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.     

Pigment binding site properties of two photosystem II antenna proteins. A resonance raman investigation

Two light-harvesting proteins associated with photosystem II of higher plants, namely the major antenna complex LHCIIb and the minor Lhcb4 protein (CP29), have been investigated by resonance Raman spectroscopy. One of the two chlorophylls b and up to five of the six chlorophylls a present in Lhcb4 are shown to adopt similar binding conformations to the (presumably) corresponding molecules in LHCIIb, whereas at least two chlorophylls in the former protein assume unique conformations relative to the bulk complex. The overall conformation of bound xanthophyll molecules is identical in the two antenna proteins, although some small differences are apparent. The pigment binding properties of these two LHCs are discussed, with particular reference to possible structural motifs within this extended family of proteins.     

Optimization and evolution of light harvesting in photosynthesis: the role of antenna chlorophyll conserved between photosystem II and photosystem I

The efficiency of oxygenic photosynthesis depends on the presence of core antenna chlorophyll closely associated with the photochemical reaction centers of both photosystem II (PSII) and photosystem I (PSI). Although the number and overall arrangement of these chlorophylls in PSII and PSI differ, structural comparison reveals a cluster of 26 conserved chlorophylls in nearly identical positions and orientations. To explore the role of these conserved chlorophylls within PSII and PSI we studied the influence of their orientation on the efficiency of photochemistry in computer simulations. We found that the native orientations of the conserved chlorophylls were not optimal for light harvesting in either photosystem. However, PSII and PSI each contain two highly orientationally optimized antenna chlorophylls, located close to their respective reaction centers, in positions unique to each photosystem. In both photosystems the orientation of these optimized bridging chlorophylls had a much larger impact on photochemical efficiency than the orientation of any of the conserved chlorophylls. The differential optimization of antenna chlorophyll is discussed in the context of competing selection pressures for the evolution of light harvesting in photosynthesis.     

AtFtsH heterocomplex-mediated degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to stresses

Chloroplastic heterocomplex consisting of AtFtsH1, 2, 5 and 8 proteases, integrally bound to thylakoid membrane was shown to play a critical role in degradation of photodamaged PsbA molecules, inherent to photosystem II (PSII) repair cycle and in plastid development. As no one thylakoid bound apoproteins besides PsbA has been identified as target for the heterocomplex-mediated degradation we investigated the significance of this protease complex in degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to various stressing conditions and in stress-related changes in overall composition of LHCII trimers of PSII-enriched membranes (BBY particles). To reach this goal a combination of approaches was applied based on immunoblotting, in vitro degradation and non-denaturing isoelectrofocusing. Exposure of Arabidopsis thaliana leaves to desiccation, cold and high irradiance led to a step-wise disappearance of Lhcb1 and Lhcb2, while Lhcb3 level remained unchanged, except for high irradiance which caused significant Lhcb3 decrease. Furthermore, it was demonstrated that stress-dependent disappearance of Lhcb1–3 is a proteolytic phenomenon for which a metalloprotease is responsible. No changes in Lhcb1–3 level were observed due to exposition of var1-1 mutant leaves to the three stresses clearly pointing to the involvement of AtFtsH heterocomplex in the desiccation, cold and high irradiance-dependent degradation of Lhcb1 and Lhcb2 and in high irradiance-dependent degradation of Lhcb3. Non-denaturing isoelectrofocusing analyses revealed that AtFtsH heterocomplex-dependent differential Lhcb1–3 disappearance behaviour following desiccation stress was accompanied by modulations in abundances of individual LHCII trimers of BBY particles and that LHCII of var1-1 resisted the modulations.     

Diurnal fluctuation in the composition of chlorophyll a/b light harvesting antenna of photosystem II in young wheat leaves

We investigated the diurnal fluctuation in the composition of the light harvesting chlorophyll a/b antenna of photosystem II in young wheat (Triticum aestivum) leaves grown under periodic day/night irradiation. By means of gel electrophoresis of the polypeptides of thylakoid membranes, we determined the amount of 25 kDa and 27 kDa polypeptides, which are the main components of the peripheral and inner antenna subpopulations, respectively. Our data show a preferential fluctuation in the amount of the 25 kDa protein relative to the 27 kDa polypeptide, in parallel to the fluctuation in the amount of chlorophyll a/b antenna of photosystem II, which suggests that the peripheral antenna plays a role in the diurnal adjustment of the antenna size.     

An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo-electron microscopy studies.

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked 'grana' regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo-electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.     

Identification of a photosystem II phosphatase involved in light acclimation in Arabidopsis

Reversible protein phosphorylation plays a major role in the acclimation of the photosynthetic apparatus to changes in light. Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STATE TRANSITION7 (STN7) phosphorylates LHCII, the light-harvesting antenna of photosystem II (PSII), to balance the activity of the two photosystems through state transitions. STN8, which is mainly involved in phosphorylation of PSII core subunits, influences folding of the thylakoid membranes and repair of PSII after photodamage. The rapid reversibility of these acclimatory responses requires the action of protein phosphatases. In a reverse genetic screen, we identified the chloroplast PP2C phosphatase, PHOTOSYSTEM II CORE PHOSPHATASE (PBCP), which is required for efficient dephosphorylation of PSII proteins. Its targets, identified by immunoblotting and mass spectrometry, largely coincide with those of the kinase STN8. The recombinant phosphatase is active in vitro on a synthetic substrate or on isolated thylakoids. Thylakoid folding is affected in the absence of PBCP, while its overexpression alters the kinetics of state transitions. PBCP and STN8 form an antagonistic kinase and phosphatase pair whose substrate specificity and physiological functions are distinct from those of STN7 and the counteracting phosphatase PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, but their activities may overlap to some degree.     

Evolution and functional properties of photosystem II light harvesting complexes in eukaryotes

Photoautotrophic organisms, the major agent of inorganic carbon fixation into biomass, convert light energy into chemical energy. The first step of photosynthesis consists of the absorption of solar energy by pigments binding protein complexes named photosystems. Within photosystems, a family of proteins called Light Harvesting Complexes (LHC), responsible for light harvesting and energy transfer to reaction centers, has evolved along with eukaryotic organisms. Besides light absorption, these proteins catalyze photoprotective reactions which allowed functioning of oxygenic photosynthetic machinery in the increasingly oxidant environment. In this work we review current knowledge of LHC proteins serving Photosystem II. Balance between light harvesting and photoprotection is critical in Photosystem II, due to the lower quantum efficiency as compared to Photosystem I. In particular, we focus on the role of each antenna complex in light harvesting, energy transfer, scavenging of reactive oxygen species, chlorophyll triplet quenching and thermal dissipation of excess energy. This article is part of a Special Issue entitled: Photosystem II.     

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.     

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.     

Hydrogen bonding in a model bacteriochlorophyll-binding site drives assembly of light harvesting complex

In this study, the contribution of intramembrane hydrogen bonding at the interface between polypeptide and cofactor is explored in the native lipid environment by use of model bacteriochlorophyll proteins. In the peripheral antenna complex, LH2, large portions of the transmembrane helices, which make up the dimeric bacteriochlorophyll-binding site, are replaced by simplified, alternating alanine-leucine stretches. Replacement of either one of the two helices with the helices containing the model sequence at a time results in the assembly of complexes with nearly native light harvesting properties. In contrast, replacement of both helices results in the loss of antenna complexes from the membrane. The assembly of such doubly modified complexes is restored by a single intramembrane serine residue at position -4 relative to the liganding histidine of the alpha-subunit. In situ analysis of the spectral properties in a series of site-directed mutants reveals a critical dependence of the model complex assembly on the side chain of the residue at this position in the helix. A hydrogen bond between the hydroxy group of the serine and the 13(1) keto group of one of the central bacteriochlorophylls of the complexes is identified by Raman spectroscopy in the model antenna complex containing one of the alanine-leucine helices. The additional OH group of the serine residue, which participates in hydrogen bonding, increases the thermal stability of the model complexes in the native membrane. Intramembrane hydrogen bonding is thus shown to be a key factor for the binding of bacteriochlorophyll and assembly of this model cofactor-polypeptide site.     

Photoprotection in plants involves a change in lutein 1 binding domain in the major light-harvesting complex of photosystem II

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).     

Spatial relationship of photosystem I, photosystem II, and the light- harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent- light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment- protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Delayed fluorescence emitted from light harvesting complex II and photosystem II of higher plants in the 100 ns-5 micros time domain

This study presents the first report on delayed fluorescence (DF) emitted from spinach thylakoids, D1/D2/Cytb-559 preparations and solubilized light harvesting complex II (LHCII) in the ns time domain after excitation with saturating laser flashes. The use of a new commercially available multichannel plate with rapid gating permitted a sufficient suppression of detector distortions due to the strong prompt fluorescence. The following results were obtained: (a) in dark-adapted thylakoids, the DF amplitudes at 100 ns and 5 micros after each flash of a train of saturating actinic pulses exhibit characteristic period four oscillations of opposite sign: the DF amplitudes at 100 ns oscillate in the same manner as the quantum yield of prompt fluorescence, whereas those at 5 micros resemble the oscillation of the micros kinetics of P680(.) reduction in samples with an intact water oxidizing complex, (b) the quantum yield of total DF emission in the range up to a few micros is estimated to be <10(-4) for thylakoids, (c) the DF of D1/D2/Cytb-559 exhibits a monophasic decay with tau approximately 50 ns, (d) DF emission is also observed in isolated LHCII with biphasic decay kinetics characterized by tau values of 65 ns and about 800 ns, (e) in contrast to thylakoids, the amplitudes of DF in D1/D2/Cytb-559 preparations and solubilized LHCII do not exhibit any oscillation pattern and (f) all spectra of DF from the different sample types are characteristic for emission from the lowest excited singlet state of chlorophyll a. The implications of these findings and problems to be addressed in future research are briefly discussed.     

Characterisation of senescence-induced changes in light harvesting complex II and photosystem I complex of thylakoids of Cucumis sativus cotyledons: age induced association of LHCII with photosystem I

Structure and function of chloroplasts are known to after during senescence. The senescence-induced specific changes in light harvesting antenna of photosystem II (PSII) and photosystem I (PSI) were investigated in Cucumis cotyledons. Purified light harvesting complex II (LHCII) and photosystem I complex were isolated from 6-day non-senescing and 27-day senescing Cucumis cotyledons. The chlorophyll a/b ratio of LHCII obtained from 6-day-old control cotyledons and their absorption, chlorophyll a fluorescence emission and the circular dichroism (CD) spectral properties were comparable to the LHCII preparations from other plants such as pea and spinach. The purified LHCII obtained from 27-day senescing cotyledons had a Chl a/b ratio of 1.25 instead of 1.2 as with 6-day LHCII and also exhibited significant changes in the visible CD spectrum compared to that of 6-day LHCII, indicating some specific alterations in the organisation of chlorophylls of LHCII. The light harvesting antenna of photosystems are likely to be altered due to aging. The room temperature absorption spectrum of LHCII obtained from 27-day senescing cotyledons showed changes in the peak positions. Similarly, comparison of 77K chlorophyll a fluorescence emission characteristics of LHCII preparation from senescing cotyledons with that of control showed a small shift in the peak position and the alteration in the emission profile, which is suggestive of possible changes in energy transfer within LHCII chlorophylls. Further, the salt induced aggregation of LHCII samples was lower, resulting in lower yields of LHCII from 27-day cotyledons than from normal cotyledons. Moreover, the PSI preparations of 6-day cotyledons showed Chl a/b ratios of 5 to 5.5, where as the PSI sample of 27-day cotyledons had a Chl a/b ratio of 2.9 suggesting LHCII association with PSI. The absorption, fluorescence emission and visible CD spectral measurements as well as the polypeptide profiles of 27-day cotyledon-PSI complexes indicated age-induced association of LHCII of PSII with PSI obtained from 27-day cotyledons. We modified our isolation protocols by increasing the duration of detergent Triton X-100 treatment for preparing the PSI and LHCII complexes from 27-day cotyledons. However, the PSI complexes isolated from senescing samples invariably proved to have significantly low Chl a/b ratio suggesting an age induced lateral movement and possible association of LHCII with PSI complexes. The analyses of polypeptide compositions of LHCII and PSI holocomplexes isolated from 6-day control and 27-day senescing cotyledons showed distinctive differences in their profiles. The presence of 26-28 kDa polypeptide in PSI complexes from 27-day cotyledons, but not in 6-day control PSI complexes is in agreement with the notion that senescence induced migration of LHCII to stroma lamellae and its possible association with PSI. We suggest that the migration of LHCII to the stroma lamellae region and its possible association with PSI might cause the destacking and flattening of grana structure during senescence of the chloroplasts. Such structural changes in light harvesting antenna are likely to alter energy transfer between two photosystems. The nature of aging induced migration and association of LHCII with PSI and its existence in other senescing systems need to be estimated in the future.