A Thylakoid Membrane Protein Harboring a DnaJ-type Zinc Finger Domain Is Required for Photosystem I Accumulation in Plants

Photosystem I (PSI) is a large pigment-protein complex and one of the two photosystems that drive electron transfer in oxygenic photosynthesis. We identified a nuclear gene required specifically for the accumulation of PSI in a forward genetic analysis of chloroplast biogenesis in maize. This gene, designated psa2, belongs to the “GreenCut” gene set, a group of genes found in green algae and plants but not in non-photosynthetic organisms. Disruption of the psa2 ortholog in Arabidopsis likewise resulted in the specific loss of PSI proteins. PSA2 harbors a conserved domain found in DnaJ chaperones where it has been shown to form a zinc finger and to have protein-disulfide isomerase activity. Accordingly, PSA2 exhibited protein-disulfide reductase activity in vitro. PSA2 localized to the thylakoid lumen and was found in a ∼250-kDa complex harboring the peripheral PSI protein PsaG but lacking several core PSI subunits. PSA2 mRNA is coexpressed with mRNAs encoding various proteins involved in the biogenesis of the photosynthetic apparatus with peak expression preceding that of genes encoding structural components. PSA2 protein abundance was not decreased in the absence of PSI but was reduced in the absence of the PSI assembly factor Ycf3. These findings suggest that a complex harboring PSA2 and PsaG mediates thiol transactions in the thylakoid lumen that are important for the assembly of PSI.     

The role of light-harvesting complex I in excitation energy transfer from LHCII to photosystem I in Arabidopsis

Photosynthesis powers nearly all life on Earth. Light absorbed by photosystems drives the conversion of water and carbon dioxide into sugars. In plants, photosystem I (PSI) and photosystem II (PSII) work in series to drive the electron transport from water to NADP⁺. As both photosystems largely work in series, a balanced excitation pressure is required for optimal photosynthetic performance. Both photosystems are composed of a core and light-harvesting complexes (LHCI) for PSI and LHCII for PSII. When the light conditions favor the excitation of one photosystem over the other, a mobile pool of trimeric LHCII moves between both photosystems thus tuning their antenna cross-section in a process called state transitions. When PSII is overexcited multiple LHCIIs can associate with PSI. A trimeric LHCII binds to PSI at the PsaH/L/O site to form a well-characterized PSI–LHCI–LHCII supercomplex. The binding site(s) of the “additional” LHCII is still unclear, although a mediating role for LHCI has been proposed. In this work, we measured the PSI antenna size and trapping kinetics of photosynthetic membranes from Arabidopsis (Arabidopsis thaliana) plants. Membranes from wild-type (WT) plants were compared to those of the ΔLhca mutant that completely lacks the LHCI antenna. The results showed that “additional” LHCII complexes can transfer energy directly to the PSI core in the absence of LHCI. However, the transfer is about two times faster and therefore more efficient, when LHCI is present. This suggests LHCI mediates excitation energy transfer from loosely bound LHCII to PSI in WT plants.

The light-harvesting antennae of photosystem I facilitate energy transfer from trimeric light-harvesting complex II to photosystem I in the stroma lamellae membrane.     

Concerning a dual function of coupled cyclic electron transport in leaves

Coupled cyclic electron transport is assigned a role in the protection of leaves against photoinhibition in addition to its role in ATP synthesis. In leaves, as in reconstituted thylakoid systems, cyclic electron transport requires “poising,” i.e. availability of electrons at the reducing side of photosystem I (PSI) and the presence of some oxidized plastoquinone between photosystem II (PSII) and PSI. Under self-regulatory poising conditions that are established when carbon dioxide limits photosynthesis at high light intensities, and particularly when stomata are partially or fully closed as a result of water stress, coupled cyclic electron transport controls linear electron transport by helping to establish a proton gradient large enough to decrease PSII activity and electron flow to PSI. This brings electron donation by PSII, and electron consumption by available electron acceptors, into a balance in which PSI becomes more oxidized than it is during fast carbon assimilation. Avoidance of overreduction of the electron transport chain is a prerequisite for the efficient protection of the photosynthetic apparatus against photoinactivation.     

A developmental study of photosystem I peripheral chlorophyll proteins

An isolated “native” photosystem I (PSI complex) contains three spectral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of these antennae (45 Chl/P₇₀₀) are associated with polypeptides of 21,500 to 24,500 daltons. The present study utilizes two developmental systems to verify this association.     

Usefulness of Midregional Proadrenomedullin to Predict Poor Outcome in Patients with Community Acquired Pneumonia

Background

midregional proadrenomedullin (MR-proADM) is a prognostic biomarker in patients with community-acquired pneumonia (CAP). We sought to confirm whether MR-proADM added to Pneumonia Severity Index (PSI) improves the potential prognostic value of PSI alone, and tested to what extent this combination could be useful in predicting poor outcome of patients with CAP in an Emergency Department (ED).

Methods

Consecutive patients diagnosed with CAP were enrolled in this prospective, single-centre, observational study. We analyzed the ability of MR-proADM added to PSI to predict poor outcome using receiver operating characteristic (ROC) curves, logistic regression and risk reclassification and comparing it with the ability of PSI alone. The primary outcome was “poor outcome”, defined as the incidence of an adverse event (ICU admission, hospital readmission, or mortality at 30 days after CAP diagnosis).

Results

226 patients were included; 33 patients (14.6%) reached primary outcome. To predict primary outcome the highest area under curve (AUC) was found for PSI (0.74 [0.64-0.85]), which was not significantly higher than for MR-proADM (AUC 0.72 [0.63-0.81, p > 0.05]). The combination of PSI and MR-proADM failed to improve the predictive potential of PSI alone (AUC 0.75 [0.65-0.85, p=0.56]). Ten patients were appropriately reclassified when the combined PSI and MR-proADM model was used as compared with the model of PSI alone. Net reclassification improvement (NRI) index was statistically significant (7.69%, p = 0.03) with an improvement percentage of 3.03% (p = 0.32) for adverse event, and 4.66% (P = 0.02) for no adverse event.

Conclusion

MR-proADM in combination with PSI may be helpful in individual risk stratification for short-term poor outcome of CAP patients, allowing a better reclassification of patients compared with PSI alone.     

Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii

Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of >1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography–tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wild-type cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures in the purified preparation measure 285 × 185 Å; these particles may represent several large oligomeric states. Pulse-chase protein labeling revealed that the PSI polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.     

Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12–15 core and 4–9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.     

Crystallization of the photosystem I reaction centre

The reaction centre of the photosynthetic membrane complex photosystem I (PSI) from the thermophilic cyanobacterium Phormidium laminosum was found to crystallize under a range of conditions. The crystallization method, which can occur in the presence of larger detergent molecules than those used previously for the crystallization of membrane proteins, is presented in this report. Several crystal forms have been observed, and some of these show birefringence and linear dichroism. Optical measurements on crystals thicker than ˜5 µm were severely restricted because of the very high chlorophyll density within the crystals, but linear dichroism measurements on thin single crystals were possible and the results are presented here. By comparing the data with earlier measurements on oriented PSI complexes, a working model for the orientation of the PSI complexes within the crystal could be proposed. The PSI reaction centre is one of the largest and most complex membrane protein units that have been crystallized to date.     

Low growth temperature-induced increase in light saturated photosystem I electron transport is cation dependent

Thylakoid membranes isolated from cold tolerant, herbaceous monocots and dicots grown at 5°C exhibit a 1.5-fold to 2.7-fold increase in light saturated rates of photosystem I (PSI) electron transport compared to thylakoids isolated from the same plant species grown at 20°C. This was observed only when either water or reduced dichlorophenolindophenol was used as an electron donor. The apparent quantum yield for PSI electron transport was not affected by growth temperature. The higher light saturated rates of PSI electron transport in 5°C thylakoids had an absolute requirement for the presence of Na⁺ and Mg⁺². The accessibility of reduced dichlorophenolindophenol to the donor site was not affected by growth temperature since 5°C and 20°C thylakoids exhibited no significant difference in the concentration of this electron donor required for half-maximal PSI activity. The cation dependent higher rates of light saturated PSI activity were also observed when rye thylakoids were developed under intermittent light conditions at 5°C. Thus, this cation effect on PSI activity appeared to be independent of light harvesting complex I and II. The extent of the in vitro reversibility of this cation effect appeared to be limited by an inherent decay process for PSI electron transport. The rate of decay for PSI activity was greatest when thylakoids were isolated in the absence of NaCl and MgCl₂. We conclude that exposure of plants to low growth temperatures induces a reorganization of thylakoid membranes which increases the light saturated rates of PSI electron transport with no change in the apparent quantum efficiency for this reaction. Cations are required to stabilize this reorganization.     

Towards spruce-type photosystem II: consequences of the loss of light-harvesting proteins LHCB3 and LHCB6 in Arabidopsis

The largest stable photosystem II (PSII) supercomplex in land plants (C₂S₂M₂) consists of a core complex dimer (C₂), two strongly (S₂) and two moderately (M₂) bound light-harvesting protein (LHCB) trimers attached to C₂ via monomeric antenna proteins LHCB4–6. Recently, we have shown that LHCB3 and LHCB6, presumably essential for land plants, are missing in Norway spruce (Picea abies), which results in a unique structure of its C₂S₂M₂ supercomplex. Here, we performed structure–function characterization of PSII supercomplexes in Arabidopsis (Arabidopsis thaliana) mutants lhcb3, lhcb6, and lhcb3 lhcb6 to examine the possibility of the formation of the “spruce-type” PSII supercomplex in angiosperms. Unlike in spruce, in Arabidopsis both LHCB3 and LHCB6 are necessary for stable binding of the M trimer to PSII core. The “spruce-type” PSII supercomplex was observed with low abundance only in the lhcb3 plants and its formation did not require the presence of LHCB4.3, the only LHCB4-type protein in spruce. Electron microscopy analysis of grana membranes revealed that the majority of PSII in lhcb6 and namely in lhcb3 lhcb6 mutants were arranged into C₂S₂ semi-crystalline arrays, some of which appeared to structurally restrict plastoquinone diffusion. Mutants without LHCB6 were characterized by fast induction of non-photochemical quenching and, on the contrary to the previous lhcb6 study, by only transient slowdown of electron transport between PSII and PSI. We hypothesize that these functional changes, associated with the arrangement of PSII into C₂S₂ arrays in thylakoids, may be important for the photoprotection of both PSI and PSII upon abrupt high-light exposure.

Photosystem II supercomplexes in Arabidopsis lacking antenna proteins LHCB3 and LHCB6 differ from their spruce counterparts and form potentially photoprotective semi-crystalline arrays in thylakoids.     

Structure and function of wild-type and subunit-depleted photosystem I in Synechocystis

The ability of photosynthetic organisms to use the sun's light as a sole source of energy sustains life on our planet. Photosystems I (PSI) and II (PSII) are large, multi-subunit, pigment–protein complexes that enable photosynthesis, but this intriguing process remains to be explained fully. Currently, crystal structures of these complexes are available for thermophilic prokaryotic cyanobacteria. The mega-Dalton trimeric PSI complex from thermophilic cyanobacterium, Thermosynechococcus elongatus, was solved at 2.5?Å resolution with X-ray crystallography. That structure revealed the positions of 12 protein subunits (PsaA-F, PsaI-M, and PsaX) and 127 cofactors.Although mesophilic organisms perform most of the world's photosynthesis, no well-resolved trimeric structure of a mesophilic organism exists. Our research model for a mesophilic cyanobacterium was Synechocystis sp. PCC6803. This study aimed to obtain well-resolved crystal structures of [1] a monomeric PSI with all subunits, [2] a trimeric PSI with a reduced number of subunits, and [3] the full, trimeric wild-type PSI complex. We only partially succeeded with the first two structures, but we successfully produced the trimeric PSI structure at 2.5?Å resolution. This structure was comparable to that of the thermophilic species, but we provided more detail. The PSI trimeric supercomplex consisted of 33 protein subunits, 72 carotenoids, 285 chlorophyll a molecules, 51 lipids, 9 iron-sulfur clusters, 6 plastoquinones, 6 putative calcium ions, and over 870 water molecules.This study showed that the structure of the PSI in Synechocystis sp. PCC6803 differed from previously described PSI structures. These findings have broadened our understanding of PSI structure.     

A Novel Bio-Sensor Based on DNA Strand Displacement

DNA strand displacement technology performs well in sensing and programming DNA segments. In this work, we construct DNA molecular systems based on DNA strand displacement performing computation of logic gates. Specifically, a class of so-called “DNA neurons” are achieved, in which a “smart” way inspired by biological neurons encoding information is developed to encode and deliver information using DNA molecules. The “DNA neuron” is bistable, that is, it can sense DNA molecules as input signals, and release “negative” or “positive” signals DNA molecules. We design intelligent DNA molecular systems that are constructed by cascading some particularly organized “DNA neurons”, which could perform logic computation, including AND, OR, XOR logic gates, automatically. Both simulation results using visual DSD (DNA strand displacement) software and experimental results are obtained, which shows that the proposed systems can detect DNA signals with high sensitivity and accretion; moreover, the systems can process input signals automatically with complex nonlinear logic. The method proposed in this work may provide a new way to construct a sensitive molecular signal detection system with neurons spiking behavior in vitro, and can be used to develop intelligent molecular processing systems in vivo.     

Relationship between biosynthesis of carotenoids and increasing complexity of photosystem I in mutant C-6D of Scenedesmus obtiquus

Dark-grown cells of the pigment mutant C-6D of Scenedesmus obliquus, strain D₃ (Gaffron 1939), contain only chlorophyll (Chl) a and carotenoid precursors. In these cells a functioning photosystem I (PSI) of basic structure was characterised by a high PSI activity and a low Chl/P700 ratio. The reaction-center complex of PSI (CPI) was shown to exist in the dark-grown cells. These findings demonstrate that the assembly of the core complex of PSI and its function are independent of the presence of carotenoids. Upon illumination, carotenoids, Ch1 b and additional Chl a were synthesized. Newly formed -carotene was shown by pigment analysis using high-performance liquid chromatography (HPLC) to be incorporated into CPI. Parallel to this process a shift of the long-wavelength fluorescence emission of PSI from 712–714 to 718–719 nm was observed. In the later stages of chloroplast differentiation, when xanthophylls and Chl b were synthesized, a higher-molecular-weight complex of PSI (CPIa) could be isolated. Pigment analysis demonstrated that CPIa contained xanthophylls and Chl b in addition to Chl a and -carotene. This indicates the formation of a light-harvesting antenna closely associated with PSI (LHCI). The addition of an LHCI to the reaction-center complex of PSI caused an increase in the absorption cross-section of PSI as shown by action spectroscopy and in-vivo fluorescence measurements. A model demonstrating the changes in the molecular organization of PSI during light-induced carotenoid biosynthesis in mutant C-6D of Scenedesmus obliquus is presented.Abbreviations Chl chlorophyll - CP chlorophyll-protein complex - LHC light-harvesting complex - HPLC high-performance liquid chromatography - PSI, II photosystem I, II - PAGE polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft and a scholarship of the Studienstiftung des deutschen Volkes to S. Römer. We thank Ms. K. Bölte for technical assistance and Mr. H. Becker for drafting the figures.     

Isolation of photosystem I complexes from octyl glucoside/sodium dodecyl sulfate solubilized spinach thylakoids : characterization and reconstitution into liposomes

We have used the nonionic detergent octyl-β-d-glucopyranoside in combination with sodium dodecyl sulfate to isolate two novel Photosystem I (PSI) complexes from spinach (Spinacea oleracea L.) thylakoid membranes. These complexes have been characterized as to their spectral properties, content of PSI reaction center chlorophyll P₇₀₀, and protein composition. PSI-B, purified from solubilized membranes by sucrose density gradient centrifugation, is a putative native PSI complex. PSI-B contains four polypeptides between 21 and 25 kilodaltons in addition to the components of the PSI antenna complex (LHCI); three of these polypeptides have not previously been associated with PSI. A second complex, CPI^*, is purified from octyl glucoside/sodium dodecyl sulfate solubilized thylakoids by two cycles of preparative gel electrophoresis under mildly denaturing conditions. Electrophoresis under these conditions releases a discrete set of polypeptides from PSI producing a complex composed only of the PSI reaction center and the LHCI antenna.     

Action of Deflocculating Agents on Saccharomyces cerevisiae Class III Brewer''s Yeast

Earlier work with a fluorescent aid indicated that flocculent brewer's yeast may have more surface lipids than nonflocculent types. Organic solvents were checked against flocculent Gilliland yeasts. It was found that those reagents which affect “free” lipids had no dispersive action, and those which remove “bound” fats had a powerful dispersive action against such yeasts. There was no indication that such an action could be correlated with other physical properties of the solvents. The uranyl ion is known for its ability to complex with phospholipids, and it was found to have a powerful dispersive action on Gilliland yeasts. Its effect was compared with that of glucose in its dispersion of yeast flocs, and possible cell “sites” were suggested. This, along with other work, suggests the possibility that lipids are directly or indirectly involved in yeast flocculation.     

Genetic and genomic analysis of a fat mass trait with complex inheritance reveals marked sex specificity

The integration of expression profiling with linkage analysis has increasingly been used to identify genes underlying complex phenotypes. The effects of gender on the regulation of many physiological traits are well documented; however, “genetical genomic” analyses have not yet addressed the degree to which their conclusions are affected by sex. We constructed and densely genotyped a large F2 intercross derived from the inbred mouse strains C57BL/6J and C3H/HeJ on an apolipoprotein E null (ApoE^−/−) background. This BXH.ApoE^−/− population recapitulates several “metabolic syndrome” phenotypes. The cross consists of 334 animals of both sexes, allowing us to specifically test for the dependence of linkage on sex. We detected several thousand liver gene expression quantitative trait loci, a significant proportion of which are sex-biased. We used these analyses to dissect the genetics of gonadal fat mass, a complex trait with sex-specific regulation. We present evidence for a remarkably high degree of sex-dependence on both the cis and trans regulation of gene expression. We demonstrate how these analyses can be applied to the study of the genetics underlying gonadal fat mass, a complex trait showing significantly female-biased heritability. These data have implications on the potential effects of sex on the genetic regulation of other complex traits.     

Micro-Macro Analysis of Complex Networks

Complex systems have attracted considerable interest because of their wide range of applications, and are often studied via a “classic” approach: study a specific system, find a complex network behind it, and analyze the corresponding properties. This simple methodology has produced a great deal of interesting results, but relies on an often implicit underlying assumption: the level of detail on which the system is observed. However, in many situations, physical or abstract, the level of detail can be one out of many, and might also depend on intrinsic limitations in viewing the data with a different level of abstraction or precision. So, a fundamental question arises: do properties of a network depend on its level of observability, or are they invariant? If there is a dependence, then an apparently correct network modeling could in fact just be a bad approximation of the true behavior of a complex system. In order to answer this question, we propose a novel micro-macro analysis of complex systems that quantitatively describes how the structure of complex networks varies as a function of the detail level. To this extent, we have developed a new telescopic algorithm that abstracts from the local properties of a system and reconstructs the original structure according to a fuzziness level. This way we can study what happens when passing from a fine level of detail (“micro”) to a different scale level (“macro”), and analyze the corresponding behavior in this transition, obtaining a deeper spectrum analysis. The obtained results show that many important properties are not universally invariant with respect to the level of detail, but instead strongly depend on the specific level on which a network is observed. Therefore, caution should be taken in every situation where a complex network is considered, if its context allows for different levels of observability.     

Problems Related to the Use of Food Additives in the United States

Major problems encountered by enforcement agencies and by regulated industries, respectively, in implementing and conforming to recent food additive laws are reviewed. Decisions as to which substances fall within the broad terms of the legal definition, and which escape by virtue of “generally recognized as safe” (GRAS) status, are often difficult and complex. Distinctions cannot be made solely on the basis of whether substances are old or new, natural or synthetic. Registration of pesticides on a “no residue” basis and establishment of “zero tolerances” for food additives have created an anomalous situation as a result of improvements in sensitivity of analytical techniques which revealed the presence of minute amounts of substances where none were believed to exist. A solution has been recommended by a specially appointed committee of the National Academy of Sciences-National Research Council (U.S.A.). Enforcement of the new food additive laws warrants revision of present labelling requirements to provide for designating chemical ingredients by functional categories rather than by confusing chemical terminology.     

Action of Deflocculating Agents on Saccharomyces cerevisiae Class III Brewer's Yeast

Earlier work with a fluorescent aid indicated that flocculent brewer''s yeast may have more surface lipids than nonflocculent types. Organic solvents were checked against flocculent Gilliland yeasts. It was found that those reagents which affect “free” lipids had no dispersive action, and those which remove “bound” fats had a powerful dispersive action against such yeasts. There was no indication that such an action could be correlated with other physical properties of the solvents. The uranyl ion is known for its ability to complex with phospholipids, and it was found to have a powerful dispersive action on Gilliland yeasts. Its effect was compared with that of glucose in its dispersion of yeast flocs, and possible cell “sites” were suggested. This, along with other work, suggests the possibility that lipids are directly or indirectly involved in yeast flocculation.