Photosystem II in different parts of the thylakoid membrane: a functional comparison between different domains

The electron transport properties of photosystem II (PSII) from five different domains of the thylakoid membrane were analyzed by flash-induced fluorescence kinetics. These domains are the entire grana, the grana core, the margins from the grana, the stroma lamellae, and the Y100 fraction (which represent more purified stroma lamellae). The two first fractions originate from appressed grana membranes and have PSII with a high proportion of O(2)-evolving centers (80-90%) and efficient electron transport on the acceptor side. About 30% of the granal PSII centers were found in the margin fraction. Two-thirds of those PSII centers evolve O(2), but the electron transfer on the acceptor side is slowed. PSII from the stroma lamellae was less active. The fraction containing the entire stroma has only 43% O(2)-evolving PSII centers and slow electron transfer on the acceptor side. In contrast, PSII centers of the Y100 fraction show no O(2) evolution and were unable to reduce Q(B). Flash-induced fluorescence decay measurements in the presence of DCMU give information about the integrity of the donor side of PSII. We were able to distinguish between PSII centers with a functional Mn cluster and without any Mn cluster, and PSII centers which undergo photoactivation and have a partially assembled Mn cluster. From this analysis, we propose the existence of a PSII activity gradient in the thylakoid membrane. The gradient is directed from the stroma lamellae, where the Mn cluster is absent or inactive, via the margins where photoactivation accelerates, to the grana core domain where PSII is fully photoactivated. The photoactivation process correlates to the PSII diffusion along the membrane and is initiated in the stroma lamellae while the final steps take place in the appressed regions of the grana core. The margin domain is seemingly very important in this process.     

Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.     

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.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Role of phosphorylation in the repair cycle and oligomeric structure of photosystem II

The role of PSII protein phosphorylation in the oligomeric structure of the complex and in the repair of photodamaged PSII centers was studied with intact thylakoids and thylakoid membrane subfractions isolated from differentially light-treated pumpkin (Cucurbita pepo L.) leaves. A combination of sucrose gradient fractionation of thylakoid protein complexes and immunodetection with phosphothreonine and protein-specific antibodies was used. We report in this study that the extent of phosphorylation of PSII core proteins is equivalent in dimers and monomers, and directly depends on light intensity. Phosphorylated PSII monomers migrate to the stroma-exposed thylakoids, probably following damage of the D1 protein and the dissociation of the light-harvesting complex of PSII. Once in the stroma lamellae, monomers are gradually dephosphorylated to allow the reparation of the complex. First, CP43 is dephosphorylated and as a consequence of this modification it detaches from the PSII core. In addition to D1, D2 is also thereafter dephosphorylated. Phosphorylation of PSII core polypeptides probably ensures the integrity of the monomers until repair can proceed. Dephosphorylation, on the other hand, might serve the need for opening the complex and coordinating D1 proteolysis and the attachment of ribosomes.     

Functional heterogeneity of photosystem II in domain specific regions of the thylakoid membrane of spinach (Spinacia oleracea L.)

A mild sonication and phase fractionation method has been used to isolate five regions of the thylakoid membrane in order to characterize the functional lateral heterogeneity of photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decay kinetics were used to determine the relative amounts of photosystem II (PSII) and photosystem I (PSI), to determine the relative PSII antenna size, and to characterize the excited-state dynamics of PSI and PSII in each fraction. Marked progressive increases in the proportion of PSI complexes were observed in the following sequence: grana core (BS), whole grana (B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decay kinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found to have only two decay components with slower lifetimes in the stroma. Results are discussed in the framework of existing models of chloroplast thylakoid membrane lateral heterogeneity and the PSII repair cycle. Kinetic modeling of the PSII fluorescence decay kinetics revealed that PSII populations in the stroma and grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populations in the grana stack.     

Structural organization of photosynthetic apparatus in agranal chloroplasts of maize

We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment.     

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.     

Fragmentation and separation analysis of the photosynthetic membrane from spinach

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.     

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.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

The structural and functional domains of plant thylakoid membranes

In plants, the stacking of part of the photosynthetic thylakoid membrane generates two main subcompartments: the stacked grana core and unstacked stroma lamellae. However, a third distinct domain, the grana margin, has been postulated but its structural and functional identity remains elusive. Here, an optimized thylakoid fragmentation procedure combined with detailed ultrastructural, biochemical, and functional analyses reveals the distinct composition of grana margins. It is enriched with lipids, cytochrome b₆f complex, and ATPase while depleted in photosystems and light‐harvesting complexes. A quantitative method is introduced that is based on Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) and dot immunoblotting for quantifying various photosystem II (PSII) assembly forms in different thylakoid subcompartments. The results indicate that the grana margin functions as a degradation and disassembly zone for photodamaged PSII. In contrast, the stacked grana core region contains fully assembled and functional PSII holocomplexes. The stroma lamellae, finally, contain monomeric PSII as well as a significant fraction of dimeric holocomplexes that identify this membrane area as the PSII repair zone. This structural organization and the heterogeneous PSII distribution support the idea that the stacking of thylakoid membranes leads to a division of labor that establishes distinct membrane areas with specific functions.     

High light induced disassembly of photosystem II supercomplexes in Arabidopsis requires STN7-dependent phosphorylation of CP29

Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light. The high light treatment of the wild type and stn8 caused specific increase in phosphorylation of Lhcb4.1 and Lhcb4.2 isoforms of the PSII linker protein CP29 at five different threonine residues. Phosphorylation of CP29 at four of these residues was not found in stn7 and stn7stn8 plants lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the membrane protein complexes revealed that the high light treatment of the wild type caused redistribution of CP29 from PSII supercomplexes to PSII dimers and monomers. A similar high-light-induced disassembly of the PSII supercomplexes occurred in stn8, but not in stn7 and stn7stn8. Transfer of the high-light-treated wild type plants to normal light relocated CP29 back to PSII supercomplexes. We postulate that disassembly of PSII supercomplexes in plants exposed to high light involves STN7-kinase-dependent phosphorylation of the linker protein CP29. Disruption of this adaptive mechanism can explain dramatically retarded growth of the stn7 and stn7stn8 mutants under fluctuating normal/high light conditions, as previously reported.     

Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

AtCYP38 ensures early biogenesis, correct assembly and sustenance of photosystem II

AtCYP38 is a thylakoid lumen protein comprising the immunophilin domain and the phosphatase inhibitor module. Here we show the association of AtCYP38 with the photosystem II (PSII) monomer complex and address its functional role using AtCYP38-deficient mutants. The dynamic greening process of etiolated leaves failed in the absence of AtCYP38, due to specific problems in the biogenesis of PSII complexes. Also the development of leaves under short-day conditions was severely disturbed. Detailed biophysical and biochemical analysis of mature AtCYP38-deficient plants from favorable growth conditions (long photoperiod) revealed: (i) intrinsic malfunction of PSII, which (ii) occurred on the donor side of PSII and (iii) was dependent on growing light intensity. AtCYP38 mutant plants also showed decreased accumulation of PSII, which was shown not to originate from impaired D1 synthesis or assembly of PSII monomers, dimers and supercomplexes as such but rather from the incorrect fine-tuning of the oxygen-evolving side of PSII. This, in turn, rendered PSII centers extremely susceptible to photoinhibition. AtCYP38 deficiency also drastically decreased the in vivo phosphorylation of PSII core proteins, probably related to the absence of the AtCYP38 phosphatase inhibitor domain. It is proposed that during PSII assembly AtCYP38 protein guides the proper folding of D1 (and CP43) into PSII, thereby enabling the correct assembly of the water-splitting Mn₄–Ca cluster even with high turnover of PSII.     

A land plant‐specific thylakoid membrane protein contributes to photosystem II maintenance in Arabidopsis thaliana

The structure and function of photosystem II (PSII) are highly susceptible to photo‐oxidative damage induced by high‐fluence or fluctuating light. However, many of the mechanistic details of how PSII homeostasis is maintained under photoinhibitory light remain to be determined. We describe an analysis of the Arabidopsis thaliana gene At5g07020, which encodes an unannotated integral thylakoid membrane protein. Loss of the protein causes altered PSII function under high‐irradiance light, and hence it is named ‘Maintenance of PSII under High light 1’ (MPH1). The MPH1 protein co‐purifies with PSII core complexes and co‐immunoprecipitates core proteins. Consistent with a role in PSII structure, PSII complexes (supercomplexes, dimers and monomers) of the mph1 mutant are less stable in plants subjected to photoinhibitory light. Accumulation of PSII core proteins is compromised under these conditions in the presence of translational inhibitors. This is consistent with the hypothesis that the mutant has enhanced PSII protein damage rather than defective repair. These data are consistent with the distribution of the MPH1 protein in grana and stroma thylakoids, and its interaction with PSII core complexes. Taken together, these results strongly suggest a role for MPH1 in the protection and/or stabilization of PSII under high‐light stress in land plants.     

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     

Jumping mode atomic force microscopy on grana membranes from spinach

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.     

Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants

In higher plants, the photosystem (PS) II core and its several light harvesting antenna (LHCII) proteins undergo reversible phosphorylation cycles according to the light intensity. High light intensity induces strong phosphorylation of the PSII core proteins and suppresses the phosphorylation level of the LHCII proteins. Decrease in light intensity, in turn, suppresses the phosphorylation of PSII core, but strongly induces the phosphorylation of LHCII. Reversible and differential phosphorylation of the PSII-LHCII proteins is dependent on the interplay between the STN7 and STN8 kinases, and the respective phosphatases. The STN7 kinase phosphorylates the LHCII proteins and to a lesser extent also the PSII core proteins D1, D2 and CP43. The STN8 kinase, on the contrary, is rather specific for the PSII core proteins. Mechanistically, the PSII-LHCII protein phosphorylation is required for optimal mobility of the PSII-LHCII protein complexes along the thylakoid membrane. Physiologically, the phosphorylation of LHCII is a prerequisite for sufficient excitation of PSI, enabling the excitation and redox balance between PSII and PSI under low irradiance, when excitation energy transfer from the LHCII antenna to the two photosystems is efficient and thermal dissipation of excitation energy (NPQ) is minimised. The importance of PSII core protein phosphorylation is manifested under highlight when the photodamage of PSII is rapid and phosphorylation is required to facilitate the migration of damaged PSII from grana stacks to stroma lamellae for repair. The importance of thylakoid protein phosphorylation is highlighted under fluctuating intensity of light where the STN7 kinase dependent balancing of electron transfer is a prerequisite for optimal growth and development of the plant. This article is part of a Special Issue entitled: Photosystem II.