Photosynthesis Research

In oxygenic photosynthetic organisms excluding angiosperms, flavodiiron proteins (FDPs) catalyze light-dependent reduction of O2 to H2O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero-oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase-like complex (NDH-1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH-1 types haven been characterized in cyanobacteria: NDH-11 and NDH-12, which function in respiration; and NDH-13 and NDH-14, which function in CO2 uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants ({Delta}flv1 and {Delta}flv3) and the double NDH-1 mutants ({Delta}d1d2, which is deficient in NDH-11,2 and {Delta}d3d4, which is deficient in NDH-13,4), we studied triple mutants lacking either one of Flv1 or Flv3, and NDH-11,2 or NDH-13,4. We show that the presence of either Flv1/3 or NDH-11,2, but not NDH-13,4, is indispensable for survival during changes in growth conditions from high CO2 /moderate light to low CO2 / high light. Our results suggest functional redundancy and crosstalk between FDPs and NDH-11,2 under the studied conditions, and demonstrate that the functions of FDPs and NDH-11,2 are dynamically coordinated for the efficient oxidation of PSI and for photoprotection under variable CO2 and light availability. One sentence summaryFlavodiiron proteins and NDH-1 complex ensure survival of cyanobacterial cells by cooperatively safeguarding the photosynthetic apparatus against excessive reduction

The acclimation of cyanobacteria to iron deficiency is crucial for their survival in natural environments. In response to iron deficiency, many cyanobacterial species induce the production of a pigment-protein complex called IsiA. IsiA proteins associate with photosystem I (PSI) and can function as light-harvesting antennas or dissipate excess energy. They may also serve as Chl storage during iron limitation. In this study we examined the functional role of IsiA in cells of Synechocystis sp. PCC 6803 grown under iron limitation conditions by measuring the cellular IsiA content and its capability to transfer energy to PSI. We specifically test the effect of the oligomeric state of PSI by comparing wild-type (WT) Synechocystis sp. PCC 6803 to mutants lacking specific subunits of PSI, namely PsaL/PsaI ({Delta}psaL mutant) and PsaF/PsaJ ({Delta}FIJL). Time-resolved fluorescence spectroscopy revealed that IsiA formed functional PSI3-IsiA18 supercomplexes, wherein IsiA effectively transfers energy to PSI on a timescale of 10 ps at room temperature - measured in isolated complexes and in vivo - confirming the primary role of IsiA as an accessory light-harvesting antenna to PSI. However, a significant fraction (40%) remained unconnected to PSI, supporting the notion of a dual functional role of IsiA. Cells with monomeric PSI under iron deficiency contained only 3-4 IsiA complexes bound to PSI. Together the results show that IsiA is capable of transferring energy to trimeric and monomeric PSI but to varying degrees and that the acclimatory production of IsiA under iron stress is controlled by its ability to perform its light-harvesting function.

Non-photochemical quenching (NPQ) is the process that protects photosynthetic organisms from photodamage by dissipating the energy absorbed in excess as heat. In the model green alga Chlamydomonas reinhardtii, NPQ was abolished in the knock-out mutants of the pigment-protein complexes LHCSR3 and LHCBM1. However, while LHCSR3 was shown to be a pH sensor and switching to a quenched conformation at low pH, the role of LHCBM1 in NPQ has not been elucidated yet. In this work, we combine biochemical and physiological measurements to study short-term high light acclimation of npq5, the mutant lacking LHCBM1. We show that while in low light in the absence of this complex, the antenna size of PSII is smaller than in its presence, this effect is marginal in high light, implying that a reduction of the antenna is not responsible for the low NPQ. We also show that the mutant expresses LHCSR3 at the WT level in high light, indicating that the absence of this complex is also not the reason. Finally, NPQ remains low in the mutant even when the pH is artificially lowered to values that can switch LHCSR3 to the quenched conformation. It is concluded that both LHCSR3 and LHCBM1 need to be present for the induction of NPQ and that LHCBM1 is the interacting partner of LHCSR3. This interaction can either enhance the quenching capacity of LHCSR3 or connect this complex with the PSII supercomplex.

The assembly of the Mn4O5Ca cluster of the photosystem II (PSII) starts from the initial binding and photooxidation of the first Mn2+ at a high affinity site (HAS). Recent cryo-EM apo-PSII structures reveal an altered geometry of amino ligands in this region and suggest the involvement of D1-Glu189 ligand in the formation of the HAS. We now find that Gln and Lys substitution mutants photoactivate with reduced quantum efficiency compared to the wild-type. However, the affinity of Mn2+ at the HAS in D1-E189K was very similar to the wild-type (~2.2 M). Thus, we conclude that D1-E189 does not form the HAS (~2.9 M) and that the reduced quantum efficiency of photoactivation in D1-E189K cannot be ascribed to the initial photooxidation of Mn2+ at the HAS. Besides reduced quantum efficiency, the D1-E189K mutant exhibits a large fraction of centers that fail to recover activity during photoactivation starting early in the assembly phase, becoming recalcitrant to further assembly. Fluorescence relaxation kinetics indicate on the presence of an alternative route for the charge recombination in Mn-depleted samples in all studied mutants and exclude damage to the photochemical reaction center as the cause for the recalcitrant centers failing to assemble and show that dark incubation of cells reverses some of the inactivation. This reversibility would explain the ability of these mutants to accumulate a significant fraction of active PSII during extended periods of cell growth. The failed recovery in the fraction of inactive centers appears to a reversible mis-assembly involving the accumulation of photooxidized, but non-catalytic high valence Mn at the donor side of photosystem II, and that a reductive mechanism exists for restoration of assembly capacity at sites incurring mis-assembly. Given the established role of Ca2+ in preventing misassembled Mn, we conclude that D1-E189K mutant impairs the ligation of Ca2+ at its effector site in all PSII centers that consequently leads to the mis-assembly resulting in accumulation of non-catalytic Mn at the donor side of PSII. Our data indicate that D1-E189 is not functionally involved in Mn2+ oxidation\binding at the HAS but rather involved in Ca2+ ligation and steps following the initial Mn2+ photooxidation.

Marine diatoms effectively photosynthesize by acclimating to the wide range of growth irradiance in the ocean. Using a pennate diatom Phaeodactylum tricornutum and a centric diatom Chaetoceros gracilis, we evaluated differences in the photosynthetic machinery under a wide range of growth irradiances. The chlorophyll a-specific amounts of the major accessory pigments remained relatively constant irrespective of growth irradiance in both diatoms. However, fluorescence spectra at 77K differed drastically depending on the growth irradiance: In P. tricornutum, fluorescence from photosystem II was dominant in high-light-grown cells and negligible in low-light-grown cells, while in C. gracilis, the opposite trend was observed. These drastic changes in fluorescence spectra were slow processes. The amounts of the two reaction centers, as assessed by specific antibodies and absorption changes in P700, remained almost constant under different irradiances. These results indicate that under dim growth irradiance, more excitation energy is diverted to photosystem I in the pennate diatom, and to photosystem II in the centric diatom. Therefore, the light-harvesting antennas balance excitation energy distribution by changing their association between photosystems I and II in different manners between P. tricornutum and C. gracilis, depending on irradiance. This phenomenon is similar to state transitions, but differs in its magnitude and duration. Differences in the preference of energy distribution in the two diatoms suggest that the dynamic state conversion--an antenna rearrangement during the long-term acclimation process in diatoms--is the species-specific strategy to achieve effective photosynthesis under the wide range of growth irradiances in the ocean.

Photosynthesis needs light energy, but that exceeding the maximal capacity of photosynthesis enhances formation of reactive oxygen species, which potentially causes photodamages. Therefore, light-harvesting complexes (Lhc) in phototrophs harbor various proteins and pigments to function in both light capture and energy dissipation. Diatom Lhcx proteins are reported to be a critical component for thermal dissipation of excess light energy, but the molecular mechanism of photoprotection is still not fully understood and the functions of each Lhcx isoform are not yet differentiated. Here, we focused on two types of Lhcx isoforms in Thalassiosira pseudonana: TpLhcx1/2, putative major components for energy-dependent fluorescence quenching (qE); and TpLhcx6_1, functionally unknown isoform uniquely conserved in Thalassiosirales. TpLhcx1/2 proteins accumulated more under high light than under low light, while the TpLhcx6_1 protein level was constitutive irrespective of light intensities and CO2 concentrations. High-light induced photodamage of photosystem II was increased in the genome-editing transformants of these Lhcx isoforms relative to the wild-type. Transformants lacking TpLhcx1/2 showed significantly lowered qE capacities, strongly suggesting that these proteins are important for the fast thermal energy dissipation. While in contrast, genome-editing transformants lacking the TpLhcx6_1 protein rather increased the qE capacity. TpLhcx6_1 transformants were further evaluated by the low-temperature time-resolved chlorophyll fluorescence measurement, showing the longer fluorescence lifetime in transformants than that in the wild type cells even at the dark-acclimated state of these cells. These results suggest that TpLhcx6_1 functions in photoprotection through non-photochemical energy dissipation in the different way from qE. One sentence summaryThe marine diatom Thalassiosira pseudonana dissipates excess light energy for photoprotection via two types of mechanisms supported by different Lhc isofoms.

Several auxiliary factors are required for the assembly of photosystem (PS) II, one of which is Psb28. While the absence of Psb28 in cyanobacteria has little effect on PSII assembly, we show here that the Chlamydomonas psb28-null mutant is severely impaired in PSII assembly, showing drastically reduced PSII supercomplexes, dimers and monomers, while overaccumulating RCII, CP43mod and D1mod. The mutant had less PSI and more Cytb6f and showed fewer thylakoid stacks and distorted chloroplast morphology. Complexome profiling of the psb28 mutant revealed that TEF5, the homolog of Arabidopsis PSB33/LIL8, co-migrated particularly with RCII. TEF5 also interacted with PSI. A Chlamydomonas tef5 null mutant is also severely impaired in PSII assembly and overaccumulates RCII and CP43mod. RC47 was not detectable in the light-grown tef5 mutant. Our data suggest a possible role for TEF5 in facilitating the assembly of CP47mod into RCII. Both the psb28 and tef5 mutants exhibited decreased synthesis of CP47 and PsbH, suggesting negative feedback regulation possibly exerted by the accumulation of RCII and/or CP43mod in both mutants. The strong effects of missing auxiliary factors on PSII assembly in Chlamydomonas suggest a more effective protein quality control system in this alga than in land plants and cyanobacteria. One-sentence summaryThe Chlamydomonas psb28 mutant is severely impaired in PSII assembly which via complexome profiling allowed identifying TEF5 as a novel PSII assembly factor that likely facilitates CP47 assembly. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: Michael Schroda (m.schroda@rptu.de).

Excess excitation energy in the light-harvesting antenna of Photosystem II (PSII) can cause irreversible damage to the photosynthetic apparatus. In periods of high light intensity, a feedback mechanism known as non-photochemical quenching (NPQ), induces the formation of quenchers which can safely dissipate excess excitation energy as heat. Although quenchers have been identified in more than one compartment of the PSII supercomplex, there is currently no quantitative description of how much NPQ is occurring at each of these locations. Here, we perform time-resolved fluorescence measurements on WT and antenna mutants lacking LHCII (NoL) and all peripheral antenna (Ch1 and Ch1lhcb5). By combining the results with those of steady-state fluorescence experiments we are able to estimate the intrinsic rate of NPQ for each plant and each PSII compartment. It is concluded that 60-70% of quenching occurs in LHCII, 15-20% in the minor antenna and 15-20% in the PSII core.

Photosynthetic organisms require acclimation mechanisms to regulate photosynthesis in response to light conditions. Here, two mutant alleles of ACCLIMATION OF PHOTOSYNTHESIS TO THE ENVIRONMENT 1 (ape1) have been characterized in Chlamydomonas reinhardtii. The ape1 mutants are photosensitive and show PSII photoinhibition during high light acclimation or under high light stress. The ape1 mutants retain more PSII super-complexes and have changes to thylakoid stacking relative to control strains during photosynthetic growth at different light intensities. The APE1 protein is found in all oxygenic phototrophs and encodes a 25 kDa thylakoid protein that interacts with the Photosystem II core complex as monomers, dimers and supercomplexes. We propose a model where APE1 bound to PSII supercomplexes releases core complexes and promotes PSII heterogeneity influencing the stacking of Chlamydomonas thylakoids. APE1 is a regulator in light acclimation and its function is to reduce over-excitation of PSII centres and avoid PSII photoinhibition to increase the resilience of photosynthesis to high light.

Binding of Psb28 to the photosystem II assembly intermediate PSII-I induces conformational changes to the PSII acceptor side that impact charge recombination and reduce the in situ production of singlet oxygen (Zabret et al. 2021, Nat. Plants 7, 524-538). A detailed fluorometric analysis of the PSII-I assembly intermediate compared with OEC-disrupted and Mn-depleted PSII complexes showed differences between their variable (OJIP) chlorophyll fluorescence induction profiles. These revealed a distinct destabilisation of the QA- state in the PSII-I assembly intermediate and inactivated PSII samples related to an increased rate of direct and safe charge recombination. Furthermore, inactivation or removal of the OEC increases the binding affinity for plastoquinone analogues like DCBQ to the different PSII complexes. These results might indicate a mechanism that further contributes to the protection of PSII during biogenesis or repair.

Flash-induced absorption changes in the Soret region, which originate from the [PD1PD2]+ state, the chlorophyll cation radical formed upon Photosystem II (PSII) excitation, were investigated in Mn-depleted Photosystem II. In wild-type PSII from Thermosynechococcus elongatus, the [PD1PD2]+-minus-[PD1PD2] difference spectrum shows a main negative feature at 434 nm and a smaller negative feature at 446 nm [Boussac et al. Photosynth Res (2023), https://doi.org/10.1007/s11120-023-01049-3]. While the main feature at 434 nm is associated with PD1+ formation, the origin of the dip at 446 nm remains to be identified. For that, we have compared the [PD1PD2]+-minus-[PD1PD2] difference spectra from the PsbA3/H198Q PSII mutant in T. elongatus and D2/H197A PSII mutant in Synechocystis sp. PCC 6803 with their respective wild type strains. By modifying the PD1 axial ligand with the H198Q mutation in the D1 protein in T. elongatus, the contribution at 434 nm was shifted to 431 nm, while the contribution at 446 nm was hardly affected. In Synechocystis sp. PCC 6803, by modifying the PD2 axial ligand with the H197A mutation in the D2 protein, the contribution at 446 nm was downshifted by [~] 3 nm to [~] 443 nm, while the main contribution at 432 nm was only slightly shifted upwards to 433 nm. This result suggests that the bleaching seen at 446 nm involves PD2. This could reflects a change in the [PD1+PD2]{longleftrightarrow}[PD1PD2+] equilibrium or a more complex mechanism.

Cyanobacteria carry out photosynthetic light-energy conversion using phycobiliproteins for light harvesting and the chlorophyll-rich photosystems for photochemistry. While most cyanobacteria only absorb visible photons, some of them can acclimate to harvest far-red light (FRL, 700-800 nm) by integrating chlorophyll f and d in their photosystems and producing red-shifted allophycocyanin. Chlorophyll f insertion enables the photosystems to use FRL but slows down charge separation, reducing photosynthetic efficiency. Here we demonstrate with time-resolved fluorescence spectroscopy that charge separation in chlorophyll-f-containing Photosystem II becomes faster in the presence of red-shifted allophycocyanin antennas. This is different from all known photosynthetic systems, where additional light-harvesting complexes slow down charge separation. Based on the available structural information, we propose a model for the connectivity between the phycobiliproteins and Photosystem II that qualitatively accounts for our spectroscopic data. This unique design is probably important for these cyanobacteria to efficiently switch between visible and far-red light.

Chlorophyll a fluorescence is a powerful indicator of photosynthetic energy conversion in plants and photosynthetic microorganisms. One of the most widely used measurement techniques is Pulse Amplitude Modulation (PAM) fluorometry. Unfortunately, parameter settings of PAM instruments are often not completely described in scientific articles although their variations, however small these may seem, can influence measurements. We show the effects of parameter settings on PAM measurements. We first simulated fluorescence signals using a previously published computational model of photosynthesis. Then, we validated our findings experimentally. Our analysis demonstrates how the kinetics of non-photochemical quenching (NPQ) induction and relaxation are affected by different settings of PAM instrument parameters. Neglecting these parameters may mislead data interpretation and derived hypotheses, hamper independent validation of the results, and cause problems for mathematical formulation of underlying processes. Given the uncertainties inflicted by this neglect, we urge PAM users to provide detailed documentation of measurement protocols. Moreover, to ensure accessibility to the required information, we advocate minimum information standards that can serve both experimental and computational biologists in our efforts to advance system-wide understanding of biological processes. Such specification will enable launching a standardized database for plant and data science communities. HighlightPAM fluorometry measurement is sensitive to instrument settings and protocols. Yet, protocols are published incompletely. We urge to reach an agreement on minimal protocol information of PAM experiments to be shared publicly.

Photosystem II uses water as the ultimate electron source of the photosynthetic electron transfer chain. Water is oxidized to dioxygen at the Oxygen Evolving Complex (OEC), a Mn4CaO5 inorganic core embedded in the lumenal side of PSII. Water-filled channels are thought to bring in substrate water molecules to the OEC, remove the substrate protons to the lumen, and may transport the product oxygen. Three water-filled channels, denoted large, narrow, and broad, that extend from the OEC towards the aqueous surface more than 15 [A] away are seen. However, the actual mechanisms of water supply to the OEC, the removal of protons to the lumen and diffusion of oxygen away from the OEC have yet to be established. Here, we combine Molecular Dynamics (MD), Multi Conformation Continuum Electrostatics (MCCE) and Network Analysis to compare and contrast the three potential proton transfer paths during the S1 to S2 transition of the OEC. Hydrogen bond network analysis shows that the three channels are highly interconnected with similar energetics for hydronium as calculated for all paths near the OEC. The channels diverge as they approach the lumen, with the water chain in the broad channel better interconnected that in the narrow and large channels, where disruptions in the network are observed at about 10 [A] from the OEC. In addition, the barrier for hydronium translocation is lower in the broad channel, suggesting that a proton from the OEC could access the paths near the OEC, and likely exit to the lumen via the broad channel, passing through PsbO.

Photosynthetic organisms harvest light using pigment-protein super-complexes. In cyanobacteria, these are water-soluble antennae known as phycobilisomes (PBSs). The light absorbed by PBS is transferred to the photosystems in the thylakoid membrane to drive photosynthesis. The energy transfer between these super-complexes implies that protein-protein interactions allow the association of PBS with the photosystems. However, the specific proteins involved in the interaction of PBS with the photosystems are not fully characterized. Here, we show that the newly discovered PBS linker protein ApcG interacts specifically with photosystem II through its N-terminal region. Growth of cyanobacteria is impaired in apcG deletion strains under light-limiting conditions. Furthermore, complementation of these strains using a phospho-mimicking version of ApcG exhibit reduced growth under normal growth conditions. Interestingly, the interaction of ApcG with photosystem II is affected when a phospho-mimicking version of ApcG is used, targeting the positively charged residues interacting with thylakoid membrane suggesting a regulatory role mediated by phosphorylation of ApcG. Low temperature fluorescence measurements showed increased photosystem I fluorescence in apcG deletion and complementation strains. The photosystem I fluorescence was the highest in the phospho-mimicking complementation strain while pull-down experiment showed no interaction of ApcG with PSI under any tested condition. Our results highlight the importance of ApcG for selectively directing energy harvested by the PBS and implies that the phosphorylation status of ApcG plays a role in regulating energy transfer from PSII to PSI.

Phycobilisomes are versatile cyanobacterial antenna complexes that harvest light energy to drive photosynthesis. These complexes can also adapt to various light conditions, dismantling under high light to prevent photo-oxidation and arranging in rows under low light to increase light harvesting efficiency. Light quality also influences phycobilisome structure and function, as observed under far-red light exposure. Here we describe a new, phycobilisome linker protein, ApcI (previously hypothetical protein sll1911), expressed specifically under red light. We characterized ApcI in Synechocystis sp. PCC 6803 using mutant strain analyses, phycobilisome binding experiments, and protein interaction studies. Mutation of apcI conferred high light tolerance to Synechocystis sp. PCC 6803 compared to wild type with reduced energy transfer from phycobilisomes to the photosystems. Binding experiments revealed that ApcI replaces the linker protein ApcG at the membrane-facing side of the phycobilisome core using a paralogous C-terminal domain. Additionally, the N-terminal extension of ApcI was found to interact with photosystem II. Our findings highlight the importance of phycobilisome remodeling for adaptation under different light conditions. The characterization of ApcI provides new insights into the mechanisms by which cyanobacteria optimize light-harvesting in response to varying light environments.

The photosynthetic reaction is driven by the cooperation of two light-excited pigment-protein supercomplexes: photosystem II (PSII) and photosystem I (PSI). The efficiency of the excitation of the two PSs relies on the exquisite organization of their light-harvesting antenna under environmental fluctuations. However, since the antenna-protein composition within cells remains elusive, the in vivo events arising from antenna variations cannot be accurately explored. Here, we implemented the single-pixel excitation-emission spectroscopy of Chlamydomonas cells under 80 K using a cryogenic optical microscope. The antenna variations of in vivo PSI can be exclusively evaluated via this low-temperature spectro-imaging method. The simultaneous acquisition of two types of fluorescence spectra enables the analysis of the intracellular correlation between the PSII/PSI intensity ratio and the chlorophyll-b/a (Chl-b/a) concentration ratio. We found that the Chl-b/a ratio hardly correlated with the PSII/PSI intensity ratio in most cases, suggesting that the in vivo PSI intensity ratio reflects the relative PSI stoichiometry rather than their antenna sizes. More importantly, the analysis of the PSI antenna-related Chl-b concentration within cells reveals a mega-antenna system that is much larger than the antenna sizes of the PSI supercomplexes whose structures have been resolved so far. Such PSI megacomplexes tended to be enriched in the region surrounding the pyrenoids of Chlamydomonas cells. We anticipate the present investigation to be a starting point for directly estimating the arrangements of antenna systems of photosystems at the single-cell scale, which is necessary for a deeper understanding of dynamic in vivo events related to the photosynthetic light-harvesting process.

Photomixotrophy is a metabolic state, which enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we show via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual reduction of the plastoquinone pool in wild-type Synechocystis, which fully downscales photosynthesis over three days of growth. This process is circumvented by deleting the gene encoding cytochrome cM (CytM), a cryptic c-type heme protein widespread in cyanobacteria. {Delta}CytM maintained active photosynthesis over the three day period, demonstrated by high photosynthetic O2 and CO2 fluxes and effective yields of Photosystem II and Photosystem I. Overall, this resulted in a higher growth rate than wild-type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localised respiratory terminal oxidases and CytM ({Delta}Cox/Cyd/CytM) displayed a similar phenotype under photomixotrophy to {Delta}CytM, demonstrating that CytM is not transferring electrons to these complexes, which has previously been suggested. In summary, the obtained data suggests that CytM may have a regulatory role in photomixotrophy by reducing the photosynthetic capacity of cells. One sentence summaryThe cryptic, highly conserved cytochrome cM completely blocks photosynthesis in Synechocystis under three days of photomixotrophy, possibly by suppressing CO2 assimilation.

We report here a technical advancement that enables time-resolved fluorescence spectroscopy in spatially resolved domains of a living cell at low temperatures. The technique is based on a combination of the self-developed cryo-confocal microscope system and the streak-camera technology. An instrumental response time of ca. 24 ps was achieved. This technique was applied to reveal the light-harvesting dynamics in local domains within single Chlamydomonas reinhardtii cells. Organisms performing oxygenic photosynthesis, like Chlamydomonas, have evolved a regulation mechanism called state transitions (ST), which maintains the excitation balance between PSI and PSII. ST relies on the shuttling of light-harvesting chlorophyll protein complex II (LHCII) between the two PSs. In the present experiment, cells were induced either to state1, where LHCII is bound to PSII, or state2, where LHCII moved and is bound to PSI. After the induction of ST, cells were immediately cooled to ca. 80 K, where PSI and PSII show clearly separated fluorescence emission bands, enabling the visualization of these components separately. Based on kinetic analyses of the time-resolved fluorescence spectra in both PSI-rich and PSII-rich local domains, we concluded that (1) the intracellular inhomogeneity in the PSII/PSI fluorescence ratio comes from that in the PSII/PSI stoichiometry, not from that in the antenna sizes of the PSs, and (2) the antenna size of PSI in state2 cells may larger in intact cells than that of the isolated PSI-LHCI-LHCII super-complex reported so far.

Cyanobacteria play a key role in primary production in both oceans and fresh waters and hold great potential for sustainable production of a large number of commodities. During their life, cyanobacteria cells need to acclimate to a multitude of challenges, including shifts in intensity and quality of incident light. Despite our increasing understanding of metabolic regulation under various light regimes, detailed insight into fitness advantages and limitations under shifting light quality has been missing. Here, we study photo-physiological acclimation in the cyanobacterium Synechocystis sp. PCC 6803 through the whole range of photosynthetically active radiation (PAR). Using LEDs with qualitatively different narrow spectra, we describe wavelength dependence of light capture, electron transport and energy transduction to main cellular pools. In addition, we describe processes fine-tuning light capture such as state transitions and efficiency of energy transfer from phycobilisomes to photosystems. We show that growth was the most limited under blue light due to inefficient light harvesting, and that many cellular processes are tightly linked to the redox state of the PQ pool, which was the most reduced under red light. The PSI-to-PSII ratio was low under blue photons, however, it was not the main growth-limiting factor, since it was even more reduced under violet and near far-red lights, where Synechocystis grew faster compared to blue light. Our results provide insight into the spectral dependence of phototrophic growth and can provide the foundation for future studies of molecular mechanisms underlying light acclimation in cyanobacteria, leading to light optimization in controlled cultivations.