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.

Circadian rhythm response and its effect on photosynthetic characteristics of the Lhcb family genes in tea plant.

BACKGROUND: The circadian clock, also known as the circadian rhythm, is responsible for predicting daily and seasonal changes in the environment, and adjusting various physiological and developmental processes to the appropriate times during plant growth and development. The circadian clock controls the expression of the Lhcb gene, which encodes the chlorophyll a/b binding protein. However, the roles of the Lhcb gene in tea plant remain unclear. RESULTS: In this study, a total of 16 CsLhcb genes were identified based on the tea plant genome, which were distributed on 8 chromosomes of the tea plant. The promoter regions of CsLhcb genes have a variety of cis-acting elements including hormonal, abiotic stress responses and light response elements. The CsLhcb family genes are involved in the light response process in tea plant. The photosynthetic parameter of tea leaves showed rhythmic changes during the two photoperiod periods (48 h). Stomata are basically open during the day and closed at night. Real-time quantitative PCR results showed that most of the CsLhcb family genes were highly expressed during the day, but were less expressed at night. CONCLUSIONS: Results indicated that CsLhcb genes were involved in the circadian clock process of tea plant, it also provided potential references for further understanding of the function of CsLhcb gene family in tea plant.

Spectral Tuning and Excitation-Energy Transfer by Unique Carotenoids in Diatom Light-Harvesting Antenna.

The light-harvesting antennae of diatoms and spinach are composed of similar chromophores; however, they exhibit different absorption wavelengths. Recent advances in cryoelectron microscopy have revealed that the diatom light-harvesting antenna fucoxanthin chlorophyll a/c-binding protein (FCPII) forms a tetramer and differs from the spinach antenna in terms of the number of protomers; however, the detailed molecular mechanism remains elusive. Herein, we report the physicochemical factors contributing to the characteristic light absorption of the diatom light-harvesting antenna based on spectral calculations using an exciton model. Spectral analysis reveals the significant contribution of unique fucoxanthin molecules (fucoxanthin-S) in FCPII to the diatom-specific spectrum, and further analysis determines their essential role in excitation-energy transfer to chlorophyll. It was revealed that the specificity of these fucoxanthin-S molecules is caused by the proximity between protomers associated with the tetramerization of FCPII. The findings of this study demonstrate that diatoms employ fucoxanthin-S to harvest energy under the ocean in the absence of long-wavelength sunlight and can provide significant information about the survival strategies of photosynthetic organisms to adjust to their living environment.

Structure of a diatom photosystem II supercomplex containing a member of Lhcx family and dimeric FCPII.

Diatoms rely on fucoxanthin chlorophyll a/c-binding proteins (FCPs) for their great success in oceans, which have a great diversity in their pigment, protein compositions, and subunit organizations. We report a unique structure of photosystem II (PSII)-FCPII supercomplex from Thalassiosira pseudonana at 2.68-Å resolution by cryo-electron microscopy. FCPIIs within this PSII-FCPII supercomplex exist in dimers and monomers, and a homodimer and a heterodimer were found to bind to a PSII core. The FCPII homodimer is formed by Lhcf7 and associates with PSII through an Lhcx family antenna Lhcx6_1, whereas the heterodimer is formed by Lhcf6 and Lhcf11 and connects to the core together with an Lhcf5 monomer through Lhca2 monomer. An extended pigment network consisting of diatoxanthins, diadinoxanthins, fucoxanthins, and chlorophylls a/c is revealed, which functions in efficient light harvesting, energy transfer, and dissipation. These results provide a structural basis for revealing the energy transfer and dissipation mechanisms and also for the structural diversity of FCP antennas in diatoms.

The location of the low-energy states in Lhca1 favors excitation energy transfer to the core in the plant PSI-LHCI supercomplex.

Lhca1 is one of the four pigment-protein complexes composing the outer antenna of plant Photosystem I-light-havesting I supercomplex (PSI-LHCI). It forms a functional dimer with Lhca4 but, differently from this complex, it does not contain 'red-forms,' i.e., pigments absorbing above 700 nm. Interestingly, the recent PSI-LHCI structures suggest that Lhca1 is the main point of delivering the energy harvested by the antenna to the core. To identify the excitation energy pathways in Lhca1, we developed a structure-based exciton model based on the simultaneous fit of the low-temperature absorption, linear dichroism, and fluorescence spectra of wild-type Lhca1 and two mutants, lacking chlorophylls contributing to the long-wavelength region of the absorption. The model enables us to define the locations of the lowest energy pigments in Lhca1 and estimate pathways and timescales of energy transfer within the complex and to the PSI core. We found that Lhca1 has a particular energy landscape with an unusual (compared to Lhca4, LHCII, and CP29) configuration of the low-energy states. Remarkably, these states are located near the core, facilitating direct energy transfer to it. Moreover, the low-energy states of Lhca1 are also coupled to the red-most state (red forms) of the neighboring Lhca4 antenna, providing a pathway for effective excitation energy transfer from Lhca4 to the core.

Isolation of fucoxanthin chlorophyll protein complexes of the centric diatom Thalassiosira pseudonana associated with the xanthophyll cycle enzyme diadinoxanthin de-epoxidase.

In the present study, low concentrations of the very mild detergent n-dodecyl-α-d-maltoside in conjunction with sucrose gradient ultracentrifugation were used to prepare fucoxanthin chlorophyll protein (FCP) complexes of the centric diatom Thalassiosira pseudonana. Two main FCP fractions were observed in the sucrose gradients, one in the upper part and one at high sucrose concentrations in the lower part of the gradient. The first fraction was dominated by the 18 kDa FCP protein band in SDS-gels. Since this fraction also contained other protein bands, it was designated as fraction enriched in FCP-A complex. The second fraction contained mainly the 21 kDa FCP band, which is typical for the FCP-B complex. Determination of the lipid composition showed that both FCP fractions contained monogalactosyl diacylglycerol as the main lipid followed by the second galactolipid of the thylakoid membrane, namely digalactosyl diacylglycerol. The negatively charged lipids sulfoquinovosyl diacylglycerol and phosphatidyl glycerol were also present in both fractions in pronounced concentrations. With respect to the pigment composition, the fraction enriched in FCP-A contained a higher amount of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt), whereas the FCP-B fraction was characterized by a lower ratio of xanthophyll cycle pigments to the light-harvesting pigment fucoxanthin. Protein analysis by mass spectrometry revealed that in both FCP fractions the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (DDE) was present. In addition, the analysis showed an enrichment of DDE in the fraction enriched in FCP-A but only a very low amount of DDE in the FCP-B fraction. In-vitro de-epoxidation assays, employing the isolated FCP complexes, were characterized by an inefficient conversion of DD to Dt. However, in line with the heterogeneous DDE distribution, the fraction enriched in FCP-A showed a more pronounced DD de-epoxidation compared with the FCP-B.

Structure-based model of fucoxanthin-chlorophyll protein complex: Calculations of chlorophyll electronic couplings.

Diatoms are a group of marine algae that are responsible for a significant part of global oxygen production. Adapted to life in an aqueous environment dominated by the blue-green light, their major light-harvesting antennae-fucoxanthin-chlorophyll protein complexes (FCPs)-exhibit different pigment compositions than of plants. Despite extensive experimental studies, until recently the theoretical description of excitation energy dynamics in these complexes was limited by the lack of high-resolution structural data. In this work, we use the recently resolved crystallographic information of the FCP complex from Phaeodactylum tricornutum diatom [Wang et al., Science 363, 6427 (2019)] and quantum chemistry-based calculations to evaluate the chlorophyll transition dipole moments, atomic transition charges from electrostatic potential, and the inter-chlorophyll couplings in this complex. The obtained structure-based excitonic couplings form the foundation for any modeling of stationary or time-resolved spectroscopic data. We also calculate the inter-pigment Förster energy transfer rates and identify two quickly equilibrating chlorophyll clusters.

Lipid-Enhanced Photoprotection of LHCII in Membrane Nanodisc by Reducing Chlorophyll Triplet Production.

Carotenoid (Car) quenching chlorophyll triplet state (3Chl a*), an unwanted photosensitizer yielding harmful reactive oxygen species, is crucial for the survival of oxygenic photosynthetic organisms. For the major light-harvesting complex of photosystem II (LHCII) in isolated form, 3Chl a* is deactivated via sub-nanosecond Chl-to-Car triplet excitation energy transfer by lutein in the central domain of LHCII; however, the mechanistic difference from LHCII in vivo remains to be explored. To investigate the intrinsic Car-photoprotection properties of LHCII in a bio-mimicking circumstance, we reconstituted trimeric spinach LHCII into the discoidal membrane of nanosize made from l-α-phosphatidylcholine and examined the triplet excited dynamics. Time-resolved optical absorption combined with circular dichroism spectroscopies revealed that, with reference to LHCII in buffer, LHCII in the membrane nanodisc shows appreciable conformational variation in the neoxanthin and the Lut621 domains and in the Chl a-terminal cluster owing to the lipid-protein interactions, which, in turn, alters the triplet population of Lut620 and Lut621 and their partition. Importantly, the unquenched 3Chl a* population in the complex was reduced by 60%, indicating that LHCII in the membrane adopts a conformation that is optimized for the alleviation of photoinhibition.

Molecular phylogeny of fucoxanthin-chlorophyll a/c proteins from Chaetoceros gracilis and Lhcq/Lhcf diversity.

Diatoms adapt to various aquatic light environments and play major roles in the global carbon cycle using their unique light-harvesting system, i.e. fucoxanthin chlorophyll a/c binding proteins (FCPs). Structural analyses of photosystem II (PSII)-FCPII and photosystem I (PSI)-FCPI complexes from the diatom Chaetoceros gracilis have revealed the localization and interactions of many FCPs; however, the entire set of FCPs has not been characterized. Here, we identify 46 FCPs in the newly assembled genome and transcriptome of C. gracilis. Phylogenetic analyses suggest that these FCPs can be classified into five subfamilies: Lhcr, Lhcf, Lhcx, Lhcz, and the novel Lhcq, in addition to a distinct type of Lhcr, CgLhcr9. The FCPs in Lhcr, including CgLhcr9 and some Lhcqs, have orthologous proteins in other diatoms, particularly those found in the PSI-FCPI structure. By contrast, the Lhcf subfamily, some of which were found in the PSII-FCPII complex, seems to be diversified in each diatom species, and the number of Lhcqs differs among species, indicating that their diversification may contribute to species-specific adaptations to light. Further phylogenetic analyses of FCPs/light-harvesting complex (LHC) proteins using genome data and assembled transcriptomes of other diatoms and microalgae in public databases suggest that our proposed classification of FCPs is common among various red-lineage algae derived from secondary endosymbiosis of red algae, including Haptophyta. These results provide insights into the loss and gain of FCP/LHC subfamilies during the evolutionary history of the red algal lineage.

Towards spruce-type photosystem II: consequences of the loss of light-harvesting proteins LHCB3 and LHCB6 in Arabidopsis.

The largest stable photosystem II (PSII) supercomplex in land plants (C2S2M2) consists of a core complex dimer (C2), two strongly (S2) and two moderately (M2) bound light-harvesting protein (LHCB) trimers attached to C2 via monomeric antenna proteins LHCB4-6. Recently, we have shown that LHCB3 and LHCB6, presumably essential for land plants, are missing in Norway spruce (Picea abies), which results in a unique structure of its C2S2M2 supercomplex. Here, we performed structure-function characterization of PSII supercomplexes in Arabidopsis (Arabidopsis thaliana) mutants lhcb3, lhcb6, and lhcb3 lhcb6 to examine the possibility of the formation of the "spruce-type" PSII supercomplex in angiosperms. Unlike in spruce, in Arabidopsis both LHCB3 and LHCB6 are necessary for stable binding of the M trimer to PSII core. The "spruce-type" PSII supercomplex was observed with low abundance only in the lhcb3 plants and its formation did not require the presence of LHCB4.3, the only LHCB4-type protein in spruce. Electron microscopy analysis of grana membranes revealed that the majority of PSII in lhcb6 and namely in lhcb3 lhcb6 mutants were arranged into C2S2 semi-crystalline arrays, some of which appeared to structurally restrict plastoquinone diffusion. Mutants without LHCB6 were characterized by fast induction of non-photochemical quenching and, on the contrary to the previous lhcb6 study, by only transient slowdown of electron transport between PSII and PSI. We hypothesize that these functional changes, associated with the arrangement of PSII into C2S2 arrays in thylakoids, may be important for the photoprotection of both PSI and PSII upon abrupt high-light exposure.

Robust Strategy for Photoprotection in the Light-Harvesting Antenna of Diatoms: A Molecular Dynamics Study.

Diatoms generate a large portion of the oxygen produced on earth due to their exceptional light-harvesting properties involving fucoxanthin and chlorophyll-binding proteins (FCP). At the same time, an efficient adaptation of these complexes to fluctuating light conditions is necessary to protect the diatoms against photodamage. So far, structural and dynamic data for the interaction between FCP and the photoprotective LHCX family of proteins in diatoms are lacking. In this computational study, we provide a structural basis for a remarkable pH-dependent adaptation at the molecular level. Upon binding of the LHCX1 protein to the FCP complex together with a change in pH, conformational changes within the FCP protein result in a variation of the electronic coupling in a specific chlorophyll-fucoxanthin pair, leading to a change in the exciton transfer rate by almost an order of magnitude. A common strategy for photoprotection between diatoms and higher plants is identified and discussed.

Identification of chlorophyll a-b binding protein AB96 as a novel TGFß1 neutralizing agent.

The discovery of compounds and proteins from plants has greatly contributed to modern medicine. Vernonia amygdalina Del. (Compositae) is used by humans and primates for a variety of conditions including parasitic infection. This paper describes the serendipitous discovery that V. amygdalina extract was able to bind to, and functionally inhibit, active TGFß1. The binding agent was isolated and identified as chlorophyll a-b binding protein AB96. Given that active TGFß1 contributes to the pathology of many infectious diseases, inhibiting these processes may explain some of the benefits associated with the ingestion of this species. This is the first plant-derived cytokine-neutralizing protein to be described and paves the way for further such discoveries.

Role of an ancient light-harvesting protein of PSI in light absorption and photoprotection.

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

A distinctive pathway for triplet-triplet energy transfer photoprotection in fucoxanthin chlorophyll-binding proteins from Cyclotella meneghiniana.

Fucoxanthin chlorophyll-binding proteins (FCPs) are the major light-harvesting complexes of diatoms. In this work, FCPs isolated from Cyclotella meneghiniana have been studied by means of optically detected magnetic resonance (ODMR) and time-resolved electron paramagnetic resonance (TR-EPR), with the aim to characterize the photoprotective mechanism based on triplet-triplet energy transfer (TTET). The spectroscopic properties of the chromophores carrying the triplet state have been interpreted on the basis of a delved analysis of the recently solved crystallographic structures of FCP. The results point toward a photoprotective role for two fucoxanthin molecules exposed to the exterior of the FCP monomers. This shows that FCP has adopted a structural strategy different from that of related light-harvesting complexes from plants and other microalgae, in which the photoprotective role is carried out by two highly conserved carotenoids in the interior of the complex.

Enhancement of excitation-energy quenching in fucoxanthin chlorophyll a/c-binding proteins isolated from a diatom Phaeodactylum tricornutum upon excess-light illumination.

Photosynthetic organisms regulate pigment composition and molecular oligomerization of light-harvesting complexes in response to solar light intensities, in order to improve light-harvesting efficiency. Here we report excitation-energy dynamics and relaxation of fucoxanthin chlorophyll a/c-binding protein (FCP) complexes isolated from a diatom Phaeodactylum tricornutum grown under high-light (HL) illumination. Two types of FCP complexes were prepared from this diatom under the HL condition, whereas one FCP complex was isolated from the cells grown under a low-light (LL) condition. The subunit composition and oligomeric states of FCP complexes under the HL condition are different from those under the LL condition. Absorption and fluorescence spectra at 77 K of the FCP complexes also vary between the two conditions, indicating modifications of the pigment composition and arrangement upon the HL illumination. Time-resolved fluorescence curves at 77 K of the FCP complexes under the HL condition showed shorter lifetime components compared with the LL condition. Fluorescence decay-associated spectra at 77 K showed distinct excitation-energy-quenching components and alterations of energy-transfer pathways in the FCP complexes under the HL condition. These findings provide insights into molecular and functional mechanisms of the dynamic regulation of FCPs in this diatom under excess-light conditions.

Pre-purification of diatom pigment protein complexes provides insight into the heterogeneity of FCP complexes.

BACKGROUND: Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants. RESULTS: In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments. CONCLUSION: The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.

Light-induced psbA translation in plants is triggered by photosystem II damage via an assembly-linked autoregulatory circuit.

The D1 reaction center protein of photosystem II (PSII) is subject to light-induced damage. Degradation of damaged D1 and its replacement by nascent D1 are at the heart of a PSII repair cycle, without which photosynthesis is inhibited. In mature plant chloroplasts, light stimulates the recruitment of ribosomes specifically to psbA mRNA to provide nascent D1 for PSII repair and also triggers a global increase in translation elongation rate. The light-induced signals that initiate these responses are unclear. We present action spectrum and genetic data indicating that the light-induced recruitment of ribosomes to psbA mRNA is triggered by D1 photodamage, whereas the global stimulation of translation elongation is triggered by photosynthetic electron transport. Furthermore, mutants lacking HCF136, which mediates an early step in D1 assembly, exhibit constitutively high psbA ribosome occupancy in the dark and differ in this way from mutants lacking PSII for other reasons. These results, together with the recent elucidation of a thylakoid membrane complex that functions in PSII assembly, PSII repair, and psbA translation, suggest an autoregulatory mechanism in which the light-induced degradation of D1 relieves repressive interactions between D1 and translational activators in the complex. We suggest that the presence of D1 in this complex coordinates D1 synthesis with the need for nascent D1 during both PSII biogenesis and PSII repair in plant chloroplasts.

Acidic pH-Induced Modification of Energy Transfer in Diatom Fucoxanthin Chlorophyll a/c-Binding Proteins.

pH influences excitation-energy-relaxation processes in photosynthetic light-harvesting complexes. Here, we report the excitation-energy dynamics by pH changes in fucoxanthin chlorophyll a/c-binding proteins (FCPs) isolated from a diatom Phaeodactylum tricornutum, probed by time-resolved fluorescence spectroscopy at 77 K. The fluorescence curve measured at pH 5.0 showed a shorter lifetime component than that measured at pH 6.5 and 8.0. The rapid decay component at pH 5.0 is supported by fluorescence decay-associated (FDA) spectra, where strong fluorescence decays relative to fluorescence rises appear in the pH-5.0 FDA spectrum with 70 ps. These results indicate that the diatom FCPs switch their function from light-harvesting to energy-quenching via arrangements of the energy-transfer pathways under acidic pHs. Based on the crystal structure of the diatom FCPs, we propose a model for the energy-quenching machinery through structural changes of the pigment environments, thus providing insights into the pH-dependent light-harvesting strategy in the diatom FCPs.

Cloning and characterization of two chlorophyll A/B binding protein genes and analysis of their gene family in Camellia sinensis.

In this study, two chlorophyll A/B binding protein (CAB) genes (CsCP1 and CsCP2) in tea plant were cloned. The proteins encoded by these genes belong to the external or internal antenna proteins of PS II, respectively. They may be the targets of physiological regulation for tea leaf cell PS II because they all contain multiple functional domains and modifiable sites. The CAB gene family in the tea genome consists of 25 homologous genes. We measured the expression patterns of ten genes in the CsCP1 and CsCP2 subfamily under six different stresses. CsCP1 expression was inhibited in response to 6 kinds of stress; CsCP2 expression was slightly upregulated only after cold stress and ABA treatment. However, the expression levels of CSA016997 and CSA030476 were upregulated significantly in the six stresses. The results suggested that the 10 CAB genes may have different functions in tea leaves. Moreover, changes in the expression of the 10 genes under stress appear to be related to ABA- and MeJA-dependent signalling pathways, and their responses to MeJA treatment is faster than those to ABA. In addition, we introduced our experiences for cloning the genes in the context of complex genomes.