Light-independent pathway of STN7 kinase activation under low temperature stress in runner bean (Phaseolus coccineus L.).

BACKGROUND: The phosphorylation of the Light-Harvesting Complex of photosystem II (LHCII) driven by STATE TRANSITION 7 (STN7) kinase is a part of one of the crucial regulatory mechanisms of photosynthetic light reactions operating in fluctuating environmental conditions, light in particular. There are evidenced that STN7 can also be activated without light as well as in dark-chilling conditions. However, the biochemical mechanism standing behind this complex metabolic pathway has not been deciphered yet. RESULTS: In this work, we showed that dark-chilling induces light-independent LHCII phosphorylation in runner bean (Phaseolus coccineus L.). In dark-chilling conditions, we registered an increased reduction of the PQ pool which led to activation of STN7 kinase, subsequent LHCII phosphorylation, and possible LHCII relocation inside the thylakoid membrane. We also presented the formation of a complex composed of phosphorylated LHCII and photosystem I typically formed upon light-induced phosphorylation. Moreover, we indicated that the observed steps were preceded by the activation of the oxidative pentose phosphate pathway (OPPP) enzymes and starch accumulation. CONCLUSIONS: Our results suggest a direct connection between photosynthetic complexes reorganization and dark-chilling-induced activation of the thioredoxin system. The proposed possible pathway starts from the activation of OPPP enzymes and further NADPH-dependent thioredoxin reductase C (NTRC) activation. In the next steps, NTRC simultaneously activates ADP-glucose pyrophosphorylase and thylakoid membrane-located NAD(P)H dehydrogenase-like complex. These results in starch synthesis and electron transfer to the plastoquinone (PQ) pool, respectively. Reduced PQ pool activates STN7 kinase which phosphorylates LHCII. In this work, we present a new perspective on the mechanisms involving photosynthetic complexes while efficiently operating in the darkness. Although we describe the studied pathway in detail, taking into account also the time course of the following steps, the biological significance of this phenomenon remains puzzling.

Structure of cryptophyte photosystem II-light-harvesting antennae supercomplex.

Cryptophytes are ancestral photosynthetic organisms evolved from red algae through secondary endosymbiosis. They have developed alloxanthin-chlorophyll a/c2-binding proteins (ACPs) as light-harvesting complexes (LHCs). The distinctive properties of cryptophytes contribute to efficient oxygenic photosynthesis and underscore the evolutionary relationships of red-lineage plastids. Here we present the cryo-electron microscopy structure of the Photosystem II (PSII)-ACPII supercomplex from the cryptophyte Chroomonas placoidea. The structure includes a PSII dimer and twelve ACPII monomers forming four linear trimers. These trimers structurally resemble red algae LHCs and cryptophyte ACPI trimers that associate with Photosystem I (PSI), suggesting their close evolutionary links. We also determine a Chl a-binding subunit, Psb-γ, essential for stabilizing PSII-ACPII association. Furthermore, computational calculation provides insights into the excitation energy transfer pathways. Our study lays a solid structural foundation for understanding the light-energy capture and transfer in cryptophyte PSII-ACPII, evolutionary variations in PSII-LHCII, and the origin of red-lineage LHCIIs.

Altered excitation energy transfer between phycobilisome and photosystems in the absence of ApcG, a small linker peptide, in Synechocystis sp. PCC 6803, a cyanobacterium.

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.

Investigating the Balance between Structural Conservation and Functional Flexibility in Photosystem I.

Photosynthesis, as the primary source of energy for all life forms, plays a crucial role in maintaining the global balance of energy, entropy, and enthalpy in living organisms. Among its various building blocks, photosystem I (PSI) is responsible for light-driven electron transfer, crucial for generating cellular reducing power. PSI acts as a light-driven plastocyanin-ferredoxin oxidoreductase and is situated in the thylakoid membranes of cyanobacteria and the chloroplasts of eukaryotic photosynthetic organisms. Comprehending the structure and function of the photosynthetic machinery is essential for understanding its mode of action. New insights are offered into the structure and function of PSI and its associated light-harvesting proteins, with a specific focus on the remarkable structural conservation of the core complex and high plasticity of the peripheral light-harvesting complexes.

Functional consequences of modification of the photosystem I/photosystem II ratio in the cyanobacterium Synechocystis sp. PCC 6803.

The stoichiometry of photosystem II (PSII) and photosystem I (PSI) varies between photoautotrophic organisms. The cyanobacterium Synechocystis sp. PCC 6803 maintains two- to fivefold more PSI than PSII reaction center complexes, and we sought to modify this stoichiometry by changing the promoter region of the psaAB operon. We thus generated mutants with varied psaAB expression, ranging from ~3% to almost 200% of the wild-type transcript level, but all showing a reduction in PSI levels, relative to wild type, suggesting a role of the psaAB promoter region in translational regulation. Mutants with 25%-70% of wild-type PSI levels were photoautotrophic, with whole-chain oxygen evolution rates on a per-cell basis comparable to that of wild type. In contrast, mutant strains with <10% of the wild-type level of PSI were obligate photoheterotrophs. Variable fluorescence yields of all mutants were much higher than those of wild type, indicating that the PSI content is localized differently than in wild type, with less transfer of PSII-absorbed energy to PSI. Strains with less PSI saturate at a higher light intensity, enhancing productivity at higher light intensities. This is similar to what is found in mutants with reduced antennae. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea present, P700+ re-reduction kinetics in the mutants were slower than in wild type, consistent with the notion that there is less cyclic electron transport if less PSI is present. Overall, strains with a reduction in PSI content displayed surprisingly vigorous growth and linear electron transport. IMPORTANCE: Consequences of reduction in photosystem I content were investigated in the cyanobacterium Synechocystis sp. PCC 6803 where photosystem I far exceeds the number of photosystem II complexes. Strains with less photosystem I displayed less cyclic electron transport, grew more slowly at lower light intensity and needed more light for saturation but were surprisingly normal in their whole-chain electron transport rates, implying that a significant fraction of photosystem I is dispensable for linear electron transport in cyanobacteria. These strains with reduced photosystem I levels may have biotechnological relevance as they grow well at higher light intensities.

Is the A-1 Pigment in Photosystem I Part of P700? A (P700+-P700) FTIR Difference Spectroscopy Study of A-1 Mutants.

The involvement of the second pair of chlorophylls, termed A-1A and A-1B, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A-1B and A-1A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from Synechocystis sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700+-P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700+-P700) FTIR DS cannot be due to only the PA and PB pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A-1 pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A-1 pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A-1A and A-1B pigments are modified upon P700+ formation. Previously, we showed that the A-1 pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.

Growth phase-dependent reorganization of cryptophyte photosystem I antennae.

Photosynthetic cryptophytes are eukaryotic algae that utilize membrane-embedded chlorophyll a/c binding proteins (CACs) and lumen-localized phycobiliproteins (PBPs) as their light-harvesting antennae. Cryptophytes go through logarithmic and stationary growth phases, and may adjust their light-harvesting capability according to their particular growth state. How cryptophytes change the type/arrangement of the photosynthetic antenna proteins to regulate their light-harvesting remains unknown. Here we solve four structures of cryptophyte photosystem I (PSI) bound with CACs that show the rearrangement of CACs at different growth phases. We identify a cryptophyte-unique protein, PsaQ, which harbors two chlorophyll molecules. PsaQ specifically binds to the lumenal region of PSI during logarithmic growth phase and may assist the association of PBPs with photosystems and energy transfer from PBPs to photosystems.

Impact of Peripheral Hydrogen Bond on Electronic Properties of the Primary Acceptor Chlorophyll in the Reaction Center of Photosystem I.

Photosystem I (PS I) is a photosynthetic pigment-protein complex that absorbs light and uses the absorbed energy to initiate electron transfer. Electron transfer has been shown to occur concurrently along two (A- and B-) branches of reaction center (RC) cofactors. The electron transfer chain originates from a special pair of chlorophyll a molecules (P700), followed by two chlorophylls and one phylloquinone in each branch (denoted as A-1, A0, A1, respectively), converging in a single iron-sulfur complex Fx. While there is a consensus that the ultimate electron donor-acceptor pair is P700+A0-, the involvement of A-1 in electron transfer, as well as the mechanism of the very first step in the charge separation sequence, has been under debate. To resolve this question, multiple groups have targeted electron transfer cofactors by site-directed mutations. In this work, the peripheral hydrogen bonds to keto groups of A0 chlorophylls have been disrupted by mutagenesis. Four mutants were generated: PsaA-Y692F; PsaB-Y667F; PsaB-Y667A; and a double mutant PsaA-Y692F/PsaB-Y667F. Contrary to expectations, but in agreement with density functional theory modeling, the removal of the hydrogen bond by Tyr → Phe substitution was found to have a negligible effect on redox potentials and optical absorption spectra of respective chlorophylls. In contrast, Tyr → Ala substitution was shown to have a fatal effect on the PS I function. It is thus inferred that PsaA-Y692 and PsaB-Y667 residues have primarily structural significance, and their ability to coordinate respective chlorophylls in electron transfer via hydrogen bond plays a minor role.

APX2 Is an Ascorbate Peroxidase-Related Protein that Regulates the Levels of Plastocyanin in Chlamydomonas.

The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.

Critical role of cyclic electron transport around photosystem I in the maintenance of photosystem I activity.

In angiosperms, cyclic electron transport around photosystem I (PSI) is mediated by two pathways that depend on the PROTON GRADIENT REGULATION 5 (PGR5) protein and the chloroplast NADH dehydrogenase-like (NDH) complex, respectively. In the Arabidopsis double mutants defective in both pathways, plant growth and photosynthesis are impaired. The pgr5-1 mutant used in the original study is a missense allele and accumulates low levels of PGR5 protein. In this study, we generated two knockout (KO) alleles, designated as pgr5-5 and pgr5-6, using the CRISPR-Cas9 technology. Although both KO alleles showed a severe reduction in P700 similar to the pgr5-1 allele, NPQ induction was less severely impaired in the KO alleles than in the pgr5-1 allele. In the pgr5-1 allele, the second mutation affecting NPQ size was mapped to ~21 cM south of the pgr5-1 locus. Overexpression of the pgr5-1 allele, encoding the glycine130-to-serine change, complemented the pgr5-5 phenotype, suggesting that the pgr5-1 mutation destabilizes PGR5 but that the mutant protein retains partial functionality. Using two KO alleles, we created the double mutants with two chlororespiratory reduction (crr) mutants defective in the NDH complex. The growth of the double mutants was notably impaired. In the double mutant seedlings that survived on the medium containing sucrose, PSI activity evaluated by the P700 oxidation was severely impaired, whereas PSII activity was only mildly impaired. Cyclic electron transport around PSI is required to maintain PSI activity.

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.

Light Wavelength as a Contributory Factor of Environmental Fitness in the Cyanobacterial Circadian Clock.

A circadian clock is an essential system that drives the 24-h expression rhythms for adaptation to day-night cycles. The molecular mechanism of the circadian clock has been extensively studied in cyanobacteria harboring the KaiC-based timing system. Nevertheless, our understanding of the physiological significance of the cyanobacterial circadian clock is still limited. In this study, we cultured wild-type Synechococcus elongatus PCC 7942 and circadian clock mutants in day-night cycles at different light qualities and found that the growth of the circadian clock mutants was specifically impaired during 12-h blue light/12-h dark (BD) cycles for the first time. The arrhythmic mutant kaiCAA was further analyzed by photosynthetic measurements. Compared with the wild type, the mutant exhibited decreases in the chlorophyll content, the ratio of photosystem I to II, net O2 evolution rate and efficiency of photosystem II photochemistry during BD cycles. These results indicate that the circadian clock is necessary for the growth and the maintenance of the optimum function of the photosynthetic apparatus in cyanobacteria under blue photoperiodic conditions.

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.

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.

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.

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.

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.