Masayuki Komura

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.

Mechanism of strong quenching of photosystem II chlorophyll fluorescence under drought stress in a lichen, Physciella melanchla, studied by subpicosecond fluorescence spectroscopy

The mechanism of the severe quenching of chlorophyll (Chl) fluorescence under drought stress was studied in a lichen Physciella melanchla, which contains a photobiont green alga, Trebouxia sp., using a streak camera and a reflection-mode fluorescence up-conversion system. We detected a large 0.31 ps rise of fluorescence at 715 and 740 nm in the dry lichen suggesting the rapid energy influx to the 715-740 nm bands from the shorter-wavelength Chls with a small contribution from the internal conversion from Soret bands. The fluorescence, then, decayed with time constants of 23 and 112 ps, suggesting the rapid dissipation into heat through the quencher. The result confirms the accelerated 40 ps decay of fluorescence reported in another lichen (Veerman et al., 2007 [36]) and gives a direct evidence for the rapid energy transfer from bulk Chls to the longer-wavelength quencher. We simulated the entire PS II fluorescence kinetics by a global analysis and estimated the 20.2 ns(-1) or 55.0 ns(-1) energy transfer rate to the quencher that is connected either to the LHC II or to the PS II core antenna. The strong quenching with the 3-12 times higher rate compared to the reported NPQ rate, suggests the operation of a new type of quenching, such as the extreme case of Chl-aggregation in LHCII or a new type of quenching in PS II core antenna in dry lichens.

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.

Shallow sink in an antenna pigment system of photosystem I of a marine centric diatom, Chaetoceros gracilis, revealed by ultrafast fluorescence spectroscopy at 17 K.

The ultrafast fluorescence dynamics of photosystem I (PS I) purified from a marine centric diatom, Chaetoceros gracilis, at 17 K was studied using fluorescence up-conversion and streak-camera setups. The experiments were done for two kinds of sample preparations containing different amounts of the peripheral antenna proteins, the fucoxanthin-chlorophyll (Chl) binding proteins associated with PS I (FCPI). Upon excitation at 430 nm, which selectively excites Chl a mainly contained in the core complex, the fluorescence dynamics of both samples was roughly expressed by four decay-associated spectra (DASs) with time constants of ca. 5, ca. 22, ca. 100, and ca. 400 ps. These DAS components have corresponding counterparts in the results of a previous study of Thermosynechococcus elongatus PS I (Shibata et al. J. Phys. Chem. B 2010, 114, 2954) except for that with a time constant of ca. 22 ps. The similar distribution of the time constants suggests a shared light-harvesting pathway by PS I of these two organisms. The DAS with a ca. 400 ps time constant has its peak wavelength at around 710 nm, suggesting the presence of antenna pigment states with slightly lower excitation energy than that of P700. This antenna state acts as a shallow sink in the core complex of the diatom PS I and causes a specific temperature dependence of its fluorescence spectrum below 77 K. Excitation energy funneling into the shallow-sink state seems to take place within 0.2 ps, suggesting an extremely efficient energy transfer. Upon the selective excitation of Chl c in FCPI by a 460 nm laser, three DAS components suggesting excitation energy transfers were obtained. The 0.2 ps DAS shows the energy transfer from Chl c to Chl a within FCPI, while the 0.7 and 40 ps DASs suggest the energy transfer from FCPI to the core complex. The excitation energy seems to be effectively transferred from FCPI to the core complex in diatom PS I because the selective excitation of Chl c in FCPI does not induce a severe retardation of the overall light-harvesting kinetics.

Drought-Induced Ultra-Fast Fluorescence Quenching in Photosystem II in Lichens Revealed by Picosecond Time-Resolved Fluorescence Spectrophotometry

Lichens survive under the extreme drought environments. It has been suggested that dried lichens convert excess light energy into heat by unknown mechanism to prevent the accumulation of harmful photoproducts. We studied 18 lichen species by their steady-state fluorescence spectra, PAM and picosecond time-resolved fluorescence decay profiles at 4–300 K. Quantitative analyses of the decay profiles were applied. We obtained the following results: (1) All dried lichens showed a low intensity of PS II fluorescence; (2) the picosecond decays of PS II fluorescence were fast (<10 ps) in most dry lichens; (3) the excitation energy transfer from LHC II to CP43/CP47 was still active; (4) the lifetime of the PS I fluorescence was little affected; (5) the changes were fully reversed within 1 min after the re-hydration; and (6) some lichens showed no fast decay of PS II fluorescence. We noticed two different types of drought-induced energy dissipation mechanisms: Most of lichens dissipated almost all the excitation energy in a few picoseconds by an unknown quencher; some lichens decreased the antenna size of PS II by the state transition mechanism. The new type of the quencher found in this study seems to be situated in the core antenna, and is different from the well-known non-photochemical quenching mechanism.