Photosynthesis Research (v.124, #1)

We celebrate distinguished photobiologist Rajni Govindjee for her pioneering research in photosynthesis and retinal proteins on the occasion of her 80th birthday.
Keywords: Bacteriorhodopsin; Biophysics; Photochemistry; Photosynthesis

Metal ion oxidation state assignment based on coordinating ligand hyperfine interaction by Paul H. Oyala; Troy A. Stich; R. David Britt (7-18).
In exchange-coupled mixed-valence spin systems, the magnitude and sign of the effective ligand hyperfine interaction (HFI) can be useful in determining the formal oxidation state of the coordinating metal ion, as well as provide information about the coordination geometry. This is due to the fact that the observed ligand HFI is a function of the projection factor (Clebsch-Gordon coefficient) that maps the site spin value S i of the local paramagnetic center onto the total spin of the exchange-coupled system, S T. Recently, this relationship has been successfully exploited in identifying the oxidation state of the Mn ion coordinated by the sole nitrogenous ligand to the oxygen-evolving complex in certain states of photosystem II. The origin and evolution of these efforts is described.
Keywords: Electron paramagnetic resonance; Photosystem II; Hyperfine; Exchange coupling; Electronic structure

The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c’ from the thermophilic purple sulfur bacterium Thermochromatium tepidum by Yukihiro Kimura; Sachiko Kasuga; Masashi Unno; Takashi Furusawa; Shinsuke Osoegawa; Yuko Sasaki; Takashi Ohno; Zheng-Yu Wang-Otomo (19-29).
A soluble cytochrome (Cyt) c’ from thermophilic purple sulfur photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits marked thermal tolerance compared with that from the closely related mesophilic counterpart Allochromatium vinosum. Here, we focused on the difference in the C-terminal region of the two Cyts c’ and examined the effects of D131 and R129 mutations on the thermal stability and local heme environment of Cyt c’ by differential scanning calorimetry (DSC) and resonance Raman (RR) spectroscopy. In the oxidized forms, D131K and D131G mutants exhibited denaturing temperatures significantly lower than that of the recombinant control Cyt c’. In contrast, R129K and R129A mutants denatured at nearly identical temperatures with the control Cyt c’, indicating that the C-terminal D131 is an important residue maintaining the enhanced thermal stability of Tch. tepidum Cyt c’. The control Cyt c’ and all of the mutants increased their thermal stability upon the reduction. Interestingly, D131K exhibited narrow DSC curves and unusual thermodynamic parameters in both redox states. The RR spectra of the control Cyt c’ exhibited characteristic bands at 1,635 and 1,625 cm−1, ascribed to intermediate spin (IS) and high spin (HS) states, respectively. The IS/HS distribution was differently affected by the D131 and R129 mutations and pH changes. Furthermore, R129 mutants suggested the lowering of their redox potentials. These results strongly indicate that the D131 and R129 residues play significant roles in maintaining the thermal stability and modulating the local heme environment of Tch. tepidum Cyt c’.
Keywords: Cytochrome c’ ; Thermochromatium tepidum ; Purple bacteria; Thermal stability; Differential scanning calorimetry; Resonance Raman spectroscopy

The dark relaxation of the yield of variable BChl fluorescence in the 10−5–10 s time range is measured after laser diode (808 nm) excitation of variable duration in intact cells of photosynthetic bacteria Rba. sphaeroides, Rsp. rubrum, and Rvx. gelatinosus under various treatments of redox agents, inhibitors, and temperature. The kinetics of the relaxation is complex and much wider extended than a monoexponential function. The longer is the excitation, the slower is the relaxation which is determined by the redox states, sizes, and accessibility of the pools of cytochrome $$c_{2}^{2+}$$ c 2 2 + and quinone for donor and acceptor side-limited bacterial strains, respectively. The kinetics of fluorescence decay reflects the opening kinetics of the closed RC. The relaxation is controlled preferentially by the rate of re-reduction of the oxidized dimer by mobile cytochrome $$c_{2}^{2+}$$ c 2 2 + in Rba. sphaeroides and Rsp. rubrum and by the rate constant of the $${ m{Q}}_{ m{A}}^{-}$$ Q A - interquinone electron transfer, (350 μs)−1 and/or the quinol/quinone exchange at the acceptor side in Rvx. gelatinosus. The commonly used acceptor side inhibitors (e.g., terbutryn) demonstrate kinetically limited block of re-oxidation of the primary quinone. The observations are interpreted in frame of a minimum kinetic and energetic model of electron transfer reactions in bacterial RC of intact cells.
Keywords: Purple photosynthetic bacteria; Reaction center; Light-induced electron transfer; Cytochromes; Quinones

Electron transport kinetics in the diazotrophic cyanobacterium Trichodesmium spp. grown across a range of light levels by Xiaoni Cai; Kunshan Gao; Feixue Fu; Douglas A. Campbell; John Beardall; David A. Hutchins (45-56).
The diazotrophic cyanobacterium Trichodesmium is a major contributor to marine nitrogen fixation. We analyzed how light acclimation influences the photophysiological performance of Trichodesmium IMS101 during exponential growth in semi-continuous nitrogen fixing cultures under light levels of 70, 150, 250, and 400 μmol photons m−2 s−1, across diel cycles. There were close correlations between growth rate, trichome length, particulate organic carbon and nitrogen assimilation, and cellular absorbance, which all peaked at 150 μmol photons m−2 s−1. Growth rate was light saturated by about 100 μmol photons m−2 s−1 and was photoinhibited above 150 μmol photons m−2 s−1. In contrast, the light level (I k) to saturate PSII electron transport (e  PSII−1 s−1) was much higher, in the range of 450–550 μmol photons m−2 s−1, and increased with growth light. Growth rate correlates with the absorption cross section as well as with absorbed photons per cell, but not to electron transport per PSII; this disparity suggests that numbers of PSII in a cell, along with the energy allocation between two photosystems and the state transition mechanism underlie the changes in growth rates. The rate of state transitions after a transfer to darkness increased with growth light, indicating faster respiratory input into the intersystem electron transport chain.
Keywords: Light; Electron transport; Trichodesmium ; State transition

Effects of overexpression of high activity-type Rubisco small subunit (RbcS) from a cold-resistant plant, timothy (Phleum pratense), on kinetic properties of Rubisco were studied in rice (Oryza sativa). The full-length mRNA sequence of timothy RbcS (PpRbcS1) was determined by 5′RACE and 3′RACE. The coding sequence of PpRbcS1 was fused to the chlorophyll a/b-binding protein promoter and introduced into rice. PpRbcS was highly expressed in leaf blade and accounted for approximately 30 % of total RbcS in homozygous transgenic lines. However, the catalytic turnover rate and K m for CO2 of Rubisco did not significantly change in these transgenic lines compared to non-transgenic rice, suggesting that PpRbcS1 is not effective for improvement of catalytic efficiency of rice Rubisco. The photosynthetic rate and growth were essentially unchanged, whereas the photosynthetic rate at low CO2 condition was marginally increased in transgenic lines. Rubisco content was significantly increased, whereas soluble protein, nitrogen, and chlorophyll contents were unchanged in transgenic lines compared to non-transgenic rice. Because the kinetic properties were similar, observed slight increase in photosynthetic rate at low CO2 is considered to be large due to increase in Rubisco content in transgenic lines. Introduction of foreign RbcS is an effective approach for the improvement of Rubisco kinetics and photosynthesis. However, in this study, it was suggested that RbcS of high activity-type Rubisco, even showing higher amino acid identity with rice RbcS, did not always enhance the catalytic turnover rate of Rubisco in rice. Thus, we should carefully select RbcS to be overexpressed before introduction.
Keywords: CO2 fixation; Cold-resistant plant; Rice; Rubisco; Timothy

Temperature dependence of in vitro Rubisco kinetics in species of Flaveria with different photosynthetic mechanisms by Juan Alejandro Perdomo; Amanda P. Cavanagh; David S. Kubien; Jeroni Galmés (67-75).
There is general consensus in the literature that plants with different photosynthetic mechanisms (i.e. C3 vs. C4) have Rubiscos characterised by different kinetic performances. However, potential differences in the temperature dependencies of Rubisco kinetic parameters between C3 and C4 plants are uncertain. Accordingly, six species of Flaveria with contrasting photosynthetic mechanisms (C3, C3/C4 and C4) were selected and their Rubisco Michaelis–Menten constants for CO2 and RuBP (K c and K RuBP), carboxylase catalytic turnover rate ( $${k_{ ext{cat}}}^{ ext{c}}$$ k cat c ) and CO2/O2 specificity factor (S c/o) were measured between 10 and 40 °C. The results confirmed different Rubisco characteristics between C3 and C4 plants. Rubisco from the C3 species had higher E a for K c and $${k_{ ext{cat}}}^{ ext{c}}$$ k cat c than that from C4 species, which were translated into differences in the temperature response of the carboxylase catalytic efficiency ( $${k_{ ext{cat}}}^{ ext{c}}$$ k cat c /K c). However, E a did not differ for S c/o or K RuBP. Although a mechanism remains uncertain, it appears that the Asp/Glu-149-Ala and Met-309-Ile substitutions lead to differences in the temperature responses of catalysis between C3 and C4 Rubiscos in Flaveria. Therefore, the above observations are consistent with the fact that C3 species have a higher photosynthetic efficiency and ecological dominance in cool environments, with respect to C4 species in temperate environments.
Keywords: C3 ; C4 ; Kinetics; Photosynthesis; Rubisco; Temperature

How do surrounding environments influence the electronic and vibrational properties of spheroidene? by Noriyuki Tonouchi; Daisuke Kosumi; Mitsuru Sugisaki; Mamoru Nango; Hideki Hashimoto (77-86).
Absorption and Raman spectra of spheroidene dissolved in various organic solvents and bound to peripheral light-harvesting LH2 complexes from photosynthetic purple bacteria Rhodobacter (Rba.) sphaeroides 2.4.1 were measured. The results showed that the peak energies of absorption and C–C and C=C stretching Raman lines are linearly proportional to the polarizability of solvents, as has already been reported. When comparing these results with those measured on LH2 complexes, it was confirmed that spheroidene is surrounded by a media with high polarizability. However, the change in the spectral width of the Raman lines, which reflect vibrational decay time, cannot be explained simply by a similar dependence of solvent polarizability. The experimental results were analyzed using a potential theoretical model. Consequently, a systematic change in the Raman line widths in the ground state can be satisfactorily explained as a function of the viscosity of the surrounding media. Even when the absorption peaks appear at the same energy, the vibrational decay time of spheroidene in the LH2 complexes is approximately 15–20 % slower than that in organic solvents.
Keywords: Carotenoid; LH2; Raman spectroscopy; Vibrational decay; Polarizability; Viscosity

Paper describes principles and application of a novel routine that enables the quantitative analysis of the photochemical O–J phase of the variable fluorescence F v associated with the reversible photo-reduction of the secondary electron acceptor QA of photosystem II (PSII) in algae and intact leaves. The kinetic parameters that determine the variable fluorescence F PP(t) associated with the release of photochemical quenching are estimated from 10 µs time-resolved light-on and light-off responses of F v induced by two subsequent light pulses of 0.25 (default) and 1000 ms duration, respectively. Application of these pulses allows estimations of (i) the actual value of the rate constants k L and k AB of the light excitation (photoreduction of QA) and of the dark re-oxidation of photoreduced QA ( $${ ext{Q}}_{ ext{A}}^{ - }$$ Q A - ), respectively, (ii) the actual maximal normalized variable fluorescence [nF v] associated with 100 % photoreduction of QA of open RCs, and (iii) the actual size β of RCs in which the re-oxidation of $${ ext{Q}}_{ ext{A}}^{ - }$$ Q A - is largely suppressed (QB-nonreducing RC with k AB ~ 0). The rate constants of the dark reversion of Fv associated with the release of photo-electrochemical quenching F PE and photo-electric stimulation F CET in the successive J–I and I–P parts of the thermal phase are in the range of (100 ms)−1 and (1 s)−1, respectively. The kinetics of fluorescence changes during and after the I–P phase are given special attention in relation to the hypothesis on the involvement of a Δµ H+-dependent effect during this phase and thereafter. Paper closes with author’s personal view on the demands that should be fulfilled for chlorophyll fluorescence methods being a correct and unchallenged signature of photosynthesis in algae and plants.
Keywords: Chlorophyll fluorescence kinetics; Quenching mechanisms; System analysis; TSTM; OJIP; Views

Time-averaged net photosynthetic rate (P n) under pulsed light (PL) is known to be affected by the PL frequency and duty ratio, even though the time-averaged photosynthetic photon flux density (PPFD) is unchanged. This phenomenon can be explained by considering that photosynthetic intermediates (PIs) are pooled during light periods and then consumed by partial photosynthetic reactions during dark periods. In this study, we developed a kinetic model to estimate P n of cos lettuce (Lactuca sativa L. var. longifolia) leaves under PL based on the dynamics of the amount of pooled PIs. The model inputs are average PPFD, duty ratio, and frequency; the output is P n. The rates of both PI accumulation and consumption at a given moment are assumed to be dependent on the amount of pooled PIs at that point. Required model parameters and three explanatory variables (average PPFD, frequency, and duty ratio) were determined for the simulation using P n values under PL based on several combinations of the three variables. The model simulation for various PL levels with a wide range of time-averaged PPFDs, frequencies, and duty ratios further demonstrated that P n under PL with high frequencies and duty ratios was comparable to, but did not exceed, P n under continuous light, and also showed that P n under PL decreased as either frequency or duty ratio was decreased. The developed model can be used to estimate P n under various light environments where PPFD changes cyclically.
Keywords: Duty ratio; Fluctuating light; Frequency; Photosynthetic intermediate; Lightfleck

The arc mutants of Arabidopsis with fewer large chloroplasts have a lower mesophyll conductance by Sean E. Weise; David J. Carr; Ashley M. Bourke; David T. Hanson; Debbie Swarthout; Thomas D. Sharkey (117-126).
Photosynthetic cells of most land plant lineages have numerous small chloroplasts even though most algae, and even the early diverging land plant group the hornworts, tend to have one or a few large chloroplasts. One constraint that small chloroplasts could improve is the resistance to CO2 diffusion from the atmosphere to the chloroplast stroma. We examined the mesophyll conductance (inverse of the diffusion resistance) of mutant Arabidopsis thaliana plants with one or only a few large chloroplasts per cell. The accumulation and replication of chloroplasts (arc) mutants of A. thaliana were studied by model fitting to gas exchange data and 13CO2 discrimination during carbon fixation. The two methods generally agreed, but the value of the CO2 compensation point of Rubisco (Γ *) used in the model had a large impact on the estimated photosynthetic parameters, including mesophyll conductance. We found that having only a few large chloroplasts per cell resulted in a 25–50 % reduction in the mesophyll conductance at ambient CO2.
Keywords: Mesophyll conductance; Gamma * ; Arc mutants; Farquhar model; Carbon isotope; Chloroplast size