Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni²⁺-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b₆f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.     

X-ray structures of the peridinin-chlorophyll-protein reconstituted with different chlorophylls

The peridinin-chlorophyll a-protein (PCP) from dinoflagellates is a soluble light harvesting antenna which gathers incoming photons mainly by the carotenoid peridinin. In PCPs reconstituted with different chlorophylls, the peridinin to chlorophyll energy transfer rates are well predicted by a Förster-like theory, but only if the pigment arrangements are identical in all PCPs. We have determined the X-ray structures of PCPs reconstituted with Chlorophyll-b (Chl-b), Chlorophyll-d (Chl-d) and Bacteriochlorophyll-a (BChl-a) to resolutions ?2 Å. In all three cases the pigment arrangements are essentially the same as in native PCP. Hydrogen bonding is not responsible for preferential incorporation of “non-native” chlorophylls over Chl-a.     

The redox state of the plastoquinone pool directly modulates minimum chlorophyll fluorescence yield in Chlamydomonas reinhardtii

The effect of the plastoquionone (PQ) pool oxidation state on minimum chlorophyll fluorescence was studied in the green alga Chlamydomonas reinhardtii. In wild type and a mutant strain that lacks both photosystems but retains light harvesting complexes, oxygen depletion induced a rise in minimum chlorophyll fluorescence. An increase in minimum fluorescence yield is also observed when the PQ pool becomes reduced in the presence of oxygen and after application of an ionophore that collapses the transmembrane proton gradient. Together these results indicate that minimum chlorophyll fluorescence is modulated by the PQ oxidation state.     

Electromagnetic and thermal evaluation of an applicator specialized to permit high‐resolution non‐perturbing optical evaluation of cells being irradiated in the W‐band

To permit epi‐illuminated, high‐resolution optical microscopy of cells in monolayer culture during unperturbed W‐band (75–110 GHz) irradiation, a new class of applicator has been developed based upon WR10 rectangular waveguide components: the cells are normally plated onto the underside of a coverslip which is then placed against the under side of a waveguide flange and receives a roughly circular exposure pattern, with the ±1 dB central spot roughly 1 mm in diameter. Constructed and tested with 94 GHz millimeter waves, water‐immersion optics, and free‐convection cooling, the applicator works robustly and permits SARs at the cell layer as high as 4500 W/kg before the steady‐state temperature rise at the cell layer exceeds 0.5 K. Bioelectromagnetics 31:140–149, 2010. © 2009 Wiley‐Liss, Inc.     

Physiological links among alternative electron transport pathways that reduce and oxidize plastoquinone in Arabidopsis

In addition to linear electron transport from water to NADP⁺, alternative electron transport pathways are believed to regulate photosynthesis. In the two routes of photosystem I (PSI) cyclic electron transport, electrons are recycled from the stromal reducing pool to plastoquinone (PQ), generating additional ΔpH (proton gradient across thylakoid membranes). Plastid terminal oxidase (PTOX) accepts electrons from PQ and transfers them to oxygen to produce water. Although both electron transport pathways share the PQ pool, it is unclear whether they interact in vivo. To investigate the physiological link between PSI cyclic electron transport‐dependent PQ reduction and PTOX‐dependent PQ oxidation, we characterized mutants defective in both functions. Impairment of PSI cyclic electron transport suppressed leaf variegation in the Arabidopsis immutans (im) mutant, which is defective in PTOX. The im variegation was more effectively suppressed in the pgr5 mutant, which is defective in the main pathway of PSI cyclic electron transport, than in the crr2‐2 mutant, which is defective in the minor pathway. In contrast to this chloroplast development phenotype, the im defect alleviated the growth phenotype of the crr2‐2 pgr5 double mutant. This was accompanied by partial suppression of stromal over‐reduction and restricted linear electron transport. We discuss the function of the alternative electron transport pathways in both chloroplast development and photosynthesis in mature leaves.     

REVIEW: Time lag between photosynthesis and carbon dioxide efflux from soil: a review of mechanisms and controls

CO₂ efflux from soil depends on the availability of organic substances respired by roots and microorganisms. Therefore, photosynthetic activity supplying carbohydrates from leaves to roots and rhizosphere is a key driver of soil CO₂. This fact has been overlooked in most soil CO₂ studies because temperature variations are highly correlated with solar radiation and mask the direct effect of photosynthesis on substrate availability in soil. This review highlights the importance of photosynthesis for rhizosphere processes and evaluates the time lag between carbon (C) assimilation and CO₂ release from soil. Mechanisms and processes contributing to the lag were evaluated. We compared the advantages and shortcomings of four main approaches used to estimate this time lag: (1) interruption of assimilate flow from leaves into the roots and rhizosphere, and analysis of the decrease of CO₂ efflux from soil, (2) time series analysis (TSA) of CO₂ fluxes from soil and photosynthesis proxies, (3) analysis of natural δ¹³C variation in CO₂ with photosynthesis‐related parameters or δ¹³C in the phloem and leaves, and (4) pulse labeling of plants in artificial ¹⁴CO₂ or ¹³CO₂ atmosphere with subsequent tracing of ¹⁴C or ¹³C in CO₂ efflux from soil. We concluded that pulse labeling is the most advantageous approach. It allows clear evaluation not only of the time lag, but also of the label dynamics in soil CO₂, and helps estimate the mean residence time of recently assimilated C in various above‐ and belowground C pools. The impossibility of tracing the phloem pressure–concentration waves by labeling approach may be overcome by its combination with approaches based on TSA of CO₂ fluxes and its δ¹³C with photosynthesis proxies. Numerous studies showed that the time lag for grasses is about 12.5±7.5 (SD) h. The time lag for mature trees was much longer (～4–5 days). Tree height slightly affected the lag, with increasing delay of 0.1 day m^?1. By evaluating bottle‐neck processes responsible for the time lag, we conclude that, for trees, the transport of assimilates in phloem is the rate‐limiting step. However, it was not possible to predict the lag based on the phloem transport rates reported in the literature. We conclude that studies of CO₂ fluxes from soil, especially in ecosystems with a high contribution of root‐derived CO₂, should consider photosynthesis as one of the main drivers of C fluxes. This calls for incorporating photosynthesis in soil C turnover models.     

Reversal of aging‐associated hippocampal synaptic plasticity deficits by reductants via regulation of thiol redox and NMDA receptor function

Deficits in learning and memory accompanied by age‐related neurodegenerative diseases are closely related to the impairment of synaptic plasticity. In this study, we investigated the role of thiol redox status in the modulation of the N‐methyl‐d ‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP) in CA1 areas of hippocampal slices. Our results demonstrated that the impaired LTP induced by aging could be reversed by acute administration of reductants that can regulate thiol redox status directly, such as dithiothreitol or β‐mercaptoethanol, but not by classical anti‐oxidants such as vitamin C or trolox. This repair was mediated by the recruitment of aging‐related deficits in NMDAR function induced by these reductants and was mimicked by glutathione, which can restore the age‐associated alterations in endogenous thiol redox status. Moreover, antioxidant prevented but failed to reverse H₂O₂‐induced impairment of NMDAR‐mediated synaptic plasticity. These results indicate that the restoring of thiol redox status may be a more effective strategy than the scavenging of oxidants in the treatment of pre‐existing oxidative injury in learning and memory.     

Cellular and biochemical parameters of exercise-induced oxidative stress: relationship with training levels

To better clarify the relationship between physical activity and oxidative stress, we determined the effects of a maximal test in 18 young subjects with different training levels (six professional Athletes and 12 non-agonists (NA)). Redox homeostasis (total antioxidant activity (TAS), vitamin C and glutathione (GSH)), oxidative damage (diene conjugation and hemolysis), lymphocyte cell death and repair systems (apoptosis, micronuclei and Hsp70 expression) were evaluated. We found that agonistic training led to a chronic oxidative insult (high baseline values of oxidized glutathione (GSSG), micronuclei and hemolysis). On the contrary, NA with the lowest level of training frequency showed a well balanced profile at rest, but they were more susceptible to exercise-induced variations (GSSG/GSH and diene increased values), respect to the NA with an higher level of training. As almost all the parameters employed in this study showed inter-individual variations, the GSSG/GSH ratio remains the most sensitive and reliable marker of oxidative stress, accordingly with other data just reported in the literature.