Photosynthesis Research (v.107, #2)

In vitro synthesis and characterization of bacteriochlorophyll-f and its absence in bacteriochlorophyll-e producing organisms by Hitoshi Tamiaki; Jun Komada; Michio Kunieda; Kazuhiro Fukai; Taichi Yoshitomi; Jiro Harada; Tadashi Mizoguchi (133-138).
Bacteriochlorophyll(BChl)-f which has not yet been found in natural phototrophs was prepared by chemically modifying chlorophyll-b. The retention time of reverse-phase high-performance liquid chromatography of the synthetic monomeric BChl-f as well as its visible absorption and fluorescence emission spectra in a solution were identified and compared with other naturally occurring chlorophyll pigments obtained from the main light-harvesting antenna systems of green sulfur bacteria, BChls-c/d/e. Based on the above data, BChl-f was below the level of detection in three strains of green photosynthetic bacteria producing BChl-e.
Keywords: Chlorobaculum ; Chlorobium ; Chlorophyll pigment; Chlorosome; Green sulfur bacterium; High-performance liquid chromatography

The three-dimensional structure of the FMO protein from Pelodictyon phaeum and the implications for energy transfer by Chadwick R. Larson; Chenda O. Seng; Lisa Lauman; Heather J. Matthies; Jianzhong Wen; Robert E. Blankenship; James P. Allen (139-150).
The Fenna–Matthews–Olson (FMO) antenna protein from the green bacterium Pelodictyon phaeum mediates the transfer of energy from the peripheral chlorosome antenna complex to the membrane-bound reaction center. The three-dimensional structure of this protein has been solved using protein crystallography to a resolution limit of 2.0 Å, with R work and R free values of 16.6 and 19.9%, respectively. The structure is a trimer of three identical subunits related by a threefold symmetry axis. Each subunit has two beta sheets that surround 8 bacteriochlorophylls. The bacteriochlorophylls are all five-coordinated, with the axial ligand being a histidine, serine, backbone carbonyl, or bound water molecule. The arrangement of the bacteriochlorophylls is generally well conserved in comparison to other FMO structures, but differences are apparent in the interactions with the surrounding protein. In this structure the position and orientation of the eighth bacteriochlorophyll is well defined and shows differences in its location and the coordination of the central Mg compared to previous models. The implications of this structure on the ability of the FMO protein to perform energy transfer are discussed in terms of the experimental optical measurements.
Keywords: Light-harvesting complex; Green bacteria; Photosynthesis; Energy transfer; Chlorosome

In this study, we evaluated how cadmium inhibitory effect on photosystem II and I electron transport may affect light energy conversion into electron transport by photosystem II. To induce cadmium effect on the photosynthetic apparatus, we exposed Chlamydomonas reinhardtii 24 h to 0–4.62 μM Cd2+. By evaluating the half time of fluorescence transients O–J–I–P at different temperatures (20–30°C), we were able to determine the photosystem II apparent activation energies for different reduction steps of photosystem II, indicated by the O–J–I–P fluorescence transients. The decrease of the apparent activation energies for PSII electron transport was found to be strongly related to the cadmium-induced inhibition of photosynthetic electron transport. We found a strong correlation between the photosystem II apparent activation energies and photosystem II oxygen evolution rate and photosystem I activity. Different levels of cadmium inhibition at photosystem II water-splitting system and photosystem I activity showed that photosystem II apparent activation energies are strongly dependent to photosystem II donor and acceptor sides. Therefore, the oxido-reduction state of whole photosystem II and I electron transport chain affects the conversion of light energy from antenna complex to photosystem II electron transport.
Keywords: Cadmium; Photosystem II; Photosystem I; Oxygen-evolving complex; Activation energy; Chlamydomonas reinhardtii

The photosynthetic oxygen evolution characteristics were examined in both vegetative (blade) and sexual reproductive (receptacle) tissues of Sargassum henslowianum (Fucales, Phaeophyta) from the Shenao bay of Nanao Island, China, to establish the mechanism of photosynthetic acquisition of inorganic carbon (Ci) in this species. In natural seawater (pH 8.1, ca. 2.2 mM Ci), irradiance-saturated net photosynthetic rate (NPR) was greater by 25.3% in blade than receptacle, whereas dark respiratory rate (DR) was 2-fold higher in receptacle than blade. NPR at pH 8.1 was nearly saturated with the 2.2 mM Ci for both blade and receptacle. However, the values of the half-saturation constant for Ci were sharply increased at pH 9.0. NPR was significantly affected, but DR was remained unchanged, with the variation of the pH values in seawater. The data from the final pH value derived from the pH-drift experiments and the comparison between the measured and theoretically estimated photosynthetic rates suggested that both blade and receptacle were capable of acquiring HCO3 in seawater. The inhibitors experiments showed that a HCO3 dehydration mechanism mediated by external carbonic anhydrase activity occurred in both the blade and receptacle tissues of S. henslowianum. The proton buffer TRIS had no inhibitory effect on NPR at normal pH value in natural seawater (pH 8.1), but it significantly depressed NPR at pH 9.0. This suggested that proton transport occurred at the outside of the plasma membrane facilitated the operation of the carbon acquisition at pH 9.0. It was proposed that the strategy of photosynthetic carbon acquisition at higher pH would prevent the alga from the damage of over-excitation and photoinhibition in case of sunshine and calm water. We concluded that the blade and receptacle tissues of S. henslowianum have similar mechanism of acquisition of exogenous Ci from seawater to drive photosynthesis; yet they are differentiated more or less with the photosynthetic properties.
Keywords: Sargassum henslowianum ; Brown algae; Inorganic carbon acquisition; Photosynthesis; Receptacle; Marine macroalgae

Polyamines stimulate non-photochemical quenching of chlorophyll a fluorescence in Scenedesmus obliquus by Nikolaos E. Ioannidis; Liliana Sfichi-Duke; Kiriakos Kotzabasis (169-175).
Polyamines (PAs) are small metabolites that are produced and oxidized in chloroplasts with an obscure mode of action. Recently, we showed that qE is stimulated by PAs in higher plants (Nicotiana tabacum) and in genetically modified plants with elevated thylakoid-associated PAs (Ioannidis and Kotzabasis Biochim Biophys Acta 1767:1371–1382, 2007; Ioannidis et al. Biochim Biophys Acta 1787:1215–1222, 2009). Here, we investigated further their quenching properties both in vivo in green algae and in vitro is isolated LHCII. In vivo spermine up-regulates NPQ in Scenedesums obliquus about 30%. In vitro putrescine—the obligatory metabolic precursor of PAs—has a marginal quenching effect, while spermidine and spermine exhibit strong quenching abilities in isolated LHCII up to 40%. Based on available 3D models of LHCII we report a special cavity of about 600 Å3 and a near-by larger pocket in the trimeric LHCII that could be of importance for the stimulation of qE by amines.
Keywords: Energization quenching; Thylakoids; Photosystem; Amines; Protons

Light-harvesting complex 2 from the anoxygenic phototrophic purple bacterium Thermochromatium tepidum was purified and studied by steady-state absorption, fluorescence and flash photolysis spectroscopy. Steady-state absorption and fluorescence measurements show that carotenoids play a negligible role as supportive energy donors and transfer excitation to bacteriochlorophyll-a with low energy transfer efficiency of ~30%. HPLC analysis determined that the dominant carotenoids in the complex are rhodopin and spirilloxanthin. Carotenoid excited triplet state formation upon direct (carotenoid) or indirect (bacteriochlorophyll-a Qx band) excitation shows that carotenoid triplets are mostly localized on spirilloxanthin. In addition, no triplet excitation transfer between carotenoids was observed. Such specific carotenoid composition and spectroscopic results strongly suggest that this organism optimized carotenoid composition in the light-harvesting complex 2 in order to maximize photoprotective capabilities of carotenoids but subsequently drastically suppressed their supporting role in light-harvesting process.
Keywords: Flash photolysis; Triplet state lifetime; Carotenoids; Light-harvesting complex

A novel membrane based process to isolate photosystem-I membrane complex from spinach by Jianguo Liu; Mengmeng Yin; Meng Wang; Xuefang Zhang; Baosheng Ge; Shuang Liu; Jianren Lu; Zhanfeng Cui (187-193).
The isolation of photosystem-I (PS-I) from spinach has been conducted using ultrafiltration with 300 kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on PS-I activity were optimized using parameter scanning ultrafiltration. These conditions included solution pH, ionic strength, stirring speed, and permeate flux. The effects of detergent (Triton X-100 and n-dodecyl-beta-D-maltoside) concentration on time dependent activity of PS-I were also studied using an O2 electrode. Under optimized conditions, the PS-I purity obtained in the retentate was about 84% and the activity recovery was greater than 94% after ultrafiltration. To our knowledge, this is the first report of the isolation of a membrane protein using ultrafiltration alone.
Keywords: Ultrafiltration; Photosystem-I; Membrane protein; Spinach; Activity

Excitation energy transfer in the LHC-II trimer: from carotenoids to chlorophylls in space and time by Jari Martiskainen; Robertas Kananavičius; Juha Linnanto; Heli Lehtivuori; Mika Keränen; Viivi Aumanen; Nikolai Tkachenko; Jouko Korppi-Tommola (195-207).
Exciton model for description of experimentally determined excitation energy transfer from carotenoids to chlorophylls in the LHC-II trimer of spinach is presented. Such an approach allows connecting the excitonic states to the spatial structure of the complex and hence descriptions of advancements of the initially created excitations in space and time. Carotenoids were excited at 490 nm and at 500 nm and induced absorbance changes probed in the Chl Qy region to provide kinetic data that were interpreted by using the results from exciton calculations. Calculations included the 42 chlorophylls and the 12 carotenoids of the complex, Soret, Qx and Qy states of the chlorophylls, and the main absorbing S2 state of the carotenoids. According to the calculations excitation at 500 nm populates mostly a mixed Lut S2 Chl a Soret state, from where excitation is transferred to the Qx and Qy states of the Chl a’s on the stromal side. Internal conversion of the mixed state to a mixed Lut S1 and Chl a Qy state provides a channel for Lut S1 to Chl a Qy energy transfer. The results from the calculations support a picture where excitation at 490 nm populates primarily a mixed neoxanthin S2 Chl b Soret state. From this state excitation from neoxanthin is transferred to iso-energetic Chl b Soret states or via internal conversion to S1 Chl b Qy states. From the Soret states excitation proceeds via internal conversion to Qy states of Chl b's mostly on the lumenal side. A rapid Chl b to Chl a transfer and subsequent transfer to the stromal side Chl a’s and to the final state completes the process after 490 nm excitation. The interpretation is further supported by the fact that excitation energy transfer kinetics after excitation of neoxanthin at 490 nm and the Chl b Qy band at 647 nm (Linnanto et al., Photosynth Res 87:267–279, 2006) are very similar.
Keywords: Excitation energy transfer; Light-harvesting complex II; Carotenoid; Exciton calculation; Femtosecond spectroscopy; Chlorophyll; Fluorescence up-conversion

The SPAD-502 meter is a hand-held device that is widely used for the rapid, accurate and non-destructive measurement of leaf chlorophyll concentrations. It has been employed extensively in both research and agricultural applications, with a range of different plant species. However, its utility has not been fully exploited in relation to the most intensively studied model organism for plant science research, Arabidopsis thaliana. Measurements with the SPAD-502 meter produce relative SPAD meter values that are proportional to the amount of chlorophyll present in the leaf. In order to convert these values into absolute units of chlorophyll concentration, calibration curves must be derived and utilized. Here, we present calibration equations for Arabidopsis that can be used to convert SPAD values into total chlorophyll per unit leaf area (nmol/cm2; R 2 = 0.9960) or per unit fresh weight of leaf tissue (nmol/mg; R 2 = 0.9809). These relationships were derived using a series of Arabidopsis chloroplast biogenesis mutants that exhibit chlorophyll deficiencies of varying severity, and were verified by the subsequent analysis of senescent or light-stressed leaves. Our results revealed that the converted SPAD values differ from photometric measurements of solvent-extracted chlorophyll by just ~6% on average.
Keywords: Arabidopsis ; Calibration curve; Chlorophyll content; Chloroplast development; SPAD meter

Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.
Keywords: Gene inactivation; Glycogen metabolism; Glycogen phosphorylase; Homologous recombination; Natural transformation; Uracil-specific excision reagent