Photosynthesis Research (v.102, #1)

I provide here a personal perspective on (i) the awards that were given to young investigators at the 2008 and 2009 Gordon Research Conferences on Biochemistry/Biophysics of Photosynthesis; and (ii) the ambiance at these conferences through some photographs, particularly of the 2009 conference.
Keywords: Ana Andrea Arteni; Libai Huang; André Klauss; Gary F. Moore; Tim Schulte; Jianzhong Wen

Photosystem II (PSII), the light-absorbing complex of photosynthesis that evolves oxygen, requires chloride for activation of the oxygen evolving complex (OEC). In this study, fluoride was characterized as an inhibitor of Cl-activated oxygen evolution in higher plant PSII. It was confirmed to be primarily a competitive inhibitor in intact PSII, with Cl-competitive inhibition constant Ki = 2 mM and uncompetitive inhibition constant $$ { ext{K}}_{ ext{i}}^{prime } $$  = 79 mM. A pH dependence study showed that fluoride inhibition was more pronounced at lower pH values. In order to determine the location of the fluoride effect, PSII preparations lacking various amounts of the PsbQ subunit were prepared. The competitive F inhibition constant and the Michaelis constant for Cl activation increased with loss of the PsbQ subunit, while the uncompetitive F inhibition constant was relatively insensitive to loss of PsbQ. The S2 state EPR signals from PSII lacking PsbQ responded to Ca2+ and Cl removal and to F treatment similar to intact PSII, with enhancement of the g = 4.1 signal and suppression of the multiline signal, but the effects were more pronounced in PSII lacking PsbQ. Together, these results support the interpretation that the PsbQ subunit has a role in retaining anions within the OEC.
Keywords: Photosystem II; Oxygen evolving complex; Chloride; Fluoride; PsbQ subunit; Electron paramagnetic resonance

Selection and use of appropriate reference genes as internal controls in real-time reverse transcription PCR (RT-PCR) assays is highly important for accurate quantification of gene expression levels. Since some photosynthetic genes are encoded in the nuclear genome and others in the chloroplast genome, we evaluated both nuclear- and plastid-encoded candidate reference genes. Six plastid-encoded candidate reference genes were derived from Arabidopsis microarray data and three plastid- and five nuclear-encoded reference genes were derived from literature. Cytokinins influence photosynthetic gene expression, so we evaluated the expression stability of the candidate reference genes in transgenic Nicotiana tabacum plants with elevated or diminished cytokinin content. We found that the most reliable strategy makes use of plastid-encoded genes for normalizing plastid photosynthetic genes and nuclear-encoded reference genes for normalizing nuclear photosynthetic genes. Compared to the use of nuclear reference genes only, this approach assimilates any effects on transcriptional activity of chloroplasts or number of chloroplast. The best expression stabilities in Nicotiana tabacum were observed for the plastid-encoded references genes Nt-RPS3, Nt-NDHI and Nt-IN1 and for the nuclear-encoded genes Nt-ACT9, Nt-αTUB and Nt-SSU. These genes may be suitable for normalization of photosynthetic genes under other experimental conditions in Nicotiana tabacum, and orthologues of these genes may be suitable candidates for normalizing photosynthetic gene expression in other species.
Keywords: Cytokinins; geNorm; Nicotiana tabacum ; Photosynthesis; Real-time PCR; Reference genes

Light absorption by isolated chloroplasts and leaves: effects of scattering and ‘packing’ by Mark N. Merzlyak; Olga B. Chivkunova; Tatiana V. Zhigalova; K. Razi Naqvi (31-41).
Light absorption was quantified in the following systems: isolated chloroplasts and leaves of spinach (Spinacea oleracea L.), a mutant of geranium (Pelargonium zonale L.) widely differing in pigment content, and coleus (Coleus blumei Benth.) at different stages of leaf ontogenesis. For these species and pea (Pisum sativum L.), scattering-compensated absorption spectra of chloroplast suspensions are presented. Comparison of leaf and chloroplast spectra showed considerable changes in the extent of the ‘package’ effect and the lengthening of the effective optical path in a leaf. The difference between leaf and isolated chloroplast absorption could be quantitatively described by adapting Duysens’s treatment of flattening. It was found that the accumulation of chlorophyll in leaves is accompanied by a monotonous enhancement of the package effect. The results are discussed with special reference to the role of light scattering in leaf optics, light utilization in photosynthesis and wavelength-dependent light gradients in a leaf.
Keywords: Chloroplasts; Leaves; Light absorption; Package effect; Photosynthesis; Scattering

Purification and characterization of cytochrome c 6 from Acaryochloris marina by Patrick D. Bell; Yueyong Xin; Robert E. Blankenship (43-51).
Cytochrome c 6 , (cyt c 6) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.
Keywords: Acaryochloris marina ; Cyanobacteria; Cytochrome c 6 ; Photosynthesis; Chlorophyll d

Photosynthetic parameters of phytoplankton and sea ice algae from landfast sea ice of the Chukchi Sea off Point Barrow, Alaska, were assessed in spring 2005 and winter through spring 2006 using Pulse Amplitude Modulated (PAM) fluorometry including estimates of maximum quantum efficiency (F v/F m), maximum relative electron transport rate (rETRmax), photosynthetic efficiency (α), and the photoadaptive index (E k). The use of centrifuged brine samples allowed to document vertical gradients in ice algal acclimation with 5 cm vertical resolution for the first time. Bottom ice algae (0–5 cm from ice–water interface) expressed low F v/F m (0.331–0.426) and low α (0.098–0.130 (μmol photons m−2s−1)−1) in December. F v/F m and α increased in March and May (0.468–0.588 and 0.141–0.438 (μmol photons m−2s−1)−1, respectively) indicating increased photosynthetic activity. In addition, increases in rETRmax (3.3–16.4 a.u.) and E k (20–88 μmol photons m−2 s−1) from December to May illustrates a higher potential for primary productivity as communities become better acclimated to under-ice light conditions. In conclusion, photosynthetic performance by ice algae (as assessed by PAM fluorometry) was tightly linked to sea ice salinity, temperature, and inorganic nutrient concentrations (mainly nitrogen).
Keywords: Arctic; Ice algae; Photosynthetic yield; Fast ice; Nutrients; Primary production

Probing of photosynthetic reactions in four phytoplanktonic algae with a PEA fluorometer by T. K. Antal; D. N. Matorin; L. V. Ilyash; A. A. Volgusheva; V. Osipov; I. V. Konyuhov; T. E. Krendeleva; A. B. Rubin (67-76).
High-resolution light-induced kinetics of chlorophyll fluorescence (OJIP transients) were recorded and analyzed in cultures of diatoms (Thalassiosira weissflogii, Chaetoceros mulleri) and dinoflagellates (Amphidinium carterae, Prorocentrum minimum). Fluorescence transients showed the rapid exponential initial rise from the point O indicating low connectivity between PS II units and high absorption cross-section of PS II antenna. Dark-adapted dinoflagellates revealed capability to maintain the PS I-mediated re-oxidation of the PQ pool at the exposure to strong actinic light that may lead to the underestimation of F M value. In OJIP transients recorded in phytoplanktonic algae the fluorescence yield at the point O exceeded F O level because QA has been already partly reduced at 50 μs after the illumination onset. PEA was also employed to study the recovery of photosynthetic reactions in T. weissflogii during incubation of nitrogen starved cells in N-replete medium. N limitation caused the impairment of electron transport between QA and PQs, accumulation of closed PS II centers, and the reduced ability to generate transmembrane ΔpH upon illumination, almost fully restored during the recovery period. The recovered cells showed much higher values of NPQ than control ones suggesting maximization of photoprotection mechanisms in the population with a ‘stress history.’
Keywords: Chlorophyll a fluorescence; OJIP transients; Phytoplankton; Nitrogen starvation

Overexpression, characterization, and crystallization of the functional domain of cytochrome c z from Chlorobium tepidum by Makoto Higuchi; Yu Hirano; Yukihiro Kimura; Hirozo Oh-oka; Kunio Miki; Zheng-Yu Wang (77-84).
Cytochrome c z is found in green sulfur photosynthetic bacteria, and is considered to be the only electron donor to the special pair P840 of the reaction center. It consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. Large scale expression of the C-terminal functional domain of the cytochrome c z (C-cyt c z) from the thermophilic bacterium Chlorobium tepidum has been achieved using the Escherichia coli expression system. The C-cyt c z expressed has been highly purified, and is stable at room temperature over 10 days of incubation for both reduced and oxidized forms. Spectroscopic measurements indicate that the heme iron in C-cyt c z is in a low-spin state and this does not change with the redox state. 1H-NMR spectra of the oxidized C-cyt c z exhibited unusually large paramagnetic chemical shifts for the heme methyl protons in comparison with those of other Class I ferric cytochromes c. Differences in the 1H-NMR linewidth were observed for some resonances, indicating different dynamic environments for these protons. Crystals of the oxidized C-cyt c z were obtained using ammonium sulfate as a precipitant. The crystals diffracted X-rays to a maximum resolution of 1.2 Å, and the diffraction data were collected to 1.3 Å resolution.
Keywords: Green sulfur bacteria; Reaction center; Electron transfer; Heme protein

A new setup for in vivo fluorescence imaging of photosynthetic activity by Xenie Johnson; Guillaume Vandystadt; Sandrine Bujaldon; Francis-André Wollman; Rémi Dubois; Pierre Roussel; Jean Alric; Daniel Béal (85-93).
Here, we describe a new imaging setup able to assess in vivo photosynthetic activity. The system specifically measures time-resolved chlorophyll fluorescence in response to light. It is composed of a fast digital camera equipped with a wide-angle lens for the analysis of samples up to 10 × 10 cm, i.e. entire plants or petri dishes. In the choice of CCD, we have opted for a 12-bits high frame rate [150 fps (frames per second)] at the expense of definition (640 × 480 pixels). Although the choice of digital camera is always a compromise between these two related features, we have designed a flexible system allowing the fast sampling of images (down to 100 μs) with a maximum spatial resolution. This image readout system, synchronized with actinic light and saturating pulses, allows a precise determination of F 0 and F M, which is required to monitor PSII activity. This new imaging system, together with image processing techniques, is useful to investigate the heterogeneity of photosynthetic activity within leaves or to screen large numbers of unicellular algal mutant colonies to identify those with subtle changes in photosynthetic electron flow.
Keywords: Chlorophyll fluorescence; Imaging techniques; Screening of algal colonies

Modification of a French pressure cell to improve microbial cell disruption by Paul R. Jaschke; Ian Drake; J. Thomas Beatty (95-97).
A procedure for modification of the valve stem of a 40 K French pressure cell is described. The modification should be done by a machinist and requires a metalworking lathe. After modification of the valve stem, a torlon 4203 plastic ball is used between the valve stem and valve seat to control the pressure within the cell. The torlon plastic ball is a key component needed to obtain the high pressures required for efficient disruption of microbial cells.
Keywords: French press; Bacterial lysis; Photosynthetic bacteria

Quantitative data on laser flash-induced variable fluorescence in the 100 ns to 1 ms time range (Belyaeva et al. in Photosynth Res 98:105–119, 2008) confirming those of others (Steffen et al. in Biochemistry 40:173–180, 2001, Biochemistry 44:3123–3132, 2005; Belyaeva et al. in Biophysics 51(6):976–990, 2006), need a substantial correction with respect to magnitude of the normalized variable fluorescence associated with single turnover-induced charge separation in RCs of PS II. Their data are conclusive with the involvement of donor side quenching, the release of which occurs with a rate constant in the range of tens of ms−1, and presumed to be associated with reduction of $$ Y_{ ext{z}}^{ + } $$ by the OEC.
Keywords: Chlorophyll a fluorescence; PS II modeling; Donor side quenching