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Plants can quickly and dynamically respond to spectral and intensity variations of the incident light. These responses include activation of developmental processes, morphological changes, and photosynthetic acclimation that ensure optimal energy conversion and minimal photoinhibition. Plant adaptation and acclimation to environmental changes have been extensively studied, but many details surrounding these processes remain elusive. The photosystem II (PSII)-associated protein PSB33 plays a fundamental role in sustaining PSII as well as in the regulation of the light antenna in fluctuating light. We investigated how PSB33 knock-out Arabidopsis plants perform under different light qualities. psb33 plants displayed a reduction of 88% of total fresh weight compared to wild type plants when cultivated at the boundary of UV-A and blue light. The sensitivity towards UV-A light was associated with a lower abundance of PSII proteins, which reduces psb33 plants’ capacity for photosynthesis. The UV-A phenotype was found to be linked to altered phytohormone status and changed thylakoid ultrastructure. Our results collectively show that PSB33 is involved in a UV-A light-mediated mechanism to maintain a functional PSII pool in the chloroplast.

In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.

In green plants, photosystem II (PSII) forms multisubunit supercomplexes (SCs) containing a dimeric core and light-harvesting complexes (LHCs). In this study, we show that Arabidopsis thaliana PsbP-like protein 1 (PPL1) is involved in the assembly of the PSII SCs and is required for adaptation to changing light intensity. PPL1 is a homolog of PsbP protein that optimizes the water-oxidizing reaction of PSII in green plants and is required for the efficient repair of photodamaged PSII; however, its exact function has been unknown. PPL1 was enriched in stroma lamellae and grana margins and associated with PSII subcomplexes including PSII monomers and PSII dimers, and several LHCII assemblies, while PPL1 was not detected in PSII–LHCII SCs. In a PPL1 null mutant (ppl1-2), assembly of CP43, PsbR and PsbW was affected, resulting in a reduced accumulation of PSII SCs even under moderate light intensity. This caused the abnormal association of LHCII in ppl1-2, as indicated by lower maximal quantum efficiency of PSII (Fv/Fm) and accelerated State 1 to State 2 transitions. These differences would lower the capability of plants to adapt to changing light environments, thereby leading to reduced growth under natural fluctuating light environments. Phylogenetic and structural analyses suggest that PPL1 is closely related to its cyanobacterial homolog CyanoP, which functions as an assembly factor in the early stage of PSII biogenesis. Our results suggest that PPL1 has a similar function, but the data also indicate that it could aid the association of LHCII with PSII.

Vascular plants have evolved a long-term light acclimation strategy primarily relying on the regulation of the relative amounts of light-harvesting complex II (LHCII) and of the two photosystems, photosystem I (PSI) and photosystem II (PSII). We investigated whether such a model is also valid in Selaginella martensii, a species belonging to the early diverging group of lycophytes. Selaginella martensii plants were acclimated to three natural light regimes (extremely low light (L), medium light (M) and full sunlight (H)) and thylakoid organization was characterized combining ultrastructural, biochemical and functional methods. From L to H plants, thylakoid architecture was rearranged from (pseudo)lamellar to predominantly granal, the PSII : PSI ratio changed in favour of PSI, and the photochemical capacity increased. However, regulation of light harvesting did not occur through variations in the amount of free LHCII, but rather resulted from the flexibility of the association of free LHCII with PSII and PSI. In lycophytes, the free interspersed LHCII serves a fixed proportion of reaction centres, either PSII or PSI, and the regulation of PSI-LHCII(-PSII) megacomplexes is an integral part of long-term acclimation. Free LHCII ensures photoprotection of PSII, allows regulated use of PSI as an energy quencher, and can also quench endangered PSI.

Redox‐regulated reversible phosphorylation of the light‐harvesting complex II (LHCII) controls the excitation energy distribution between photosystem (PS) II and PSI. The PsaL and PsaH subunits of PSI enable the association of pLHCII to PSI. Here, we show that the failure of the psal mutant to dock pLHCII to PSI induces excessive phosphorylation of LHCII, primarily due to a marked downregulation of the TAP38/PPH1 phosphatase occurring at post‐transcriptional level. TAP38/PPH1 is shown to be associated with megacomplex that contains both photosystems in a light‐ and LHCII‐PSII core‐phosphorylation‐dependent manner. It is suggested that proper megacomplex‐related association of TAP38/PPH1 protects it against degradation.

STN7 kinase catalyzes the phosphorylation of the globally most common membrane proteins, the light-harvesting complex II (LHCII) in plant chloroplasts. STN7 itself possesses one serine (Ser) and two threonine (Thr) phosphosites. We show that phosphorylation of the Thr residues protects STN7 against degradation in darkness, low light and red light, whereas increasing light intensity and far red illumination decrease phosphorylation and induce STN7 degradation. Ser phosphorylation, in turn, occurs under red and low intensity white light, coinciding with the client protein (LHCII) phosphorylation. Through analysis of the counteracting LHCII phosphatase mutant tap38/pph1, we show that Ser phosphorylation and activation of the STN7 kinase for subsequent LHCII phosphorylation are heavily affected by pre-illumination conditions. Transitions between the three activity states of the STN7 kinase (deactivated in darkness and far red light, activated in low and red light, inhibited in high light) are shown to modulate the phosphorylation of the STN7 Ser and Thr residues independently of each other. Such dynamic regulation of STN7 kinase phosphorylation is crucial for plant growth and environmental acclimation.

Photosystem (PS) II is a multisubunit thylakoid membrane pigment–protein complex responsible for light-driven oxidation of water and reduction of plastoquinone. Currently more than 40 proteins are known to associate with PSII, either stably or transiently. The inherent feature of the PSII complex is its vulnerability in light, with the damage mainly targeted to one of its core proteins, the D1 protein. The repair of the damaged D1 protein, i.e. the repair cycle of PSII, initiates in the grana stacks where the damage generally takes place, but subsequently continues in non-appressed thylakoid domains, where many steps are common for both the repair and de novo assembly of PSII. The sequence of the (re)assembly steps of genuine PSII subunits is relatively well-characterized in higher plants. A number of novel findings have shed light into the regulation mechanisms of lateral migration of PSII subcomplexes and the repair as well as the (re)assembly of the complex. Besides the utmost importance of the PSII repair cycle for the maintenance of PSII functionality, recent research has pointed out that the maintenance of PSI is closely dependent on regulation of the PSII repair cycle. This review focuses on the current knowledge of regulation of the repair cycle of PSII in higher plant chloroplasts. Particular emphasis is paid on sequential assembly steps of PSII and the function of the number of PSII auxiliary proteins involved both in the biogenesis and repair of PSII. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

Photosystem II (PSII) core and light-harvesting complex II (LHCII) proteins in plant chloroplasts undergo reversible phosphorylation upon changes in light intensity (being under control of redox-regulated STN7 and STN8 kinases and TAP38/PPH1 and PSII core phosphatases). Shift of plants from growth light to high light results in an increase of PSII core phosphorylation, whereas LHCII phosphorylation concomitantly decreases. Exactly the opposite takes place when plants are shifted to lower light intensity. Despite distinct changes occurring in thylakoid protein phosphorylation upon light intensity changes, the excitation balance between PSII and photosystem I remains unchanged. This differs drastically from the canonical-state transition model induced by artificial states 1 and 2 lights that concomitantly either dephosphorylate or phosphorylate, respectively, both the PSII core and LHCII phosphoproteins. Analysis of the kinase and phosphatase mutants revealed that TAP38/PPH1 phosphatase is crucial in preventing state transition upon increase in light intensity. Indeed, tap38/pph1 mutant revealed strong concomitant phosphorylation of both the PSII core and LHCII proteins upon transfer to high light, thus resembling the wild type under state 2 light. Coordinated function of thylakoid protein kinases and phosphatases is shown to secure balanced excitation energy for both photosystems by preventing state transitions upon changes in light intensity. Moreover, PROTON GRADIENT REGULATION5 (PGR5) is required for proper regulation of thylakoid protein kinases and phosphatases, and the pgr5 mutant mimics phenotypes of tap38/pph1. This shows that there is a close cooperation between the redox- and proton gradient-dependent regulatory mechanisms for proper function of the photosynthetic machinery.

In plant chloroplasts, the two photosystems (PSII and PSI) are enriched in different thylakoid domains and, according to the established view, are regarded as energetically segregated from each other. A specific fraction of the light harvesting complex II (LHCII) has been postulated to get phosphorylated by the STN7 kinase and subsequently to migrate from PSII to PSI as part of a process called ‘state transition’. Nevertheless, the thylakoid membrane incorporates a large excess of LHCII not present in the isolatable PSII–LHCII and PSI–LHCII complexes. Moreover, LHCII phosphorylation is not limited to a specific LHCII pool and “state 2” condition, but is found in all thylakoid domains in any constant light condition. Here, using a targeted solubilization of pigment–protein complexes from different thylakoid domains, we demonstrate that even a minor detachment of LHCII leads to markedly increased fluorescence emission from LHCII and PSII both in grana core and non-appressed thylakoid membranes and the effect of the detergent to detach LHCII is enhanced in the absence of LHCII phosphorylation. These findings provide evidence that PSII and PSI are energy traps embedded in the same energetically connected LHCII lake. In the lake, PSI and LHCII are energetically connected even in the absence of LHCII phosphorylation, yet the phosphorylation enhances the interaction required for efficient energy transfer to PSI in the grana margin regions.

The chloroplast protein PSB33 is primarily located in non-appressed thylakoid regions, and is vital for growth of plants in fluctuating light through regulation of PSII quality control., On Earth, solar irradiance varies as the sun rises and sets over the horizon, and sunlight is thus in constant fluctuation, following a slow dark–low–high–low–dark curve. Optimal plant growth and development are dependent on the capacity of plants to acclimate and regulate photosynthesis in response to these changes of light. Little is known of regulative processes for photosynthesis during nocturnal events. The nucleus-encoded plant lineage-specific protein PSB33 has been described as stabilizing the photosystem II complex, especially under light stress conditions, and plants lacking PSB33 have a dysfunctional state transition. To clarify the localization and function of this protein, we used phenomic, biochemical and proteomics approaches in the model plant Arabidopsis. We report that PSB33 is predominantly located in non-appressed thylakoid regions and dynamically associates with a thylakoid protein complex in a light-dependent manner. Moreover, plants lacking PSB33 show an accelerated D1 protein degradation in nocturnal periods, and show severely stunted growth when challenged with fluctuating light. We further show that the function of PSB33 precedes the STN7 kinase to regulate or balance the excitation energy of photosystems I and II in fluctuating light conditions.

Oxygenic photosynthetic organisms experience strong fluctuations in light intensity in their natural terrestrial and aquatic growth environments. Recent studies with both plants and cyanobacteria have revealed that Photosystem (PS) I is the potential target of damage upon abrupt changes in light intensity. Photosynthetic organisms have, however, developed powerful mechanisms in order to protect their photosynthetic apparatus against such potentially hazardous light conditions. Although the electron transfer chain has remained relatively unchanged in both plant chloroplasts and their cyanobacterial ancestors, the photoprotective and regulatory mechanisms of photosynthetic light reactions have experienced conspicuous evolutionary changes. In cyanobacteria, the specific flavodiiron proteins (Flv1 and Flv3) are responsible for safeguarding PSI under rapidly fluctuating light intensities, whilst the thylakoid located terminal oxidases are involved in the protection of PSII during 12h diurnal cycles involving abrupt, square-wave, changes from dark to high light. Higher plants such as Arabidopsis thaliana have evolved different protective mechanisms. In particular, the PGR5 protein controls electron flow during sudden changes in light intensity by allowing the regulation mostly via the Cytochrome b6f complex. Besides the function of PGR5, plants have also acquired other dynamic regulatory mechanisms, among them the STN7-related LHCII protein phosphorylation that is similarly responsible for protection against rapid changes in the light environment. The green alga Chlamydomonas reinhardtii, as an evolutionary intermediate between cyanobacteria and higher plants, probably possesses both protective mechanisms. In this review, evolutionarily different photoprotective mechanisms under fluctuating light conditions are described and their contributions to cyanobacterial and plant photosynthesis are discussed.

Photosynthetic light harvesting in plants is regulated by phosphorylation-driven state transitions: functional redistributions of the major trimeric light-harvesting complex II (LHCII) to balance the relative excitation of photosystem I and photosystem II. State transitions are driven by reversible LHCII phosphorylation by the STN7 kinase and PPH1/TAP38 phosphatase. LHCII trimers are composed of Lhcb1, Lhcb2, and Lhcb3 proteins in various trimeric configurations. Here, we show that despite their nearly identical amino acid composition, the functional roles of Lhcb1 and Lhcb2 are different but complementary. Arabidopsis thaliana plants lacking only Lhcb2 contain thylakoid protein complexes similar to wild-type plants, where Lhcb2 has been replaced by Lhcb1. However, these do not perform state transitions, so phosphorylation of Lhcb2 seems to be a critical step. In contrast, plants lacking Lhcb1 had a more profound antenna remodeling due to a decrease in the amount of LHCII trimers influencing thylakoid membrane structure and, more indirectly, state transitions. Although state transitions are also found in green algae, the detailed architecture of the extant seed plant light-harvesting antenna can now be dated back to a time after the divergence of the bryophyte and spermatophyte lineages, but before the split of the angiosperm and gymnosperm lineages more than 300 million years ago.

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.

PsaI represents one of three low molecular weight peptides of PSI. Targeted inactivation of the plastid PsaI gene in Nicotiana tabacum has no measurable effect on photosynthetic electron transport around PSI or on accumulation of proteins involved in photosynthesis. Instead, the lack of PsaI destabilizes the association of PsaL and PsaH to PSI, both forming the light-harvesting complex (LHC)II docking site of PSI. These alterations at the LHCII binding site surprisingly did not prevent state transition but led to an increased incidence of PSI-LHCII complexes, coinciding with an elevated phosphorylation level of the LHCII under normal growth light conditions. Remarkably, LHCII was rapidly phosphorylated in ΔpsaI in darkness even after illumination with far-red light. We found that this dark phosphorylation also occurs in previously described mutants impaired in PSI function or state transition. A prompt shift of the plastoquinone (PQ) pool into a more reduced redox state in the dark caused an enhanced LHCII phosphorylation in ΔpsaI Since the redox status of the PQ pool is functionally connected to a series of physiological, biochemical, and gene expression reactions, we propose that the shift of mutant plants into state 2 in darkness represents a compensatory and/or protective metabolic mechanism. This involves an increased reduction and/or reduced oxidation of the PQ pool, presumably to sustain a balanced excitation of both photosystems upon the onset of light.

Chloroplasts play an important role in the cellular sensing of abiotic and biotic stress. Signals originating from photosynthetic light reactions, in the form of redox and pH changes, accumulation of reactive oxygen and electrophile species or stromal metabolites are of key importance in chloroplast retrograde signaling. These signals initiate plant acclimation responses to both abiotic and biotic stresses. To reveal the molecular responses activated by rapid fluctuations in growth light intensity, gene expression analysis was performed with Arabidopsis thaliana wild type and the tlp18.3 mutant plants, the latter showing a stunted growth phenotype under fluctuating light conditions (Biochem. J, 406, 415-425). Expression pattern of genes encoding components of the photosynthetic electron transfer chain did not differ between fluctuating and constant light conditions, neither in wild type nor in tlp18.3 plants, and the composition of the thylakoid membrane protein complexes likewise remained unchanged. Nevertheless, the fluctuating light conditions repressed in wild-type plants a broad spectrum of genes involved in immune responses, which likely resulted from shade-avoidance responses and their intermixing with hormonal signaling. On the contrary, in the tlp18.3 mutant plants there was an imperfect repression of defense-related transcripts upon growth under fluctuating light, possibly by signals originating from minor malfunction of the photosystem II (PSII) repair cycle, which directly or indirectly modulated the transcript abundances of genes related to light perception via phytochromes. Consequently, a strong allocation of resources to defense reactions in the tlp18.3 mutant plants presumably results in the stunted growth phenotype under fluctuating light.

Regulation of photosynthetic electron transport provides efficient performance of oxygenic photosynthesis in plants. During the last 15 years, the molecular bases of various photosynthesis short-term regulatory processes have been elucidated, however the wild type-like phenotypes of mutants lacking of State Transitions, Non Photochemical Quenching, or Cyclic Electron Transport, when grown under constant light conditions, have also raised doubts about the acclimatory significance of these short-regulatory mechanisms on plant performance. Interestingly, recent studies performed by growing wild type and mutant plants under field conditions revealed a prominent role of State Transitions and Non Photochemical Quenching on plant fitness, with almost no effect on vegetative plant growth. Conversely, the analysis of plants lacking the regulation of electron transport by the cytochrome b6f complex, also known as Photosynthesis Control, revealed the fundamental role of this regulatory mechanism in the survival of young, developing seedlings under fluctuating light conditions.

The comparative study of photosynthetic regulation in the thylakoid membrane of different phylogenetic groups can yield valuable insights into mechanisms, genetic requirements and redundancy of regulatory processes. This review offers a brief summary on the current understanding of light harvesting and photosynthetic electron transport regulation in different photosynthetic eukaryotes, with a special focus on the comparison between higher plants and unicellular algae of secondary endosymbiotic origin. The foundations of thylakoid structure, light harvesting, reversible protein phosphorylation and PSI-mediated cyclic electron transport are traced not only from green algae to vascular plants but also at the branching point between the "green" and the "red" lineage of photosynthetic organisms. This approach was particularly valuable in revealing processes that (1) are highly conserved between phylogenetic groups, (2) serve a common physiological role but nevertheless originate in divergent genetic backgrounds or (3) are missing in one phylogenetic branch despite their unequivocal importance in another, necessitating a search for alternative regulatory mechanisms and interactions.

The DnaJ proteins (also called as J proteins, J domain proteins or HSP40 proteins) function as molecular co-chaperones for the HSP70 proteins. We assessed the expression of the small chloroplast-targeted DnaJ protein, the AtJ8 protein, by subjecting the wild type Arabidopsis plants to different illumination conditions. It is shown that the expression of the transcripts and proteins of the ATJ8 gene is primarily regulated at the level of transcription. When plants were incubated under high light for 3h, both the transcripts and proteins were completely abolished. Upon transfer of plants to darkness, the transcripts started rapidly accumulating, and subsequently, the AtJ8 protein became visible after 2h in darkness. Conversely, incubation of plants in darkness or under low light intensities induced expression of the ATJ8 transcripts and proteins. Feeding plants with sugars clearly decreased the transcript and protein levels, and incubation with cycloheximide revealed a rapid turnover for AtJ8 in darkness. Moreover, the AtJ8 protein was found to be nearly missing from the var1 mutant, which lacks the FTSH5 protease. It is concluded that AtJ8 is expressed mainly in darkness, is prone to a rapid turnover but is partially stabilized by the FTSH proteases.

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.

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI–LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI–LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.

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.

Plants need tight regulation of photosynthetic electron transport for survival and growth under environmental and metabolic conditions. For this purpose, the linear electron transport (LET) pathway is supplemented by a number of alternative electron transfer pathways and valves. In Arabidopsis, cyclic electron transport (CET) around photosystem I (PSI), which recycles electrons from ferrodoxin to plastoquinone, is the most investigated alternative route. However, the interdependence of LET and CET and the relative importance of CET remain unclear, largely due to the difficulties in precise assessment of the contribution of CET in the presence of LET, which dominates electron flow under physiological conditions. We therefore generated Arabidopsis mutants with a minimal water-splitting activity, and thus a low rate of LET, by combining knockout mutations in PsbO1, PsbP2, PsbQ1, PsbQ2, and PsbR loci. The resulting Δ5 mutant is viable, although mature leaves contain only ∼ 20% of wild-type naturally less abundant PsbO2 protein. Δ5 plants compensate for the reduction in LET by increasing the rate of CET, and inducing a strong non-photochemical quenching (NPQ) response during dark-to-light transitions. To identify the molecular origin of such a high-capacity CET, we constructed three sextuple mutants lacking the qE component of NPQ (Δ5 npq4-1), NDH-mediated CET (Δ5 crr4-3), or PGR5-PGRL1-mediated CET (Δ5 pgr5). Their analysis revealed that PGR5-PGRL1-mediated CET plays a major role in ΔpH formation and induction of NPQ in C3 plants. Moreover, while pgr5 dies at the seedling stage under fluctuating light conditions, Δ5 pgr5 plants are able to survive, which underlines the importance of PGR5 in modulating the intersystem electron transfer.

Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens. We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV’s biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV’s role was taken by other alternative electron routes.

Vascular plants have evolved a long-term light acclimation strategy primarily relying on the regulation of the relative amounts of light-harvesting complex II (LHCII) and of the two photosystems, photosystem I (PSI) and photosystem II (PSII). We investigated whether such a model is also valid in Selaginella martensii, a species belonging to the early diverging group of lycophytes. Selaginella martensii plants were acclimated to three natural light regimes (extremely low light (L), medium light (M) and full sunlight (H)) and thylakoid organization was characterized combining ultrastructural, biochemical and functional methods. From L to H plants, thylakoid architecture was rearranged from (pseudo)lamellar to predominantly granal, the PSII : PSI ratio changed in favour of PSI, and the photochemical capacity increased. However, regulation of light harvesting did not occur through variations in the amount of free LHCII, but rather resulted from the flexibility of the association of free LHCII with PSII and PSI. In lycophytes, the free interspersed LHCII serves a fixed proportion of reaction centres, either PSII or PSI, and the regulation of PSI-LHCII(-PSII) megacomplexes is an integral part of long-term acclimation. Free LHCII ensures photoprotection of PSII, allows regulated use of PSI as an energy quencher, and can also quench endangered PSI.

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The DeltaPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y(D)(ox) radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S(2)Q(A)(-) and S(2)Q(B)(-) charge recombinations were stabilized in DeltaPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y(D)(+)Q(A)(-) recombination, pointed to the donor side modifications in DeltaPsbR. EPR measurements revealed that S(1)-to-S(2)-transition and S(2)-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q(A) to Q(B) electron transfer in DeltaPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.

In Arabidopsis thaliana, the chloroplast-targeted enzyme ferredoxin-NADP+-oxidoreductase (FNR) exists as two isoforms, AtLFNR1 and AtLFNR2, encoded by the genes At5g66190 and At1g20020, respectively. Both isoforms are evenly distributed between the thylakoids and soluble stroma, and they are separated by two-dimensional electrophoresis in four distinct spots, suggesting post-translational modification of both isoforms. To reveal the functional specificity of AtLFNR1, we have characterized the T-DNA insertion mutants with an interrupted At5g66190 gene. Absence of AtLFNR1 resulted in a reduced size of the rosette with pale green leaves, which was accompanied by a low content of chlorophyll and light-harvesting complex proteins. Also the photosystem I/photosystem II (PSI/PSII) ratio was significantly lower in the mutant, but the PSII activity, measured as the F(V)/F(M) ratio, remained nearly unchanged and the excitation pressure of PSII was lower in the mutants than in the wild type. A slow re-reduction rate of P700 measured in the mutant plants suggested that AtLFNR1 is involved in PSI-dependent cyclic electron flow. Impaired function of FNR also resulted in decreased capacity for carbon fixation, whereas nitrogen metabolism was upregulated. In the absence of AtLFNR1, we found AtLFNR2 exclusively in the stroma, suggesting that AtLFNR1 is required for membrane attachment of FNR. Structural modeling supports the formation of a AtLFNR1-AtLFNR2 heterodimer that would mediate the membrane attachment of AtLFNR2. Dimer formation, in turn, might regulate the distribution of electrons between the cyclic and linear electron transfer pathways according to environmental cues.

Photosynthetic electron flow operates in two modes, linear and cyclic. In cyclic electron flow (CEF), electrons are recycled around photosystem I. As a result, a transthylakoid proton gradient (ΔpH) is generated, leading to the production of ATP without concomitant production of NADPH, thus increasing the ATP/NADPH ratio within the chloroplast. At least two routes for CEF exist: a PROTON GRADIENT REGULATION5-PGRL1-and a chloroplast NDH-like complex mediated pathway. This review focuses on recent findings concerning the characteristics of both CEF routes in higher plants, with special emphasis paid on the crucial role of CEF in under challenging environmental conditions and developmental stages.

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.

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.

To study the synthesis and assembly of multisubunit thylakoid protein complexes, we performed [35S]Met pulse and chase experiments with isolated chloroplasts and intact leaves of spinach (Spinacia oleracea L.), followed by Blue Native gel separation of the (sub)complexes and subsequent identification of the newly synthesized and assembled protein subunits. PSII (photosystem II) core subunits were the most intensively synthesized proteins, particularly in vitro and at high light intensities in vivo, and could be sequestered in several distinct PSII subassemblies. Newly synthesized D1 was first found in the reaction centre complex that also contained labelled D2 and two labelled low-molecular-mass proteins. The next biggest PSII subassembly contained CP47 also. Then PsbH was assembled together with at least two other labelled chloroplast-encoded low-molecular-mass subunits, PsbM and PsbTc, and a nuclear-encoded PsbR. Subsequently, CP43 was inserted into the PSII complex concomitantly with PsbK. These assembly steps seemed to be essential for the dimerization of PSII core monomers. Intact PSII core monomer was the smallest subcomplex harbouring the newly synthesized 33 kDa oxygen-evolving complex protein PsbO. Nuclear-encoded PsbW was synthesized only at low light intensities concomitantly with Lhcb polypeptides and was distinctively present in PSII–LHCII (where LHC stands for light-harvesting complex) supercomplexes. The PsbH protein, on the contrary, was vigorously synthesized and incorporated into PSII core monomers together with the D1 protein, suggesting an intrinsic role for PsbH in the photoinhibition-repair cycle of PSII.

Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition-repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII–light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in ΔpsbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII–LHCII supercomplexes. The ΔpsbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.

Thylakoid energy metabolism is crucial for plant growth, development and acclimation. Non-appressed thylakoids harbor several high molecular mass pigment-protein megacomplexes that have flexible compositions depending upon the environmental cues. This composition is important for dynamic energy balancing in photosystems (PS) I and II. We analysed the megacomplexes of Arabidopsis wild type (WT) plants and of several thylakoid regulatory mutants. The stn7 mutant, which is defective in phosphorylation of the light-harvesting complex (LHC) II, possessed a megacomplex composition that was strikingly different from that of the WT. Of the nine megacomplexes in total for the non-appressed thylakoids, the largest megacomplex in particular was less abundant in the stn7 mutant under standard growth conditions. This megacomplex contains both PSI and PSII and was recently shown to allow energy spillover between PSII and PSI (Nat. Commun., 6, 2015, 6675). The dynamics of the megacomplex composition was addressed by exposing plants to different light conditions prior to thylakoid isolation. The megacomplex pattern in the WT was highly dynamic. Under darkness or far red light it showed low levels of LHCII phosphorylation and resembled the stn7 pattern; under low light, which triggers LHCII phosphorylation, it resembled that of the tap38/pph1 phosphatase mutant. In contrast, solubilization of the entire thylakoid network with dodecyl maltoside, which efficiently solubilizes pigment-protein complexes from all thylakoid compartments, revealed that the pigment-protein composition remained stable despite the changing light conditions or mutations that affected LHCII (de)phosphorylation. We conclude that the composition of pigment-protein megacomplexes specifically in non-appressed thylakoids undergoes redox-dependent changes, thus facilitating maintenance of the excitation balance between the two photosystems upon changes in light conditions.

Summary Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light-harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non-photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1-2. This protein, which showed light intensity-dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light-induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI–PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.

†These authors contributed equally to this work. SUMMARY The oxygen-evolving complex of eukaryotic photosystem II (PSII) consists of four extrinsic subunits, PsbO (33 kDa), PsbP (23 kDa), PsbQ (17 kDa) and PsbR (10 kDa), encoded by seven nuclear genes, PsbO1 (At5g66570), PsbO2 (At3g50820), PsbP1 (At1g06680), PsbP2 (At2g30790), PsbQ1 (At4g21280), PsbQ2 (At4g05180) and PsbR (At1g79040). Using Arabidopsis insertion mutant lines, we show that PsbP1, but not PsbP2, is essential for photoautotrophic growth, whereas plants lacking both forms of PsbQ and/or PsbR show normal growth rates. Complete elimination of PsbQ has a minor effect on PSII function, but plants lacking PsbR or both PsbR and PsbQ are characterized by more pronounced defects in PSII activity. Gene expression and immunoblot analyses indicate that accumulation of each of these proteins is highly dependent on the presence of the others, and is controlled at the post-transcriptional level, whereas PsbO stability appears to be less sensitive to depletion of other subunits of the oxygen-evolving complex. In addition, comparison of levels of the PSII super-complex in wild-type and mutant leaves reveals the importance of the individual subunits of the oxygen-evolving complex for the supramolecular organization of PSII and their influence on the rate of state transitions.

Thylakoid membrane-bound FtsH proteases have a well-characterized role in degradation of the photosystem II (PSII) reaction center protein D1 upon repair of photodamaged PSII. Here, we show that the Arabidopsis (Arabidopsis thaliana) var1 and var2 mutants, devoid of the FtsH5 and FtsH2 proteins, respectively, are capable of normal D1 protein turnover under moderate growth light intensity. Instead, they both demonstrate a significant scarcity of PSI complexes. It is further shown that the reduced level of PSI does not result from accelerated photodamage of the PSI centers in var1 or var2 under moderate growth light intensity. On the contrary, radiolabeling experiments revealed impaired synthesis of the PsaA/B reaction center proteins of PSI, which was accompanied by the accumulation of PSI-specific assembly factors. psaA/B transcript accumulation and translation initiation, however, occurred in var1 and var2 mutants as in wild-type Arabidopsis, suggesting problems in later stages of PsaA/B protein expression in the two var mutants. Presumably, the thylakoid membrane-bound FtsH5 and FtsH2 have dual functions in the maintenance of photosynthetic complexes. In addition to their function as a protease in the degradation of the photodamaged D1 protein, they also are required, either directly or indirectly, for early assembly of the PSI complexes.

In a plant's natural environment, the intensity of light can change rapidly due to sunflecks, cloudiness and intermittent shading. Fluctuations between high and low illumination phases expose the photosynthetic machinery to rapidly changing signals that can be overlapping or contradictory, and accordingly plants have developed astute acclimation strategies to maintain optimal photosynthetic performance in these conditions. Continuous exposure to high light induces an array of protective mechanisms at anatomical, chemical and molecular levels, but when high light phases are short, such as under fluctuating light conditions, the protective strategies that afford protection to constant high light are not employed by plants. One mechanism that is engaged under both constant and fluctuating high light is the photosynthetic control of the Cyt b 6f complex, which prevents hyper-reduction of the electron transfer chain in order to protect PSI from photodamage. The PGR5 protein was recently shown to play an indispensable role in this protective mechanism. This review revisits the findings of earlier studies into photosynthetic control and places PGR5 within the broader context of photoprotection and light acclimation strategies.

Phosphorylation of the major photosynthetic light harvesting antenna proteins by STN7 kinase balances excitation between PSII and PSI. Phosphorylation of such abundant proteins is unique, differing distinctively from conventional tasks of protein kinases in phosphorylation of low abundance proteins in signalling cascades. Excitation balance between PSII and PSI is critical for redox homeostasis between the plastoquinone and plastocyanin pools and PSI electron acceptors, determining the capacity of the thylakoid membrane to produce reactive oxygen species (ROS) that operate as signals relaying information between chloroplasts and other cellular compartments. STN7 has also been proposed to be a conventional signalling kinase, instigating the phosphorylation cascade required for coordinated expression of photosynthesis genes and assembly of the photosynthetic machinery. The absence of STN7 kinase, however, does not prevent plants from sensing redox imbalance and adjusting the stoichiometry of the photosynthetic machinery to restore redox homeostasis. This suggests that STN7 is not essential for signalling between the chloroplast and the nucleus. Here we discuss the evolution and functions of the STN7 and other thylakoid protein kinases and phosphatases, and the inherent difficulties in analysing signalling cascades initiated from the photosynthetic machinery. Based on our analyses of literature and publicly available expression data, we conclude that STN7 exerts it signalling effect primarily by controlling chloroplast ROS homeostasis through maintaining steady-state phosphorylation of the light-harvesting II proteins and the redox balance in the thylakoid membrane. ROS are important signalling molecules with a direct effect on the development of jasmonate, which in turn relays information out from the chloroplast. We propose that thylakoid membrane redox homeostasis, regulated by SNT7, sends cell-wide signals that reprogram the entire hormonal network in the cell.

Light-harvesting complex II (LHCII) contains three highly homologous chlorophyll-a/b-binding proteins (Lhcb1, Lhcb2 and Lhcb3), which can be assembled into both homo- and heterotrimers. Lhcb1 and Lhcb2 are reversibly phosphorylated by the action of STN7 kinase and PPH1/TAP38 phosphatase in the so-called state-transition process. We have developed antibodies that are specific for the phosphorylated forms of Lhcb1 and Lhcb2. We found that Lhcb2 is more rapidly phosphorylated than Lhcb1: 10sec of state 2 light' results in Lhcb2 phosphorylation to 30% of the maximum level. Phosphorylated and non-phosphorylated forms of the proteins showed no difference in electrophoretic mobility and dephosphorylation kinetics did not differ between the two proteins. In state 2, most of the phosphorylated forms of Lhcb1 and Lhcb2 were present in super- and mega-complexes that comprised both photosystem (PS)I and PSII, and the state 2-specific PSI-LHCII complex was highly enriched in the phosphorylated forms of Lhcb2. Our results imply distinct and specific roles for Lhcb1 and Lhcb2 in the regulation of photosynthetic light harvesting. This work was supported by the European Union project FP7-PEO-PLE-ITN-2008 'HARVEST: Control of Light Use Efficiency in Plants and Algae From Light to Harvest', the Swedish Research Council (VR), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas), the Academy of Finland (project Nos 118637 and 138703) and C. Leoni by a 1-year fellowship from the University of Bari (Rectoral Decree No. 1598). The authors declare no conflict of interest.

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.

Photosynthetic machinery requires balanced distribution of excitation energy from the light-harvesting complexes to photosystems. The efficiency of light-harvesting is regulated by thermal dissipation of excess energy, while the distribution of energy between photosystems is dependent on STN7 kinase and phosphorylation of thylakoid proteins. The regulation of excitation energy transfer has been linked to the lateral segregation of photosynthetic complexes along the thylakoid membrane. The study of photosynthetic regulation mechanisms using Arabidopsis mutants, which have been available for the last ten years, has challenged traditional views on regulation of excitation energy distribution. Here, we discuss an urgent need to create a holistic view of the dynamics of the thylakoid membrane using systematic research of the mutants available today.

Photosynthetic machinery in the thylakoid membrane is prone to modifications depending on -environmental, developmental, and morphological parameters. Such plasticity in the composition of the thylakoid membrane protein complexes guarantees efficient function of the photosynthetic machinery. In this chapter, we describe methods for separation of thylakoid membrane protein complexes at high resolution by two-dimensional gel electrophoretic systems. Solubilization of the thylakoid membrane protein complexes either by dodecylmaltoside or digitonin is described first. Then, two partially overlapping -methods, blue native gel electrophoresis and high-resolution clear native gel electrophoresis, are demonstrated to separate the individual protein complexes. Finally, denaturing SDS-polyacrylamide gel electrophoresis is used to reveal the protein composition of each complex. Critical points in all protocols are addressed and representative examples of the composition of Arabidopsis thaliana thylakoid membrane protein complexes are shown.

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.

Photosynthetic machinery in the thylakoid membrane is prone to modifications depending on -environmental, developmental, and morphological parameters. Such plasticity in the composition of the thylakoid membrane protein complexes guarantees efficient function of the photosynthetic machinery. In this chapter, we describe methods for separation of thylakoid membrane protein complexes at high resolution by two-dimensional gel electrophoretic systems. Solubilization of the thylakoid membrane protein complexes either by dodecylmaltoside or digitonin is described first. Then, two partially overlapping -methods, blue native gel electrophoresis and high-resolution clear native gel electrophoresis, are demonstrated to separate the individual protein complexes. Finally, denaturing SDS-polyacrylamide gel electrophoresis is used to reveal the protein composition of each complex. Critical points in all protocols are addressed and representative examples of the composition of Arabidopsis thaliana thylakoid membrane protein complexes are shown.

Background DnaJ proteins participate in many metabolic pathways through dynamic interactions with various components of these processes. The role of three small chloroplast-targeted DnaJ proteins, AtJ8 (At1 g80920), AtJ11 (At4 g36040) and AtJ20 (At4 g13830), was investigated here using knock-out mutants of Arabidopsis thaliana. Photochemical efficiency, capacity of CO2 assimilation, stabilization of Photosystem (PS) II dimers and supercomplexes under high light illumination, energy distribution between PSI and PSII and phosphorylation of PSII-LHCII proteins, global gene expression profiles and oxidative stress responses of these DnaJ mutants were analyzed. Results Knockout of one of these proteins caused a series of events including a decrease in photosynthetic efficiency, destabilization of PSII complexes and loss of control for balancing the redox reactions in chloroplasts. Data obtained with DNA microarray analysis demonstrated that the lack of one of these DnaJ proteins triggers a global stress response and therefore confers the plants greater tolerance to oxidative stress induced by high light or methyl viologen treatments. Expression of a set of genes encoding enzymes that detoxify reactive oxygen species (ROS) as well as a number of stress-related transcription factors behaved in the mutants at growth light similarly to that when wild-type (WT) plants were transferred to high light. Also a set of genes related to redox regulation were upregulated in the mutants. On the other hand, although the three DnaJ proteins reside in chloroplasts, the expression of most genes encoding thylakoid membrane proteins was not changed in the mutants. Conclusion It is proposed that the tolerance of the DnaJ protein knockout plants to oxidative stress occurs at the expense of the flexibility of photosynthetic reactions. Despite the fact that the effects of the individual protein knockout on the response of plants to high light treatment are quite similar, it is conceivable that both specific- and cross-talk functions exist between the three small chloroplast-targeted DnaJ proteins, AtJ8, AtJ11 and AtJ20.

Chloroplast proteins that regulate the biogenesis, performance and acclimation of the photosynthetic protein complexes are currently under intense research. Dozens, possibly even hundreds, of such proteins in the stroma, thylakoid membrane and the lumen assist the biogenesis and constant repair of the water splitting photosystem (PS) II complex. During the repair cycle, assistance is required at several levels including the degradation of photodamaged D1 protein, de novo synthesis, membrane insertion, folding of the nascent protein chains and the reassembly of released protein subunits and different co-factors into PSII in order to guarantee the maintenance of the PSII function. Here we review the present knowledge of the auxiliary proteins, which have been reported to be involved in the biogenesis and maintenance of PSII.

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.

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 ΔTLP18.3 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 ΔTLP18.3 plants showed no clear visual phenotype under standard growth conditions, but when challenged by fluctuating light during growth, the retarded growth of ΔTLP18.3 plants was evident.

The oxygen-evolving complex (OEC) of higher plant photosystem II (PSII) consists of an inorganic Mn(4)Ca cluster and three nuclear-encoded proteins, PsbO, PsbP and PsbQ. In this review, we focus on the assembly of these OEC proteins, and especially on the role of the small intrinsic PSII proteins and recently found "novel" PSII proteins in the assembly process. The numerous auxiliary functions suggested during the past few years for the OEC proteins will likewise be discussed. For example, besides being a manganese-stabilizing protein, PsbO has been found to bind calcium and GTP and possess a carbonic anhydrase activity. In addition, specific roles have been suggested for the two isoforms of the PsbO protein in Arabidopsis thaliana. PsbP and PsbQ seem to play an additional role in the formation of PSII supercomplexes and in grana stacking, besides their originally recognized role in providing a proper calcium and chloride ion concentration for water splitting.