Photoinactivation and photoprotection of photosystem II in nature

Photosystem II plays a central role not only in energy transduction, but also in monitoring the molecular redox mechanisms involved in signal transduction for acclimation to environmental stresses. Central to the regulation of photosystem II (PSII) function as a light-driven molecular machine in higher plant leaves, is an inevitable photo-inactivation of one PSII after 10⁶–10⁷ photons have been delivered to the leaf, although the act of photoinactivation per se requires only one photon. PSII function in acclimated pea leaves shows a reciprocity between irradiance and the time of illumination, demonstrating that the photoinactivation of PSII is a light dosage effect, depending on the number of photons absorbed rather than the rate of photon absorption. Hence, PSII photoinactivation will occur at low as well as high irradiance. There is a heterogeneity of PSII functional stability, possibly with less stable PSII monomers being located in grana margins and more stable PSII dimers in appressed granal domains. Matching the inevitable photoinactivation of PSII, green plants have an intrinsic capacity for D1 protein synthesis to restore PSII function which is saturated at very low light. Photoinhibition of PSII in vivo is often a photoprotective strategy rather than a damaging process.     

The roles of specific xanthophylls in light utilization

To evaluate the role of specific xanthophylls in light utilization, wild-type and xanthophyll mutant plants (npq1, npq2, lut2, lut2npq1 and lut2npq2) from Arabidopsis thaliana were grown under three different light regimes: 30 (low light, LL), 150 (medium light, ML) and 450 (high light, HL) μmol photons m⁻² s⁻¹. We studied the pigment content, growth rate, xanthophyll cycle activity, chlorophyll fluorescence parameters and the response to photoinhibition. All genotypes differed strongly in the growth rates and the resistance against photoinhibition. In particular, replacement of lutein (Lut) by violaxanthin (Vx) in the lut2npq1 mutant did not affect the growth at non-saturating light intensities (LL and ML), but led to a pronounced reduction of growth under HL conditions, indicating an important photoprotective role of Lut. This was further supported by a much higher sensitivity of all Lut-deficient plants to photoinhibition in comparison with the wild type. In contrast, replacement of Lut by zeaxanthin (Zx) in lut2npq2 led to a pronounced reduction of growth under all light regimes, most likely related to the permanent non-photochemical dissipation of excitation energy by Zx at Vx-binding sites and the destabilization of antenna proteins by binding of Zx to Lut-binding sites. The high susceptibility of lut2npq2 to photoinhibition in comparison with npq2 further indicated that the photoprotective function of Zx is abolished in the absence of Lut. Thus, it can be concluded from our work that neither Vx nor Zx is able to fulfil the essential photoprotective function at Lut-binding sites under in vivo conditions.     

The orange carotenoid protein in photoprotection of photosystem II in cyanobacteria

Photoprotective mechanisms have evolved in photosynthetic organisms to cope with fluctuating light conditions. Under high irradiance, the production of dangerous oxygen species is stimulated and causes photo-oxidative stress. One of these photoprotective mechanisms, non photochemical quenching (qE), decreases the excess absorbed energy arriving at the reaction centers by increasing thermal dissipation at the level of the antenna. In this review we describe results leading to the discovery of this process in cyanobacteria (qE(cya)), which is mechanistically distinct from its counterpart in plants, and recent progress in the elucidation of this mechanism. The cyanobacterial photoactive soluble orange carotenoid protein is essential for the triggering of this photoprotective mechanism. Light induces structural changes in the carotenoid and the protein leading to the formation of a red active form. The activated red form interacts with the phycobilisome, the cyanobacterial light-harvesting antenna, and induces a decrease of the phycobilisome fluorescence emission and of the energy arriving to the reaction centers. The orange carotenoid protein is the first photoactive protein to be identified that contains a carotenoid as the chromophore. Moreover, its photocycle is completely different from those of other photoactive proteins. A second protein, called the Fluorescence Recovery Protein encoded by the slr1964 gene in Synechocystis PCC 6803, plays a key role in dislodging the red orange carotenoid protein from the phycobilisome and in the conversion of the free red orange carotenoid protein to the orange, inactive, form. This protein is essential to recover the full antenna capacity under low light conditions after exposure to high irradiance. This article is part of a Special Issue entitled: Photosystem II.     

Biogenesis and assembly of photosystem I

Photosystem I (PS I) is a multisubunit membrane protein complex consisting of 11 to 14 different subunits. In addition, several cofactors, such as chlorophylls, phylloquinones, carotenoids and iron-sulfur clusters are bound by this complex. We now have a detailed understanding of the structural basics, yet we know very little about the molecular details of the assembly process that finally yields functional PS I. Moreover, not much is known about the molecular dynamics of PS I in the thylakoid membrane or its regulated degradation. These areas have become the focus of recent work and first results have emerged. In this minireview we describe the latest findings in this fascinating and rapidly evolving field.     

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.     

Reaction centre quenching of excess light energy and photoprotection of photosystem II

In addition to the energy dissipation of excess light occurring in PSII antenna via the xanthophyll cycle, there is mounting evidence of a zeaxanthin-independent pathway for non-photochemical quenching based within the PSII reaction centre (reaction centre quenching) that may also play a significant role in photoprotection. It has been demonstrated that acclimation of higher plants, green algae and cyanobacteria to low temperature or high light conditions which potentially induce an imbalance between energy supply and energy utilization is accompanied by the development of higher reduction state of Q_A and higher resistance to photoinhibition (Huner et al., 1998). Although this is a fundamental feature of all photoautotrophs, and the acquisition of increased tolerance to photoinhibition has been ascribed to growth and development under high PSII excitation pressure, the precise mechanism controlling the redox state of Q_A and its physiological significance in developing higher resistance to photoinhibition has not been fully elucidated. In this review we summarize recent data indicating that the increased resistance to high light in a broad spectrum of photosynthetic organisms acclimated to high excitation pressure conditions is associated with an increase probability for alternative non-radiative P680⁺Q_A ^- radical pair recombination pathway for energy dissipation within the reaction centre of PSII. The various molecular mechanisms that could account for non-photochemical quenching through PSII reaction centre are also discussed.     

Molecular design of the photosystem II light-harvesting antenna: photosynthesis and photoprotection

The photosystem II (PSII) light-harvesting system carries out two essential functions, the efficient collection of light energy for photosynthesis, and the regulated dissipation of excitation energy in excess of that which can be used. This dual function requires structural and functional flexibility, in which light-harvesting proteins respond to an external signal, the thylakoid DeltapH, to induce feedback control. This process, referred to as non-photochemical quenching (NPQ) depends upon the xanthophyll cycle and the PsbS protein. In nature, NPQ is heterogeneous in terms of kinetics and capacity, and this adapts photosynthetic systems to the specific dynamic features of the light environment. The molecular features of the thylakoid membrane which may enable this flexibility and plasticity are discussed.     

Photochemical behavior of xanthophylls in the recombinant photosystem II antenna complex, CP26

The steady state absorption and fluorescence spectroscopic properties of the xanthophylls, violaxanthin, zeaxanthin, and lutein, and the efficiencies of singlet energy transfer from the individual xanthophylls to chlorophyll have been investigated in recombinant CP26 protein overexpressed in Escherichia coli and then refolded in vitro with purified pigments. Also, the effect of the different xanthophylls on the extents of static and dynamic quenching of chlorophyll fluorescence has been investigated. Absorption, fluorescence, and fluorescence excitation demonstrate that the efficiency of light harvesting from the xanthophylls to chlorophyll a is relatively high and insensitive to the particular xanthophyll that is present. A small effect of the different xanthophylls is observed on the extent of quenching of Chl fluorescence. The data provide the precise wavelengths of the absorption and fluorescence features of the bound pigments in the highly congested spectral profiles from these light-harvesting complexes. This information is important in assessing the mechanisms by which higher plants dissipate excess energy in light-harvesting proteins.     

Role of charge recombination processes in photodamage and photoprotection of the photosystem II complex

Light-induced damage of the photosynthetic apparatus is an important and complex phenomenon, which affects primarily the photosystem II (PSII) complex. Here, the author summarizes the current state of understanding, which concerns the role of charge recombination reactions in photodamage and photoprotection. The main mechanism of photodamage induced by visible light appears to be mediated by acceptor side modifications, which develop under light intensity conditions when the capacity of light-independent photosynthetic processes limits the utilization of electrons produced in the initial photoreactions. This situation facilitates triplet chlorophyll formation and singlet oxygen production in the reaction center of PSII, which initiates the damage of electron transport components and protein structure. This mechanism is an important, but not exclusive, pathway of photodamage, and light-induced inactivation of the Mn cluster of water oxidation may occur in parallel with the singlet oxygen-dependent pathway.     

The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II

Photoprotection of photosystem II (PSII) is essential to avoid the light-induced damage of the photosynthetic apparatus due to the formation of reactive oxygen species (=photo-oxidative stress) under excess light. Carotenoids are known to play a crucial role in these processes based on their property to deactivate triplet chlorophyll (3Chl*) and singlet oxygen (1O?*). Xanthophylls are further assumed to be involved either directly or indirectly in the non-photochemical quenching (NPQ) of excess light energy in the antenna of PSII. This review gives an overview on recent progress in the understanding of the photoprotective role of the xanthophylls zeaxanthin (which is formed in the light in the so-called xanthophyll cycle) and lutein with emphasis on the NPQ processes associated with PSII of higher plants. The current knowledge supports the view that the photoprotective role of Lut is predominantly restricted to its function in the deactivation of 3Chl*, while zeaxanthin is the major player in the deactivation of excited singlet Chl (1Chl*) and thus in NPQ (non-photochemical quenching). Additionally, zeaxanthin serves important functions as an antioxidant in the lipid phase of the membrane and is likely to act as a key component in the memory of the chloroplast with respect to preceding photo-oxidative stress. This article is part of a Special Issue entitled: Photosystem II.     

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.     

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.     

Photoinactivation of photosystem II complexes and photoprotection by non-functional neighbours in Capsicum annuum L. leaves

Leaf segments from Capsicum annuum plants grown at 100 micromol photons m(-2) s(-1) (low light) or 500 micromol photons m(-2) s(-1) (high light) were illuminated at three irradiances and three temperatures for several hours. At various times, the remaining fraction (f) of functional photosystem II (PS II) complexes was measured by a chlorophyll fluorescence parameter (1/Fo -1/Fm, where Fo and Fm are the fluorescence yields corresponding to open and closed PS II traps, respectively), which was in turn calibrated by the oxygen yield per saturating single-turnover flash. During illumination of leaf segments in the presence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis, the decline of f from 1.0 to about 0.3 was mono-exponential. Thereafter, f declined much more slowly, the remaining fraction (approximately equals 0.2) being able to survive prolonged illumination. The results can be interpreted as being in support of the hypothesis that photoinactivated PS II complexes photoprotect functional neighbours (G. Oquist et al. 1992, Planta 186: 450-460), provided it is assumed that a photoinactivated PS II is initially only a weak quencher of excitation energy, but becomes a much stronger quencher during prolonged illumination when a substantial fraction of PS II complexes has also been photoinactivated. In the absence of lincomycin, photoinactivation and repair of PS II occur in parallel, allowing f to reach a steady-state value that is determined by the treatment irradiance, temperature and growth irradiance. The results obtained in the presence and absence of lincomycin are analysed according to a simple kinetic model which formally incorporates a conversion from weak to strong quenchers, yielding the rate coefficients of photoinactivation and of repair for various conditions, as well as gaining an insight into the influence off on the rate coefficient of photoinactivation. They demonstrate that the method is a convenient alternative to the use of radiolabelled amino acids for quantifying photoinactivation and repair of PS II in leaves.     

Important roles of tyrosines in photosystem II and cytochrome oxidase

Theoretical studies (B3LYP) on models of the active sites in Photosystem II (PSII) and cytochrome oxidase are discussed. The role of a tyrosyl radical in the O-O bond formation in PSII is investigated, as well as the tyrosyl radical formation. In cytochrome oxidase, mechanisms for O-O bond cleavage involving tyrosyl radical formation are investigated, together with possible roles for the tyrosine in the proton translocation.     

The assembly of protein subunits and cofactors in photosystem I

The recently determined crystal structures of photosystems I and II at 2.5 A and 3.8 A resolution, respectively, have improved the structural basis for understanding the processes of light trapping, exciton transfer and electron transfer occurring in the primary steps of oxygenic photosynthesis. Understanding the assembly of the 12 protein subunits and 128 cofactors in photosystem I allows us to study the possible functions of the individual players in this protein-cofactor complex.     

Biogenesis,assembly and turnover of photosystem II units

Assembly of photosystem II, a multiprotein complex embedded in the thylakoid membrane, requires stoichiometric production of over 20 protein subunits. Since part of the protein subunits are encoded in the chloroplast genome and part in the nucleus, a signalling network operates between the two genetic compartments in order to prevent wasteful production of proteins. Coordinated synthesis of proteins also takes place among the chloroplast-encoded subunits, thus establishing a hierarchy in the protein components that allows a stepwise building of the complex. In addition to this dependence on assembly partners, other factors such as the developmental stage of the plastid and various photosynthesis-related parameters exert a strict control on the accumulation, membrane targeting and assembly of the PSII subunits. Here, we briefly review recent results on this field obtained with three major approaches: biogenesis of photosystem II during the development of chloroplasts from etioplasts, use of photosystem II-specific mutants and photosystem II turnover during its repair cycle.     

The interplay of anthocyanin biosynthesis and chlorophyll catabolism in senescing leaves and the question of photosystem II photoprotection

Fully exposed, senescing leaves of Cornus sanguinea and Parthenocissus quinquefolia display during autumn considerable variation in both anthocyanin and chlorophyll (Chl) concentrations. They were used in this study to test the hypothesis that anthocyanins may have a photoprotective function against photosystem II (PSII) photoinhibitory damage. The hypothesis could not be confirmed with field sampled leaves since maximum photochemical efficiency (F_v/F_m) of PSII was negatively correlated to anthocyanin concentration and the possible effects of anthocyanins were also confounded by a decrease in F_v/F_m with Chl loss. However, after short-term laboratory photoinhibitory trials, the percent decrease of F_v/F_m was independent of Chl concentration. In this case, a slight alleviation of PSII damage with increasing anthocyanins was observed in P. quinquefolia, while a similar trend in C. sanguinea was not statistically significant. It is inferred that the assumed photoprotection, if addressed to PSII, may be of limited advantage and only under adverse environmental conditions.