Thylakoid protein FPB1 synergistically cooperates with PAM68 to promote CP47 biogenesis and Photosystem II assembly.

In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68. FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex.

The thylakoid proton antiporter KEA3 regulates photosynthesis in response to the chloroplast energy status.

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.

How Did Thylakoids Emerge in Cyanobacteria, and How Were the Primary Chloroplast and Chromatophore Acquired?

The emergence of thylakoid membranes in cyanobacteria is a key event in the evolution of all oxygenic photosynthetic cells, from prokaryotes to eukaryotes. Recent analyses show that they could originate from a unique lipid phase transition rather than from a supposed vesicular budding mechanism. Emergence of thylakoids coincided with the great oxygenation event, more than two billion years ago. The acquisition of semi-autonomous organelles, such as the mitochondrion, the chloroplast, and, more recently, the chromatophore, is a critical step in the evolution of eukaryotes. They resulted from primary endosymbiotic events that seem to share general features, i.e., an acquisition of a bacterium/cyanobacteria likely via a phagocytic membrane, a genome reduction coinciding with an escape of genes from the organelle to the nucleus, and, finally, the appearance of an active system translocating nuclear-encoded proteins back to the organelles. An intense mobilization of foreign genes of bacterial origin, via horizontal gene transfers, plays a critical role. Some third partners, like Chlamydia, might have facilitated the transition from cyanobacteria to the early chloroplast. This chapter further details our current understanding of primary endosymbiosis, focusing on primary chloroplasts, thought to have appeared over a billion years ago, and the chromatophore, which appeared around a hundred years ago.

Purification of Chloroplast Envelope, Thylakoids, and Stroma from Angiosperm Leaves.

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been studied, and its involvement in state transitions and PSII repair is known. Yet, little is known about the phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, we applied proteomics analysis to generate a map of the phosphorylation sites of the PSI-LHCI proteins in Chlorella ohadii cells that were grown under low or extreme high-light intensities (LL and HL). Furthermore, we analyzed the content of oxidized tryptophans and PSI-LHCI protein degradation products in these cells, to estimate the light-induced damage to PSI-LHCI. Our work revealed the phosphorylation of 17 of 22 PSI-LHCI subunits. The analyses detected the extensive phosphorylation of the LHCI subunits Lhca6 and Lhca7, which is modulated by growth light intensity. Other PSI-LHCI subunits were phosphorylated to a lesser extent, including PsaE, where molecular dynamic simulation proposed that a phosphoserine stabilizes ferredoxin binding. Additionally, we show that HL-grown cells accumulate less oxidative damage and degradation products of PSI-LHCI proteins, compared with LL-grown cells. The significant phosphorylation of Lhca6 and Lhca7 at the interface with other LHCI subunits suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other subunits.

Inter-subunit energy transfer processes in a minimal plant photosystem II supercomplex.

Photosystem II (PSII) is an integral part of the photosynthesis machinery, in which several light-harvesting complexes rely on inter-complex excitonic energy transfer (EET) processes to channel energy to the reaction center. In this paper, we report on a direct observation of the inter-complex EET in a minimal PSII supercomplex from plants, containing the trimeric light-harvesting complex II (LHCII), the monomeric light-harvesting complex CP26, and the monomeric PSII core complex. Using two-dimensional (2D) electronic spectroscopy, we measure an inter-complex EET timescale of 50 picoseconds for excitations from the LHCII-CP26 peripheral antenna to the PSII core. The 2D electronic spectra also reveal that the transfer timescale is nearly constant over the pump spectrum of 600 to 700 nanometers. Structure-based calculations reveal the contribution of each antenna complex to the measured inter-complex EET time. These results provide a step in elucidating the full inter-complex energy transfer network of the PSII machinery.

Thylakoid membrane stacking controls electron transport mode during the dark-to-light transition by adjusting the distances between PSI and PSII.

The balance between linear electron transport (LET) and cyclic electron transport (CET) plays an essential role in plant adaptation and protection against photo-induced damage. This balance is largely maintained by phosphorylation-driven alterations in the PSII-LHCII assembly and thylakoid membrane stacking. During the dark-to-light transition, plants shift this balance from CET, which prevails to prevent overreduction of the electron transport chain and consequent photo-induced damage, towards LET, which enables efficient CO2 assimilation and biomass production. Using freeze-fracture cryo-scanning electron microscopy and transmission electron microscopy of Arabidopsis leaves, we reveal unique membrane regions possessing characteristics of both stacked and unstacked regions of the thylakoid network that form during this transition. A notable consequence of the morphological attributes of these regions, which we refer to as 'stacked thylakoid doublets', is an overall increase in the proximity and connectivity of the two photosystems (PSI and PSII) that drive LET. This, in turn, reduces diffusion distances and barriers for the mobile carriers that transfer electrons between the two PSs, thereby maximizing LET and optimizing the plant's ability to utilize light energy. The mechanics described here for the shift between CET and LET during the dark-to-light transition are probably also used during chromatic adaptation mediated by state transitions.

The role of EGY2 protease in response to high light stress.

In this study, we investigated the importance of one of the intramembrane proteases, EGY2, for the proper functioning of PSII under short-term high light stress conditions. EGY2 is a chloroplast intramembrane protease of the S2P family, whose absence in Arabidopsis thaliana affects PSII protein composition. The egy2 mutants exhibited a slower degradation of PsbA and decreased content of PsbC and PsbD. During exposure to high light stress, these stoichiometric changes affect the functional state of PSII, leading to its higher sensitivity to photoinhibition of the PSII reaction centre and increased heat dissipation. Furthermore, we explored the relationship between EGY2 and the pTAC16 transcription factor, which is a potential EGY2 substrate. Under light stress, WT plants showed decreased levels of pTAC16, while it remained unchanged in the egy2 mutants. This finding suggests that EGY2 may release pTAC16 from thylakoid membranes through proteolytic cleavage. We also confirmed the physical interaction between EGY2 and pTAC16 using the yeast two-hybrid system, providing evidence of EGY2's involvement in the regulation of PsbA and PsbC/PsbD operons by releasing pTAC16 from the thylakoid membrane.

Cold priming on pathogen susceptibility in the Arabidopsis eds1 mutant background requires a functional stromal Ascorbate Peroxidase.

24 h cold exposure (4°C) is sufficient to reduce pathogen susceptibility in Arabidopsis thaliana against the virulent Pseudomonas syringae pv. tomato (Pst) strain even when the infection occurs five days later. This priming effect is independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) and can be observed in the immune-compromised eds1-2 null mutant. In contrast, cold priming-reduced Pst susceptibility is strongly impaired in knock-out lines of the stromal and thylakoid ascorbate peroxidases (sAPX/tAPX) highlighting their relevance for abiotic stress-related increased immune resilience. Here, we extended our analysis by generating an eds1 sapx double mutant. eds1 sapx showed eds1-like resistance and susceptibility phenotypes against Pst strains containing the effectors avrRPM1 and avrRPS4. In comparison to eds1-2, susceptibility against the wildtype Pst strain was constitutively enhanced in eds1 sapx. Although a prior cold priming exposure resulted in reduced Pst titers in eds1-2, it did not alter Pst resistance in eds1 sapx. This demonstrates that the genetic sAPX requirement for cold priming of basal plant immunity applies also to an eds1 null mutant background.

Oldest thylakoids in fossil cells directly evidence oxygenic photosynthesis.

Today oxygenic photosynthesis is unique to cyanobacteria and their plastid relatives within eukaryotes. Although its origin before the Great Oxidation Event is still debated1-4, the accumulation of O2 profoundly modified the redox chemistry of the Earth and the evolution of the biosphere, including complex life. Understanding the diversification of cyanobacteria is thus crucial to grasping the coevolution of our planet and life, but their early fossil record remains ambiguous5. Extant cyanobacteria include the thylakoid-less Gloeobacter-like group and the remainder of cyanobacteria that acquired thylakoid membranes6,7. The timing of this divergence is indirectly estimated at between 2.7 and 2.0 billion years ago (Ga) based on molecular clocks and phylogenies8-11 and inferred from the earliest undisputed fossil record of Eoentophysalis belcherensis, a 2.018-1.854 Ga pleurocapsalean cyanobacterium preserved in silicified stromatolites12,13. Here we report the oldest direct evidence of thylakoid membranes in a parallel-to-contorted arrangement within the enigmatic cylindrical microfossils Navifusa majensis from the McDermott Formation, Tawallah Group, Australia (1.78-1.73 Ga), and in a parietal arrangement in specimens from the Grassy Bay Formation, Shaler Supergroup, Canada (1.01-0.9 Ga). This discovery extends their fossil record by at least 1.2 Ga and provides a minimum age for the divergence of thylakoid-bearing cyanobacteria at roughly 1.75 Ga. It allows the unambiguous identification of early oxygenic photosynthesizers and a new redox proxy for probing early Earth ecosystems, highlighting the importance of examining the ultrastructure of fossil cells to decipher their palaeobiology and early evolution.

CAH3 from Chlamydomonas reinhardtii: Unique Carbonic Anhydrase of the Thylakoid Lumen.

CAH3 is the only carbonic anhydrase (CA) present in the thylakoid lumen of the green algae Chlamydomonas reinhardtii. The monomer of the enzyme has a molecular weight of ~29.5 kDa with high CA activity. Through its dehydration activity, CAH3 can be involved either in the carbon-concentrating mechanism supplying CO2 for RuBisCO in the pyrenoid or in supporting the maximal photosynthetic activity of photosystem II (PSII) by accelerating the removal of protons from the active center of the water-oxidizing complex. Both proposed roles are considered in this review, together with a description of the enzymatic parameters of native and recombinant CAH3, the crystal structure of the protein, and the possible use of lumenal CA as a tool for increasing biomass production in higher plants. The identified involvement of lumenal CAH3 in the function of PSII is still unique among green algae and higher plants and can be used to understand the mechanism(s) of the functional interconnection between PSII and the proposed CA(s) of the thylakoid lumen in other organisms.

Biosynthesis of phosphatidylglycerol in photosynthetic organisms.

Phosphatidylglycerol (PG) is a unique phospholipid class with its indispensable role in photosynthesis and growth in land plants, algae, and cyanobacteria. PG is the only major phospholipid in the thylakoid membrane of cyanobacteria and plant chloroplasts and a main lipid component in photosynthetic protein-cofactor complexes such as photosystem I and photosystem II. In plants and algae, PG is also essential as a substrate for the biosynthesis of cardiolipin, which is a unique lipid present only in mitochondrial membranes and crucial for the functions of mitochondria. PG biosynthesis pathways in plants include three membranous organelles, plastids, mitochondria, and the endoplasmic reticulum in a complex manner. While the molecular biology underlying the role of PG in photosynthetic functions is well established, many enzymes responsible for the PG biosynthesis are only recently cloned and functionally characterized in the model plant species including Arabidopsis thaliana and Chlamydomonas reinhardtii and cyanobacteria such as Synechocystis sp. PCC 6803. The characterization of those enzymes helps understand not only the metabolic flow for PG production but also the crosstalk of biosynthesis pathways between PG and other lipids. This review aims to summarize recent advances in the understanding of the PG biosynthesis pathway and functions of involved enzymes.

A thylakoid biogenesis BtpA protein is required for the initial step of tetrapyrrole biosynthesis in cyanobacteria.

Biogenesis of the photosynthetic apparatus requires complicated molecular machinery, individual components of which are either poorly characterized or unknown. The BtpA protein has been described as a factor required for the stability of photosystem I (PSI) in cyanobacteria; however, how the BtpA stabilized PSI remains unexplained. To clarify the role of BtpA, we constructed and characterized the btpA-null mutant (ΔbtpA) in the cyanobacterium Synechocystis sp. PCC 6803. The mutant contained only c. 1% of chlorophyll and nearly no thylakoid membranes. However, this strain, growing only in the presence of glucose, was genetically unstable and readily generated suppressor mutations that restore the photoautotrophy. Two suppressor mutations were mapped into the hemA gene encoding glutamyl-tRNA reductase (GluTR) - the first enzyme of tetrapyrrole biosynthesis. Indeed, the GluTR was not detectable in the ΔbtpA mutant and the suppressor mutations restored biosynthesis of tetrapyrroles and photoautotrophy by increased GluTR expression or by improved GluTR stability/processivity. We further demonstrated that GluTR associates with a large BtpA oligomer and that BtpA is required for the stability of GluTR. Our results show that the BtpA protein is involved in the biogenesis of photosystems at the level of regulation of tetrapyrrole biosynthesis.

Quantifying the Energy Spillover between Photosystems II and I in Cyanobacterial Thylakoid Membranes and Cells.

The spatial separation of photosystems I and II (PSI and PSII) is thought to be essential for efficient photosynthesis by maintaining a balanced flow of excitation energy between them. Unlike the thylakoid membranes of plant chloroplasts, cyanobacterial thylakoids do not form tightly appressed grana stacks that enforce strict lateral separation. The coexistence of the two photosystems provides a ground for spillover-excitation energy transfer from PSII to PSI. Spillover has been considered as a pathway of energy transfer from the phycobilisomes to PSI and may also play a role in state transitions as means to avoid overexcitation of PSII. Here, we demonstrate a significant degree of energy spillover from PSII to PSI in reconstituted membranes and isolated thylakoid membranes of Thermosynechococcus (Thermostichus) vulcanus and Synechocystis sp. PCC 6803 by steady-state and time-resolved fluorescence spectroscopy. The quantum yield of spillover in these systems was determined to be up to 40%. Spillover was also found in intact cells but to a considerably lower degree (20%) than in isolated thylakoid membranes. The findings support a model of coexistence of laterally separated microdomains of PSI and PSII in the cyanobacterial cells as well as domains where the two photosystems are energetically connected. The methodology presented here can be applied to probe spillover in other photosynthetic organisms.

Unravelling the fluorescence kinetics of light-harvesting proteins with simulated measurements.

The plant light-harvesting pigment-protein complex LHCII is the major antenna sub-unit of PSII and is generally (though not universally) accepted to play a role in photoprotective energy dissipation under high light conditions, a process known Non-Photochemical Quenching (NPQ). The underlying mechanisms of energy trapping and dissipation within LHCII are still debated. Various models have been proposed for the underlying molecular detail of NPQ, but they are often based on different interpretations of very similar transient absorption measurements of isolated complexes. Here we present a simulated measurement of the fluorescence decay kinetics of quenched LHCII aggregates to determine whether this relatively simple measurement can discriminate between different potential NPQ mechanisms. We simulate not just the underlying physics (excitation, energy migration, quenching and singlet-singlet annihilation) but also the signal detection and typical experimental data analysis. Comparing this to a selection of published fluorescence decay kinetics we find that: (1) Different proposed quenching mechanisms produce noticeably different fluorescence kinetics even at low (annihilation free) excitation density, though the degree of difference is dependent on pulse width. (2) Measured decay kinetics are consistent with most LHCII trimers becoming relatively slow excitation quenchers. A small sub-population of very fast quenchers produces kinetics which do not resemble any observed measurement. (3) It is necessary to consider at least two distinct quenching mechanisms in order to accurately reproduce experimental kinetics, supporting the idea that NPQ is not a simple binary switch.

A structure of the relict phycobilisome from a thylakoid-free cyanobacterium.

Phycobilisomes (PBS) are antenna megacomplexes that transfer energy to photosystems II and I in thylakoids. PBS likely evolved from a basic, inefficient form into the predominant hemidiscoidal shape with radiating peripheral rods. However, it has been challenging to test this hypothesis because ancestral species are generally inaccessible. Here we use spectroscopy and cryo-electron microscopy to reveal a structure of a "paddle-shaped" PBS from a thylakoid-free cyanobacterium that likely retains ancestral traits. This PBS lacks rods and specialized ApcD and ApcF subunits, indicating relict characteristics. Other features include linkers connecting two chains of five phycocyanin hexamers (CpcN) and two core subdomains (ApcH), resulting in a paddle-shaped configuration. Energy transfer calculations demonstrate that chains are less efficient than rods. These features may nevertheless have increased light absorption by elongating PBS before multilayered thylakoids with hemidiscoidal PBS evolved. Our results provide insights into the evolution and diversification of light-harvesting strategies before the origin of thylakoids.

The photosystem-II repair cycle: updates and open questions.

MAIN CONCLUSION: The photosystem-II (PSII) repair cycle is essential for the maintenance of photosynthesis in plants. A number of novel findings have illuminated the regulatory mechanisms of the PSII repair cycle. Photosystem II (PSII) is a large pigment-protein complex embedded in the thylakoid membrane. It plays a vital role in photosynthesis by absorbing light energy, splitting water, releasing molecular oxygen, and transferring electrons for plastoquinone reduction. However, PSII, especially the PsbA (D1) core subunit, is highly susceptible to oxidative damage. To prevent irreversible damage, plants have developed a repair cycle. The main objective of the PSII repair cycle is the degradation of photodamaged D1 and insertion of newly synthesized D1 into the PSII complex. While many factors are known to be involved in PSII repair, the exact mechanism is still under investigation. In this review, we discuss the primary steps of PSII repair, focusing on the proteolytic degradation of photodamaged D1 and the factors involved.

Energetic driving force for LHCII clustering in plant membranes.

Plants capture and convert solar energy in a complex network of membrane proteins. Under high light, the luminal pH drops and induces a reorganization of the protein network, particularly clustering of the major light-harvesting complex (LHCII). While the structures of the network have been resolved in exquisite detail, the thermodynamics that control the assembly and reorganization had not been determined, largely because the interaction energies of membrane proteins have been inaccessible. Here, we describe a method to quantify these energies and its application to LHCII. Using single-molecule measurements, LHCII proteoliposomes, and statistical thermodynamic modeling, we quantified the LHCII-LHCII interaction energy as ~-5 kBT at neutral pH and at least -7 kBT at acidic pH. These values revealed an enthalpic thermodynamic driving force behind LHCII clustering. Collectively, this work captures the interactions that drive the organization of membrane protein networks from the perspective of equilibrium statistical thermodynamics, which has a long and rich tradition in biology.

Locations of membrane protein production in a cyanobacterium.

Cyanobacteria show an unusually complex prokaryotic cell structure including a distinct intracytoplasmic membrane system, the thylakoid membranes that are the site of the photosynthetic light reactions. The thylakoid and plasma membranes have sharply distinct proteomes, but the mechanisms that target proteins to a specific membrane remain poorly understood. Here, we investigate the locations of translation of thylakoid and plasma membrane proteins in the model unicellular cyanobacterium Synechococcus elongatus PCC 7942. We use fluorescent in situ hybridization to probe the locations of mRNAs encoding membrane-integral proteins, plus Green Fluorescent Protein tagging of the RplL subunit to reveal the location of ribosomes under different conditions. We show that membrane-integral thylakoid and plasma membrane proteins are translated in different locations. Thylakoid membrane proteins are translated in patches at the innermost thylakoid membrane surface facing the nucleoid. However, different proteins are translated in different patches, even when they are subunits of the same multiprotein complex. This implies that translation is distributed over the proximal thylakoid surface, with newly inserted proteins migrating within the membrane prior to incorporation into complexes. mRNAs encoding plasma membrane proteins form patches at the plasma membrane. Ribosomes can be observed at similar locations near the thylakoid and plasma membranes, with more ribosomes near the plasma membrane when conditions force rapid production of plasma membrane proteins. There must be routes for ribosomes and mRNAs past the thylakoids to the plasma membrane. We infer a system to chaperone plasma membrane mRNAs to prevent their translation prior to arrival at the correct membrane. IMPORTANCE Cyanobacteria have a complex and distinct membrane system within the cytoplasm, the thylakoid membranes that house the photosynthetic light reactions. The thylakoid and plasma membranes contain distinct sets of proteins, but the steps that target proteins to the two membranes remain unclear. Knowledge of the protein sorting rules will be crucial for the biotechnological re-engineering of cyanobacterial cells, and for understanding the evolutionary development of the thylakoids. Here, we probe the subcellular locations of the mRNAs that encode cyanobacterial membrane proteins and the ribosomes that translate them. We show that thylakoid and plasma membrane proteins are produced at different locations, providing the first direct evidence for a sorting mechanism that operates prior to protein translation.