The chloroplast‐localized small heat shock protein Hsp21 associates with the thylakoid membranes in heat‐stressed plants

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β‐sandwich fold domain defining the family, the α‐crystallin domain, whereas the N‐terminal and C‐terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non‐metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat‐stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic ¹⁵N‐isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat‐stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered.     

The role of lipids in photosystem II

The thylakoid membranes of photosynthetic organisms, which are the sites of oxygenic photosynthesis, are composed of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG). The identification of many genes involved in the biosynthesis of each lipid class over the past decade has allowed the generation and isolation of mutants of various photosynthetic organisms incapable of synthesizing specific lipids. Numerous studies using such mutants have revealed that deficiency of these lipids primarily affects the structure and function of photosystem II (PSII) but not of photosystem I (PSI). Recent X-ray crystallographic analyses of PSII and PSI complexes from Thermosynechococcus elongatus revealed the presence of 25 and 4 lipid molecules per PSII and PSI monomer, respectively, indicating the enrichment of lipids in PSII. Therefore, lipid molecules bound to PSII may play special roles in the assembly and functional regulation of the PSII complex. This review summarizes our present understanding of the biochemical and physiological roles of lipids in photosynthesis, with a special focus on PSII. This article is part of a Special Issue entitled: Photosystem II.     

Does the chloroplast small heat shock protein protect photosystem II during heat stress in vitro?

It has been suggested that the function of the chloroplast-localized small heat shock protein (sHsp) is to protect photosystem II (PSII) from heat inactivation. This paper reports that addition of purified sHsp protein to isolated thylakoid membranes gave no protection of PSII and questions that there is any direct effect of the sHsp on PSII. The opinion is forwarded that the primary role for the chloroplast-localized sHsp may not even be protection of PSII.     

Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas

The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.     

Involvement of sulfoquinovosyl diacylglycerol in the structural integrity and heat-tolerance of photosystem II

To examine the role of sulfoquinovosyl diacylglycerol (SQDG) in thylakoid membranes, we compared the structural and functional properties of photosystem II (PSII) between a mutant of Chlamydomonas reinhardtii defective in SQDG ( hf-2) and the wild type. The PSII core complex of hf-2, as compared with that of the wild type, showed structural fragility when solubilized with a detergent, dodecyl beta- d-maltoside, suggesting that the physical properties of the PSII complex were altered by the loss of SQDG. On the other hand, exposure of the cells to 41 degrees C for 120 min in the dark decreased the PSII activity to 70% and 50% of the initial levels in the wild type and hf-2, respectively, which implies that the PSII activity, in the absence of SQDG, becomes less stable under heat-stress conditions. PSII inactivated to 60% of the initial level by dark incubation at 41 degrees C was reactivated by following illumination even at 41 degrees C to more than 90% in the wild type, but only to 70% in hf-2. These results suggest that PSII inactivated by heat recovers through some mechanism dependent on light, and that SQDG participates in functioning of the mechanism. The conformational disorder of PSII caused by the defect in SQDG might be correlated with the increased susceptibility of its activity to heat-stress.     

Decrease in the efficiency of the electron donation to tyrosine Z of photosystem II in an SQDG-deficient mutant of Chlamydomonas

Photosystem (PS) II activity of a sulfoquinovosyl diacylglycerol (SQDG)-deficient mutant (hf-2) of Chlamydomonas was partially decreased compared with that of wild-type. The susceptibility to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modified in the mutant. Photometric measurements in the isolated thylakoid membranes of hf-2 revealed that the lowered activity in the mutant was derived from a decrease in the efficiency of the electron donation from water to tyrosine Z, not from the efficiency of the electron transport from Q(A) to Q(B). This result was confirmed by the decay kinetics of chlorophyll fluorescence determined in vivo. We conclude that SQDG contributes to maintaining the conformation of PSII complexes, particularly that of D1 polypeptides, which are necessary for maximum activities in Chlamydomonas.     

Anionic lipids are required for chloroplast structure and function in Arabidopsis

Photosynthetic membranes of plants primarily contain non-phosphorous glycolipids. The exception is phosphatidylglycerol (PG), which is an acidic/anionic phospholipid. A second major anionic lipid in chloroplasts is the sulfolipid sulfoquinovosyldiacylglycerol (SQDG). It is hypothesized that under severe phosphate limitation, SQDG substitutes for PG, ensuring a constant proportion of anionic lipids even under adverse conditions. A newly constructed SQDG and PG-deficient double mutant supports this hypothesis. This mutant, sqd2 pgp1-1, carries a T-DNA insertion in the structural gene for SQDG synthase (SQD2) and a point mutation in the structural gene for phosphatidylglycerolphosphate synthase (PGP1). In the sqd2 pgp1-1 double mutant, the fraction of total anionic lipids is reduced by approximately one-third, resulting in pale yellow cotyledons and leaves with reduced chlorophyll content. Photoautotrophic growth of the double mutant is severely compromised, and its photosynthetic capacity is impaired. In particular, photosynthetic electron transfer at the level of photosystem II (PSII) is affected. Besides these physiological changes, the mutant shows altered leaf structure, a reduced number of mesophyll cells, and ultrastructural changes of the chloroplasts. All observations on the sqd2 pgp1-1 mutant lead to the conclusion that the total content of anionic thylakoid lipids is limiting for chloroplast structure and function, and is critical for overall photoautotrophic growth and plant development.     

The lipid dependence of diadinoxanthin de-epoxidation presents new evidence for a macrodomain organization of the diatom thylakoid membrane

The present study shows that thylakoid membranes of the diatom Cyclotella meneghiniana contain much higher amounts of negatively charged lipids than higher plant or green algal thylakoids. Based on these findings, we examined the influence of SQDG on the de-epoxidation reaction of the diadinoxanthin cycle and compared it with results from the second negatively charged thylakoid lipid PG. SQDG and PG exhibited a lower capacity for the solubilization of the hydrophobic xanthophyll cycle pigment diadinoxanthin than the main membrane lipid MGDG. Although complete pigment solubilization took place at higher concentrations of the negatively charged lipids, SQDG and PG strongly suppressed the de-epoxidation of diadinoxanthin in artificial membrane systems. In in vitro assays employing the isolated diadinoxanthin cycle enzyme diadinoxanthin de-epoxidase, no or only a very weak de-epoxidation reaction was observed in the presence of SQDG or PG, respectively. In binary mixtures of the inverted hexagonal phase forming lipid MGDG with the negatively charged bilayer lipids, comparable suppression took place. This is in contrast to binary mixtures of MGDG with the neutral bilayer lipids DGDG and PC, where rapid and efficient de-epoxidation was observed. In complex lipid mixtures resembling the lipid composition of the native diatom thylakoid membrane, we again found strong suppression of diadinoxanthin de-epoxidation due to the presence of SQDG or PG. We conclude that, in the native thylakoids of diatoms, a strict separation of the MGDG and SQDG domains must occur; otherwise, the rapid diadinoxanthin de-epoxidation observed in intact cells upon illumination would not be possible.     

Role of sulfoquinovosyl diacylglycerol for the maintenance of photosystem II in Chlamydomonas reinhardtii.

The physiological role of sulfoquinovosyl diacylglycerol (SQDG) in photosynthesis was investigated with a SQDG defective mutant (hf-2) of Chlamydomonas reinhardtii that did not have any detectable amount of SQDG. The mutant showed a lower rate of photosystem II (PSII) activity by approximately 40% and also a lower growth rate than those of the wild-type. Results of genetical analysis of hf-2 strongly suggest that the SQDG defect and the lowered PSII activity are due to a single gene mutation. The supplementation of SQDG to hf-2 cells restored the lowered PSII activity to the same level as that of wild-type cells, and also enabled the mutant to grow even in the presence of 135 nm 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Moreover, the incubation of isolated thylakoid membranes of hf-2 with SQDG raised the lowered PSII activity. Chemical modifications of SQDG impaired the recovery of PSII activity. The results suggest that SQDG is indispensable for PSII activity in Chlamydomonas by maintaining PSII complexes in their proper state.     

Gross morphological changes in thylakoid membrane structure are associated with photosystem I deletion in Synechocystis sp. PCC 6803

Cells of Synechocystis sp. PCC 6803 lacking photosystem I (PSI-less) and containing only photosystem II (PSII) or lacking both photosystems I and II (PSI/PSII-less) were compared to wild type (WT) cells to investigate the role of the photosystems in the architecture, structure, and number of thylakoid membranes. All cells were grown at 0.5μmol photons m(-2)s(-1). The lumen of the thylakoid membranes of the WT cells grown at this low light intensity were inflated compared to cells grown at higher light intensity. Tubular as well as sheet-like thylakoid membranes were found in the PSI-less strain at all stages of development with organized regular arrays of phycobilisomes on the surface of the thylakoid membranes. Tubular structures were also found in the PSI/PSII-less strain, but these were smaller in diameter to those found in the PSI-less strain with what appeared to be a different internal structure and were less common. There were fewer and smaller thylakoid membrane sheets in the double mutant and the phycobilisomes were found on the surface in more disordered arrays. These differences in thylakoid membrane structure most likely reflect the altered composition of photosynthetic particles and distribution of other integral membrane proteins and their interaction with the lipid bilayer. These results suggest an important role for the presence of PSII in the formation of the highly ordered tubular structures.     

Effect of monogalactosyldiacylglycerol on the interaction between photosystem II core complex and its antenna complexes in liposomes of thylakoid lipids

The non-bilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant type of lipid in the thylakoid membrane and plays an important role in regulating the structure and function of photosynthetic membrane proteins. In this study, we have reconstituted the isolated major light-harvesting complexes of photosystem II (PSII) (LHCIIb) and a preparation consisting of PSII core complexes and minor LHCII of PSII (PSIICC) into liposomes that consisted of digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), with or without MGDG. Transmission electron microscopy and freeze-fracture studies showed unilamellar proteoliposomes, and demonstrated that most of the MGDG is incorporated into bilayer structures. The impact of MGDG on the functional interaction between LHCIIb and PSIICC was investigated by low temperature (77 K) fluorescence emission spectra and the photochemical activity of PSII. The additional incorporation of LHCIIb into liposomes containing PSIICC markedly increased oxygen evolution of PSIICC. Excitation at 480 nm of chlorophyll (Chl) b in LHCIIb stimulated a characteristic fluorescence emission of the Chl a in PSII (684.2 nm), rather than that of the Chl a in LHCIIb (680 nm) in the LHCIIb–PSIICC proteoliposomes, which indicated that the energy was transferred from LHCIIb to PSIICC in liposome membranes. Increasing the percentage of MGDG in the PSIICC–LHCIIb proteoliposomes enhanced the photochemical activity of PSII, due to a more efficient energy transfer from LHCIIb to PSIICC and, thus, an enlarged antenna cross section of PSII.     

Photosynthetic pigments, photosynthesis and plastid ultrastructure in RbcS antisense DNA mutants of tobacco (Nicotiana tabacum)

RbcS antisense DNA mutants of tobacco have reduced amounts of ribulose bisphosphate carboxylase oxygenase (Rubisco). We found that carotenoid and chlorophyll contents decrease in parallel as Rubisco is decreased, however, pigment levels are not significantly altered until Rubisco levels are reduced sharply. The mutants have normal Chl a/Chl b ratios and normal plastid ultrastructures, suggesting that reductions in Rubisco do not dramatically alter the composition of the thylakoid membranes. Nevertheless, chlorophyll fluorescence measurements, in which developmentally homogenous leaves were sampled, showed that there is reduced photosynthetic capacity of PSII and an enhanced photosensitivity in the mutants, especially in transgenics with severe reductions in Rubisco content. Support for this conclusion comes from several observations: 1) light saturation occurs at a lower light intensity in the mutants, resulting in an earlier closure of PS II (lower photochemical quenching); 2) the mutants have reduced photosynthetic efficiency (lower deltaF/Fm'); and 3) the mutants have a slower recovery of Fv/Fm. We found that acclimation to increasing light intensies in the mutants appears to involve an enhanced inactivation of PSII reaction centers as well as an increased activation of photoprotective mechanisms, notably an engagement of the xanthophyll cycle at lower than normal light intensities. We conclude that the photosensitivity of the antisense mutants is due, in part, to a limitation in Rubisco activation state.     

Detection of oligomerisation and substrate recognition sites of small heat shock proteins by peptide arrays

Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central alpha-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS(191-205)) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.     

Site-directed mutations within the core "alpha-crystallin" domain of the small heat-shock protein, human alphaB-crystallin, decrease molecular chaperone functions.

Site-directed mutagenesis was used to evaluate the effects on structure and function of selected substitutions within and N-terminal to the core "alpha-crystallin" domain of the small heat-shock protein (sHsp) and molecular chaperone, human alphaB-crystallin. Five alphaB-crystallin mutants containing single amino acid substitutions within the core alpha-crystallin domain displayed a modest decrease in chaperone activity in aggregation assays in vitro and in protecting cell viability of E. coli at 50 degrees C in vivo. In contrast, seven alphaB-crystallin mutants containing substitutions N-terminal to the core alpha-crystallin domain generally resembled wild-type alphaB-crystallin in chaperone activity in vitro and in vivo. Size-exclusion chromatography, ultraviolet circular dichroism spectroscopy and limited proteolysis were used to evaluate potential structural changes in the 12 alphaB-crystallin mutants. The secondary, tertiary and quaternary structures of mutants within and N-terminal to the core alpha-crystallin domain were similar to wild-type alphaB-crystallin. SDS-PAGE patterns of chymotryptic digestion were also similar in the mutant and wild-type proteins, indicating that the mutations did not introduce structural modifications that altered the exposure of proteolytic cleavage sites in alphaB-crystallin. On the basis of the similarities between the sequences of human alphaB-crystallin and the sHsp Mj HSP16.5, the only sHsp for which there exists high resolution structural information, a three-dimensional model for alphaB-crystallin was constructed. The mutations at sites within the core alpha-crystallin domain of alphaB-crystallin identify regions that may be important for the molecular chaperone functions of sHsps.     

Structural and functional consequences of galactolipids on thylakoid membrane organization

Photosynthetic membranes of higher plant chloroplasts are composed primarily of polar, but uncharged, galactolipids unlike most mammalian membranes which contain large amounts of phosphatidylcholine. It is unclear what role(s) the galactolipids play in maintaining the differentiated thylakoid membranes, or in stabilizing the photosynthetically active enzyme complexes. Some of the membrane complexes show no lipid selectivity for maintaining structural or functional integrity. Others are poisoned or dissociated in the presence of high concentrations of a trace lipid class. The efficiency of energy transfer and the reconstitution of protein complexes into liposomes are dependent on the lipid class employed. The lipids are asymmetrically arranged along and across the thylakoid membranes but not as distinctly as the proteins.Abbreviations DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SQDG sulfoquinovosyldiglyceride - PG phosphatidylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PSI photosystem I - PSII photosystem II - LHC chlorophylla/b lightharvesting complex - cytb ₆ f cytochromeb ₆ f complex - CF₀/CF₁ coupling factor ATPase - DCIP 2,6-dichlorophenolindophenol - LRa galactolipase fromRhizopus arrhis     

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.     

Conformational changes resulting from pseudophosphorylation of mammalian small heat shock proteins--a two-hybrid study

The human genome codes for 10 so-called mammalian small heat shock or stress proteins (sHsp) with the various tissues expressing characteristic sets of sHsps. Most sHsps interact with each other and form homo- and heterooligomeric complexes. Some of the sHsps are phosphoproteins in vivo, and phosphorylation has been implicated in the regulation of complex size and composition. In this study, we analyze, by the 2-hybrid method, the reporter gene activation pattern of several sHsp pairs that previously have been demonstrated to interact. We show that pseudophosphorylation (mimicry of phosphorylation) of the homologous phosphorylation sites Ser15 and Ser16 in Hsp27 and Hsp20, respectively, modulates characteristics of these sHsps that can be detected by their ability to activate reporter genes in suitable 2-hybrid assays. Pseudophosphorylation of the separated N-terminus of Hsp27 alone is not sufficient for the activation of the reporter genes, whereas the separated C-terminus is sufficient. We conclude that pseudophosphorylation of Hsp27 and Hsp20 at their N-termini results in conformational changes that can be detected by their interaction with other sHsps. Pseudophosphorylation of alphaB-crystallin at Ser19, in contrast, had no detectable consequences.     

Influence of thylakoid membrane lipids on the structure and function of the plant photosystem II core complex

Main conclusion

MGDG leads to a dimerization of isolated, monomeric PSII core complexes. SQDG and PG induce a detachment of CP43 from the PSII core, thereby disturbing the intrinsic PSII electron transport. The influence of the four thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure and function of isolated monomeric photosystem (PS) II core complexes was investigated. Incubation with the negatively charged lipids SQDG and PG led to a loss of the long-wavelength 77 K fluorescence emission at 693 nm that is associated with the inner antenna proteins. The neutral galactolipids DGDG and MGDG had no or only minor effects on the fluorescence emission spectra of the PSII core complexes, respectively. Pigment analysis, absorption and 77 K fluorescence excitation spectroscopy showed that incubation with SQDG and PG led to an exposure of chlorophyll molecules to the surrounding medium followed by conversion to pheophytin under acidic conditions. Size-exclusion chromatography and polypeptide analysis corroborated the findings of the spectroscopic measurements and pigment analysis. They showed that the negatively charged lipid SQDG led to a dissociation of the inner antenna protein CP43 and the 27- and 25-kDa apoproteins of the light-harvesting complex II, that were also associated with a part of the PSII core complexes used in the present study. Incubation of PSII core complexes with MGDG, on the other hand, induced an almost complete dimerization of the monomeric PSII. Measurements of the fast PSII fluorescence induction demonstrated that MGDG and DGDG only had a minor influence on the reduction kinetics of plastoquinone Q_A and the artificial PSII electron acceptor 2,5-dimethyl-p-benzoquinone (DMBQ). SQDG and, to a lesser extent, PG perturbed the intrinsic PSII electron transport significantly.     

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.