Conformation of Light-Harvesting Complex II Trimer Depends upon Its Binding Site.

The light-harvesting complex II (LHCII) trimer in plants functions as a major antenna complex and a quencher to protect it from photooxidative damage. Theoretical studies on the structure of an LHCII trimer have demonstrated that excitation energy transfer between chlorophylls (Chls) in LHCII can be modulated by its exquisite conformational fluctuation. However, conformational changes depending on its binding location have not yet been investigated, even though reorganization of protein complexes occurs by physiological regulations. In this study, we investigated conformational differences in LHCII by comparing published structures of an identical LHCII trimer in the three different photosystem supercomplexes from the green alga Chlamydomonas reinhardtii. Our results revealed distinct differences in Chl configurations as well as polypeptide conformations of the LHCII trimers depending on its binding location. We propose that these configurational differences readily modulate the function of LHCII and possibly lead to a change in excitation-energy flow over the photosynthetic supercomplex.

An ancient function of PGR5 in iron delivery?

In all phototrophic organisms, the photosynthetic apparatus must be protected from light-induced damage. One important mechanism that mitigates photodamage in plants is antimycin A (AA)-sensitive cyclic electron flow (CEF), the evolution of which remains largely obscure. Here we show that proton gradient regulation 5 (PGR5), a key protein involved in AA-sensitive CEF, displays intriguing commonalities - including sequence and structural features - with a group of ferritin-like proteins. We therefore propose that PGR5 may originally have been involved in prokaryotic iron mobilization and delivery, which facilitated a primordial type of CEF as a side effect. The abandonment of the bacterioferritin system during the transformation of cyanobacterial endosymbionts into chloroplasts might have allowed PGR5 to functionally specialize in CEF.

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.

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 .

Diversification of the light-harvesting complex gene family via intra- and intergenic duplications in the coral symbiotic alga Symbiodinium.

The light-harvesting complex (LHC) is an essential component in light energy capture and transduction to facilitate downstream photosynthetic reactions in plant and algal chloroplasts. The unicellular dinoflagellate alga Symbiodinium is an endosymbiont of cnidarian animals, including corals and sea anemones, and provides carbohydrates generated through photosynthesis to host animals. Although Symbiodinium possesses a unique LHC gene family, called chlorophyll a-chlorophyll c2-peridinin protein complex (acpPC), its genome-level diversity and evolutionary trajectories have not been investigated. Here, we describe a phylogenetic analysis revealing that many of the LHCs are encoded by highly duplicated genes with multi-subunit polyprotein structures in the nuclear genome of Symbiodinium minutum. This analysis provides an extended list of the LHC gene family in a single organism, including 80 loci encoding polyproteins composed of 145 LHC subunits recovered in the phylogenetic tree. In S. minutum, 5 phylogenetic groups of the Lhcf-type gene family, which is exclusively conserved in algae harboring secondary plastids of red algal origin, were identified. Moreover, 5 groups of the Lhcr-type gene family, of which members are known to be associated with PSI in red algal plastids and secondary plastids of red algal origin, were identified. Notably, members classified within a phylogenetic group of the Lhcf-type (group F1) are highly duplicated, which may explain the presence of an unusually large number of LHC genes in this species. Some gene units were homologous to other units within single loci of the polyprotein genes, whereas intergenic homologies between separate loci were conspicuous in other cases, implying that gene unit 'shuffling' by gene conversion and/or genome rearrangement might have been a driving force for diversification. These results suggest that vigorous intra- and intergenic gene duplication events have resulted in the genomic framework of photosynthesis in coral symbiont dinoflagellate algae.

Transcriptional regulation of the stress-responsive light harvesting complex genes in Chlamydomonas reinhardtii.

Dissipating excess energy of light is critical for photosynthetic organisms to keep the photosynthetic apparatus functional and less harmful under stressful environmental conditions. In the green alga Chlamydomonas reinhardtii, efficient energy dissipation is achieved by a process called non-photochemical quenching (NPQ), in which a distinct member of light harvesting complex, LHCSR, is known to play a key role. Although it has been known that two very closely related genes (LHCSR3.1 and LHCSR3.2) encoding LHCSR3 protein and another paralogous gene LHCSR1 are present in the C. reinhardtii genome, it is unclear how these isoforms are differentiated in terms of transcriptional regulation and functionalization. Here, we show that transcripts of both of the isoforms, LHCSR3.1 and LHCSR3.2, are accumulated under high light stress. Reexamination of the genomic sequence and gene models along with survey of sequence motifs suggested that these two isoforms shared an almost identical but still distinct promoter sequence and a completely identical polypeptide sequence, with more divergent 3'-untranscribed regions. Transcriptional induction under high light condition of both isoforms was suppressed by treatment with a photosystem II inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and a calmodulin inhibitor W7. Despite a similar response to high light, the inhibitory effects of DCMU and W7 to the LHCSR1 transcript accumulation were limited compared to LHCSR3 genes. These results suggest that the transcription of LHCSR paralogs in C. reinhardtii are regulated by light signal and differentially modulated via photosynthetic electron transfer and calmodulin-mediated calcium signaling pathway(s).

Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis.

Photosynthetic light reactions establish electron flow in the chloroplast's thylakoid membranes, leading to the production of the ATP and NADPH that participate in carbon fixation. Two modes of electron flow exist-linear electron flow (LEF) from water to NADP(+) via photosystem (PS) II and PSI in series and cyclic electron flow (CEF) around PSI (ref. 2). Although CEF is essential for satisfying the varying demand for ATP, the exact molecule(s) and operational site are as yet unclear. In the green alga Chlamydomonas reinhardtii, the electron flow shifts from LEF to CEF on preferential excitation of PSII (ref. 3), which is brought about by an energy balancing mechanism between PSII and PSI (state transitions). Here, we isolated a protein supercomplex composed of PSI with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), the cytochrome b(6)f complex (Cyt bf), ferredoxin (Fd)-NADPH oxidoreductase (FNR), and the integral membrane protein PGRL1 (ref. 5) from C. reinhardtii cells under PSII-favouring conditions. Spectroscopic analyses indicated that on illumination, reducing equivalents from downstream of PSI were transferred to Cyt bf, whereas oxidised PSI was re-reduced by reducing equivalents from Cyt bf, indicating that this supercomplex is engaged in CEF (Supplementary Fig. 1). Thus, formation and dissociation of the PSI-LHCI-LHCII-FNR-Cyt bf-PGRL1 supercomplex not only controlled the energy balance of the two photosystems, but also switched the mode of photosynthetic electron flow.

Molecular remodeling of photosystem II during state transitions in Chlamydomonas reinhardtii.

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.

Identification of the mobile light-harvesting complex II polypeptides for state transitions in Chlamydomonas reinhardtii.

State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and photosystem II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II (LHCII) antenna proteins is thought to migrate from PSII to PSI, but biochemical evidence for a physical association between LHCII proteins and PSI in state 2 is weak. Here, using the green alga Chlamydomonas reinhardtii, which has a high capacity for state transitions, we report the isolation of PSI-light-harvesting complex I (LHCI) super-complexes from cells locked into state 1 and state 2. We solubilized the thylakoid membranes with a mild detergent, separated the proteins by sucrose density gradient centrifugation, and subjected gradient fractions to gel-filtration chromatography. Three LHCII polypeptides were associated with a PSI-LHCI supercomplex only in state 2; we identified them as two minor monomeric LHCII proteins (CP26 and CP29) and one previously unreported major LHCII protein type II, or LhcbM5. These three LHCII proteins, in addition to the major trimeric LHCII proteins, were phosphorylated upon transition to state 2. The corresponding phylogenetic tree indicates that among the LHCII proteins associated with PSII, these three LHCII proteins are the most similar to the LHC proteins for PSI (LHCI). Our results are important because CP26, CP29, and LhcbM5, which have been viewed as belonging solely to the PSII complex, are now postulated to shuttle between PSI and PSII during state transitions, thereby acting as docking sites for the trimeric LHCII proteins in both PSI and PSII.