Three structures of PSI-LHCI from Chlamydomonas reinhardtii suggest a resting state re-activated by ferredoxin.

Photosystem I (PSI) from the green alga Chlamydomonas reinhardtii, with various numbers of membrane bound antenna complexes (LHCI), has been described in great detail. In contrast, structural characterization of soluble binding partners is less advanced. Here, we used X-ray crystallography and single particle cryo-EM to investigate three structures of the PSI-LHCI supercomplex from Chlamydomonas reinhardtii. An X-ray structure demonstrates the absence of six chlorophylls from the luminal side of the LHCI belts, suggesting these pigments were either physically absent or less stably associated with the complex, potentially influencing excitation transfer significantly. CryoEM revealed extra densities on luminal and stromal sides of the supercomplex, situated in the vicinity of the electron transfer sites. These densities disappeared after the binding of oxidized ferredoxin to PSI-LHCI. Based on these structures, we propose the existence of a PSI-LHCI resting state with a reduced active chlorophyll content, electron donors docked in waiting positions and regulatory binding partners positioned at the electron acceptor site. The resting state PSI-LHCI supercomplex would be recruited to its active form by the availability of oxidized ferredoxin.

Light-independent regulation of algal photoprotection by CO2 availability.

Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2 conditions. When light energy exceeds CO2 fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2 Concentrating Mechanism (CCM). How light and CO2 signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2 concentrations and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2 and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2 levels.

Small RNA-mediated silencing of phototropin suppresses the induction of photoprotection in the green alga Chlamydomonas reinhardtii.

Small RNAs (sRNAs) form complexes with Argonaute proteins and bind to transcripts with complementary sequences to repress gene expression. sRNA-mediated regulation is conserved in a diverse range of eukaryotes and is involved in the control of various physiological functions. sRNAs are present in the unicellular green alga Chlamydomonas reinhardtii, and genetic analyses revealed that the core sRNA biogenesis and action mechanisms are conserved with those of multicellular organisms. However, the roles of sRNAs in this organism remain largely unknown. Here, we report that Chlamydomonas sRNAs contribute to the induction of photoprotection. In this alga, photoprotection is mediated by LIGHT HARVESTING COMPLEX STRESS-RELATED 3 (LHCSR3), whose expression is induced by light signals through the blue-light receptor phototropin (PHOT). We demonstrate here that sRNA-defective mutants showed increased PHOT abundance leading to greater LHCSR3 expression. Disruption of the precursor for two sRNAs predicted to bind to the PHOT transcript also increased PHOT accumulation and LHCSR3 expression. The induction of LHCSR3 in the mutants was enhanced by light containing blue wavelengths, but not by red light, indicating that the sRNAs regulate the degree of photoprotection via regulation of PHOT expression. Our results suggest that sRNAs are involved not only in the regulation of photoprotection but also in biological phenomena regulated by PHOT signaling.

State transition is quiet around pyrenoid and LHCII phosphorylation is not essential for thylakoid deformation in Chlamydomonas 137c.

Photosynthetic organisms have developed a regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between the two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the dynamic transformations of the thylakoid membrane with ST, evidence of that remains elusive. To clarify the above-mentioned coupling in a model organism Chlamydomonas, here we used two advanced microscope techniques, the excitation-spectral microscope (ESM) developed recently by us and the superresolution imaging based on structured-illumination microscopy (SIM). The ESM observation revealed ST-dependent spectral changes upon repeated ST inductions. Surprisingly, it clarified a less significant ST occurrence in the region surrounding the pyrenoid, which is a subcellular compartment specialized for the carbon-fixation reaction, than that in the other domains. Further, we found a species dependence of this phenomenon: 137c strain showed the significant intracellular inhomogeneity of ST occurrence, whereas 4A+ strain hardly did. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations caused by the ST-inducing illumination. This fine, irreversible thylakoid transformation was also observed in the STT7 kinase-lacking mutant. This result revealed that the fine thylakoid transformation is not induced solely by the LHCII phosphorylation, suggesting the highly susceptible nature of the thylakoid ultrastructure to the photosynthetic light reactions.

The four-celled Volvocales green alga Tetrabaena socialis exhibits weak photobehavior and high-photoprotection ability.

Photo-induced behavioral responses (photobehaviors) are crucial to the survival of motile phototrophic organisms in changing light conditions. Volvocine green algae are excellent model organisms for studying the regulatory mechanisms of photobehavior. We recently reported that unicellular Chlamydomonas reinhardtii and multicellular Volvox rousseletii exhibit similar photobehaviors, such as phototactic and photoshock responses, via different ciliary regulations. To clarify how the regulatory systems have changed during the evolution of multicellularity, we investigated the photobehaviors of four-celled Tetrabaena socialis. Surprisingly, unlike C. reinhardtii and V. rousseletii, T. socialis did not exhibit immediate photobehaviors after light illumination. Electrophysiological analysis revealed that the T. socialis eyespot does not function as a photoreceptor. Instead, T. socialis exhibited slow accumulation toward the light source in a photosynthesis-dependent manner. Our assessment of photosynthetic activities showed that T. socialis chloroplasts possess higher photoprotection abilities against strong light than C. reinhardtii. These data suggest that C. reinhardtii and T. socialis employ different strategies to avoid high-light stress (moving away rapidly and gaining photoprotection, respectively) despite their close phylogenetic relationship.

Characterization of Chlamydomonas reinhardtii Mutants That Exhibit Strong Positive Phototaxis.

The most motile phototrophic organisms exhibit photo-induced behavioral responses (photobehavior) to inhabit better light conditions for photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study photobehavior. Several years ago, we found that C. reinhardtii cells reverse their phototactic signs (i.e., positive and negative phototaxis) depending on the amount of reactive oxygen species (ROS) accumulated in the cell. However, its molecular mechanism is unclear. In this study, we isolated seven mutants showing positive phototaxis, even after the induction of negative phototaxis (ap1~7: always positive) to understand the ROS-dependent regulatory mechanism for the phototactic sign. We found no common feature in the mutants regarding their growth, high-light tolerance, and photosynthetic phenotypes. Interestingly, five of them grew faster than the wild type. These data suggest that the ROS-dependent regulation of the phototactic sign is not a single pathway and is affected by various cellular factors. Additionally, the isolation and analyses of mutants with defects in phototactic-sign regulation may provide clues for their application to the efficient cultivation of algae.

Structural basis of LhcbM5-mediated state transitions in green algae.

In green algae and plants, state transitions serve as a short-term light-acclimation process in the regulation of the light-harvesting capacity of photosystems I and II (PSI and PSII, respectively). During the process, a portion of light-harvesting complex II (LHCII) is phosphorylated, dissociated from PSII and binds with PSI to form the supercomplex PSI-LHCI-LHCII. Here, we report high-resolution structures of PSI-LHCI-LHCII from Chlamydomonas reinhardtii, revealing the mechanism of assembly between the PSI-LHCI complex and two phosphorylated LHCII trimers containing all four types of LhcbM protein. Two specific LhcbM isoforms, namely LhcbM1 and LhcbM5, directly interact with the PSI core through their phosphorylated amino terminal regions. Furthermore, biochemical and functional studies on mutant strains lacking either LhcbM1 or LhcbM5 indicate that only LhcbM5 is indispensable in supercomplex formation. The results unravel the specific interactions and potential excitation energy transfer routes between green algal PSI and two phosphorylated LHCIIs.

High-Speed Excitation-Spectral Microscopy Uncovers In Situ Rearrangement of Light-Harvesting Apparatus in Chlamydomonas during State Transitions at Submicron Precision.

Photosynthetic organisms adjust to fluctuating natural light under physiological ambient conditions through flexible light-harvesting ability of light-harvesting complex II (LHCII). A process called state transition is an efficient regulation mechanism to balance the excitations between photosystem II (PSII) and photosystem I (PSI) by shuttling mobile LHCII between them. However, in situ observation of the migration of LHCII in vivo remains limited. In this study, we investigated the in vivo reversible changes in the intracellular distribution of the chlorophyll (Chl) fluorescence during the light-induced state transitions in Chlamydomonas reinhardtii. The newly developed noninvasive excitation-spectral microscope provided powerful spectral information about excitation-energy transfer between Chl-a and Chl-b. The excitation spectra were detected through the fluorescence emission in the 700-750-nm spectral range, where PSII makes the main contribution, though PSI still makes a non-negligible contribution at room temperature. The technique is sensitive to the Chl-b spectral component specifically bound to LHCII. Using a PSI-specific 685-nm component also provided visualization of the local relative concentration of PSI within a chloroplast at room temperature. The decrease in the relative intensity of the Chl-b band in state 2 was more conspicuous in the PSII-rich region than in the PSI-rich region, reflecting the dissociation of LHCII from PSII. We observed intracellular redistributions of the Chl-b-related light-harvesting abilities within a chloroplast during the state transitions. This observation implies the association of the state transitions with the morphological changes in the thylakoid membrane.

UV-A/B radiation rapidly activates photoprotective mechanisms in Chlamydomonas reinhardtii.

Conversion of light energy into chemical energy through photosynthesis in the chloroplasts of photosynthetic organisms is essential for photoautotrophic growth, and non-photochemical quenching (NPQ) of excess light energy prevents the generation of reactive oxygen species and maintains efficient photosynthesis under high light. In the unicellular green alga Chlamydomonas reinhardtii, NPQ is activated as a photoprotective mechanism through wavelength-specific light signaling pathways mediated by the phototropin (blue light) and ultra-violet (UV) light photoreceptors, but the biological significance of photoprotection activation by light with different qualities remains poorly understood. Here, we demonstrate that NPQ-dependent photoprotection is activated more rapidly by UV than by visible light. We found that induction of gene expression and protein accumulation related to photoprotection was significantly faster and greater in magnitude under UV treatment compared with that under blue- or red-light treatment. Furthermore, the action spectrum of UV-dependent induction of photoprotective factors implied that C. reinhardtii senses relatively long-wavelength UV (including UV-A/B), whereas the model dicot plant Arabidopsis (Arabidopsis thaliana) preferentially senses relatively short-wavelength UV (mainly UV-B/C) for induction of photoprotective responses. Therefore, we hypothesize that C. reinhardtii developed a UV response distinct from that of land plants.

Characterization of a Giant PSI Supercomplex in the Symbiotic Dinoflagellate Symbiodiniaceae.

Symbiodiniaceae are symbiotic dinoflagellates that provide photosynthetic products to corals. Because corals are distributed across a wide range of depths in the ocean, Symbiodiniaceae species must adapt to various light environments to optimize their photosynthetic performance. However, as few biochemical studies of Symbiodiniaceae photosystems have been reported, the molecular mechanisms of photoadaptation in this algal family remain poorly understood. Here, to investigate the photosynthetic machineries in Symbiodiniaceae, we purified and characterized the PSI supercomplex from the genome-sequenced Breviolum minutum (formerly Symbiodinium minutum). Mass spectrometry analysis revealed 25 light-harvesting complexes (LHCs), including both LHCF and LHCR families, from the purified PSI-LHC supercomplex. Single-particle electron microscopy showed unique giant supercomplex structures of PSI that were associated with the LHCs. Moreover, the PSI-LHC supercomplex contained a significant amount of the xanthophyll cycle pigment diadinoxanthin. Upon high light treatment, B. minutum cells showed increased nonphotochemical quenching, which was correlated with the conversion of diadinoxanthin to diatoxanthin, occurring preferentially in the PSI-LHC supercomplex. The possible role of PSI-LHC in photoprotection in Symbiodiniaceae is discussed.

Light regulation of light-harvesting antenna size substantially enhances photosynthetic efficiency and biomass yield in green algae†.

One of the major factors limiting biomass productivity in algae is the low thermodynamic efficiency of photosynthesis. The greatest thermodynamic inefficiencies in photosynthesis occur during the conversion of light into chemical energy. At full sunlight the light-harvesting antenna captures photons at a rate nearly 10 times faster than the rate-limiting step in photosynthetic electron transport. Excess captured energy is dissipated by non-productive pathways including the production of reactive oxygen species. Substantial improvements in photosynthetic efficiency have been achieved by reducing the optical cross-section of the light-harvesting antenna by selectively reducing chlorophyll b levels and peripheral light-harvesting complex subunits. Smaller light-harvesting antenna, however, may not exhibit optimal photosynthetic performance in low or fluctuating light environments. We describe a translational control system to dynamically adjust light-harvesting antenna sizes for enhanced photosynthetic performance. By expressing a chlorophyllide a oxygenase (CAO) gene having a 5' mRNA extension encoding a Nab1 translational repressor binding site in a CAO knockout line it was possible to continuously alter chlorophyll b levels and correspondingly light-harvesting antenna sizes by light-activated Nab1 repression of CAO expression as a function of growth light intensity. Significantly, algae having light-regulated antenna sizes had substantially higher photosynthetic rates and two-fold greater biomass productivity than the parental wild-type strains as well as near wild-type ability to carry out state transitions and non-photochemical quenching. These results have broad implications for enhanced algae and plant biomass productivity.

Four distinct trimeric forms of light-harvesting complex II isolated from the green alga Chlamydomonas reinhardtii.

Light-harvesting complex II (LHCII) absorbs light energy and transfers it primarily to photosystem II in green algae and land plants. Although the trimeric structure of LHCII is conserved between the two lineages, its subunit composition and function are believed to differ significantly. In this study, we purified four LHCII trimers from the green alga Chlamydomonas reinhardtii and analyzed their biochemical properties. We used several preparation methods to obtain four distinct fractions (fractions 1-4), each of which contained an LHCII trimer with different contents of Type I, III, and IV proteins. The pigment compositions of the LHCIIs in the four fractions were similar. The absorption and fluorescence spectra were also similar, although the peak positions differed slightly. These results indicate that this green alga contains four types of LHCII trimer with different biochemical and spectroscopic features. Based on these findings, we discuss the function and structural organization of green algal LHCII antennae.

The CONSTANS flowering complex controls the protective response of photosynthesis in the green alga Chlamydomonas.

Light is essential for photosynthesis, but the amounts of light that exceed an organism's assimilation capacity can result in oxidative stress and even cell death. Plants and microalgae have developed a photoprotective response mechanism, qE, that dissipates excess light energy as thermal energy. In the green alga Chlamydomonas reinhardtii, qE is regulated by light-inducible photoprotective proteins, but the pathway from light perception to qE is not fully understood. Here, we show that the transcription factors CONSTANS and Nuclear transcription Factor Ys (NF-Ys) form a complex that governs light-dependent photoprotective responses in C. reinhardtii. The qE responses do not occur in CONSTANS or NF-Y mutants. The signal from light perception to the CONSTANS/NF-Ys complex is directly inhibited by the SPA1/COP1-dependent E3 ubiquitin ligase. This negative regulation mediated by the E3 ubiquitin ligase and the CONSTANS/NF-Ys complex is common to photoprotective response in algal photosynthesis and flowering in plants.

Structural determination of the large photosystem II-light-harvesting complex II supercomplex of Chlamydomonas reinhardtii using nonionic amphipol.

In photosynthetic organisms, photosystem II (PSII) is a large membrane protein complex, consisting of a pair of core complexes surrounded by an array of variable numbers of light-harvesting complex (LHC) II proteins. Previously reported structures of the PSII-LHCII supercomplex of the green alga Chlamydomonas reinhardtii exhibit significant structural heterogeneity, but recently improved purification methods employing ionic amphipol A8-35 have enhanced supercomplex stability, providing opportunities for determining a more intact structure. Herein, we present a 5.8 Å cryo-EM map of the C. reinhardtii PSII-LHCII supercomplex containing six LHCII trimers (C2S2M2L2). Utilizing a newly developed nonionic amphipol-based purification and stabilizing method, we purified the largest photosynthetic supercomplex to the highest percentage of the intact configuration reported to date. We found that the interprotein distances within the light-harvesting complex array in the green algal photosystem are larger than those previously observed in higher plants, indicating that the potential route of energy transfer in the PSII-LHCII supercomplex in green algae may be altered. Interestingly, we also observed an asymmetric PSII-LHCII supercomplex structure comprising C2S2M1L1 in the same sample. Moreover, we found a new density adjacent to the PSII core complex, attributable to a single-transmembrane helix. It was previously unreported in the cryo-EM maps of PSII-LHCII supercomplexes from land plants.

Amphipol-assisted purification method for the highly active and stable photosystem II supercomplex of Chlamydomonas reinhardtii.

Photosystem II (PSII) splits water and drives electron transfer to plastoquinone via photochemical reactions using light energy. It is surrounded by light-harvesting complex II (LHCII) to form the PSII-LHCII supercomplex. Complete characterization of its structure and function has, however, been hampered due to instability of the complex in the presence of detergent. To overcome this problem, we developed a new procedure for purifying the PSII-LHCII supercomplexes of Chlamydomonas reinhardtii employing amphipol A8-35. The obtained supercomplexes showed little LHCII dissociation even 4 days after purification. Oxygen-evolving activity was retained within amphipol if the extrinsic polypeptides were kept associated by betaine. Electron microscopy revealed that this method also improved structural uniformity and that the major organization was C2 S2 M2 L2 .

Isolation of photoprotective signal transduction mutants by systematic bioluminescence screening in Chlamydomonas reinhardtii.

In photosynthetic organisms, photoprotection to avoid overexcitation of photosystems is a prerequisite for survival. Green algae have evolved light-inducible photoprotective mechanisms mediated by genes such as light-harvesting complex stress-related (LHCSR). Studies on the light-dependent regulation of LHCSR expression in the green alga Chlamydomonas reinhardtii have revealed that photoreceptors for blue light (phototropin) and ultraviolet light perception (UVR8) play key roles in initiating photoprotective signal transduction. Although initial light perception via phototropin or UVR8 is known to result in increased LHCSR3 and LHCSR1 gene expression, respectively, the mechanisms of signal transduction from the input (light perception) to the output (gene expression) remain unclear. In this study, to further elucidate the signal transduction pathway of the photoprotective response of green algae, we established a systematic screening protocol for UV-inducible LHCSR1 gene expression mutants using a bioluminescence reporter assay. Following random mutagenesis screening, we succeeded in isolating mutants deficient in LHCSR1 gene and protein expression after UV illumination. Further characterization revealed that the obtained mutants could be separated into 3 different phenotype groups, the "UV-specific", "LHCSR1-promoter/transcript-specific" and "general photoprotective" mutant groups, which provided further insight into photoprotective signal transduction in C. reinhardtii.

Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii.

Photosystem I (PSI) is a large pigment-protein complex mediating light-driven charge separation and generating a highly negative redox potential, which is eventually utilized to produce organic matter. In plants and algae, PSI possesses outer antennae, termed light-harvesting complex I (LHCI), which increase the energy flux to the reaction center. The number of outer antennae for PSI in the green alga Chlamydomonas reinhardtii is known to be larger than that of land plants. However, their exact number and location remain to be elucidated. Here, applying a newly established sample purification procedure, we isolated a highly pure PSI-LHCI supercomplex containing all nine LHCA gene products under state 1 conditions. Single-particle cryo-EM revealed the 3D structure of this supercomplex at 6.9 Å resolution, in which the densities near the PsaF and PsaJ subunits were assigned to two layers of LHCI belts containing eight LHCIs, whereas the densities between the PsaG and PsaH subunits on the opposite side of the LHCI belt were assigned to two extra LHCIs. Using single-particle cryo-EM, we also determined the 2D projection map of the lhca2 mutant, which confirmed the assignment of LHCA2 and LHCA9 to the densities between PsaG and PsaH. Spectroscopic measurements of the PSI-LHCI supercomplex suggested that the bound LHCA2 and LHCA9 proteins have the ability to increase the light-harvesting energy for PSI. We conclude that the PSI in C. reinhardtii has a larger and more distinct outer-antenna organization and higher light-harvesting capability than that in land plants.

Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms.

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein.

LHCSR1-dependent fluorescence quenching is mediated by excitation energy transfer from LHCII to photosystem I in Chlamydomonas reinhardtii.

Photosynthetic organisms are frequently exposed to light intensities that surpass the photosynthetic electron transport capacity. Under these conditions, the excess absorbed energy can be transferred from excited chlorophyll in the triplet state (3Chl*) to molecular O2, which leads to the production of harmful reactive oxygen species. To avoid this photooxidative stress, photosynthetic organisms must respond to excess light. In the green alga Chlamydomonas reinhardtii, the fastest response to high light is nonphotochemical quenching, a process that allows safe dissipation of the excess energy as heat. The two proteins, UV-inducible LHCSR1 and blue light-inducible LHCSR3, appear to be responsible for this function. While the LHCSR3 protein has been intensively studied, the role of LHCSR1 has been only partially elucidated. To investigate the molecular functions of LHCSR1 in C. reinhardtii, we performed biochemical and spectroscopic experiments and found that the protein mediates excitation energy transfer from light-harvesting complexes for Photosystem II (LHCII) to Photosystem I (PSI), rather than Photosystem II, at a low pH. This altered excitation transfer allows remarkable fluorescence quenching under high light. Our findings suggest that there is a PSI-dependent photoprotection mechanism that is facilitated by LHCSR1.