Utilization of nitrogen in legume-based mobile green manures stored as compost or silage

The utilization of nitrogen (N) in green manure leys can be improved by harvesting, storage and spreading of the plant material as manure in other crops. By green manure storage as silage, storage losses of N are lower than by composting. Also, a relatively high fertilizer value of silage N is achievable depending on the C/N ratio of the material. Nitrogen availability in green manure leys is higher after storage as silage compared to composting. Use of mobile green manures is mainly relevant in arable cropping systems without livestock where utilization of the roughage for animal feed or biogas production is impossible, as costs for ley/roughage harvest and transport can be relatively high. Our study showed that surface application of green manure silage to growing crops can damage plants and is therefore not recommended, whereas incorporation of silage before sowing has significant positive effects on crop yields

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

The promiscuous activity of the enzyme o-succinylbenzoate synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both density functional theory and semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylamino acids, with N-succinyl-R-phenylglycine being the best substrate. We investigated the molecular mechanisms for both reactions exploring the potential energy surface. Then, molecular dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.This work was supported by the Spanish Ministerio de Economia y Competitividad project CTQ2012-36253-C03-03 ́ and FEDER funds. K.S. thanks the Polish National Science Center (NCN) for Grant 2011/02/A/ST4/00246. The authors acknowledge computational facilities of the Servei d’Informatica ̀ de la Universitat de Valencia in the ̀ “Tirant” supercomputer, which is part of the Spanish Supercomputing Network

Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination

Plant ARGONAUTEs: Features, Functions and Unknowns

ARGONAUTEs (AGOs) are the effector proteins in eukaryotic small RNA(sRNA)– based gene silencing pathways controlling gene expression and transposon activity. In plants, AGOs regulate key biological processes such as development, response to stress, genome structure and integrity, and pathogen defense. Canonical functions of plant AGO–sRNA complexes include the endonucleolytic cleavage or translational inhibition of target RNAs, and the methylation of target DNAs. Here, I provide a brief update on the major features, molecular functions and biological roles of plant AGOs. A special focus is given to the more recent discoveries related to emerging molecular or biological functions of plant AGOs, as well as to the major unknowns in the plant AGO field.This work was supported by an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 655841 to A.C.Carbonell Olivares, A. (2017). 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Antiviral Silencing and Suppression of Gene Silencing in Plants

RNA silencing is an evolutionary conserved sequence-specific gene inactivation mechanism that contributes to the control of development, maintains heterochromatin, acts in stress responses, DNA repair and defends against invading nucleic acids like transposons and viruses. In plants RNA silencing functions as one of the main immune systems. RNA silencing process involves the small RNAs and trans factor components like Dicers, Argonautes and RNA-dependent RNA poly- merases. To deal with host antiviral silencing responses viruses evolved mecha- nisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Due to the overlap between endogenous and antiviral silencing pathways while blocking antiviral pathways viruses also impact endogenous silencing processes. Here we provide an overview of antiviral silencing pathway, host factors implicated in it and the crosstalk between antiviral and endogenous branches of silencing. We summarize the current status of knowledge about the viral counter-defense strategies acting at various steps during virus infection in plants with the focus on representative, well studied silencing suppres- sor proteins. Finally we discuss future challenges of the antiviral silencing and counter-defense research field

Basic Density And Strength Properties Of Pines In Uganda

Basic density and strength properties and their variations in 25 year - old Pinus caribaea, P. oocarpa and P. kesiya grown in Katugo, Uganda were investigated. Six trees for each species were selected representing three diameter classes. The trees were felled after measuring their diameter at breast height. Three disks were cut from each tree at 1.3 m, 45% and 75% of total tree height for growth ring width and basic density assessment. For strength properties determination, a central plank measuring 65 mm thick was extracted from each sample tree starting at breast height upwards. Basic density was determined using water displacement. Tests of various strength properties were done in accordance with standard procedures. The data were analyzed using the Statistical Analysis System. Results for the studied properties of Pinus caribaea, P. kesiya and P. oocarpa respectively were as follows: Basic density: 424, 431,and 444 kg/m3; Modulus of rupture: 54.51, 56.31 and 60.69 N/mm2; Modulus of elasticity: 8590,8752 and 8825 N/mm2; Work to maximum load: 0.088. 0.099 and 0.101 mmN/mm3; Maximum compression strength: 33.38, 33.83and 34.66 N/mm2; Maximum shear strength: 9.43, 9.80 and 10.37 N/mm2 and cleavage: 11.79,12.15 and 12.48 n/mm. There were no significant differences in all wood properties between the three species. Between tree variations in wood properties were statistically insignificant (

Comparison of <i>Alicyclobacillus acidocaldarius</i> <i>o</i>‑Succinylbenzoate Synthase to Its Promiscuous <i>N</i>‑Succinylamino Acid Racemase/<i>o</i>‑Succinylbenzoate Synthase Relatives

Studying the evolution of catalytically promiscuous enzymes like those from the <i>N</i>-succinylamino acid racemase/<i>o</i>-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, <i>Alicyclobacillus acidocaldarius</i> OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds <i>N</i>-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 10² M^–1 s^–1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects <i>k</i>_cat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily