et al., 1992), which binds to proton-active residues in hydrophobic environments. 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 that are candidate proton- and/or DCCD-binding sites (Fig. 3) (Li et al., 2002c). These eight residues are arranged as 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 mutagenesis in Arabidopsis, none of these mutations affected a potential proton-binding site. Therefore, a site-directed mutagenesis 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, A535, 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).
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PsbS and qE capacity |
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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).
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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; Demmig-Adams, 1998). 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 intraspecies variation for qE in Arabidopsis (Fig. 5). However, selected high and low qE accessions appeared to have the same level of PsbS expression (H-S Jung and KK Niyogi, unpublished results). Analysis of F2 plants 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, unpublished results). The extent to which this conclusion can be generalized to other Arabidopsis accessions and to other species remains to be determined.
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PsbS in algae |
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Is PsbS a key player in qE in other photosynthetic organisms? Besides angiosperms like Arabidopsis and tobacco, genes encoding PsbS have been identified in a moss (Physcomitrella patens) and in two green algae (C. reinhardtii and 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. Although nearly all photosynthetic eukaryotes have qE, it is becoming clear from genome sequencing projects that not 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 zeaxanthin and antheraxanthin), but the first completely sequenced genome of a diatom, Thalassiosira pseudonana (http://genome.jgi-psf.org/thaps1/thaps1.home.html), lacks a psbS gene. There are, however, many genes encoding other members of the LHC protein superfamily, so it is possible that another member of the family performs the function of PsbS in diatoms. As genome sequence information becomes available for other diverse photosynthetic eukaryotes, a challenge 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.
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Conclusion |
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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 mechanism 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 complicated 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 et al., 2002b; Hieber et 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 periphery 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 reconstitution of qE in a much simpler system than isolated thylakoid 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 methodological advances in spectroscopy and structural biology, it will then be possible to obtain a full picture of the mechanism of qE.
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Acknowledgements |
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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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Footnotes |
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Abbreviations: DCCD, N,N'-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 epoxidase.
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References |
|---|
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, 1193–1204.
Andersson J, Wentworth M, Walters RG, Howard CA, Ruban AV, Horton P, Jansson S. 2003. Absence of the 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.[CrossRef][ISI][Medline]
Anwaruzzaman M, Chin BL, Li X-P, Lohr M, Martinez DA, Niyogi KK. 2004. Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in Chlamydomonas reinhardtii. Photosynthesis Research (in press).
Aspinall-O'Dea M, Wentworth M, Pascal A, Robert B, Ruban A, Horton P. 2002. In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants. Proceedings of the National Academy of Sciences, USA 99, 16331–16335.
Bassi R, Pineau B, Dainese P, Marquardt J. 1993. Carotenoid-binding proteins of photosystem II. European Journal of Biochemistry 212, 297–303.[ISI][Medline]
Bassi R, Croce R, Cugini D, Sandonà D. 1999. Mutational analysis of a higher plant antenna protein provides identification of chromophores bound in multiple sites. Proceedings of the National Academy of Sciences, USA 96, 10056–10061.
Bergantino E, Segalla A, Brunetta A, Teardo E, Rigoni F, Giacometti GM, Szabó I. 2003. Light- and pH-dependent structural changes in the PsbS subunit of photosystem II. Proceedings of the National Academy of Sciences, USA 100, 15265–15270.
Berthold DA, Babcock GT, Yocum CF. 1981. A highly resolved, oxygen-evolving photosystem II preparation from spinach thylakoid membranes: EPR and electron-transport properties. FEBS Letters 134, 231–234.[CrossRef]
Bowlby NR, Yocum CF. 1993. Effects of cholate on photosystem II: selective extraction of a 22 kDa polypeptide and modification of QB-site activity. Biochimica et Biophysica Acta 1144, 271–277.[CrossRef]
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.
Croce R, Weiss S, Bassi R. 1999. Carotenoid-binding sites of the major light-harvesting complex II of higher plants. Journal of Biological Chemistry 274, 29613–29623.
Demmig-Adams B. 1990. Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochimica et Biophysica Acta 1020, 1–24.[CrossRef]
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-Adams B, Adams III WW. 1992. Carotenoid composition in sun and shade leaves of plants with different life forms. Plant, Cell and Environment 15, 411–419.[CrossRef]
Demmig-Adams B, Adams III WW. 1994. Capacity for energy dissipation in the pigment bed in leaves with different xanthophyll cycle pools. Australian Journal of Plant Physiology 21, 575–588.[CrossRef]
Demmig-Adams B, Adams III WW. 1996. The role of xanthophyll cycle carotenoids in the protection of photosynthesis. Trends in Plant Science 1, 21–26.
Demmig-Adams B, Adams III WW, Ebbert V, Logan BA. 1999. Ecophysiology of the xanthophyll cycle. In: Frank HA, Young AJ, Britton G, Cogdell RJ, eds. The photochemistry of carotenoids. Dordrecht: Kluwer Academic Publishers, 245–269.
Demmig-Adams B, Adams III WW, Heber U, Neimanis S, Winter K, Krüger A, Czygan F-C, Bilger W, Björkman O. 1990. Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts. Plant Physiology 92, 293–301.
Dominici P, Caffarri S, Armenante F, Ceoldo S, Crimi M, Bassi R. 2002. Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant. Journal of Biological Chemistry 277, 22750–22758.
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.
Funk C, Schröder WP, Green BR, Renger G, Andersson B. 1994. The intrinsic 22 kDa protein is a chlorophyll-binding subunit of photosystem II. FEBS Letters 342, 261–266.[CrossRef][ISI][Medline]
Funk C, Adamska I, Green BR, Andersson B, Renger G. 1995a. The nuclear-encoded chlorophyll-binding photosystem II-S protein is stable in the absence of pigments. Journal of Biological Chemistry 270, 30141–30147.
Funk C, Schröder WP, Napiwotzki A, Tjus SE, Renger G, Andersson B. 1995b. The PSII-S protein of higher plants: a new type of pigment-binding protein. Biochemistry 34, 11133–11141.[CrossRef][Medline]
Ghanotakis DF, Waggoner CM, Bowlby NR, Demetriou DM, Babcock GT, Yocum CF. 1987. Comparative structural and catalytic properties of oxygen-evolving photosystem II preparations. Photosynthesis Research 14, 191–199.
Gilmore AM, Yamamoto HY. 1993. Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin-independent quenching. Photosynthesis Research 35, 67–78.[CrossRef]
Gilmore AM. 1997. Mechanistic aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves. Physiologia Plantarum 99, 197–209.[CrossRef]
Graßes T, Pesaresi P, Schiavon F, Varotto C, Salamini F, Jahns P, Leister D. 2002. The role of pH-dependent dissipation of excitation energy in protecting photosystem II against light-induced damage in Arabidopsis thaliana. Plant Physiology and Biochemistry 40, 41–49.[CrossRef][ISI]
Green BR, Pichersky E. 1994. Hypothesis for the evolution of three-helix Chl a/b and Chl a/c light-harvesting proteins from two-helix and four-helix ancestors. Photosynthesis Research 39, 149–162.[CrossRef]
Green BR, Durnford DG. 1996. The chlorophyll–carotenoid proteins of oxygenic photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 47, 685–714.[CrossRef][ISI][Medline]
Hager A, Holocher K. 1994. Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) pH decrease. Planta 192, 581–589.
Haldrup A, Lunde C, Scheller HV. 2003. Arabidopsis thaliana plants lacking the PSI-D subunit of photosystem I suffer severe photoinhibition, have unstable photosystem I complexes, and altered redox homeostasis in the chloroplast stroma. Journal of Biological Chemistry 278, 33276–33283.
Hankamer B, Morris E, Nield J, Gerle C, Barber J. 2001. Three-dimensional structure of the photosystem II core dimer of higher plants determined by electron microscopy. Journal of Structural Biology 135, 262–269.[CrossRef][ISI][Medline]
Harrer R, Bassi R, Testi MG, Schäfer C. 1998. Nearest-neighbor analysis of a photosystem II complex from Marchantia polymorpha L. (liverwort), which contains reaction centre and antenna proteins. European Journal of Biochemistry 255, 196–205.[ISI][Medline]
Hieber AD, Kawabata O, Yamamoto HY. 2004. Significance of the lipid phase in the dynamics and functions of the xanthophyll cycle as revealed by PsbS overexpression in tobacco and in vitro de-epoxidation in monogalactosyldiacylglycerol micelles. Plant and Cell Physiology 45, 92–102.
Holt NE, Fleming GR, Niyogi KK. 2004. Toward an understanding of the mechanism of non-photochemical quenching in green plants. Biochemistry 43, 8281–8289.[CrossRef][Medline]
Horton P, Ruban AV. 1992. Regulation of photosystem II. Photosynthesis Research 34, 375–385.[CrossRef]
Horton P, Ruban AV, Wentworth M. 2000. Allosteric regulation of the light-harvesting system of photosystem II. Philosophical Transactions of the Royal Society of London B, Biological Sciences 355, 1361–1370.
Jahns P, Schweig S. 1995. Energy-dependent fluorescence quenching in thylakoids from intermittent light-grown pea plants: evidence for an interaction of zeaxanthin and the chlorophyll a/b binding protein CP26. Plant Physiology and Biochemistry 33, 683–687.
Jansson S. 1999. A guide to the Lhc genes and their relatives in Arabidopsis. Trends in Plant Science 4, 236–240.[CrossRef][ISI][Medline]
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.[CrossRef]
Kim S, Pichersky E, Yocum CF. 1994. Topological studies of spinach 22 kDa protein of photosystem II. Biochimica et Biophysica Acta 1188, 339–348.[Medline]
Kim S, Sandusky P, Bowlby NR, Aebersold R, Green BR, Vlahakis S, Yocum CF, Pichersky E. 1992. Characterization of a spinach psbS cDNA encoding the 22 kDa protein of photosystem II. FEBS Letters 314, 67–71.[CrossRef][ISI][Medline]
Kramer DM, Sacksteder CA, Cruz JA. 1999. How acidic is the lumen? Photosynthesis Research 60, 151–163.[CrossRef]
Kühlbrandt W, Wang DN, Fujiyoshi Y. 1994. Atomic model of plant light-harvesting complex by electron crystallography. Nature 367, 614–621.[CrossRef][Medline]
Külheim C, Ågren J, Jansson S. 2002. Rapid regulation of light harvesting and plant fitness in the field. Science 297, 91–93.
Li X-P, Björkman 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.[CrossRef][Medline]
Li X-P, Gilmore AM, Caffarri S, Bassi R, Golan T, Kramer D, Niyogi KK. 2004. Regulation of photosynthetic light harvesting involves intrathylakoid lumen pH sensing by the PsbS protein. Journal of Biological Chemistry 279, 22866–22874.
Li X-P, Gilmore AM, Niyogi KK. 2002a. Molecular and global time-resolved analysis of a psbS gene dosage effect on pH- and xanthophyll cycle-dependent non-photochemical quenching in photosystem II. Journal of Biological Chemistry 277, 33590–33597.
Li X-P, Müller-Moulé P, Gilmore AM, Niyogi KK. 2002b. PsbS-dependent enhancement of feedback de-excitation protects photosystem II from photoinhibition. Proceedings of the National Academy of Sciences, USA 99, 15222–15227.
Li X-P, Phippard A, Pasari J, Niyogi KK. 2002c. Structure–function analysis of photosystem II subunit S (PsbS) in vivo. Functional Plant Biology 29, 1131–1139.[CrossRef]
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.[CrossRef][Medline]
Ljungberg U, Åkerlund H-E, Larsson C, Andersson B. 1984. Identification of polypeptides associated with the 23 and 33 kDa proteins of photosynthetic oxygen evolution. Biochimica et Biophysica Acta 767, 145–152.[CrossRef]
Ljungberg U, Åkerlund H-E, Andersson B. 1986. Isolation and characterization of the 10 kDa and 22 kDa polypeptides of higher plant photosystem II. European Journal of Biochemistry 158, 477–482.[ISI][Medline]
Lokstein H, Härtel H, Hoffmann P, Renger G. 1993. Comparison of chlorophyll fluorescence quenching in leaves of wild-type with a chlorophyll-b-less mutant of barley (Hordeum vulgare L.). Journal of Photochemistry and Photobiology B 19, 217–225.[CrossRef]
Ma Y-Z, Holt NE, Li X-P, 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.
Maxwell K, Johnson GN. 2000. Chlorophyll fluorescence: a practical guide. Journal of Experimental Botany 51, 659–668.
Morosinotto T, Baronio R, Bassi R. 2002. Dynamics of chromophore binding to Lhc proteins in vivo and in vitro during operation of the xanthophyll cycle. Journal of Biological Chemistry 277, 36913–36920.
Müller P, Li X-P, Niyogi KK. 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiology 125, 1558–1566.
Nield J, Funk C, Barber J. 2000. Supermolecular structure of photosystem II and location of the PsbS protein. Philosophical Transactions of the Royal Society of London B Biological Sciences 355, 1337–1344.
Niyogi KK, Björkman O, Grossman AR. 1997. Chlamydomonas xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching. The Plant Cell 9, 1369–1380.[Abstract]
Niyogi KK, Grossman AR, Björkman O. 1998. Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. The Plant Cell 10, 1121–1134.
Pesaresi P, Sandona D, Giuffra E, Bassi R. 1997. A single point mutation (E166Q) prevents dicyclohexylcarbodiimide binding to the photosystem II subunit CP29. FEBS Letters 402, 151–156.[CrossRef][ISI][Medline]
Peterson RB, Havir EA. 2000. A non-photochemical-quenching-deficient mutant of Arabidopsis thaliana possessing normal pigment composition and xanthophyll-cycle activity. Planta 210, 205–214.[CrossRef][ISI][Medline]
Peterson RB, Havir EA. 2001. Photosynthetic properties of an Arabidopsis thaliana mutant possessing a defective PsbS gene. Planta 214, 142–152.[CrossRef][ISI][Medline]
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, Walters RG, Horton P. 1992. The molecular mechanism of the control of excitation energy dissipation in chloroplast membranes: inhibition of pH-dependent quenching of chlorophyll fluorescence by dicyclohexylcarbodiimide. FEBS Letters 309, 175–179.[CrossRef][ISI][Medline]
Shikanai T, Munekage Y, Shimizu K, Endo T, Hashimoto T. 1999. Identification and characterization of Arabidopsis mutants with reduced quenching of chlorophyll fluorescence. Plant and Cell Physiology 40, 1134–1142.
Thayer SS, Björkman O. 1990. Leaf xanthophyll content and composition in sun and shade determined by HPLC. Photosynthesis Research 23,