Two-Dimensional Electronic Spectroscopy Characterization of Fucoxanthin-Chlorophyll Protein Reveals Excitonic Carotenoid-Chlorophyll Interactions.

Fucoxanthin Chlorophyll Protein (FCP) is a Light Harvesting Complex found in diatoms and brown algae. It is particularly interesting for its efficiency in capturing the blue-green part of the light spectrum due to the presence of specific chromophores (fucoxanthin, chlorophyll a, and chlorophyll c). Recently, the crystallographic structure of FCP was solved, revealing the 3D arrangement of the pigments in the protein scaffold. While this information is helpful for interpreting the spectroscopic features of FCP, it has also raised new questions about the potential interactions between fucoxanthin and chlorophyll c. These interactions were suggested by their spatial closeness but have never been experimentally observed. To investigate this possible interaction mechanism, in this work, two-dimensional electronic spectroscopy (2DES) has been applied to study the ultrafast relaxation dynamics of FCP. The experiments captured an instantaneous delocalization of the excitation among fucoxanthin and chlorophyll c, suggesting the presence of a non-negligible coupling between the chromophores.

Fluorescence quenching in aggregates of fucoxanthin-chlorophyll protein complexes: Interplay of fluorescing and dark states.

Diatoms, a major group of algae, account for about a quarter of the global primary production on Earth. These photosynthetic organisms face significant challenges due to light intensity variations in their underwater habitat. To avoid photodamage, they have developed very efficient non-photochemical quenching (NPQ) mechanisms. These mechanisms originate in their light-harvesting antenna - the fucoxanthin-chlorophyll protein (FCP) complexes. Spectroscopic studies of NPQ in vivo are often hindered by strongly overlapping signals from the photosystems and their antennae. Fortunately, in vitro FCP aggregates constitute a useful model system to study fluorescence (FL) quenching in diatoms. In this work, we present streak-camera FL measurements on FCPa and FCPb complexes, isolated from a centric diatom Cyclotella meneghiniana, and their aggregates. We find that spectra of non-aggregated FCP are dominated by a single fluorescing species, but the FL spectra of FCP aggregates additionally contain contributions from a redshifted emissive state. We relate this red state to a charge transfer state between chlorophyll c and chlorophyll a molecules. The FL quenching, on the other hand, is due to an additional dark state that involves incoherent energy transfer to the fucoxanthin carotenoids. Overall, the global picture of energy transfer and quenching in FCP aggregates is very similar to that of major light-harvesting complexes in higher plants (LHCII), but microscopic details between FCPs and LHCIIs differ significantly.

Bicarbonate activation of the monomeric photosystem II-PsbS/Psb27 complex.

In thylakoid membranes, photosystem II (PSII) monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while PSII monomers (PSIIm) from granal regions lack these subunits. Here, we have isolated and characterized these 2 types of PSII complexes in tobacco (Nicotiana tabacum). PSIIm-S/27 showed enhanced fluorescence, the near absence of oxygen evolution, and limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•- couple, which controls the charge recombination route, and this limits chlorophyll triplet-mediated 1O2 formation. These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit using a bicarbonate-mediated switch and protective mechanism.

Photoacclimation impacts the molecular features of photosystem supercomplexes in the centric diatom Thalassiosira pseudonana.

In diatoms, light-harvesting processes take place in a specific group of proteins, called fucoxanthin chlorophyll a/c proteins (FCP). This group includes many members and represents the major characteristic of the diatom photosynthetic apparatus, with specific pigments bound (chlorophyll c, fucoxanthin, diadino- and diatoxanthin besides chlorophyll a). In thylakoids, FCP and photosystems (PS) form multimeric supercomplexes. In this study, we compared the biochemical properties of PS supercomplexes isolated from Thalassiosira pseudonana cells grown under low light or high light conditions, respectively. High light acclimation changed the molecular features of the PS and their ratio in thylakoids. In PSII, no obvious changes in polypeptide composition were observed, whereas for PSI changes in one specific group of FCP proteins were detected. As reported before, the amount of xanthophyll cycle pigments and their de-epoxidation ratio was increased in PSI under HL. In PSII, however, no additional xanthophyll cycle pigments occurred, but the de-epoxidation ratio was increased as well. This comparison suggests how mechanisms of photoprotection might take place within and in the proximity of the PS, which gives new insights into the capacity of diatoms to adapt to different conditions and in different environments.

Structure-based model of fucoxanthin-chlorophyll protein complex: Calculations of chlorophyll electronic couplings.

Diatoms are a group of marine algae that are responsible for a significant part of global oxygen production. Adapted to life in an aqueous environment dominated by the blue-green light, their major light-harvesting antennae-fucoxanthin-chlorophyll protein complexes (FCPs)-exhibit different pigment compositions than of plants. Despite extensive experimental studies, until recently the theoretical description of excitation energy dynamics in these complexes was limited by the lack of high-resolution structural data. In this work, we use the recently resolved crystallographic information of the FCP complex from Phaeodactylum tricornutum diatom [Wang et al., Science 363, 6427 (2019)] and quantum chemistry-based calculations to evaluate the chlorophyll transition dipole moments, atomic transition charges from electrostatic potential, and the inter-chlorophyll couplings in this complex. The obtained structure-based excitonic couplings form the foundation for any modeling of stationary or time-resolved spectroscopic data. We also calculate the inter-pigment Förster energy transfer rates and identify two quickly equilibrating chlorophyll clusters.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

The C-terminus of a diatom plant-like cryptochrome influences the FAD redox state and binding of interaction partners.

A plant-like cryptochrome of diatom microalgae, CryP, acts as a photoreceptor involved in transcriptional regulation. It contains FAD and 5,10-methenyltetrahydrofolate as chromophores. Here, we demonstrate that the unstructured C-terminal extension (CTE) of CryP has an influence on the redox state of the flavin. In CryP lacking the CTE, the flavin is in the oxidized state (FADox), whereas it is a neutral radical (FADH•) in the full-length protein. When the CTE of CryP is coupled to another diatom cryptochrome that naturally binds FADox, this chimera also binds FADH•. In full-length CryP, FADH• is the most stable redox state and oxidation to FADox is extremely slow, whereas reduction to FADH2 is reversible in the dark in approximately 1 h. We also identified novel interaction partners of this algal CRY and characterized two of them in depth regarding their binding activities. BolA, a putative transcription factor, binds to monomeric and to dimeric CryP via the CTE, independent of the redox state of the flavin. In contrast, an unknown protein, ID42612, which occurs solely in heterokont algae, binds only to CryP dimers. This binding is independent of the CTE and shows slight differences in strength depending on the flavin's redox state.

Revealing the architecture of the photosynthetic apparatus in the diatom Thalassiosira pseudonana.

Diatoms are a large group of marine algae that are responsible for about one-quarter of global carbon fixation. Light-harvesting complexes of diatoms are formed by the fucoxanthin chlorophyll a/c proteins and their overall organization around core complexes of photosystems (PSs) I and II is unique in the plant kingdom. Using cryo-electron tomography, we have elucidated the structural organization of PSII and PSI supercomplexes and their spatial segregation in the thylakoid membrane of the model diatom species Thalassiosira pseudonana. 3D sub-volume averaging revealed that the PSII supercomplex of T. pseudonana incorporates a trimeric form of light-harvesting antenna, which differs from the tetrameric antenna observed previously in another diatom, Chaetoceros gracilis. Surprisingly, the organization of the PSI supercomplex is conserved in both diatom species. These results strongly suggest that different diatom classes have various architectures of PSII as an adaptation strategy, whilst a convergent evolution occurred concerning PSI and the overall plastid structure.

Confronting FCP structure with ultrafast spectroscopy data: evidence for structural variations.

Diatoms are a major group of algae, responsible for a quarter of the global primary production on our planet. Their adaptation to marine environments is ensured by their light-harvesting antenna - the fucoxanthin-chlorophyll protein (FCP) complex, which absorbs strongly in the blue-green spectral region. Although these essential proteins have been the subject of many studies, for a long time their comprehensive description was not possible in the absence of structural data. Last year, the 3D structures of several FCP complexes were revealed. The structure of an FCP dimer was resolved by crystallography for the pennate diatom Phaeodactylum tricornutum [W. Wang et al., Science, 2019, 363, 6427] and the structure of the PSII supercomplex from the centric diatom Chaetoceros gracilis, containing several FCPs, was obtained by electron microscopy [X. Pi et al., Science, 2019, 365, 6452; R. Nagao et al., Nat. Plants, 2019, 5, 890]. In this Perspective article, we evaluate how precisely these structures may account for previously published ultrafast spectroscopy results, describing the excitation energy transfer in the FCP from another centric diatom Cyclotella meneghiniana. Surprisingly, we find that the published FCP structures cannot explain several observations obtained from ultrafast spectroscopy. Using the available structures, and results from electron microscopy, we construct a trimer-based FCP model for Cyclotella meneghiniana, consistent with ultrafast experimental data. As a whole, our observations suggest that the structures from the proteins belonging to the FCP family display larger variations than the equivalent LHC proteins in plants, which may reflect species-specific adaptations or original strategies for adapting to rapidly changing marine environments.

A distinctive pathway for triplet-triplet energy transfer photoprotection in fucoxanthin chlorophyll-binding proteins from Cyclotella meneghiniana.

Fucoxanthin chlorophyll-binding proteins (FCPs) are the major light-harvesting complexes of diatoms. In this work, FCPs isolated from Cyclotella meneghiniana have been studied by means of optically detected magnetic resonance (ODMR) and time-resolved electron paramagnetic resonance (TR-EPR), with the aim to characterize the photoprotective mechanism based on triplet-triplet energy transfer (TTET). The spectroscopic properties of the chromophores carrying the triplet state have been interpreted on the basis of a delved analysis of the recently solved crystallographic structures of FCP. The results point toward a photoprotective role for two fucoxanthin molecules exposed to the exterior of the FCP monomers. This shows that FCP has adopted a structural strategy different from that of related light-harvesting complexes from plants and other microalgae, in which the photoprotective role is carried out by two highly conserved carotenoids in the interior of the complex.

Specific Lhc Proteins Are Bound to PSI or PSII Supercomplexes in the Diatom Thalassiosira pseudonana.

Despite the ecological relevance of diatoms, many aspects of their photosynthetic machinery remain poorly understood. Diatoms differ from the green lineage of oxygenic organisms by their photosynthetic pigments and light-harvesting complex (Lhc) proteins, the latter of which are also called fucoxanthin-chlorophyll proteins (FCP). These are composed of three groups of proteins: Lhcf as the main group, Lhcr that are PSI associated, and Lhcx that are involved in photoprotection. The FCP complexes are assembled in trimers and higher oligomers. Several studies have investigated the biochemical properties of purified FCP complexes, but limited knowledge is available about their interaction with the photosystem cores. In this study, isolation of stable supercomplexes from the centric diatom Thalassiosira pseudonana was achieved. To preserve in vivo structure, the separation of thylakoid complexes was performed by native PAGE and sucrose density centrifugation. Different subpopulations of PSI and PSII supercomplexes were isolated and their subunits identified. Analysis of Lhc antenna composition identified Lhc(s) specific for either PSI (Lhcr 1, 3, 4, 7, 10-14, and Lhcf10) or PSII (Lhcf 1-7, 11, and Lhcr2). Lhcx6_1 was reproducibly found in PSII supercomplexes, whereas its association with PSI was unclear. No evidence was found for the interaction between photosystems and higher oligomeric FCPs, comprising Lhcf8 as the main component. Although the subunit composition of the PSII supercomplexes in comparison with that of the trimeric FCP complexes indicated a close mutual association, the higher oligomeric pool is only weakly associated with the photosystems, albeit its abundance in the thylakoid membrane.

Light harvesting complexes in chlorophyll c-containing algae.

Besides the so-called 'green lineage' of eukaryotic photosynthetic organisms that include vascular plants, a huge variety of different algal groups exist that also harvest light by means of membrane intrinsic light harvesting proteins (Lhc). The main taxa of these algae are the Cryptophytes, Haptophytes, Dinophytes, Chromeridae and the Heterokonts, the latter including diatoms, brown algae, Xanthophyceae and Eustigmatophyceae amongst others. Despite the similarity in Lhc proteins between vascular plants and these algae, pigmentation is significantly different since no Chl b is bound, but often replaced by Chl c, and a large diversity in carotenoids functioning in light harvesting and/or photoprotection is present. Due to the presence of Chl c in most of the taxa the name 'Chl c-containing organisms' has become common, however, Chl b-less is more precise since some harbour Lhc proteins that only bind one type of Chl, Chl a. In recent years huge progress has been made about the occurrence and function of Lhc in diatoms, so-called fucoxanthin chlorophyll proteins (FCP), where also the first molecular structure became available recently. In addition, especially energy transfer amongst the unusual pigments bound was intensively studied in many of these groups. This review summarises the present knowledge about the molecular structure, the arrangement of the different Lhc in complexes, the excitation energy transfer abilities and the involvement in photoprotection of the different Lhc systems in the so-called Chl c-containing organisms. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.

Development of an automated on-line purification HPLC single cell-ICP-MS approach for fast diatom analysis.

The most challenging part in performing a single cell ICP-MS (sc-ICP-MS) approach is the sample preparation, in particular the reduction of the ionic background. This step is, in many cases, time-consuming and required for each sample separately. Furthermore, sc-ICP-MS measurements are mostly carried out "manually", given the fact that present systems are not allowing for an automated change of samples. Thus, within this work, we developed an approach based on a HPLC system coupled on-line with sc-ICP-MS via a set of switching valves as well as an in-line filter for automated cell washing. This set-up enables the ionic background removal as well as analysis of single cells completely automated without any manual sample pretreatment. Our approach was applied for the analysis of the single celled diatom species Cyclotella meneghiniana, a marine diatom species, on the basis of 24Mg and facilitates testing in 11 min per sample, requiring only around 10,000 cells in a volume of 10 µL and approx. 10 mL of a 5% MeOH/95% deionized water (v/v) mixture. Even at extremely saline culturing media concentrations (up to 1000 mg L-1 magnesium) our on-line approach worked sufficiently allowing for distinction of ionic and particulate fractions. Furthermore, a set of diatom samples was analyzed completely automated without the need for changing samples manually. So, utilizing this approach enables analyzing a high quantity of samples in a short time and therefore in future the investigation of ecotoxicological effects is simplified for example in terms of metal accumulation by taking biovariability into account.

Multi-endpoint toxicological assessment of polystyrene nano- and microparticles in different biological models in vitro.

Nanoplastics (NP) and microplastics (MP) accumulate in our environment as a consequence of the massive consumption of plastics. Huge knowledge-gaps exist regarding uptake and fate of plastic particles in micro- and nano-dimensions in humans as well as on their impact on human health. This study investigated the transport and effects of 50 nm and 0.5 µm COOH-modified polystyrene (PS) particles, as representatives for NP and MP, in different biological models in vitro. Acute toxicity and potential translocation of the particles were studied at the human intestinal and placental barrier using advanced in vitro co-culture models. Furthermore, embryotoxicity and genotoxicity were investigated as highly sensitive endpoints. Polystyrene was not acutely toxic in both sizes (nano- and microparticles). No transport across the intestinal and placental barrier but a cellular uptake and intracellular accumulation of PS nano- and microparticles were determined. The particles were identified as weak embryotoxic and non-genotoxic. In contrast to single-organ studies, this multi-endpoint study is providing a data-set with the exact same type of particles to compare organ-specific outcomes. Our study clearly shows the need to investigate other types of plastics as well as towards long-term or chronic effects of plastic particles in different biological models in vitro.

Fucoxanthin-Chlorophyll Protein Complexes of the Centric Diatom Cyclotella Meneghiniana Differ in Lhcx1 and Lhcx6_1 Content.

The ecological success of diatoms, key contributors to photosynthesis, is partly based on their ability to perfectly balance efficient light harvesting and photoprotection. Diatoms contain higher numbers of antenna proteins than vascular plants for light harvesting and for photoprotection. These proteins are arranged in fucoxanthin-chlorophyll protein (FCP) complexes. The number of FCP complexes, their subunit composition, and their interactions in the thylakoid membranes remain elusive in different diatoms. We used the recently available genome sequence of the centric diatom Cyclotella cryptica to analyze gene sequences for putative light-harvesting proteins in C. meneghiniana, and to elucidate the FCP complex composition. We analyzed two pools of FCP complexes that were trimeric (FCPa) and nonameric (FCPb). FCPa was composed of four different trimeric subtypes. Two different nonameric FCPb complexes were present. All were distinguished by their polypeptide composition and partly by pigmentation. With use of a milder purification method, two fractions composed of different FCP complexes were isolated. One was enriched in FCPs incorporating the photoprotective subunit Lhcx1, such as the newly identified nonameric FCPb2 and the major trimeric FCPa4 complex, which are predetermined to be involved in energy-dependent nonphotochemical quenching. The other fraction contained mainly FCPs that were devoid of Lhcx1, FCPa3, and FCPb1. Both fractions also included small amounts of trimeric FCPa complexes with the centric diatom-specific Lhcx protein, Lhcx6_1, as subunit. Thus, the antenna organization of centric diatoms, as well as the distribution of different photoprotective Lhcx proteins, differs from that of other diatoms, as well as from plants.

Cation-dependent changes in the thylakoid membrane appression of the diatom Thalassiosira pseudonana.

Diatoms show a special organisation of their plastid membranes, such that their thylakoids span the entire plastid in bands of three. While in higher plants the interaction of the light harvesting complex II and photosystem II with divalent cations (especially Mg2+) was found to take part in the interplay of electrostatic attraction and repulsion in grana membrane appression, for diatoms the key players in maintaining proper membrane distances were not identified so far. In this work, we investigated the changes in the thylakoid architecture of Thalassiosira pseudonana in reaction to different salts by using circular dichroism and fluorescence spectroscopy in combination with other techniques. We show that divalent cations have an important influence on optimal pigment organisation and thus also on maintaining membrane appression. Thereby, monovalent cations are far less effective. The concentration needed is in a physiological range and fits well with the values obtained for higher plant grana stacking, despite the fact that strict protein segregation as seen in higher plant grana is missing.

Knock-Down of a ligIV Homologue Enables DNA Integration via Homologous Recombination in the Marine Diatom Phaeodactylum tricornutum.

Genetic engineering of Phaeodactylum tricornutum as a model organism for diatoms is the basis of molecular and biochemical research, and can also be used in biotechnological approaches. So far, integration of foreign DNA into the genome happens randomly by nonhomologous end joining (NHEJ), if the classical method of particle bombardment is used, with the danger of negative physiological side effects. Here we show that a putative gene for a DNA ligase IV homologue ( ligIV) in P. tricornutum codes for a functional LigIV. The knock-down of ligIV in P. tricornutum via antisense RNA drastically enhances homologous recombination (HR) by interfering with the NHEJ pathway at its central DNA ligation step done by LigIV. This enables a specific integration of DNA at desired locations, greatly enhanced transformation rates and provides a new way of specifically altering the genome of P. tricornutum.

Energy dissipation mechanisms in the FCPb light-harvesting complex of the diatom Cyclotella meneghiniana.

Transient absorption spectroscopy has been applied to investigate the energy dissipation mechanisms in the nonameric fucoxanthin-chlorophyll-a,c-binding protein FCPb of the centric diatom Cyclotella meneghiniana. FCPb complexes in their unquenched state were compared with those in two types of quenching environments, namely aggregation-induced quenching by detergent removal, and clustering via incorporation into liposomes. Applying global and target analysis, in combination with a fluorescence lifetime study and annihilation calculations, we were able to resolve two quenching channels in FCPb that involve chlorophyll-a pigments for FCPb exposed to both quenching environments. The fast quenching channel operates on a timescale of tens of picoseconds and exhibits similar spectral signatures as the unquenched state. The slower quenching channel operates on a timescale of tens to hundreds of picoseconds, depending on the degree of quenching, and is characterized by enhanced population of low-energy states between 680 and 710 nm. The results indicate that FCPb is, in principle, able to function as a dissipater of excess energy and can do this in vitro even more efficiently than the homologous FCPa complex, the sole complex involved in fast photoprotection in these organisms. This indicates that when a complex displays photoprotection-related spectral signatures in vitro it does not imply that the complex participates in photoprotection in vivo. We suggest that FCPa is favored over FCPb as the sole energy-regulating complex in diatoms because its composition can more easily establish the balance between light-harvesting and quenching required for efficient photoprotection.