Kazuhiko Satoh

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (approximately 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A1 instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.

Photosystem II complex in vivo is a monomer

Photosystem II (PS II) complexes are membrane protein complexes that are composed of >20 distinct subunit proteins. Similar to many other membrane protein complexes, two PS II complexes are believed to form a homo-dimer whose molecular mass is ∼650 kDa. Contrary to this well known concept, we propose that the functional form of PS II in vivo is a monomer, based on the following observations. Deprivation of lipids caused the conversion of PS II from a monomeric form to a dimeric form. Only a monomeric PS II was detected in solubilized cyanobacterial and red algal thylakoids using blue-native polyacrylamide gel electrophoresis. Furthermore, energy transfer between PS II units, which was observed in the purified dimeric PS II, was not detected in vivo. Our proposal will lead to a re-evaluation of many crystallographic models of membrane protein complexes in terms of their oligomerization status.

Responses to desiccation stress in lichens are different from those in their photobionts.

In order to clarify the role of symbiotic association in desiccation tolerance of photosynthetic partners in lichens, responses to air-drying and hypertonic treatments in a green-algal lichen (a chlorolichen, Ramalina yasudae Räsänen) and its green algal photobiont (freshly released and cultured Trebouxia sp.) were studied. Responses to dehydration in the isolated Trebouxia sp. were different from those in the lichen, R. yasudae, i.e. (i) the PSII reaction was totally inhibited in R. yasudae when photosynthesis was completely inhibited by desiccation, but it remained partially active in isolated Trebouxia sp; (ii) dehydration-induced quenching of PSII fluorescence was less in the isolated Trebouxia sp. compared with that in R. yasudae, suggesting that a substance(s) or a mechanism(s) to dissipate absorbed light energy to heat was lost by the isolation of the photobiont; and (iii) the air-dried isolated Trebouxia sp. showed a higher sensitivity to photoinhibition than R. yasudae. These results support the idea that association of the photobionts with the mycobionts increases tolerance to photoinhibition under drying conditions.

Mechanisms to Avoid Photoinhibition in a Desiccation-Tolerant Cyanobacterium, Nostoc commune

A desiccation-tolerant cyanobacterium, Nostoc commune, shows unique responses to dehydration. These responses are: (i) loss of PSII activity in parallel with the loss of photosynthesis; (ii) loss of PSI activity; and (iii) dissipation of light energy absorbed by pigment-protein complexes. In this study, the deactivation of PSII is shown to be important in avoiding photoinhibition when the Calvin-Benson cycle is repressed by dehydration. Furthermore, our evidence suggests that dissipation of light energy absorbed by PSII blocks photoinhibition under strong light in dehydrated states.

Chloroplast-encoded polypeptide PsbT is involved in the repair of primary electron acceptor QA of photosystem II during photoinhibition in Chlamydomonas reinhardtii

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the D1/D2 heterodimer of the photosystem II (PSII) reaction center and is required for the efficient post-translational repair of photodamaged PSII. Here we addressed that role in detail in Chlamydomonas reinhardtii wild type and ΔpsbT cells by analyzing the activities of PSII, the assembly of PSII proteins, and the redox components of PSII during photoinhibition and repair. Strong illumination of cells for 15 min decreased the activities of electron transfer through PSII and QA photoreduction by 50%, and it reduced the amount of atomic manganese by 20%, but it did not affect the steady-state level of PSII proteins, photoreduction of pheophytin (pheoD1), and the amount of bound plastoquinone (QA), indicating that the decrease in PSII activity resulted mainly from inhibition of the electron transfer from pheoD1 to QA. In wild type cells, we observed parallel recovery of electron transfer activity through PSII and QA photoreduction, suggesting that the recovery of QA activity is one of the rate-limiting steps of PSII repair. In ΔpsbT cells, the repairs of electron transfer activity through PSII and of QA photoreduction activity were both impaired, but PSII protein turnover was unaffected. Moreover, about half the QA was lost from the PSII core complex during purification. Since PsbT is intimately associated with the QA-binding region on D2, we propose that this polypeptide enhances the efficient recovery of QA photoreduction by stabilizing the structure of the QA-binding region.

Two types of fucoxanthin-chlorophyll-binding proteins I tightly bound to the photosystem I core complex in marine centric diatoms

Intact fucoxanthin (Fucox)-chlorophyll (Chl)-binding protein I-photosystem I supercomplexes (FCPI-PSIs) were prepared by a newly developed simple fast procedure from centric diatoms Chaetoceros gracilis and Thalassiosira pseudonana to study the mechanism of their efficient solar energy accumulation. FCPI-PSI purified from C. gracilis contained 252 Chl a, 23 Chl c, 56 Fucox, 34 diadinoxanthin+diatoxanthin, 1 violaxanthin, 21 ß-carotene, and 2 menaquinone-4 per P700. The complex showed a high electron transfer activity at 185,000μmolmg Chl a(-1)·h(-1) to reduce methyl viologen from added cytochrome c6. We identified 14 and 21 FCP proteins in FCPI-PSI of C. gracilis and T. pseudonana, respectively, determined by N-terminal and internal amino acid sequences and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. PsaO and a red lineage Chla/b-binding-like protein (RedCAP), Thaps3:270215, were also identified. Severe detergent treatment of FCPI-PSI released FCPI-1 first, leaving the FCPI-2-PSI-core complex. FCPI-1 contained more Chl c and showed Chl a fluorescence at a shorter wavelength than FCPI-2, suggesting an excitation-energy transfer from FCPI-1 to FCPI-2 and then to the PSI core. Fluorescence emission spectra at 17K in FCPI-2 varied depending on the excitation wavelength, suggesting two independent energy transfer routes. We formulated a model of FCPI-PSI based on the biochemical assay results.

Multiple dissipation components of excess light energy in dry lichen revealed by ultrafast fluorescence study at 5 K

A time-resolved fluorescence study of living lichen thalli at 5xa0K was conducted to clarify the dynamics and mechanism of the effective dissipation of excess light energy taking place in lichen under extreme drought conditions. The decay-associated spectra obtained from the experiment at 5xa0K were characterized by a drastically sharpened spectral band which could not be resolved by experiments at higher temperatures. The present results indicated the existence of two distinct dissipation components of excess light energy in desiccated lichen; one is characterized as rapid fluorescence decay with a time constant of 27xa0ps in the far-red region that was absent in wet lichen thalli, and the other is recognized as accelerated fluorescence decay in the 685–700xa0nm spectral region. The former energy-dissipation component with extremely high quenching efficiency is most probably ascribed to the emergence of a rapid quenching state in the peripheral-antenna system of photosystem II (PS II) on desiccation. This is an extremely effective protection mechanism of PS II under desiccation, which lichens have developed to survive in the severely desiccated environments. The latter, which is less efficient at 5xa0K, might have a supplementary role and take place either in the core antenna of PS II or aggregated peripheral antenna of PS II.

Arabitol Provided by Lichenous Fungi Enhances Ability to Dissipate Excess Light Energy in a Symbiotic Green Alga under Desiccation

Lichens are drought-resistant symbiotic organisms of mycobiont fungi and photobiont green algae or cyanobacteria, and have an efficient mechanism to dissipate excess captured light energy into heat in a picosecond time range to avoid photoinhibition. This mechanism can be assessed as drought-induced non-photochemical quenching (d-NPQ) using time-resolved fluorescence spectroscopy. A green alga Trebouxia sp., which lives within a lichen Ramalina yasudae, is one of the most common green algal photobionts. This alga showed very efficient d-NPQ under desiccation within the lichen thallus, whereas it lost d-NPQ ability when isolated from R. yasudae, indicating the importance of the interaction with the mycobiont for d-NPQ ability. We analyzed the water extracts from lichen thalli that enhanced d-NPQ in Trebouxia. Of several sugar compounds identified in the water extracts by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography (GC) analyses, only d-arabitol recovered d-NPQ in isolated Trebouxia to a level similar to that detected for R. yasudae thallus. Other sugar compounds did not help the expression of d-NPQ at the same concentrations. Thus, arabitol is essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. The relationship between mycobionts and photobionts is, therefore, not commensalism, but mutualism with each other, as shown by d-NPQ expression.

COMPARATIVE STUDY ON THE PHOTOSYNTHETIC PROPERTIES OF PRASIOLA (CHLOROPHYCEAE) AND NOSTOC (CYANOPHYCEAE) FROM ANTARCTIC AND NON‐ANTARCTIC SITES1

The terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault occurs worldwide, including in Japan and on the Antarctic continent. The terrestrial green alga Prasiola crispa (Lightf.) Kütz. is also distributed in Antarctica. These two species need to acclimate to the severe Antarctic climate including low ambient temperature and desiccation under strong light conditions. To clarify this acclimation process, the physiological characteristics of the photosynthetic systems of these two Antarctic terrestrial organisms were assessed. The relative rate of photosynthetic electron flow in N. commune collected in Japan and in Antarctica reached maxima at 900 and 1,100u2003μmol photonsu2003·u2003m−2u2003·u2003s−1, respectively. The difference seemed to reflect the presence of high amounts of UV‐absorbing substances within the Antarctic cyanobacterium. On the other hand, the optimal temperatures for photosynthesis at the two locations were 30°C–35°C and 20°C–25°C, respectively. This finding suggested a decreased photosynthetic thermotolerance in the Antarctic strain. P. crispa exhibited desiccation tolerance and dehydration‐induced quenching of PSII fluorescence. Re‐reduction of the photooxidized PSI reaction center, P700, was also inhibited at fully dry states. Photosynthetic electron flow in P. crispa reached a maximum at 20°C–25°C and at a light intensity of 700u2003μmol photonsu2003ḃu2003m−2u2003ḃu2003s−1. Interestingly, the osmolarity of P. crispa cells suggested that photosynthesis is performed using water absorbed in a liquid form rather than water absorbed from the air. Overall, these data suggest that these two species have acclimated to optimally photosynthesize under conditions of the highest light intensity and the highest temperature for their habitat in Antarctica.

Evidence for a stable association of Psb30 (Ycf12) with photosystem II core complex in the cyanobacterium Synechocystis sp. PCC 6803

Ycf12 (Psb30) is a small hydrophobic subunit of photosystem II (PS II) complexes found in the cyanobacterium, Thermosynechococcus elongatus. However, earlier intense proteomic analysis on the PS II complexes from the cyanobacterium, Synechocystis 6803, could not detect Psb30. In this work, we generated a mutant of Synechocystis 6803 in which a hexa-histidine tag was fused to the C-terminus of Synechocystis Psb30. The mutant accumulated fully functional PS II complexes. Purification of Psb30 by metal affinity chromatography from thylakoid extracts resulted in co-purification of an oxygen-evolving PS II complex with normal subunit composition. This result indicates that Psb30 is expressed and stably associated with the PS II complex in Synechocystis. The histidine-tagged Psb30 in the purified PS II complex was not detected by staining or anti-polyhistidine antibodies. We also generated a mutant in which ycf12 was disrupted. The mutant grew photosynthetically and showed no significant phenotype under moderate growth conditions. Purified PS II complexes from the disruptant showed an oxygen-evolving activity comparable to wild type under low irradiance. However, it showed a remarkably lower activity than wild type under high irradiance. Thus Psb30 is required for the efficient function of PS II complexes, particularly under high irradiance conditions.