High-Resolution Frequency-Domain Spectroscopic and Modeling Studies of Photosystem I (PSI), PSI Mutants and PSI Supercomplexes.

Photosystem I (PSI) is one of the two main pigment-protein complexes where the primary steps of oxygenic photosynthesis take place. This review describes low-temperature frequency-domain experiments (absorption, emission, circular dichroism, resonant and non-resonant hole-burned spectra) and modeling efforts reported for PSI in recent years. In particular, we focus on the spectral hole-burning studies, which are not as common in photosynthesis research as the time-domain spectroscopies. Experimental and modeling data obtained for trimeric cyanobacterial Photosystem I (PSI3), PSI3 mutants, and PSI3-IsiA18 supercomplexes are analyzed to provide a more comprehensive understanding of their excitonic structure and excitation energy transfer (EET) processes. Detailed information on the excitonic structure of photosynthetic complexes is essential to determine the structure-function relationship. We will focus on the so-called "red antenna states" of cyanobacterial PSI, as these states play an important role in photochemical processes and EET pathways. The high-resolution data and modeling studies presented here provide additional information on the energetics of the lowest energy states and their chlorophyll (Chl) compositions, as well as the EET pathways and how they are altered by mutations. We present evidence that the low-energy traps observed in PSI are excitonically coupled states with significant charge-transfer (CT) character. The analysis presented for various optical spectra of PSI3 and PSI3-IsiA18 supercomplexes allowed us to make inferences about EET from the IsiA18 ring to the PSI3 core and demonstrate that the number of entry points varies between sample preparations studied by different groups. In our most recent samples, there most likely are three entry points for EET from the IsiA18 ring per the PSI core monomer, with two of these entry points likely being located next to each other. Therefore, there are nine entry points from the IsiA18 ring to the PSI3 trimer. We anticipate that the data discussed below will stimulate further research in this area, providing even more insight into the structure-based models of these important cyanobacterial photosystems.

Energy Transfer and Radical-Pair Dynamics in Photosystem I with Different Red Chlorophyll a Pigments.

We establish a general kinetic scheme for the energy transfer and radical-pair dynamics in photosystem I (PSI) of Chlamydomonas reinhardtii, Synechocystis PCC6803, Thermosynechococcus elongatus and Spirulina platensis grown under white-light conditions. With the help of simultaneous target analysis of transient-absorption data sets measured with two selective excitations, we resolved the spectral and kinetic properties of the different species present in PSI. WL-PSI can be described as a Bulk Chl a in equilibrium with a higher-energy Chl a, one or two Red Chl a and a reaction-center compartment (WL-RC). Three radical pairs (RPs) have been resolved with very similar properties in the four model organisms. The charge separation is virtually irreversible with a rate of ≈900 ns-1. The second rate, of RP1 → RP2, ranges from 70-90 ns-1 and the third rate, of RP2 → RP3, is ≈30 ns-1. Since RP1 and the Red Chl a are simultaneously present, resolving the RP1 properties is challenging. In Chlamydomonas reinhardtii, the excited WL-RC and Bulk Chl a compartments equilibrate with a lifetime of ≈0.28 ps, whereas the Red and the Bulk Chl a compartments equilibrate with a lifetime of ≈2.65 ps. We present a description of the thermodynamic properties of the model organisms at room temperature.

Anoxygenic phototroph of the Chloroflexota uses a type I reaction centre.

Scientific exploration of phototrophic bacteria over nearly 200 years has revealed large phylogenetic gaps between known phototrophic groups that limit understanding of how phototrophy evolved and diversified1,2. Here, through Boreal Shield lake water incubations, we cultivated an anoxygenic phototrophic bacterium from a previously unknown order within the Chloroflexota phylum that represents a highly novel transition form in the evolution of photosynthesis. Unlike all other known phototrophs, this bacterium uses a type I reaction centre (RCI) for light energy conversion yet belongs to the same bacterial phylum as organisms that use a type II reaction centre (RCII) for phototrophy. Using physiological, phylogenomic and environmental metatranscriptomic data, we demonstrate active RCI-utilizing metabolism by the strain alongside usage of chlorosomes3 and bacteriochlorophylls4 related to those of RCII-utilizing Chloroflexota members. Despite using different reaction centres, our phylogenomic data provide strong evidence that RCI-utilizing and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor. The Chloroflexota phylum preserves an evolutionary record of the use of contrasting phototrophic modes among genetically related bacteria, giving new context for exploring the diversification of phototrophy on Earth.

Access to the Antenna System of Photosystem I via Single-Molecule Excitation-Emission Spectroscopy.

In the development of single-molecule spectroscopy, the simultaneous detection of the excitation and emission spectra has been limited. The fluorescence excitation spectrum based on background-free signals is compatible with the fluorescence-emission-based detection of single molecules and can provide insight into the variations in the input energy of the different terminal emitters. Here, we implement single-molecule excitation-emission spectroscopy (SMEES) for photosystem I (PSI) via a cryogenic optical microscope. To this end, we extended our line-focus-based excitation-spectral microscope system to the cryogenic temperature-compatible version. PSI is one of the two photosystems embedded in the thylakoid membrane in oxygen-free photosynthetic organisms. PSI plays an essential role in electron transfer in the photosynthesis reaction. PSIs of many organisms contain a few red-shifted chlorophylls (Chls) with much lower excitation energies than ordinary antenna Chls. The fluorescence emission spectrum originates primarily from the red-shifted Chls, whereas the excitation spectrum is sensitive to the antenna Chls that are upstream of red-shifted Chls. Using SMEES, we obtained the inclining two-dimensional excitation-emission matrix (2D-EEM) of PSI particles isolated from a cyanobacterium, Thermosynechococcus vestitus (equivalent to elongatus), at about 80 K. Interestingly, by decomposing the inclining 2D-EEMs within time course observation, we found prominent variations in the excitation spectra of the red-shifted Chl pools with different emission wavelengths, strongly indicating the variable excitation energy transfer (EET) pathway from the antenna to the terminal emitting pools. SMEES helps us to directly gain information about the antenna system, which is fundamental to depicting the EET within pigment-protein complexes.

Architecture of symbiotic dinoflagellate photosystem I-light-harvesting supercomplex in Symbiodinium.

Symbiodinium are the photosynthetic endosymbionts for corals and play a vital role in supplying their coral hosts with photosynthetic products, forming the nutritional foundation for high-yield coral reef ecosystems. Here, we determine the cryo-electron microscopy structure of Symbiodinium photosystem I (PSI) supercomplex with a PSI core composed of 13 subunits including 2 previously unidentified subunits, PsaT and PsaU, as well as 13 peridinin-Chl a/c-binding light-harvesting antenna proteins (AcpPCIs). The PSI-AcpPCI supercomplex exhibits distinctive structural features compared to their red lineage counterparts, including extended termini of PsaD/E/I/J/L/M/R and AcpPCI-1/3/5/7/8/11 subunits, conformational changes in the surface loops of PsaA and PsaB subunits, facilitating the association between the PSI core and peripheral antennae. Structural analysis and computational calculation of excitation energy transfer rates unravel specific pigment networks in Symbiodinium PSI-AcpPCI for efficient excitation energy transfer. Overall, this study provides a structural basis for deciphering the mechanisms governing light harvesting and energy transfer in Symbiodinium PSI-AcpPCI supercomplexes adapted to their symbiotic ecosystem, as well as insights into the evolutionary diversity of PSI-LHCI among various photosynthetic organisms.

The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs.

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Enhanced Reduction of Ferredoxin in PGR5-Deficient Mutant of Arabidopsis thaliana Stimulated Ferredoxin-Dependent Cyclic Electron Flow around Photosystem I.

The molecular entity responsible for catalyzing ferredoxin (Fd)-dependent cyclic electron flow around photosystem I (Fd-CEF) remains unidentified. To reveal the in vivo molecular mechanism of Fd-CEF, evaluating ferredoxin reduction-oxidation kinetics proves to be a reliable indicator of Fd-CEF activity. Recent research has demonstrated that the expression of Fd-CEF activity is contingent upon the oxidation of plastoquinone. Moreover, chloroplast NAD(P)H dehydrogenase does not catalyze Fd-CEF in Arabidopsis thaliana. In this study, we analyzed the impact of reduced Fd on Fd-CEF activity by comparing wild-type and pgr5-deficient mutants (pgr5hope1). PGR5 has been proposed as the mediator of Fd-CEF, and pgr5hope1 exhibited a comparable CO2 assimilation rate and the same reduction-oxidation level of PQ as the wild type. However, P700 oxidation was suppressed with highly reduced Fd in pgr5hope1, unlike in the wild type. As anticipated, the Fd-CEF activity was enhanced in pgr5hope1 compared to the wild type, and its activity further increased with the oxidation of PQ due to the elevated CO2 assimilation rate. This in vivo research clearly demonstrates that the expression of Fd-CEF activity requires not only reduced Fd but also oxidized PQ. Importantly, PGR5 was found to not catalyze Fd-CEF, challenging previous assumptions about its role in this process.

Transcriptomic and proteomic analyses of distinct Arabidopsis organs reveal high PSI-NDH complex accumulation in stems.

In addition to leaves, the main site of photosynthetic reactions, active photosynthesis also takes place in stems, siliques and tree trunks. Although non-foliar photosynthesis has a marked effect on plant growth and yield, only limited information on the expression patterns of photosynthesis-related genes and the structure of photosynthetic machinery in different plant organs has been available. Here, we report the results of transcriptomic analysis of various organs of Arabidopsis thaliana and compare the gene expression profiles of young and mature leaves with a special focus on photosynthetic genes. Further, we analyzed the composition and organization of the photosynthetic electron transfer machinery in leaves, stems and green siliques at the protein level using BN-PAGE. RNA-Seq analysis revealed unique gene expression profiles in different plant organs and showed major differences in the expression of photosynthesis-related genes in young as compared to mature rosettes. Gel-based proteomic analysis of the thylakoid protein complex organization further showed that all studied plant organs contain the necessary components of the photosynthetic electron transfer chain. Intriguingly, stems accumulate high amounts of PSI-NDH complex, which has previously been implicated in cyclic electron transfer.

Thylakoid membrane stacking controls electron transport mode during the dark-to-light transition by adjusting the distances between PSI and PSII.

The balance between linear electron transport (LET) and cyclic electron transport (CET) plays an essential role in plant adaptation and protection against photo-induced damage. This balance is largely maintained by phosphorylation-driven alterations in the PSII-LHCII assembly and thylakoid membrane stacking. During the dark-to-light transition, plants shift this balance from CET, which prevails to prevent overreduction of the electron transport chain and consequent photo-induced damage, towards LET, which enables efficient CO2 assimilation and biomass production. Using freeze-fracture cryo-scanning electron microscopy and transmission electron microscopy of Arabidopsis leaves, we reveal unique membrane regions possessing characteristics of both stacked and unstacked regions of the thylakoid network that form during this transition. A notable consequence of the morphological attributes of these regions, which we refer to as 'stacked thylakoid doublets', is an overall increase in the proximity and connectivity of the two photosystems (PSI and PSII) that drive LET. This, in turn, reduces diffusion distances and barriers for the mobile carriers that transfer electrons between the two PSs, thereby maximizing LET and optimizing the plant's ability to utilize light energy. The mechanics described here for the shift between CET and LET during the dark-to-light transition are probably also used during chromatic adaptation mediated by state transitions.

The protein phosphorylation landscape in photosystem I of the desert algae Chlorella sp.

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been studied, and its involvement in state transitions and PSII repair is known. Yet, little is known about the phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, we applied proteomics analysis to generate a map of the phosphorylation sites of the PSI-LHCI proteins in Chlorella ohadii cells that were grown under low or extreme high-light intensities (LL and HL). Furthermore, we analyzed the content of oxidized tryptophans and PSI-LHCI protein degradation products in these cells, to estimate the light-induced damage to PSI-LHCI. Our work revealed the phosphorylation of 17 of 22 PSI-LHCI subunits. The analyses detected the extensive phosphorylation of the LHCI subunits Lhca6 and Lhca7, which is modulated by growth light intensity. Other PSI-LHCI subunits were phosphorylated to a lesser extent, including PsaE, where molecular dynamic simulation proposed that a phosphoserine stabilizes ferredoxin binding. Additionally, we show that HL-grown cells accumulate less oxidative damage and degradation products of PSI-LHCI proteins, compared with LL-grown cells. The significant phosphorylation of Lhca6 and Lhca7 at the interface with other LHCI subunits suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other subunits.

NADH dehydrogenase-like complex L subunit improves salt tolerance by enhancing photosynthetic electron transport.

Cyclic electron transport (CET) around photosystem I (PSI) mediated by the NADH dehydrogenase-like (NDH) complex is closely related to plant salt tolerance. However, whether overexpression of a core subunit of the NDH complex affects the photosynthetic electron transport under salt stress is currently unclear. Here, we expressed the NDH complex L subunit (Ndhl) genes ZmNdhl1 and ZmNdhl2 from C4 plant maize (Zea mays) or OsNdhl from C3 plant rice (Oryza sativa) using a constitutive promoter in rice. Transgenic rice lines expressing ZmNdhl1, ZmNdhl2, or OsNdhl displayed enhanced salt tolerance, as indicated by greater plant height, dry weight, and leaf relative water content, as well as lower malondialdehyde content compared to wild-type plants under salt stress. Fluorescence parameters such as post-illumination rise (PIR), the prompt chlorophyll a fluorescence transient (OJIP), modulated 820-nm reflection (MR), and delayed chlorophyll a fluorescence (DF) remained relatively normal in transgenic plants during salt stress. These results indicate that expression of ZmNdhl1, ZmNdhl2, or OsNdhl increases cyclic electron transport activity, slows down damage to linear electron transport, alleviates oxidative damage to the PSI reaction center and plastocyanin, and reduces damage to electron transport on the receptor side of PSI in rice leaves under salt stress. Thus, expression of Ndhl genes from maize or rice improves salt tolerance by enhancing photosynthetic electron transport in rice. Maize and rice Ndhl genes played a similar role in enhancing salinity tolerance and avoiding photosynthetic damage.

Fluctuating light induces a significant photoinhibition of photosystem I in maize.

In nature, light intensity usually fluctuates and a sudden shade-sun transition can induce photodamage to photosystem I (PSI) in many angiosperms. Photosynthetic regulation in fluctuating light (FL) has been studied extensively in C3 plants; however, little is known about how C4 plants cope FL to prevent PSI photoinhibition. We here compared photosynthetic responses to FL between maize (Zea mays, C4) and tomato (Solanum lycopersicum, C3) grown under full sunlight. Maize leaves had significantly higher cyclic electron flow (CEF) activity and lower photorespiration activity than tomato. Upon a sudden shade-sun transition, maize showed a significant stronger transient PSI over-reduction than tomato, resulting in a significant greater PSI photoinhibition in maize after FL treatment. During the first seconds upon shade-sun transition, CEF was stimulated in maize at a much higher extent than tomato, favoring the rapid formation of trans-thylakoid proton gradient (ΔpH), which was helped by a transient down-regulation of chloroplast ATP synthase activity. Therefore, modulation of ΔpH by regulation of CEF and chloroplast ATP synthase adjusted PSI redox state at donor side, which partially compensated for the deficiency of photorespiration. We propose that C4 plants use different photosynthetic strategies for coping with FL as compared with C3 plants.

Structures and organizations of PSI-AcpPCI supercomplexes from red tidal and coral symbiotic photosynthetic dinoflagellates.

Marine photosynthetic dinoflagellates are a group of successful phytoplankton that can form red tides in the ocean and also symbiosis with corals. These features are closely related to the photosynthetic properties of dinoflagellates. We report here three structures of photosystem I (PSI)-chlorophylls (Chls) a/c-peridinin protein complex (PSI-AcpPCI) from two species of dinoflagellates by single-particle cryoelectron microscopy. The crucial PsaA/B subunits of a red tidal dinoflagellate Amphidinium carterae are remarkably smaller and hence losing over 20 pigment-binding sites, whereas its PsaD/F/I/J/L/M/R subunits are larger and coordinate some additional pigment sites compared to other eukaryotic photosynthetic organisms, which may compensate for the smaller PsaA/B subunits. Similar modifications are observed in a coral symbiotic dinoflagellate Symbiodinium species, where two additional core proteins and fewer AcpPCIs are identified in the PSI-AcpPCI supercomplex. The antenna proteins AcpPCIs in dinoflagellates developed some loops and pigment sites as a result to accommodate the changed PSI core, therefore the structures of PSI-AcpPCI supercomplex of dinoflagellates reveal an unusual protein assembly pattern. A huge pigment network comprising Chls a and c and various carotenoids is revealed from the structural analysis, which provides the basis for our deeper understanding of the energy transfer and dissipation within the PSI-AcpPCI supercomplex, as well as the evolution of photosynthetic organisms.

Transcriptome analysis of the bloom-forming dinoflagellate Prorocentrum donghaiense exposed to Ginkgo biloba leaf extract, with an emphasis on photosynthesis.

Ginkgo biloba leaf extract (GBE) can effectively treat bloom-forming freshwater algae. However, there is limited information about the underlying suppression mechanism of the marine bloom-forming Prorocentrum donghaiense-the most dominant algal bloom species in the East China Sea. We investigated the effect of GBE on P. donghaiense in terms of its response to photosynthesis at the molecular/omic level. In total, 93,743 unigenes were annotated using six functional databases. Furthermore, 67,203 differentially expressed genes (DEGs) were identified in algae treated with 1.8 g∙L-1 GBE. Among these DEGs, we identified the genes involved in photosynthesis. PsbA, PsbB and PsbD in photosystem II, PsaA in photosystem I, and PetB and PetD in the cytochrome b6/f complex were downregulated. Other related genes, such as PsaC, PsaE, and PsaF in photosystem I; PetA in the cytochrome b6/f complex; and atpA, atpD, atpH, atpG, and atpE in the F-type H+-ATPase were upregulated. These results suggest that the structure and activity of the complexes were destroyed by GBE, thereby inhibiting the electron flow between the primary and secondary quinone electron acceptors, primary quinone electron acceptor, and oxygen-evolving complex in the PSII complex, and interrupting the electron flow between PSII and PSI, ultimately leading to a decline in algal cell photosynthesis. These findings provide a basis for understanding the molecular mechanisms underlying P. donghaiense exposure to GBE and a theoretical basis for the prevention and control of harmful algal blooms.

Coloring Outside the Lines: Exploiting Pigment-Protein Synergy for Far-Red Absorption in Plant Light-Harvesting Complexes.

Plants are designed to utilize visible light for photosynthesis. Expanding this light absorption toward the far-red could boost growth in low-light conditions and potentially increase crop productivity in dense canopies. A promising strategy is broadening the absorption of antenna complexes to the far-red. In this study, we investigated the capacity of the photosystem I antenna protein Lhca4 to incorporate far-red absorbing chlorophylls d and f and optimize their spectra. We demonstrate that these pigments can successfully bind to Lhca4, with the protein environment further red-shifting the chlorophyll d absorption, markedly extending the absorption range of this complex above 750 nm. Notably, chlorophyll d substitutes the canonical chlorophyll a red-forms, resulting in the most red-shifted emission observed in a plant light-harvesting complex. Using ultrafast spectroscopy, we show that the introduction of these novel chlorophylls does not interfere with the excited state decay or the energy equilibration processes within the complex. The results demonstrate the feasibility of engineering plant antennae to absorb deeper into the far-red region while preserving their functional and structural integrity, paving the way for innovative strategies to enhance photosynthesis.

Proton Gradient Regulation 5 is required to avoid photosynthetic oscillations during light transitions.

The production of ATP and NADPH by the light reactions of photosynthesis and their consumption by the Calvin-Benson-Bassham (CBB) cycle and other downstream metabolic reactions requires careful regulation. Environmental shifts perturb this balance, leading to photo-oxidative stress and losses in CO2 assimilation. Imbalances in the production and consumption of ATP and NADPH manifest themselves as transient instability in the chlorophyll fluorescence, P700, electrochromic shift, and CO2 uptake signals recorded on leaves. These oscillations can be induced in wild-type plants by sudden shifts in CO2 concentration or light intensity; however, mutants exhibiting increased oscillatory behaviour have yet to be reported. This has precluded an understanding of the regulatory mechanisms employed by plants to suppress oscillations. Here we show that the Arabidopsis pgr5 mutant, which is deficient in Proton Gradient Regulation 5 (PGR5)-dependent cyclic electron transfer (CET), exhibits increased oscillatory behaviour. In contrast, mutants lacking the NADH-dehydrogenase-like-dependent CET are largely unaffected. The absence of oscillations in the hope2 mutant which, like pgr5, lacks photosynthetic control and exhibits high ATP synthase conductivity, ruled out loss of these photoprotective mechanisms as causes. Instead, we observed slower formation of the proton motive force and, by inference, ATP synthesis in pgr5 following environmental perturbation, leading to the transient reduction of the electron transfer chain and photosynthetic oscillations. PGR5-dependent CET therefore plays a major role in damping the effect of environmental perturbations on photosynthesis to avoid losses in CO2 fixation.

Immunophilin FKB20-2 participates in oligomerization of Photosystem I in Chlamydomonas.

PSI is a sophisticated photosynthesis protein complex that fuels the light reaction of photosynthesis in algae and vascular plants. While the structure and function of PSI have been studied extensively, the dynamic regulation on PSI oligomerization and high light response is less understood. In this work, we characterized a high light-responsive immunophilin gene FKB20-2 (FK506-binding protein 20-2) required for PSI oligomerization and high light tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Biochemical assays and 77-K fluorescence measurement showed that loss of FKB20-2 led to the reduced accumulation of PSI core subunits and abnormal oligomerization of PSI complexes and, particularly, reduced PSI intermediate complexes in fkb20-2. It is noteworthy that the abnormal PSI oligomerization was observed in fkb20-2 even under dark and dim light growth conditions. Coimmunoprecipitation, MS, and yeast 2-hybrid assay revealed that FKB20-2 directly interacted with the low molecular weight PSI subunit PsaG, which might be involved in the dynamic regulation of PSI-light-harvesting complex I supercomplexes. Moreover, abnormal PSI oligomerization caused accelerated photodamage to PSII in fkb20-2 under high light stress. Together, we demonstrated that immunophilin FKB20-2 affects PSI oligomerization probably by interacting with PsaG and plays pivotal roles during Chlamydomonas tolerance to high light.