Effects on gene expression during maize-Azospirillum interaction in the presence of a plant-specific inhibitor of indole-3-acetic acid production.

Amongst the sustainable alternatives to increase maize production is the use of plant growth-promoting bacteria (PGPB). Azospirillum brasilense is one of the most well-known PGPB being able to fix nitrogen and produce phytohormones, especially indole-3-acetic acid - IAA. This work investigated if there is any contribution of the bacterium to the plant's IAA levels, and how it affects the plant. To inhibit plant IAA production, yucasin, an inhibitor of the TAM/YUC pathway, was applied. Plantlets' IAA concentration was evaluated through HPLC and dual RNA-Seq was used to analyze gene expression. Statistical differences between the group treated with yucasin and the other groups showed that A. brasilense inoculation was able to prevent the phenotype caused by yucasin concerning the number of lateral roots. Genes involved in the auxin and ABA response pathways, auxin efflux transport, and the cell cycle were regulated by the presence of the bacterium, yucasin, or both. Genes involved in the response to biotic/abiotic stress, plant disease resistance, and a D-type cellulose synthase changed their expression pattern among two sets of comparisons in which A. brasilense acted as treatment. The results suggest that A. brasilense interferes with the expression of many maize genes through an IAA-independent pathway.

Metataxonomic and metagenomic analysis of mangrove microbiomes reveals community patterns driven by salinity and pH gradients in Paranaguá Bay, Brazil.

While environmental drivers regulate the structure of mangrove microbial communities, their exact nature and the extent of their influence require further elucidation. By means of 16S rRNA gene-based sequencing, we determined the microbial taxonomic profiles of mangroves in the subtropical Paranaguá Bay, Brazil, considering as potential drivers: salinity, as represented by two sectors in the extremes of a salinity gradient (<5 PSU and >30 PSU); proximity to/absence of the prevailing plants, Avicennia schaueriana, Laguncularia racemosa, Rhizophora mangle, and Spartina alterniflora; and the chemical composition of the sediments. Salinity levels within the estuary had the strongest influence on microbial structure, and pH was important to separate two communities within the high salinity environment. About one fourth of the total variation in community structure resulted from covariation of salinity and the overall chemical composition, which might indicate that the chemical profile was also related to salinity. The most prevalent bacterial phyla associated with the mangrove soils analyzed included Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, Acidobacteria, and Cyanobacteria. Taxonomic and functional comparisons of our results for whole-genome sequencing with available data from other biomes showed that the studied microbiomes cluster first according to biome type, then to matrix type and salinity status. Metabolic functions were more conserved than organisms within mangroves and across all biomes, indicating that core functions are preserved in any of the given conditions regardless of the specific organisms harboring them.

Modulation of defence and iron homeostasis genes in rice roots by the diazotrophic endophyte Herbaspirillum seropedicae.

Rice is staple food of nearly half the world's population. Rice yields must therefore increase to feed ever larger populations. By colonising rice and other plants, Herbaspirillum spp. stimulate plant growth and productivity. However the molecular factors involved are largely unknown. To further explore this interaction, the transcription profiles of Nipponbare rice roots inoculated with Herbaspirillum seropedicae were determined by RNA-seq. Mapping the 104 million reads against the Oryza sativa cv. Nipponbare genome produced 65 million unique mapped reads that represented 13,840 transcripts each with at least two-times coverage. About 7.4% (1,014) genes were differentially regulated and of these 255 changed expression levels more than two times. Several of the repressed genes encoded proteins related to plant defence (e.g. a putative probenazole inducible protein), plant disease resistance as well as enzymes involved in flavonoid and isoprenoid synthesis. Genes related to the synthesis and efflux of phytosiderophores (PS) and transport of PS-iron complexes were induced by the bacteria. These data suggest that the bacterium represses the rice defence system while concomitantly activating iron uptake. Transcripts of H. seropedicae were also detected amongst which transcripts of genes involved in nitrogen fixation, cell motility and cell wall synthesis were the most expressed.

Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ.

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

Cellulose production increases sorghum colonization and the pathogenic potential of Herbaspirillum rubrisubalbicans M1.

Three species of the ß-Proteobacterial genus Herbaspirillum are able to fix nitrogen in endophytic associations with such important agricultural crops as maize, rice, sorghum, sugar-cane and wheat. In addition, Herbaspirillum rubrisubalbicans causes the mottled-stripe disease in susceptible sugar-cane cultivars as well as the red-stripe disease in some sorghum cultivars. The xylem of these cultivars exhibited a massive colonisation of mucus-producing bacteria leading to blocking the vessels. A cluster of eight genes (bcs) are involved in cellulose synthesis in Herbaspirillum rubrisubalbicans. Mutation of bcsZ, that encodes a 1,4-endoglucanase, impaired the exopolysaccharide production, the ability to form early biofilm and colonize sorghum when compared to the wild-type strain M1. This mutation also impaired the ability of Herbaspirillum rubrisubalbicans M1 to cause the red-stripe disease in Sorghum bicolor. We show cellulose synthesis is involved in the biofilm formation and as a consequence significantly modulates bacterial-plant interactions, indicating the importance of cellulose biosynthesis in this process.

Sugarcane apoplast fluid modulates the global transcriptional profile of the diazotrophic bacteria Paraburkholderia tropica strain Ppe8.

The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium

Abstract Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75 Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

The genomes of three Bradyrhizobium sp. isolated from root nodules of Lupinus albescens grown in extremely poor soils display important genes for resistance to environmental stress.

Lupinus albescens is a resistant cover plant that establishes symbiotic relationships with bacteria belonging to the Bradyrhizobium genus. This symbiosis helps the development of these plants in adverse environmental conditions, such as the ones found in arenized areas of Southern Brazil. This work studied three Bradyrhizobium sp. (AS23, NAS80 and NAS96) isolated from L. albescens plants that grow in extremely poor soils (arenized areas and adjacent grasslands). The genomes of these three strains were sequenced in the Ion Torrent platform using the IonXpress library preparation kit, and presented a total number of bases of 1,230,460,823 for AS23, 1,320,104,022 for NAS80, and 1,236,105,093 for NAS96. The genome comparison with closest strains Bradyrhizobium japonicum USDA6 and Bradyrhizobium diazoefficiens USDA110 showed important variable regions (with less than 80% of similarity). Genes encoding for factors for resistance/tolerance to heavy metal, flagellar motility, response to osmotic and oxidative stresses, heat shock proteins (present only in the three sequenced genomes) could be responsible for the ability of these microorganisms to survive in inhospitable environments. Knowledge about these genomes will provide a foundation for future development of an inoculant bioproduct that should optimize the recovery of degraded soils using cover crops.

Co-expression, purification and characterization of the lipase and foldase of Burkholderia contaminans LTEB11.

Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase subfamily I.2. The genes lipA and lipB were amplified and cloned into expression vectors pET28a(+) and pT7-7, respectively. His-tagged LipBC and native LifBC were co-expressed in Escherichia coli and purified. LipBC and LifBC have molecular weights of 35.9 kDa and 37 kDa, respectively, and remain complexed after purification. The Lip-LifBC complex was active and stable over a wide range of pH values (6.5-10) and temperatures (25-45 °C), with the highest specific activity (1426 U mg-1) being against tributyrin. The Lip-LifBC complex immobilized on Sepabeads was able to catalyze the synthesis of ethyl-oleate in n­hexane with an activity of 4 U g-1, maintaining high conversion (>80%) over 5 reaction cycles of 6 h at 45 °C. The results obtained in this work provide a basis for the development of applications of recombinant LipBC in biocatalysis.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium.

Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH.

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candidarugosa lipase, metagenomic lipase, ß-galactosidase and ß-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

Labeled Azospirillum brasilense wild type and excretion-ammonium strains in association with barley roots.

Soil bacteria colonization in plants is a complex process, which involves interaction between many bacterial characters and plant responses. In this work, we labeled Azospirillum brasilense FP2 (wild type) and HM053 (excretion-ammonium) strains by insertion of the reporter gene gusA-kanamycin into the dinitrogenase reductase coding gene, nifH, and evaluated bacteria colonization in barley (Hordeum vulgare). In addition, we determined inoculation effect based on growth promotion parameters. We report an uncommon endophytic behavior of A. brasilense Sp7 derivative inside the root hair cells of barley and highlight the promising use of A. brasilense HM053 as plant growth-promoting bacterium.

Serum Fluorescent Advanced Glycation End (F-AGE) products in gestational diabetes patients

ABSTRACT Objectives Advanced glycation end products (AGEs) are involved in the pathogenesis and complications of diabetes mellitus (DM). Gestational DM (GDM) is characterized by increased glycemia and oxidative stress, which are factors associated with high serum AGE concentrations. The aim of this study was to evaluate the utility of a serum fluorescence AGE (F-AGE) method as a screening tool for gestational diabetes. Subjects and methods Serum samples from 225 GDM patients and 217 healthy pregnant women (healthy controls) were diluted 50-fold in phosphate-buffered saline, and the AGEs were estimated by fluorometric analysis (λEx 350 nm/ λEm 440 nm). Results No significant (P > 0.05) differences in AGE concentrations, expressed in Arbitrary Units (UA/mL × 104), were observed in the women with GDM or in the healthy controls. Furthermore, F-AGE concentrations did not change significantly during the pregnancy (12-32 weeks of gestation). Only the GDM group had a positive correlation (r = 0.421; P < 0.001) between F-AGEs and serum creatinine concentrations. Conclusion It was not possible to distinguish women with gestational diabetes from the healthy controls on the basis of serum F-AGE concentrations.

Phylogenomic relationship of feijoa (Acca sellowiana (O.Berg) Burret) with other Myrtaceae based on complete chloroplast genome sequences.

Given their distribution, importance, and richness, Myrtaceae species comprise a model system for studying the evolution of tropical plant diversity. In addition, chloroplast (cp) genome sequencing is an efficient tool for phylogenetic relationship studies. Feijoa [Acca sellowiana (O. Berg) Burret; CN: pineapple-guava] is a Myrtaceae species that occurs naturally in southern Brazil and northern Uruguay. Feijoa is known for its exquisite perfume and flavorful fruits, pharmacological properties, ornamental value and increasing economic relevance. In the present work, we reported the complete cp genome of feijoa. The feijoa cp genome is a circular molecule of 159,370 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC 88,028 bp) and a Small Single Copy region (SSC 18,598 bp) separated by Inverted Repeat regions (IRs 26,372 bp). The genome structure, gene order, GC content and codon usage are similar to those of typical angiosperm cp genomes. When compared to other cp genome sequences of Myrtaceae, feijoa showed closest relationship with pitanga (Eugenia uniflora L.). Furthermore, a comparison of pitanga synonymous (Ks) and nonsynonymous (Ka) substitution rates revealed extremely low values. Maximum Likelihood and Bayesian Inference analyses produced phylogenomic trees identical in topology. These trees supported monophyly of three Myrtoideae clades.

Serum Fluorescent Advanced Glycation End (F-AGE) products in gestational diabetes patients.

OBJECTIVES: Advanced glycation end products (AGEs) are involved in the pathogenesis and complications of diabetes mellitus (DM). Gestational DM (GDM) is characterized by increased glycemia and oxidative stress, which are factors associated with high serum AGE concentrations. The aim of this study was to evaluate the utility of a serum fluorescence AGE (F-AGE) method as a screening tool for gestational diabetes. SUBJECTS AND METHODS: Serum samples from 225 GDM patients and 217 healthy pregnant women (healthy controls) were diluted 50-fold in phosphate-buffered saline, and the AGEs were estimated by fluorometric analysis (λEx 350 nm/ λEm 440 nm). RESULTS: No significant (P > 0.05) differences in AGE concentrations, expressed in Arbitrary Units (UA/mL × 104), were observed in the women with GDM or in the healthy controls. Furthermore, F-AGE concentrations did not change significantly during the pregnancy (12-32 weeks of gestation). Only the GDM group had a positive correlation (r = 0.421; P < 0.001) between F-AGEs and serum creatinine concentrations. CONCLUSION: It was not possible to distinguish women with gestational diabetes from the healthy controls on the basis of serum F-AGE concentrations.

The rs10885122 polymorphism of the adrenoceptor alpha 2A (ADRA2A) gene in Euro-Brazilians with type 2 diabetes mellitus

Objective To investigate the association of the rs10885122G>T polymorphism in the ADRA2A gene in a Euro-Brazilian sample of healthy (controls) and type 2 diabetic (T2D) subjects. Subjects and methods We used fluorescent probes (TaqMan) to genotype 241 subjects, that is, 121 healthy and 120 T2D subjects, who were classified based on the Brazilian Diabetes Association (2013) and American Diabetes Association (2014) criteria. Results The genotype and allele frequencies showed no significant (P > 0.05) difference between the two studied groups. The minor allele (T) frequencies (95%CI) for rs10885122 were 19% (14-24%) and 20% (15-26%) for healthy and T2D groups, respectively. Carriers of the T allele (genotypes GT+TT) were significantly associated (P = 0.016) with approximately a 7-kg body weight reduction compared with the genotype GG, which was only found in the T2D group. Conclusion The rs10885122G>T polymorphism of the ADRA2A gene was not associated with T2D in Euro-Brazilians, and carriers of the T allele had lower body weight in the presence of T2D. Arch Endocrinol Metab. 2015;59(1):29-33 .

RAGE receptor and its soluble isoforms in diabetes mellitus complications / O receptor RAGE e suas isoformas solúveis nas complicações do diabetes mellitus

Chronic hyperglycemia, which is present in all types of diabetes, increases the formation of advanced glycation end-products (AGEs). The interaction of AGEs with receptor of advanced glycation end-products (RAGE) initiates a cascade of pro-inflammatory and pro-coagulant processes that result in oxidative stress, stimulating the formation and accumulation of more AGE molecules. This cyclic process, denominated metabolic memory, may explain the persistency of diabetic vascular complications in patients with satisfactory glycemic control. The RAGE found in several cell membranes is also present in soluble isoforms (esRAGE and cRAGE), which are generated by alternative deoxyribonucleic acid splicing or by proteolytic cleavage. This review focuses on new research into these mediators as potential biomarkers for vascular complications in diabetes.

A hiperglicemia crônica, presente em todas as formas de diabetes, favorece a formação de produtos finais de glicação avançada (AGEs). A interação de AGEs com o receptor de produtos finais glicosilados (RAGE) inicia uma cascata de processos pró-inflamatórios e pró-coagulantes que resultam em estresse oxidativo, o qual estimula a formação e o acúmulo de maior quantidade de moléculas de AGEs. Esse processo cíclico, denominado memória metabólica, pode explicar por que, mesmo após um período de bom controle glicêmico, as complicações vasculares associadas ao diabetes não regridem. O RAGE fixado nas membranas de várias células também está presente em isoformas solúveis (esRAGE e cRAGE), geradas por processamento alternativo do ácido desoxirribonucleico (DNA) ou por clivagem proteolítica. Esta revisão aborda os novos estudos sobre a função desses mediadores como potenciais biomarcadores para as complicações vasculares no diabetes.

The involvement of PUMP from mitochondria of Araucaria angustifolia embryogenic cells in response to cold stress.

In this study, the responses of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX) in mitochondria from embryogenic cells of A. angustifolia subjected to cold stress (4°C for 24 h or 48 h) is reported. In the mitochondria of stressed cells, PUMP activity increased by approximately 45% (at 24h and 48 h), which was determined by measuring the oxygen consumption after the addition of linoleic acid and the inhibition by BSA and ATP. PUMP activation was confirmed using transmembrane electrical potential (Δψ) assays. Immunoblot assays showed an increase of PUMP expression by 40% and 150% after 24h and 48 h of cold stress, respectively. AOX activity, measured under conditions similar to those of the PUMP assays, was only slightly increased in the mitochondria from stressed cells (at 24h and 48 h), as demonstrated by oxygen consumption experiments. Cell viability was unaffected by cold stress, indicating that the effects on PUMP and AOX were not caused by cell death. These results show that the main response of this gymnosperm to cold stress is the activation of PUMP, which suggests that this protein may be involved in the control of reactive oxygen species generation, which has been previously associated with this condition.