Protective responses of tolerant and sensitive wheat seedlings to systemic and local zearalenone application – Electron paramagnetic resonance studies

Background Mycotoxins are among the environmental stressors whose oxidative action is currently widely studied. The aim of this paper was to investigate the response of seedling leaves to zearalenone (ZEA) applied to the leaves (directly) and to the grains (indirectly) in tolerant and sensitive wheat cultivars. Results Biochemical analyses of antioxidant activity were performed for chloroplasts and showed a similar decrease in this activity irrespective of plant sensitivity and the way of ZEA application. On the other hand, higher amounts of superoxide radical (microscopic observations) were generated in the leaves of plants grown from the grains incubated in ZEA solution and in the sensitive cultivar. Electron paramagnetic resonance (EPR) studies showed that upon ZEA treatment greater numbers of Mn - aqua complexes were formed in the leaves of the tolerant wheat cultivar than in those of the sensitive one, whereas the degradation of Fe-protein complexes occurred independently of the cultivar sensitivity. Conclusion The changes in the quantity of stable, organic radicals formed by stabilizing reactive oxygen species on biochemical macromolecules, indicated greater potential for their generation in leaf tissues subjected to foliar ZEA treatment. This suggested an important role of these radical species in protective mechanisms mainly against direct toxin action. The way the defense mechanisms were activated depended on the method of the toxin application.

Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco

Integration of prokaryotic nitrogen fixation (nif) genes into the plastid genome for expression of functional nitrogenase components could render plants capable of assimilating atmospheric N2 making their crops less dependent of nitrogen fertilizers. The nitrogenase Fe protein component (NifH) has been used as proxy for expression and targeting of Nif proteins within plant and yeast cells. Here we use tobacco plants with the Azotobacter vinelandii nifH and nifM genes integrated into the plastid genome. NifH and its maturase NifM were constitutively produced in leaves, but not roots, during light and dark periods. Nif protein expression in transplastomic plants was stable throughout development. Chloroplast NifH was soluble, but it only showed in vitro activity when isolated from leaves collected at the end of the dark period. Exposing the plant extracts to elevated temperatures precipitated NifM and apo-NifH protein devoid of [Fe4S4] clusters, dramatically increasing the specific activity of remaining NifH protein. Our data indicate that the chloroplast endogenous [Fe-S] cluster biosynthesis was insufficient for complete NifH maturation, albeit a negative effect on NifH maturation due to excess NifM in the chloroplast cannot be excluded. NifH and NifM constitutive expression in transplastomic plants did not affect any of the following traits: seed size, germination time, germination ratio, seedling growth, emergence of the cotyledon and first leaves, chlorophyll content and plant height throughout development.

Molecular and Biochemical Characterization of nifHDK Genes in Klebsiella pneumoniae

Nitrogen fixation is carried by an enzyme complex called nitrogenase which consists of two main components, a dinitrogenase that is encoded by nifD and nifK and an iron containing reductase, also called Fe protein which is encoded by nifH. Nitrogen-free medium was used to detect the ability of nitrogen fixation by Klebsiella pneumonia, then DNA was extracted and overlap extension polymerase chain reaction of nifH, nifD and nifK. To obtain nucleotide sequences of these genes, sequencing of the PCR products was one. The reverse sequence of nifH and the forward sequences of nifD and nifK were converted into amino acids using online translation tool. Homology modeling was carried out using SWISS-MODEL. The modeled amino acids sequences was validated using ERRAT and PROCHECK. The modeled sequences were reliable and of quality higher than 90%. The two subunits of Fe protein were constructed and tertiary structure was predicted together with the binding sites for prosthetic group and ADP molecule in Fe protein. The following amino acids Asp11, Lys13, Asn157, Ser158, Val183, Pro184, Arg185, Asp186, Val189, Gln190 and Glu193 seem to participate in the ADP binding. The complexity of this enzyme makes it difficult to be cloned in plants.

Nitrogen Fixation and Diazotrophs – A Review

Nitrogen fixation involves formation of ammonium from N2, which needs a high input of energy. Biological nitrogen fixation utilizes the enzyme nitrogenase and ATP to fix nitrogen. Nitrogenase contains a Fe-protein and a Mo-Fe-protein and other metal cofactors. Soil diazotrophs possess the function of fixing atmospheric N2 into biologically available ammonium in ecosystems. In Aechaea, nitrogen fixation has been reported in some methanogens such as Methanobacteriales, Methanococcales, and Methanosarcinales. Community structure and diversity of diazotrophic are correlated with soil pH. All known organisms which involve in nitrogen-fixing which are called diazatrophs are prokaryotes, and both bacterial and archaeal domains are responsible for that. Diazotrophs are categorized into two main groups namely: root-nodule bacteria and plant growth-promoting rhizobacteria. Diazotrophs include free living bacteria, such as Azospirillum, Cupriavidus, and some sulfate reducing bacteria, and symbiotic diazotrophs such Rhizobium and Frankia. Two important parameters which may affect diazotroph communities are temperature and soil moisture in different seasons. To have sustainable agriculture, replacing expensive chemical nitrogen fertilizers with environmentally friendly ways is the most accepted practice.

Protective Reactions of Seedlings Against Systemic and Local Zearalenone Application to Tolerant and Sensitive Wheat Varieties – Epr Studies

BACKGROUNDAmong the environmental stressors, which oxidative action is currently widely studied, are mycotoxins. The aim of presented studies was to investigate the response of leaves of seedlings to zearalenone (ZEN) applied directly to leaves and to grains (indirectly) of tolerant and sensitive wheat cultivars.RESULTS Biochemical analyses of antioxidative activity were performed for chloroplasts and showed the similar decrease of this activity independently of plant sensitivity and the way of ZEN application. On the other hand, higher amounts of superoxide radicals (microscopic observation) were formed in leaves of plants grown from grains incubated in solution of ZEN and in sensitive variety. Electron paramagnetic resonance (EPR) studies showed that upon ZEN treatment larger amount of Mn - aqua complexes was formed in leaves of tolerant wheat cultivar than in those of sensitive one whereas the degradation of Fe-protein complexes occurred independently of the sensitivity of plant varieties. CONCLUSIONThe changes in the quantity of organic stable radicals, which were formed by stabilizing reactive oxygen species on biochemical macromolecules, pointed to the higher ability to their generation in leaf tissues subjected to foliar ZEN treatment indicating the important role of these radical species in protective mechanism mainly against direct toxin action. The way of activation the defense mechanisms depended on the method of applications toxin.

Mechanical coupling in the nitrogenase complex

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue β-188Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: (i) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; (ii) within each half of the complex, the switch regions I and II are coupled to the loop containing β-188Ser; (iii) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing β-188Ser) are important for communication between the two halves.

Synthesis of Nitrogenase by Paenibacillus Sabinae T27 in Presence of High Levels of Ammonia During Anaerobic Fermentation

BackgroundBiological nitrogen fixation catalyzed by nitrogenase is a high energy-intensive process, and thus nitrogenase synthesis and activity are inhibited by ammonium (NH4+). Microorganism fix nitrogen at high ammonium (30-300 mM) concentration has not been reported before.ResultsPaenibacillus sabinae T27, a Gram-positive, spore-forming diazotroph (N2-fixing microorganism, showed nitrogenase activities not only in low (0-4 mM) concentration of NH4+, but also in high (30-300 mM) concentration of NH4+, no matter whether the cells of this bacterium were grown in flask or in fermentor on scale cultivation. qRT-PCR and western blotting analysis supported that Fe protein and MoFe protein were synthesized under both low (0-4 mM) and high (30-300 mM) concentration of NH4+. Liquid chromatography-mass spectrometry（LC-MS）analysis revealed that MoFe protein purified form cultures grown in nitrogen-limited condition or nitrogen-excess condition was encoded by nifDK and Fe protein was encoded by both nifH and nifH2. The cross-reaction suggested the purified Fe and MoFe components from P. sabinae T27 grown in both nitrogen-limited and -excess conditions were active.ConclusionsOur results indicate that N2 fixation occurs in presence of high (30-300 mM) concentration of NH4+ in P. sabinae T27. Nitrogen fixation under both low and high concentration of NH4+ was catalyzed by the same nitrogenases and the Fe protein was encoded by both nifH and nifH2. Our study will provide a clue for studying the mechanisms on nitrogen fixation in presence of the high concentration of NH4+.

The association between nutrient patterns and metabolic syndrome among Iranian adults: cross-sectional analysis of Shahedieh cohort study

Objective: To assess the association between patterns of nutrient intake and metabolic syndrome (MetS) in a large sample of Iranian adults. Design: Baseline data from the Shahedieh cohort study were used in the current cross-sectional study. Dietary intakes were assessed through the use of a validated semi-quantitative FFQ. Nutrient patterns (NP) were derived using factor analysis. The MetS was defined according to criteria introduced from the National Cholesterol Education Program Adult Treatment Panel III, modified for Iranian adults. Setting: Yazd, Iran. Participants: A total of 7325 Iranian adults aged between 30 and 75 years. Results: Three NP were identified. A significant positive association was found between adherence to semi-plant NP (characterised by the high intakes of P; vitamins B1, B3, B6 and B5; Se; Mg; Fe; protein; Cr; Cu; fibre; biotin; Mn; Zn and Na) and odds of MetS (OR 1·68, 95 % CI 1·43, 1·98). However, after adjusting for potential confounders, this association became non-significant. In addition, after taking potential confounders into account, individuals in the highest quintile of the semi-animal NP, rich in Ca; K; vitamins B2, B12, A, D, K and C; SFA; dietary cholesterol and trans-fatty acid, were 26 % more likely to have MetS compared with those in the lowest quintile (OR 1·26, 95 % CI 1·05, 1·51). No significant association was seen between adherence to the high-carbohydrate/low-fat NP and odds of MetS. Conclusions: We found that adherence to a semi-animal NP was associated with increased odds of MetS.