Proton Gradient Regulation5-Like1-Mediated Cyclic Electron Flow Is Crucial for Acclimation to Anoxia and Complementary to Nonphotochemical Quenching in Stress Adaptation

To investigate the functional importance of Proton Gradient Regulation5-Like1 (PGRL1) for photosynthetic performances in the moss Physcomitrella patens, we generated a pgrl1 knockout mutant. Functional analysis revealed diminished nonphotochemical quenching (NPQ) as well as decreased capacity for cyclic electron flow (CEF) in pgrl1. Under anoxia, where CEF is induced, quantitative proteomics evidenced severe down-regulation of photosystems but up-regulation of the chloroplast NADH dehydrogenase complex, plastocyanin, and Ca²⁺ sensors in the mutant, indicating that the absence of PGRL1 triggered a mechanism compensatory for diminished CEF. On the other hand, proteins required for NPQ, such as light-harvesting complex stress-related protein1 (LHCSR1), violaxanthin de-epoxidase, and PSII subunit S, remained stable. To further investigate the interrelation between CEF and NPQ, we generated a pgrl1 npq4 double mutant in the green alga Chlamydomonas reinhardtii lacking both PGRL1 and LHCSR3 expression. Phenotypic comparative analyses of this double mutant, together with the single knockout strains and with the P. patens pgrl1, demonstrated that PGRL1 is crucial for acclimation to high light and anoxia in both organisms. Moreover, the data generated for the C. reinhardtii double mutant clearly showed a complementary role of PGRL1 and LHCSR3 in managing high light stress response. We conclude that both proteins are needed for photoprotection and for survival under low oxygen, underpinning a tight link between CEF and NPQ in oxygenic photosynthesis. Given the complementarity of the energy-dependent component of NPQ (qE) and PGRL1-mediated CEF, we suggest that PGRL1 is a capacitor linked to the evolution of the PSII subunit S-dependent qE in terrestrial plants.The conversion of solar energy into chemical energy and building material by oxygenic photosynthesis, as performed by plants, green algae, and cyanobacteria, supports much of the life on our planet. The production of oxygen and the assimilation of carbon dioxide into organic matter determines, to a large extent, the composition of our atmosphere. Plant photosynthesis is achieved thanks to a series of reactions that occur mainly in the chloroplast, resulting in light-dependent water oxidation, NADP⁺ reduction, and ATP formation (Whatley et al., 1963). Two separate photosystems (PSI and PSII) and an ATP synthase (ATPase) embedded in the thylakoid membrane catalyze these reactions. The ATPase produces ATP at the expense of the proton motive force that is generated by the light reactions (Mitchell, 1961). The cytochrome (cyt) b₆f complex assures the link between the two photosystems by transferring electrons from the membrane-bound plastoquinone to a soluble carrier, plastocyanin, or cyt c₆ and functions in the pumping of protons. NADPH and ATP that are produced by linear electron flow from PSII to PSI are fueled into the Calvin Benson Bassham cycle (Bassham et al., 1950) to fix CO₂. In parallel, cyclic electron flow (CEF) between the cyt b₆f complex and PSI may occur, which would solely lead to the production of ATP. CEF around PSI has been first recognized by Arnon (1959) and is involved in the reequilibration of the ATP poise and prevention of overreduction of the PSI acceptor side (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). In microalgae and vascular plants, CEF operates via an NAD(P)H dehydrogenase-like complex (NDH)-dependent and/or PROTON GRADIENT REGULATION5 (PGR5)-related pathway (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). The thylakoid protein Proton Gradient Regulation5-Like1 (PGRL1; DalCorso et al., 2008) has been first discovered as a novel component for the PGR5-dependent CEF pathway in Arabidopsis (Arabidopsis thaliana), as its knockout causes a PGR5-like photosynthetic phenotype and is suggested to operate as a ferredoxin-plastoquinone reductase (Hertle et al., 2013). PGRL1 is also important for efficient CEF in the green alga Chlamydomonas reinhardtii, which becomes particularly evident under settings where CEF is induced, such as in acclimation to iron deficiency, high light (HL), or anaerobic growth conditions (Petroutsos et al., 2009; Iwai et al., 2010; Tolleter et al., 2011; Terashima et al., 2012). Remarkably, a CEF protein supercomplex composed of PSI-light-harvesting complex I (LHCI), LHCII, the cyt b₆f complex, ferredoxin-NADPH oxidoreductase, and PGRL1 was isolated from state 2 conditions (Iwai et al., 2010). Under anaerobic conditions, the Ca²⁺ sensor CAS and Anaerobic response1 (ANR1) were shown to interact with PGRL1 in vivo (Terashima et al., 2012) and were found to be associated with the C. reinhardtii CEF supercomplex. Consistently, depletion of CAS and ANR1 by artificial microRNA expression in C. reinhardtii resulted in strong inhibition of CEF under anoxia, which could be partially rescued by an increase in the extracellular Ca²⁺ concentration, inferring that CEF is Ca²⁺ dependent (Terashima et al., 2012). Notably, the regulation of the proton motive force by a two-pore potassium channel in the thylakoid membrane of Arabidopsis (AtTPK3), is also Ca²⁺ dependent (Carraretto et al., 2013), suggesting that Ca²⁺-dependent activation of CEF and the channel may work hand in hand.qE, the energy-dependent component of nonphotochemical quenching (NPQ) that occurs due to thermal dissipation of excess absorbed light energy (Li et al., 2000; Peers et al., 2009), is dependent on rapid luminal acidification upon photosynthetic electron transfer (Wraight and Crofts, 1970; Li et al., 2000). Thus, processes such as CEF that contribute to the pH gradient across the thylakoid membrane are interrelated to NPQ, as an acidified lumen is required for efficient qE (Joliot and Finazzi, 2010). In vascular plants, PSII subunit S (PSBS) is essential for efficient qE (Li et al., 2000), whereas qE induction in the green alga C. reinhardtii is mediated by light-harvesting complex stress-related protein3 (LHCSR3), an ancient light-harvesting protein that is missing in vascular plants (Peers et al., 2009). The moss Physcomitrella patens, which possesses genes encoding for PSBS and LHCSR proteins, utilizes both types of regulatory proteins to operate qE (Alboresi et al., 2010), suggesting that land plants evolved a novel PSBS-dependent qE mechanism before losing the ancestral LHCSR-dependent qE found in algae. This makes mosses a very interesting subject for investigating the interrelation and evolution of the CEF and NPQ molecular effectors.Mosses diverged from vascular plants early after land colonization and are one of the oldest groups of land plants present on earth. This places the moss model system P. patens (Rensing et al., 2008) evolutionarily in the middle between algae and vascular plants and makes it an ideal model organism for the study of the evolution of photosynthetic organisms. Analysis of photosynthesis in P. patens can provide insights into the events leading to adaptation to the harsher physicochemical conditions of the terrestrial environment (Rensing et al., 2008), as evidenced by the presence and functional overlap of LHCSRs and PSBS (Alboresi et al., 2010).To obtain insights into the interrelation and evolution of CEF and NPQ, we knocked out the PGRL1 gene from P. patens and analyzed functional phenotypic consequences. Moreover, we compared these phenotypes with phenotypic analyses of C. reinhardtii pgrl1, npq4, and pgrl1 npq4 single and double mutants lacking PGRL1, LHCSR3, and both PGRL1 and LHCSR3, respectively. The data provided strong evidence that the green cut protein PGRL1 (Karpowicz et al., 2011) is required for acclimation to anoxia both in algae and mosses. Moreover, an involvement of PGRL1 in the evolution of PSBS-dependent qE in terrestrial plants is implied.     

A Novel Pex14 Protein-interacting Site of Human Pex5 Is Critical for Matrix Protein Import into Peroxisomes

Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14. A systematic screening of a Pex5 peptide library by ligand blot analysis revealed a novel Pex5-Pex14 interaction site of Pex5. The novel motif composes the sequence LVAEF with the evolutionarily conserved consensus sequence LVXEF. Replacement of the amino acid LVAEF sequence by alanines strongly affects matrix protein import into peroxisomes in vivo. The NMR structure of a complex of Pex5-(57–71) with the Pex14-N-terminal domain showed that the novel motif binds in a similar α-helical orientation as the WXXX(F/Y) motif but that the tryptophan pocket is now occupied by a leucine residue. Surface plasmon resonance analyses revealed 33 times faster dissociation rates for the LVXEF ligand when compared with a WXXX(F/Y) motif. Surprisingly, substitution of the novel motif with the higher affinity WXXX(F/Y) motif impairs protein import into peroxisomes. These data indicate that the distinct kinetic properties of the novel Pex14-binding site in Pex5 are important for processing of the peroxisomal targeting signal 1 receptor at the peroxisomal membrane. The novel Pex14-binding site may represent the initial tethering site of Pex5 from which the cargo-loaded receptor is further processed in a sequential manner.     

Female Remating Behavior in a Lekking Moth

In polyandrous species, male reproductive success will at least partly be determined by males' success in sperm competition. To understand the potential for post‐mating sexual selection, it is therefore important to assess the extent of female remating. In the lekking moth Achroia grisella, male mating success is strongly determined by female choice based on the attractiveness of male ultrasonic songs. Although observations have indicated that some females will remate, only little is known about the level of sperm competition. In many species, females are more likely to remate if their first mating involved an already mated male than if the first male was virgin. Potentially, this is because mated males are less well able to provide an adequate sperm supply, nutrients, or substances inhibiting female remating. This phenomenon will effectively reduce the strength of pre‐copulatory sexual selection because attractive males with high mating success will be more susceptible to sperm competition. We therefore performed an experiment designed both to provide a more precise estimate of female remating probability and simultaneously to test the hypothesis that female remating is influenced by male mating history. Overall, approximately one of five females remated with a second male. Yet, although females mated to non‐virgin males were somewhat more prone to remate, the effect of male mating history was not significant. The results revealed, however, that heavier females were more likely to remate. Furthermore, we found that females' second copulations were longer, suggesting that, in accordance with theory, males may invest more sperm in situations with an elevated risk of sperm competition.     

In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells

An efficient isolation of mitochondria from cells under physiological conditions is crucial for many studies in life sciences but still challenging in many cases such as in metabolic characterization of mitochondria. In this work, four methods for the disruption of Chinese hamster ovary cells were evaluated regarding their influence on mitochondrial integrity and yield. After cell disruption, mitochondria released from cells were separated from the remaining cell homogenate by differential centrifugation. Sonication was shown to be a rapid and sensitive isolation method. Yields of 14.0 ± 0.3 mg raw mitochondrial protein per 10⁸ cells were obtained. The mitochondria were morphologically intact, with membrane integrities of 67% (outer membrane) to 94% (inner membrane). Compared with the methods using Dounce homogenization, digitonin permeabilization, or electroporation for cell disruption the ultrasound method provided the highest yield of isolated mitochondria. Furthermore, this method is rapid (≈ 45 s for disruption), more robust than Dounce homogenization regarding their influence on mitochondrial integrity and especially suitable for preparing a relatively large amount of mitochondria. The results of this work can be helpful for quantitative and dynamic studies of molecular processes related to mitochondria under physiological conditions for many questions in both biomedicine and biotechnology.     

Evaluation of the "Cellscreen" system for proliferation studies on liver progenitor cells

Proliferation studies on mammalian cells have been disadvantaged by the limited availability of non-invasive assays as the majority of approaches are based on chemical treatment, sampling or staining of cells removed from culture. In this study, we utilised the Cellscreen system (Innovatis AG, Bielefeld, Germany), a non-invasive automated technique for measuring proliferation of adherent and suspension cells over time. We have evaluated the ability of the Cellscreen system to monitor and quantify growth of adherent liver progenitor cells over time and tested several applications, (i) serum reduction or (ii) treatment with a cytokine. Our results demonstrate that the Cellscreen system reproducibly documents pro- and anti-proliferative effects of cytokines and growth factors and quantifies changes by providing cell-doubling times for control and test cultures. However, we found that for the conversion of cell density values into absolute cell numbers different conversion factors, which better suit the individual growth phases, need to be established. Collectively, these findings reveal that the Cellscreen system is applicable for the determination of cell proliferation of adherent and suspension cells in response to a variety of (growth) factors. It minimises operator participation and thus enables more rapid and larger screens and, being non-invasive, permits multiple assays on the same culture of cells. Hence, this technique proves superior to the common proliferation assays opening up new dimensions of proliferation studies in cell biology.     

Assessment of sources of uncertainty in macrophyte surveys and the consequences for river classification

The application of macrophytes in freshwater monitoring is still relatively limited and studies on their intercalibration and sources of variation are required. Therefore, the aim of the study was to compare selected indices and metrics based on macrophytes and to quantify their variability. During the STAR project, several aspects influencing uncertainty in estimation of the ecological quality of river were assessed. Results showed that several metrics based on the indicative value of plant species can be used in evaluation of the ecological status of rivers. Among estimated sources of variance in metric values the inter-surveyor differences had the lowest effect and slightly stronger were the influences of temporal variation (years and seasons) and shading. The impact of habitat modification was the most important factor. Analysis showed that some of macrophyte-based metrics (notably MTR and IBMR) are of sufficient precision in terms of sampling uncertainty, that they could be useful for estimating the ecological status of rivers in accordance with the aims of the Water Framework Directive.     

Separase: a universal trigger for sister chromatid disjunction but not chromosome cycle progression

Separase is a protease whose liberation from its inhibitory chaperone Securin triggers sister chromatid disjunction at anaphase onset in yeast by cleaving cohesin's kleisin subunit. We have created conditional knockout alleles of the mouse Separase and Securin genes. Deletion of both copies of Separase but not Securin causes embryonic lethality. Loss of Securin reduces Separase activity because deletion of just one copy of the Separase gene is lethal to embryos lacking Securin. In embryonic fibroblasts, Separase depletion blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication. Thus, fibroblasts lacking Separase become highly polyploid. Hepatocytes stimulated to proliferate in vivo by hepatectomy also become unusually large and polyploid in the absence of Separase but are able to regenerate functional livers. Separase depletion in bone marrow causes aplasia and the presumed death of hematopoietic cells other than erythrocytes. Destruction of sister chromatid cohesion by Separase may be a universal feature of mitosis in eukaryotic cells.     

Site-specific attachment of polyethylene glycol-like oligomers to proteins and peptides

Modification of proteins with polymers is a viable method to tune protein properties, e.g., to render them more water-soluble by using hydrophilic polymers. We have utilized precision-length, polyethylene glycol-based oligomers carrying a thioester moiety in transthioesterification and native chemical ligation reactions with internal and N-terminal cysteine residues in proteins and peptides. These reactions lead to uniquely modified proteins with an increased solubility in chaotrope- and detergent-free aqueous systems. Polymer modification of internal cysteines is fully reversible and allows generation of stable protein-polymer conjugates for enzymatic manipulations as demonstrated by proteolytic cleavage of a protein construct that was only soluble in buffers incompatible with protease activity before polymer modification. The permanent polymer modification of a Rab protein at its N-terminal cysteine produced a fully active Rab variant that was efficiently prenylated. Thus, PEGylation of prenylated proteins might be a viable route to increase water solubility of such proteins in order to carry out experiments in detergent- and lipid-free systems.