Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

Further studies of the relationship between cation-induced chlorophyll fluorescence and thylakoid membrane stacking changes

Salt-induced changes in thylakoid stacking and chlorophyll fluorescence do not occur with granal membranes obtained by treatment of stacked thylakoids with digitonin. In contrast to normal untreated thylakoids, digitonin prepared granal membranes remain stacked under all ionic conditions and exhibit a constant high level of chlorophyll fluorescence. However, unstacking of these granal membranes is possible if they are pretreated with either acetic anhydride or linolenic acid.Trypsin treatment of the thylakoids inhibits the salt induced chlorophyll fluorescence and stacking changes but stacking of these treated membranes does occur when the pH is lowered, with the optimum being at about pH 4.5. This type of stacking is due to charge neutralization and does not require the presence of the 2000 dalton fragment of the polypeptide associated with the $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b}$ light harvesting complex and known to be lost during treatment with trypsin (Mullet, J.E. and Arntzen, C.J. (1980) Biochim. Biophys. Acta 589, 100–117).Using the method of 9-aminoacridine fluorescence quenching it is argued that the surface charge density, on a chlorophyll basis, of unstacked thylakoid membranes is intermediate between digitonin derived granal and stromal membranes, with granal having the lowest value.The results are discussed in terms of the importance of surface negative charges in controlling salt induced chlorophyll fluorescence and thylakoid stacking changes. In particular, emphasis is placed on a model involving lateral diffusion of different types of chlorophyll protein complex within the thylakoid lipid matrix.     

Acclimation of leaves to low light produces large grana: the origin of the predominant attractive force at work

Photosynthetic membrane sacs (thylakoids) of plants form granal stacks interconnected by non-stacked thylakoids, thereby being able to fine-tune (i) photosynthesis, (ii) photoprotection and (iii) acclimation to the environment. Growth in low light leads to the formation of large grana, which sometimes contain as many as 160 thylakoids. The net surface charge of thylakoid membranes is negative, even in low-light-grown plants; so an attractive force is required to overcome the electrostatic repulsion. The theoretical van der Waals attraction is, however, at least 20-fold too small to play the role. We determined the enthalpy change, in the spontaneous stacking of previously unstacked thylakoids in the dark on addition of Mg²⁺, to be zero or marginally positive (endothermic). The Gibbs free-energy change for the spontaneous process is necessarily negative, a requirement that can be met only by an increase in entropy for an endothermic process. We conclude that the dominant attractive force in thylakoid stacking is entropy-driven. Several mechanisms for increasing entropy upon stacking of thylakoid membranes in the dark, particularly in low-light plants, are discussed. In the light, which drives the chloroplast far away from equilibrium, granal stacking accelerates non-cyclic photophosphorylation, possibly enhancing the rate at which entropy is produced.     

Insights into the complex 3-D architecture of thylakoid membranes in the unicellular cyanobacterium Cyanothece sp. ATCC 51142

In cyanobacteria and chloroplasts, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. In plant chloroplasts, thylakoids are differentiated into a highly interconnected system of stacked grana and unstacked stroma membranes. In contrast, in cyanobacteria, the evolutionary progenitors of chloroplasts, thylakoids do not routinely form stacked and unstacked regions, and the architecture of the thylakoid membrane systems is only now being described in detail in these organisms. We used electron tomography to examine the thylakoid membrane systems in one cyanobacterium, Cyanothece sp. ATCC 51142. Our data showed that thylakoids form a complicated branched network with a rudimentary quasi-helical architecture in this organism. A well accepted helical model of grana-stroma architecture of plant thylakoids describes an organization in which stroma thylakoids wind around stacked granum in right-handed spirals. Here we present data showing that the simplified helical architecture in Cyanothece 51142 is lefthanded in nature. We propose a model comparing the thylakoid membranes in plants and this cyanobacterium in which the system in Cyanothece 51142 is composed of non-stacked membranes linked by fret-like connections to other membrane components of the system in a limited left-handed arrangement.Key words: cyanobacteria, Cyanothece 51142, thylakoid membrane, electron tomography, chloroplast     

Thylakoid and grana formation during the development of pea chloroplasts,illuminated by white,red, and blue low intensity light

Summary We analyzed the formation of thylakoids and grana during the development of pea chloroplasts, illuminated by white, red and blue low intensity light. The total length of granal and intergranal thylakoids, and the length of granal thylakoids per unit area of plastid section were measured. Initially the greatest increase in length of granal thylakoids and the highest incidence of grana with large thylakoid content occurred in red light. On the other hand, with illumination times of over 12 hours blue light appeared to be more efficient in stimulating grana formation and thylakoid growth.     

The stacking of chloroplast thylakoids: Evidence for segregation of charged groups into nonstacked regions

Small particles derived from the digitonin treatment of chloroplast thylakoid membranes in either the stacked (grana-containing) or unstacked condition, as determined by cation concentration, have been used to study the aggregation of thylakoid membranes. At pH values above 5, the small particles from stacked chloroplasts do not aggregate in the presence of Mg²⁺ or other screening cations at concentrations sufficient to cause the restacking of thylakoids from low-salt chloroplasts. However, the small particles from stacked chloroplasts are aggregated either by lowering the pH to 4.6 or adding the binding cation La³⁺. In contrast, the small particles obtained on digitonin treatment of unstacked chloroplasts were aggregated by cations at neutral pH. Large particles (mainly grana) derived from digitonin treatment of stacked chloroplasts could not be unstacked by transfer to media of low cation concentration. It is concluded that the nonappressed regions of the chloroplast thylakoid membranes under stacking conditions carry higher than average negative surface charge densities under physiological pH conditions. Transfer of chloroplasts to media of low cation concentration causes a time-dependent lateral redistribution of charge between the appressed and nonappressed regions, but this redistribution is prevented by prior digitonin treatment of stacked chloroplasts.     

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes

ABSTRACT: BACKGROUND: The thylakoid system in plant chloroplasts is organized into two distinct domains: granaarranged in stacks of appressed membranes and non-appressed membranes consisting ofstroma thylakoids and margins of granal stacks. It is argued that the reason for thedevelopment of appressed membranes in plants is that their photosynthetic apparatus need tocope with and survive ever-changing environmental conditions. It is not known however,why different plant species have different arrangements of grana within their chloroplasts. Itis important to elucidate whether a different arrangement and distribution of appressed andnon-appressed thylakoids in chloroplasts are linked with different qualitative and/orquantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranesand whether this arrangement influences the photosynthetic efficiency. RESULTS: Our results from TEM and in situ CLSM strongly indicate the existence of differentarrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids areregularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, whileirregular appressed thylakoid membranes within bean chloroplasts correspond to smaller andless distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show adistinct spatial separation of stacked thylakoids from stromal spaces whereas spatial divisionof stroma and thylakoid areas in bean chloroplasts are more complex. Structural differencesinfluenced the PSII photochemistry, however without significant changes in photosyntheticefficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well asspectroscopic investigations indicated a similar proportion between PSI and PSII corecomplexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones.Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSIsupercomplexes between species are suggested. CONCLUSIONS: Based on proteomic and spectroscopic investigations we postulate that the differences in thechloroplast structure between the analyzed species are a consequence of quantitativeproportions between the individual CP complexes and its arrangement inside membranes.Such a structure of membranes induced the formation of large stacked domains in pea, orsmaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with eachother and not always parallel to each other.     

Dynamic flexibility in the structure and function of photosystem II in higher plant thylakoid membranes: the grana enigma

Grana are not essential for photosynthesis, yet they are ubiquitous in higher plants and in the recently evolved Charaphyta algae; hence grana role and its need is still an intriguing enigma. This article discusses how the grana provide integrated and multifaceted functional advantages, by facilitating mechanisms that fine-tune the dynamics of the photosynthetic apparatus, with particular implications for photosystem II (PSII). This dynamic flexibility of photosynthetic membranes is advantageous in plants responding to ever-changing environmental conditions, from darkness or limiting light to saturating light and sustained or intermittent high light. The thylakoid dynamics are brought about by structural and organizational changes at the level of the overall height and number of granal stacks per chloroplast, molecular dynamics within the membrane itself, the partition gap between appressed membranes within stacks, the aqueous lumen encased by the continuous thylakoid membrane network, and even the stroma bathing the thylakoids. The structural and organizational changes of grana stacks in turn are driven by physicochemical forces, including entropy, at work in the chloroplast. In response to light, attractive van der Waals interactions and screening of electrostatic repulsion between appressed grana thylakoids across the partition gap and most probably direct protein interactions across the granal lumen (PSII extrinsic proteins OEEp-OEEp, particularly PsbQ-PsbQ) contribute to the integrity of grana stacks. We propose that both the light-induced contraction of the partition gap and the granal lumen elicit maximisation of entropy in the chloroplast stroma, thereby enhancing carbon fixation and chloroplast protein synthesizing capacity. This spatiotemporal dynamic flexibility in the structure and function of active and inactive PSIIs within grana stacks in higher plant chloroplasts is vital for the optimization of photosynthesis under a wide range of environmental and developmental conditions.     

Bizonoplast, a unique chloroplast in the epidermal cells of microphylls in the shade plant Selaginella erythropus (Selaginellaceae)

Study of the unique leaf anatomy and chloroplast structure in shade-adapted plants will aid our understanding of how plants use light efficiently in low light environments. Unusual chloroplasts in terms of size and thylakoid membrane stacking have been described previously in several deep-shade plants. In this study, a single giant cup-shaped chloroplast, termed a bizonoplast, was found in the abaxial epidermal cells of the dorsal microphylls and the adaxial epidermal cells of the ventral microphylls in the deep-shade spike moss Selaginella erythropus. Bizonoplasts are dimorphic in ultrastructure: the upper zone is occupied by numerous layers of 2-4 stacked thylakoid membranes while the lower zone contains both unstacked stromal thylakoids and thylakoid lamellae stacked in normal grana structure oriented in different directions. In contrast, other cell types in the microphylls contain chloroplasts with typical structure. This unique chloroplast has not been reported from any other species. The enlargement of epidermal cells into funnel-shaped, photosynthetic cells coupled with specific localization of a large bizonoplast in the lower part of the cells and differential modification in ultrastructure within the chloroplast may allow the plant to better adapt to low light. Further experiments are required to determine whether this shade-adapted organism derives any evolutionary or ecophysiological fitness from these unique chloroplasts.     

Diffusion of a membrane protein, Tat subunit Hcf106, is highly restricted within the chloroplast thylakoid network

The thylakoid membrane forms stacked thylakoids interconnected by ‘stromal’ lamellae. Little is known about the mobility of proteins within this system. We studied a stromal lamellae protein, Hcf106, by targeting an Hcf106-GFP fusion protein to the thylakoids and photobleaching. We find that even small regions fail to recover Hcf106-GFP fluorescence over periods of up to 3 min after photobleaching. The protein is thus either immobile within the thylakoid membrane, or its diffusion is tightly restricted within distinct regions. Autofluorescence from the photosystem II light-harvesting complex in the granal stacks likewise fails to recover. Integral membrane proteins within both the stromal and granal membranes are therefore highly constrained, possibly forming ‘microdomains’ that are sharply separated.     

Pigment-protein complexes are organized into stable microdomains in cyanobacterial thylakoids

Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the interactions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that have been defined as a domain-like structures characteristic by different biochemical composition and function (Albertsson P-Å. 2001,Trends Plant Science 6: 349–354). We explored this complex organization of thylakoid pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These microcompartments are of sub-micrometer in sizes (~0.5–1.5 μm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evolutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids.     

Plants under Climatic Stress: II. Low Temperature, High Light Effects on Chloroplast Ultrastructure

Mesophyll chloroplasts of the C₄-pathway grasses Sorghum and Paspalum and of the C₃-pathway legume soybean undergo ultrastructural changes under moderate light intensities (170 w·m⁻², 400-700 nanometers) at a tme when photosynthesis is much reduced by low temperature (10 C). The pattern of ultrastructural change was similar in these species, despite some differences in the initial sites of low temperature action on photosynthesis and differences in their mechanisms of CO₂ fixation. Starch grains in the chloroplasts rapidly reduce in size when chilling stress is applied. At or before the time starch grains completely disappear the membranes of the individual stromal thylakoids close together, reducing the intraspace between them while the chloroplast as a whole begins to swell. Extensive granal stacking appears to hold the thylakoids in position for some time, causing initial swelling to occur in the zone of the peripheral reticulum, when present. At more advanced stages of swelling the thylakoid system unravels while the thylakoid intraspaces dilate markedly. Initial thylakoid intraspace contraction is tentatively ascribed to an increase in the transmembrane hydrogen ion gradient causing movement of cations and undissociated organic acids from the thylakoid intraspace to the stroma. Chloroplast swelling may be caused by a hold-up of some osmotically active photosynthetic product in the chloroplast stroma. After granal unraveling and redilation of the thylakoid intraspaces, chloroplasts appear similar to those isolated in low salt hypotonic media. At the initial stages of stress-induced ultrastructural change, a marked gradient in degree of chloroplast swelling is seen within and between cells, being most pronounced near the surface of the leaf directly exposed to light.     

The importance of grana stacking for xanthophyll cycle-dependent NPQ in the thylakoid membranes of higher plants

In the present study we have examined the effects of grana stacking on the rate of violaxanthin (Vx) de-epoxidation and the extent of non-photochemical quenching of chlorophyll a fluorescence (NPQ) in isolated thylakoid membranes of spinach. Our results show that partial and complete unstacking of thylakoids in reaction media devoid of sorbitol and MgCl₂ did not significantly affect the efficiency of Vx de-epoxidation. Under high light (HL) illumination we found slightly higher values of Vx conversion in stacked membranes, whereas in thylakoids incubated at pH 5.2 in the dark, representing the pH-optimum of Vx de-epoxidase, de-epoxidation was slightly increased in the unstacked membranes. Partial and complete unstacking of grana membranes, however, had a dramatic effect on the HL-induced NPQ. High NPQ values could only be achieved in stacked thylakoid membranes in the presence of MgCl₂ and sorbitol. In unstacked membranes NPQ was drastically decreased. The effects of grana stacking on the xanthophyll cycle-dependent component of NPQ were even more pronounced, and complete unstacking of thylakoid membranes led to a total loss of this quenching component. Our data imply that grana stacking in the thylakoid membranes of higher plants is of high importance for the process of overall NPQ. For the xanthophyll cycle-dependent component of NPQ it may even be essential. Possible effects of grana stacking on the mechanism of zeaxanthin-dependent quenching are discussed.     

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.     

Electron and proton transport in chloroplasts taking into account lateral heterogeneity of thylakoids. Mathematical model]

A mathematical model of a chloroplast was constructed, which takes into account the inhomogeneous distribution of complexes of photosystems I and II between granal and intergranal thylakoids. The structural and functional complexes of photosystems I and II, which are localized in intergranal and granal thylakoids, respectively, and the b/f complex, which is uniformly distributed in thylakoid membranes, are assumed to be immobile. The interactions between spatially distant electron transport complexes are provided by plastoquinone and plastocyanine, which diffuse in the thylakoid membrane and intrathylakoid space, respectively. The main stages of proton transport associated with the functioning of photosystem II and oxidation-reduction transformations of plastoquinone are considered. The model takes into account the interactions of protons with membrane-bound buffer groups, the lateral diffusion of hydrogen ions in the intrathylakoid space and in the lumen between adjacent granal thylakoids, and the transmembrane proton transport associated with the function of ATP synthase and passive leakage of protons from thylakoids outside. The numerical integration of two systems of differential equations describing the behavior of some variables in two different regions: granal and intergranal thylakoids was performed. The model describes adequately the kinetics of processes being studied and predicts the occurrence of inhomogeneous lateral profiles of proton potentials and redox state of electron carriers. Modeling the electron and proton transport with allowance for the topological features of chloroplasts (lateral heterogeneity of thylakoids) is important for correct interpretation of "power-flux" interactions and the experimentally measured kinetic parameters averaged over the entire spatially inhomogeneous thylakoid system.     

The stacking of chloroplast thylakoids: Effects of cation screening and binding,studied by the digitonin method

The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions.     

High Light-Induced Unstacking of Thylakoids and Randomization of Pigment-Protein Complexes of Chloroplast

Using absorbance changes at 540 nm and the per cent chlorophyll in 10K pellet after digitonin fractionation as the criteria of thylakoid stacking, high light induced unstacking of thylakoids and randomization of pigment protein complexes of chloroplast was characterized. Cation-induced increase in absorbance at 540 nm was abolished when chloroplasts were exposed to high light. Also, the high light induced decrease in absorbance at 540 nm was more pronounced In low salt chloroplasts as compared to the decrease in 540 nm absorbance in chloroplasts isolated in high salt. The decrease In the fraction of chlorophyll in digitonin fractionated 10K pellet was much more than the decrease in chlorophyll in 10K pellet from high salt chloroplasts exposed to high light These results suggest that high light causes unstacking of thylakoids.     

Photosystem reaction centers and structural organization of chloroplasts in chlorotica mutants of Pisum sativum L

Chlorophyll-protein complexes of photosystems (PS), as well as ultrastructural arrangement of chloroplasts in pea leaves of the primary cultivar Torsdag and mutants chlorotica 2004 and 2014 were studied. It was shown that both mutants accumulated 80 and 55% of chlorophyll, respectively, and were able to synthesize all of four types of photosystems complexes. The value of the light-harvesting antenna in mutant 2014 was close to the control one, and in mutant 2004 it increased significantly (by 30%). These changes were caused by a proportional decrease, 40-50%, of any complexes in mutant 2014, whereas the number of PS I reaction centers in mutant 2004 decreased by 50% and the reaction centers of PS II complexes were almost completely retained. It was established that the proportional decrease of PS I and PS II complexes in mutant chlorotica 2014 was followed by a partial reduction of the entire membrane system in chloroplasts, but with a good development of both granal and intergranal sites of thylakoids. On the contrary, the loss of complexes of PS I reaction centers in mutant chlorotica 2004 led to a reduction of unstacked sites of thylakoids in chloroplasts. It was concluded that the disturbance of the lateral orientation of the membrane system of chloroplasts is associated with the loss of complexes of reaction centers of PS I, which is predominantly localized in unstacked sites of thylakoids.     

Ultrastructure organization of chloroplasts in chlorotica mutants of Pisum sativum L

A study was made of chlorophyll-protein complexes of photosystems, and of ultrastructural organization of chloroplasts in pea leaves of the primary cultivar Torsdag and of its mutants, chlorotica 2004 and 2014. It has been shown that mutants accumulated 80 and 55% chlorophyll, respectively, and were able to synthesize all four types of photosystem complexes. The value of the light-harvesting antenna in mutant 2014 was close to the control one, and in mutant 2004 it increased significantly (by 30%). These changes were caused by a proportional decrease (40-50%) in any complexes in mutant 2014, whereas the number of PS-I reaction centre complexes, decreased by 50% in mutant 2004 at nearly complete storage of PS-I reaction centre complexes, decreased by 50% in mutant 2004 at nearly complete storage of PS-II complexes. The proportional decrease of PS-I and PS-II complexes in mutant chlorotica 2014 was followed by partial reduction of the entire membrane system in chloroplasts, but with a normal development of both granal and intergranal thylakoids. On the contrary, the loss of PS-I reaction centre complexes in mutant chlorotica 2004 leads to reduction of unstacked sites of thylakoids in chloroplasts. It is concluded that this effect may be associated with localization of PS-I complexes mainly in unstacked sites of thylakoids.     

Action spectra and functional antenna sizes of Photosystems I and II in relation to the thylakoid membrane organization and pigment composition

The functional organization of competent photosynthetic units in developing thylakoids from intermittent-light grown pea as well as in the unstacked, stacked and phosphorylated stacked thylakoids from its mature chloroplasts was characterized by polarographic measurements of action spectra, reaction centre contents and optical cross-sections for PS I-mediated O2 uptake and PS II-mediated O2 evolution. The minimum antenna sizes of 60 and 37 chlorophyll a molecules for PS I and PS II, respectively, were determined in developing thylakoids with a ratio of Chl a/Chl b>50. In mature chloroplasts, the embedded light-harvesting chlorophyll a/b-binding (LHC) protein complexes increased the PS I and PS II effective antenna sizes by 3–6 times depending on the thylakoid membrane organization. In unstacked thylakoids, a randomization of PS I, PS II and LHC II led to the most uniform spectral distribution of light harvesting between the two photosystems but caused the maximal difference of their antenna sizes to be 370 and 100 Chls for the competent PS I and PS II units, respectively. Following the Mg2+-induced stacking of thylakoids, opposite complementary changes of the action spectra, antenna sizes and Chl a/Chl b ratios indicated a redistribution of a LHC II pool of 100 Chl ( a + b) molecules from PS I to PS II. Unlike to the stroma-exposed PS II in unstacked thylakoids, the granal PS II units of 200 Chls demonstrated an additional 2-fold increase of the effective antenna size due to energy transfer within PS II dimers under strong background illumination, which closed >90% of reaction centres. Protein phosphorylation of the stacked thylakoids induced a significant inactivation of the O2-evolving PS II centres but did not cause complementary changes of the action spectra and antenna sizes of the competent PS I and PS II. In this case, light harvesting parameters of the O2-evolving PS II units were nearly unaffected, whereas the obvious relative increase of the PS I activity at 650 nm and its decrease at >700 nm both in the action spectrum and optical cross-section measurements might suggest a substitution of PS I units in the O2-reducing fraction by another distinct fraction of -type which in turn is not the same to PS I units in unstacked thylakoids.