Structural changes of the thylakoid membrane network induced by high light stress in plant chloroplasts

Land plants live in a challenging environment dominated by unpredictable changes. A particular problem is fluctuation in sunlight intensity that can cause irreversible damage of components of the photosynthetic apparatus in thylakoid membranes under high light conditions. Although a battery of photoprotective mechanisms minimize damage, photoinhibition of the photosystem II (PSII) complex occurs. Plants have evolved a multi-step PSII repair cycle that allows efficient recovery from photooxidative PSII damage. An important feature of the repair cycle is its subcompartmentalization to stacked grana thylakoids and unstacked thylakoid regions. Thus, understanding the crosstalk between stacked and unstacked thylakoid membranes is essential to understand the PSII repair cycle. This review summarizes recent progress in our understanding of high-light-induced structural changes of the thylakoid membrane system and correlates these changes to the efficiency of the PSII repair cycle. The role of reversible protein phosphorylation for structural alterations is discussed. It turns out that dynamic changes in thylakoid membrane architecture triggered by high light exposure are central for efficient repair of PSII.     

Structural variability of plant photosystem II megacomplexes in thylakoid membranes

Plant photosystem II (PSII) is organized into large supercomplexes with variable levels of membrane‐bound light‐harvesting proteins (LHCIIs). The largest stable form of the PSII supercomplex involves four LHCII trimers, which are specifically connected to the PSII core dimer via monomeric antenna proteins. The PSII supercomplexes can further interact in the thylakoid membrane, forming PSII megacomplexes. So far, only megacomplexes consisting of two PSII supercomplexes associated in parallel have been observed. Here we show that the forms of PSII megacomplexes can be much more variable. We performed single particle electron microscopy (EM) analysis of PSII megacomplexes isolated from Arabidopsis thaliana using clear‐native polyacrylamide gel electrophoresis. Extensive image analysis of a large data set revealed that besides the known PSII megacomplexes, there are distinct groups of megacomplexes with non‐parallel association of supercomplexes. In some of them, we have found additional LHCII trimers, which appear to stabilize the non‐parallel assemblies. We also performed EM analysis of the PSII supercomplexes on the level of whole grana membranes and successfully identified several types of megacomplexes, including those with non‐parallel supercomplexes, which strongly supports their natural origin. Our data demonstrate a remarkable ability of plant PSII to form various larger assemblies, which may control photochemical usage of absorbed light energy in plants in a changing environment.     

Towards elucidation of dynamic structural changes of plant thylakoid architecture

Long-term acclimation of shade versus sun plants modulates the composition, function and structural organization of the architecture of the thylakoid membrane network. Significantly, these changes in the macroscopic structural organization of shade and sun plant chloroplasts during long-term acclimation are also mimicked following rapid transitions in irradiance: reversible ultrastructural changes in the entire thylakoid membrane network increase the number of grana per chloroplast, but decrease the number of stacked thylakoids per granum in seconds to minutes in leaves. It is proposed that these dynamic changes depend on reversible macro-reorganization of some light-harvesting complex IIb and photosystem II supracomplexes within the plant thylakoid network owing to differential phosphorylation cycles and other biochemical changes known to ensure flexibility in photosynthetic function in vivo. Some lingering grana enigmas remain: elucidation of the mechanisms involved in the dynamic architecture of the thylakoid membrane network under fluctuating irradiance and its implications for function merit extensive further studies.     

The grana margins of plant thylakoid membranes

Plant thylakoid membranes contain three structurally distinct domains: the planar appressed membranes of the grana; the planar non-appressed stroma thylakoids; and the highly curved, non-appressed margins of the grana. Evidence is presented to suggest that the grana margins form a significant structural domain, which has hitherto been neglected. If indeed the grana margins contain some of the cytochrome b/f complex, photosystem (PS) I complex and ATP synthase, they form a third functional domain of the laterally heterogeneous continuous thylakoid membrane network. The consequences of grana margins containing complexes are explored with respect to linear electron transport under light-saturating and light-limiting conditions, non-cyclic vs cyclic photophorylation, and the regulation of light energy distribution to both PS I and PS II.     

Lateral heterogeneity of plant thylakoid protein complexes: early reminiscences

The concept that the two photosystems of photosynthesis cooperate in series, immortalized in Hill and Bendall''s Z scheme, was still a black box that defined neither the structural nor the molecular organization of the thylakoid membrane network into grana and stroma thylakoids. The differentiation of the continuous thylakoid membrane into stacked grana thylakoids interconnected by single stroma thylakoids is a morphological reflection of the non-random distribution of photosystem II/light-harvesting complex of photosystem II, photosystem I and ATP synthase, which became known as lateral heterogeneity.     

Counter-current distribution of sonicated inside-out thylakoid vesicles

Summary Inside-out thylakoid vesicles were isolated from spinach chloroplasts, and fragmented by sonication. Different fragments were separated by counter-current distribution and analyzed for chlorophyll and P700. The inside-out vesicles had a chlorophyll a/b ratio of 2.2–2.4 (original chloroplasts 2.8–3.0). After further fragmentation of the inside-out vesicles by sonication and separation by countercurrent distribution three populations of vesicles were obtained having chlorophyll a/b ratios of 1.7, 1.9 and 2.5 respectively. The P-700 was depleted in fractions with lower chlorophyll a/b ratio and was nearly absent in the fraction having a chlorophyll a/b ratio of 1.7 (chlorophyll/P700 > 4500 mol/mol). That PSII membrane vesicles, with such a low chlorophyll a/b ratio and lacking PSI, can be prepared by a non-detergent method provides strong support for the notion that PSI and PSII are segregated along the thylakoid membrane.A plot of P700 per chlorophyll against chlorophyll b/(a+b) fits a straight line connecting the pure PSI membrane (chlorophyll a/b = 6; P700/chlorophyll = 5.6 mmol/mol) with the pure PSII membrane (chlorophyll a/b = 1.7; P700 = 0). These two membranes can be considered as separate phases of a two-dimensional phase system. Models for the thylakoid membrane are discussed.Abbreviations PSI Photosystem I - PSII Photosystem II - PEG Polyethylene Glycol - P700 Reaction Center of PSI     

Expression of 5-amino levulinic acid induced photodynamic damage to the thylakoid membranes in dark sensitized by brief pre-illumination

The 5-amino levulinic acid treated cucumber (Cucumis sativus L., CV. Pointsette) plants upon exposure to light (≃30,000 lux) wilted within 6 h and died after 36 h due to photodynamic reactions. Thylakoid membranes, the site of accumulation of porphyrins, were damaged due to photodynamic reactions leading to the inhibition of membrane linked functions of photosystem II, photosystem I and the whole chain electron transport. Photosystem II was more susceptible to photodynamic damage than photosystem I. The exogenous electron donors Mn²⁺, diphenyl carbazide and NH₂OH failed to donate electrons to photosystem II suggesting that the damage has taken place close to P680. The 5-amino levulinic acid treated plants exposed to 30 min of light did not show any damage to the thylakoid membranes. However, when the above plants were transferred to dark for 12 h there was substantial damage to the thylakoid membrane system.     

The Arabidopsis thylakoid transporter PHT4;1 influences phosphate availability for ATP synthesis and plant growth

The Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high‐phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO₂ fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non‐photochemical quenching than the wild type, in line with the increased contribution of ΔpH to the proton‐motive force across thylakoids. Small‐angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long‐range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild‐type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro‐organization of complexes and induction of photoprotective mechanisms.     

The use of polyclonal antibodies to identify peptides exposed on the stroma side of the spinach thylakoid

We have raised polyclonal antibodies against an oxygen-evolving photosystem II preparation. Western Blot analysis of the whole serum revaals antibodies specific for at least 15 Coomassie visible bands ranging from 59 to 11 kDa. These antibodies are specific for proteins located on both sides of the membrane. Included are antibodies specific for Tris-removable peptides (33, 25 and 18 kda), which are thought to be exposed on the lumen surface of the PS II complex. Since the whole serum agglutinates thylakoids, antibodies specific for the stroma side of the PS II complex are also present. A sub-population of antibodies can be isolated by allowing the antibodies in whole serum to bind to EDTA-treated thylakoid membranes. The antibodies which specifically bind are cross-reactive with peptides with Mr of 59, 57, 34, 28, 27, 26, and 23 kDa. Our data indicate that these peptides have antigenic determinants exposed on the stroma side of the thylakoid membrane.     

The action of lipases on chloroplast membranes I. The release of plastocyanin from galactolipase-treated thylakoid membranes

Bean chloroplasts treated with galactolipase (lipolytic acyl hydrolase) isolated from bean leaves showed an inhibition of photosystem I activity as measured by methyl viologen-mediated oxygen uptake and NADP⁺ photoreduction. This inhibition was partially reversed by exogenous plastocyanin added to galactolipase-treated thylakoid membranes. Galactolipase released substantial amounts of endogenous plastocyanin (about 40%) from bean chloroplasts. The results are discussed with regard to the localization of plastocyanin in thylakoid membranes.Abbreviations chlf chlorophyll - DCMU 3-(2,4-dichlorophenyl)-1,1-dimethylurea - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - MV methyl viologen - NADP⁺ nicotinamide dinucleotide phosphate - PC phosphatidylcholine - PG phosphatidylglycerol - PE phosphatidylethanolamine - PI phosphatidylinositol - SQDG sulphoquinovosyldiacylglycerol - SDS sodium dodecyl sulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethane     

Influence of structural and physical properties of the thylakoid membrane on QA - oxidation

Using isolated pea thylakoids, the relative rate of Q_A ^- oxidation has been estimated under various conditions, from the restoration of the induction curves following a dark period and from light 1-induced changes in modulated chlorophyll fluorescence excited by light 2.Alterations of _QinfA ^sup– oxidation rates were observed under conditions which affected the degree of thylakoid stacking, the lipid fluidity and the integrity of the membranes. The results are discussed in terms of the interactions between Q_A ^- and the plastoquinone pool with particular emphasis on lateral diffusion.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA Ethylenediaminetetracetate - Hepes N-2-hydroxyethyl piperazine-N-2-ethanesulphonic acid - NADP nicotinamide adenine dinucleotide phosphate     

Identification and characterization of a stable intermediate in photosystem I assembly in tobacco

Photosystem I (PSI) is the most efficient bioenergetic nanomachine in nature and one of the largest membrane protein complexes known. It is composed of 18 protein subunits that bind more than 200 co‐factors and prosthetic groups. While the structure and function of PSI have been studied in great detail, very little is known about the PSI assembly process. In this work, we have characterized a PSI assembly intermediate in tobacco plants, which we named PSI*. We found PSI* to contain only a specific subset of the core subunits of PSI. PSI* is particularly abundant in young leaves where active thylakoid biogenesis takes place. Moreover, PSI* was found to overaccumulate in PsaF‐deficient mutant plants, and we show that re‐initiation of PsaF synthesis promotes the maturation of PSI* into PSI. The attachment of antenna proteins to PSI also requires the transition from PSI* to mature PSI. Our data could provide a biochemical entry point into the challenging investigation of PSI biogenesis and allow us to improve the model for the assembly pathway of PSI in thylakoid membranes of vascular plants.     

The case for chloroplast thylakoid carbonic anhydrase

Washed thylakoid membranes and photosystem II-enriched membrane fragments from cyanobacteria, green algae, and chloroplasts from both C₃ and C₄ plants possess the ability to reversibly hydrate CO₂. That is, the membranes have an intrinsic carbonic anhydrase activity. The present review outlines the discovery of thylakoid carbonic anhydrase and presents the evidence that it is a unique isozyme, distinct from other cellular carbonic anhydrases. It appears that at least some thylakoid carbonic anhydrase is closely associated with photosystem II and may be required for electron transport. This would explain why all inhibitors of carbonic anhydrase also inhibit photosystem II. Several speculative functions of thylakoid carbonic anhydrase are discussed. These include a possible role in carbon metabolism, in the protonation of plastoquinone, and/or in oxygen evolution.     

Grana stacking and protection of Photosystem II in thylakoid membranes of higher plant leaves under sustained high irradiance: An hypothesis

We propose yet another function for the unique appressed thylakoids of grana stacks of higher plants, namely that during prolonged high light, the non-functional, photoinhibited PS II centres accumulate as D1 protein degradation is prevented and may act as dissipative conduits to protect other functional PS II centres. The need for this photoprotective mechanism to prevent high D1 protein turnover under excess photons in higher plants, especially those grown in shade, is due to conflicting demands between efficient use of low irradiance and protection from periodic exposure to excessive irradiance.     

A current assessment of photosystem II structure

This review covers the recent progress in the elucidation of the structure of photosystem II (PSII). Because much of the structural information for this membrane protein complex has been revealed by electron microscopy (EM), the review will also consider the specific technical and interpretation problems that arise with EM where they are of particular relevance to the structural data. Most recent reviews of photosystem II structure have concentrated on molecular studies of the PSII genes and on the likely roles of the subunits that they encode or they were mainly concerned with the biophysical data and fast absorption spectroscopy largely relating to electron transfer in various purified PSII preparations. In this review, we will focus on the approaches to the three-dimensional architecture of the complex and the lipid bilayer in which it is located (the thylakoid membrane) with special emphasis placed upon electron microscopical studies of PSII-containing thylakoid membranes. There are a few reports of 3D crystals of PSII and of associated X-ray diffraction measurements and although little structural information has so far been obtained from such studies (because of the lack of 3D crystals of sufficient quality), the prospects for such studies are also assessed.Abbreviations ATP adenosine triphosphate - Chl chlorophyll - CP chlorophyll-binding protein - EM electron microscopy - LHC light harvesting complex - NADP nicotinamide adenine dinucleotide phosphate - OEC oxygen evolution enhancing complex - PS photosystem - Tris tris-hydroxymethyl aminomethane     

The molecular anatomy and function of thylakoid proteins

Abstract. The structure of chloroplast membrane proteins and their organization into photosynthetically-active multimeric complexes is described. Extensive use has been made of information derived from gene sequencing and other biochemical studies to predict likely protein conformations. These predictions have been assimilated into structural models of the various thylakoid complexes. The enzymatic activities of the complexes have also been described and where possible related to individual polypeptides.     

Functional organization of thylakoid membranes in viable pea mutants with low chlorophyll content

The functional organizations of thylakoid membranes from wild type pea ( Pisum sativum L. cv. Kapital) and two viable mutants with low chlorophyll (Chl) contents were compared. Nuclear mutations in mutants 7 and 42 led to two- and three-fold decrease in total chlorophyll content, respectively. In spite of low Chl content mutants showed 80% photosynthetic activity, biological productivity, and seed production. It has been shown that mutant membranes differed from that of wild type by Chl distribution between the pigment-protein complexes and by stoichiometry of the main electrontransport complexes. The ratio photosystem I (PSI): photosystem II (PSII): cytochrome (Cyt) bjf complex: Chl was 1:1.1:1.2:650 in wild type chloroplasts, 1:1.8:1.7:600 in mutant 7 , and 1:1.5:1.9:350 in mutant 42 . PSI- and PSII-dependent electron-transport activities were enhanced in the mutants per mg Chl in proportion to number of reaction centers. The activity of the non-cyclic electron-transport chain increased in proportion to PSII and Cyt bjf complexes. The amount of ATP synthetase per unit of Chl as estimated by H⁻ATPase activity was much greater in mutant thylakoids, which is favorable for photosynthetic energy transduction. The low content of the light-harvesting complexes (LHC) in mutants is compensated by an increase of the number of PSII and Cyt bjf complexes, which eliminates the bottleneck at the site of plastoquinone oxidation.     

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes*

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

The phase behavior of lipids in photosynthetic membranes

The phase behavior of the main classes of polar lipids found in the photosynthetic membranes of higher plants and algae is reviewed and compared to that of binary lipid mixtures and total lipid extracts of such membranes. Particular interest is paid to the way in which factors such as temperature and acyl chain saturation influence the phase behavior of these lipids and the implications this has in terms of the ability of photosynthetic membranes to resist environmental stress.     

Light‐harvesting superstructures of green plant chloroplasts lacking photosystems

The light‐harvesting antenna of higher plant photosystem II (LHCII) is the major photosynthetic membrane component encoded by an entire family of homologous nuclear genes. On the contrary, the great majority of proteins of photosystems and electron transport components are encoded by the chloroplast genome. In this work, we succeeded in gradually inhibiting the expression of the chloroplast genes that led to the disappearance of the photosystem complexes, mimicking almost total photoinhibition. The treated plants, despite displaying only some early signs of senescence, sustained their metabolism and growth for several weeks. The only major remaining membrane component was LHCII antenna that formed superstructures – stacks of dozens of thylakoids or supergrana. Freeze‐fracture electron microscopy revealed specific organization, directly displaying frequently bifurcated membranes with reduced or totally absent photosystem II (PSII) reaction centre complexes. Our findings show that it is possible to accumulate large amounts of light‐harvesting membranes, organized into three‐dimensional structures, in the absence of reaction centre complexes. This points to the reciprocal role of LHCII and PSII in self‐assembly of the three‐dimensional matrix of the photosynthetic membrane, dictating its size and flexible adaptation to the light environment.