Enzymatic methylation of DNA and poly(dG-dC) X poly(dG-dC) modified by 4-acetoxyaminoquinoline-1-oxide, the ultimate carcinogen of 4-nitroquinoline-1-oxide

Both the initial velocity and the overall methylation of Ac-4HAQO modified DNA by a calf brain DNA (cytosine-5-)-methyltransferase are increased as compared to native DNA. The affinity of the modified DNA for the enzyme decreases as a function of the extent of the modification. Heat-denatured, single-stranded DNA shows exactly the opposite results: the more it is modified, the less it is methylated. The poly(dG-dC) X poly(dG-dC) modified by 4NQO is as well methylated as the non-modified one. The carcinogen may induce a tertiary structure favouring the 'walking' of the enzyme along the DNA. The hypermethylation caused by this carcinogen could have a significance in gene activity and cellular differentiation.     

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP⁺ oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments.Unicellular photosynthetic organisms such as the green alga Chlamydomonas reinhardtii frequently experience anoxic conditions in their natural habitat, especially during the night when the microbial community consumes the available oxygen. Under anoxia, lack of ATP synthesis by F₁F_O ATP synthase (EC 3.6.3.14) due to the absence of mitochondrial respiration is compensated by the activity of various plant- and bacterial-type fermentative enzymes that drive a sustained glycolytic activity (Mus et al., 2007; Terashima et al., 2010; Grossman et al., 2011; Yang et al., 2014). In C. reinhardtii, upstream glycolytic enzymes, including the reversible glyceraldehyde 3-P dehydrogenase, are located in the chloroplast (Johnson and Alric, 2012). This last enzyme is shared by the glycolysis (oxidative activity) and the Calvin-Benson-Bassham (CBB) cycle (reductive activity; Johnson and Alric, 2013). In dark anoxic conditions, the CBB cycle is inactive, thus avoiding wasteful using up of available ATP and depletion of the required intermediates for glycolysis. On the other side, ability of microalgae to perform photosynthetic carbon fixation when transferred from dark to light in the absence of oxygen might also be critical for adaptation to their environment. In such conditions, not only the linear electron flow (LEF) to Rubisco, but also alternative electron flow (AEF) toward oxygen (chlororespiration, Mehler reaction, and mitochondrial respiration; for review, see Miyake, 2010; Peltier et al., 2010; Cardol et al., 2011) is impaired. Thus, cells need to circumvent a paradoxical situation: the activity of the CBB cycle requires the restoration of the cellular ATP, but the chloroplastic F₁F_O ATP synthase activity is compromised by the impairment of most of the photosynthetic electron flows that usually generate the proton motive force in oxic conditions. Other AEFs, specific to anoxic conditions, should therefore be involved to promote ATP synthesis without net synthesis of NADPH and explain the light-induced restoration of CBB cycle activity.Among enzymes expressed in anoxia, the oxygen-sensitive hydrogenases (HYDA1 and HYDA2 in C. reinhardtii) catalyze the reversible reduction of protons into molecular hydrogen from the oxidation of reduced ferredoxins (FDXs; Florin et al., 2001). Although hydrogen metabolism in microalgae has been largely studied in the last 15 years in perspective of promising future renewable energy carriers (Melis et al., 2000; Kruse et al., 2005; Ghirardi et al., 2009), the physiological role of such an oxygen-sensitive enzyme linked to the photosynthetic pathway has been poorly considered. The 40-year-old proposal that H₂ evolution by hydrogenase is involved in induction of photosynthetic electron transfer after anoxic incubation (Kessler, 1973; Schreiber and Vidaver, 1974) has been only recently demonstrated in C. reinhardtii. Gas exchange measurements showed that H₂ evolution occurs prior to CO₂ fixation upon illumination (Cournac et al., 2002). At light onset after a prolonged period in dark anoxic conditions, the photosynthetic electron flow is mainly a LEF toward hydrogenase (Godaux et al., 2013), and lack of hydrogenase activity in hydrogenase maturation factor EF (hydEF) mutant strain deficient in hydrogenases maturation (Posewitz et al., 2004) induces a lag in induction of PSII activity (Ghysels et al., 2013). In cyanobacteria, the bidirectional Ni-Fe hydrogenase might also work as an electron valve for disposal of electrons generated at the onset of illumination of cells (Cournac et al., 2004) or when excess electrons are generated during photosynthesis, preventing the slowing of the electron transport chain under stress conditions (Appel et al., 2000; Carrieri et al., 2011). The bidirectional Ni-Fe hydrogenase could also dispose of excess of reducing equivalents during fermentation in dark anaerobic conditions, helping to generate ATP and maintaining homeostasis (Barz et al., 2010). A similar role for hydrogenase in setting the redox poise in the chloroplast of C. reinhardtii in anoxia has been recently uncovered (Clowez et al., 2015).Still, the physiological and evolutionary advantages of hydrogenase activity have not been demonstrated so far, and the mechanism responsible for the cessation of hydrogen evolution remains unclear. In this respect, at least three hypotheses have been formulated: (1) the inhibition of hydrogenase by O₂ produced by water photolysis (Ghirardi et al., 1997; Cohen et al., 2005), (2) the competition between ferredoxin-NADP⁺ oxidoreductase (FNR) and hydrogenase activity for reduced FDX (Yacoby et al., 2011), and (3) the inhibition of electron supply to hydrogenases by the proton gradient generated by another AEF, the cyclic electron flow around PSI (PSI-CEF; Tolleter et al., 2011). First described by Arnon (1955), PSI-CEF consists in a reinjection of electrons from reduced FDX or NADPH pool in the plastoquinone (PQ) pool. By generating an additional transthylakoidal proton gradient without producing reducing power, this AEF thus contributes to adjust the ATP/NADPH ratio for carbon fixation in various energetic unfavorable conditions including anoxia (Tolleter et al., 2011; Alric, 2014), high light (Tolleter et al., 2011; Johnson et al., 2014), or low CO₂ (Lucker and Kramer, 2013). In C. reinhardtii, two pathways have been suggested to be involved in PSI-CEF: (1) a type II NAD(P)H dehydrogenase (NDA2; Jans et al., 2008) driving the electrons from NAD(P)H to the PQ pool and (2) a pathway involving Proton Gradient Regulation (PGR) proteins where electrons from reduced FDXs return to the PQ pool or cytochrome b₆f. Not fully understood, this latter pathway comprises at least Proton Gradient Regulation5 (PGR5) and Proton-Gradient Regulation-Like1 (PGRL1) proteins (Iwai et al., 2010; Tolleter et al., 2011; Johnson et al., 2014) and is the major route for PSI-CEF in C. reinhardtii cells placed in anoxia (Alric, 2014).In this work, we took advantage of specific C. reinhardtii mutants defective in hydrogenase activity and PSI-CEF to study photosynthetic electron transfer after a period of dark anoxic conditions. Based on biophysical and physiological complementary studies, we demonstrate that at least hydrogenase activity or PSI-CEF is compulsory for the activity of the CBB cycle and for the survival of the cells submitted to anoxic conditions in their natural habitat.     

First evidence of dinosaurian secondary cartilage in the post-hatching skull of Hypacrosaurus stebingeri (Dinosauria, Ornithischia)

Bone and calcified cartilage can be fossilized and preserved for hundreds of millions of years. While primary cartilage is fairly well studied in extant and fossilized organisms, nothing is known about secondary cartilage in fossils. In extant birds, secondary cartilage arises after bone formation during embryonic life at articulations, sutures and muscular attachments in order to accommodate mechanical stress. Considering the phylogenetic inclusion of birds within the Dinosauria, we hypothesized a dinosaurian origin for this "avian" tissue. Therefore, histological thin sectioning was used to investigate secondary chondrogenesis in disarticulated craniofacial elements of several post-hatching specimens of the non-avian dinosaur Hypacrosaurus stebingeri (Ornithischia, Lambeosaurinae). Secondary cartilage was found on three membrane bones directly involved with masticatory function: (1) as nodules on the dorso-caudal face of a surangular; and (2) on the bucco-caudal face of a maxilla; and (3) between teeth as islets in the alveolar processes of a dentary. Secondary chondrogenesis at these sites is consistent with the locations of secondary cartilage in extant birds and with the induction of the cartilage by different mechanical factors - stress generated by the articulation of the quadrate, stress of a ligamentous or muscular insertion, and stress of tooth formation. Thus, our study reveals the first evidence of "avian" secondary cartilage in a non-avian dinosaur. It pushes the origin of this "avian" tissue deep into dinosaurian ancestry, suggesting the creation of the more appropriate term "dinosaurian" secondary cartilage.     

Seed spillage from grain trailers on road verges during oilseed rape harvest: an experimental survey

Context

Anthropogenic vectors enhance the natural dispersal capacity of plant seeds significantly in terms of quantity and distance. Human-mediated seed dispersal (i.e. anthropochory) greatly increases the dispersal of crop species across agroecosystems. In the case of oilseed rape (OSR), spillage of seeds from grain trailers during harvest has never been quantified.

Methods

Our experimental approach involved establishing 85 seed trap-sites on the road verges of an agricultural area around the grain silo of Selommes (Loir-et-Cher, France). We recorded OSR spillage during harvest and applied a linear model to the data.

Results

The amount of seed spilled was related positively to the area of the OSR fields served by the road, whereas the amount of seed spilled decreased with other variables, such as distance from the trap-site to the verge of the road and to the nearest field.The distance to the grain silo, through local and regional effects, affected seed loss. Local effects from fields adjacent to the road resulted in a cumulative spillage on one-lane roads. On two-lane roads, spillage was nearly constant whatever the distance to the silo due to a mixture of these local effects and of grain trailers that joined the road from more distant fields.From the data, we predicted the number of seeds lost from grain trailers on one road verge in the study area. We predicted a total spillage of 2.05×10⁶ seeds (±4.76×10⁵) along the road length, which represented a mean of 404±94 seeds per m².

Conclusion

Containment of OSR seeds will always be challenging. However, seed spillage could be reduced if grain trailers were covered and filled with less seed. Reducing distances travelled between fields and silos could also limit seed loss.     

The mechanism of cyclic electron flow

Apart from the canonical light-driven linear electron flow (LEF) from water to CO₂, numerous regulatory and alternative electron transfer pathways exist in chloroplasts. One of them is the cyclic electron flow around Photosystem I (CEF), contributing to photoprotection of both Photosystem I and II (PSI, PSII) and supplying extra ATP to fix atmospheric carbon. Nonetheless, CEF remains an enigma in the field of functional photosynthesis as we lack understanding of its pathway. Here, we address the discrepancies between functional and genetic/biochemical data in the literature and formulate novel hypotheses about the pathway and regulation of CEF based on recent structural and kinetic information.