Xanthophyll cycle and energy-dependent fluorescence quenching in leaves from pea plants grown under intermittent light

The possible role of zeaxanthin formation and antenna proteins in energy-dependent chlorophyll fluorescence quenching (qE) has been investigated. Intermittent-light-grown pea (Pisum sativum L.) plants that lack most of the chlorophyll a/b antenna proteins exhibited a significantly reduced qE upon illumination with respect to control plants. On the other hand, the violaxanthin content related to the number of reaction centers and to xanthophyll cycle activity, i.e. the conversion of violaxanthin into zeaxanthin, was found to be increased in the antenna-protein-depleted plants. Western blot analyses indicated that, with the exception of CP 26, the content of all chlorophyll a/b-binding proteins in these plants is reduced to less than 10% of control values. The results indicate that chlorophyll a/b-binding antenna proteins are involved in the energy-dependent fluorescence quenching but that only a part of qE can be attributed to quenching by chlorophyll a/b-binding proteins. It seems very unlikely that xanthophylls are exclusively responsible for the qE mechanism.Abbreviations CAB chlorophyll a/b-binding - Chl chlorophyll - F_V variable fluorescence - IML intermittent light - LHC light harvesting complex - PFD photon flux density - qP photochemical quenching of chlorophyll fluoresence - qN non-photochemical quenching - qE energy-dependent quenching - qI photoinhibitory quenching - qT quenching by state transition     

Characterization of the fast and slow reversible components of non-photochemical quenching in isolated pea thylakoids by picosecond time-resolved chlorophyll fluorescence analysis.

The fast and slow reversible components of non-photochemical chlorophyll fluorescence quenching commonly assigned to the qE and the qI mechanism have been studied in isolated pea thylakoids which were prepared from leaves after a moderate photoinhibitory treatment. Chlorophyll fluorescence decays were measured at picosecond resolution and analyzed on the basis of the heterogeneous exciton/radical pair equilibrium model. Our results show that the fast reversible non-photochemical quenching is completely assigned to the PS II antenna and is related to zeaxanthin. The slow reversible qI type quenching is located at the PS II reaction center and involves enhanced nonradiative decay of the primary charge separated state to its ground state and/or triplet excited state. Apart from its independence from the proton gradient, the qI quenching shows striking similarities to a particular form of qE quenching which is also located at the PS II reaction center and has resently been resolved in isolated thylakoids from dark-adapted leaves [Wagner, B., et al. (1996) J. Photochem. Photobiol., B 36, 339-350]. Our data suggest that during exposure to the supersaturating light the reaction center qE component was replaced by qI quenching. This qE to qI transition is supposed to be part of the mechanism of the long-term downregulation of PS II during photoinhibition. It is also evident that under the conditions used in our study zeaxanthin-dependent antenna quenching is not involved in the slow reversible downregulation of PS II but that it retains its dependence on the proton gradient during exposure to strong light.     

Thermostability and photostability of photosystem II of the resurrection plant Haberlea rhodopensis studied by chlorophyll fluorescence

The stability of PSII in leaves of the resurrection plant Haberlea rhodopensis to high temperature and high light intensities was studied by means of chlorophyll fluorescence measurements. The photochemical efficiency of PSII in well-hydrated Haberlea leaves was not significantly influenced by temperatures up to 40 degrees C. Fo reached a maximum at 50 degrees C, which is connected with blocking of electron transport in reaction center II. The intrinsic efficiency of PSII photochemistry, monitored as Fv/Fm was less vulnerable to heat stress than the quantum yield of PSII electron transport under illumination (phiPSII). The reduction of phiPSII values was mainly due to a decrease in the proportion of open PSII centers (qP). Haberlea rhodopensis was very sensitive to photoinhibition. The light intensity of 120 micromol m(-2) s(-1) sharply decreased the quantum yield of PSII photochemistry and it was almost fully inhibited at 350 micromol m(-2) s(-1). As could be expected decreased photochemical efficiency of PSII was accompanied by increased proportion of thermal energy dissipation, which is considered as a protective effect regulating the light energy distribution in PSII. When differentiating between the three components of qN it was evident that the energy-dependent quenching, qE, was prevailing over photoinhibitory quenching, qI, and the quenching related to state 1-state 2 transitions, qT, at all light intensities at 25 degrees C. However, the qE values declined with increasing temperature and light intensities. The qI was higher than qE at 40 degrees C and it was the major part of qN at 45 degrees C, indicating a progressing photoinhibition of the photosynthetic apparatus.     

草莓叶片光合作用对强光的响应及其机理研究

用便携式调制叶绿素荧光仪和光合仪研究了强光下草莓叶片荧光参数及表观量子效率的变化.结果表明,Fm、Fv/Fm、PSⅡ无活性反应中心数量和Q_A的还原速率在强光下降低,在暗恢复时升高;而PSⅡ反应中心非还原性Q_B的比例在强光下增加,在暗恢复时降低.上述荧光参数的变化幅度均以强光胁迫或暗恢复的前10 min最大.强光下Φ_PSII、ETR和qP先升高后降低,但qN先大幅度降低,然后小幅回升.强光处理4 h后,丰香和宝交早生的表观量子效率(AQY)分别降低了20.9%和37.5%;qE(能量依赖的非光化学猝灭)为NPQ(非光化学猝灭)的最主要成分.强光胁迫下丰香的Fo、Fm、Fv/Fm、Φ_PSII、ETR和AQY的变化幅度均明显比宝交早生小.DTT处理后,草莓叶片的Fm和Fv/Fm明显降低,Fo显著升高.可以认为,依赖叶黄素循环和类囊体膜质子梯度两种非辐射能量耗散在草莓叶片防御光损伤方面起着重要作用,丰香的光合机构比宝交早生更耐强光.     

The Kinetics of Zeaxanthin Formation Is Retarded by Dicyclohexylcarbodiimide

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth → Zeax) of the reaction sequence seemed to be more affected than the violaxanthin → Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.     

Kinetic correlation of recovery from photoinhibition and zeaxanthin epoxidation

The generation of non-photochemical fluorescence quenching under photoinhibitory illumination and its relaxation under subsequent low light illumination in leaves from intermittent-light-grown pea (Pisum sativum L.) plants (IML-plants) has been investigated. In parallel, we studied (i) the activity of the xanthophyll cycle with emphasis on zeaxanthin formation and reconversion to violaxanthin and (ii) the degradation rate of D1 protein. In comparison to control plants grown in continuous light, IML-plants were much more susceptible to photoinhibition as determined from the increase of slowly (halftimes > 20 min) relaxing quenching (qI) of variable chlorophyll fluorescence. The relaxation (recovery) kinetics of qI (under weak light) in both types of plant depended on the photon flux density, temperature and duration of pre-illumination. The recovery time generally increased with an increasing degree of qI. In IML-plants, relaxation of qI was kinetically closely related to the epoxidation of zeaxanthin. At high degrees of photosystem II inhibition the kinetics resembled those of D1 degradation. The results are discussed in terms of the mechanisms of photosystem II inactivation in vivo.     

De-epoxidation of violaxanthin in the minor antenna proteins of photosystem II, LHCB4, LHCB5, and LHCB6

The conversion of violaxanthin to zeaxanthin is essentially required for the pH-regulated dissipation of excess light energy in the antenna of photosystem II. Violaxanthin is bound to each of the antenna proteins of both photosystems. Former studies with recombinant Lhcb1 and different Lhca proteins implied that each antenna protein contributes specifically to violaxanthin conversion related to protein-specific affinities of the different violaxanthin binding sites. We investigated the violaxanthin de-epoxidation in the minor antenna proteins of photosystem II, Lhcb4-6. Recombinant proteins were reconstituted with different xanthophyll mixtures to study the conversion of violaxanthin at different xanthophyll binding sites in these proteins. The extent and kinetics of violaxanthin de-epoxidation were found to be dependent on the respective protein and, for each protein, also on the binding site of violaxanthin. In particular, violaxanthin bound to Lhcb4 was nearly inconvertible for de-epoxidation, whereas violaxanthin bound to Lhcb5 was fully convertible but with slow kinetics. Lhcb6 exhibited heterogeneous violaxanthin conversion characteristics, which could be assigned to different populations of reconstituted Lhcb6 complexes with respect to violaxanthin binding sites. The results support the proposed different binding affinities of violaxanthin to the three putative violaxanthin binding sites (V1, N1, and L2) in antenna proteins. Under consideration of former studies with Lhcb1 and Lhca proteins, the data imply that violaxanthin bound to the V1 and N1 binding site of antenna proteins is easily accessible for de-epoxidation in all antenna proteins, whereas violaxanthin bound to L2 is either only slowly or not convertible to zeaxanthin, depending on the respective protein.     

The Lhcb protein and xanthophyll composition of the light harvesting antenna controls the DeltapH-dependency of non-photochemical quenching in Arabidopsis thaliana

Nonphotochemical quenching (NPQ) is the photoprotective dissipation of energy in photosynthetic membranes. The hypothesis that the DeltapH-dependent component of NPQ (qE) component of non-photochemical quenching is controlled allosterically by the xanthophyll cycle has been tested using Arabidopsis mutants with different xanthophyll content and composition of Lhcb proteins. The titration curves of qE against DeltapH were different in chloroplasts containing zeaxanthin or violaxanthin, proving their roles as allosteric activator and inhibitor, respectively. The curves differed in mutants deficient in lutein and specific Lhcb proteins. The results show that qE is determined by xanthophyll occupancy and the structural interactions within the antenna that govern allostericity.     

Differences in light energy utilisation and dissipation between dipterocarp rain forest tree seedlings

 The light environment within tropical rain forests varies considerably both spatially and temporally, and photon flux density (PFD) is considered to be an important factor determining the growth and survival of rain forest tree seedlings. In this paper we examine the ability of four ecologically contrasting dipterocarps (Dryobalanops lanceolata, Shorea leprosula, Hopea nervosa and Vatica oblongifolia) to utilise and dissipate light energy when grown in different light environments in lowland dipterocarp rain forest in the Danum Valley Conservation Area, Sabah, East Malaysia. Specifically we report (i) photosynthetic light response curves and associated fluorescence characteristics, including quantum yield (ΦPSII) and non-photochemical quenching (qN) and (ii) the extent to which photoinhibition occurs when plants grown in either high or low light are exposed to short bursts of high PFD. When grown in low light (artificial or forest shade) all four species had low light saturated rates of photosynthesis which were achieved at low PFDs. In addition, values of ΦPSII and qN were similar over a range of measurement PFDs. D. lanceolata and S. leprosula were also grown at high PFD and showed marked differences in their responses. S. leprosula demonstrated an ability to increase its rate of photosynthesis and there was a small increase in capacity to dissipate excess light energy non-photochemically at high PFDs. Partitioning of this qN into its fast, photo-protective (qE) and slow, photoinhibitory (qI) components indicated that there was an increase in qE quenching. In contrast, although D. lanceolata survived in the high light environment, greater rates of photosynthesis were not observed and the plants showed a greater capacity to dissipate energy non-photochemically. Partitioning of qN revealed that the majority of this increase was attributable to the slower relaxing phases. Received: 10 February 1996 / Accepted: 14 June 1996     

Analysis of non-photochemical energy dissipating processes in wild type <Emphasis Type="Italic">Dunaliella salina</Emphasis> (green algae) and in <Emphasis Type="Italic">zea1</Emphasis>, a mutant constitutively accumulating zeaxanthin

Generally there is a correlation between the amount of zeaxanthin accumulated within the chloroplast of oxygenic photosynthetic organisms and the degree of non-photochemical quenching (NPQ). Although constitutive accumulation of zeaxanthin can help protect plants from photo-oxidative stress, organisms with such a phenotype have been reported to have altered rates of NPQ induction. In this study, basic fluorescence principles and the routinely used NPQ analysis technique were employed to investigate excitation energy quenching in the unicellular green alga Dunaliella salina, in both wild type (WT) and a mutant, zea1, constitutively accumulating zeaxanthin under all growth conditions. The results showed that, in D. salina, NPQ is a multi-component process consisting of energy- or ΔpH-dependent quenching (qE), state-transition quenching (qT), and photoinhibition quenching (qI). Despite the vast difference in the amount of zeaxanthin in WT and the zea1 mutant grown under low light, the overall kinetics of NPQ induction were almost the same. Only a slight difference in the relative contribution of each quenching component could be detected. Of all the NPQ subcomponents, qE seemed to be the primary NPQ operating in this alga in response to short-term exposure to excessive irradiance. Whenever qE could not operate, i.e., in the presence of nigericin, or under conditions where the level of photon flux is beyond its quenching power, qT and/or qI could adequately compensate its photoprotective function.     

Restoration of rapidly reversible photoprotective energy dissipation in the absence of PsbS protein by enhanced DeltapH

Variations in the light environment require higher plants to regulate the light harvesting process. Under high light a mechanism known as non-photochemical quenching (NPQ) is triggered to dissipate excess absorbed light energy within the photosystem II (PSII) antenna as heat, preventing photodamage to the reaction center. The major component of NPQ, known as qE, is rapidly reversible in the dark and dependent upon the transmembrane proton gradient (ΔpH), formed as a result of photosynthetic electron transport. Using diaminodurene and phenazine metasulfate, mediators of cyclic electron flow around photosystem I, to enhance ΔpH, it is demonstrated that rapidly reversible qE-type quenching can be observed in intact chloroplasts from Arabidopsis plants lacking the PsbS protein, previously believed to be indispensible for the process. The qE in chloroplasts lacking PsbS significantly quenched the level of fluorescence when all PSII reaction centers were in the open state (F(o) state), protected PSII reaction centers from photoinhibition, was modulated by zeaxanthin and was accompanied by the qE-typical absorption spectral changes, known as ΔA(535). Titrations of the ΔpH dependence of qE in the absence of PsbS reveal that this protein affects the cooperativity and sensitivity of the photoprotective process to protons. The roles of PsbS and zeaxanthin are discussed in light of their involvement in the control of the proton-antenna association constant, pK, via regulation of the interconnected phenomena of PSII antenna reorganization/aggregation and hydrophobicity.     

Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis

The induction and relaxation of non-photochemical quenching (NPQ) under steady-state conditions, i.e. during up to 90 min of illumination at saturating light intensities, was studied in Arabidopsis thaliana. Besides the well-characterized fast qE and the very slow qI component of NPQ, the analysis of the NPQ dynamics identified a zeaxanthin (Zx) dependent component which we term qZ. The formation (rise time 10-15 min) and relaxation (lifetime 10-15 min) of qZ correlated with the synthesis and epoxidation of Zx, respectively. Comparative analysis of different NPQ mutants from Arabidopsis showed that qZ was clearly not related to qE, qT or qI and thus represents a separate, Zx-dependent NPQ component.     

Kinetic Studies on the Xanthophyll Cycle in Barley Leaves (Influence of Antenna Size and Relations to Nonphotochemical Chlorophyll Fluorescence Quenching)

Xanthophyll-cycle kinetics as well as the relationship between the xanthophyll de-epoxidation state and Stern-Volmer type nonphotochemical chlorophyll (Chl) fluorescence quenching (qN) were investigated in barley (Hordeum vulgare L.) leaves comprising a stepwise reduced antenna system. For this purpose plants of the wild type (WT) and the Chl b-less mutant chlorina 3613 were cultivated under either continuous (CL) or intermittent light (IML). Violaxanthin (V) availability varied from about 70% in the WT up to 97 to 98% in the mutant and IML-grown plants. In CL-grown mutant leaves, de-epoxidation rates were strongly accelerated compared to the WT. This is ascribed to a different accessibility of V to the de-epoxidase due to the existence of two V pools: one bound to light-harvesting Chl a/b-binding complexes (LHC) and the other one not bound. Epoxidation rates (k) were decreased with reduction in LHC protein contents: kWT > kmutant >> kIML plants. This supports the idea that the epoxidase activity resides on certain LHC proteins. Irrespective of huge zeaxanthin and antheraxanthin accumulation, the capacity to develop qN was reduced stepwise with antenna size. The qN level obtained in dithiothreitol-treated CL- and IML-grown plants was almost identical with that in untreated IML-grown plants. The findings provide evidence that structural changes within the LHC proteins, mediated by xanthophyll-cycle operation, render the basis for the development of a major proportion of qN.     

A revised energy partitioning approach to assess the yields of non-photochemical quenching components

Non-photochemical quenching (NPQ) is a complex and still unclear mechanism essential for higher plants. The intensive research on this subject has highlighted three main components of NPQ: energy-dependent process (qE); state transitions to balance the excitation of PSII and PSI (qT); and photoinhibitory processes (qI). Recently, these components have been resolved as quantum yields according to the energy partitioning approach that takes into account the rate constants of every process involved in the quenching mechanisms of excited chlorophylls. In this study a fully extended quantum yield approach and the introduction of novel equations to assess the yields of each NPQ component are presented. Furthermore, a complete analysis of the yield of NPQ in Beta vulgaris exposed to different irradiances has been carried out. In agreement with experimental results here it is shown that the previous approach may amplify the yield of qE component and flatten the quantitative results of fluorescence analysis. Moreover, the significance of taking into account the physiological variability of NPQ for a correct assessment of energy partitioning is demonstrated.     

The Zeaxanthin-Independent and Zeaxanthin-Dependent qE Components of Nonphotochemical Quenching Involve Common Conformational Changes within the Photosystem II Antenna in Arabidopsis

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 (ΔA₅₃₅) 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). The regulation of energy flow occurs by control of the thermal dissipation of excess excitation within the PSII antenna, a process termed nonphotochemical quenching (NPQ). NPQ is heterogeneous, comprising a slowly reversible qI component and a rapidly reversible qE component (Horton et al., 1996). The trigger for qE is the buildup of the transmembrane proton gradient or ΔpH (Briantais et al., 1979). The ΔpH is sensed by the PsbS protein (Li et al., 2004), without which the rapidly reversible behavior of NPQ is lost (Li et al., 2000). Full expression of qE in vivo is associated with the enzymatic deepoxidation of the epoxy-xanthophyll violaxanthin to zeaxanthin, via the action of the xanthophyll cycle (Demmig-Adams, 1990). The majority of the photoconvertible xanthophyll cycle pool is associated with trimeric LHCII, bound at the external V1 binding site (Ruban et al., 1999, 2002a; Caffarri et al., 2001; Liu et al., 2004). Trimeric LHCII binds two other types of xanthophylls internally: two all-trans-luteins at the L1 and L2 sites associated with the central membrane-spanning α-helices; and a 9-cis-neoxanthin at the N1 site associated with the C-helix chlorophyll b domain (Liu et al., 2004). The minor monomeric complexes CP24, CP26, and CP29 all bind lutein at the L1 site. In addition, CP29 binds two xanthophyll cycle carotenoids and one-half to one neoxanthin, CP24 binds two xanthophyll cycle carotenoids, while CP26 binds one xanthophyll cycle carotenoid and one neoxanthin (Peter and Thornber, 1991; Bassi et al., 1993; Ruban et al., 1994, 1999; Morosinotto et al., 2002).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). Recently, evidence was obtained that formation of the zeaxanthin radical cation occurs exclusively at the L2 binding site of the minor antenna complexes (Ahn et al., 2008; Avenson et al., 2008), quenching therefore requiring reversible insertion of zeaxanthin into this internal site. Because the effect of this cation on the excited-state lifetime of the minor antenna complexes was found to be very small, it was suggested that in vivo, under the influence of the ΔpH, a large population of complexes would adopt a conformation in which this species could form (Avenson et al., 2008). Evidence was also obtained that a zeaxanthin radical cation may form in trimeric LHCII (Amarie et al., 2007). Again, the effect on the chlorophyll excited-state lifetime was very small, leading these authors to conclude that the type I mechanism could not be responsible for qE (Amarie et al., 2007; Dreuw and Wormit, 2008).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; Ilioaia et al., 2008). Evidence for a type II mechanism came from studies of trimeric LHCII aggregates (Ruban et al., 2007). Here, it was concluded that energy dissipation occurs by energy transfer from chlorophyll a to the S₁ state (2Ag¹) of lutein bound at the L1 site. Notably, this quenching mechanism decreases the chlorophyll excited-state lifetime by a magnitude sufficient to fully account for qE in vivo. A change in the conformation of another LHCII-bound xanthophyll (neoxanthin) correlates with the extent of quenching. This conformational change takes place in vivo with an amplitude that correlates with the amount of qE. In the model for type II quenching proposed by Horton and coworkers (1991, 2005), zeaxanthin acts not as a quencher but as an allosteric modulator of the ΔpH sensitivity of this intrinsic LHCII quenching process.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 ΔpH-triggered, PsbS-mediated conformational change (Ruban et al., 2007; Ahn et al., 2008). Indeed, it is possible that both mechanisms contribute to in vivo qE, since the process occurs in both the presence and absence of zeaxanthin (Adams et al., 1990; Crouchman et al., 2006). The crucial question is whether zeaxanthin-dependent and zeaxanthin-independent qE arise from the same mechanism (type II) or from two different ones (types I and II, respectively). The kinetics of NPQ formation upon the illumination of dark-adapted leaves comprise two components: the first forms rapidly and is zeaxanthin independent; the second, slower component correlates with violaxanthin deepoxidation and therefore is described as zeaxanthin dependent (Adams et al., 1990; Ruban and Horton, 1999). The two components of NPQ formation are of the qE type: both relax rapidly upon darkening (Adams et al., 1990); both are dependent upon PsbS (Li et al., 2000); and both are enhanced by PsbS overexpression (Li et al., 2002; Crouchman et al., 2006). Investigation of these kinetics provides an opportunity to determine whether a single mechanism can account for qE and to give clues to which type of mechanism is involved. Here, we test the hypothesis that the two components arise from different mechanisms: the zeaxanthin-dependent component arising in the minor monomeric antenna by a type I mechanism (Gilmore et al., 1998; Ahn et al., 2008; Avenson et al., 2008), and the zeaxanthin-independent component arising in the major trimeric LHCII by the type II mechanism. An alternative explanation for zeaxanthin-independent qE, at least under low-light conditions, when qE forms transiently, is that it is caused by quenching in the PSII reaction center (Finazzi et al., 2004). Several predictions emerge from this hypothesis. First, the removal of certain Lhcb proteins by mutation would differentially affect the two components of qE. Second, because the two components would be additive and could not compensate for the loss of one another (Niyogi et al., 1998; Pogson et al., 1998), they should each contribute a discrete component to the kinetics of qE formation and relaxation. Third, in mutants lacking lutein, the capacity of the type II mechanism would be reduced, while the zeaxanthin-dependent component would be unaffected. Finally, the two components may be expected to be characterized by different absorption changes in the Soret region, which reflect changes in the absorption spectra of bound pigments brought about by conformational changes within the PSII antenna upon qE formation (Ruban et al., 1993a, 1993b, 2002b; Bilger and Björkman, 1994). We tested this hypothesis by analysis of qE kinetics, fluorescence lifetimes, and qE-related absorption difference spectra. Contrary to the above predictions, the data indicated that both steady-state and transient qE arise from a common mechanism within the PSII antenna, in both the presence and absence of zeaxanthin.     

Elevated ΔpH restores rapidly reversible photoprotective energy dissipation in <Emphasis Type="Italic">Arabidopsis</Emphasis> chloroplasts deficient in lutein and xanthophyll cycle activity

The xanthophylls of the light-harvesting complexes of photosystem II (LHCII), zeaxanthin, and lutein are thought to be essential for non-photochemical quenching (NPQ). NPQ is a process of photoprotective energy dissipation in photosystem II (PSII). The major rapidly reversible component of NPQ, qE, is activated by the transmembrane proton gradient, and involves the quenching of antenna chlorophyll excited states by the xanthophylls lutein and zeaxanthin. Using diaminodurene (DAD), a mediator of cyclic electron flow around photosystem I, to enhance ΔpH we demonstrate that qE can still be formed in the absence of lutein and light-induced formation of zeaxanthin in chloroplasts derived from the normally qE-deficient lut2npq1 mutant of Arabidopsis. The qE induced by high ΔpH in lut2npq1 chloroplasts quenched the level of fluorescence when all PSII reaction centers were in the open state (F _o state), protected PSII reaction centers from photoinhibition, was sensitive to the uncoupler nigericin, and was accompanied by absorption changes in the 410–565 nm region. Titrations show the ΔpH threshold for activation of qE in lut2npq1 chloroplasts lies outside the normal physiological range and is highly cooperative. Comparison of quenching in isolated trimeric (LHCII) and monomeric (CP26) light-harvesting complexes from lut2npq1 plants revealed a similarly shifted pH dependency compared with wild-type LHCII. The implications for the roles of lutein and zeaxanthin as direct quenchers of excitation energy are discussed. Furthermore, we argue that the control over the proton-antenna association constant, pK, occurs via influence of xanthophyll structure on the interconnected phenomena of light-harvesting antenna reorganization/aggregation and hydrophobicity.     

Positive correlation between levels of retained zeaxanthin + antheraxanthin and degree of photoinhibition in shade leaves of Schefflera arboricola (Hayata) Merrill

Attached intact leaves of Schefflera arboricola grown at three different photon flux densities (PFDs) were subjected to 24-h exposures to a high PFD and subsequent recovery at a low PFD. While sun leaves showed virtually no sustained effects on photosystem II (PSII), shade-grown leaves exhibited pronounced photoinhibition of PSII that required several days at low PFD to recover. Upon transfer to high PFD, levels of nonphotochemical quenching in PSII as well as levels of zeaxanthin were initially low in shade leaves but continued to increase gradually during the 24-h exposure. The xanthophyll cycle pool size rose gradually during and also subsequent to the photoinhibitory treatment in shade leaves. Upon return to low PFD, a marked and extremely long-lasting retention of zeaxanthin and antheraxanthin was observed in shade but not sun leaves. During recovery, changes in the conversion state of the xanthophyll cycle therefore closely mirrored the slow increases in PSII efficiency. This novel report of a close association between zeaxanthin retention and lasting PSII depressions in these shade leaves clearly suggests a role for zeaxanthin in photoinhibition of shade leaves. In addition, there was a decrease in β-carotene levels, some decrease in chlorophyll, but no change in lutein and neoxanthin (all per leaf area) in the shade leaves during and subsequent to the photoinhibitory treatment. These data may be consistent with a degradation of a portion of core complexes but not of peripheral light-harvesting complexes. A possible conversion of β-carotene to form additional zeaxanthin is discussed. Received: 24 October 1997 / Accepted: 12 November 1997     

The Photosystem II Light-Harvesting Protein Lhcb3 Affects the Macrostructure of Photosystem II and the Rate of State Transitions in Arabidopsis

The main trimeric light-harvesting complex of higher plants (LHCII) consists of three different Lhcb proteins (Lhcb1-3). We show that Arabidopsis thaliana T-DNA knockout plants lacking Lhcb3 (koLhcb3) compensate for the lack of Lhcb3 by producing increased amounts of Lhcb1 and Lhcb2. As in wild-type plants, LHCII-photosystem II (PSII) supercomplexes were present in Lhcb3 knockout plants (koLhcb3), and preservation of the LHCII trimers (M trimers) indicates that the Lhcb3 in M trimers has been replaced by Lhcb1 and/or Lhcb2. However, the rotational position of the M LHCII trimer was altered, suggesting that the Lhcb3 subunit affects the macrostructural arrangement of the LHCII antenna. The absence of Lhcb3 did not result in any significant alteration in PSII efficiency or qE type of nonphotochemical quenching, but the rate of transition from State 1 to State 2 was increased in koLhcb3, although the final extent of state transition was unchanged. The level of phosphorylation of LHCII was increased in the koLhcb3 plants compared with wild-type plants in both State 1 and State 2. The relative increase in phosphorylation upon transition from State 1 to State 2 was also significantly higher in koLhcb3. It is suggested that the main function of Lhcb3 is to modulate the rate of state transitions.