Phylogenetic viewpoints on regulation of light harvesting and electron transport in eukaryotic photosynthetic organisms

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.     

Photochemical competence of assembled photosystem II core complex in cyanobacterial plasma membrane

Cyanobacterial cells have two autonomous internal membrane systems, plasma membrane and thylakoid membrane. In these oxygenic photosynthetic organisms the assembly of the large membrane protein complex photosystem II (PSII) is an intricate process that requires the recruitment of numerous protein subunits and cofactors involved in excitation and electron transfer processes. Precise control of this assembly process is necessary because electron transfer reactions in partially assembled PSII can lead to oxidative damage and degradation of the protein complex. In this communication we demonstrate that the activation of PSII electron transfer reactions in the cyanobacterium Synechocystis sp. PCC 6803 takes place sequentially. In this organism partially assembled PSII complexes can be detected in the plasma membrane. We have determined that such PSII complexes can undergo light-induced charge separation and contain a functional electron acceptor side but not an assembled donor side. In contrast, PSII complexes in thylakoid membrane are fully assembled and capable of multiple turnovers. We conclude that PSII reaction center cores assembled in the plasma membrane are photochemically competent and can catalyze single turnovers. We propose that upon transfer of such PSII core complexes to the thylakoid membrane, additional proteins are incorporated followed by binding and activation of various donor side cofactors. Such a stepwise process protects cyanobacterial cells from potentially harmful consequences of performing water oxidation in a partially assembled PSII complex before it reaches its final destination in the thylakoid membrane.     

The yeast split-ubiquitin system to study chloroplast membrane protein interactions

Each photosynthetic complex within the thylakoid membrane consists of several different subunits. During formation of these complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. Interactions between transport/assembly factors and their specific polypeptides occur in a membraneous environment and are usually transient and short-lived. Thus, a detailed analysis of the underlying molecular mechanisms by biochemical techniques is often difficult to perform. Here, we report on the suitability of a genetic system, i.e. the yeast split-ubiquitin system, to investigate protein–protein interactions of thylakoid membrane proteins. The data confirm the previously established binding of the cpSec-translocase subunits, cpSecY and cpSecE, and the interaction of the cpSec-translocase from Arabidopsis thaliana with Alb3, a factor required for the insertion of the light-harvesting chlorophyll-binding proteins into the thylakoid membrane. In addition, the proposed interaction between D1, the reaction center protein of photosystem II and the soluble periplasmic PratA factor from Synechocystis sp. PCC 6803 was verified. A more comprehensive analysis of Alb3-interacting proteins revealed that Alb3 is able to form dimers or oligomers. Interestingly, Alb3 was also shown to bind to the PSII proteins D1, D2 and CP43, to the PSI reaction center protein PSI-A and the ATP synthase subunit CF₀III, suggesting an important role of Alb3 in the assembly of photosynthetic thylakoid membrane complexes.     

Pigment ligation to proteins of the photosynthetic apparatus in higher plants

Ligation of pigments to proteins of the thylakoid membrane is a central step in the assembly of the photosynthetic apparatus in higher plants. Because of the potentially damaging photooxidative activity of chlorophylls, it is likely that between their biosynthesis and final assembly, chlorophylls will always be bound to protein complexes in which photooxidation is prevented by quenchers such as carotenoids. Such complexes may include chlorophyll carriers and/or membrane receptors involved in protein insertion into the membrane. Many if not all pigment-protein complexes of the thylakoid are stabilised towards protease attack by bound pigments. The major light-harvesting chlorophyll a/b protein (Lhebl,2) folds into its native structure in vitro only when it binds pigments. Pigment-induced folding may also be a general feature of chlorophyll-carotenoid proteins of the photosynthetic apparatus.     

The role of lipids in photosystem II

The thylakoid membranes of photosynthetic organisms, which are the sites of oxygenic photosynthesis, are composed of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG). The identification of many genes involved in the biosynthesis of each lipid class over the past decade has allowed the generation and isolation of mutants of various photosynthetic organisms incapable of synthesizing specific lipids. Numerous studies using such mutants have revealed that deficiency of these lipids primarily affects the structure and function of photosystem II (PSII) but not of photosystem I (PSI). Recent X-ray crystallographic analyses of PSII and PSI complexes from Thermosynechococcus elongatus revealed the presence of 25 and 4 lipid molecules per PSII and PSI monomer, respectively, indicating the enrichment of lipids in PSII. Therefore, lipid molecules bound to PSII may play special roles in the assembly and functional regulation of the PSII complex. This review summarizes our present understanding of the biochemical and physiological roles of lipids in photosynthesis, with a special focus on PSII. This article is part of a Special Issue entitled: Photosystem II.     

Glycerolipids in photosynthesis: Composition,synthesis and trafficking

Glycerolipids constituting the matrix of photosynthetic membranes, from cyanobacteria to chloroplasts of eukaryotic cells, comprise monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol and phosphatidylglycerol. This review covers our current knowledge on the structural and functional features of these lipids in various cellular models, from prokaryotes to eukaryotes. Their relative proportions in thylakoid membranes result from highly regulated and compartmentalized metabolic pathways, with a cooperation, in the case of eukaryotes, of non-plastidic compartments. This review also focuses on the role of each of these thylakoid glycerolipids in stabilizing protein complexes of the photosynthetic machinery, which might be one of the reasons for their fascinating conservation in the course of evolution. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

PPIase activities and interaction partners of FK506-binding proteins in the wheat thylakoid

FK506-binding proteins (FKBPs) and cyclophilins, collectively called immunophilins, conserve peptidyl-prolyl cis/trans isomerase (PPIase) active sites, although many lack PPIase activity. The chloroplast thylakoid contains a large proportion of the plant immunophilin family, but their functions within this compartment are unclear. Some lumenal immunophilins are important for assembly of photosynthetic complexes, implicating them in the maintenance and turnover of the photosynthetic apparatus during acclimation processes. In this investigation into the functions of three FKBPs localized to the thylakoid of Triticum aestivum (wheat), we present the first evidence of PPIase activity in the thylakoid of a cereal plant, and also show that PPIase activity is not conserved in all lumenal FKBPs. Using yeast two-hybrid analysis we found that the PPIase-active FKBP13 interacts with the globular domain of the wheat Rieske protein, with potential impact on photosynthetic electron transfer. Specific interaction partners for PPIase-deficient FKBP16-1 and FKBP16-3 link these isoforms to photosystem assembly.     

Modular antenna of photosystem I in secondary plastids of red algal origin: a <Emphasis Type="Italic">Nannochloropsis oceanica</Emphasis> case study

Photosystem I (PSI) is a multi-subunit integral pigment–protein complex that performs light-driven electron transfer from plastocyanin to ferredoxin in the thylakoid membrane of oxygenic photoautotrophs. In order to achieve the optimal photosynthetic performance under ambient irradiance, the absorption cross section of PSI is extended by means of peripheral antenna complexes. In eukaryotes, this role is played mostly by the pigment–protein complexes of the LHC family. The structure of the PSI-antenna supercomplexes has been relatively well understood in organisms harboring the primary plastid: red algae, green algae and plants. The secondary endosymbiotic algae, despite their major ecological importance, have so far received less attention. Here we report a detailed structural analysis of the antenna-PSI association in the stramenopile alga Nannochloropsis oceanica (Eustigmatophyceae). Several types of PSI-antenna assemblies are identified allowing for identification of antenna docking sites on the PSI core. Instances of departure of the stramenopile system from the red algal model of PSI-Lhcr structure are recorded, and evolutionary implications of these observations are discussed.     

Synthesis, assembly and degradation of thylakoid membrane proteins

The thylakoid membrane of chloroplasts contains four major protein complexes, involved in the photosynthetic electron transfer chain and in ATP synthesis. These complexes are built from a large number of polypeptide subunits encoded either in the nuclear or in the plastid genome. In this review, we are considering the mechanism that couples assembly (association of the polypeptides with each other and with their cofactors) with the upstream and downstream steps of the biogenetic pathway, translation and proteolytic degradation. We present the contrasting images of assembly that have emerged from a variety of approaches (studies of photosynthesis mutants, developmental studies and direct biochemical analysis of the kinetics of assembly). We develop the concept of control by epistasy of synthesis, through which the translation of certain subunits is controlled by the state of assembly of the complex and address the question of its mechanisms. We describe additional factors that assist in the integration and assembly of thylakoid membrane proteins.     

Mechanisms of protein import into thylakoids of chloroplasts

The thylakoid membrane of chloroplasts contains the major photosynthetic complexes, which consist of several either nuclear or chloroplast encoded subunits. The biogenesis of these thylakoid membrane complexes requires coordinated transport and subsequent assembly of the subunits into functional complexes. Nuclear-encoded thylakoid proteins are first imported into the chloroplast and then directed to the thylakoid using different sorting mechanisms. The cpSec pathway and the cpTat pathway are mainly involved in the transport of lumenal proteins, whereas the spontaneous pathway and the cpSRP pathway are used for the insertion of integral membrane proteins into the thylakoid membrane. While cpSec-, cpTat- and cpSRP-mediated targeting can be classified as 'assisted' mechanisms involving numerous components, 'unassisted' spontaneous insertion does not require additional targeting factors. However, even the assisted pathways differ fundamentally with respect to stromal targeting factors, the composition of the translocase and energy requirements.     

Changes in the composition of the photosynthetic apparatus in the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana.

The glycerolipid digalactosyl diacylglycerol (DGDG) is exclusively associated with photosynthetic membranes and thus may play a role in the proper assembly and maintenance of the photosynthetic apparatus. Here we employ a genetic approach based on the dgd1 mutant of Arabidopsis thaliana to investigate the function of DGDG in thylakoid membranes. The primary defect in the genetically well-characterized dgd1 mutant resulted in a 90% reduction of the DGDG content. The mutant showed a decreased photosystem II (PSII) to photosystem I ratio. In vivo room- and low-temperature (77 K) chlorophyll fluorescence measurements with thylakoid preparations are in agreement with a drastically altered excitation energy allocation to the reaction centers. Quantification of pigment-binding apoproteins and pigments supports an altered stoichiometry of individual pigment-protein complexes in the mutant. Most strikingly, an increase in the amount of peripheral light-harvesting complexes of PSII relative to the inner antenna complexes and the PSII reaction center/core complexes was observed. Regardless of the severe alterations in thylakoid organization, photosynthetic oxygen evolution was virtually not compromised in dgd1 mutant leaves.     

Phenotypes of Alb3p and carotenoid synthesis mutants show similarities regarding light sensitivity, thylakoid structure and protein stability

Chloroplast proteins of the Alb3/YidC/Oxa1p family are necessary for assembly of photosynthetic complexes in the thylakoid membranes. Alb3p in Arabidopsis thaliana is essential for posttranslational LHCII-integration into thylakoid membranes and participates in cotranslational assembly of D1. However, the pleiotropic defects of an Alb3p mutant, albino3, suggest additional functions for Alb3p. To obtain an impression of such potential further Alb3p activities from phenotypic manifestations, properties of mutants disturbed in thylakoid membrane protein transport or carotenoid biosynthesis were compared with the albino3 mutant. Specific defects observed in albino3 were similar to those in a carotenoid synthesis mutant. While this correlation did not provide tangible evidence for Alb3p being involved in the integration of carotenoids in photosynthetic complexes, it suggests a possible avenue for future investigations.     

Emergence of new regulatory mechanisms in the Benson-Calvin pathway via protein-protein interactions: a glyceraldehyde-3-phosphate dehydrogenase/CP12/phosphoribulokinase complex

Protein-protein interactions are involved in many metabolic pathways. This review will focus on the role of such associations in CO2 assimilation (Benson-Calvin cycle) and especially on the involvement of a GAPDH/CP12/PRK complex which has been identified in many photosynthetic organisms and may have an important role in the regulation of CO2 assimilation. The emergence of new kinetic and regulatory properties as a consequence of protein-protein interactions will be addressed as well as some of the questions raised by the existence of these supramolecular complexes such as composition, function, and assembly pathways. The presence and role of small intrinsically unstructured proteins like the 8.5 kDa protein CP12, involved in the regulation and/or assembly of these complexes will be discussed.     

Supramolecular organization of photosystem II in green plants

Green plant photosystem II (PSII) is involved in the light reactions of photosynthesis, which take place in the thylakoid membrane of the chloroplast. PSII is organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. These supercomplexes are dimeric and contain usually 2-4 copies of trimeric LHCII complexes and have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. This review focuses on the overall composition and structure of the PSII supercomplex of green plants and its organization and interactions within the photosynthetic membrane. Further, we present the current knowledge how the thylakoid membrane is three-dimensionally organized within the chloroplast. We also discuss how the supramolecular organization in the thylakoid membrane and the PSII flexibility may play roles in various short-term regulatory mechanisms of green plant photosynthesis. This article is part of a Special Issue entitled: Photosystem II.     

The chloroplast signal recognition particle (CpSRP) pathway as a tool to minimize chlorophyll antenna size and maximize photosynthetic productivity

The concept of the Truncated Light-harvesting chlorophyll Antenna (TLA) size, as a tool by which to maximize sunlight utilization and photosynthetic productivity in microalgal mass cultures or high-density plant canopies, is discussed. TLA technology is known to improve sunlight-to-product energy conversion efficiencies and is hereby exemplified by photosynthetic productivity estimates of wild type and a TLA strain under simulated mass culture conditions. Recent advances in the generation of TLA-type mutants by targeting genes of the chloroplast signal-recognition particle (CpSRP) pathway, affecting the thylakoid membrane assembly of light-harvesting proteins, are also summarized. Two distinct CpSRP assembly pathways are recognized, one entailing post-translational, the other a co-translational mechanism. Differences between the post-translational and co-translational integration mechanisms are outlined, as these pertain to the CpSRP-mediated assembly of thylakoid membrane protein complexes in higher plants and green microalgae. The applicability of the CpSRP pathway genes in efforts to generate TLA-type strains with enhanced solar energy conversion efficiency in photosynthesis is evaluated.     

A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana.

To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear- and plastome-encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome-encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T-DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark-grown seedlings and its levels do not increase dramatically during light-induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo- and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial-like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.     

Time Line of Discoveries: Anoxygenic Bacterial Photosynthesis

A time line of important research relating to anoxygenic photosynthetic organisms is presented. The time line includes discoveries of organisms, metabolic capabilities, molecular complexes and genetic systems. It also pinpoints important milestones in our understanding of the structure, function, organization, assembly and regulation of photosynthetic complexes.     

N-Terminal Structure of Maize Ferredoxin:NADP+ Reductase Determines Recruitment into Different Thylakoid Membrane Complexes

To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.     

A permease-like protein involved in ER to thylakoid lipid transfer in Arabidopsis

In eukaryotes, enzymes of different subcellular compartments participate in the assembly of membrane lipids. As a consequence, interorganelle lipid transfer is extensive in growing cells. A prominent example is the transfer of membrane lipid precursors between the endoplasmic reticulum (ER) and the photosynthetic thylakoid membranes in plants. Mono- and digalactolipids are typical photosynthetic membrane lipids. In Arabidopsis, they are derived from one of two pathways, either synthesized de novo in the plastid, or precursors are imported from the ER, giving rise to distinct molecular species. Employing a high-throughput robotic screening procedure generating arrays of spot chromatograms, mutants of Arabidopsis were isolated, which accumulated unusual trigalactolipids. In one allelic mutant subclass, trigalactosyldiacylglycerol1, the primary defect caused a disruption in the biosynthesis of ER-derived thylakoid lipids. Secondarily, a processive galactosyltransferase was activated, leading to the accumulation of oligogalactolipids. Mutations in a permease-like protein of the outer chloroplastic envelope are responsible for the primary biochemical defect. It is proposed that this protein is part of a lipid transfer complex.