Significance of phosphorus deficiency for the mitigation of mercury toxicity in the Robinia pseudoacacia L.- rhizobia symbiotic association.

Nitrogen (N2)-fixing legumes can be used for phytoremediation of toxic heavy metal Mercury (Hg) contaminated soil, but N2-fixation highly relies on phosphorus (P) availability for nodule formation and functioning. Here, we characterized the significance of P deficiency for Hg accumulation and toxicity in woody legume plants. Consequences for foliar and root traits of rhizobia inoculation, Hg exposure (+Hg) and low P (-P) supply, individually and in combination were characterized at both the metabolite and transcriptome levels in seedlings of two Robinia pseudoacacia L. provenances originating from contrasting climate and soil backgrounds, i.e., GS in northwest and the DB in northeast China. Our results reveal that depleted P mitigates the toxicity of Hg at the transcriptional level. In leaves of Robinia depleted P reduced oxidative stress and improved the utilization strategy of C, N and P nutrition; in roots depleted P regulated the expression of genes scavenging oxidative stress and promoting cell membrane synthesis. Rhizobia inoculation significantly improved the performance of both Robinia provenances under individual and combined +Hg and -P by promoting photosynthesis, increasing foliar N and P content and reducing H2O2 and MDA accumulation despite enhanced Hg uptake. DB plants developed more nodules, had higher biomass and accumulated higher Hg amounts than GS plants and thus are suggested as the high potential Robinia provenance for future phytoremediation of Hg contaminated soils with P deficiency.

Antagonistic effect of mercury and excess nitrogen exposure reveals provenance-specific phytoremediation potential of black locust-rhizobia symbiosis.

Interaction of different environmental constrains pose severe threats to plants that cannot be predicted from individual stress exposure. In this context, mercury (Hg), as a typical toxic and hazardous heavy metal, has recently attracted particular attention. Nitrogen (N2)-fixing legumes can be used for phytoremediation of Hg accumulation, whereas N availability could greatly affect its N2-fixation efficiency. However, information on the physiological responses to combined Hg exposure and excess N supply of woody legume species is still lacking. Here, we investigated the interactive effects of rhizobia inoculation, Hg exposure (+Hg), and high N (+N) supply, individually and in combination (+N*Hg), on photosynthesis and biochemical traits in Robinia pseudoacacia L. seedlings of two provenances, one from Northeast (DB) and one from Northwest (GS) China. Our results showed antagonistic effects of combined + N*Hg exposure compared to the individual treatments that were provenance-specific. Compared to individual Hg exposure, combined + N*Hg stress significantly increased foliar photosynthesis (+50.6%) of inoculated DB seedlings and resulted in more negative (-137.4%) δ15N abundance in the roots. Furthermore, combined + N*Hg stress showed 47.7% increase in amino acid N content, 39.4% increase in NR activity, and 14.8% decrease in MDA content in roots of inoculated GS seedlings. Inoculation with rhizobia significantly promoted Hg uptake in both provenances, reduced MDA contents of leaves and roots, enhanced photosynthesis and maintained the nutrient balance of Robinia. Among the two Robinia provenances investigated, DB seedlings formed more nodules, had higher biomass and Hg accumulation than GS seedlings. For example, total Hg concentrations in leaves and roots and total biomass of inoculated DB seedlings were 1.3,1.9 and 3.4 times higher than in inoculated GS seedlings under combined + N*Hg stress, respectively. Therefore, the DB provenance is considered to possess a higher potential for phytoremediation of Hg contamination compared to the GS provenance in environments subjected to N deposition.

Selenium- and chitosan-modified biochars reduce methylmercury contents in rice seeds with recruiting Bacillus to inhibit methylmercury production.

Biochar could reshape microbial communities, thereby altering methylmercury (MeHg) concentrations in rice rhizosphere and seeds. However, it remains unclear whether and how biochar amendment perturbs microbe-mediated MeHg production in mercury (Hg) contaminated paddy soil. Here, we used pinecone-derived biochar and its six modified biochars to reveal the disturbance. Results showed that selenium- and chitosan-modified biochar significantly reduced MeHg concentrations in the rhizosphere by 85.83% and 63.90%, thereby decreasing MeHg contents in seeds by 86.37% and 75.50%. The two modified bicohars increased the abundance of putative Hg-resistant microorganisms Bacillus, the dominant microbe in rhizosphere. These reductions about MeHg could be facilitated by biochar sensitive microbes such as Oxalobacteraceae and Subgroup_7. Pinecone-derived biochar increased MeHg concentration in rhizosphere but unimpacted MeHg content in seeds was observed. This biochar decreased the abundance in Bacillus but enhanced in putative Hg methylator Desulfovibrio. The increasing MeHg concentration in rhizosphere could be improved by biochar sensitive microbes such as Saccharimonadales and Clostridia. Network analysis showed that Saccharimonadales and Clostridia were the most prominent keystone taxa in rhizosphere, and the three biochars manipulated abundances of the microbes related to MeHg production in rhizosphere by those biochar sensitive microbes. Therefore, selenium- and chitosan-modified biochar could reduce soil MeHg production by these microorganisms, and is helpful in controlling MeHg contamination in rice.

Impact of Assimilable Nitrogen Supplementation on Saccharomyces cerevisiae Metabolic Response and Aromatic Profile of Moschofilero Wine.

The concentration of nitrogen in must is critical to yeast fermentation efficiency and wine aroma profile. The present work determined the effect of the amount of yeast assimilable nitrogen (YAN) on fermentation kinetics, aroma production, and gene expression patterns of the wine yeast Saccharomyces cerevisiae. Fermentations were performed under two different YAN concentrations of must. Acetate esters, linalool, and nerol appeared to be clearly affected by the different YAN levels. Real-time-PCR results revealed that the genes involved in ethyl and acetate esters production recorded, in general, higher transcript levels under high nitrogen supplementation. In addition, an up-regulation of the BGL2 and EXG1 genes, which are related to terpenes production, was observed in the case of high nitrogen content and it is well corresponded to the terpenol concentration found. Our study revealed the impact of nitrogen supplementation on yeast metabolism and its importance to adjust wine's aromatic composition and sensory profile.

Effect of a Carotenoid Extract from Citrus reticulata By-Products on the Immune-Oxidative Status of Broilers.

Although carotenoids generally possess antimicrobial and antioxidant properties, the in vivo synergistic action of carotenoid blends derived from plant-based by-products has not been thoroughly studied. Therefore, the carotenoid characterization and antimicrobial potential of Citrus reticulata extract as well as the impact of this carotenoid-rich extract (CCE) dietary supplementation on the performance, meat quality, and immune-oxidative status of broiler chickens were determined. One hundred and twenty one-day-old hatched chicks (Ross 308) were allocated to two dietary groups, with four replicate pens of 15 birds each. Birds were fed either a basal diet (CON) or the basal diet supplemented with 0.1% CCE (25 mg carotenoid extract included in 1 g of soluble starch) for 42 d. ß-Cryptoxanthin, ß-Carotene, Zeaxanthin, and Lutein were the prevailing carotenoid compounds in the Citrus reticulata extract. The CCE feed additive exerted inhibitory properties against both Gram-positive (Staphylococcus aureus) and negative (Klebsiella oxytoca, Escherichia coli, and Salmonella typhimurium) bacteria. Both the broiler performance and meat quality did not substantially differ, while the breast muscle malondialdehyde (MDA) concentration tended to decrease (p = 0.070) in the CCE-fed broilers. The inclusion of CCE decreased the alanine aminotransferase and MDA concentration, and the activity of glutathione peroxidase, while the activity of superoxide dismutase was increased in the blood. Catalase and NADPH oxidase 2 relative transcript levels were significantly downregulated in the livers of the CCE-fed broilers. Additionally, Interleukin 1ß and tumor necrosis factor (TNF) relative transcript levels were downregulated in the livers of the CCE- fed broilers, while TNF and interferon γ (IFNG) tended to decrease in the spleens and bursa of Fabricius, respectively. The present study provided new insights regarding the beneficial properties of carotenoids contained in Citrus reticulata in broilers' immune-oxidative status. These promising outcomes could be the basis for further research under field conditions.

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.

The effects of dietary supplementation with rumen-protected amino acids on the expression of several genes involved in the immune system of dairy sheep.

Amino acids might be a tool to transform animals from a pro- to an anti-inflammatory phenotype through the downregulation of several genes (TLR-4, NF-κB, TNFa, IL-1ß, IL-2, IL-6, IL-8, CCL-5 and CXCL-16) whose expression increases during inflammation. To examine this possibility, each of sixty Chios dairy sheep was assigned to one of the following five dietary treatments: A: basal diet (control group); B: basal diet +6.0 g/head rumen-protected methionine (MetaSmart™ ); C: basal diet +5.0 g/head rumen-protected lysine (LysiGEM™ ); D: basal diet +6.0 g/head MetaSmart™  + 5.0 g/head LysiGEM™ ; and E: basal diet +12.0 g/head MetaSmart™  + 5.0 g/head LysiGEM. The results showed a significant downregulation in the expression of the TLR-4 gene in both the macrophages and the neutrophils of ewes fed rumen-protected amino acids. Significantly lower mRNA transcript accumulation was also observed for the TNFa, IL-1ß and CXCL-16 genes in the macrophages and for the IL-1ß gene in the neutrophils of ewes supplemented with amino acids. The ewes that received dietary supplementation with rumen-protected lysine alone (C) had significantly lower CCL-5 transcript levels in their macrophages than the ewes fed the other supplemented diets. Diet D enhanced the mRNA expression of the IL-2 gene in ewe neutrophils. Negative correlations were found between: a. TLR-4, TNFa, IL-1ß and CXCL-16 gene expression in macrophages and the milk fat and total solids content; b. CCL-5 gene expression in neutrophils and the milk yield and FCM(6%) ; and c. CXCL-16 gene expression and the milk protein content. Moreover, positive correlations were found between the BHBA concentration and the expression of the TLR-4 and CXCL-16 genes in macrophages. In conclusion, the rumen-protected amino acids improved sheep metabolism (as indicated by reduced blood BHBA and urea concentrations), milk chemical composition and immune system function.

Cloning and functional characterization of LjPLT4, a plasma membrane xylitol H(+)- symporter from Lotus japonicus.

Polyols are compounds that play various physiological roles in plants. Here we present the identification of four cDNA clones of the model legume Lotus japonicus, encoding proteins of the monosaccharide transporter-like (MST) superfamily that share significant homology with previously characterized polyol transporters (PLTs). One of the transporters, named LjPLT4, was characterized functionally after expression in yeast. Transport assays revealed that LjPLT4 is a xylitol-specific H(+)-symporter (K (m), 0.34 mM). In contrast to the previously characterized homologues, LjPLT4 was unable to transport other polyols, including mannitol, sorbitol, myo-inositol and galactitol, or any of the monosaccharides tested. Interestingly, some monosaccharides, including fructose and xylose, inhibited xylitol uptake, although no significant uptake of these compounds was detected in the LjPLT4 transformed yeast cells, suggesting interactions with the xylitol binding site. Subcellular localization of LjPLT4-eYFP fusions expressed in Arabidopsis leaf epidermal cells indicated that LjPLT4 is localized in the plasma membrane. Real-time RT-PCR revealed that LjPLT4 is expressed in all major plant organs, with maximum transcript accumulation in leaves correlating with maximum xylitol levels there, as determined by GC-MS. Thus, LjPLT4 is the first plasma membrane xylitol-specific H(+)-symporter to be characterized in plants.

A root- and hypocotyl-specific gene coding for copper-containing amine oxidase is related to cell expansion in soybean seedlings.

Polyamines are considered to participate in various processes of plant development. In this study, the possible implication of putrescine catabolism by the copper-containing amine oxidases (CuAOs, EC 1.4.3.6) in the development of roots and hypocotyls was examined. For this purpose, two cDNA clones of Glycine max (L.) Merr. cv. Williams, designated as GmCuAO1 and GmCuAO2, exhibiting extensive similarity with previously characterized CuAO clones from other plants, have been isolated and characterized. The expression of the GmCuAO1 gene is root- and hypocotyl-specific, while GmCuAO2 seems not to be expressed in a tissue-specific manner. Moreover, the GmCuAO1 gene is predominantly expressed in tissues which are characterized by rapid extension growth, such as the apical segments of etiolated hypocotyls. Using convex and concave segments of the etiolated hypocotyl apical hook it has been demonstrated that GmCuAO1 is strongly expressed in expanding cells of the concave part when exposed to light, while the same pattern is also followed by the activity of enzymes involved in putrescine catabolism. In dark and photoperiodically grown hypocotyls, activity measurements of the enzymes involved in putrescine catabolism have shown that the activity of these enzymes is several-fold higher in rapidly growing tissues. Furthermore, the cellular and tissue distribution of GmCuAO1 gene transcripts in the root axis and in hypocotyls confirmed their abundance in developing tissues and expanding cells. The results provide evidence suggesting that a tissue-specific gene coding for CuAO is correlated with cell expansion in fast-growing tissues of root and hypocotyls.

The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus.

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.

Lotus japonicus gene Ljsbp is highly conserved among plants and animals and encodes a homologue to the mammalian selenium-binding proteins.

We have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones homologous to LjSBP were also isolated from soybean, Medicago sativa, and Arabidopsis thaliana. Comparative expression studies in L japonicus and A. thaliana showed that sbp transcripts are present in various tissues and at different levels. Especially in L japonicus nodules and seedpods and A. thaliana siliques, sbp expression appears to be developmentally up-regulated. sbp Gene transcripts were localized by in situ hybridization in the infected cells and vascular bundles of young nodules, while in mature nodules, low levels of expression were only detected in the parenchymatous cells. Expression of sbp transcripts in young seedpods and siliques was clearly visible in vascular tissues and embryos, while in embryos, low levels of expression were detected in the root epidermis and the vascular bundles. Polyclonal antibodies raised against a truncated LjSBP recombinant protein recognized a polypeptide of about 60 kDa in nodule extracts. Immunohistochemical experiments showed that accumulation of LjSBP occurred in root hairs, in the root epidermis above the nodule primordium, in the phloem of the vasculature, and abundantly in the infected cells of young nodules. Irrespective of the presence of rhizobia, expression of SBP was also observed in root tips, where it was confined in the root epidermis and protophloem cells. We hypothesize that LjSBP may have more than one physiological role and can be implicated in controlling the oxidation/reduction status of target proteins, in vesicular Golgi transport, or both.