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Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2's ß-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.

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In the field of photosynthesis, only a limited number of approaches of super-resolution fluorescence microscopy can be used, as the functional architecture of the thylakoid membrane in chloroplasts is probed through the natural fluorescence of chlorophyll molecules. In this work, we have used a custom-built fluorescence microscopy method called Single Pixel Reconstruction Imaging (SPiRI) that yields a 1.4 gain in lateral and axial resolution relative to confocal fluorescence microscopy, to obtain 2D images and 3D-reconstucted volumes of isolated chloroplasts, obtained from pea (Pisum sativum), spinach (Spinacia oleracea) and Arabidopsis thaliana. In agreement with previous studies, SPiRI images exhibit larger thylakoid grana diameters when extracted from plants under low-light regimes. The three-dimensional thylakoid architecture, revealing the complete network of the thylakoid membrane in intact, non-chemically-fixed chloroplasts can be visualized from the volume reconstructions obtained at high resolution. From such reconstructions, the stromal connections between each granum can be determined and the fluorescence intensity in the stromal lamellae compared to those of neighboring grana.

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In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the posttranslational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the cotranslational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for posttranslational transport, while a ribosome-associated pool coordinates its cotranslational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis thaliana cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in posttranslational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear- as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate.

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Cyanobacteriota, the sole prokaryotes capable of oxygenic photosynthesis (OxyP), occupy a unique and pivotal role in Earth's history. While the notion that OxyP may have originated from Cyanobacteriota is widely accepted, its early evolution remains elusive. Here, by using both metagenomics and metatranscriptomics, we explore 36 metagenome-assembled genomes from hot spring ecosystems, belonging to two deep-branching cyanobacterial orders: Thermostichales and Gloeomargaritales. Functional investigation reveals that Thermostichales encode the crucial thylakoid membrane biogenesis protein, vesicle-inducing protein in plastids 1 (Vipp1). Based on the phylogenetic results, we infer that the evolution of the thylakoid membrane predates the divergence of Thermostichales from other cyanobacterial groups and that Thermostichales may be the most ancient lineage known to date to have inherited this feature from their common ancestor. Apart from OxyP, both lineages are potentially capable of sulfide-driven AnoxyP by linking sulfide oxidation to the photosynthetic electron transport chain. Unexpectedly, this AnoxyP capacity appears to be an acquired feature, as the key gene sqr was horizontally transferred from later-evolved cyanobacterial lineages. The presence of two D1 protein variants in Thermostichales suggests the functional flexibility of photosystems, ensuring their survival in fluctuating redox environments. Furthermore, all MAGs feature streamlined phycobilisomes with a preference for capturing longer-wavelength light, implying a unique evolutionary trajectory. Collectively, these results reveal the photosynthetic flexibility in these early-diverging cyanobacterial lineages, shedding new light on the early evolution of Cyanobacteriota and their photosynthetic processes.

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The plastid stroma-localized chaperone HSP90C plays a crucial role in maintaining optimal proteostasis within chloroplasts and participates in protein translocation processes. While existing studies have revealed HSP90C's direct interaction with the Sec translocase-dependent client pre-protein PsbO1 and the SecY1 subunit of the thylakoid membrane-bound Sec1 translocase channel system, its direct involvement with the extrinsic homodimeric Sec translocase subunit, SecA1, remains elusive. Employing bimolecular fluorescence complementation (BiFC) assay and other in vitro analyses, we unraveled potential interactions between HSP90C and SecA1. Our investigation revealed dynamic interactions between HSP90C and SecA1 at the thylakoid membrane and stroma. The thylakoid membrane localization of this interaction was contingent upon active HSP90C ATPase activity, whereas their stromal interaction was associated with active SecA1 ATPase activity. Furthermore, we observed a direct interaction between these two proteins by analyzing their ATP hydrolysis activities, and their interaction likely impacts their respective functional cycles. Additionally, using PsbO1, a model Sec translocase client pre-protein, we studied the intricacies of HSP90C's possible involvement in pre-protein translocation via the Sec1 system in chloroplasts. The results suggest a complex nature of the HSP90C-SecA1 interaction, possibly mediated by the Sec client protein. Our studies shed light on the nuanced aspects of HSP90C's engagement in orchestrating pre-protein translocation, and we propose a potential collaborative role of HSP90C with SecA1 in actively facilitating pre-protein transport across the thylakoid membrane.

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Cyanobacteria and chloroplasts of algae and plants harbor specialized thylakoid membranes that convert sunlight into chemical energy. These membranes house photosystems II and I, the vital protein-pigment complexes that drive oxygenic photosynthesis. In the course of their evolution, thylakoid membranes have diversified in structure. However, the core machinery for photosynthetic electron transport remained largely unchanged, with adaptations occurring primarily in the light-harvesting antenna systems. Whereas thylakoid membranes in cyanobacteria are relatively simple they become more complex in algae and plants. The chloroplasts of vascular plants contain intricate networks of stacked grana and unstacked stroma thylakoids. This review provides an in-depth view of thylakoid membrane architectures in phototrophs, and the determinants that shape their forms, as well as presenting recent insights into the spatial organization of their biogenesis and maintenance. Its overall goal is to define the underlying principles that have guided the evolution of these bioenergetic membranes.

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The chloroplast thylakoid membrane is composed of membrane lipids and photosynthetic protein complexes, and the orchestration of thylakoid lipid biosynthesis and photosynthesis-associated protein accumulation is considered important for thylakoid development. Galactolipids consist of ∼80% of the thylakoid lipids, and their biosynthesis is fundamental for chloroplast development. We previously reported that the suppression of galactolipid biosynthesis decreased the expression of photosynthesis-associated nuclear-encoded genes (PhAPGs) and photosynthesis-associated plastid-encoded genes (PhAPGs). However, the mechanism for coordinative regulation between galactolipid biosynthesis in plastids and the expression of PhANGs and PhAPGs remains largely unknown. To elucidate this mechanism, we investigated the gene expression patterns in galactolipid-deficient Arabidopsis seedlings during the de-etiolation process. We found that galactolipids are crucial for inducing both the transcript accumulation of PhANGs and PhAPGs and the accumulation of plastid-encoded photosynthesis-associated proteins in developing chloroplasts. Genetic analysis indicates the contribution of the GENOMES UNCOUPLED1 (GUN1)-mediated plastid-to-nucleus signaling pathway to PhANG regulation in response to galactolipid levels. Previous studies suggested that the accumulation of GUN1 reflects the state of protein homeostasis in plastids and alters the PhANG expression level. Thus, we propose a model that galactolipid biosynthesis determines the protein homeostasis in plastids in the initial phase of de-etiolation and optimizes GUN1-dependent signaling to regulate the PhANG expression. This mechanism might contribute to orchestrating the biosynthesis of lipids and proteins for the biogenesis of functional chloroplasts in plants.

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The biological role of lipids goes far beyond the formation of a structural membrane bilayer platform for membrane proteins and controlling fluxes across the membranes. For example, in photosynthetic thylakoid membranes, lipids occupy well-defined binding niches within protein complexes and determine the structural organization of membrane proteins and their function by controlling generic physicochemical membrane properties. In this chapter, two-dimensional thin-layer chromatography (2D TLC) and gas chromatography (GC) techniques are presented for quantitative analysis of lipid classes and fatty acids in thylakoid membranes. In addition, lipid extraction methods from isolated thylakoid membranes and leaves are described together with a procedure for the derivatization of fatty acids to fatty acid methyl esters (FAME) that is required for GC analysis.

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The light-induced transthylakoid membrane potential (ΔΨm) can function as a driving force to help catalyzing the formation of ATP molecules, proving a tight connection between ΔΨm and the ATP synthase. Naturally, a question can be raised on the effects of altered functioning of ATP synthases on regulating ΔΨm, which is attractive in the area of photosynthetic research. Lots of findings, when making efforts of solving this difficulty, can offer an in-depth understanding into the mechanism behind. However, the functional network on modulating ΔΨm is highly interdependent. It is difficult to comprehend the consequences of altered activity of ATP synthases on adjusting ΔΨm because parameters that have influences on ΔΨm would themselves be affected by ΔΨm. In this work, a computer model was applied to check the kinetic changes in polarization/depolarization across the thylakoid membrane (TM) regulated by the modified action of ATP synthases. The computing data revealed that under the extreme condition by numerically "switching off" the action of the ATP synthase, the complete inactivation of ATP synthase would markedly impede proton translocation at the cytb6f complex. Concurrently, the KEA3 (CLCe) porter, actively pumping protons into the stroma, further contributes to achieving a sustained low level of ΔΨm. Besides, the quantitative consequences on every particular component of ΔΨm adjusted by the modified functioning of ATP synthases were also explored. By employing the model, we bring evidence from the theoretical perspective that the ATP synthase is a key factor in forming a transmembrane proton loop thereby maintaining a propriate steady-state ΔΨm to meet variable environmental conditions.

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Light is required for photosynthesis, but plants are often exposed to excess light, which can lead to photodamage and eventually cell death. To prevent this, they evolved photoprotective feedback mechanisms that regulate photosynthesis and trigger processes that dissipate light energy as heat, called non-photochemical quenching (NPQ). In excess light conditions, the light reaction and activity of Photosystem II (PSII) generates acidification of the thylakoid lumen, which is sensed by special pH-sensitive proteins called Photosystem II Subunit S (PsbS), actuating a photoprotective "switch" in the light-harvesting antenna. Despite its central role in regulating photosynthetic energy conversion, the molecular mechanism of PsbS as well as its interaction with partner proteins are not well understood. This review summarizes the current knowledge on the molecular structure and mechanistic aspects of the light-stress sensor PsbS and addresses open questions and challenges in the field regarding a full understanding of its functional mechanism and role in NPQ.

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Membrane-bound FtsH proteases are universally present in prokaryotes and in mitochondria and chloroplasts of eukaryotic cells. These metalloproteases are often critical for viability and play both protease and chaperone roles to maintain cellular homeostasis. In contrast to most bacteria bearing a single ftsH gene, cyanobacteria typically possess four FtsH proteases (FtsH1-4) forming heteromeric (FtsH1/3 and FtsH2/3) and homomeric (FtsH4) complexes. The functions and substrate repertoire of each complex are however poorly understood. To identify substrates of the FtsH4 protease complex we established a trapping assay in the cyanobacterium Synechocystis PCC 6803 utilizing a proteolytically inactivated trapFtsH4-His. Around 40 proteins were specifically enriched in trapFtsH4 pulldown when compared with the active FtsH4. As the list of putative FtsH4 substrates contained Ycf4 and Ycf37 assembly factors of Photosystem I (PSI), its core PsaB subunit and the IsiA chlorophyll-binding protein that associates with PSI during iron stress, we focused on these PSI-related proteins. Therefore, we analysed their degradation by FtsH4 in vivo in Synechocystis mutants and in vitro using purified substrates. The data confirmed that FtsH4 degrades Ycf4, Ycf37, IsiA, and also the individual PsaA and PsaB subunits in the unassembled state but not when assembled within the PSI complexes. A possible role of FtsH4 in the PSI life-cycle is discussed.

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Cyanobacteria were among the oldest organisms to undertake oxygenic photosynthesis and have an essential impact on the atmosphere and carbon/nitrogen cycles on the planet. The thylakoid membrane of cyanobacteria represents an intricate compartment that houses a variety of multi-component (pigment-)protein complexes, assembly factors, and regulators, as well as transporters involved in photosynthetic light reactions, and respiratory electron transport. How these protein components are incorporated into membranes during thylakoid formation and how individual complexes are regulated to construct the functional machinery remains elusive. Here, we carried out an in-depth statistical analysis of the thylakoid proteome data obtained during light-induced thylakoid membrane biogenesis in the model cyanobacterium Synechococcus elongatus PCC 7942. A total of 1581 proteins were experimentally quantified, among which 457 proteins demonstrated statistically significant variations in abundance at distinct thylakoid biogenesis stages. Gene Ontology and KEGG enrichment analysis revealed that predominantly photosystems, light-harvesting antennae, ABC transporters, and pathway enzymes involved in oxidative stress responses and protein folding exhibited notable alternations in abundance between high light and growth light. Moreover, through cluster analysis the 1581 proteins were categorized into six distinct clusters that have significantly different trajectories of the change in their abundance during thylakoid development. Our study provides insights into the physiological regulation for the membrane integration of protein components and functionally linked complexes during the cyanobacterial TM biogenesis process. The findings and analytical methodologies developed in this study may be valuable for studying the global responses of TM biogenesis and photosynthetic acclimation in plants and algae.

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ALBINO3 (ALB3) protein functions in the insertion and assembly of thylakoid membrane protein complexes and plays a critical role for chloroplast development in Arabidopsis. However, the biological function of ALB3 homologs in rice, OsALB3, remains elusive. Here, we identified a rice mutant, yellow leaf and lethal1 (yll1), that displayed yellow leaves and died at the seedling stage. The content of chlorophyll in yll1, compared with wild type, was significantly decreased. Transmission electron microscopy observation shows that the chloroplast of yll1 lacks thylakoid membranes. The causal mutation, which is located in OsALB3, was isolated by Mutmap+ combined with a simple mutation filtering process. Knockout of OsALB3 leads to yellow leaves and seedling lethality, mimicking the phenotype of yll1. OsALB3 is widely expressed and OsALB3 is chloroplast-localized. Moreover, the content of light-harvesting chlorophyll-binding proteins in yll1 is reduced. Together, our study demonstrated the essential role of OsALB3 in chloroplast development and provided clues to the possible conserved molecular function of ALB3 in rice.

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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.

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Rare earth elements (REEs) are emerging as an anticipated pollution in the environment due to their active use in many areas. However, the effects of REEs on the photosynthesis of rice have not been thoroughly explored. Therefore, this study emphasizes how high levels of La(III) affect the thylakoid membrane of rice seedlings, thereby inhibiting photosynthesis and growth. Here, we reported that rice plants treated with La(III) exhibited an increase in La accumulation in the leaves, accompanied by a decrease in chlorophyll content and photosynthetic capacity. La(III) exposure decreased Mg content in leaves, but possibly increased other nutrients including Cu, Mn, and Zn through systemic endocytosis. K-band and L-band appeared in the fluorescence OJIP transients, indicating La(III) stress destroyed the donor and receptor sides of photosystem II (PSII). Numerous reaction centers (RC/CSm) were inactivated by La(III) treatment, which resulted in a reduction in electron transport capacity (decreased ETo/RC and ETo/CSm) and an increase in the dissipation of the excess excitation energy by heat (increased DIo/RC and DIo/CSm). The BN-PAGE analysis of thylakoid membrane protein complexes showed that La(III) induced the degradation of supercomplexes, PSII core, LHCII, PSI core, LHCI, and F1-ATPase binding Cyt b6f complex. Collectively, this study revealed that La(III) causes significant degradation of thylakoid membrane proteins, thereby promoting the decomposition of photosynthetic complexes, ultimately destroying the chloroplast structure and reducing the photosynthetic performance of rice seedlings.

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The spectral composition of light is an important factor for the metabolism of photosynthetic organisms. Several blue light-regulated metabolic processes have already been identified in the industrially relevant microalga Monoraphidium braunii. However, little is known about the spectral impact on this species' growth, fatty acid (FA), and pigment composition. In this study, M. braunii was cultivated under different light spectra (white light: 400-700 nm, blue light: 400-550 nm, green light: 450-600 nm, and red light: 580-700 nm) at 25°C for 96 h. The growth was monitored daily. Additionally, the FA composition, and pigment concentration was analyzed after 96 h. The highest biomass production was observed upon white light and red light irradiation. However, green light also led to comparably high biomass production, fueling the scientific debate about the contribution of weakly absorbed light wavelengths to microalgal biomass production. All light spectra (white, blue, and green) that comprised blue-green light (450-550 nm) led to a higher degree of FA unsaturation and a greater concentration of all identified pigments than red light. These results further contribute to the growing understanding that blue-green light is an essential trigger for maximized pigment concentration and FA unsaturation in green microalgae.

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The chloroplast lumen contains at least 80 proteins whose function and regulation are not yet fully understood. Isolating the chloroplast lumen enables the characterization of the lumenal proteins. The lumen can be isolated in several ways through thylakoid disruption using a Yeda press or sonication, or through thylakoid solubilization using a detergent. Here, we present a simple procedure to isolate thylakoid lumen by sonication using leaves of the plant Arabidopsis thaliana. The step-by-step procedure is as follows: thylakoids are isolated from chloroplasts, loosely associated thylakoid surface proteins from the stroma are removed, and the lumen fraction is collected in the supernatant following sonication and centrifugation. Compared to other procedures, this method is easy to implement and saves time, plant material, and cost. Lumenal proteins are obtained in high quantity and purity; however, some stromal membrane-associated proteins are released to the lumen fraction, so this method could be further adapted if needed by decreasing sonication power and/or time.

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In biomembranes, lipids form bilayer structures that serve as the fluid matrix for membrane proteins and other hydrophobic compounds. Additionally, lipid molecules associate with membrane proteins and impact their structures and functions. In both cyanobacteria and the chloroplasts of plants and algae, the lipid bilayer of the thylakoid membrane consists of four distinct glycerolipid classes: monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol. These lipids are also integral components of photosynthetic complexes such as photosystem II and photosystem I. The lipid-binding sites within the photosystems, as well as the lipid composition in the thylakoid membrane, are highly conserved between cyanobacteria and photosynthetic eukaryotes, and each lipid class has specific roles in oxygenic photosynthesis. This review aims to shed light on the potential evolutionary implications of lipid utilization in membrane lipid bilayers and photosynthetic complexes in oxygenic photosynthetic organisms.

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The barley cultivar Sarab 1 (SRB1) can continue photosynthesis despite its low Fe acquisition potential via roots and dramatically reduced amounts of photosystem I (PSI) reaction-center proteins under Fe-deficient conditions. We compared the characteristics of photosynthetic electron transfer (ET), thylakoid ultrastructure, and Fe and protein distribution on thylakoid membranes among barley cultivars. The Fe-deficient SRB1 had a large proportion of functional PSI proteins by avoiding P700 over-reduction. An analysis of the thylakoid ultrastructure clarified that SRB1 had a larger proportion of non-appressed thylakoid membranes than those in another Fe-tolerant cultivar, Ehimehadaka-1 (EHM1). Separating thylakoids by differential centrifugation further revealed that the Fe-deficient SRB1 had increased amounts of low/light-density thylakoids with increased Fe and light-harvesting complex II (LHCII) than did EHM1. LHCII with uncommon localization probably prevents excessive ET from PSII leading to elevated NPQ and lower PSI photodamage in SRB1 than in EHM1, as supported by increased Y(NPQ) and Y(ND) in the Fe-deficient SRB1. Unlike this strategy, EHM1 may preferentially supply Fe cofactors to PSI, thereby exploiting more surplus reaction center proteins than SRB1 under Fe-deficient conditions. In summary, SRB1 and EHM1 support PSI through different mechanisms during Fe deficiency, suggesting that barley species have multiple strategies for acclimating photosynthetic apparatus to Fe deficiency.

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The knockout of the At2g28210 gene encoding α-carbonic anhydrase 2 (α-CA2) in Arabidopsis thaliana (Columbia) led to alterations in photosynthetic processes. The effective quantum yields of both photosystem II (PSII) and photosystem I (PSI) were higher in α-carbonic anhydrase 2 knockout plants (α-CA2-KO), and the reduction state of plastoquinone pool was lower than in wild type (WT). The electron transport rate in the isolated thylakoids measured with methyl viologen was higher in α-CA2-KO plants. The amounts of reaction centers of PSII and PSI were similar in WT and α-CA2-KO plants. The non-photochemical quenching of chlorophyll a fluorescence in α-CA2-KO leaves was lower at the beginning of illumination, but became slightly higher than in WT leaves when the steady state was achieved. The degree of state transitions in the leaves was lower in α-CA2-KO than in WT plants. Measurements of the electrochromic carotenoid absorbance shift (ECS) revealed that the light-dependent pH gradient (ΔpH) across the thylakoid membrane was lower in the leaves of α-CA2-KO plants than in WT plants. The starch content in α-CA2-KO leaves was lower than in WT plants. The expression levels of the genes encoding chloroplast CAs in α-CA2-KO changed noticeably, whereas the expression levels of genes of cytoplasmic CAs remained almost the same. It is proposed that α-CA2 may be situated in the chloroplasts.