Photoprotective energy dissipation involves the reorganization of photosystem II light-harvesting complexes in the grana membranes of spinach chloroplasts

Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on ΔpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.     

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.     

Origin of absorption changes associated with photoprotective energy dissipation in the absence of zeaxanthin

To prevent photo-oxidative damage to the photosynthetic membrane in strong light, plants dissipate excess absorbed light energy as heat in a mechanism known as non-photochemical quenching (NPQ). NPQ is triggered by the trans-membrane proton gradient (ΔpH), which causes the protonation of the photosystem II light-harvesting antenna (LHCII) and the PsbS protein, as well as the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin. The combination of these factors brings about formation of dissipative pigment interactions that quench the excess energy. The formation of NPQ is associated with certain absorption changes that have been suggested to reflect a conformational change in LHCII brought about by its protonation. The light-minus-dark recovery absorption difference spectrum is characterized by a series of positive and negative bands, the best known of which is ΔA(535). Light-minus-dark recovery resonance Raman difference spectra performed at the wavelength of the absorption change of interest allows identification of the pigment responsible from its unique vibrational signature. Using this technique, the origin of ΔA(535) was previously shown to be a subpopulation of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin), a proportion of NPQ remains, and the ΔA(535) change is blue-shifted to 525 nm (ΔA(525)). Using resonance Raman spectroscopy, it is shown that the ΔA(525) absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted subpopulation of violaxanthin molecules formed during NPQ. The presence of the same ΔA(535) and ΔA(525) Raman signatures in vitro in aggregated LHCII, containing zeaxanthin and violaxanthin, respectively, leads to a new proposal for the origin of the xanthophyll red shifts associated with NPQ.     

The functional significance of the monomeric and trimeric states of the photosystem II light harvesting complexes

The main light harvesting complex of photosystem II in plants, LHCII, exists in a trimeric state. To understand the biological significance of trimerization, a comparison has been made been LHCII trimers and LHCII monomers prepared by treatment with phospholipase. The treatment used caused no loss of chlorophyll, but there was a difference in carotenoid composition, together with the previously observed alterations in absorption spectrum. It was found that, when compared to monomers, LHCII trimers showed increased thermal stability and a reduced structural flexibility as determined by the decreased rate and amplitude of fluorescence quenching in low-detergent concentration. It is suggested that LHCII should be considered as having two interacting domains: the lutein 1 domain, the site of fluorescence quenching [Wentworth et al. (2003) J. Biol. Chem. 278, 21845-21850], and the lutein 2 domain. The lutein 2 domain faces the interior of the trimer, the differences in absorption spectrum and carotenoid binding in trimers compared to monomers indicating that the trimeric state modulates the conformation of this domain. It is suggested that the lutein 2 domain controls the conformation of the lutein 1 domain, thereby providing allosteric control of fluorescence quenching in LHCII. Thus, the pigment configuration and protein conformation in trimers is adapted for efficient light harvesting and enhanced protein stability. Furthermore, trimers exhibit the optimum level of control of energy dissipation by modulating the development of the quenched state of the complex.     

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.     

EPR characterization of photosystem II from different domains of the thylakoid membrane

We report electron paramagnetic resonance (EPR) studies on photosystem II (PSII) from higher plants in five different domains of the thylakoid membrane prepared by sonication and two-phase partitioning. The domains studied were the grana core, the entire grana stack, the grana margins, the stroma lamellae and the purified stromal fraction, Y100. The electron transport properties of both donor and acceptor sides of PSII such as oxygen evolution, cofactors Y D, Q A, the CaMn 4-cluster, and Cytb 559 were investigated. The PSII content was estimated on the basis of oxidized Y D and Q A (-) Fe (2+) signal from the acceptor side vs Chl content (100% in the grana core fraction). It was found to be about 82% in the grana, 59% in the margins, 35% in the stroma and 15% in the Y100 fraction. The most active PSII centers were found in the granal fractions as was estimated from the rates of electron transfer and the S 2 state multiline EPR signal. In the margin and stroma fractions the multiline signal was smaller (40 and 33%, respectively). The S 2 state multiline could not be induced in the Y100 fraction. In addition, the oxidized LP Cytb 559 prevailed in the stromal fractions while the HP form dominated in the grana core. The margins and entire grana fractions have Cytb 559 in both potential forms. These data together with previous analyses indicate that the sequence of activation of the PSII properties can be represented as: PSII content > oxygen evolution > reduced Cytb 559 > dimerization of PSII centers in all fractions of the thylakoid membrane with the gradual increase from stromal fractions via margin to the grana core fraction. The results further support the existence of a PSII activity gradient which reflects lateral movement and photoactivation of PSII centers in the thylakoid membrane. The possible role of the PSII redox components in this process is discussed.     

The photoprotective molecular switch in the photosystem II antenna

We have reviewed the current state of multidisciplinary knowledge of the photoprotective mechanism in the photosystem II antenna underlying non-photochemical chlorophyll fluorescence quenching (NPQ). The physiological need for photoprotection of photosystem II and the concept of feed-back control of excess light energy are described. The outline of the major component of nonphotochemical quenching, qE, is suggested to comprise four key elements: trigger (ΔpH), site (antenna), mechanics (antenna dynamics) and quencher(s). The current understanding of the identity and role of these qE components is presented. Existing opinions on the involvement of protons, different LHCII antenna complexes, the PsbS protein and different xanthophylls are reviewed. The evidence for LHCII aggregation and macrostructural reorganization of photosystem II and their role in qE are also discussed. The models describing the qE locus in LHCII complexes, the pigments involved and the evidence for structural dynamics within single monomeric antenna complexes are reviewed. We suggest how PsbS and xanthophylls may exert control over qE by controlling the affinity of LHCII complexes for protons with reference to the concepts of hydrophobicity, allostery and hysteresis. Finally, the physics of the proposed chlorophyll-chlorophyll and chlorophyll-xanthophyll mechanisms of energy quenching is explained and discussed. This article is part of a Special Issue entitled: Photosystem II.     

On photoprotective mechanisms of carotenoids in light harvesting complex

Carotenoids in light harvesting complex (LHC) play an important role in preventing plants photodamage caused by excess light. Non-photochemical quenching (NPQ) is an important mechanism adopted by plants to deal with high light intensity and the major component is referred to as energy dependent quenching (qE). Despite numerous studies have been devoted to investigating the site and mechanism of qE, there are still much debate on these topics. In this article, we discussed the possible site and underlying mechanism of qE based on the structural similarity of carotenoids. Moreover, being as good antioxidants, carotenoids’ potential protective effects against LHC photo-oxidation by quenching active oxygen species or triplet excited state chlorophyll are also discussed.     

Protein phosphorylation and Mg2+ influence light harvesting and electron transport in chloroplast thylakoid membrane material containing only the chlorophyll-a/b-binding light-harvesting complex of photosystem II and photosystem I.

A material containing only photosystem I (PSI) and the chlorophyll-a/b-binding light-harvesting complex of PSII (LHC-II) has been isolated from the chloroplast thylakoid membrane by solubilization with Triton X-100. Fluorescence spectroscopy shows that, within the material, LHC-II is coupled to PSI for excitation-energy transfer and that this coupling is decreased by the presence of Mg2+, which also decreased PSI electron transport specifically at limiting light intensity. Inclusion of phosphorylated LHC-II within the material did not alter its structure, but gave decreased energy transfer to PSI and inhibition of electron transport which was independent of light intensity, implying effects of phosphorylation on both light harvesting and directly on electron transport. Inclusion of Mg2+ within the phosphorylated material gave decreased energy transfer, but slightly increased PSI electron transport. A cation-induced direct promotion of PSI electron transport was also observed in isolated PSI particles. The PSI/LHC-II material represents a model system for examining protein interactions during light-state adaptations and the possibility that LHC-II can contribute to the antenna of PSI in light state 2 in vivo is discussed.     

Recovery of photoinactivated photosystem II in leaves: retardation due to restricted mobility of photosystem II in the thylakoid membrane

The functionality of photosystem II (PS II) following high-light pre-treatment of leaf segments at a chilling temperature was monitored as F(v)/F(m), the ratio of variable to maximum chlorophyll fluorescence in the dark-adapted state and a measure of the optimal photochemical efficiency in PS II. Recovery of PS II functionality in low light (LL) and at a favourable temperature was retarded by (1) water stress and (2) growth in LL, in both spinach and Alocasia macrorrhiza L. In spinach leaf segments, water stress per se affected neither F(v)/F(m) nor the ability of the adenosine triphosphate (ATP) synthase to be activated by far-red light for ATP synthesis, but it induced chloroplast shrinkage as observed in frozen and fractured samples by scanning electron microscopy. A common feature of water stress and growth of plants in LL is the enhanced anchoring of PS II complexes, either across the shrunken lumen in water-stress conditions or across the partition gap in larger grana due to growth in LL. We suggest that such enhanced anchoring restricts the mobility of PS II complexes in the thylakoid membrane system, and hence hinders the lateral migration of photoinactivated PS II reaction centres to the stroma-located ribosomes for repair.     

TAKs, thylakoid membrane protein kinases associated with energy transduction

The phosphorylation of proteins within the eukaryotic photosynthetic membrane is thought to regulate a number of photosynthetic processes in land plants and algae. Both light quality and intensity influence protein kinase activity via the levels of reductants produced by the thylakoid electron transport chain. We have isolated a family of proteins called TAKs, Arabidopsis thylakoid membrane threonine kinases that phosphorylate the light harvesting complex proteins. TAK activity is enhanced by reductant and is associated with the photosynthetic reaction center II and the cytochrome b6f complex. TAKs are encoded by a gene family that has striking similarity to transforming growth factor beta receptors of metazoans. Thus thylakoid protein phosphorylation may be regulated by a cascade of reductant-controlled membrane-bound protein kinases.     

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes

Oxygenic photosynthesis produces various radicals and activeoxygen species with harmful effects on photosystem II (PSII).Such photodamage occurs at all light intensities. Damaged PSIIcentres, however, do not usually accumulate in the thylakoidmembrane due to a rapid and efficient repair mechanism. Theexcellent design of PSII gives protection to most of the proteincomponents and the damage is most often targeted only to thereaction centre D1 protein. Repair of PSII via turnover of thedamaged protein subunits is a complex process involving (i)highly regulated reversible phosphorylation of several PSIIcore subunits, (ii) monomerization and migration of the PSIIcore from the grana to the stroma lamellae, (iii) partial disassemblyof the PSII core monomer, (iv) highly specific proteolysis ofthe damaged proteins, and finally (v) a multi-step replacementof the damaged proteins with de novo synthesized copies followedby (vi) the reassembly, dimerization, and photoactivation ofthe PSII complexes. These processes will shortly be reviewedpaying particular attention to the damage, turnover, and assemblyof the PSII complex in grana and stroma thylakoids during thephotoinhibition–repair cycle of PSII. Moreover, a two-dimensionalBlue-native gel map of thylakoid membrane protein complexes,and their modification in the grana and stroma lamellae duringa high-light treatment, is presented. Key words: Arabidopsis thylakoid membrane proteome, assembly of photosystem II, D1 protein, light stress, photosystem II photoinhibition, repair of photosystem II     

Dimeric and monomeric organization of photosystem II. Distribution of five distinct complexes in the different domains of the thylakoid membrane

The supramolecular organization of photosystem II (PSII) was characterized in distinct domains of the thylakoid membrane, the grana core, the grana margins, the stroma lamellae, and the so-called Y100 fraction. PSII supercomplexes, PSII core dimers, PSII core monomers, PSII core monomers lacking the CP43 subunit, and PSII reaction centers were resolved and quantified by blue native PAGE, SDS-PAGE for the second dimension, and immunoanalysis of the D1 protein. Dimeric PSII (PSII supercomplexes and PSII core dimers) dominate in the core part of the thylakoid granum, whereas the monomeric PSII prevails in the stroma lamellae. Considerable amounts of PSII monomers lacking the CP43 protein and PSII reaction centers (D1-D2-cytochrome b559 complex) were found in the stroma lamellae. Our quantitative picture of the supramolecular composition of PSII, which is totally different between different domains of the thylakoid membrane, is discussed with respect to the function of PSII in each fraction. Steady state electron transfer, flash-induced fluorescence decay, and EPR analysis revealed that nearly all of the dimeric forms represent oxygen-evolving PSII centers. PSII core monomers were heterogeneous, and a large fraction did not evolve oxygen. PSII monomers without the CP43 protein and PSII reaction centers showed no oxygen-evolving activity.     

A few molecules of zeaxanthin per reaction centre of photosystem II permit effective thermal dissipation of light energy in photosystem II of a poikilohydric moss

The relationship between thermal dissipation of light energy (as indicated by the quenching of chlorophyll fluorescence), zeaxanthin availability and protonation reactions was investigated in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. In the absence of zeaxanthin and actinic illumination, acidification by 20% CO₂ in air was incapable of quenching basal, so-called F ₀ fluorescence either in the moss or in spinach (Spinacia oleracea L.) leaves. However, 1-s light pulses given either every 40, 60 or 200 s increased thermal dissipation as indicated by F ₀ and F _m quenching in the presence of 20% CO₂ in air in the moss, but not in spinach while reaction centres of photosystem II (PSII) were photochemically open. In the moss, a few short light pulses, which were separated by prolonged dark times, were sufficient to raise zeaxanthin levels in the presence of 20% CO₂ in air. Simultaneously, quantum efficiency of charge separation in PSII was decreased. Increasing the CO₂ concentration beyond 20% further decreased quantum efficiency even in the absence of short light pulses. Under conditions optimal for fluorescence quenching, one molecule of zeaxanthin per reaction centre of PSII was sufficient to decrease quantum efficiency of charge separation in PSII by 50%. Thus, in combination with a protonation reaction, one molecule of zeaxanthin was as efficient at capturing excitation energy as a photochemically open reaction centre. The data are discussed in relation to the interaction between zeaxanthin and thylakoid protonation, which enables effective thermal dissipation of light energy in the antennae of PSII in the moss but not in higher plants when actinic illumination is absent. Received: 8 April 2000 / Accepted: 31 August 2000     

Energy dissipation in photosystem 2 complexes of peanut leaves subjected to light pulses

To increase crop yields, intercropping agricultural cultivation approach (IACA) and relay cropping agricultural cultivation approach (RACA) were introduced into cultivation of peanut (Arachis hypogaca L.), one of the most important oil crops in the world. IACA/RACA improves the yield of the crop per unit area, but rather decreases the yield of a single crop compared with mono-cropping agricultural cultivation approach. In peanut IACA/RACA, peanut plants would grow under low light and high light pulses (HLP) before maize/wheat harvest. Energy dissipation in photosystem 2 (PS2) complexes was evaluated in peanut leaves after being induced by light pulses and continuous light with different light intensities. Actual photochemical efficiency of PS2 (ΦPS2) and non-photochemical quenching induced by light pulses in peanut leaves generally were significantly lower compared with those induced by continuous light. In addition, the degree of PS2 reaction center closure (1-qP) induced by light pulses in peanut leaves was significantly higher compared with those induced by continuous light. Different results were obtained only for ΦPS2 and 1-qP induced by 20 s HLP. In methyl viologen (MV) treated samples, ΦPS2 and the quantum yield of light induced thermal dissipation by non-functional PS2 (Φ_NF) were similar to those observed in non-MV treatments. These results implied that light pulses could induce photoinactivation of PS2 reaction centers but not the xanthophyll cycle to dissipate excess energy.     

Differential detergent-solubilization of integral thylakoid membrane complexes in spinach chloroplasts. Localization of photosystem II, cytochrome b6-f complex and photosystem I

Progressive solubilization of spinach chloroplast thylakoids by Triton X-100 was employed to investigate the domain organization of the electron transport complexes in the thylakoid membrane. Triton/chlorophyll ratios of 1:1 were sufficient to disrupt fully the continuity of the thylakoid membrane network, but not sufficient to solubilize either photosystem I (PSI), photosystem II (PSII) or the cytochrome b6-f(Cyt b6-f) complex. Progressive with the Triton concentration increase (Triton/Chl greater than 1:1), a differential solubilization of the three electron transport complexes was observed. Solubilization of the Cyt b6-f complex from the thylakoid membrane preceded that of PSI and apparently occurred early in the solubilization of stroma-exposed segments of the chloroplast lamellae. The initial removal of chlorophyll (up to 40% of the total) occurred upon solubilization of PSI from the stroma-exposed lamella regions in which PSI is localized. The tightly appressed membrane of the grana partition regions was markedly resistant to solubilization by Triton X-100. Thus, solubilization of PSII from this membrane region was initiated only after all Cyt b6-f and PSI complexes were removed from the chloroplast lamellae. The results support the notion of extreme lateral heterogeneity in the organization of the electron transport complexes in higher plant chloroplasts and suggest a Cyt b6-f localization in the membrane of the narrow fret regions which serve as a continuum between the grana and stroma lamellae.     

Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii

The turnover of photosystem-II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae-CP-29 and light-harvesting-complex-II polypeptides and the water-oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis-saturating light (2500 W/m2 for 90 min) at 25 degrees C. While 80% of photosystem-II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold-labeled whole-cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition. We propose that the light-dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem-II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.     

The excess light energy that is neither utilized in photosynthesis nor dissipated by photoprotective mechanisms determines the rate of photoinactivation in photosystem II

Photoinactivation of PSII is thought to be caused by the excessive light energy that is neither used for photosynthetic electron transport nor dissipated as heat. However, the relationship between the photoinactivation rate and excess energy has not been quantitatively evaluated. Chenopodium album L. plants grown under high-light and high-nitrogen (HL-HN) conditions show higher tolerance to photoinactivation and have higher photosynthetic capacity than the high-light and low-nitrogen (HL-LN)- and low-light and high-nitrogen (LL-HN)-grown plants. The rate of photoinactivation in the LL-HN plants was faster than that in the HL-LN, which was similar to that in the HL-HN plants, while the LL-HN and HL-LN plants had similar photosynthetic capacities [Kato et al. (2002b) Funct. Plant Biol. 29: 787]. We quantified partitioning of light energy between the electron transport and heat dissipation at the light intensities ranging from 300 to 1,800 micromol m(-2) s(-1). The maximum electron transport rate was highest in the HL-HN plants, heat dissipation was greatest in the HL-LN plants, and the excess energy, which was neither consumed for electron transport nor dissipated as heat, was greatest in the LL-HN plants. The first-order rate constant of the PSII photoinactivation was proportional to the magnitude of excess energy, with a single proportional constant for all the plants, irrespective of their growth conditions. Thus the excess energy primarily determines the rate of PSII photoinactivation. A large photosynthetic capacity in the HL-HN plants and a large heat dissipation capacity in the HL-LN plants both contribute to the protection of PSII against photoinactivation.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700(+) and Y(D)( .-), respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIbeta) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIalpha) 300, PSI (PSIbeta) in stroma lamellae 214, PSII in grana core (PSIIalpha) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700⁺ and Y_D, respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIβ) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIα) 300, PSI (PSIβ) in stroma lamellae 214, PSII in grana core (PSIIα) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.