Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes

Xanthophylls have a crucial role in the structure and function of the light harvesting complexes of photosystem II (LHCII) in plants. The binding of xanthophylls to LHCII has been investigated, particularly with respect to the xanthophyll cycle carotenoids violaxanthin and zeaxanthin. It was found that most of the violaxanthin pool was loosely bound to the major complex and could be removed by mild detergent treatment. Gentle solubilization of photosystem II particles and thylakoids allowed the isolation of complexes, including a newly described oligomeric preparation, enriched in trimers, that retained all of the in vivo violaxanthin pool. It was estimated that each LHCII monomer can bind at least one violaxanthin. The extent to which different pigments can be removed from LHCII indicated that the relative strength of binding was chlorophyll b > neoxanthin > chlorophyll a > lutein > zeaxanthin > violaxanthin. The xanthophyll binding sites are of two types: internal sites binding lutein and peripheral sites binding neoxanthin and violaxanthin. In CP29, a minor LHCII, both a lutein site and the neoxanthin site can be occupied by violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest de-epoxidation state was found for the main LHCII component and the lowest for CP29, suggesting that only violaxanthin loosely bound to LHCII is available for de-epoxidation.     

A specific binding site for neoxanthin in the monomeric antenna proteins CP26 and CP29 of Photosystem II

The location of the neoxanthin binding site in CP26 and CP29 was investigated by site-directed mutagenesis. The crystallographic structure of LHCII shows that the binding of neoxanthin to the N1 site is stabilised by an H bond with a tyrosine in the lumenal loop. This residue is conserved in CP26 and CP29. Mutation of this tyrosine into phenylalanine induced specific loss of neoxanthin without affecting violaxanthin binding. In contrast to previous proposals, it is thus concluded that also in these minor antenna complexes neoxanthin is accommodated in the N1 site. The characteristics of this binding site in the different antenna complexes are discussed.     

Chromophore organization in the higher-plant photosystem II antenna protein CP26

The chlorophyll a/b-xanthophyll-protein CP26 complex belongs to the Lhc protein family. It binds nine chlorophylls and two xanthophylls per 26.6 kDa polypeptide. Determination of the characteristics of each binding site is needed for the understanding of functional organization of individual proteins belonging to the photosystem II supramolecular complex. The biochemical and spectroscopic features of native CP26 are presented here together with identification of pigment binding and energy transitions in different sites. The analysis has been performed via a new approach using recombinant CP26 complexes in which the chromophore content has been experimentally modified. Data were interpreted on the basis of homology with CP29 and LHCII complexes, for which detailed knowledge is available from mutation analysis. We propose that one additional Chl b is present in CP26 as compared to CP29 and that it is located in site B2. We also found that in CP26 three chlorophyll binding sites are selective for Chl a, one of them being essential for the folding of the pigment-protein complex. Two xanthophyll binding sites were identified, one of which (L1) is essential for protein folding and specifically binds lutein. The second site (L2) has lower selectivity and can bind any of the xanthophyll species present in thylakoids.     

Energy transfer pathways in the minor antenna complex CP29 of photosystem II: a femtosecond study of carotenoid to chlorophyll transfer on mutant and WT complexes

The energy transfer processes between carotenoids and Chls have been studied by femtosecond transient absorption in the CP29-WT complex, which contains only two carotenoids per polypeptide located in the L1 and L2 sites, and in the CP29-E166V mutant in which only the L1 site is occupied. The comparison of these two samples allowed us to discriminate between the energy transfer pathways from the two carotenoid binding sites and thus to obtain detailed information on the Chl organization in CP29 and to assign the acceptor chlorophylls. For both samples, the main transfer occurs from the S(2) state of the carotenoid. In the case of the L1 site the energy acceptor is the Chl a 680 nm (A2), whereas the Chl a 675 nm (A4-A5) and the Chl b 652 nm (B6) are the acceptors from the xanthophyll in the L2 site. These transfers occur with lifetimes of 80-130 fs. Two additional transfers are observed with 700-fs and 8- to 20-ps lifetimes. Both these transfers originate from the carotenoid S(1) states. The faster lifetime is due to energy transfer from a vibrationally unrelaxed S(1) state, whereas the 8- to 20-ps component is due to a transfer from the S(1,0) state of violaxanthin and/or neoxanthin located in site L2. A comparison between the carotenoid to Chl energy transfer pathways in CP29 and LHCII is presented and differences in the structural organization in the two complexes are discussed.     

Molecular configuration of xanthophyll cycle carotenoids in photosystem II antenna complexes

The molecular configuration of the xanthophyll cycle carotenoids, violaxanthin and zeaxanthin, was studied in various isolated photosystem II antenna components in comparison to intact photosystem II membranes using resonance Raman combined with low-temperature absorption spectroscopy. The molecular configurations of zeaxanthin and violaxanthin in thylakoids and isolated photosystem II membranes were found to be the same within an isolated oligomeric LHCII antenna, confirming our recent conclusion that these molecules are not freely located in photosynthetic membranes (Ruban, A. V., Pascal, A. A., Robert, B., and Horton, P. (2001) J. Biol. Chem. 276, 24862-24870). In contrast, xanthophyll cycle carotenoids bound to LHCII trimers had largely lost their in vivo configuration, suggesting their partial dissociation from the binding locus. Violaxanthin and zeaxanthin associated with the minor antenna complexes, CP26 and CP29, were also found to be in a relaxed configuration, similar to that of free pigment. The origin of the characteristic C-H vibrational bands of violaxanthin and zeaxanthin in vivo is discussed by comparison with those of neoxanthin and lutein in oligomeric and trimeric LHCII respectively.     

Carotenoid-binding sites of the major light-harvesting complex II of higher plants.

Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in (1)O(2)(*) scavenging.     

Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes

We have studied the time-resolved fluorescence properties of the light-harvesting complexes (Lhc) of photosystem II (Lhcb) in order to obtain information on the mechanism of energy dissipation (non-photochemical quenching) which is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCII, CP29, CP26, and CP24 in detergent solution is mostly determined by two lifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the faster component is higher in CP29, CP26, and CP24 with respect to LHCII. The xanthophyll composition of Lhc proteins affects the ratio of the lifetime components: when zeaxanthin is bound into the site L2 of LHCII, the relative amplitude of the faster component is increased and, consequently, the chlorophyll fluorescence quenching is enhanced. Analysis of quenching in mutants of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxanthin in their Lhc proteins, shows that the extent of quenching is enhanced in the presence of zeaxanthin. The origin of the two fluorescence lifetimes was analyzed by their temperature dependence: since lifetime heterogeneity was not affected by cooling to 77 K, it is concluded that each lifetime component corresponds to a distinct conformation of the Lhc proteins. Upon incorporation of Lhc proteins into liposomes, a quenching of chlorophyll fluorescence was observed due to shortening of all their lifetime components: this indicates that the equilibrium between the two conformations of Lhcb proteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein density in the liposomes, and therefore the probability of protein-protein interactions, a further decrease of fluorescence lifetimes takes place down to values typical of quenched leaves. We conclude that at least two major factors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions within the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.     

Mechanistic aspects of the xanthophyll dynamics in higher plant thylakoids

Plant thylakoids have a highly conserved xanthophyll composition, consisting of β-carotene, lutein, neoxanthin and a pool of violaxanthin that can be converted to antheraxanthin and zeaxanthin in excess light conditions. Recent work has shown that xanthophylls undergo dynamic changes, not only in their composition but also in their distribution among Lhc proteins. Xanthophylls are released from specific binding site in the major trimeric LHCII complex of photosystem II and are subsequently bound to different sites into monomeric Lhcb proteins and dimeric Lhca proteins. In this work we review available evidence from in vivo and in vitro studies on the structural determinants that control xanthophyll exchange in Lhc proteins. We conclude that the xanthophyll exchange rate is determined by the structure of individual Lhc gene products and it is specifically controlled by the lumenal pH independently from the activation state of the violaxanthin de-epoxidase enzyme. The xanthophyll exchange induces important modifications in the organization of the antenna system of Photosystem II and, possibly of Photosystem I. Major changes consist into a modulation of the light harvesting efficiency and an increase of the protection from lipid peroxidation. The xanthophyll cycle thus appears to be a signal transduction system for co-ordinated regulation of the photoprotection mechanisms under persistent stress from excess light.     

Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant

The biochemical properties of PsbS protein, a nuclear-encoded Photosystem II subunit involved in the high energy quenching of chlorophyll fluorescence, have been studied using preparations purified from chloroplasts or obtained by overexpression in bacteria. Despite the homology with chlorophyll a/b/xanthophyll-binding proteins of the Lhc family, native PsbS protein does not show any detectable ability to bind chlorophylls or carotenoids in conditions in which Lhc proteins maintain full pigment binding. The recombinant protein, when refolded in vitro in the presence of purified pigments, neither binds chlorophylls nor xanthophylls, differently from the homologous proteins LHCII, CP26, and CP29 that refold into stable pigment-binding complexes. Thus, it is concluded that if PsbS is a pigment-binding protein in vivo, the binding mechanism must be different from that present in other Lhc proteins. Primary sequence analysis provides evidence for homology of PsbS helices I and III with the central 2-fold symmetric core of chlorophyll a/b-binding proteins. Moreover, a structural homology owed to the presence of acidic residues in each of the two lumen-exposed loops is found with the dicyclohexylcarbodiimide/Ca(2+)-binding domain of CP29. Consistently, both native and recombinant PsbS proteins showed [(14)C]dicyclohexylcarbodiimide binding, thus supporting a functional basis for its homology with CP29 on the lumen-exposed loops. This domain is suggested to be involved in sensing low luminal pH.     

Different roles of alpha- and beta-branch xanthophylls in photosystem assembly and photoprotection

Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively alpha-branch (chy1chy2lut5) or beta-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of beta-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that alpha-branch (lutein) and beta-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection.     

Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions

The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI-PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.     

Xanthophyll Cycle Pigment Localization and Dynamics during Exposure to Low Temperatures and Light Stress in Vinca major

The distribution of xanthophyll cycle pigments (violaxanthin plus antheraxanthin plus zeaxanthin [VAZ]) among photosynthetic pigment-protein complexes was examined in Vinca major before, during, and subsequent to a photoinhibitory treatment at low temperature. Four pigment-protein complexes were isolated: the core of photosystem (PS) II, the major light-harvesting complex (LHC) protein of PSII (LHCII), the minor light-harvesting proteins (CPs) of PSII (CP29, CP26, and CP24), and PSI with its LHC proteins (PSI-LHCI). In isolated thylakoids 80% of VAZ was bound to protein independently of the de-epoxidation state and was found in all complexes. Plants grown outside in natural sunlight had higher levels of VAZ (expressed per chlorophyll), compared with plants grown in low light in the laboratory, and the additional VAZ was mainly bound to the major LHCII complex, apparently in an acid-labile site. The extent of de-epoxidation of VAZ in high light and the rate of reconversion of Z plus A to V following 2.5 h of recovery were greatest in the free-pigment fraction and varied among the pigment-protein complexes. Photoinhibition caused increases in VAZ, particularly in low-light-acclimated leaves. The data suggest that the photoinhibitory treatment caused an enrichment in VAZ bound to the minor CPs caused by de novo synthesis of the pigments and/or a redistribution of VAZ from the major LHCII complex.     

The effect of zeaxanthin as the only xanthophyll on the structure and function of the photosynthetic apparatus in Arabidopsis thaliana

In green plants, the xanthophyll carotenoid zeaxanthin is synthesized transiently under conditions of excess light energy and participates in photoprotection. In the Arabidopsis lut2 npq2 double mutant, all xanthophylls were replaced constitutively by zeaxanthin, the only xanthophyll whose synthesis was not impaired. The relative proportions of the different chlorophyll antenna proteins were strongly affected with respect to the wild-type strain. The major antenna, LHCII, did not form trimers, and its abundance was strongly reduced as was CP26, albeit to a lesser extent. In contrast, CP29, CP24, LHCI proteins, and the PSI and PSII core complexes did not undergo major changes. PSII-LHCII supercomplexes were not detectable while the PSI-LHCI supercomplex remained unaffected. The effect of zeaxanthin accumulation on the stability of the different Lhc proteins was uneven: the LHCII proteins from lut2 npq2 had a lower melting temperature as compared with the wild-type complex while LHCI showed increased resistance to heat denaturation. Consistent with the loss of LHCII, light-state 1 to state 2 transitions were suppressed, the photochemical efficiency in limiting light was reduced and photosynthesis was saturated at higher light intensities in lut2 npq2 leaves, resulting in a photosynthetic phenotype resembling that of high light-acclimated leaves. Zeaxanthin functioned in vivo as a light-harvesting accessory pigment in lut2 npq2 chlorophyll antennae. As a whole, the in vivo data are consistent with the results obtained by using recombinant Lhc proteins reconstituted in vitro with purified zeaxanthin. While PSII photoinhibition was similar in wild type and lut2 npq2 exposed to high light at low temperature, the double mutant was much more resistant to photooxidative stress and lipid peroxidation than the wild type. The latter observation is consistent with an antioxidant and lipid protective role of zeaxanthin in vivo.     

Energy transfer in light-harvesting complexes LHCII and CP29 of spinach studied with three pulse echo peak shift and transient grating

Three pulse echo peak shift and transient grating (TG) measurements on the plant light-harvesting complexes LHCII and CP29 are reported. The LHCII complex is by far the most abundant light-harvesting complex in higher plants and fulfills several important physiological functions such as light-harvesting and photoprotection. Our study is focused on the light-harvesting function of LHCII and the very similar CP29 complex and reveals hitherto unresolved excitation energy transfer processes. All measurements were performed at room temperature using detergent isolated complexes from spinach leaves. Both complexes were excited in their Chl b band at 650 nm and in the blue shoulder of the Chl a band at 670 nm. Exponential fits to the TG and three pulse echo peak shift decay curves were used to estimate the timescales of the observed energy transfer processes. At 650 nm, the TG decay can be described with time constants of 130 fs and 2.2 ps for CP29, and 300 fs and 2.8 ps for LHCII. At 670 nm, the TG shows decay components of 230 fs and 6 ps for LHCII, and 300 fs and 5 ps for CP29. These time constants correspond to well-known energy transfer processes, from Chl b to Chl a for the 650 nm TG and from blue (670 nm) Chl a to red (680 nm) Chl a for the 670 nm TG. The peak shift decay times are entirely different. At 650 nm we find times of 150 fs and 0.5-1 ps for LHCII, and 360 fs and 3 ps for CP29, which we can associate mainly with Chl b <--> Chl b energy transfer. At 670 nm we find times of 140 fs and 3 ps for LHCII, and 3 ps for CP29, which we can associate with fast (only in LHCII) and slow transfer between relatively blue Chls a or Chl a states. From the occurrence of both fast Chl b <--> Chl b and fast Chl b --> Chl a transfer in CP29, we conclude that at least two mixed binding sites are present in this complex. A detailed comparison of our observed rates with exciton calculations on both CP29 and LHCII provides us with more insight in the location of these mixed sites. Most importantly, for CP29, we find that a Chl b pair must be present in some, but not all, complexes, on sites A(3) and B(3). For LHCII, the observed rates can best be understood if the same pair, A(3) and B(3), is involved in both fast Chl b <--> Chl b and fast Chl a <--> Chl a transfer. Hence, it is likely that mixed sites also occur in the native LHCII complex. Such flexibility in chlorophyll binding would agree with the general flexibility in aggregation form and xanthophyll binding of the LHCII complex and could be of use for optimizing the role of LHCII under specific circumstances, for example under high-light conditions. Our study is the first to provide spectroscopic evidence for mixed binding sites, as well as the first to show their existence in native complexes.     

Photoprotection in the antenna complexes of photosystem II: role of individual xanthophylls in chlorophyll triplet quenching

In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O(2) to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O(2). As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins.     

Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii

Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.     

Xanthophylls as modulators of membrane protein function

This review discusses the structural aspect of the role of photosynthetic antenna xanthophylls. It argues that xanthophyll hydrophobicity/polarity could explain the reason for xanthophyll variety and help to understand their recently emerging function - control of membrane organization and the work of membrane proteins. The structure of a xanthophyll molecule is discussed in relation to other amphiphilic compounds like lipids, detergents, etc. Xanthophyll composition of membrane proteins, the role of their variety in protein function are discussed using as an example for the major light harvesting antenna complex of photosystem II, LHCII, from higher plants. A new empirical parameter, hydrophobicity parameter (H-parameter), has been introduced as an effective measure of the hydrophobicity of the xanthophyll complement of LHCII from different xanthophyll biosynthesis mutants of Arabidopsis. Photosystem II quantum efficiency was found to correlate well with the H-parameter of LHCII xanthophylls. PSII down-regulation by non-photochemical chlorophyll fluorescence quenching, NPQ, had optimum corresponding to the wild-type xanthophyll composition, where lutein occupies intrinsic sites, L1 and L2. Xanthophyll polarity/hydrophobicity alteration by the activity of the xanthophyll cycle explains the allosteric character of NPQ regulation, memory of illumination history and the hysteretic nature of the relationship between the triggering factor, ΔpH, and the energy dissipation process.     

Protein dynamics and lipid affinity of monomeric,zeaxanthin-binding LHCII in thylakoid membranes

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from ¹³C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.     

Dephosphorylation of photosystem II proteins and phosphorylation of CP29 in barley photosynthetic membranes as a response to water stress

Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.     

Photosystem II protein phosphorylation follows four distinctly different regulatory patterns induced by environmental cues

Differential redox regulation of thylakoid phosphoproteins was studied in winter rye plants in vivo. The redox state of chloroplasts was modulated by growing plants under different light/temperature conditions and by transient shifts to different light/temperature regimes. Phosphorylation of PSII reaction centre proteins D1 and D2, the chlorophyll a binding protein CP43, the major chlorophyll a/b binding proteins Lhcb1 and Lhcb2 (LHCII) and the minor light‐harvesting antenna protein CP29 seem to belong to four distinct regulatory groups. Phosphorylation of D1 and D2 was directly dependent on the reduction state of the plastoquinone pool. CP43 protein phosphorylation generally followed the same pattern, but often remained phosphorylated even in darkness. Phosphorylation of CP29 occurred upon strong reduction of the plastoquinone pool, and was further enhanced by low temperatures. In vitro studies further demonstrated that CP29 phosphorylation is independent of the redox state of both the cytochrome b₆/f complex and the thiol compounds. Complete phosphorylation of Lhcb1 and 2 proteins, on the contrary, required only modest reduction of the plastoquinone pool, and was subject to inhibition upon increase in the thiol redox state of the stroma. Furthermore, the reversible phosphorylation of Lhcb1 and 2 proteins appeared to be an extremely dynamic process, being rapidly modulated by short‐term fluctuations in chloroplast redox conditions.