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Phycobilisomes play a crucial role in the light-harvesting mechanisms of cyanobacteria, red algae and glaucophytes, but the molecular mechanism of their regulation is largely unknown. In the cyanobacterium, Synechocystis sp. PCC 6803, we identified slr0244 as a phycobilisome-related gene using phylogenetic profiling analysis, a method used to predict gene function based on comparative genomics. To investigate the physiological function of the slr0244 gene, we characterized slr0244 mutants spectroscopically. Disruption of the slr0244 gene impaired state transition, a process by which the distribution of light energy absorbed by the phycobilisomes between two photosystems is regulated in response to the changes in light conditions. The Slr0244 protein seems to act in the process of state transition, somewhere at or downstream of the sensing step of the redox state of the plastoquinone (PQ) pool. These findings, together with past reports describing the interaction of this gene product with thioredoxin and glutaredoxin, suggest that the slr0244 gene is a novel state-transition regulator that integrates the redox signal of PQ pools with that of the photosystem I-reducing side. The protein has two universal stress protein (USP) motifs in tandem. The second motif has two conserved cysteine residues found in USPs of other cyanobacteria and land plants. These redox-type USPs with conserved cysteines may function as redox regulators in various photosynthetic organisms. Our study also shows the efficacy of phylogenetic profiling analysis in predicting the function of cyanobacterial genes that have not been annotated so far., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Cyanobacteria, red algae, and cryptophytes produce 2 classes of proteins for light harvesting: water-soluble phycobiliproteins (PBP) and membrane-intrinsic proteins that bind chlorophylls (Chls) and carotenoids. In cyanobacteria, red algae, and glaucophytes, phycobilisomes (PBS) are complexes of brightly colored PBP and linker (assembly) proteins. To date, 6 structural classes of PBS have been described: hemiellipsoidal, block-shaped, hemidiscoidal, bundle-shaped, paddle-shaped, and far-red-light bicylindrical. Two additional antenna complexes containing single types of PBP have also been described. Since 2017, structures have been reported for examples of all of these complexes except bundle-shaped PBS by cryogenic electron microscopy. PBS range in size from about 4.6 to 18 mDa and can include ∼900 polypeptides and bind >2000 chromophores. Cyanobacteria additionally produce membrane-associated proteins of the PsbC/CP43 superfamily of Chl a/b/d-binding proteins, including the iron-stress protein IsiA and other paralogous Chl-binding proteins (CBP) that can form antenna complexes with Photosystem I (PSI) and/or Photosystem II (PSII). Red and cryptophyte algae also produce CBP associated with PSI but which belong to the Chl a/b-binding protein superfamily and which are unrelated to the CBP of cyanobacteria. This review describes recent progress in structure determination for PBS and the Chl proteins of cyanobacteria, red algae, and cryptophytan algae., Competing Interests: Conflict of Interest Statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Phycobilisomes (PBs) are giant antenna supercomplexes of cyanobacteria that use phycobilin pigments to capture sunlight and transfer the collected energy to membrane-bound photosystems. In the PB core, phycobilins are bound to particular allophycocyanin (APC) proteins. Some phycobilins are thought to be terminal emitters (TEs) with red-shifted fluorescence. However, the precise identification of TEs is still under debate. In this work, we employ multiscale quantum-mechanical calculations to disentangle the excitation energy landscape of PB cores. Using the recent atomistic PB structures from Synechoccoccus PCC 7002 and Synechocystis PCC 6803, we compute the spectral properties of different APC trimers and assign the low-energy pigments. We show that the excitation energy of APC phycobilins is determined by geometric and electrostatic factors and is tuned by the specific protein-protein interactions within the core. Our findings challenge the simple picture of a few red-shifted bilins in the PB core and instead suggest that the red-shifts are established by the entire TE-containing APC trimers. Our work provides a theoretical microscopic basis for the interpretation of energy migration and time-resolved spectroscopy in phycobilisomes.

Cyanobacteria hold great potential to revolutionize conventional industries and farming practices with their light-driven chemical production. To fully exploit their photosynthetic capacity and enhance product yield, it is crucial to investigate their intricate interplay with the environment including the light intensity and spectrum. Mathematical models provide valuable insights for optimizing strategies in this pursuit. In this study, we present an ordinary differential equation-based model for the cyanobacterium Synechocystis sp. PCC 6803 to assess its performance under various light sources, including monochromatic light. Our model can reproduce a variety of physiologically measured quantities, e.g. experimentally reported partitioning of electrons through four main pathways, O2 evolution, and the rate of carbon fixation for ambient and saturated CO2. By capturing the interactions between different components of a photosynthetic system, our model helps in understanding the underlying mechanisms driving system behavior. Our model qualitatively reproduces fluorescence emitted under various light regimes, replicating Pulse-amplitude modulation (PAM) fluorometry experiments with saturating pulses. Using our model, we test four hypothesized mechanisms of cyanobacterial state transitions for ensemble of parameter sets and found no physiological benefit of a model assuming phycobilisome detachment. Moreover, we evaluate metabolic control for biotechnological production under diverse light colors and irradiances. We suggest gene targets for overexpression under different illuminations to increase the yield. By offering a comprehensive computational model of cyanobacterial photosynthesis, our work enhances the basic understanding of light-dependent cyanobacterial behavior and sets the first wavelength-dependent framework to systematically test their producing capacity for biocatalysis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pfennig et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cyanobacteria are photoautotrophic organisms that use light and water as a source of energy and electrons, respectively, to fix atmospheric carbon dioxide and release oxygen as a by-product during photosynthesis. However, photosynthesis and fitness of organisms are challenged by seasonal and diurnal fluctuations in light environments. Also, the distribution of cyanobacteria in a water column is subject to changes in the light regime. The quality and quantity of light change significantly in low and bright light environments that either limit photochemistry or result in photoinhibition due to an excess amount of light reaching reaction centers. Therefore, cyanobacteria have to adjust their light-harvesting machinery and cell morphology for the optimal harvesting of light. This adjustment of light-harvesting involves remodeling of the light-harvesting complex called phycobilisome or incorporation of chlorophyll molecules such as chlorophyll d and f into their light-harvesting machinery. Thus, photoacclimation responses of cyanobacteria at the level of pigment composition and cell morphology maximize their photosynthetic ability and fitness under a dynamic light environment. Cyanobacteria exhibit different types of photoacclimation responses that are commonly known as chromatic acclimation (CA). In this work, we discuss different types of CA reported in cyanobacteria and present a molecular mechanism of well-known type 3 CA where phycoerythrin and phycocyanin of phycobilisome changes according to light signals. We also include other aspects of type 3 CA that have been recently studied at a molecular level and highlight the importance of morphogenes, cytoskeleton, and carboxysome proteins. In summary, CA gives a unique competitive benefit to cyanobacteria by increasing their resource utilization ability and fitness., (© 2024 Scandinavian Plant Physiology Society.)

Photosynthesis converting solar energy to chemical energy is one of the most important chemical reactions on earth. In cyanobacteria, light energy is captured by antenna system phycobilisomes (PBSs) and transferred to photosynthetic reaction centers of photosystem II (PSII) and photosystem I (PSI). While most of the protein complexes involved in photosynthesis have been characterized by in vitro structural analyses, how these protein complexes function together in vivo is not well understood. Here we implemented STAgSPA, an in situ structural analysis strategy, to solve the native structure of PBS-PSII supercomplex from the cyanobacteria Arthrospira sp. FACHB439 at resolution of ~3.5 Å. The structure reveals coupling details among adjacent PBSs and PSII dimers, and the collaborative energy transfer mechanism mediated by multiple super-PBS in cyanobacteria. Our results provide insights into the diversity of photosynthesis-related systems between prokaryotic cyanobacteria and eukaryotic red algae but are also a methodological demonstration for high-resolution structural analysis in cellular or tissue samples., (© 2024. The Author(s).)

Phycobilisomes (PBSs) are light-harvesting antenna complexes in cyanobacteria that adapt to diverse light environments through the use of phycobiliproteins within the PBS structures. Freshwater cyanobacteria, such as Synechococcus elongatus PCC 7942, thrive under red light because of the presence of phycocyanin (PC) and its chromophore, phycocyanobilin (PCB), in the PBS. Cyanobacteria in shorter-wavelength light environments such as green light, employ phycoerythrin paired with phycoerythrobilin (PEB) along with PC in the PBS. Synthetic biology studies have shown that PEB production can be achieved by expression of the heterologous PEB synthases 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA) and PEB:ferredoxin oxidoreductase (PebB), leading to PEB accumulation and cellular browning. This approach is genetically unstable, and the properties of the resulting PEB-bound PBS complexes remain uncharacterized. In this study, we engineered a novel strain of Synechococcus 7942 PEB1 with finely tuned control of PEB biosynthesis. PEB1 exhibited a reversible change in the color of the culture from green to brown and pink based on PebA and PebB induction levels. High induction led to complete PCB-to-PEB substitution, causing the disassembly of the PBS rod complex. In contrast, low induction levels of PebA and PebB resulted in the formation of a stable chimeric PBS complex with partial PCB-to-PEB substitution. This acclimation enabled efficient light harvesting in the green spectrum and energy transfer to the photosynthetic reaction center. These findings, which improve our understanding of PBS and highlight the structural importance of the bilin composition, provide a foundation for future studies on PBS adaptation in bioengineering, synthetic biology, and renewable energy.

Cyanobacteria were the first microorganisms that released oxygen into the atmosphere billions of years ago. To do it safely under intense sunlight, they developed strategies that prevent photooxidation in the photosynthetic membrane, by regulating the light-harvesting activity of their antenna complexes-the phycobilisomes-via the orange-carotenoid protein (OCP). This water-soluble protein interacts with the phycobilisomes and triggers nonphotochemical quenching (NPQ), a mechanism that safely dissipates overexcitation in the membrane. To date, the mechanism of action of OCP in performing NPQ is unknown. In this work, we performed ultrafast spectroscopy on a minimal NPQ system composed of the active domain of OCP bound to the phycobilisome core. The use of this system allowed us to disentangle the signal of the carotenoid from that of the bilins. Our results demonstrate that the binding to the phycobilisomes modifies the structure of the ketocarotenoid associated with OCP. We show that this molecular switch activates NPQ, by enabling excitation-energy transfer from the antenna pigments to the ketocarotenoid.

Phycobilisome (PBS) is a large pigment-protein complex in cyanobacteria and red algae responsible for capturing sunlight and transferring its energy to photosystems (PS). Spectroscopic and structural properties of various PBSs have been widely studied, however, the nature of so-called complex-complex interactions between PBS and PSs remains much less explored. In this work, we have investigated the function of a newly identified PBS linker protein, ApcG, some domain of which, together with a loop region (PB-loop in ApcE), is possibly located near the PBS-PS interface. Using Synechocystis sp. PCC 6803, we generated an ApcG deletion mutant and probed its deletion effect on the energetic coupling between PBS and photosystems. Steady-state and time-resolved spectroscopic characterization of the purified ΔApcG-PBS demonstrated that ApcG removal weakly affects the photophysical properties of PBS for which the spectroscopic properties of terminal energy emitters are comparable to those of PBS from wild-type strain. However, analysis of fluorescence decay imaging datasets reveals that ApcG deletion induces disruptions within the allophycocyanin (APC) core, resulting in the emergence (splitting) of two spectrally diverse subgroups with some short-lived APC. Profound spectroscopic changes of the whole ΔApcG mutant cell, however, emerge during state transition, a dynamic process of light scheme adaptation. The mutant cells in State I show a substantial increase in PBS-related fluorescence. On the other hand, global analysis of time-resolved fluorescence demonstrates that in general ApcG deletion does not alter or inhibit state transitions interpreted in terms of the changes of the PSII and PSI fluorescence emission intensity. The results revealed yet-to-be discovered mechanism of ApcG-docking induced excitation energy transfer regulation within PBS or to Photosystems., Competing Interests: Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

CpcL-phycobilisomes (CpcL-PBSs) are a reduced type of phycobilisome (PBS) found in several cyanobacteria. They lack the traditional PBS terminal energy emitters, but still show the characteristic red-shifted fluorescence at ~670 nm. We established a method of assembling in vitro a rod-membrane linker protein, CpcL, with phycocyanin, generating complexes with the red-shifted spectral features of CpcL-PBSs. The red-shift arises from the interaction of a conserved key glutamine, Q57 of CpcL in Synechocystis sp. PCC 6803, with a single phycocyanobilin chromophore of trimeric phycocyanin at one of the three β82-sites. This chromophore is the terminal energy acceptor of CpcL-PBSs and donor to the photosystem(s). This mechanism also operates in PBSs from Acaryochloris marina MBIC11017. We then generated multichromic complexes harvesting light over nearly the complete visible range via the replacement of phycocyanobilin chromophores at sites α84 and β153 of phycocyanins by phycoerythrobilin and/or phycourobilin. The results demonstrate the rational design of biliprotein-based light-harvesting elements by engineering CpcL and phycocyanins, which broadens the light-harvesting range and accordingly improves the light-harvesting capacity and may be potentially applied in solar energy harvesting., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cyanobacteria harvest light by using architecturally complex, soluble, light-harvesting complexes known as phycobilisomes (PBSs). PBS diversity includes specialized subunit paralogs that are tuned to specific regions of the light spectrum; some cyanobacterial lineages can even absorb far-red light. In a recent issue of the Journal of Biological Chemistry, Gisriel et al. reported the cryo-electron microscopic structure of a far-red PBS core, showing how bilin binding in the α-subunits of allophycocyanin paralogs can modify the bilin-binding site to red shift the absorbance spectrum. This work helps explain how cyanobacteria can grow in environments where most of the visible light has been filtered out., Competing Interests: Conflict of interest The author declares no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cyanobacteria use large antenna complexes called phycobilisomes (PBSs) for light harvesting. However, intense light triggers non-photochemical quenching, where the orange carotenoid protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood. Using cryo-electron microscopy, we unveiled the OCP-PBS complex structure at 1.6- to 2.1-angstrom resolution, showcasing its inherent flexibility. Using multiscale quantum chemistry, we disclosed the quenching mechanism. Identifying key protein residues, we clarified how canthaxanthin's transition dipole moment in its lowest-energy dark state becomes large enough for efficient energy transfer from phycocyanobilins. Our energy transfer model offers a detailed understanding of the atomic determinants of light harvesting regulation and antenna architecture in cyanobacteria.

Phycobilisomes (PBs) play an important role in cyanobacterial photosynthesis. They capture light and transfer excitation energy to the photosynthetic reaction centres. PBs are also central to some photoprotective and photoregulatory mechanisms that help sustain photosynthesis under non-optimal conditions. Amongst the mechanisms involved in excitation energy dissipation that are activated in response to excessive illumination is a recently discovered light-induced mechanism that is intrinsic to PBs and has been the least studied. Here, we used single-molecule spectroscopy and developed robust data analysis methods to explore the role of a terminal emitter subunit, ApcE, in this intrinsic, light-induced mechanism. We isolated the PBs from WT Synechocystis PCC 6803 as well as from the ApcE-C190S mutant of this strain and compared the dynamics of their fluorescence emission. PBs isolated from the mutant (i.e., ApcE-C190S-PBs), despite not binding some of the red-shifted pigments in the complex, showed similar global emission dynamics to WT-PBs. However, a detailed analysis of dynamics in the core revealed that the ApcE-C190S-PBs are less likely than WT-PBs to enter quenched states under illumination but still fully capable of doing so. This result points to an important but not exclusive role of the ApcE pigments in the light-induced intrinsic excitation energy dissipation mechanism in PBs., (© 2024. The Author(s).)

The chromophorylated PBLcm domain of the ApcE linker protein in the cyanobacterial phycobilisome (PBS) serves as a bottleneck for Förster resonance energy transfer (FRET) from the PBS to the antennal chlorophyll of photosystem II (PS II) and as a redirection point for energy distribution to the orange protein ketocarotenoid (OCP), which is excitonically coupled to the PBLcm chromophore in the process of non-photochemical quenching (NPQ) under high light conditions. The involvement of PBLcm in the quenching process was first directly demonstrated by measuring steady-state fluorescence spectra of cyanobacterial cells at different stages of NPQ development. The time required to transfer energy from the PBLcm to the OCP is several times shorter than the time it takes to transfer energy from the PBLcm to the PS II, ensuring quenching efficiency. The data obtained provide an explanation for the different rates of PBS quenching in vivo and in vitro according to the half ratio of OCP/PBS in the cyanobacterial cell, which is tens of times lower than that realized for an effective NPQ process in solution., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

In response to fluctuation in light intensity and quality, oxygenic photosynthetic organisms modify their light-harvesting and excitation energy-transfer processes to maintain optimal photosynthetic activity. Glaucophytes, which are a group of primary symbiotic algae, possess light-harvesting antennas called phycobilisomes (PBSs) consistent with cyanobacteria and red algae. However, compared with cyanobacteria and red algae, glaucophytes are poorly studied and there are few reports on the regulation of photosynthesis in the group. In this study, we examined the long-term light adaptation of light-harvesting functions in a glaucophyte, Cyanophora paradoxa, grown under different light conditions. Compared with cells grown under white light, the relative number of PBSs to photosystems (PSs) increased in blue-light-grown cells and decreased in green-, yellow-, and red-light-grown cells. Moreover, the PBS number increased with increment in the monochromatic light intensity. More energy was transferred from PBSs to PSII than to PSI under blue light, whereas energy transfer from PBSs to PSII was reduced under green and yellow lights, and energy transfer from the PBSs to both PSs decreased under red light. Decoupling of PBSs was induced by intense green, yellow, and red lights. Energy transfer from PSII to PSI (spillover) was observed, but the contribution of the spillover did not distinctly change depending on the culture light intensity and quality. These results suggest that the glaucophyte C. paradoxa modifies the light-harvesting abilities of both PSs and excitation energy-transfer processes between the light-harvesting antennas and both PSs during long-term light adaption., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Photosynthetic organisms harvest light using pigment-protein complexes. In cyanobacteria, these are water-soluble antennae known as phycobilisomes (PBSs). The light absorbed by PBS is transferred to the photosystems in the thylakoid membrane to drive photosynthesis. The energy transfer between these complexes implies that protein-protein interactions allow the association of PBS with the photosystems. However, the specific proteins involved in the interaction of PBS with the photosystems are not fully characterized. Here, we show in Synechocystis sp. PCC 6803 that the recently discovered PBS linker protein ApcG (sll1873) interacts specifically with PSII through its N-terminal region. Growth of cyanobacteria is impaired in apcG deletion strains under light-limiting conditions. Furthermore, complementation of these strains using a phospho-mimicking version of ApcG causes reduced growth under normal growth conditions. Interestingly, the interaction of ApcG with PSII is affected when a phospho-mimicking version of ApcG is used, targeting the positively charged residues interacting with the thylakoid membrane, suggesting a regulatory role mediated by phosphorylation of ApcG. Low-temperature fluorescence measurements showed decreased PSI fluorescence in apcG deletion and complementation strains. The PSI fluorescence was the lowest in the phospho-mimicking complementation strain, while the pull-down experiment showed no interaction of ApcG with PSI under any tested condition. Our results highlight the importance of ApcG for selectively directing energy harvested by the PBS and imply that the phosphorylation status of ApcG plays a role in regulating energy transfer from PSII to PSI., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phycobilisomes (PBSs) are giant water-soluble light-harvesting complexes of cyanobacteria and red algae, consisting of hundreds of phycobiliproteins precisely organized to deliver the energy of absorbed light to chlorophyll chromophores of the photosynthetic electron-transport chain. Quenching the excess of excitation energy is necessary for the photoprotection of photosynthetic apparatus. In cyanobacteria, quenching of PBS excitation is provided by the Orange Carotenoid Protein (OCP), which is activated under high light conditions. In this work, we describe parameters of anti-Stokes fluorescence of cyanobacterial PBSs in quenched and unquenched states. We compare the fluorescence readout from entire phycobilisomes and their fragments. The obtained results revealed the heterogeneity of conformations of chromophores in isolated phycobiliproteins, while such heterogeneity was not observed in the entire PBS. Under excitation by low-energy quanta, we did not detect a significant uphill energy transfer from the core to the peripheral rods of PBS, while the one from the terminal emitters to the bulk allophycocyanin chromophores is highly probable. We show that this direction of energy migration does not eliminate fluorescence quenching in the complex with OCP. Thus, long-wave excitation provides new insights into the pathways of energy conversion in the phycobilisome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2023. The Author(s).)

Gloeobacter violaceus is an ancient cyanobacterium as it branches out from the basal position in the phylogenic tree of cyanobacteria. It lacks thylakoid membranes and its unique bundle-shaped type of phycobilisomes (PBS) for light harvesting in photosynthesis are located on the interior side of cytoplasmic membranes. The PBS from G. violaceus have two large linker proteins that are not present in any other PBS, Glr2806, and Glr1262, which are encoded by the genes glr2806 and glr1262, respectively. The location and functions of the linkers Glr2806 and Glr1262 are currently unclear. Here, we report the studies of mutagenetic analysis of glr2806 and the genes of cpeBA, encoding the β and α subunits of phycoerythrin (PE), respectively. In the mutant lacking glr2806, the length of the PBS rods remains unchanged, but the bundles are less tightly packed as examined by electron microscopy with negative staining. It is also shown that two hexamers are missing in the peripheral area of the PBS core, strongly suggesting that the linker Glr2806 is located in the core area instead of the rods. In the mutant lacking the cpeBA genes, PE is no longer present and the PBS rods have only three layers of phycocyanin hexamers. The construction of deletional mutants in G. violaceus, achieved for the first time, provides critical information for our understanding of its unique PBS and should be useful in studies of other aspects of this interesting organism as well., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Phycobilisomes (PBSs), which are huge pigment-protein complexes displaying distinctive color variations, bind to photosystem cores for excitation-energy transfer. It is known that isolation of supercomplexes consisting of PBSs and photosystem I (PSI) or PBSs and photosystem II is challenging due to weak interactions between PBSs and the photosystem cores. In this study, we succeeded in purifying PSI-monomer-PBS and PSI-dimer-PBS supercomplexes from the cyanobacterium Anabaena sp. PCC 7120 grown under iron-deficient conditions by anion-exchange chromatography, followed by trehalose density gradient centrifugation. The absorption spectra of the two types of supercomplexes showed apparent bands originating from PBSs, and their fluorescence-emission spectra exhibited characteristic peaks of PBSs. Two-dimensional blue-native (BN)/SDS-PAGE of the two samples showed a band of CpcL, which is a linker protein of PBS, in addition to PsaA/B. Since interactions of PBSs with PSI are easily dissociated during BN-PAGE using thylakoids from this cyanobacterium grown under iron-replete conditions, it is suggested that iron deficiency for Anabaena induces tight association of CpcL with PSI, resulting in the formation of PSI-monomer-PBS and PSI-dimer-PBS supercomplexes. Based on these findings, we discuss interactions of PBSs with PSI in Anabaena., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Our planet is sustained by sunlight, the primary energy source made accessible to all life forms by photoautotrophs. Photoautotrophs are equipped with light-harvesting complexes (LHCs) that enable efficient capture of solar energy, particularly when light is limiting. However, under high light, LHCs can harvest photons in excess of the utilization capacity of cells, causing photodamage. This damaging effect is most evident when there is a disparity between the amount of light harvested and carbon available. Cells strive to circumvent this problem by dynamically adjusting the antenna structure in response to the changing light signals, a process known to be energetically expensive. Much emphasis has been laid on elucidating the relationship between antenna size and photosynthetic efficiency and identifying strategies to synthetically modify antennae for optimal light capture. Our study is an effort in this direction and investigates the possibility of modifying phycobilisomes, the LHCs present in cyanobacteria, the simplest of photoautotrophs. We systematically truncate the phycobilisomes of Synechococcus elongatus UTEX 2973, a widely studied, fast-growing model cyanobacterium and demonstrate that partial truncation of its antenna can lead to a growth advantage of up to 36% compared to the wild type and an increase in sucrose titer of up to 22%. In contrast, targeted deletion of the linker protein which connects the first phycocyanin rod to the core proved detrimental, indicating that the core alone is not enough, and it is essential to maintain a minimal rod-core structure for efficient light harvest and strain fitness. IMPORTANCE Light energy is essential for the existence of life on this planet, and only photosynthetic organisms, equipped with light-harvesting antenna protein complexes, can capture this energy, making it readily accessible to all other life forms. However, these light-harvesting antennae are not designed to function optimally under extreme high light, a condition which can cause photodamage and significantly reduce photosynthetic productivity. In this study, we attempt to assess the optimal antenna structure for a fast-growing, high-light tolerant photosynthetic microbe with the goal of improving its productivity. Our findings provide concrete evidence that although the antenna complex is essential, antenna modification is a viable strategy to maximize strain performance under controlled growth conditions. This understanding can also be translated into identifying avenues to improve light harvesting efficiency in higher photoautotrophs., Competing Interests: The authors declare no conflict of interest.

Orange carotenoid protein (OCP) is a photoactive protein that participates in the photoprotection of cyanobacteria. There are 2 full-length OCP proteins, 4 N-terminal paralogs (helical carotenoid protein [HCP]), and 1 C-terminal domain-like carotenoid protein (CCP) found in Nostoc flagelliforme, a desert cyanobacterium. All HCPs (HCP1 to 3 and HCP6) from N. flagelliforme demonstrated their excellent singlet oxygen quenching activities, in which HCP2 was the strongest singlet oxygen quencher compared with others. Two OCPs, OCPx1 and OCPx2, were not involved in singlet oxygen scavenging; instead, they functioned as phycobilisome fluorescence quenchers. The fast-acting OCPx1 showed more effective photoactivation and stronger phycobilisome fluorescence quenching compared with OCPx2, which behaved differently from all reported OCP paralogs. The resolved crystal structure and mutant analysis revealed that Trp111 and Met125 play essential roles in OCPx2, which is dominant and long acting. The resolved crystal structure of OCPx2 is maintained in a monomer state and showed more flexible regulation in energy quenching activities compared with the packed oligomer of OCPx1. The recombinant apo-CCP obtained the carotenoid pigment from holo-HCPs and holo-OCPx1 of N. flagelliforme. No such carotenoid transferring processes were observed between apo-CCP and holo-OCPx2. The close phylogenetic relationship of OCP paralogs from subaerial Nostoc species indicates an adaptive evolution toward development of photoprotection: protecting cellular metabolism against singlet oxygen damage using HCPs and against excess energy captured by active phycobilisomes using 2 different working modes of OCPx., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phycobilisomes (PBS) are the major light harvesting complexes of photosynthesis in the cyanobacteria and red algae. CpcL-PBS is a type of small PBS in cyanobacteria that transfers energy directly to photosystem I without the core structure. Here we report the cryo-EM structure of the CpcL-PBS from the cyanobacterium Synechocystis sp. PCC 6803 at 2.6-Å resolution. The structure shows the CpcD domain of ferredoxin: NADP + oxidoreductase is located at the distal end of CpcL-PBS, responsible for its attachment to PBS. With the evidence of ultrafast transient absorption and fluorescence spectroscopy, the roles of individual bilins in energy transfer are revealed. The bilin 1I β 82 2 located near photosystem I has an enhanced planarity and is the red-bilin responsible for the direct energy transfer to photosystem I., (© 2023. The Author(s).)

The phycobilisome (PBS) is an antenna protein complex in cyanobacteria, Glaucocystophytes, and red algae. In the standard PBS, the rod-core PBS, the rods are connected to the core by the rod-core linker protein CpcG. The rod-core PBS transfers the light energy mainly to photosystem (PS) II and to a lesser extent to PSI. Cyanobacteria assemble another type of PBS, the CpcL-PBS, which consists of only one rod. This rod-type PBS is connected to the thylakoid membrane by the linker protein CpcL and is a PSI-specific antenna. In the filamentous heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120, the CpcL-PBS forms a complex with the tetrameric PSI (PBS-PSI supercomplex). The CpcL-PBS and the rod part of the rod-core PBS are identical except for the linker proteins CpcL and CpcG. How cells control the accumulation of the two different types of PBS is unknown. Here, we analyzed two mutant strains which either lack the major rod-core linker CpcG4 or overexpress the rod-membrane linker CpcL. In both mutant strains, more and larger PBS-PSI supercomplexes accumulated compared to the wild type. Our results suggest that CpcL and CpcG4 compete for the same phycobiliprotein pool, and therefore the CpcL/CpcG4 ratio determines the levels of PBS-PSI supercomplexes. We propose that the CpcL-PBS and the rod-core PBS fulfill distinct functions in light harvesting., (© 2023. The Author(s).)

The phycobilisome (PBS) is the major light-harvesting apparatus in cyanobacteria and red algae. It is a large multi-subunit protein complex of several megadaltons that is found on the stromal side of thylakoid membranes in orderly arrays. Chromophore lyases catalyse the thioether bond between apoproteins and phycobilins of PBSs. Depending on the species, composition, spatial assembly, and, especially, the functional tuning of different phycobiliproteins mediated by linker proteins, PBSs can absorb light between 450 and 650 nm, making them efficient and versatile light-harvesting systems. However, basic research and technological innovations are needed, not only to understand their role in photosynthesis but also to realise the potential applications of PBSs. Crucial components including phycobiliproteins, phycobilins, and lyases together make the PBS an efficient light-harvesting system, and these provide a scheme to explore the heterologous synthesis of PBS. Focusing on these topics, this review describes the essential components needed for PBS assembly, the functional basis of PBS photosynthesis, and the applications of phycobiliproteins. Moreover, key technical challenges for heterologous biosynthesis of phycobiliproteins in chassis cells are discussed.

Phytochromes are linear tetrapyrrole-binding photoreceptors in eukaryotes and bacteria, primarily responding to red and far-red light signals reversibly. Among the GAF domain-based phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show various optical properties covering the entire visible region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here, we utilize ancestral sequence reconstruction and biochemical verification to show that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore is crucial to perceive green light. The ancestral cyanobacteriochromes show only modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense the incident green/red light ratio. Many cyanobacteria can utilize green light for photosynthesis using phycobilisome light-harvesting complexes. The green/red sensing cyanobacteriochromes may have allowed better acclimation to changing light environments by rearranging the absorption capacity of the phycobilisome through chromatic acclimation., (© 2023. The Author(s).)

The phycobilisome is the primary light-harvesting antenna in cyanobacterial and red algal oxygenic photosynthesis. It maintains near-unity efficiency of energy transfer to reaction centers despite relying on slow exciton hopping along a relatively sparse network of highly fluorescent phycobilin chromophores. How the complex maintains this high efficiency remains unexplained. Using a two-dimensional electronic spectroscopy polarization scheme that enhances energy transfer features, we directly watch energy flow in the phycobilisome complex of Synechocystis sp. PCC 6803 from the outer phycocyanin rods to the allophycocyanin core. The observed downhill flow of energy, previously hidden within congested spectra, is faster than timescales predicted by Förster hopping along single rod chromophores. We attribute the fast, 8 ps energy transfer to interactions between rod-core linker proteins and terminal rod chromophores, which facilitate unidirectionally downhill energy flow to the core. This mechanism drives the high energy transfer efficiency in the phycobilisome and suggests that linker protein-chromophore interactions have likely evolved to shape its energetic landscape.

The aim of this study was to examine how light intensity and quality affect the photosynthetic apparatus of Cyanidioschyzon merolae cells by modulating the structure and function of phycobilisomes. Cells were grown in equal amounts of white, blue, red, and yellow light of low (LL) and high (HL) intensity. Biochemical characterization, fluorescence emission, and oxygen exchange were used to investigate selected cellular physiological parameters. It was found that the allophycocyanin content was sensitive only to light intensity, whereas the phycocynin content was also sensitive to light quality. Furthermore, the concentration of the PSI core protein was not affected by the intensity or quality of the growth light, but the concentration of the PSII core D1 protein was. Finally, the amount of ATP and ADP was lower in HL than LL. In our opinion, both light intensity and quality are main factors that play an important regulatory role in acclimatization/adaptation of C. merolae to environmental changes, and this is achieved by balancing the amounts of thylakoid membrane and phycobilisome proteins, the energy level, and the photosynthetic and respiratory activity. This understanding contributes to the development of a mix of cultivation techniques and genetic changes for a future large-scale synthesis of desirable biomolecules.

Efforts to stably over-express recombinant proteins in cyanobacteria are hindered due to cellular proteasome activity that efficiently degrades foreign proteins. Recent work from this lab showed that a variety of exogenous genes from plants, humans, and bacteria can be successfully and stably over-expressed in cyanobacteria, as fusion constructs with the abundant β-subunit of phycocyanin (the cpcB gene product) in quantities up to 10-15% of the total cell protein. The CpcB*P fusion proteins did not simply accumulate in a soluble free-floating form in the cell but, rather, they assembled as functional (α,β*P) 3 CpcG1 heterohexameric light-harvesting phycocyanin antenna discs, where α is the CpcA α-subunit of phycocyanin, β*P is the CpcB*P fusion protein, the asterisk denoting fusion, and CpcG1 is the 28.9 kDa phycocyanin disc linker polypeptide (Hidalgo Martinez et al., 2022). The present work showed that the CpcA α-subunit of phycocyanin and the CpcG1 28.9 kDa phycocyanin disc linker polypeptide can also successfully serve as leading sequences in functional heterohexameric (α*P,β) 3 CpcG1 and (α,β) 3 CpcG1*P fusion constructs that permit stable recombinant protein over-expression and accumulation. These were shown to form a residual light-harvesting antenna and to contribute to photosystem-II photochemistry in the cyanobacterial cells. The work suggested that cyanobacterial cells need phycocyanin for light absorption, photosynthesis, and survival and, therefore, may tolerate the presence of heterologous recombinant proteins, when the latter are in a fusion construct configuration with essential cellular proteins, e.g., phycocyanin, thus allowing their substantial and stable accumulation., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Phycobilisomes (PBSs) are the major photosynthetic light-harvesting complexes in cyanobacteria and red algae. PBS, a multisubunit protein complex, has two major interfaces that comprise intrinsically disordered regions (IDRs): rod-core and core-membrane. IDRs do not form regular, three-dimensional structures on their own. Their presence in the photosynthetic pigment-protein complexes portends their structural and functional importance. A recent model suggests that PB-loop, an IDR located on the PBS subunit ApcE and C-terminal extension (CTE) of the PBS subunit ApcG, forms a structural protrusion on the PBS core-membrane side, facing the thylakoid membrane. Here, the structural synergy between the rod-core region and the core-membrane region was investigated using quantitative mass spectrometry (MS). The AlphaFold-predicted CpcG-CTE structure was first modeled onto the PBS rod-core region, guided and justified by the isotopically encoded structural MS data. Quantitative cross-linking MS analysis revealed that the structural proximity of the PB-loop in ApcE and ApcG-CTE is significantly disturbed in the absence of six PBS rods, which are attached to PBS via CpcG-CTE, indicative of drastic conformational changes and decreased structural integrity. These results suggest that CpcG-rod attachment on the PBS rod-core side is essentially required for the PBS core-membrane structural assembly. The hypothesized long-range synergy between the rod-core interface (where the orange carotenoid protein also functions) and the terminal energy emitter of PBS must have important regulatory roles in PBS core assembly, light-harvesting, and excitation energy transmission. These data also lend strategies that genetic truncation of the light-harvesting antennas aimed for improved photosynthetic productivity must rely on an in-depth understanding of their global structural integrity.

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis 1,2 . The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs) 3 . Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS-PSII-PSI-LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis 4 , providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS-PSII-PSI-LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Thermophilic cyanobacteria are cosmopolitan and abundant in the thermal environment. Their light-harvesting complexes, phycobilisomes (PBS), are highly important in photosynthesis. To date, there is limited information on the PBS composition of thermophilic cyanobacteria whose habitats are challenging for survival. Herein, genome-based methods were used to investigate the molecular components of PBS in 19 well-described thermophilic cyanobacteria. These cyanobacteria are from the genera Leptolyngbya , Leptothermofonsia , Ocullathermofonsia , Thermoleptolyngbya , Trichothermofonsia , Synechococcus , Thermostichus , and Thermosynechococcus . According to the phycobiliprotein (PBP) composition of the rods, two pigment types are observed in these thermophiles. The amino acid sequence analysis of different PBP subunits suggests several highly conserved cysteine residues in these thermophiles. Certain amino acid contents in the PBP of thermophiles are significantly higher than their mesophilic counterparts, highlighting the potential roles of specific substitutions of amino acid in the adaptive thermostability of light-harvesting complexes in thermophilic cyanobacteria. Genes encoding PBS linker polypeptides vary among the thermophiles. Intriguingly, motifs in linker apcE indicate a photoacclimation of a far-red light by Leptolyngbya JSC-1, Leptothermofonsia E412, and Ocullathermofonsia A174. The composition pattern of phycobilin lyases is consistent among the thermophiles, except for Thermostichus strains that have extra homologs of cpcE , cpcF , and cpcT . In addition, phylogenetic analyses of genes coding for PBPs, linkers, and lyases suggest extensive genetic diversity among these thermophiles, which is further discussed with the domain analyses. Moreover, comparative genomic analysis suggests different genomic distributions of PBS-related genes among the thermophiles, indicating probably various regulations of expression. In summary, the comparative analysis elucidates distinct molecular components and organization of PBS in thermophilic cyanobacteria. These results provide insights into the PBS components of thermophilic cyanobacteria and fundamental knowledge for future research regarding structures, functions, and photosynthetic improvement.

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, are a resource for photosynthetic, photonic and fluorescence labeling elements. They cover an exceptionally broad spectral range, but the complex superstructure and assembly have been an obstacle. By replacing in Synechocystis sp. PCC 6803 the biliverdin reductases, we studied the role of chromophores in the assembly of the phycobilisome core. Introduction of the green-absorbing phycoerythrobilin instead of the red-absorbing phycocyanobilin inhibited aggregation. A novel, trimeric allophycocyanin (Dic-APC) was obtained. In the small (110 kDa) unit, the two chromophores, phycoerythrobilin and phytochromobilin, cover a wide spectral range (550 to 660 nm). Due to efficient energy transfer, it provides an efficient artificial light-harvesting element. Dic-APC was generated in vitro by using the contained core-linker, L C , for template-assisted purification and assembly. Labeling the linker provides a method for targeting Dic-APC., (© 2022 Wiley-VCH GmbH.)

Several cyanobacteria can adjust their light-harvesting machinery in response to existing light signals in a process called chromatic acclimation (CA) which permits the utilization of available light resources for photosynthesis. CA involves alteration in the pigment composition of a major light-harvesting complex called phycobilisome (PBS) and allows some cyanobacteria to utilize green light (GL) to drive photosynthesis. However, cyanobacteria, in contrast with eukaryotic algae and higher plants, can not utilize blue light (BL) for photosynthesis due to their dependency on PBS. Here, we studied a black-colored soil crust that was composed of a single cyanobacterium identified and named Oscillatoria sp. Malviya-1 after phenotypic and phylogenetic analyses. The black-colored crust can absorb light from almost all parts of photosynthetically active radiation (400-700 nm) and ultraviolet radiation (280-400 nm) due to the presence of photosynthetic pigments and microbial sunscreens such as chlorophyll ɑ, carotenoids, phycoerythrin, phycocyanin, allophycocyanin, mycosporine-like amino acids, and scytonemin. Unlike other cyanobacteria, Oscillatoria sp. Malviya-1 can grow using GL, BL, and red light (RL) in addition to white light (WL) which was accompanied by the different colors of the mat under different light conditions. The presence of CA and sunscreens compounds can maximize the fitness of soil crust under a dynamic light environment, UVR, and desiccation. Detailed study of Oscillatoria sp. Malviya-1 will provide information on the mechanism of CA in cyanobacterial soil crust and its unique ability to use both GL and BL., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Eukaryotic photosynthesis originated in the course of evolution as a result of the uptake of some unstored cyanobacterium and its transformation to chloroplasts by an ancestral heterotrophic eukaryotic cell. The pigment apparatus of Archaeplastida and other algal phyla that emerged later turned out to be arranged in the same way. Pigment-protein complexes of photosystem I (PS I) and photosystem II (PS II) are characterized by uniform structures, while the light-harvesting antennae have undergone a series of changes. The phycobilisome (PBS) antenna present in cyanobacteria was replaced by Chl a/b - or Chl a/c -containing pigment-protein complexes in most groups of photosynthetics. In the form of PBS or phycobiliprotein aggregates, it was inherited by members of Cyanophyta, Cryptophyta, red algae, and photosynthetic amoebae. Supramolecular organization and architectural modifications of phycobiliprotein antennae in various algal phyla in line with the endosymbiotic theory of chloroplast origin are the subject of this review.

This study assesses the ability of a new active fluorometer, the LabSTAF, to diagnostically assess the physiology of freshwater cyanobacteria in a reservoir exhibiting annual blooms. Specifically, we analyse the correlation of relative cyanobacteria abundance with photosynthetic parameters derived from fluorescence light curves (FLCs) obtained using several combinations of excitation wavebands, photosystem II (PSII) excitation spectra and the emission ratio of 730 over 685 nm (Fo(730/685)) using excitation protocols with varying degrees of sensitivity to cyanobacteria and algae. FLCs using blue excitation (B) and green−orange−red (GOR) excitation wavebands capture physiology parameters of algae and cyanobacteria, respectively. The green−orange (GO) protocol, expected to have the best diagnostic properties for cyanobacteria, did not guarantee PSII saturation. PSII excitation spectra showed distinct response from cyanobacteria and algae, depending on spectral optimisation of the light dose. Fo(730/685), obtained using a combination of GOR excitation wavebands, Fo(GOR, 730/685), showed a significant correlation with the relative abundance of cyanobacteria (linear regression, p-value < 0.01, adjusted R2 = 0.42). We recommend using, in parallel, Fo(GOR, 730/685), PSII excitation spectra (appropriately optimised for cyanobacteria versus algae), and physiological parameters derived from the FLCs obtained with GOR and B protocols to assess the physiology of cyanobacteria and to ultimately predict their growth. Higher intensity LEDs (G and O) should be considered to reach PSII saturation to further increase diagnostic sensitivity to the cyanobacteria component of the community.

The light environment in a mixing water column is arguably the most erratic condition under which photosynthesis functions. Shifts in light intensity, by an order of magnitude, can occur over the time scale of hours. In marine Synechococcus, light is harvested by massive, membrane attached, phycobilisome chromophore-protein complexes (PBS). We examined the ability of a phycobilisome-containing marine Synechococcus strain (WH8102) to acclimate to illumination perturbations on this scale. Although changes in pigment composition occurred gradually over the course of days, we did observe significant and reversible changes in the pigment's fluorescence emission spectra on a time scale of hours. Upon transition to ten-fold higher intensities, we observed a decrease in the energy transferred to Photosystem II. At the same time, the spectral composition of PBS fluorescence emission shifted. Unlike fluorescence quenching mechanisms, this phenomenon resulted in increased fluorescence intensities. These data suggest a mechanism by which marine Synechococcus WH8102 detaches hexamers from the phycobilisome structure. The fluorescence yield of these uncoupled hexamers is high. The detachment process does not require protein synthesis as opposed to reattachment. Hence, the most likely process would be the degradation and resynthesis of labile PBS linker proteins. Experiments with additional species yielded similar results, suggesting that this novel mechanism might be broadly used among PBS-containing organisms., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Despite a global prevalence of photosynthetic organisms in the ocean's mesophotic zone (30-200+ m depth), the mechanisms that enable photosynthesis to proceed in this low light environment are poorly defined. Red coralline algae are the deepest known marine benthic macroalgae - here we investigated the light harvesting mechanism and mesophotic acclimatory response of the red coralline alga Lithothamnion glaciale., Results: Following initial absorption by phycourobilin and phycoerythrobilin in phycoerythrin, energy was transferred from the phycobilisome to photosystems I and II within 120 ps. This enabled delivery of 94% of excitations to reaction centres. Low light intensity, and to a lesser extent a mesophotic spectrum, caused significant acclimatory change in chromophores and biliproteins, including a 10% increase in phycoerythrin light harvesting capacity and a 20% reduction in chlorophyll-a concentration and photon requirements for photosystems I and II. The rate of energy transfer remained consistent across experimental treatments, indicating an acclimatory response that maintains energy transfer., Conclusions: Our results demonstrate that responsive light harvesting by phycobilisomes and photosystem functional acclimation are key to red algal success in the mesophotic zone., (© 2022. The Author(s).)

The phycobilisome is an oligomeric chromoprotein complex that serves as the principal mid-visible light-harvesting system in cyanobacteria. Here we report the observation of excitation-energy-transfer pathways involving delocalized optical excitations of the bilin (linear tetrapyrrole) chromophores in intact phycobilisomes isolated from Fremyella diplosiphon. By using broadband multidimensional electronic spectroscopy with 6.7-fs laser pulses, we are able to follow the progress of excitation energy from the phycoerythrin disks at the ends of the phycobilisome's rods to the C-phycocyanin disks along their length in <600 fs. Oscillation maps show that coherent wavepacket motions prominently involving the hydrogen out-of-plane vibrations of the bilins mediate non-adiabatic relaxation of a manifold of vibronic exciton states. However, the charge-transfer character of the bilins in the allophycocyanin-containing segments localizes the excitations in the core of the phycobilisome, yielding a kinetic bottleneck that enables photoregulatory mechanisms to operate efficiently on the >10-ps timescale., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Nonphotochemical quenching acts as a frontline response to prevent excitation energy from reaching the photochemical reaction center of photosystem II before photodamage occurs. Strong fluorescence quenching after merely one multi-turnover saturating light pulse characterizes a unique feature of nonphotochemical quenching in red algae. Several mechanisms underlying red algal nonphotochemical quenching have been proposed, yet which process(es) dominantly account for the strong fluorescence quenching is still under discussion. Here we assessed multiple nonphotochemical quenching processes in the extremophilic red alga Cyanidioschyzon merolae under light pulse and continuous illumination conditions. To assess the nonphotochemical quenching processes that might display different kinetics, fluorescence emission spectra at 77 K were measured after different periods of light treatments, and external fluorophores were added for normalization of the fluorescence level. The phycobilisome- and photosystem II-related nonphotochemical quenching processes were distinguished by light preferentially absorbed by phycobilisomes and photosystems, respectively. Multiple nonphotochemical quenching processes, including the energetic decoupling of phycobilisomes from photosystem II, the energy spillover from phycobilisomes to photosystem I and from photosystem II to photosystem I, were identified along with the previously identified intrinsic quenching within photosystem II. The ability to use multiple nonphotochemical quenching processes appears to maximize the light harvesting efficiency for photochemistry and to provide the flexibility of the energy redistribution between photosystem II and photosystem I. The effect of the various ionophores on the nonphotochemical quenching level suggests that nonphotochemical quenching is modulated by transmembrane gradients of protons and other cations., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Light harvesting is fundamental for production of ATP and reducing equivalents for CO 2 fixation during photosynthesis. However, electronic energy transfer (EET) through a photosystem can harm the photosynthetic apparatus when not balanced with CO 2 . Here, we show that CO 2 binding to the light-harvesting complex modulates EET in photosynthetic cyanobacteria. More specifically, CO 2 binding to the allophycocyanin alpha subunit of the light-harvesting complex regulates EET and its fluorescence quantum yield in the cyanobacterium Synechocystis sp. PCC 6803. CO 2 binding decreases the inter-chromophore distance in the allophycocyanin trimer. The result is enhanced EET in vitro and in live cells. Our work identifies a direct target for CO 2 in the cyanobacterial light-harvesting apparatus and provides insights into photosynthesis regulation., (© 2022. The Author(s).)

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae 1,2 . These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching 3-6 . Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystis sp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS. For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP-PBS complex, quenching is achieved through the binding of four 34 kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60 Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties 7 , we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs) for capturing solar energy and conveying it to photosynthetic reaction centers. PBSs are compositionally and structurally diverse, and exceedingly complex, all of which pose a challenge for a comprehensive understanding of their function. To date, three detailed architectures of PBSs by cryo-electron microscopy (cryo-EM) have been described: a hemiellipsoidal type, a block-type from rhodophytes, and a cyanobacterial hemidiscoidal-type. Here, we report cryo-EM structures of a pentacylindrical allophycocyanin core and phycocyanin-containing rod of a thermophilic cyanobacterial hemidiscoidal PBS. The structures define the spatial arrangement of protein subunits and chromophores, crucial for deciphering the energy transfer mechanism. They reveal how the pentacylindrical core is formed, identify key interactions between linker proteins and the bilin chromophores, and indicate pathways for unidirectional energy transfer., (© 2022. The Author(s).)

In cyanobacteria, phycobilisomes (PBS) serve as peripheral light-harvesting complexes of the two photosystems, extending their antenna size and the wavelength range of photons available for photosynthesis. The abundance of PBS, the number of phycobiliproteins they contain, and their light-harvesting function are dynamically adjusted in response to the physiological conditions. PBS are also thought to be involved in state transitions that maintain the excitation balance between the two photosystems. Unlike its eukaryotic counterpart, PSI is trimeric in many cyanobacterial species and the physiological significance of this is not well understood. Here, we compared the composition and light-harvesting function of PBS in cells of Synechocystis sp. PCC 6803, which has primarily trimeric PSI, and the ΔpsaL mutant, which lacks the PsaL subunit of PSI and is unable to form trimers. We also investigated a mutant additionally lacking the PsaJ and PsaF subunits of PSI. Both strains with monomeric PSI accumulated significantly more allophycocyanin per chlorophyll, indicating higher abundance of PBS. On the other hand, a higher phycocyanin:allophycocyanin ratio in the wild type suggests larger PBS or the presence of APC-less PBS (CpcL-type) that are not assembled in cells with monomeric PSI. Steady-state and time-resolved fluorescence spectroscopy at room temperature and 77 K revealed that PSII receives more energy from the PBS at the expense of PSI in cells with monomeric PSI, regardless of the presence of PsaF. Taken together, these results show that the oligomeric state of PSI impacts the excitation energy flow in Synechocystis., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The orange carotenoid protein (OCP) is responsible for nonphotochemical quenching (NPQ) in cyanobacteria, a defense mechanism against potentially damaging effects of excess light conditions. This soluble two-domain protein undergoes profound conformational changes upon photoactivation, involving translocation of the ketocarotenoid inside the cavity followed by domain separation. Domain separation is a critical step in the photocycle of OCP because it exposes the N-terminal domain (NTD) to perform quenching of the phycobilisomes. Many details regarding the mechanism and energetics of OCP domain separation remain unknown. In this work, we apply metadynamics to elucidate the protein rearrangements that lead to the active, domain-separated, form of OCP. We find that translocation of the ketocarotenoid canthaxanthin has a profound effect on the energetic landscape and that domain separation only becomes favorable following translocation. We further explore, characterize, and validate the free energy surface (FES) using equilibrium simulations initiated from different states on the FES. Through pathway optimization methods, we characterize the most probable path to domain separation and reveal the barriers along that pathway. We find that the free energy barriers are relatively small (<5 kcal/mol), but the overall estimated kinetic rate is consistent with experimental measurements (>1 ms). Overall, our results provide detailed information on the requirement for canthaxanthin translocation to precede domain separation and an energetically feasible pathway to dissociation., (© 2022 The Protein Society.)

Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins., (© 2022. The Author(s).)

Phycobilisomes are light-harvesting antenna complexes of cyanobacteria and red algae that are comprised of chromoproteins called phycobiliproteins. PBS core structures are made up of allophycocyanin subunits. Halomicronema hongdechloris (H. hongdechloris) is one of the cyanobacteria that produce chlorophyll f (Chl f) under far-red light and is regulated by the Far-Red Light Photoacclimation gene cluster. There are five genes encoding APC in this specific gene cluster, and they are responsible for assembling the red-shifted PBS in H. hongdechloris grown under far-red light. In this study, the five apc genes located in the FaRLiP gene cluster were heterologously expressed in an Escherichia coli reconstitution system. The canonical APC-encoding genes were also constructed in the same system for comparison. Additionally, five annotated phycobiliprotein lyase-encoding genes (cpcS) from the H. hongdechloris genome were phylogenetically classified and experimentally tested for their catalytic properties including their contribution to the shifted absorption of PBS. Through analysis of recombinant proteins, we determined that the heterodimer of CpcS-I and CpcU are able to ligate a chromophore to the APC-α/APC-β subunits. We discuss some hypotheses towards understanding the roles of the specialised APC and contributions of PBP lyases., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Single-molecule fluorescence spectroscopy allows direct, real-time observation of dynamic photophysical changes in light harvesting complexes. The Anti-Brownian ELectrokinetic (ABEL) trap is one such single-molecule method with useful advantages. This approach is particularly well-suited to make detailed spectroscopic measurements of pigment-protein complexes in a solution phase because it enables extended-duration single-molecule observation by counteracting Brownian motion. This Perspective summarizes recent contributions by the authors and others that have utilized the unique capabilities of the ABEL trap to advance our understanding of phycobiliproteins and the phycobilisome complex, the primary light-harvesting apparatus of cyanobacteria. Monitoring the rich spectroscopic data from these measurements, which include brightness, fluorescence lifetime, polarization, and emission spectra, among other measurable parameters, has provided direct characterization of pigments and energy transfer pathways in the phycobilisome, spanning scales from single pigments and monomeric phycobiliproteins to higher order oligomers and protein-protein interactions of the phycobilisome complex. Importantly, new photophysical states and photodynamics were observed to modulate the flow of energy through the phycobilisome and suggest a previously unknown complexity in phycobilisome light harvesting and energy transport with a possible link to photoadaptive or photoprotective functions in cyanobacteria. Beyond deepening our collective understanding of natural light-harvesting systems, these and future discoveries may serve as inspiration for engineering improved artificial light-harvesting technologies.

Under nitrogen (N)-limited conditions, the non-N2-fixing cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) actively grows during early stages of starvation by performing photosynthesis but gradually stops the growth and eventually enters dormancy to withstand long-term N limitation. During N limitation, Synechocystis 6803 cells degrade the large light-harvesting antenna complex phycobilisomes (PBSs) presumably to avoid excess light absorption and to reallocate available N to essential functions for growth and survival. These two requirements may be driving forces for PBS degradation during N limitation, but how photosynthesis and cell growth affect PBS degradation remains unclear. To address this question, we examined involvements of photosynthesis and cell growth in PBS degradation during N limitation. Treatment of photosynthesis inhibitors and shading suppressed PBS degradation and caused non-bleaching of cells during N limitation. Limitations of photosynthesis after initial gene responses to N limitation suppressed PBS degradation, implying that photosynthesis affects PBS degradation in a post-translational manner. In addition, limitations of cell growth by inhibition of peptidoglycan and fatty acid biosynthesis, low growth temperature and phosphorous starvation suppressed PBS degradation during N limitation. Because decreased photosynthetic activity led to decreased cell growth, and vice versa, photosynthesis and cell growth would inseparably intertwine each other and affect PBS degradation during N limitation in a complex manner. Our data shed light on the coordination mechanisms among photosynthesis, cell growth and PBS degradation during N limitation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Under nitrogen (N)-limited conditions, the non-N2-fixing cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) actively grows during early stages of starvation by performing photosynthesis but gradually stops the growth and eventually enters dormancy to withstand long-term N limitation. During N limitation, Synechocystis 6803 cells degrade the large light-harvesting antenna complex phycobilisomes (PBSs) presumably to avoid excess light absorption and to reallocate available N to essential functions for growth and survival. These two requirements may be driving forces for PBS degradation during N limitation, but how photosynthesis and cell growth affect PBS degradation remains unclear. To address this question, we examined involvements of photosynthesis and cell growth in PBS degradation during N limitation. Treatment of photosynthesis inhibitors and shading suppressed PBS degradation and caused non-bleaching of cells during N limitation. Limitations of photosynthesis after initial gene responses to N limitation suppressed PBS degradation, implying that photosynthesis affects PBS degradation in a post-translational manner. In addition, limitations of cell growth by inhibition of peptidoglycan and fatty acid biosynthesis, low growth temperature and phosphorous starvation suppressed PBS degradation during N limitation. Because decreased photosynthetic activity led to decreased cell growth, and vice versa, photosynthesis and cell growth would inseparably intertwine each other and affect PBS degradation during N limitation in a complex manner. Our data shed light on the coordination mechanisms among photosynthesis, cell growth and PBS degradation during N limitation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)