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First published online November 14, 2008; 10.1104/pp.108.129957 Plant Physiology 149:1061-1075 (2009) © 2009 American Society of Plant Biologists
The Zeaxanthin-Independent and Zeaxanthin-Dependent qE Components of Nonphotochemical Quenching Involve Common Conformational Changes within the Photosystem II Antenna in Arabidopsis1,[W]School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom (M.P.J., A.Z., A.V.R.); and Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom (M.L.P.-B., P.H.)
The light-harvesting antenna of higher plant photosystem II (LHCII) has the intrinsic capacity to dissipate excess light energy as heat in a process termed nonphotochemical quenching (NPQ). Recent studies suggest that zeaxanthin and lutein both contribute to the rapidly relaxing component of NPQ, qE, possibly acting in the minor monomeric antenna complexes and the major trimeric LHCII, respectively. To distinguish whether zeaxanthin and lutein act independently as quenchers at separate sites, or alternatively whether zeaxanthin fulfills an allosteric role regulating lutein-mediated quenching, the kinetics of qE and the qE-related conformational changes (  A535) were compared in Arabidopsis (Arabidopsis thaliana) mutant/antisense plants with altered contents of minor antenna (kolhcb6, aslhcb4), trimeric LHCII (aslhcb2), lutein (lut2, lut2npq1, lut2npq2), and zeaxanthin (npq1, npq2). The kinetics of the two components of NPQ induction arising from zeaxanthin-independent and zeaxanthin-dependent qE were both sensitive to changes in the protein composition of the photosystem II antenna. The replacement of lutein by zeaxanthin or violaxanthin in the internal Lhcb protein-binding sites affected the kinetics and relative amplitude of each component as well as the absolute chlorophyll fluorescence lifetime. Both components of qE were characterized by a conformational change leading to nearly identical absorption changes in the Soret region that indicated the involvement of the LHCII lutein 1 domain. Based on these observations, we suggest that both components of qE arise from a common quenching mechanism based upon a conformational change within the photosystem II antenna, optimized by Lhcb subunit-subunit interactions and tuned by the synergistic effects of external and internally bound xanthophylls.
The chlorophyll a/b-binding light-harvesting antenna of photosystem II (PSII of higher plants is responsible for the efficient collection and transfer of excitation energy to the reaction center. The PSII antenna comprises the main trimeric light-harvesting complex, LHCII, which is composed of the Lhcb1 to -3 polypeptides, and the minor light-harvesting complexes, CP29, CP26, and CP24, composed of Lhcb4, -5, and -6, respectively. In Arabidopsis (Arabidopsis thaliana), four LHCII trimers associate with two copies each of CP24, CP26, and CP29 and a core dimer of PSII (CP43/D1/D2/CP47) to form the C2S2M2 LHCII-PSII supercomplex (Dekker and Boekema, 2005  ). In addition, depending upon the growth conditions, two or three extra LHCII trimers per PSII may be present in LHCII-only regions of the grana, providing additional light-harvesting capacity.
The PSII antenna is a highly dynamic system that is able to tune the amount of excitation delivered to the PSII reaction center to match physiological need (Horton et al., 1996
Although there is strong evidence that qE occurs in the PSII antenna light-harvesting proteins and that xanthophylls are involved, the mechanism of energy dissipation remains unclear. There is evidence for two distinct quenching mechanisms, one involving zeaxanthin (type I) and the other lutein (type II). In the type I mechanism, it is proposed that qE obligatorily depends upon zeaxanthin acting as a quencher of excited chlorophyll via the formation of a charge transfer state. Evidence for type I is the formation of a carotenoid radical cation absorbing at approximately 1,000 nm that correlates with the extent of qE (Holt et al., 2005
In the type II mechanism, qE is an inbuilt property of LHCII proteins; a protein conformational change alters the configuration of bound pigments and results in the xanthophyll bound at the L1 site (normally lutein) becoming an effective quencher of chlorophyll excited states (Ruban et al., 2007
Although the type I and type II mechanisms involve different xanthophylls operating at different sites, there are similarities: in particular, both are proposed to involve a
Both Zeaxanthin-Independent and Zeaxanthin-Dependent Components of qE Are Sensitive to Changes in the Protein Composition of the PSII Antenna
NPQ was analyzed in dark-adapted plants lacking the proteins of the major trimeric LHCII (aslhcb2; Andersson et al., 2003
To test whether the zeaxanthin-independent or zeaxanthin-dependent components of qE were affected in the Lhcb mutant/antisense plants, the kinetics of NPQ were recorded after infiltration of leaves with dithiothreitol (DTT). HPLC analysis of the leaves revealed no detectable increase in the contents of antheraxanthin or zeaxanthin compared with the dark-adapted leaves, indicating that DTT had completely inhibited deepoxidation (Table I). In wild-type leaves infiltrated with DTT, NPQ formed rapidly, but to a level only 40% of that found in untreated leaves (Fig. 1B). As was the case in the noninfiltrated leaves, in the presence of DTT, rates of NPQ formation in aslhcb2 and aslhcb4 were slower than in the wild type but faster than in kolhcb6 (Fig. 1B; Table II). Significantly, in the presence of DTT, the amplitude of NPQ in each of the mutant/antisense plants was less than in the wild type. Furthermore, the amplitude of the zeaxanthin-dependent NPQ (calculated by the subtraction of NPQ + DTT from the total NPQ [Adams et al., 1990 ]) was also reduced in each mutant. Hence, both the zeaxanthin-dependent and zeaxanthin-independent components of NPQ were sensitive to changes in the PSII antenna protein composition, irrespective of whether the mutation/antisense affected the major trimeric or minor monomeric antenna complexes. The relaxation of the qE component of NPQ was also monitored in the wild-type and mutant/antisense leaves in both the presence and absence of DTT. qE relaxed with similar kinetics in the wild type and in aslhcb4, but it relaxed more slowly in aslhcb2 and more rapidly in kolhcb6 (Fig. 1, C and D; Table II). In all cases, the relaxation of qE was faster in the presence of DTT, but most importantly, the differences between the mutant/antisense leaves and wild-type leaves were still observed. In all cases, the major fraction of the zeaxanthin-independent NPQ was rapidly reversible (Fig. 1D), showing it to be of the qE type. The kinetics of the reformation of NPQ were tested in wild-type and mutant/antisense leaves that had previously been preilluminated for 10 min at 700 µmol photons m–2 s–1, before being given 5 min of dark to relax qE. In the wild type and each mutant, the NPQ formation kinetics were accelerated compared with the first illumination cycle (Fig. 2A ; Table II), yet the differences between them were maintained. The aslhcb4 and aslhcb2 formed NPQ more slowly than the wild type, while in kolhcb6 NPQ formed faster. A similar result was also observed for DTT-infiltrated leaves (Fig. 2B; Table II), but interestingly, NPQ now formed more slowly than in noninfiltrated leaves, except in the case of kolhcb6, where there was no difference between the two conditions. Therefore, differences in Lhcb protein composition affect in the same way the formation and relaxation of qE both in the presence and in the absence of deepoxidation.
Investigation of the rate of zeaxanthin formation and comparison with the rate of formation of NPQ allows the sensitivity of NPQ to zeaxanthin in each mutant to be determined. The change in absorption at 505 nm measured relative to a 565-nm reference ( A505-565) has a linear dependence on zeaxanthin concentration (Bilger et al., 1989). Therefore, the kinetics of A505-565 following illumination of dark-adapted leaves were monitored (Fig. 3A
). The rate of zeaxanthin formation was significantly faster in kolhcb6 (half-time [t ] = 140 ± 4 s) than in either the wild type (t = 170 ± 4 s) or aslhcb4 (t = 170 ± 2 s), but in aslhcb2 leaves the kinetics were much slower (t = 245 ± 8 s). In each case, the deepoxidation states were maximal within approximately 10 min, further illumination producing no additional zeaxanthin formation (data not shown). HPLC analysis of pigments extracted from illuminated plants revealed that the final deepoxidation states were 42% in aslhcb2, 45% in aslhcb4, 46% in the wild type, and 53% in kolhcb6 (Table I). When the kinetics of NPQ formation were plotted against the 505-nm absorption kinetics for the wild-type plants, a nonlinear relationship was found, which may be attributed to the zeaxanthin-independent component of NPQ. Subtraction of the zeaxanthin-independent component of NPQ (wild type minus wild type + DTT [Adams et al., 1990]) yields the relationship between zeaxanthin formation and the zeaxanthin-dependent component of NPQ. In the wild type, this relationship was initially linear, as found previously (Demmig-Adams et al., 1989), but saturated at high concentrations of zeaxanthin (Fig. 3B). This procedure was repeated for the mutant plants. In the case of aslhcb4, the nonlinearity at high zeaxanthin content was slightly more pronounced. In aslhcb2, the slope of the NPQ versus A505-565 plot was significantly reduced, indicating an apparently reduced sensitivity of NPQ to zeaxanthin. In contrast, the kolhcb6 mutant appeared to be initially the most sensitive to zeaxanthin, but the NPQ response was saturated at relatively low levels of zeaxanthin. Thus, changes in the composition of the proteins of both trimeric LHCII and the minor antenna complexes affect the sensitivity of NPQ to zeaxanthin, implicating a role for both sets of proteins in the zeaxanthin-dependent component of qE.
qE-Related Conformational Changes in the PSII Antenna Are Dependent upon Lhcb Subunit Composition
The formation of qE is correlated with a positive absorption change in leaves at 535 nm (
Absorption difference spectra (5 min of light minus 5 min of dark relaxation) were recorded in the Soret region (420–565 nm) to provide information on the pigment changes resulting from the qE-related conformational changes (Fig. 4B). In the wild type, the difference spectrum was similar to that reported previously (Ruban et al., 1993b ), with a broad positive band at 535 nm, attributed to zeaxanthin (Ruban et al., 2002b), and negative bands at 495, 468, and 438 nm. The same bands were found in the difference spectra obtained from the kolhcb6, aslhcb2 and aslhcb4 plants, but they were of smaller amplitude, consistent with their reduced levels of qE. In the case of the kolhcb6 mutant, an attempt was made to record a second difference spectrum for the transient signal (1 min of light minus 5 min of dark relaxation). Although it was impossible to obtain a completely accurate spectrum during the transient changes in absorption, a positive band in the 535-nm region and negative bands below 500 nm were observed, the same features found in the steady-state spectrum (data not shown).
Inhibition of zeaxanthin formation by treatment with DTT resulted in a reduction in the amplitude of
Absorption difference spectra revealed the nature of the absorption changes that accompany the zeaxanthin-independent component of qE. All of the features present in the difference spectra in untreated leaves were found, although the amplitudes of all bands were smaller, consistent with the reduced level of NPQ in the absence of zeaxanthin (Fig. 4D). The peak position of the positive band was down-shifted from 535 to 525 nm, as reported previously (Noctor et al., 1993
The effect of varying xanthophyll composition within the PSII antenna on the kinetics of the two components of qE was examined in wild-type and xanthophyll mutant leaves. The pigment composition in light-treated and dark-adapted leaves of these mutants (Table III
) was similar to the previously reported values (Niyogi et al., 1998
Upon the second illumination, qE formed rapidly in the wild-type leaves, reaching saturation in less than 20 s (Fig. 5B; Table IV), and was now faster than that observed in the npq1 mutant. In contrast, npq2 still displayed faster kinetics of NPQ formation than the wild type (Fig. 5B; Table IV). The effect of lutein replacement on qE was dramatic: qE formed more slowly and with reduced amplitude in lut2npq2 compared with npq2. Similarly, the replacement of lutein by violaxanthin in lut2 reduced the rate and amplitude of qE formation compared with the wild type, despite the fact that the deepoxidation state was similar to that in the wild type (Fig. 5B; Table IV). Hence, the replacement of lutein in the internal binding sites of the Lhcb proteins by either violaxanthin or zeaxanthin resulted in a reduction in the rate and amplitude of qE formation independent of differences in the deepoxidation state. It was also tested whether the qE transiently formed at low light intensity, which is suggested to occur in the PSII reaction center (Finazzi et al., 2004 ), was similarly affected by alteration in the xanthophyll composition. The same dependence on xanthophyll composition was found for transient qE occurring at low light intensity as for the steady-state qE generated at high light intensity, with strong reductions in amplitude observed in the npq1, lut2, and lut2npq2 mutants and a complete absence in the lut2npq1 mutant (Supplemental Fig. S1).
The lower values of NPQ in the two zeaxanthin-accumulating mutants have been suggested to arise from the prequenching of Fm (for definitions of fluorescence terms, see "Materials and Methods") caused by sustained zeaxanthin-mediated NPQ (Dall'Osto et al., 2005
qE Relaxation Kinetics Are Composed of a Single Component and Are Modulated by Zeaxanthin The kinetics of qE relaxation were also analyzed in the xanthophyll mutants. In all cases, the relaxation could be fitted to a single hyperbolic decay (Fig. 5C; Table IV). The rate of relaxation of qE in the npq1 mutant, which contains only the zeaxanthin-independent component of qE, was twice as fast as in the wild type. The comparison of npq1 with npq2 relaxation kinetics provided clues about whether one or two mechanisms are involved in qE. In the case of npq2, if separate type I and type II mechanisms were present, then qE should have relaxed with two components, one of which is fast (as in npq1) and the second of which is slower (because of the presence of zeaxanthin). In contrast to this prediction, it was observed that the additional zeaxanthin in npq2 slowed the relaxation of all of qE, and not just the zeaxanthin-dependent component. Indeed, the rate of relaxation of qE in npq2 was found to nearly triple that found in the wild type (Fig. 5C; Table IV). The replacement of lutein by additional zeaxanthin in lut2npq2 was associated with a further slowing of the relaxation of qE. In contrast, the relaxation of qE in lut2 was marginally faster than in the wild type (Fig. 5C; Table IV). Hence, the amount of zeaxanthin appeared to modulate the rate of qE relaxation, which behaved kinetically as a single process.
The qE-related conformational changes were investigated in dark-adapted leaves of wild-type and xanthophyll mutant plants by measurement of the kinetics of
The absorption difference spectra of the qE-related conformational changes were recorded in the xanthophyll mutants in order to provide clues about which xanthophylls were involved (Fig. 6, C and D). The difference spectrum for npq1 was nearly identical to that of the wild type (Fig. 6C), with negative bands at 438, 468, and 495 nm. The positive band was shifted to around 525 nm, as found in DTT-treated wild-type leaves (Fig. 4C). Consistent with the much reduced qE in this mutant, the amplitudes of all bands were much less than those found in the wild-type spectrum. The difference spectrum of npq2 showed some differences from the wild type. The amplitude of the positive 535-nm band was the same as in the wild type but was slightly red shifted to around 537 nm. In contrast, the negative bands in this spectrum were all of lower amplitude than in the spectrum for the wild type, with a slight broadening and a red shift of the 495-nm band. The negative and positive bands in the lut2 difference spectrum were all of smaller amplitude than in the wild-type spectrum, consistent with the reduced qE in this mutant (Fig. 6D). The 495-nm negative band was again red shifted slightly to around 497 nm. In the difference spectrum of the lut2npq2 mutant, the amplitude of the 535-nm band was enhanced relative to the band in the lut2 mutant, consistent with the A535 data. As in npq2, the wavelength maximum was red shifted to around 537 nm. Interestingly, however, the negative bands were smaller and there was a strong red shift of the 495-nm band to around 501 nm. The complete absence of either the negative or the positive band in the lut2npq1 difference spectrum demonstrated that all of the features of the spectrum are entirely related to qE (Fig. 6D) and that the electrochromic shift (A = 518 nm) makes negligible contribution to the steady-state spectra, as found previously (Li et al., 2000). The transient qE generated at low light intensity was also found to be associated with a transient increase in 535-nm absorption. The light-minus-recovery spectrum of this transient absorption change was virtually identical to the steady-state spectrum observed at high light intensity (Supplemental Fig. S2).
We have tested the hypothesis that zeaxanthin-dependent and zeaxanthin-independent components of NPQ arise from two separate mechanisms (type I and type II, respectively), occurring in different parts of the LHCII/PSII complexes. Contrary to the predictions of this hypothesis, it was found that both NPQ components shared a similar sensitivity to changes in the protein compositions of both the minor monomeric and the major trimeric LHCII antenna. Thus, the amplitudes of both NPQ components were reduced in kolhcb6, aslhcb4, and aslhcb2 plants compared with wild-type plants. The observation that the zeaxanthin-dependent NPQ was reduced in these plants is consistent with the fact that xanthophyll cycle pigments are bound to the minor monomeric antenna and trimeric LHCII (Ruban et al., 1999 , 2002a) and with the previously documented effects of these xanthophylls on quenching of all of these complexes in vitro (Ruban et al., 1996, 1997; Wentworth et al., 2000). The data also suggest that qE is unlikely to arise solely in the minor monomeric antenna proteins of PSII, as proposed many times (Gilmore et al., 1998; Ahn et al., 2008). These conclusions are consistent with the finding that, while no individual Lhcb protein appears to be obligatory for qE, each one may play a role in shaping the PSII macrostructure for the optimal combination of light harvesting and photoprotection (Horton et al., 2008) and with studies on the chlorophyll b-less mutants with severely reduced PSII antenna size that showed strong reductions in NPQ capacity (Härtel et al., 1996; Gilmore et al., 2000). The disruptive effect of the depletion of Lhcb proteins on the PSII macrostructure is well documented (Horton et al., 2008). Indeed, even in aslhcb2 plants, in which the lateral C2S2M2 arrangement of PSII is maintained (Ruban et al., 2003), the replacement of Lhcb1 and -2 by Lhcb5 leads to weaker grana stacking forces (E.-H. Kim, W.S. Chow, and J.M. Anderson, unpublished data). Thus, the proper arrangement of PSII and grana stacking appears to be essential for fully functional NPQ. Additional insight into the structural basis of NPQ has been obtained in this investigation by analysis of the in vivo absorption change at A535, which arises from the conformational changes within the PSII antenna that accompany the formation of the quenched state (Ruban et al., 1993b; Bilger and Björkman, 1994). As found for the PSII macrostructure, A535 was sensitive to the loss of both minor monomeric and trimeric LHCII proteins. Again, A535 was correlated with both the zeaxanthin-dependent and zeaxanthin-independent components of qE, the shift to shorter wavelength in the latter reflecting the involvement of xanthophylls in this change. Deficiencies in Lhcb proteins affected not only the amplitude of NPQ but also the kinetics of formation and relaxation. The rate of relaxation of NPQ was faster in the absence of Lhcb6 and slower in the absence of Lhcb1 and -2. In neither case was there evidence of two components to the relaxation kinetics that may have arisen if zeaxanthin-dependent and zeaxanthin-independent quenching were caused by different type I and type II mechanisms and/or within different complexes. In dark-adapted leaves, the differences in the formation kinetics of the zeaxanthin-dependent qE arise at least in part from the altered kinetics of deepoxidation. In kolhcb6, the rate of deepoxidation was accelerated compared with the wild type, while in aslhcb2, it was retarded. The reasons for the differences in deepoxidation kinetics are unclear at present, but we suggest that they are also a consequence of the altered macrostructure and antenna protein composition.
The differences in kinetics of zeaxanthin-dependent quenching cannot be fully explained by altered deepoxidation rates. Thus, the plots of the rates of violaxanthin deepoxidation against the rate of development of the zeaxanthin-dependent component of NPQ were not the same in the mutants and the wild type (i.e. the sensitivity to zeaxanthin was variable). Thus, while aslhcb4 had a similar sensitivity to zeaxanthin as the wild type, aslhcb2 was less sensitive and kolhcb6 was more sensitive. Reduced sensitivity of qE to the
Investigation of mutants deficient in specific xanthophylls confirmed the conclusions that arose from the study of the Lhcb mutants. As argued above, if type I and type II mechanisms both contribute to qE, there would be discrete kinetic components from each mechanism. In contrast to this prediction, only one component of qE relaxation was observed, the rate of which was regulated by the level of zeaxanthin. In the type I mechanism, zeaxanthin is the quencher and its level would not affect the relaxation kinetics. In contrast, the role of zeaxanthin as an allosteric regulator, proposed in the type II mechanism, readily explains the changes in relaxation kinetics. The effect of zeaxanthin in slowing qE relaxation has been reported previously (Noctor et al., 1991
Measurement of fluorescence lifetimes confirmed the suggestion that the extra zeaxanthin in npq2 and lut2npq2 caused a prequenching of Fm (Dall'Osto et al., 2005
The perturbation of the capacity and kinetics of NPQ formation in lutein-deficient mutants, observed previously and repeated here, lends some support for the presence of a type II mechanism. Both zeaxanthin-dependent and zeaxanthin-independent qE were affected, the latter being almost completely eliminated. However, the presence of zeaxanthin-dependent qE in the absence of lutein, albeit kinetically different in the mutants compared with the wild type, indicated the lack of an obligatory requirement for lutein. It is clear that when either zeaxanthin or violaxanthin replaces lutein in the internal binding sites in Lhcb proteins, quenching still occurs, but both the dynamics and amplitude of qE are affected. These data, therefore, add to previous work demonstrating the importance of lutein for fully functional qE (Pogson et al., 1998
Exploration of the absorption changes associated with qE formation gave some insight into how the xanthophyll composition perturbed the conformational changes. The constitutive presence of zeaxanthin in npq2 and lut2npq2 increased the amplitude of the
Improved resolution of the negative bands (495, 468, and 438 nm) in the qE-related absorption difference spectra provides an indication of the complexity of the changes in pigment configurations upon the establishment of qE. Consistent with this finding, light-minus-recovery Raman difference spectra of leaves and chloroplasts show loss of resonance below 500 nm, while it is gained above 500 nm, reflecting exactly the same negative and positive features of the qE absorption difference spectra (Ruban et al., 2002b ). The position of the 495-nm band was red shifted when lutein was replaced by zeaxanthin or violaxanthin in lut2npq2 and lut2 mutants, consistent with the assignment of this band to lutein (Ruban et al., 2000). In contrast to the behavior of the 535-nm band, the amplitudes of these bands were reduced in lut2npq2 compared with the wild type, therefore being better correlated with qE (Fig. 7B). Thus, it is suggested that this observation provides support for the involvement of the L1 domain in qE, as in the type II mechanism.
Analysis of the 535-nm absorption change also suggested that the qE that is transiently formed at low light intensity also involves conformational changes within the PSII antenna. Previously, qE formed under these conditions was ascribed to quenching in the PSII reaction center (Finazzi et al., 2004
In summary, the evidence that both the zeaxanthin-independent and zeaxanthin-dependent components of qE arise from the same mechanism within the PSII antenna has been strengthened. Both are enhanced by PsbS overexpression (Li et al., 2002
Plants and Growth Conditions
Arabidopsis (Arabidopsis thaliana ‘Columbia’) and mutant and transgenic lines derived from it were grown for 8 to 9 weeks in Sanyo plant growth cabinets with an 8-h photoperiod at a light intensity of 100 µmol photons m–2 s–1 and day/night temperatures of 22°C/15°C. The xanthophyll mutants used were as follows: npq1 (mutated in violaxanthin deepoxidase and therefore unable to synthesize zeaxanthin in excess light); npq2 (mutated in zeaxanthin epoxidase and constitutively containing zeaxanthin even in low light, while lacking violaxanthin and neoxanthin; Niyogi et al., 1998
Chlorophyll fluorescence was measured with a Dual PAM 100 chlorophyll fluorescence photosynthesis analyzer (Heinz Walz). The plants were adapted in the dark for 30 min prior to measurement. Actinic illumination (700 µmol photons m–2 s–1) was provided by arrays of 635-nm light-emitting diodes illuminating both the adaxial and abaxial surfaces of the leaf. Fo (the fluorescence level with PSII reaction centers open) was measured in the presence of a measuring beam of 10 µmol photons m–2 s–1. The maximum fluorescence in the dark-adapted state (Fm), during the course of actinic illumination (Fm'), and in the subsequent dark relaxation periods was determined using a 0.8-s saturating light pulse (4,000 µmol photons m–2 s–1). The quantum yield of PSII (Fv/Fm) was defined as (Fm – Fo)/Fm, and NPQ was defined as (Fm – Fm')/Fm'. The reversible component of NPQ (relaxing within 5 min) was assigned to energy-dependent NPQ (qE) and calculated as (Fm/Fm') – (Fm/Fm''), where Fm'' is the maximal yield of fluorescence after 5 min of dark relaxation following the actinic illumination. Leaves were vacuum infiltrated with a 5 mM DTT solution in order to completely inhibit violaxanthin deepoxidation. Analysis of the kinetics of NPQ was carried out using a SigmaPlot software curve-fitting procedure (SPSS).
Time-correlated single photon counting measurements were performed using a FluoTime 200 picosecond fluorometer (PicoQuant). Detached leaves were vacuum infiltrated with 50 µM nigericin to completely inhibit NPQ. Excitation at a 10-MHz repetition rate was provided by a 470-nm laser diode, which was carefully adjusted to completely close all PSII reaction centers without causing photoinhibitory quenching of Fm and to be far below the onset of singlet-singlet exciton annihilation. For measurement of Fm' values, NPQ was induced at 700 µmol photons m–2 s–1 by arrays of 635-nm light-emitting diodes illuminating both the adaxial and abaxial surfaces of the leaf. Fluorescence was detected at 685 nm with 2-nm slit width. The instrumental response function was in the range of 50 ps. For lifetime analysis, FluoFit software (PicoQuant) was used.
Absorption changes in the 410- to 565-nm region were measured using a SLM DW2000 dual-wavelength spectrophotometer. Whole Arabidopsis leaves were detached from plants dark adapted for 30 min, and the petioles were wrapped in moist filter paper. The actinic light (700 µmol photons m–2 s–1) illuminated the leaf at 45° and was defined using a Corning 5-58 filter. The photomultiplier was protected using a Corning 4-96 filter and an OCLl Cyan T400-570 mirror. The instrument slit width was 5 nm, and the scan rate was 2 nm s–1.
Pigment composition was determined by reverse-phase HPLC using a LiChrospher 100 RP-18 column (Merck) and a Dionex Summit chromatography system as described previously (Johnson et al., 2008
The following materials are available in the online version of this article.
We thank Patrick Romano for assistance isolating the SALK T-DNA knockout of lut2, Stefan Jansson and Jakob Damkjaer for providing seeds of aslhcb4, aslhcb2, and kolhcb6, and Kris Niyogi for providing seeds of lut2npq1 and lut2npq2 mutants. Received September 16, 2008; accepted November 11, 2008; published November 14, 2008.
1 This work was supported by grants to A.V.R. and P.H. from the Biotechnology and Biological Sciences Research Council and by the Intro2 Marie Curie Research Training Network of the European Union.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Alexander V. Ruban (a.ruban{at}qmul.ac.uk).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.129957 * Corresponding author; e-mail a.ruban{at}qmul.ac.uk.
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-helices; and a 9-cis-neoxanthin at the N1 site associated with the C-helix chlorophyll b domain (Liu et al., 2004
-cyclase and lacking lutein; Pérez-Bueno and Horton, 2008
