Lhcb Proteins Research Articles

Arabidopsis thaliana plastidial methionine sulfoxide reductases B, MSRBs, account for most leaf peptide MSR activity and are essential for growth under environmental constraints through a role in the preservation of photosystem antennae

Methionine oxidation to methionine sulfoxide (MetSO) is reversed by two types of methionine sulfoxide reductases (MSRs), A and B, specific to MetSO S- and R-diastereomers, respectively. Two MSRB isoforms, MSRB1 and MSRB2, are present in chloroplasts of Arabidopsis thaliana. To assess their physiological role, we characterized Arabidopsis mutants knockout for the expression of MSRB1, MSRB2 or both genes. Measurements of MSR activity in leaf extracts revealed that the two plastidial MSRB enzymes account for the major part of leaf peptide MSR capacity. Under standard conditions of light and temperature, plants lacking one or both plastidial MSRBs do not exhibit any phenotype, regarding growth and development. In contrast, we observed that the concomitant absence of both proteins results in a reduced growth for plants cultivated under high light or low temperature. In contrast, double mutant lines restored for MSRB2 expression display no phenotype. Under environmental constraints, the MetSO level in leaf proteins is higher in plants lacking both plastidial MSRBs than in Wt plants. The absence of plastidial MSRBs is associated with an increased chlorophyll a/b ratio, a reduced content of Lhca1 and Lhcb1 proteins and an impaired photosynthetic performance. Finally, we show that MSRBs are able to use as substrates, oxidized cpSRP43 and cpSRP54, the two main components involved in the targeting of Lhc proteins to the thylakoids. We propose that plastidial MSRBs fulfil an essential function in maintaining vegetative growth of plants during environmental constraints, through a role in the preservation of photosynthetic antennae.

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.

Lutein accumulation in the absence of zeaxanthin restores nonphotochemical quenching in the Arabidopsis thaliana npq1 mutant.

Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high transthylakoid DeltapH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthin-less1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they accumulate more lutein and alpha-carotene than the wild type. szl1 contains a point mutation in the lycopene beta-cyclase (LCYB) gene. Based on the pigment analysis, LCYB appears to be the major lycopene beta-cyclase and is not involved in neoxanthin synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type, whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.

Light-induced Dissociation of an Antenna Hetero-oligomer Is Needed for Non-photochemical Quenching Induction

PsbS plays a major role in activating the photoprotection mechanism known as "non-photochemical quenching," which dissipates chlorophyll excited states exceeding the capacity for photosynthetic electron transport. PsbS activity is known to be triggered by low lumenal pH. However, the molecular mechanism by which this subunit regulates light harvesting efficiency is still unknown. Here we show that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins (CP29 and CP24) and the trimeric LHCII-M. Dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light, strongly suggesting that protein subunits catalyzing the reaction of heat dissipation are buried into the complex and thus not available for interaction with PsbS. Consistently, we showed that knock-out mutants on two subunits participating to the B4C complex were strongly affected in heat dissipation. Direct observation by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpreted these results to mean that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions.

Photosystem II Supercomplexes Of Higher Plants: Isolation And Determination Of The Structural And Functional Organization

Photosystem II is a supercomplex composed of 27-28 different subunits and it represents the most important machinery of the plants photosynthetic appara- tus, having the ability to split water into oxygen, protons and electrons. In the last few years the structures of most of the photosynthetic complexes have been resolved, allowing to organize in a ‘‘visual framework’’ the large body of information obtained by genetics, biochemical and spectroscopic methods about the function and organization of the complexes. Only the struc- ture of PSII-LHCII from higher plants is still lacking due to the impossibility to obtain a homogeneous and stable preparation of the supercomplex, which has also prevented functional and spectroscopic studies. In this work homogeneous and stable Photosystem II supercomplexes with dif- ferent antenna size were isolated. A full gallery of complexes, from the core to the largest C2S2M2, was characterized by electron microscopy and biochemi- cal and spectroscopic methods, allowing to relate for the first time the supramo- lecular organization to the protein and pigment content and the energy transfer processes. A new complex containing a monomeric core, a trimeric LHCII (S) and a monomeric CP26 was isolated, showing that the antenna proteins can bind to the monomeric core in contrast to the current belief. The comparison of the supercomplexes obtained from WT plants and knock out mutants of sev- eral Lhcb proteins allowed determining the hierarchy of the assembly and to suggest a role for the individual subunits. The data also provides information about the organization of the oxygen evolving complex. For the first time it was possible to study the energy transfer process in the supercomplexes with the use of picosecond fluorescence spectroscopy. The functional implication of these results on photoinhibition, state transition and energy transfer are discussed.

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

Nonphotochemical quenching (NPQ) is the photoprotective dissipation of energy in photosynthetic membranes. The hypothesis that the ΔpH-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 ΔpH 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.

Interactions between the Photosystem II Subunit PsbS and Xanthophylls Studied in Vivo and in Vitro

The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.

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.

The Mobile Thylakoid Phosphoprotein TSP9 Interacts with the Light-harvesting Complex II and the Peripheries of Both Photosystems

The localization of the plant-specific thylakoid-soluble phosphoprotein of 9 kDa, TSP9, within the chloroplast thylakoid membrane of spinach has been established by the combined use of fractionation, immunoblotting, cross-linking, and mass spectrometry. TSP9 was found to be exclusively confined to the thylakoid membranes, where it is enriched in the stacked grana membrane domains. After mild solubilization of the membranes, TSP9 migrated together with the major light-harvesting antenna (LHCII) of photosystem II (PSII) and with PSII-LHCII supercomplexes upon separation of the protein complexes by either native gel electrophoresis or sucrose gradient centrifugation. Studies with a cleavable cross-linking agent revealed the interaction of TSP9 with both major and minor LHCII proteins as identified by mass spectrometric sequencing. Cross-linked complexes that in addition to TSP9 contain the peripheral PSII subunits CP29, CP26, and PsbS, which form the interface between LHCII and the PSII core, were found. Our observations also clearly suggest an interaction of TSP9 with photosystem I (PSI) as shown by both immunodetection and mass spectrometry. Sequencing identified the peripheral PSI subunits PsaL, PsaF, and PsaE, originating from cross-linked protein complexes of around 30 kDa that also contained TSP9. The distribution of TSP9 among the cross-linked forms was found to be sensitive to conditions such as light exposure. An association of TSP9 with LHCII as well as the peripheries of the photosystems suggests its involvement in regulation of photosynthetic light harvesting.

Dependence of reaction center-type energy-dependent quenching on photosystem II antenna size

The effects of photosystem II antenna size on reaction center-type energy-dependent quenching (qE) were examined in rice plants grown under two different light intensities using both wild type and qE-less (OsPsbS knockout) mutant plants. Reaction center-type qE was detected by measuring non-photochemical quenching at 50 μmol photons m − 2 s − 1 white light intensity. We observed that in low light-grown rice plants, reaction center-type qE was higher than in high light-grown plants, and the amount of reaction center-type qE did not depend on zeaxanthin accumulation. This was confirmed in Arabidopsis npq1–2 mutant plants that lack zeaxanthin due to a mutation in the violaxanthin de-epoxidase enzyme. Although the electron transport rate measured at a light intensity of 50 μmol photons m − 2 s − 1 was the same in high light- and low light-grown wild type and mutant plants lacking PsbS protein, the generation of energy-dependent quenching was completely impaired only in mutant plants. Analyses of the pigment content, Lhcb proteins and D1 protein of PSII showed that the antenna size was larger in low light-grown plants, and this correlated with the amount of reaction center-type qE. Our results mark the first time that the reaction center-type qE has been shown to depend on photosystem II antenna size and, although it depends on the existence of PsbS protein, the extent of reaction center-type qE does not correlate with the transcript levels of PsbS protein. The presence of reaction center-type energy-dependent quenching, in addition to antenna-type quenching, in higher plants for dissipation of excess light energy demonstrates the complexity and flexibility of the photosynthetic apparatus of higher plants to respond to different environmental conditions.

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.

State transitions revisited—a buffering system for dynamic low light acclimation of Arabidopsis

The mobile part of the light-harvesting chlorophyll (chl) a/b protein complex (LHCII), composed of the Lhcb1 and Lhcb2 proteins, is the basic unit of chloroplast state transitions--the short term tuning system in balancing the excitation energy between Photosystem (PS) II and PSI. State transitions are catalysed by the thylakoid associated STN7 kinase, and we show here that besides the phosphorylation of the Lhcb1 and Lhcb2 proteins, also the phosphorylation of Lhcb4.2 (CP29) is under the control of the STN7 kinase. Upon growth of Arabidopsis WT and stn7 mutant plants under low and moderate light conditions, the WT plants favoured state 2 whereas stn7 was locked in state 1. The lack of the STN7 kinase and state transitions in stn7 also modified the thylakoid protein contents upon long-term low light acclimation resulting, for example, in low Lhcb1 and in elevated Lhca1 and Lhca2 protein amounts as compared to WT. Adjustments of thylakoid protein contents probably occurred at post-transcriptional level since the DNA microarray experiments from each growth condition did not reveal any significant differences between stn7 and WT transcriptomes. The resulting high Lhcb2/Lhcb1 ratio in stn7 upon growth at low light was accompanied by lower capacity for NPQ than in WT. On the contrary, higher amounts of PsbS in stn7 under moderate and high light growth conditions resulted in higher NPQ compared to WT and consequently also in a protection of PSII against photoinhibition. STN7 kinase and the state transitions are suggested to have a physiological significance for dynamic acclimation to low but fluctuating growth light conditions. They are shown to function as a buffering system upon short high light illumination peaks by shifting the thylakoids from state 2 to state 1 and thereby down regulating the induction of stress-responsive genes, a likely result from transient over-reduction of PSI acceptors.

Differential Expression and Localization of Early Light-Induced Proteins in Arabidopsis

The early light-induced proteins (Elips) in higher plants are nuclear-encoded, light stress-induced proteins located in thylakoid membranes and related to light-harvesting chlorophyll (LHC) a/b-binding proteins. A photoprotective function was proposed for Elips. Here we showed that after 2 h exposure of Arabidopsis (Arabidopsis thaliana) leaves to light stress Elip1 and Elip2 coisolate equally with monomeric (mLhcb) and trimeric (tLhcb) populations of the major LHC from photosystem II (PSII) as based on the Elip:Lhcb protein ratio. A longer exposure to light stress resulted in increased amounts of Elips in tLhcb as compared to mLhcb, due to a reduction of tLhcb amounts. We demonstrated further that the expression of Elip1 and Elip2 transcripts was differentially regulated in green leaves exposed to light stress. The accumulation of Elip1 transcripts and proteins increased almost linearly with increasing light intensities and correlated with the degree of photoinactivation and photodamage of PSII reaction centers. A stepwise accumulation of Elip2 was induced when 40% of PSII reaction centers became photodamaged. The differential expression of Elip1 and Elip2 occurred also in light stress-preadapted or senescent leaves exposed to light stress but there was a lack of correlation between transcript and protein accumulation. Also in this system the accumulation of Elip1 but not Elip2 correlated with the degree of PSII photodamage. Based on pigment analysis, measurements of PSII activity, and assays of the oxidation status of proteins we propose that the discrepancy between amounts of Elip transcripts and proteins in light stress-preadapted or senescent leaves is related to a presence of photoprotective anthocyanins or to lower chlorophyll availability, respectively.

A Chlorophyll a/b-binding Protein Homolog That Is Induced by Iron Deficiency Is Associated with Enlarged Photosystem I Units in the Eucaryotic Alga Dunaliella salina

Adaptation of the halotolerant alga Dunaliella salina to iron deprivation involves extensive changes of chloroplast morphology, photosynthetic activities, and induction of a major 45-kDa chloroplast protein termed Tidi. Partial amino acid sequencing of proteolytic peptides suggested that Tidi resembles chlorophyll a/b-binding proteins which compose light-harvesting antenna complexes (LHC) (Varsano, T., Kaftan, D., and Pick, U. (2003) J. Plant Nutr. 26, 2197-2210). Here we show that Tidi shares the highest amino acid sequence similarity with light-harvesting I chlorophyll a/b-binding proteins from higher plants but has an extended proline-rich N-terminal domain. The accumulation of Tidi is reversed by iron supplementation, and its level is inversely correlated with photosystem I (PS-I) reaction center proteins. In native gel electrophoresis, Tidi co-migrates with enlarged PS-I-LHC-I super-complexes. Single particle electron microscopy analysis revealed that PS-I units from iron-deficient cells are larger (31 and 37 nm in diameter) than PS-I units from control cells (22 nm). The 77 K chlorophyll fluorescence emission spectra of isolated complexes suggest that the Tidi-LHC-I antenna are functionally coupled to the reaction centers of PS-I. These findings indicate that Tidi acts as an accessory antenna of PS-I. The enlargement of PS-I antenna in algae and in cyanobacteria under iron deprivation suggests a common limitation that requires rebalancing of the energy distribution between the two photosystems.

Differential adaptation of two varieties of common bean to abiotic stress

The photosynthetic characteristics of two contrasting varieties of common bean (Phaseolus vulgaris) have been determined. These varieties, Arroz and Orfeo, differ in their productivity under stress conditions, resistance to drought stress, and have distinctly different stomatal behaviour. When grown under conditions of high irradiance and high temperature, both varieties displayed evidence of photosynthetic acclimation at the chloroplast level-there was an increase in chlorophyll a/b ratio, a decreased content of Lhcb proteins, and an increased xanthophyll cycle pool size. Both varieties also showed reduced chlorophyll content on a leaf area basis and a decrease in leaf area. Both varieties showed an increase in leaf thickness but only Arroz showed the characteristic elongated palisade cells in the high light-grown plants; Orfeo instead had a larger number of smaller, rounded cells. Differences were found in stomatal development: whereas Arroz showed very little change in stomatal density, Orfeo exhibited a large increase, particularly on the upper leaf surface. It is suggested that these differences in leaf cell structure and stomatal density give rise to altered rates of photosynthesis and stomatal conductance. Whereas, Arroz had the same photosynthetic rate in plants grown at both low and high irradiance, Orfeo showed a higher photosynthetic capacity at high irradiance. It is suggested that the higher yield of Orfeo compared with Arroz under stress conditions can be explained, in part, by these cellular differences.

Differential accumulation ofLhcbgene products in thylakoid membranes ofZea maysplants grown under contrasting light and temperature conditions

In higher plants many different genes encode Lhcb proteins that belong to a highly conserved protein family. Evolutionary conservation of this genetic redundancy suggests that individual gene products play different roles in light harvesting and photoprotection depending on environmental conditions. We have tested the hypothesis that expression/accumulation of individual light harvesting complex (Lhc) proteins depends on plant growth conditions. Zea mays plants were grown in different temperature (13 degrees C vs. 24 degrees C) and light (high vs. low) conditions. The thylakoid membranes were isolated and fractionated by sucrose gradient and the protein content of the different bands was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences were found in the accumulation of both the major light harvesting complex of photosystem II (LHCII) complexes and the minor antenna chlorophyll proteins CP29, CP26 and CP24. In particular, temperature seems to play a major role in driving the expression/accumulation of the different proteins: the LHCII/minor antenna ratio increases with decreasing temperature. The pigment composition and the spectroscopic properties of LHCII complexes isolated from low temperature grown plants are significantly different from those of LHCII purified from high temperature grown plants. Two-dimensional maps show that different LHCII proteins are accumulated at different levels depending on growth conditions. Moreover the low temperature/high light grown plants show an increased value of nonphotochemical quenching. These results suggest a specific role of different LHCII complexes in the organization of the potosystem II and photoprotection.

A look within LHCII: differential analysis of the Lhcb1-3 complexes building the major trimeric antenna complex of higher-plant photosynthesis.

The major antenna complex of higher-plant photosynthesis, LHCII, is composed by the products of three genes, namely, Lhcb1-2-3. In this paper, the biochemical and spectroscopic properties of each of the three gene products were investigated. The three complexes were obtained by overexpression of the apoproteins in bacteria and refolding in vitro with purified pigments, thus allowing detection of differences in the structure/function of the pigment-binding gene products. The analyses showed that Lhcb1 and Lhcb2 complexes have similar pigment binding properties, although not identical, while Lhcb3 is clearly different with respect to both pigment binding and spectral properties and cannot produce homotrimers in vitro. Heterotrimers containing Lhcb3 together with Lhcb1 and/or -2 proteins were obtained upon assembly with Lhcb proteins purified from thylakoids. The major functional characteristics of Lhcb3 with respect to Lhcb1 and -2 consisted in (i) a red-shift of one specific chlorophyll a chromophore, strongly affecting the red-most region of the absorption spectrum and (ii) a different specificity for xanthophylls binding to sites L2 and N1. These properties make Lhcb3 a relative sink for excitation energy in isolated heterotrimers with Lhcb1 + Lhcb2, and potentially, a preferential site of regulation of the antenna function in excess light conditions.

De-epoxidation of Violaxanthin in Light-harvesting Complex I Proteins

The conversion of violaxanthin (Vx) to zeaxanthin (Zx) in the de-epoxidation reaction of the xanthophyll cycle plays an important role in the protection of chloroplasts against photooxidative damage. Vx is bound to the antenna proteins of both photosystems. In photosystem II, the formation of Zx is essential for the pH-dependent dissipation of excess light energy as heat. The function of Zx in photosystem I is still unclear. In this work we investigated the de-epoxidation characteristics of light-harvesting complex proteins of photosystem I (LHCI) under in vivo and in vitro conditions. Recombinant LHCI (Lhcal-4) proteins were reconstituted with Vx and lutein, and the convertibility of Vx was studied in an in vitro assay using partially purified Vx de-epoxidase isolated from spinach thylakoids. All four LHCI proteins exhibited unique de-epoxidation characteristics. An almost complete Vx conversion to Zx was observed only in Lhca3, whereas Zx formation in the other LHCI proteins decreased in the order Lhca4 > Lhca1 > Lhca2. Most likely, these differences in Vx de-epoxidation were related to the different accessibility of the respective carotenoid binding sites in the distinct antenna proteins. The results indicate that Vx bound to site V1 and N1 is easily accessible for de-epoxidation, whereas Vx bound to L2 is only partially and/or with the slower kinetics convertible to Zx. The de-epoxidation properties determined for the monomeric recombinant proteins were consistent with those obtained for isolated native LHCI-730 and LHCI-680 in the same in vitro assay and the de-epoxidation state found under in vivo conditions in native LHCIs.

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.

Proteomics of Chlamydomonas reinhardtii light-harvesting proteins.

With the recent development of techniques for analyzing transmembrane thylakoid proteins by two-dimensional gel electrophoresis, systematic approaches for proteomic analyses of membrane proteins became feasible. In this study, we established detailed two-dimensional protein maps of Chlamydomonas reinhardtii light-harvesting proteins (Lhca and Lhcb) by extensive tandem mass spectrometric analysis. We predicted eight distinct Lhcb proteins. Although the major Lhcb proteins were highly similar, we identified peptides which were unique for specific lhcbm gene products. Interestingly, lhcbm6 gene products were resolved as multiple spots with different masses and isoelectric points. Gene tagging experiments confirmed the presence of differentially N-terminally processed Lhcbm6 proteins. The mass spectrometric data also revealed differentially N-terminally processed forms of Lhcbm3 and phosphorylation of a threonine residue in the N terminus. The N-terminal processing of Lhcbm3 leads to the removal of the phosphorylation site, indicating a potential novel regulatory mechanism. At least nine different lhca-related gene products were predicted by comparison of the mass spectrometric data against Chlamydomonas expressed sequence tag and genomic databases, demonstrating the extensive variability of the C. reinhardtii Lhca antenna system. Out of these nine, three were identified for the first time at the protein level. This proteomic study demonstrates the complexity of the light-harvesting proteins at the protein level in C. reinhardtii and will be an important basis of future functional studies addressing this diversity.