The impact of rumen-protected amino acids on the expression of key- genes involved in the innate immunity of dairy sheep

Rumen protected amino acids inclusion in ewes’ diets has been proposed to enhance their innate immunity. The objective of this work was to determine the impact of dietary supplementation with rumen-protected methionine or lysine, as well as with a combination of these amino acids in two different ratios, on the expression of selected key-genes (NLRs, MyD88, TRIF, MAPK-1, IRF-3, JunD, TRAF-3, IRF-5, IL-1α, IL-10, IKK-α, STAT-3 and HO-1). Thus, sixty Chios dairy ewes (Ovis aries) were assigned to one of the following five dietary treatments (12 animals/ treatment): A: basal diet consist of concentrates, wheat straw and alfalfa hay (control group); B: basal diet +6.0 g/head rumen-protected methionine; C: basal diet + 5.0 g/head rumen-protected lysine; D: basal diet +6.0 g/head rumen-protected methionine + 5.0 g/head rumen-protected lysine and E: basal diet +12.0 g/head rumen-protected methionine + 5.0 g/head rumen-protected lysine. The results revealed a significant downregulation of relative transcript level of the IL-1α gene in the neutrophils of C and in monocytes of D ewes compared with the control. Significantly lower mRNA transcript accumulation was also observed for the MyD88 gene in the neutrophils of ewes fed with lysine only (C). The mRNA relative expression levels of JunD gene were highly induced in the monocytes, while those of IL-10 and HO-1 genes were declined in the neutrophils of ewes fed with the C and D diets compared with the control. Lower transcript levels of STAT-3 gene were observed in the neutrophils of ewes fed with either C or with E diets in comparison with the control. In conclusion, our results suggest that the dietary supplementation of ewes with rumen-protected amino acids, down regulate the expression of some genes involved in the pro-inflammatory signalling

Morphological, Physiological and Metabolomic Response of transgenic tobacco plants (N. tabacum L.) overexpressing GmGSTU4 under Drought Stress

GSTs appear to have a significant role in plants’ adaptation under abiotic stress as many isoenzymes are found to be differentially expressed under these conditions yet, little is known about the regulatory functions of GSTs. Wild type and transgenic tobacco plants over-expressing the soybean GmGSTU4 of cultivars Basmas, Burley and Virginia were grown in vitro under 100 and 200mM mannitol or in soil (plant pots) by withholding watering for 15 days. However, GmGSTU4 plants did not exhibit significant differences in drought tolerance compared to wild-type plants. Morphological (shoot length, total and root fresh weight) and physiological (chlorophyll content, relative water content and photosynthetic capacity) parameters of transgenic plants did not differ from the wild-type in the presence of 100 or 200mM mannitol or in the soil when watering was halted. Metabolite profiling was used to understand the dynamics between the wild-type and transgenic tobacco response to drought stress. Different metabolic pathways are involved in production of osmoprotectants. These molecules accumulate in plants under stress conditions as adaptive mechanism, which can provide stress tolerance. GmGSTU4 plants did not exhibit difference in drought tolerance compared to wild-type plants, however metabolomics analysis indicated alterations in metabolite profile and increased concentration of sorbitol, glycerol and pyruvic acid. In conclusion, overexpression of GmGSTU4 in transgenic plants did not affect their drought stress tolerance although it has altered their metabolite profile possible because of diverse effects on plant stress tolerance mechanism

Maintenance of metabolic homeostasis and induction of cytoprotectants and secondary metabolites in alachlor-treated GmGSTU4-overexpressing tobacco plants, as resolved by metabolomics

Herbicides are an invaluable tool for agricultural production scaling up. However, their continuous and intensive use has led to an increased incidence of herbicide resistant weeds and environmental pollution. Plant glutathione transferases (GSTs) are tightly connected with crop and weed herbicide tolerance capacitating their efficient metabolic detoxification, thus GSTs can be biotechnologically exploited towards addressing those issues. However, information on their effects at a “systems” level in response to herbicides is lacking. Here, we aimed to study the effects of the chloroacetanilide herbicide alachlor on the metabolome of wild-type and tobacco plants overexpressing the soybean tau class glutathione transferase GmGSTU4. Alachlor-treated wild-type plants This system, naturally serving the detoxification of endogenous exhibited an abiotic stress-like response with increased abundance of compatible solutes, decrease in TCA cycle intermediates and decreased sugar and amino acid content. Transgenic plants responded distinctly, exhibiting an increased induction of abiotic stress responsive metabolites, accumulation of secondary metabolites and its precursors, and metabolic detoxification by-products compared to wild-type plants. These results suggest that the increased metabolic capacity of GmGSTU4 overexpressing plants is accompanied by pleiotropic metabolic alterations, which could be the target for further manipulation in order to develop herbicide resistant crops, plants with increased phytoremediation potential, as well as efficient management of non-target site, GST induced, herbicide resistance in weeds

Analysis of promoter activity of the early nodulin Enod40 in Lotus japonicus

Microbial Biotechnolog

Lotus japonicus contains two distinct ENOD40 genes that are expressed in symbiotic, nonsymbiotic, and embryonic tissues

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis, We have isolated two distinct ENOD40 genes from Lotus japonicus, The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology, Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules, Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls, A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly In the phloem and epithelium, as well as in globular stage embryos.Animal science

Absolute quantification of Medicago truncatula sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry

Mass spectrometry (MS) has become increasingly important for tissue specific protein quantification at the isoform level, as well as for the analysis of protein post-translational regulation mechanisms and turnover rates. Thanks to the development of high accuracy mass spectrometers, peptide sequencing without prior knowledge of the amino acid sequence—de novo sequencing—can be performed. In this work, absolute quantification of a set of key enzymes involved in carbon and nitrogen metabolism in Medicago truncatula ‘Jemalong A17’ root nodules is presented. Among them, sucrose synthase (SuSy; EC 2.4.1.13), one of the central enzymes in sucrose cleavage in root nodules, has been further characterized and the relative phosphorylation state of the three most abundant isoforms has been quantified. De novo sequencing provided sequence information of a so far unidentified peptide, most probably belonging to SuSy2, the second most abundant isoform in M. truncatula root nodules. TiO2-phosphopeptide enrichment led to the identification of not only a phosphorylation site at Ser11 in SuSy1, but also of several novel phosphorylation sites present in other root nodule proteins such as alkaline invertase (AI; EC 3.2.1.26) and an RNA-binding protein

Tissue-specific down-regulation of LjAMT1;1 compromises nodule function and enhances nodulation in Lotus japonicus

Plant ammonium transporters of the AMT1 family are involved in N-uptake from the soil and ammonium transport, and recycling within the plant. Although AMT1 genes are known to be expressed in nitrogen-fixing nodules of legumes, their precise roles in this specialized organ remain unknown. We have taken a reverse-genetic approach to decipher the physiological role of LjAMT1;1 in Lotus japonicus nodules. LjAMT1;1 is normally expressed in both the infected zone and the vascular tissue of Lotus nodules. Inhibition of LjAMT1;1 gene expression, using an antisense gene construct driven by a leghemoglobin promoter resulted in a substantial reduction of LjAMT1;1 transcript in the infected tissue but not the vascular bundles of transgenic plants. As a result, the nitrogen-fixing activity of nodules was partially impaired and nodule number increased compared to control plants. Expression of LjAMT1;1-GFP fusion protein in plant cells indicated a plasma-membrane location for the LjAMT1;1 protein. Taken together, the results are consistent with a role of LjAMT1;1 in retaining ammonium derived from symbiotic nitrogen fixation in plant cells prior to its assimilation

Vitamins A & D Inhibit the Growth of Mycobacteria in Radiometric Culture

The role of vitamins in the combat of disease is usually conceptualized as acting by modulating the immune response of an infected, eukaryotic host. We hypothesized that some vitamins may directly influence the growth of prokaryotes, particularly mycobacteria. complex).Vitamins A and D cause dose-dependent inhibition of all three mycobacterial species studied. Vitamin A is consistently more inhibitory than vitamin D. The vitamin A precursor, β-carotene, is not inhibitory, whereas three vitamin A metabolites cause inhibition. Vitamin K has no effect. Vitamin E causes negligible inhibition in a single strain.We show that vitamin A, its metabolites Retinyl acetate, Retinoic acid and 13-cis Retinoic acid and vitamin D directly inhibit mycobacterial growth in culture. These data are compatible with the hypothesis that complementing the immune response of multicellular organisms, vitamins A and D may have heretofore unproven, unrecognized, independent and probable synergistic, direct antimycobacterial inhibitory activity

Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere

Oxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling.The ISME Journal advance online publication, 24 October 2017; doi:10.1038/ismej.2017.148