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.

Formation of a Stable PSI-PSII Megacomplex in Rice That Conducts Energy Spillover.

In green plants, photosystem I (PSI) and photosystem II (PSII) bind to their respective light-harvesting complexes (LHCI and LHCII) to form the PSI-LHCI supercomplex and the PSII-LHCII supercomplex, respectively. These supercomplexes further form megacomplexes, like PSI-PSII and PSII-PSII in Arabidopsis (Arabidopsis thaliana) and spinach to modulate their light-harvesting properties, but not in the green alga Chlamydomonas reinhardtii. Here, we fractionated and characterized the stable rice PSI-PSII megacomplex. The delayed fluorescence from PSI (lifetime ∼25 ns) indicated energy transfer capabilities between the two photosystems (energy spillover) in the rice PSI-PSII megacomplex. Fluorescence lifetime analysis revealed that the slow PSII to PSI energy transfer component was more dominant in the rice PSI-PSII supercomplexes than in Arabidopsis ones, suggesting that PSI and PSII in rice form a megacomplex not directly but through LHCII molecule(s), which was further confirmed by the negatively stained electron microscopy analysis. Our results suggest species diversity in the formation and stability of photosystem megacomplexes, and the stable PSI-PSII supercomplex in rice may reflect its structural adaptation.

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.

A blue-light photoreceptor mediates the feedback regulation of photosynthesis.

In plants and algae, light serves both as the energy source for photosynthesis and a biological signal that triggers cellular responses via specific sensory photoreceptors. Red light is perceived by bilin-containing phytochromes and blue light by the flavin-containing cryptochromes and/or phototropins (PHOTs), the latter containing two photosensory light, oxygen, or voltage (LOV) domains. Photoperception spans several orders of light intensity, ranging from far below the threshold for photosynthesis to values beyond the capacity of photosynthetic CO2 assimilation. Excess light may cause oxidative damage and cell death, processes prevented by enhanced thermal dissipation via high-energy quenching (qE), a key photoprotective response. Here we show the existence of a molecular link between photoreception, photosynthesis, and photoprotection in the green alga Chlamydomonas reinhardtii. We show that PHOT controls qE by inducing the expression of the qE effector protein LHCSR3 (light-harvesting complex stress-related protein 3) in high light intensities. This control requires blue-light perception by LOV domains on PHOT, LHCSR3 induction through PHOT kinase, and light dissipation in photosystem II via LHCSR3. Mutants deficient in the PHOT gene display severely reduced fitness under excessive light conditions, indicating that the sensing, utilization, and dissipation of light is a concerted process that plays a vital role in microalgal acclimation to environments of variable light intensities.

Green fluorescence from cnidarian hosts attracts symbiotic algae.

Reef-building corals thrive in nutrient-poor marine environments because of an obligate symbiosis with photosynthetic dinoflagellates of the genus Symbiodinium Symbiosis is established in most corals through the uptake of Symbiodinium from the environment. Corals are sessile for most of their life history, whereas free-living Symbiodinium are motile; hence, a mechanism to attract Symbiodinium would greatly increase the probability of encounter between host and symbiont. Here, we examined whether corals can attract free-living motile Symbiodinium by their green fluorescence, emitted by the excitation of endogenous GFP by purple-blue light. We found that Symbiodinium have positive and negative phototaxis toward weak green and strong purple-blue light, respectively. Under light conditions that cause corals to emit green fluorescence, (e.g., strong blue light), Symbiodinium were attracted toward live coral fragments. Symbiodinium were also attracted toward an artificial green fluorescence dye with similar excitation and emission spectra to coral-GFP. In the field, more Symbiodinium were found in traps painted with a green fluorescence dye than in controls. Our results revealed a biological signaling mechanism between the coral host and its potential symbionts.

Multimeric and monomeric photosystem II supercomplexes represent structural adaptations to low- and high-light conditions.

An intriguing molecular architecture called the "semi-crystalline photosystem II (PSII) array" has been observed in the thylakoid membranes in vascular plants. It is an array of PSII-light-harvesting complex II (LHCII) supercomplexes that only appears in low light, but its functional role has not been clarified. Here, we identified PSII-LHCII supercomplexes in their monomeric and multimeric forms in low light-acclimated spinach leaves and prepared them using sucrose-density gradient ultracentrifugation in the presence of amphipol A8-35. When the leaves were acclimated to high light, only the monomeric forms were present, suggesting that the multimeric forms represent a structural adaptation to low light and that disaggregation of the PSII-LHCII supercomplex represents an adaptation to high light. Single-particle EM revealed that the multimeric PSII-LHCII supercomplexes are composed of two ("megacomplex") or three ("arraycomplex") units of PSII-LHCII supercomplexes, which likely constitute a fraction of the semi-crystalline PSII array. Further characterization with fluorescence analysis revealed that multimeric forms have a higher light-harvesting capability but a lower thermal dissipation capability than the monomeric form. These findings suggest that the configurational conversion of PSII-LHCII supercomplexes may serve as a structural basis for acclimation of plants to environmental light.

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching.

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

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.

Plant and Algal PSII-LHCII Supercomplexes: Structure, Evolution and Energy Transfer.

Photosynthesis is the process conducted by plants and algae to capture photons and store their energy in chemical forms. The light-harvesting, excitation transfer, charge separation and electron transfer in photosystem II (PSII) are the critical initial reactions of photosynthesis and thereby largely determine its overall efficiency. In this review, we outline the rapidly accumulating knowledge about the architectures and assemblies of plant and green algal PSII-light harvesting complex II (LHCII) supercomplexes, with a particular focus on new insights provided by the recent high-resolution cryo-electron microscopy map of the supercomplexes from a green alga Chlamydomonas reinhardtii. We make pair-wise comparative analyses between the supercomplexes from plants and green algae to gain insights about the evolution of the PSII-LHCII supercomplexes involving the peripheral small PSII subunits that might have been acquired during the evolution and about the energy transfer pathways that define their light-harvesting and photoprotective properties.

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.

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.

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.

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.

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.

Structural characterization of the photosystems in the green alga Chlorella sorokiniana.

MAIN CONCLUSION: The supramolecular organization of the photosystem supercomplexes in the green alga Chlorella sorokiniana belonging to Trebouxiophyceae are essentially the same as those of Chlamydomonas reinhardtii belonging to Chlorophyceae. The photosynthetic conversion of light energy into chemical energy is performed by photosystems II and I (PSII and PSI) embedded within the thylakoid membranes. In plants and green algae, PSII and PSI comprise the core complex and light-harvesting complexes (LHCII and LHCI), forming PSII-LHCII and PSI-LHCI supercomplexes, respectively. The structural information about photosystem supercomplexes of green algae has been limited to chlorophytic algae. Here, to obtain an insight into the evolution of Chlorophyta, we determined the supramolecular organization of the PSII-LHCII and PSI-LHCI supercomplexes from the freshwater green alga Chlorella sorokiniana, which belongs to Trebouxiophyceae. The obtained results showed that the supramolecular organizations of the photosystem supercomplexes in C. sorokiniana were essentially the same as those of the model green alga C. reinhardtii, which belongs to Chlorophyceae, namely PSII-LHCII supercomplex formed the C2S2M2L2 configuration and PSI-LHCI supercomplex was associated with 10 LHCI subunits.

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.

Chloroplast-mediated regulation of CO2-concentrating mechanism by Ca2+-binding protein CAS in the green alga Chlamydomonas reinhardtii.

Aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii, induce a CO2-concentrating mechanism (CCM) to maintain photosynthetic activity in CO2-limiting conditions by sensing environmental CO2 and light availability. Previously, a novel high-CO2-requiring mutant, H82, defective in the induction of the CCM, was isolated. A homolog of calcium (Ca2+)-binding protein CAS, originally found in Arabidopsis thaliana, was disrupted in H82 cells. Although Arabidopsis CAS is reported to be associated with stomatal closure or immune responses via a chloroplast-mediated retrograde signal, the relationship between a Ca2+ signal and the CCM associated with the function of CAS in an aquatic environment is still unclear. In this study, the introduction of an intact CAS gene into H82 cells restored photosynthetic affinity for inorganic carbon, and RNA-seq analyses revealed that CAS could function in maintaining the expression levels of nuclear-encoded CO2-limiting-inducible genes, including the HCO3- transporters high-light activated 3 (HLA3) and low-CO2-inducible gene A (LCIA). CAS changed its localization from dispersed across the thylakoid membrane in high-CO2 conditions or in the dark to being associated with tubule-like structures in the pyrenoid in CO2-limiting conditions, along with a significant increase of the fluorescent signals of the Ca2+ indicator in the pyrenoid. Chlamydomonas CAS had Ca2+-binding activity, and the perturbation of intracellular Ca2+ homeostasis by a Ca2+-chelator or calmodulin antagonist impaired the accumulation of HLA3 and LCIA. These results suggest that Chlamydomonas CAS is a Ca2+-mediated regulator of CCM-related genes via a retrograde signal from the pyrenoid in the chloroplast to the nucleus.

Eyespot-dependent determination of the phototactic sign in Chlamydomonas reinhardtii.

The biflagellate green alga Chlamydomonas reinhardtii exhibits both positive and negative phototaxis to inhabit areas with proper light conditions. It has been shown that treatment of cells with reactive oxygen species (ROS) reagents biases the phototactic sign to positive, whereas that with ROS scavengers biases it to negative. Taking advantage of this property, we isolated a mutant, lts1-211, which displays a reduction-oxidation (redox) dependent phototactic sign opposite to that of the wild type. This mutant has a single amino acid substitution in phytoene synthase, an enzyme that functions in the carotenoid-biosynthesis pathway. The eyespot contains large amounts of carotenoids and is crucial for phototaxis. Most lts1-211 cells have no detectable eyespot and reduced carotenoid levels. Interestingly, the reversed phototactic-sign phenotype of lts1-211 is shared by other eyespot-less mutants. In addition, we directly showed that the cell body acts as a convex lens. The lens effect of the cell body condenses the light coming from the rear onto the photoreceptor in the absence of carotenoid layers, which can account for the reversed-phototactic-sign phenotype of the mutants. These results suggest that light-shielding property of the eyespot is essential for determination of phototactic sign.

Fluorescence lifetime analyses reveal how the high light-responsive protein LHCSR3 transforms PSII light-harvesting complexes into an energy-dissipative state.

In green algae, light-harvesting complex stress-related 3 (LHCSR3) is responsible for the pH-dependent dissipation of absorbed light energy, a function vital for survival under high-light conditions. LHCSR3 binds the photosystem II and light-harvesting complex II (PSII-LHCII) supercomplex and transforms it into an energy-dissipative form under acidic conditions, but the molecular mechanism remains unclear. Here we show that in the green alga Chlamydomonas reinhardtii, LHCSR3 modulates the excitation energy flow and dissipates the excitation energy within the light-harvesting complexes of the PSII supercomplex. Using fluorescence decay-associated spectra analysis, we found that, when the PSII supercomplex is associated with LHCSR3 under high-light conditions, excitation energy transfer from light-harvesting complexes to chlorophyll-binding protein CP43 is selectively inhibited compared with that to CP47, preventing excess excitation energy from overloading the reaction center. By analyzing femtosecond up-conversion fluorescence kinetics, we further found that pH- and LHCSR3-dependent quenching of the PSII-LHCII-LHCSR3 supercomplex is accompanied by a fluorescence emission centered at 684 nm, with a decay time constant of 18.6 ps, which is equivalent to the rise time constant of the lutein radical cation generated within a chlorophyll-lutein heterodimer. These results suggest a mechanism in which LHCSR3 transforms the PSII supercomplex into an energy-dissipative state and provide critical insight into the molecular events and characteristics in LHCSR3-dependent energy quenching.