Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo

Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation.     

Thermoluminescence and P700 redox kinetics as complementary tools to investigate the cyclic/chlororespiratory electron pathways in stress conditions in barley leaves

Cyclic electron flow around photosystem I drives additional proton pumping into the thylakoid lumen, which enhances the protective non-photochemical quenching and increases ATP synthesis. It involves several pathways activated independently. In whole barley leaves, P700 oxidation under far-red illumination and subsequent P700(+) dark reduction kinetics provide a major probe of the activation of cyclic pathways. Two 'intermediate' and 'slow' exponential reduction phases are always observed and they become faster after high light illumination, but dark inactivation of the Benson-Calvin cycle causes the emergence of both a transient in the P700 oxidation and a 'fast' phase in the P700(+) reduction. We investigate here the afterglow (AG) thermoluminescence emission as another tool to detect the activation of cyclic electron pathways from stroma reductants to the acceptor side of photosystem II. This transfer is activated by warming, yielding an AG band at about 45°C. However, treatments that accelerate the 'intermediate' and 'slow' P700(+) reduction phases (brief anoxia, hexose infiltration, fast dehydration of excised leaves) also produced a downshift of this AG band. This pathway ascribable to NADPH dehydrogenase (NDH) would be triggered by a deficit in ATP, while the 'fast' reduction phase corresponding to the ferredoxin plastoquinone reductase pathway is triggered by an overreduction of the photosystem I acceptor pool and is undetected in thermoluminescence. Contrastingly, slow dehydration of unwatered plants did not cause faster reduction of P700(+) nor temperature downshift of the AG band, that is no induction of the NDH pathway, whereas an increased intensity of the AG band indicated a strong NADPH + ATP assimilatory potential.     

Effects of dark- and light-induced proton gradients in thylakoids on the Q and B thermoluminescence bands

Thermoluminescence experiments have been carried out to study the effect of a transmembrane proton gradient on the recombination properties of the S₂ and S₃ states of the oxygen evolving complex with Q_A ^- and Q_B ^-, the reduced electron acceptors of Photosystem II. We first determined the properties of the S₂Q_A ^- (Q band), S₂Q_B ^- and S₃Q_B ^- (B bands) recombinations in the pH range 5.5 to 9.0, using uncoupled thylakoids. The, a proton gradient was created in the dark, using the ATP-hydrolase function of ATPases, in coupled unfrozen thylakoids. A shift towards low temperature of both Q and B bands was observed to increase with the magnitude of the proton gradient measured by the fluorescence quenching of 9-aminoacridine. This downshift was larger for S₃Q_B ^- than for S₂Q_B ^- and it was suppressed by nigericin, but not by valinomycin. Similar results were obtained when a proton gradient was formed by photosystem I photochemistry. When Photosystem II electron transfer was induced by a flash sequence, the reduction of the plastoquinone pool also contributed to the downshift in the absence of an electron acceptor. In leaves submitted to a flash sequence above 0°C, a downshift was also observed, which was supressed by nigericin infiltration. Thus, thermoluminescence provides direct evidence on the enhancing effect of lumen acidification on the S₃S₂ and S₂S₁ reverse-transitions. Both reduction of the plastoquinone pool and lumen acidification induce a shift of the Q and B bands to lower temperature, with a predominance of lumen acidification in non-freezing, moderate light conditions.Abbreviations 9-AA 9-aminoacridine - E_A activation energy - F₀ constant fluorescence level - F_M maximum fluorescence, when all PS-II centers are closed - F_V variable fluorescence (F_M–F₀) - PS I, PS II Photosystem I, photosystem II - PQ plastoquinone - TL thermoluminescence     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Chlorophyll thermoluminescence of leaf discs: simple instruments and progress in signal interpretation open the way to new ecophysiological indicators

Luminescence from photosynthetic material observed in darkness following illumination is a delayed fluorescence produced by a recombination of charge pairs stored in photosystem II, i.e. the back-reaction of photosynthetic charge separation. Thermoluminescence (TL) is a technique consisting of a rapid cooling followed by the progressive warming of a preilluminated sample to reveal the different types of charge pairs as successive emission bands, which are resolved better than the corresponding decay phases recorded at constant temperatures. Progress in thermoelectric Peltier elements and in compact light detectors made the development of simple, affordable and transportable instruments possible. These instruments take advantage of multifurcated light guides for combined TL, fluorescence and absorbance/reflectance measurements. Meanwhile, experiments on unfrozen leaf discs, with excitation by single turn-over flashes or far red light and infiltration by specific inhibitors/uncouplers, have led to a better understanding of in vivo TL signals. Much like chlorophyll fluorescence and in a complementary way, TL in the 0-60 degrees C temperature range not only informs on the state of photosystem II in leaf tissues and its possible alterations, but also gives a broader insight into the energetic state inside the chloroplast by probing (1) the light-induced or dark-stable thylakoid proton gradient through the protonation of the Mn oxygen-evolving complex, (2) the induction of cyclic/chlororespiratory electron flow towards the plastoquinone pool, (3) the [NADPH+ATP] assimilatory potential. By a different mechanism, warming above 60 degrees C without preillumination reveals chemiluminescence high temperature thermoluminescence (HTL) bands due to the radiative thermolysis of peroxides, which are indicators of oxidative stress in leaves.     

A Mutation in the D-de Loop of D1 Modifies the Stability of the S2QA- and S2QB- States in Photosystem II

Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three site-specific mutants of the D1 polypeptide of Synechocystis PCC 6803: E243K, E229D, and CA1 (deletion of three glutamates 242-244 and a substitution, glutamine-241 to histidine). The phenotypes of the E229D and E243K mutants were similar to that of the control strain (AR) in all of the studied aspects. The characteristics of CA1 were very different. Formate, which inhibits the QA- to QB- reaction, was severalfold less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, G. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson [1993] Plant Mol Biol 22: 1-12). A change in the redox potential of the QA/QA- couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA1. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D1 could influence the properties of the couple QA/QA- in D2 and the mechanism of recovery from photoinhibition.