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Efficient solar energy conversion is ensured by the organization, physical association, and physiological coordination of various protein complexes in photosynthetic membranes. Here, we visualize the native architecture and interactions of photosynthetic complexes within the thylakoid membranes from a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) using high-resolution atomic force microscopy. In the Syn2973 thylakoid membranes, both photosystem I (PSI)-enriched domains and crystalline photosystem II (PSII) dimer arrays were observed, providing favorable membrane environments for photosynthetic electron transport. The high light (HL)-adapted thylakoid membranes accommodated a large amount of PSI complexes, without the incorporation of iron-stress-induced protein A (IsiA) assemblies and formation of IsiA–PSI supercomplexes. In the iron deficiency (Fe−)-treated thylakoid membranes, in contrast, IsiA proteins densely associated with PSI, forming the IsiA–PSI supercomplexes with varying assembly structures. Moreover, type-I NADH dehydrogenase-like complexes (NDH-1) were upregulated under the HL and Fe− conditions and established close association with PSI complexes to facilitate cyclic electron transport. Our study provides insight into the structural heterogeneity and plasticity of the photosynthetic apparatus in the context of their native membranes in Syn2973 under environmental stress. Advanced understanding of the photosynthetic membrane organization and adaptation will provide a framework for uncovering the molecular mechanisms of efficient light harvesting and energy conversion.

Cryptophyte plastids originated from a red algal ancestor through secondary endosymbiosis. Cryptophyte photosystem I (PSI) associates with transmembrane alloxanthin-chlorophyll a/c proteins (ACPIs) as light-harvesting complexes (LHCs). Here, we report the structure of the photosynthetic PSI–ACPI supercomplex from the cryptophyte Chroomonas placoidea at 2.7-Å resolution obtained by crygenic electron microscopy. Cryptophyte PSI–ACPI represents a unique PSI–LHCI intermediate in the evolution from red algal to diatom PSI–LHCI. The PSI–ACPI supercomplex is composed of a monomeric PSI core containing 14 subunits, 12 of which originated in red algae, 1 diatom PsaR homolog, and an additional peptide. The PSI core is surrounded by 14 ACPI subunits that form 2 antenna layers: an inner layer with 11 ACPIs surrounding the PSI core and an outer layer containing 3 ACPIs. A pigment-binding subunit that is not present in any other previously characterized PSI–LHCI complexes, ACPI-S, mediates the association and energy transfer between the outer and inner ACPIs. The extensive pigment network of PSI–ACPI ensures efficient light harvesting, energy transfer, and dissipation. Overall, the PSI–LHCI structure identified in this study provides a framework for delineating the mechanisms of energy transfer in cryptophyte PSI–LHCI and for understanding the evolution of photosynthesis in the red lineage, which occurred via secondary endosymbiosis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Background QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. Results We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. Conclusions We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

The N6-methyladenosine (m6A) modification is the most common internal post-transcriptional modification, with important regulatory effects on RNA export, splicing, stability, and translation. Studies on the m6A modifications in plants have focused on Arabidopsis thaliana growth and development. However, A. thaliana is a salt-sensitive and model plant species. Thus, studies aimed at characterizing the role of the m6A modification in the salt stress responses of highly salt-tolerant crop species are needed. Sweet sorghum is cultivated as an energy and forage crop, which is highly suitable for growth on saline-alkaline land. Exploring the m6A modification in sweet sorghum may be important for elucidating the salt-resistance mechanism of crops. In this study, we mapped the m6A modifications in two sorghum genotypes (salt-tolerant M-81E and salt-sensitive Roma) that differ regarding salt tolerance. The m6A modification in sweet sorghum under salt stress was drastically altered, especially in Roma, where the m6A modification on mRNAs of some salt-resistant related transcripts increased, resulting in enhanced mRNA stability, which in turn was involved in the regulation of salt tolerance in sweet sorghum. Although m6A modifications are important for regulating sweet sorghum salt tolerance, the regulatory activity is limited by the initial m6A modification level. Additionally, in M-81E and Roma, the differences in the m6A modifications were much greater than the differences in gene expression levels and are more sensitive. Our study suggests that the number and extent of m6A modifications on the transcripts of salt-resistance genes may be important factors for determining and assessing the salt tolerance of crops.

Plant chloroplasts have complex membrane systems. Among these, thylakoids serve as the sites for photosynthesis and photosynthesis-related adaptation. In addition to the photosynthetic membrane complexes and associated molecules, lipids in the thylakoid membranes, are predominantly composed of MGDG (monogalactosyldiacylglycerol), DGDG (digalactosyldiacylglycerol), SQDG (sulfoquinovosyldiacylglycerol) and PG (phosphatidylglycerol), play essential roles in shaping the thylakoid architecture, electron transfer, and photoregulation. In this review, we discuss the effect of abiotic stress on chloroplast structure, the changes in membrane lipid composition, and the degree of unsaturation of fatty acids. Advanced understanding of the mechanisms regulating chloroplast membrane lipids and unsaturated fatty acids in response to abiotic stresses is indispensable for improving plant resistance and may inform the strategies of crop breeding.

Cyanobacteria are autotrophs whose photosynthetic process is similar to that of higher plants, although the photosynthetic apparatus is slightly different. They have been widely used for decades as model systems for studying the principles of photosynthesis, especially the effects of environmental stress on photosynthetic activities. Salt stress, which is the most common abiotic stress in nature, combines ionic and osmotic stresses. High cellular ion concentrations and osmotic stress can alter normal metabolic processes and photosynthesis. Additionally, salt stress increases the intracellular reactive oxygen species (ROS) contents. Excessive amounts of ROS will damage the photosynthetic apparatus, inhibit the synthesis of photosystem-related proteins, including the D1 protein, and destroy the thylakoid membrane structure, leading to inhibited photosynthesis. In this review, we mainly introduce the effects of salt stress on the cyanobacterial membranes and photosynthetic apparatus. We also describe specific salt tolerance mechanisms. A thorough characterization of the responses of membranes and photosynthetic apparatus to salt stress may be relevant for increasing agricultural productivity.

The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. Furthermore, we functionally tagged PSI, PSII, Cyt b6f, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes., The thylakoid membrane is the site for photosynthetic reactions and responses in cyanobacteria, algae and higher plants. Here, we performed direct visualization of the native organization and mobility of photosynthetic complexes in cyanobacterial thylakoid membranes, using atomic force, confocal, and total internal reflection fluorescence microscopy. Knowledge of the cyanobacterial thylakoid membrane could be extended to chloroplast and mitochondrial membranes.

Sweet sorghum is a C4 crop with the characteristic of fast-growth and high-yields. It is a good source for food, feed, fiber, and fuel. On saline land, sweet sorghum can not only survive, but increase its sugar content. Therefore, it is regarded as a potential source for identifying salt-related genes. Here, we review the physiological and biochemical responses of sweet sorghum to salt stress, such as photosynthesis, sucrose synthesis, hormonal regulation, and ion homeostasis, as well as their potential salt-resistance mechanisms. The major advantages of salt-tolerant sweet sorghum include: 1) improving the Na+ exclusion ability to maintain ion homeostasis in roots under salt-stress conditions, which ensures a relatively low Na+ concentration in shoots; 2) maintaining a high sugar content in shoots under salt-stress conditions, by protecting the structures of photosystems, enhancing photosynthetic performance and sucrose synthetase activity, as well as inhibiting sucrose degradation. To study the regulatory mechanism of such genes will provide opportunities for increasing the salt tolerance of sweet sorghum by breeding and genetic engineering.

The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO2fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single β-carboxysomes in the model cyanobacteriumSynechococcus elongatusPCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO2and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.One Sentence SummaryDetermination of absolute protein stoichiometry reveals the organizational variability of carboxysomes in response to microenvironmental changesThe authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Author (www.plantcell.org) is: Lu-Ning Liu (luning.liu@liverpool.ac.uk).

Bicarbonate transporters play essential roles in pH homeostasis in mammals and photosynthesis in aquatic photoautotrophs. A number of bicarbonate transporters have been characterized, among which is BicA-a low-affinity, high-flux SLC26-family bicarbonate transporter involved in cyanobacterial CO2-concentrating mechanisms (CCMs) that accumulate CO2 and improve photosynthetic carbon fixation. Here, we report the three-dimensional structure of BicA from Synechocystis sp. PCC6803. Crystal structures of the transmembrane domain (BicATM) and the cytoplasmic STAS domain (BicASTAS) of BicA were solved. BicATM was captured in an inward-facing HCO3--bound conformation and adopts a '7+7' fold monomer. HCO3- binds to a cytoplasm-facing hydrophilic pocket within the membrane. BicASTAS is assembled as a compact homodimer structure and is required for the dimerization of BicA. The dimeric structure of BicA was further analysed using cryo-electron microscopy and physiological analysis of the full-length BicA, and may represent the physiological unit of SLC26-family transporters. Comparing the BicATM structure with the outward-facing transmembrane domain structures of other bicarbonate transporters suggests an elevator transport mechanism that is applicable to the SLC26/4 family of sodium-dependent bicarbonate transporters. This study advances our knowledge of the structures and functions of cyanobacterial bicarbonate transporters, and will inform strategies for bioengineering functional BicA in heterologous organisms to increase assimilation of CO2.

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla and provide a means for compartmentalizing specific metabolic pathways. They sequester catalytic enzymes from the cytoplasm, using an icosahedral proteinaceous shell with selective permeability to metabolic molecules and substrates, to enhance metabolic efficiency. Carboxysomes were the first BMCs discovered and their unprecedented capacity of CO2 fixation allows cyanobacteria to make a significant contribution to global carbon fixation. There is an increasing interest in utilizing synthetic biology to construct synthetic carboxysomes in new hosts, i.e., higher plants, to enhance carbon fixation and productivity. Here, we report the construction of a synthetic operon of the β-carboxysome from the cyanobacterium Synechococcus elongatus PCC7942 to generate functional β-carboxysome-like structures in Escherichia coli. The protein expression, structure, assembly, and activity of synthetic β-carboxysomes were characterized in depth using confocal, electron and atomic force microscopy, proteomics, immunoblot analysis, and enzymatic assays. Furthermore, we examined the in vivo interchangeability of β-carboxysome building blocks with other BMC components. To our knowledge, this is the first production of functional β-carboxysome-like structures in heterologous organisms. It provides important information for the engineering of fully functional carboxysomes and CO2-fixing modules in higher plants. The study strengthens our synthetic biology toolbox for generating BMC-based organelles with tunable activities and new scaffolding biomaterials for metabolic improvement and molecule delivery.

Cyanobacteria have evolved effective adaptive mechanisms to improve photosynthesis and CO2 fixation. The central CO2-fixing machinery is the carboxysome, which is composed of an icosahedral proteinaceous shell encapsulating the key carbon fixation enzyme, Rubisco, in the interior. Controlled biosynthesis and ordered organization of carboxysomes are vital to the CO2-fixing activity of cyanobacterial cells. However, little is known about how carboxysome biosynthesis and spatial positioning are physiologically regulated to adjust to dynamic changes in the environment. Here, we used fluorescence tagging and live-cell confocal fluorescence imaging to explore the biosynthesis and subcellular localization of β-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation. We demonstrated that β-carboxysome biosynthesis is accelerated in response to increasing light intensity, thereby enhancing the carbon fixation activity of the cell. Inhibition of photosynthetic electron flow impairs the accumulation of carboxysomes, indicating a close coordination between β-carboxysome biogenesis and photosynthetic electron transport. Likewise, the spatial organization of carboxysomes in the cell correlates with the redox state of photosynthetic electron transport chain. This study provides essential knowledge for us to modulate the β-carboxysome biosynthesis and function in cyanobacteria. In translational terms, the knowledge is instrumental for design and synthetic engineering of functional carboxysomes into higher plants to improve photosynthesis performance and CO2 fixation. Compartmentalization of metabolic pathways in cells provides the fundamental basis for enhancing and modulating the cellular metabolism. Many prokaryotes have evolved specialized metabolic organelles, known as bacterial microcompartments, to sequester key metabolic pathways and thereby improve the efficiency of metabolic activities (for reviews, see Kerfeld et al., 2010; Bobik et al., 2015). Unlike eukaryotic organelles, bacterial microcompartments are assembled entirely by proteins. These organelles consist of interior enzymes that catalyze sequential metabolic reactions (Yeates et al., 2010), surrounded by a single-layer proteinaceous shell (Kerfeld et al., 2005; Tsai et al., 2007; Tanaka et al., 2008; Sutter et al., 2016). The shell facets are composed of hexameric and pentameric proteins, resulting in an overall shell architecture resembling an icosahedral viral capsid (Kinney et al., 2011; Hantke et al., 2014; Kerfeld and Erbilgin, 2015). Interactions between shell proteins are important for the self-assembly of the shell (Sutter et al., 2016). The selectively permeable shell serves to concentrate enzymes and substrates, mediate flux of metabolites, modulate the redox state, and prevent toxic intermediates from diffusing into the cytoplasm (Havemann et al., 2002; Yeates et al., 2008). Carboxysomes were the first bacterial microcompartments to be discovered and are widely distributed among cyanobacteria and some chemoautotrophs as the central machinery for the fixation of CO2 (Shively et al., 1973). Two different types of carboxysomes have been identified (α- and β-carboxysomes), according to the types of the CO2-fixing enzyme, Rubisco (form 1A and form 1B), possessed in cyanobacteria. In most β-cyanobacteria, Rubisco is sequestered in the β-carboxysome lumen by a shell that is composed of shell and shell-associated proteins encoded by a ccmKLMNO operon (Omata et al., 2001; Long et al., 2010; Rae et al., 2012). The carboxysomal carbonic anhydrase is colocalized with Rubisco in the β-carboxysome, serving to create a CO2-rich microenvironment to favor the Rubisco activity. Some cyanobacterial species do not have the carboxysomal β-carbonic anhydrase (CcaA) homologs; instead, the N-terminal domain of CcmM functions as an active γ-carbonic anhydrase (Peña et al., 2010). The shell facets act as a selective barrier that allows the diffusion of HCO3− and retains CO2 in the interior (Dou et al., 2008). Through these mechanisms, carboxysomes elevate the CO2 concentration in the vicinity of Rubisco and thereby enhance the efficiency of carbon fixation. Supported by this nanoscale CO2-fixing machinery, cyanobacteria contribute more than 25% of global carbon fixation (Field et al., 1998; Liu et al., 1999). The efficiency of carboxysomes in enhancing carbon fixation has attracted tremendous interest in engineering the CO2-fixing organelle in other organisms. For example, introducing β-carboxysomes into higher plants that use the ancestral C3 pathway of photosynthesis could potentially enhance photosynthetic carbon fixation and crop production (Lin et al., 2014a, 2014b). However, engineering of functional carboxysomes requires extensive understanding about the principles underlying the formation of β-carboxysomes and the physiological integration of β-carboxysomes into the cellular metabolism. Indeed, cyanobacterial cells have evolved comprehensive systems to regulate the biosynthesis and spatial organization of carboxysomes, allowing them to modulate the capacity for photosynthetic carbon fixation. Recent studies elucidated that the β-carboxysome assembly is initiated from the packing of Rubisco enzymes, followed by the encapsulation of peripheral shell proteins (Cameron et al., 2013; Chen et al., 2013). In the model rod-shaped cyanobacterium Synechococcus elongatus PCC7942 (hereafter Synechococcus), three to four β-carboxysomes were observed to be evenly spaced along the centerline of the longitudinal axis of cells, ensuring the equal segregation of the machinery between daughter cells (Savage et al., 2010). Such specific organization of carboxysomes within cyanobacterial cells is likely to be determined by the interaction between carboxysomes and the cytoskeleton (Savage et al., 2010). Advanced understanding of the functions and assembly of β-carboxysome proteins has recently led to the construction of a chimeric protein that can functionally replace four native proteins (CcmM58, CcmM35, CcaA, and CcmN) required for carboxysome formation (Gonzalez-Esquer et al., 2015). These findings outlined the self-assembly nature and integration of carboxysomes in the cell. However, how β-carboxysome biosynthesis and organization are physiologically regulated in cyanobacteria in response to environmental changes remains poorly understood. Here, using a combination of live-cell confocal fluorescence microscopy and biochemical and physiological approaches, we investigated the formation and spatial positioning of β-carboxysomes in Synechococcus under varying light intensities. Our study provides new insights into the regulation of β-carboxysome biosynthesis by light and the roles of photosynthetic electron flow in the carboxysome assembly. Knowledge obtained from this work is fundamental to the bioengineering and modulation of functional carboxysomes to boost photosynthetic carbon fixation in dynamic and diverse environments.

In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 × 41 × 34 nm (length × width × height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria. Electronic supplementary material The online version of this article (doi:10.1007/s11120-007-9264-z) contains supplementary material, which is available to authorized users.

Red algae represent an evolutionarily important group that gave rise to the whole red clade of photosynthetic organisms. They contain a unique combination of light-harvesting systems represented by a membrane-bound antenna and by phycobilisomes situated on thylakoid membrane surfaces. So far, very little has been revealed about the mobility of their phycobilisomes and the regulation of their light-harvesting system in general. Therefore, we carried out a detailed analysis of phycobilisome dynamics in several red alga strains and compared these results with the presence (or absence) of photoprotective mechanisms. Our data conclusively prove phycobilisome mobility in two model mesophilic red alga strains, Porphyridium cruentum and Rhodella violacea. In contrast, there was almost no phycobilisome mobility in the thermophilic red alga Cyanidium caldarium that was not caused by a decrease in lipid desaturation in this extremophile. Experimental data attributed this immobility to the strong phycobilisome-photosystem interaction that highly restricted phycobilisome movement. Variations in phycobilisome mobility reflect the different ways in which light-harvesting antennae can be regulated in mesophilic and thermophilic red algae. Fluorescence changes attributed in cyanobacteria to state transitions were observed only in mesophilic P. cruentum with mobile phycobilisomes, and they were absent in the extremophilic C. caldarium with immobile phycobilisomes. We suggest that state transitions have an important regulatory function in mesophilic red algae; however, in thermophilic red algae, this process is replaced by nonphotochemical quenching.

International audience; Photosynthetic processes, including light capture, electron transfer, and energy conversion, are not only ensured by the activities of individual photosynthetic complexes but also substantially determined and regulated by the composition and assembly of the overall photosynthetic apparatus at the supramolecular level. In recent years, atomic force microscopy (AFM) has matured as a unique and powerful tool for directly assessing the supramolecular assembly of integral membrane protein complexes in their native membrane environment at submolecular resolution. This review highlights the major contributions and advances of AFM studies to our understanding of the structure of the bacterial pho-tosynthetic machinery and its regulatory arrangement during chromatic adaptation. AFM topographs of other biological membrane systems and potential future applications of AFM are also discussed. Bacterial photosynthetic apparatus Photosynthesis, performed by green plants, algae, and some bacteria, is the major source of energy on the planet. Today, it is rationalised that important lessons can be learned from nature concerning the capture and conversion of abundant solar energy for the development of renewable energy resources. Ideas comprise the use of natural photosynthesis for biofuel generation, devices that partially consist of biological materials, or bio-inspired technology. In all cases, a profound knowledge of how nature designed photosynthetic machineries is a prerequisite for developments. Indeed, through photosynthesis carried out in specialised membrane systems – the photo-synthetic membranes – all green plants, algae, and some bacteria can convert solar radiant energy with high efficiency into chemical energy [1,2]. This energy is then used to power cellular metabolisms. The purple bacterial pho-tosynthetic membranes are intracytoplasmic membranes (ICMs, see Glossary, sometimes also termed chromato-phore), where absorption and conversion of light energy is efficiently accomplished by a multicomponent assembly of pigment–protein photosynthetic complexes. These complexes include light-harvesting complexes (LH2 and LH1) and reaction centres (RCs), which together form the pho-tosynthetic unit (PSU), cytochrome (cyt) bc 1 complexes, and ATP synthases (ATPases) [3]. The physiological arrangement and strong cooperation of these protein constituents are fundamental to efficient light capture and energy transfer mechanisms.

Rubisco is the essential enzyme mediating the fixation of atmospheric CO(2) during photosynthesis. In cyanobacteria, Rubisco enzymes are densely packed and encapsulated in a specialized organelle known as the carboxysome. Well-defined Rubisco assembly and carboxysome formation are pivotal for efficient CO(2) fixation. Numerous chaperone proteins, including RbcX, are essential for proper protein folding and Rubisco assembly. In this study, we investigated the in vivo function of RbcX in the cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942) using molecular, biochemical, and live-cell fluorescence imaging approaches. Our results show that genetic deletion of the rbcX gene affects Rubisco abundance, as well as carboxysome formation and spatial distribution. Moreover, RbcX appears as one component of the carboxysome and shows a dynamic interaction with Rubisco enzymes. These in vivo observations provide insight into the role of RbcX from Syn7942 in mediating carboxysome assembly. Understanding the molecular mechanism underlying Rubisco assembly and carboxysome biogenesis will provide essential information required for engineering functional CO(2)-fixing complexes in heterogeneous organisms, especially plants, with the aim of boosting photosynthesis and agricultural productivity.