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Cyanobacteria, red algae, and cryptophytes produce two classes of proteins for light-harvesting: water-soluble phycobiliproteins and membrane-intrinsic proteins that bind chlorophylls and carotenoids. In cyanobacteria, red algae, and glaucophytes, phycobilisomes (PBS) are complexes of brightly colored phycobiliproteins and linker (assembly) proteins. To date, six structural classes of phycobilisomes have been described: hemiellipsoidal, block-shaped, hemidiscoidal, bundle-shaped, paddle-shaped, and far-red-light bicylindrical. Two additional antenna complexes containing single types of phycobiliproteins have also been described. Since 2017, structures have been reported for examples of all of these complexes except bundle-shaped phycobilisomes by cryogenic electron microscopy. Phycobilisomes 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 chlorophyll-binding proteins that can form antenna complexes with Photosystem I and/or Photosystem II. Red and cryptophyte algae also produce chlorophyll-binding proteins associated with Photosystem I but which belong to the chlorophyll a/b-binding (CAB) protein superfamily and which are unrelated to the chlorophyll-binding proteins (CBP) of cyanobacteria. This review describes recent progress in structure determination for phycobilisomes and the chlorophyll proteins of cyanobacteria, red algae, and cryptophytan algae.

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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.

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Marine life is populated by a huge diversity of organisms with an incredible range of colour. While structural colour mechanisms and functions are usually well studied in marine animal species, there is a huge knowledge gap regarding the marine macroalgae (red, green and brown seaweeds) that have structural coloration and the biological significance of this phenomenon in these photosynthetic organisms. Here we show that structural colour in the gametophyte life history phase of the red alga Chondrus crispus plays an important role as a photoprotective mechanism in synergy with the other pigments present. In particular, we have demonstrated that blue structural coloration attenuates the more energetic light while simultaneously favouring green and red light harvesting through the external antennae (phycobilisomes) which possess an intensity-dependent photoprotection mechanism. These insights into the relationship between structural colour and photosynthetic light management further our understanding of the mechanisms involved.

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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.

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Membrane lipids play essential roles in regulating physiological properties in higher plants and algae. Monogalactosyldiacylglycerol (MGDG) is a major thylakoid membrane lipid, and it is an important source of polyunsaturated fatty acids for cells, plays a key role in the biogenesis of plastids, and maintains the function of the photosynthetic machinery. Several studies have indicated that the knockdown of MGDG synthase results in membrane lipid remodeling, albino seedlings, and changes in photosynthetic performance. However, the effects of MGDG synthase (MGD) inhibitors on lipids in macroalgae have not yet been clarified. Here, we characterized the effects of MGD inhibitors (ortho-phenanthroline and N-ethylmaleimide) on the composition of the fatty acids observed in MGDG and digalactosyldiacylglycerol (DGDG) in Gracilariopsis lemaneiformis using electrospray ionization-mass spectrometry. The most abundant MGDG species contained 16:0/18:1 (sn-1/sn-2) fatty acids, and the most dominant DGDG species contained 20:5/16:0 (sn-1/sn-2) fatty acids. Measurements of photosynthetic pigments and photosynthetic parameters revealed that photosynthesis of G. lemaneiformis was impaired. Principal component analysis and Spearman's correlation analysis revealed interactions between specific MGDG structural composition patterns and key metabolites involved in photosynthesis, indicating that 20:4/16:0 (sn-1/sn-2) MGDG and 16:0/18:1 (sn-1/sn-2) MGDG affect the structure and function of phycobilisomes and thus the color of G. lemaneiformis. Three genes (GlMGD1, GlMGD2, and GlMGD3) were cloned and identified. The addition of N-ethylmaleimide to G. lemaneiformis did not affect the abundance of GlMGD mRNA, and the abundance of transcripts was significantly decreased by ortho-phenanthroline.

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The phycobilisome antennas, which contain phycobilin pigments instead of chlorophyll, are crucial for the photosynthetic activity of Cyanidioschyzon merolae cells, which thrive in an acidic and hot water environment. The accessible light intensity and quality, temperature, acidity, and other factors in this environment are quite different from those in the air available for terrestrial plants. Under these conditions, adaptation to the intensity and quality of light, as well as temperature, which are key factors in photosynthesis of higher plants, also affects this process in Cyanidioschyzon merolae cells. Adaptation to varying light conditions requires fast remodeling and re-tuning of their light-harvesting antennas (phycobilisomes) at multiple levels, from regulation of gene expression to structural reorganization of protein-pigment complexes. This review presents selected data on the structure of phycobilisomes, the genetic engineering of the constituent proteins, and the latest results and opinions on the adaptation of phycobilisomes to light intensity and quality, and temperature to photosynthetic activities. We pay special attention to the latest results of the C. merolae research.

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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.

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Cyanobacteria, the evolutionary originators of oxygenic photosynthesis, have the capability to convert CO2, water, and minerals into biomass using solar energy. This process is driven by intricate bioenergetic mechanisms that consist of interconnected photosynthetic and respiratory electron transport chains coupled. Over the last few decades, advances in physiochemical analysis, molecular genetics, and structural analysis have enabled us to gain a more comprehensive understanding of cyanobacterial bioenergetics. This includes the molecular understanding of the primary energy conversion mechanisms as well as photoprotective and other dissipative mechanisms that prevent photodamage when the rates of photosynthetic output, primarily in the form of ATP and NADPH, exceed the rates that cellular assimilatory processes consume these photosynthetic outputs. Despite this progress, there is still much to learn about the systems integration and the regulatory circuits that control expression levels for optimal cellular abundance and activity of the photosynthetic complexes and the cellular components that convert their products into biomass. With an improved understanding of these regulatory principles and mechanisms, it should be possible to optimally modify cyanobacteria for enhanced biotechnological purposes.

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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.

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The phycobilisomes function as the primary light-harvesting antennae in cyanobacteria and red algae, effectively harvesting and transferring excitation energy to both photosystems. Here we investigate the direct energy transfer route from the phycobilisomes to photosystem I at room temperature in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that lacks photosystem II. The excitation dynamics are studied by picosecond time-resolved fluorescence measurements in combination with global and target analysis. Global analysis revealed several fast equilibration time scales and a decay of the equilibrated system with a time constant of ≈220 ps. From simultaneous target analysis of measurements with two different excitations of 400 nm (chlorophyll a) and 580 nm (phycobilisomes) a transfer rate of 42 ns-1 from the terminal emitter of the phycobilisome to photosystem I was estimated.

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Imbalanced light absorption by photosystem I (PSI) and photosystem II (PSII) in oxygenic phototrophs leads to changes in interaction of photosystems altering the linear electron flow. In plants and green algae, this imbalance is mitigated by a partial migration of the chlorophyll a/b containing light-harvesting antenna between the two photosystem core complexes. This migration is registered as fluorescence changes of the pigment apparatus and is termed the reverse transitions between States 1 and 2. By contrast, the molecular mechanism of State 1/2 transitions in phycobilisome (PBS)-containing photosynthetics, cyanobacteria and red algae, is still insufficiently understood. The suggested hypotheses - PBS movement along the surface of thylakoid membrane between PSI and PSII complexes, reversible PBS detachment from the dimeric PSII complex, and spillover - have some limitations as they do not fully explain the accumulated data. Here, we have recorded changes in the stationary fluorescence emission spectra of red algae and cyanobacteria in States 1/2 at room temperature, which allowed us to offer an explanation of the existing contradictions. The change of room temperature fluorescence of chlorophyll belonged to PSII was revealed, while the fluorescence of PBS associated with the PSII complexes remained during States 1/2 transitions at the stable level. Only the reversible dissociation of PBS from the monomeric PSI was revealed earlier which implied different degree of surface contact of PBS with the two photosystems. The detachment of PBS from the PSI corresponds to ferredoxin oxidation as electron carrier and the increase of cyclic electron transport in the pigment apparatus in State I.

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Photosynthesis is the major natural process that can harvest and harness solar energy into chemical energy. Photosynthesis is performed by a vast number of organisms from single cellular bacteria to higher plants and to make the process efficient, all photosynthetic organisms possess a special type of pigment protein complex(es) that is (are) capable of trapping light energy, known as photosynthetic light-harvesting antennae. From an evolutionary point of view, simpler (unicellular) organisms typically have a simple antenna, whereas higher plants possess complex antenna systems. The higher complexity of the antenna systems provides efficient fine tuning of photosynthesis. This relationship between the complexity of the antenna and the increasing complexity of the organism is mainly related to the remarkable acclimation capability of complex organisms under fluctuating environmental conditions. These antenna complexes not only harvest light, but also provide photoprotection under fluctuating light conditions. In this review, the evolution, structure, and function of different antenna complexes, from single cellular organisms to higher plants, are discussed in the context of the ability to acclimate and adapt to cope under fluctuating environmental conditions.

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Phycobilisomes are the major pigment-protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte, and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded following an intricate genetic program. Here, we describe the role of NblD, a cysteine-rich, small protein in this process in cyanobacteria. Deletion of the nblD gene in the cyanobacterium Synechocystis sp. PCC 6803 prevented the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype, which could be complemented by a plasmid-localized gene copy. Competitive growth experiments between the ΔnblD and the wild-type strain provided direct evidence for the physiological importance of NblD under nitrogen-limited conditions. Ectopic expression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation. Transcriptome analysis indicated increased nblA1/2 transcript levels in the ΔnblD strain during nitrogen starvation, implying that NblD does not act as a transcriptional (co)regulator. However, immunoprecipitation and far-western experiments identified the chromophorylated (holo form) of the phycocyanin ß-subunit (CpcB) as its target, while apo-CpcB was not bound. The addition of recombinant NblD to isolated phycobilisomes caused a reduction in phycocyanin absorbance and a broadening and shifting of the peak to lower wavelengths, indicating the occurrence of structural changes. These data demonstrate that NblD plays a crucial role in the coordinated dismantling of phycobilisomes and add it as a factor to the genetically programmed response to nitrogen starvation.

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Phycobilisomes (PBS), the major light-harvesting antenna in cyanobacteria, are supramolecular complexes of colorless linkers and heterodimeric, pigment-binding phycobiliproteins. Phycocyanin and phycoerythrin commonly comprise peripheral rods, and a multi-cylindrical core is principally assembled from allophycocyanin (AP). Each AP subunit binds one phycocyanobilin (PCB) chromophore, a linear tetrapyrrole that predominantly absorbs in the orange-red region of the visible spectrum (600-700 nm). AP facilitates excitation energy transfer from PBS peripheral rods or from directly absorbed red light to accessory chlorophylls in the photosystems. Paralogous forms of AP that bind PCB and are capable of absorbing far-red light (FRL; 700-800 nm) have recently been identified in organisms performing two types of photoacclimation: FRL photoacclimation (FaRLiP) and low-light photoacclimation (LoLiP). The FRL-absorbing AP (FRL-AP) from the thermophilic LoLiP strain Synechococcus sp. A1463 was chosen as a platform for site-specific mutagenesis to probe the structural differences between APs that absorb in the visible region and FRL-APs and to identify residues essential for the FRL absorbance phenotype. Conversely, red light-absorbing allophycocyanin-B (AP-B; ~ 670 nm) from the same organism was used as a platform for creating a FRL-AP. We demonstrate that the protein environment immediately surrounding pyrrole ring A of PCB on the alpha subunit is mostly responsible for the FRL absorbance of FRL-APs. We also show that interactions between PCBs bound to alpha and beta subunits of adjacent protomers in trimeric AP complexes are responsible for a large bathochromic shift of about ~ 20 nm and notable sharpening of the long-wavelength absorbance band.

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Photosynthetic light reactions proceed in thylakoid membranes (TMs) due to the activity of pigment-protein complexes. These complexes are heterogeneously organized into granal/stromal thylakoids (in plants) or into recently identified cyanobacterial microdomains (MDs). MDs are characterized by specific ratios of photosystem I (PSI), photosystem II (PSII), and phycobilisomes (PBS) and they are visible as sub-micrometer sized areas with different fluorescence ratios. In this report, the process of long-term plasticity in cyanobacterial thylakoid MDs has been explored under variable growth light conditions using Synechocystis sp. PCC6803 expressing YFP tagged PSI. TM organization into MDs has been observed for all categorized shapes of cells independently of their stage in cell cycle. The heterogeneous PSI, PSII, and PBS thylakoid areas were also identified under two types of growth conditions: at continuous light (CL) and at light-dark (L-D) cycle. The acclimation from CL to L-D cycle changed spatial distribution of photosystems, in particular PSI became more evenly distributed in thylakoids under L-D cycle. The process of the spatial PSI (and partially also PSII) redistribution required 1 week and was accompanied by temporal appearance of PBS decoupling probably caused by the re-organization of photosystems. The overall acclimation we observed was defined as TM plasticity as it resembles higher plants grana/stroma reorganization at variable growth light conditions. In addition, we observed large cell to cell variability in the actual MDs organization. It leads us to suggest that the plasticity, and cell to cell variability in MDs could be a manifestation of phenotypic heterogeneity, a recently broadly discussed phenomenon for prokaryotes.

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Phycobiliproteins (PBPs) are pigment proteins that comprise phycobilisomes (PBS), major light-harvesting antenna complexes of cyanobacteria and red algae. PBS core substructures are made up of allophycocyanins (APs), a subfamily of PBPs. Five paralogous AP subunits are encoded by the Far-Red Light Photoacclimation (FaRLiP) gene cluster, which is transcriptionally activated in cells grown in far-red light (FRL; λ = 700 to 800 nm). FaRLiP gene expression enables some terrestrial cyanobacteria to remodel their PBS and photosystems and perform oxygenic photosynthesis in far-red light (FRL). Paralogous AP genes encoding a putative, FRL-absorbing AP (FRL-AP) are also found in an operon associated with improved low-light growth (LL; < 50 µmol photons m-2 s-1) in some thermophilic Synechococcus spp., a phenomenon termed low-light photoacclimation (LoLiP). In this study, apc genes from FaRLiP and LoLiP gene clusters were heterologously expressed individually and in combinations in Escherichia coli. The resulting novel FRL-APs were characterized and identified as major contributors to the FRL absorbance observed in whole cells after FaRLiP and potentially LoLiP. Post-translational modifications of native FRL-APs from FaRLiP cyanobacterium, Leptolyngbya sp. strain JSC-1, were analyzed by mass spectrometry. The PBP complexes made in two FaRLiP organisms were compared, revealing strain-specific diversity in the FaRLiP responses of cyanobacteria. Through analyses of native and recombinant proteins, we improved our understanding of how different cyanobacterial strains utilize specialized APs to acclimate to FRL and LL. We discuss some insights into structural changes that may allow these APs to absorb longer light wavelengths than their visible-light-absorbing paralogs.

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Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been studied by picosecond time-resolved fluorescence spectroscopy at room temperature. Global analysis of the time-resolved fluorescence kinetics revealed two lifetimes of spectral equilibration in the isolated PBS, 30-35 ps and 110-130 ps, assigned primarily to energy transfer within the rods and between the rods and the allophycocyanin core, respectively. An additional intrinsic kinetic component with a lifetime of 500-700 ps was found, representing non-radiative decay or energy transfer in the core. Isolated tetrameric PSI complexes exhibited biexponential fluorescence decay kinetics with lifetimes of about 10 ps and 40 ps, representing equilibration between the bulk antenna chlorophylls with low-energy "red" states and trapping of the equilibrated excitations, respectively. The cascade of EET in the PBS and in PSI could be resolved in intact filaments as well. Virtually all energy absorbed by the PBS was transferred to the photosystems on a timescale of 180-190 ps.

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Some terrestrial cyanobacteria acclimate to and utilize far-red light (FRL; λ = 700-800 nm) for oxygenic photosynthesis, a process known as far-red light photoacclimation (FaRLiP). A conserved, 20-gene FaRLiP cluster encodes core subunits of Photosystem I (PSI) and Photosystem II (PSII), five phycobiliprotein subunits of FRL-bicylindrical cores, and enzymes for synthesis of chlorophyll (Chl) f and possibly Chl d. Deletion mutants for each of the five apc genes of the FaRLiP cluster were constructed in Synechococcus sp. PCC 7335, and all had similar phenotypes. When the mutants were grown in white (WL) or red (RL) light, the cells closely resembled the wild-type (WT) strain grown under the same conditions. However, the WT and mutant strains were very different when grown under FRL. Mutants grown in FRL were unable to assemble FRL-bicylindrical cores, were essentially devoid of FRL-specific phycobiliproteins, but retained RL-type phycobilisomes and WL-PSII. The transcript levels for genes of the FaRLiP cluster in the mutants were similar to those in WT. Surprisingly, the Chl d contents of the mutant strains were greatly reduced (~ 60-99%) compared to WT and so were the levels of FRL-PSII. We infer that Chl d may be essential for the assembly of FRL-PSII, which does not accumulate to normal levels in the mutants. We further infer that the cysteine-rich subunits of FRL allophycocyanin may either directly participate in the synthesis of Chl d or that FRL bicylindrical cores stabilize FRL-PSII to prevent loss of Chl d.

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Phycobilisomes (PBSs) are large (3-5 megadalton) pigment-protein complexes in cyanobacteria that associate with thylakoid membranes and harvest light primarily for photosystem II. PBSs consist of highly ordered assemblies of pigmented phycobiliproteins (PBPs) and linker proteins that can account for up to half of the soluble protein in cells. Cyanobacteria adjust to changing environmental conditions by modulating PBS size and number. In response to nutrient depletion such as nitrogen (N) deprivation, PBSs are degraded in an extensive, tightly controlled, and reversible process. In Synechococcus elongatus UTEX 2973, a fast-growing cyanobacterium with a doubling time of two hours, the process of PBS degradation is very rapid, with 80% of PBSs per cell degraded in six hours under optimal light and CO2 conditions. Proteomic analysis during PBS degradation and re-synthesis revealed multiple proteoforms of PBPs with partially degraded phycocyanobilin (PCB) pigments. NblA, a small proteolysis adaptor essential for PBS degradation, was characterized and validated with targeted mass spectrometry. NblA levels rose from essentially 0 to 25,000 copies per cell within 30 min of N depletion, and correlated with the rate of decrease in phycocyanin (PC). Implications of this correlation on the overall mechanism of PBS degradation during N deprivation are discussed.

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Ca2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca2+ signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca2+-binding protein which was called the Ca2+ Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca2+, which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes.

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