Accounting for photosystem I photoinhibition sheds new light on seasonal acclimation strategies of boreal conifers.

The photosynthetic acclimation of boreal evergreen conifers is controlled by regulatory and photoprotective mechanisms that allow conifers to cope with extreme environmental changes. However, the underlying dynamics of photosystem II (PSII) and photosystem I (PSI) remain unresolved. Here, we investigated the dynamics of PSII and PSI during the spring recovery of photosynthesis in Pinus sylvestris and Picea abies using a combination of chlorophyll a fluorescence, P700 difference absorbance measurements, and quantification of key thylakoid protein abundances. In particular, we derived a new set of PSI quantum yield equations, correcting for the effects of PSI photoinhibition. Using the corrected equations, we found that the seasonal dynamics of PSII and PSI photochemical yields remained largely in balance, despite substantial seasonal changes in the stoichiometry of PSII and PSI core complexes driven by PSI photoinhibition. Similarly, the previously reported seasonal up-regulation of cyclic electron flow was no longer evident, after accounting for PSI photoinhibition. Overall, our results emphasize the importance of considering the dynamics of PSII and PSI to elucidate the seasonal acclimation of photosynthesis in overwintering evergreens. Beyond the scope of conifers, our corrected PSI quantum yields expand the toolkit for future studies aimed at elucidating the dynamic regulation of PSI.

The Arabidopsis thylakoid chloride channel ClCe regulates ATP availability for light-harvesting complex II protein phosphorylation.

Coping with changes in light intensity is challenging for plants, but well-designed mechanisms allow them to acclimate to most unpredicted situations. The thylakoid K+/H+ antiporter KEA3 and the voltage-dependent Cl- channel VCCN1 play important roles in light acclimation by fine-tuning electron transport and photoprotection. Good evidence exists that the thylakoid Cl- channel ClCe is involved in the regulation of photosynthesis and state transitions in conditions of low light. However, a detailed mechanistic understanding of this effect is lacking. Here we report that the ClCe loss-of-function in Arabidopsis thaliana results in lower levels of phosphorylated light-harvesting complex II (LHCII) proteins as well as lower levels of the photosystem I-LHCII complexes relative to wild type (WT) in low light conditions. The phosphorylation of the photosystem II core D1/D2 proteins was less affected either in low or high light conditions. In low light conditions, the steady-state levels of ATP synthase conductivity and of the total proton flux available for ATP synthesis were lower in ClCe loss-of-function mutants, but comparable to WT at standard and high light intensity. As a long-term acclimation strategy, expression of the ClCe gene was upregulated in WT plants grown in light-limiting conditions, but not in WT plants grown in standard light even when exposed for up to 8 h to low light. Taken together, these results suggest a role of ClCe in the regulation of the ATP synthase activity which under low light conditions impacts LHCII protein phosphorylation and state transitions.

Higher order photoprotection mutants reveal the importance of ΔpH-dependent photosynthesis-control in preventing light induced damage to both photosystem II and photosystem I.

Although light is essential for photosynthesis, when in excess, it may damage the photosynthetic apparatus, leading to a phenomenon known as photoinhibition. Photoinhibition was thought as a light-induced damage to photosystem II; however, it is now clear that even photosystem I may become very vulnerable to light. One main characteristic of light induced damage to photosystem II (PSII) is the increased turnover of the reaction center protein, D1: when rate of degradation exceeds the rate of synthesis, loss of PSII activity is observed. With respect to photosystem I (PSI), an excess of electrons, instead of an excess of light, may be very dangerous. Plants possess a number of mechanisms able to prevent, or limit, such damages by safe thermal dissipation of light energy (non-photochemical quenching, NPQ), slowing-down of electron transfer through the intersystem transport chain (photosynthesis-control, PSC) in co-operation with the Proton Gradient Regulation (PGR) proteins, PGR5 and PGRL1, collectively called as short-term photoprotection mechanisms, and the redistribution of light between photosystems, called state transitions (responsible of fluorescence quenching at PSII, qT), is superimposed to these short term photoprotective mechanisms. In this manuscript we have generated a number of higher order mutants by crossing genotypes carrying defects in each of the short-term photoprotection mechanisms, with the final aim to obtain a direct comparison of their role and efficiency in photoprotection. We found that mutants carrying a defect in the ΔpH-dependent photosynthesis-control are characterized by photoinhibition of both photosystems, irrespectively of whether PSBS-dependent NPQ or state transitions defects were present or not in the same individual, demonstrating the primary role of PSC in photoprotection. Moreover, mutants with a limited capability to develop a strong PSBS-dependent NPQ, were characterized by a high turnover of the D1 protein and high values of Y(NO), which might reflect energy quenching processes occurring within the PSII reaction center.

The Role of Phosphorylation Dynamics of CURVATURE THYLAKOID 1B in Plant Thylakoid Membranes.

Thylakoid membranes in land plant chloroplasts are organized into appressed and nonappressed membranes, which contribute to the control of energy distribution between the two photosystems (PSI and PSII) from the associated light-harvesting complexes (LHCs). Under fluctuating light conditions, fast reversible phosphorylation of the N-terminal thylakoid protein domains and changes in electrostatic forces induce modifications in thylakoid organization. To gain insight into the role and dynamics of thylakoid protein phosphorylation, we used targeted proteomics to quantify amounts of the structural proteins CURVATURE THYLAKOID1 (CURT1), including the levels of CURT1B N terminus phosphorylation and acetylation, after short-term fluctuating light treatments of Arabidopsis (Arabidopsis thaliana). The CURT1B protein was localized to a specific curvature domain separated from the margin domain, and specifically depleted of chlorophyll-binding protein complexes. The acetylation and phosphorylation of the CURT1B N terminus were mutually exclusive. The level of CURT1B phosphorylation, but not of acetylation, increased upon light shifts that also led to an increase in PSII core protein phosphorylation. These dynamics were largely absent in the knockout mutant of PSII core protein kinase SER/THR PROTEIN KINASE8 (STN8). Moreover, in mutants impaired in interaction between phosphorylated LHCII and PSI, the phosphorylation dynamics of CURT1B and the amount of the other CURT1 proteins were misregulated, indicating a functional interaction between CURT1B and PSI-LHCII complexes in grana margins. The complex relationships between phosphorylation of PSII, LHCII, and CURT1B support the dynamics of thylakoid protein complexes that are crucial in the optimization of photosynthesis under fluctuating light intensities.

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis.

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

Separation of Thylakoid Protein Complexes with Two-dimensional Native-PAGE.

The hierarchical composition and interactions of the labile thylakoid protein complexes can be assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic detergent digitonin is used to solubilize labile protein super-and megacomplexes, which are then separated with first-dimension blue native polyacrylamide gel electrophoresis (1D-BN-PAGE). The digitonin derived protein complexes are further solubilized with stronger detergent, ß-DM, and subsequently separated on an orthogonal 2D-BN-PAGE to release smaller protein subcomplexes from the higher-order supercomplexes. Here we describe a detailed method for 2D-BN-PAGE analysis of thylakoid protein complexes from Arabidopsis thaliana.

Dark-adapted spinach thylakoid protein heterogeneity offers insights into the photosystem II repair cycle.

In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Steady-state phosphorylation of light-harvesting complex II proteins preserves photosystem I under fluctuating white light.

According to the "state transitions" theory, the light-harvesting complex II (LHCII) phosphorylation in plant chloroplasts is essential to adjust the relative absorption cross section of photosystem II (PSII) and PSI upon changes in light quality. The role of LHCII phosphorylation upon changes in light intensity is less thoroughly investigated, particularly when changes in light intensity are too fast to allow the phosphorylation/dephosphorylation processes to occur. Here, we demonstrate that the Arabidopsis (Arabidopsis thaliana) stn7 (for state transition7) mutant, devoid of the STN7 kinase and LHCII phosphorylation, shows a growth penalty only under fluctuating white light due to a low amount of PSI. Under constant growth light conditions, stn7 acquires chloroplast redox homeostasis by increasing the relative amount of PSI centers. Thus, in plant chloroplasts, the steady-state LHCII phosphorylation plays a major role in preserving PSI upon rapid fluctuations in white light intensity. Such protection of PSI results from LHCII phosphorylation-dependent equal distribution of excitation energy to both PSII and PSI from the shared LHCII antenna and occurs in cooperation with nonphotochemical quenching and the proton gradient regulation5-dependent control of electron flow, which are likewise strictly regulated by white light intensity. LHCII phosphorylation is concluded to function both as a stabilizer (in time scales of seconds to minutes) and a dynamic regulator (in time scales from tens of minutes to hours and days) of redox homeostasis in chloroplasts, subject to modifications by both environmental and metabolic cues. Exceeding the capacity of LHCII phosphorylation/dephosphorylation to balance the distribution of excitation energy between PSII and PSI results in readjustment of photosystem stoichiometry.

PROTON GRADIENT REGULATION5 is essential for proper acclimation of Arabidopsis photosystem I to naturally and artificially fluctuating light conditions.

In nature, plants are challenged by constantly changing light conditions. To reveal the molecular mechanisms behind acclimation to sometimes drastic and frequent changes in light intensity, we grew Arabidopsis thaliana under fluctuating light conditions, in which the low light periods were repeatedly interrupted with high light peaks. Such conditions had only marginal effect on photosystem II but induced damage to photosystem I (PSI), the damage being most severe during the early developmental stages. We showed that PROTON GRADIENT REGULATION5 (PGR5)-dependent regulation of electron transfer and proton motive force is crucial for protection of PSI against photodamage, which occurred particularly during the high light phases of fluctuating light cycles. Contrary to PGR5, the NAD(P)H dehydrogenase complex, which mediates cyclic electron flow around PSI, did not contribute to acclimation of the photosynthetic apparatus, particularly PSI, to rapidly changing light intensities. Likewise, the Arabidopsis pgr5 mutant exhibited a significantly higher mortality rate compared with the wild type under outdoor field conditions. This shows not only that regulation of PSI under natural growth conditions is crucial but also the importance of PGR5 in PSI protection.

Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes.

Gel-based analysis of thylakoid membrane protein complexes represents a valuable tool to monitor the dynamics of the photosynthetic machinery. Native-PAGE preserves the components and often also the conformation of the protein complexes, thus enabling the analysis of their subunit composition. Nevertheless, the literature and practical experimentation in the field sometimes raise confusion owing to a great variety of native-PAGE and thylakoid-solubilization systems. In the present paper, we describe optimized methods for separation of higher plant thylakoid membrane protein complexes by native-PAGE addressing particularly: (i) the use of detergent; (ii) the use of solubilization buffer; and (iii) the gel electrophoresis method. Special attention is paid to separation of high-molecular-mass thylakoid membrane super- and mega-complexes from Arabidopsis thaliana leaves. Several novel super- and mega-complexes including PS (photosystem) I, PSII and LHCs (light-harvesting complexes) in various combinations are reported.

Two proteins homologous to PsbQ are novel subunits of the chloroplast NAD(P)H dehydrogenase.

The PsbQ-like (PQL) proteins 1 and 2, previously shown to be located in the thylakoid lumen of Arabidopsis thaliana, are homologous to PSII oxygen-evolving complex protein PsbQ. Nevertheless, pql mutants showed no defects in PSII but instead the activity of the chloroplast NAD(P)H dehydrogenease (NDH) complex was severely impaired. In line with this observation, the NDH subunits were low in abundance in pql mutants, and, conversely, ndh mutants strongly down-regulated the accumulation of the PQL proteins. In addition, the PQL2 protein was up-regulated in mutant plants deficient in the PSI complex or the thylakoid membrane-bound ferredoxin-NADP(+) oxidoreductase, whereas in pql mutants the PSI complex was slightly up-regulated. Taken together, the two PQL proteins are shown to be novel subunits of the lumenal protuberance of the NDH complex.

Light regulation of CaS, a novel phosphoprotein in the thylakoid membrane of Arabidopsis thaliana.

Exposure of Arabidopsis thaliana plants to high levels of light revealed specific phosphorylation of a 40 kDa protein in photosynthetic thylakoid membranes. The protein was identified by MS as extracellular calcium-sensing receptor (CaS), previously reported to be located in the plasma membrane. By confocal laser scanning microscopy and subcellular fractionation, it was demonstrated that CaS localizes to the chloroplasts and is enriched in stroma thylakoids. The phosphorylation level of CaS responded strongly to light intensity. The light-dependent thylakoid protein kinase STN8 is required for CaS phosphorylation. The phosphorylation site was mapped to the stroma-exposed Thr380, located in a motif for interaction with 14-3-3 proteins and proteins with forkhead-associated domains, which suggests the involvement of CaS in stress responses and signaling pathways. The knockout Arabidopsis lines revealed a significant role for CaS in plant growth and development.

Expression of protein complexes and individual proteins upon transition of etioplasts to chloroplasts in pea (Pisum sativum).

The protein complexes of pea (Pisum sativum L.) etioplasts, etio-chloroplasts and chloroplasts were examined using 2D Blue Native/SDS-PAGE. The most prominent protein complexes in etioplasts were the ATPase and the Clp and FtsH protease complexes which probably have a crucial role in the biogenesis of etioplasts and chloroplasts. Also the cytochrome b(6)f (Cyt b(6)f) complex was assembled in the etioplast membrane, as well as Rubisco, at least partially, in the stroma. These complexes are composed of proteins encoded by both the plastid and nuclear genomes, indicating that a functional cross-talk exists between pea etioplasts and the nucleus. In contrast, the proteins and protein complexes that bind chlorophyll, with the PetD subunit and the entire Cyt b(6)f complex as an exception, did not accumulate in etioplasts. Nevertheless, some PSII core components such as PsbE and the luminal oxygen-evolvong complex (OEC) proteins PsbO and PsbP accumulated efficiently in etioplasts. After 6 h de-etiolation, a complete PSII core complex appeared with 40% of the maximal photochemical efficiency, but a fully functional PSII was recorded only after 24 h illumination. Similarly, the core complex of PSI was assembled after 6 h illumination, whereas the PSI-light-harvesting complex I was stably assembled only in chloroplasts illuminated for 24 h. Moreover, a battery of proteins responsible for defense against oxidative stress accumulated particularly in etioplasts, including the stromal and thylakoidal forms of ascorbate peroxidase, glutathione reductase and PsbS.

TLP18.3, a novel thylakoid lumen protein regulating photosystem II repair cycle.

A proteome analysis of Arabidopsis thaliana thylakoid-associated polysome nascent chain complexes was performed to find novel proteins involved in the biogenesis, maintenance and turnover of thylakoid protein complexes, in particular the PSII (photosystem II) complex, which exhibits a high turnover rate. Four unknown proteins were identified, of which TLP18.3 (thylakoid lumen protein of 18.3 kDa) was selected for further analysis. The Arabidopsis mutants (SALK_109618 and GABI-Kat 459D12) lacking the TLP18.3 protein showed higher susceptibility of PSII to photoinhibition. The increased susceptibility of DeltaTLP18.3 plants to high light probably originates from an inefficient reassembly of PSII monomers into dimers in the grana stacks, as well as from an impaired turnover of the D1 protein in stroma exposed thylakoids. Such dual function of the TLP18.3 protein is in accordance with its even distribution between the grana and stroma thylakoids. Notably, the lack of the TLP18.3 protein does not lead to a severe collapse of the PSII complexes, suggesting a redundancy of proteins assisting these particular repair steps to assure functional PSII. The DeltaTLP18.3 plants showed no clear visual phenotype under standard growth conditions, but when challenged by fluctuating light during growth, the retarded growth of DeltaTLP18.3 plants was evident.

Dimeric and monomeric organization of photosystem II. Distribution of five distinct complexes in the different domains of the thylakoid membrane.

The supramolecular organization of photosystem II (PSII) was characterized in distinct domains of the thylakoid membrane, the grana core, the grana margins, the stroma lamellae, and the so-called Y100 fraction. PSII supercomplexes, PSII core dimers, PSII core monomers, PSII core monomers lacking the CP43 subunit, and PSII reaction centers were resolved and quantified by blue native PAGE, SDS-PAGE for the second dimension, and immunoanalysis of the D1 protein. Dimeric PSII (PSII supercomplexes and PSII core dimers) dominate in the core part of the thylakoid granum, whereas the monomeric PSII prevails in the stroma lamellae. Considerable amounts of PSII monomers lacking the CP43 protein and PSII reaction centers (D1-D2-cytochrome b559 complex) were found in the stroma lamellae. Our quantitative picture of the supramolecular composition of PSII, which is totally different between different domains of the thylakoid membrane, is discussed with respect to the function of PSII in each fraction. Steady state electron transfer, flash-induced fluorescence decay, and EPR analysis revealed that nearly all of the dimeric forms represent oxygen-evolving PSII centers. PSII core monomers were heterogeneous, and a large fraction did not evolve oxygen. PSII monomers without the CP43 protein and PSII reaction centers showed no oxygen-evolving activity.

Chloroplast-mediated regulation of nuclear genes in Arabidopsis thaliana in the absence of light stress.

Chloroplast signaling involves mechanisms to relay information from chloroplasts to the nucleus, to change nuclear gene expression in response to environmental cues. Aside from reactive oxygen species (ROS) produced under stress conditions, changes in the reduction/oxidation state of photosynthetic electron transfer components or coupled compounds in the stroma and the accumulation of photosynthesis-derived metabolites are likely origins of chloroplast signals. We attempted to investigate the origin of the signals from chloroplasts in mature Arabidopsis leaves by differentially modulating the redox states of the plastoquinone pool and components on the reducing side of photosystem I, as well as the rate of CO2 fixation, while avoiding the production of ROS by excess light. Differential expression of several nuclear photosynthesis genes, including a set of Calvin cycle enzymes, was recorded. These responded to the stromal redox conditions under prevailing light conditions but were independent of the redox state of the plastoquinone pool. The steady-state CO2 fixation rate was reflected in the orchestration of the expression of a number of genes encoding cytoplasmic proteins, including several glycolysis genes and the trehalose-6-phosphate synthase gene, and also the chloroplast-targeted chaperone DnaJ. Clearly, in mature leaves, the redox state of the compounds on the reducing side of photosystem I is of greater importance in light-dependent modulation of nuclear gene expression than the redox state of the plastoquinone pool, particularly at early signaling phases. It also became apparent that photosynthesis-mediated generation of metabolites or signaling molecules is involved in the relay of information from chloroplast to nucleus.

PsbR, a missing link in the assembly of the oxygen-evolving complex of plant photosystem II.

The oxygen-evolving complex of eukaryotic photosystem II (PSII) consists of three extrinsic nuclear-encoded subunits, PsbO (33 kDa), PsbP (23 kDa), and PsbQ (17 kDa). Additionally, the 10-kDa PsbR protein has been found in plant PSII and anticipated to play a role in water oxidation, yet the physiological significance of PsbR has remained obscure. Using the Arabidopsis psbR mutant, we showed that the light-saturated rate of oxygen evolution is strongly reduced in the absence of PsbR, particularly in low light-grown plants. Lack of PsbR also induced a reduction in the content of both the PsbP and the PsbQ proteins, and a near depletion of these proteins was observed under steady state low light conditions. This regulation occurred post-transcriptionally and likely involves a proteolytic degradation of the PsbP and PsbQ proteins in the absence of an assembly partner, proposed to be the PsbR protein. Stable assembly of PsbR in the PSII core complex was, in turn, shown to require a chloroplast-encoded intrinsic low molecular mass PSII subunit PsbJ. Our results provided evidence that PsbR is an important link in the PSII core complex for stable assembly of the oxygen-evolving complex protein PsbP, whereas the effects on the assembly of PsbQ are probably indirect. The physiological role of the PsbR, PsbP, and PsbQ proteins is discussed in light of their peculiar expression in response to growth light conditions.

A previously found thylakoid membrane protein of 14kDa (TMP14) is a novel subunit of plant photosystem I and is designated PSI-P.

We show that the thylakoid membrane phosphoprotein TMP14 is a novel subunit of plant photosystem I (PSI). Blue native/SDS-PAGE and sucrose gradient fractionation demonstrated the association of the protein exclusively with PSI. We designate the protein PSI-P. The presence of PSI-P subunit in Arabidopsis mutants lacking other PSI subunits was analyzed and suggested a location in the proximity of PSI-L, -H and -O subunits. The PSI-P protein was not differentially phosphorylated in state 1 and state 2.

Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1.

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Towards functional proteomics of membrane protein complexes in Synechocystis sp. PCC 6803.

The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b6f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the protein complexes was studied by culturing the wild type and several mutant strains under various growth modes (photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO2, iron, or salt. The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of CP43' protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the CO2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein complexes. Our results give the first direct evidence, to our knowledge, that the NdhF3, NdhD3, and CupA proteins assemble together to form a small low CO2-induced protein complex and further demonstrate the presence of a fourth subunit, Sll1735, in this complex. The two bigger NDH-1 complexes contained a different set of NDH-1 polypeptides and are likely to function in respiratory and cyclic electron transfer. Pulse labeling experiments demonstrated the requirement of PSII activity for de novo synthesis of the NDH-1 complexes.