Helmut Kirchhoff

Dynamic control of protein diffusion within the granal thylakoid lumen

The machinery that conducts the light-driven reactions of oxygenic photosynthesis is hosted within specialized paired membranes called thylakoids. In higher plants, the thylakoids are segregated into two morphological and functional domains called grana and stroma lamellae. A large fraction of the luminal volume of the granal thylakoids is occupied by the oxygen-evolving complex of photosystem II. Electron microscopy data we obtained on dark- and light-adapted Arabidopsis thylakoids indicate that the granal thylakoid lumen significantly expands in the light. Models generated for the organization of the oxygen-evolving complex within the granal lumen predict that the light-induced expansion greatly alleviates restrictions imposed on protein diffusion in this compartment in the dark. Experiments monitoring the redox kinetics of the luminal electron carrier plastocyanin support this prediction. The impact of the increase in protein mobility within the granal luminal compartment in the light on photosynthetic electron transport rates and processes associated with the repair of photodamaged photosystem II complexes is discussed.

Protein Diffusion and Macromolecular Crowding in Thylakoid Membranes

The photosynthetic light reactions of green plants are mediated by chlorophyll-binding protein complexes located in the thylakoid membranes within the chloroplasts. Thylakoid membranes have a complex structure, with lateral segregation of protein complexes into distinct membrane regions known as the grana and the stroma lamellae. It has long been clear that some protein complexes can diffuse between the grana and the stroma lamellae, and that this movement is important for processes including membrane biogenesis, regulation of light harvesting, and turnover and repair of the photosynthetic complexes. In the grana membranes, diffusion may be problematic because the protein complexes are very densely packed (approximately 75% area occupation) and semicrystalline protein arrays are often observed. To date, direct measurements of protein diffusion in green plant thylakoids have been lacking. We have developed a form of fluorescence recovery after photobleaching that allows direct measurement of the diffusion of chlorophyll-protein complexes in isolated grana membranes from Spinacia oleracea. We show that about 75% of fluorophores are immobile within our measuring period of a few minutes. We suggest that this immobility is due to a protein network covering a whole grana disc. However, the remaining fraction is surprisingly mobile (diffusion coefficient 4.6 ± 0.4 × 10−11 cm2 s−1), which suggests that it is associated with mobile proteins that exchange between the grana and stroma lamellae within a few seconds. Manipulation of the protein-lipid ratio and the ionic strength of the buffer reveals the roles of macromolecular crowding and protein-protein interactions in restricting the mobility of grana proteins.

Architectural switch in plant photosynthetic membranes induced by light stress

Unavoidable side reactions of photosynthetic energy conversion can damage the water-splitting photosystem II (PSII) holocomplex embedded in the thylakoid membrane system inside chloroplasts. Plant survival is crucially dependent on an efficient molecular repair of damaged PSII realized by a multistep repair cycle. The PSII repair cycle requires a brisk lateral protein traffic between stacked grana thylakoids and unstacked stroma lamellae that is challenged by the tight stacking and low protein mobility in grana. We demonstrated that high light stress induced two main structural changes that work synergistically to improve the accessibility between damaged PSII in grana and its repair machinery in stroma lamellae: lateral shrinkage of grana diameter and increased protein mobility in grana thylakoids. It follows that high light stress triggers an architectural switch of the thylakoid network that is advantageous for swift protein repair. Studies of the thylakoid kinase mutant stn8 and the double mutant stn7/8 demonstrate the central role of protein phosphorylation for the structural alterations. These findings are based on the elaboration of mathematical tools for analyzing confocal laser-scanning microscopic images to study changes in the sophisticated thylakoid architecture in intact protoplasts.

The Role of Plastocyanin in the Adjustment of the Photosynthetic Electron Transport to the Carbon Metabolism in Tobacco

We investigated adaptive responses of the photosynthetic electron transport to a decline in the carbon assimilation capacity. Leaves of different ages from wild-type tobacco (Nicotiana tabacum) L. var Samsun NN and young mature leaves of tobacco transformants with impaired photoassimilate export were used. The assimilation rate decreased from 280 in young mature wild-type leaves to below 50 mmol electrons mol chlorophyll−1 s−1 in older wild-type leaves or in transformants. The electron transport capacity, measured in thylakoids isolated from the different leaves, closely matched the leaf assimilation rate. The numbers of cytochrome (cyt)-bf complexes and plastocyanin (PC) decreased with the electron transport and assimilation capacity, while the numbers of photosystem I (PSI), photosystem II, and plastoquinone remained constant. The PC to PSI ratio decreased from five in leaves with high assimilation rates, to values below one in leaves with low assimilation rates, and the PC versus flux correlation was strictly proportional. Redox kinetics of cyt-f, PC, and P700 suggest that in leaves with low electron fluxes, PC is out of the equilibrium with P700 and cyt-f and the cyt-f reoxidation rate is restricted. It is concluded that the electron flux is sensitive to variations in the number of PC, relative to PSI and cyt-bf, and PC, in concert with cyt-bf, is a key component that adjusts to control the electron transport rate. PC dependent flux control may serve to adjust the electron transport rate under conditions where the carbon assimilation is diminished and thereby protects PSI against over-reduction and reactive oxygen production.

Molecular crowding and order in photosynthetic membranes.

The integrity and maintenance of the photosynthetic apparatus in thylakoid membranes of higher plants requires lateral mobility of their components between stacked grana thylakoids and unstacked stroma lamellae. Computer simulations based on realistic protein densities suggest serious problems for lateral protein and plastoquinone diffusion especially in grana membranes, owing to strong retardation by protein complexes. It has been suggested that three structural features of grana thylakoids ensure efficient lateral transport: the organization of protein complexes into supercomplexes; the arrangement of supercomplexes into structured assemblies, which facilitates diffusion process in crowded membranes; the limitation of the diameter of grana discs to less than approximately 500 nm, which keeps diffusion times short enough to support regulation of light harvesting and repair of photodamaged photosystem II.

Efficient Light Harvesting by Photosystem II Requires an Optimized Protein Packing Density in Grana Thylakoids

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Aggregation and fluorescence quenching of chlorophyll a of the light-harvesting complex II from spinach in vitro

The salt-induced aggregation of the light-harvesting complex (LHC) II isolated from spinach and its correlation with fluorescence quenching of chlorophyll a is reported. Two transitions with distinctly different properties were observed. One transition related to salt-induced fluorescence quenching takes place at low salt concentration and is dependent both on temperature and detergent concentration. This transition seems to be related to a change in the lateral microorganization of LHCII. The second transition occurs at higher salt concentration and involves aggregation. It is independent of temperature and of detergent at sub-cmc concentrations. During the latter transition the small LHCII sheets (approximately 100 nm in diameter) are stacked to form larger aggregates of approximately 3 microm diameter. Based on the comparison between the physical properties of the transition and theoretical models, direct and specific binding of cations can practically be ruled out as driving force for the aggregation. It seems that in vitro aggregation of LHCII is caused by a complex mixture of different effects such as dielectric and electrostatic properties of the solution and surface charges.

Architectural switches in plant thylakoid membranes

Recent progress in elucidating the structure of higher plants photosynthetic membranes provides a wealth of information. It allows generation of architectural models that reveal well-organized and complex arrangements not only on whole membrane level, but also on the supramolecular level. These arrangements are not static but highly responsive to the environment. Knowledge about the interdependency between dynamic structural features of the photosynthetic machinery and the functionality of energy conversion is central to understanding the plasticity of photosynthesis in an ever-changing environment. This review summarizes the architectural switches that are realized in thylakoid membranes of green plants.

Diffusion of molecules and macromolecules in thylakoid membranes

The survival and fitness of photosynthetic organisms is critically dependent on the flexible response of the photosynthetic machinery, harbored in thylakoid membranes, to environmental changes. A central element of this flexibility is the lateral diffusion of membrane components along the membrane plane. As demonstrated, almost all functions of photosynthetic energy conversion are dependent on lateral diffusion. The mobility of both small molecules (plastoquinone, xanthophylls) as well as large protein supercomplexes is very sensitive to changes in structural boundary conditions. Knowledge about the design principles that govern the mobility of photosynthetic membrane components is essential to understand the dynamic response of the photosynthetic machinery. This review summarizes our knowledge about the factors that control diffusion in thylakoid membranes and bridges structural membrane alterations to changes in mobility and function. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.

Compartmentalization of the protein repair machinery in photosynthetic membranes

Significance The fitness and robustness of plants crucially depend on the molecular repair of the vulnerable photosystem II (PS II) supercomplex, embedded in photosynthetic thylakoid membranes. To maintain photosynthetic performance, plants evolved an efficient multistep PS II repair cycle. The PS II repair cycle relies on a well-defined order of reactions and partial separation of individual repair steps. By combining biochemical, spectroscopic, and ultrastructural techniques, we discover that plants establish reaction order and separation by confinement of the enzymes that catalyze the individual steps to spatially separated thylakoid subcompartments—grana, grana margins, and stroma lamellae—formed by the stacked membranes. Structural flexibility of the thylakoid architecture allows controlled access of the damaged PS II by the repair machinery. A crucial component of protein homeostasis in cells is the repair of damaged proteins. The repair of oxygen-evolving photosystem II (PS II) supercomplexes in plant chloroplasts is a prime example of a very efficient repair process that evolved in response to the high vulnerability of PS II to photooxidative damage, exacerbated by high-light (HL) stress. Significant progress in recent years has unraveled individual components and steps that constitute the PS II repair machinery, which is embedded in the thylakoid membrane system inside chloroplasts. However, an open question is how a certain order of these repair steps is established and how unwanted back-reactions that jeopardize the repair efficiency are avoided. Here, we report that spatial separation of key enzymes involved in PS II repair is realized by subcompartmentalization of the thylakoid membrane, accomplished by the formation of stacked grana membranes. The spatial segregation of kinases, phosphatases, proteases, and ribosomes ensures a certain order of events with minimal mutual interference. The margins of the grana turn out to be the site of protein degradation, well separated from active PS II in grana core and de novo protein synthesis in unstacked stroma lamellae. Furthermore, HL induces a partial conversion of stacked grana core to grana margin, which leads to a controlled access of proteases to PS II. Our study suggests that the origin of grana in evolution ensures high repair efficiency, which is essential for PS II homeostasis.