Regulation of hsnT, nodF and nodE genes in Rhizobium tropici CIAT 899 and their roles in the synthesis of Nod factors and in the symbiosis.

Rhizobium tropici strain CIAT 899 possesses outstanding agronomic properties as it displays tolerance to environmental stresses, a broad host range and high effectiveness in fixing nitrogen with the common bean (Phaseolus vulgaris L.); in addition, it carries intriguing features such as five copies of the regulatory nodD gene, and the capacity to synthesize a variety of nodulation factors (NFs), even in a flavonoid-independent manner, when submitted to abiotic stresses. However, the roles of several nod genes of the repertoire of CIAT 899 remain to be determined. In this study, we obtained mutants for the hsnT, nodF and nodE genes of CIAT 899 and investigated their expression, NF structures and symbiotic properties. Either in the presence of the flavonoid apigenin, or of salt the expression of hsnT, nodF and nodE in wild-type CIAT 899 was highly up-regulated in comparison to the mutants of all five copies of nodD, indicating the roles that regulatory nodD genes play in the activation of hsnT, nodF and nodE; however, NodD1 was recognized as the main inducer. In total, 29 different NF structures were synthesized by wild-type CIAT 899 induced by apigenin, and 36 when induced by salt, being drastically reduced by mutations in hsnT, nodF and nodE, especially under osmotic stress, with specific changes related to each gene, indicating that the three genes participate in the synthesis of NFs. Mutations in hsnT, nodF and nodE affected differently symbiotic performance (nodule number and shoot dry weight), according to the host plant. Our results indicate that the expression of hsnT, nodF and nodE genes of CIAT 899 is mediated by nodD genes, and although these three genes do not belong to the main set of genes controlling nodulation, they contribute to the synthesis of NFs that will impact symbiotic performance and host specificity.

Revealing the roles of y4wF and tidC genes in Rhizobium tropici CIAT 899: biosynthesis of indolic compounds and impact on symbiotic properties.

Rhizobium tropici CIAT 899 is a strain known by its ability to nodulate a broad range of legume species, to synthesize a variety of Nod factors, its tolerance of abiotic stresses, and its high capacity to fix atmospheric N2, especially in symbiosis with common bean (Phaseolus vulgaris L.). Genes putatively related to the synthesis of indole acetic acid (IAA) have been found in the symbiotic plasmid of CIAT 899, in the vicinity of the regulatory nodulation gene nodD5, and, in this study, we obtained mutants for two of these genes, y4wF and tidC (R. tropiciindole-3-pyruvic acid decarboxylase), and investigated their expression in the absence and presence of tryptophan (TRP) and apigenin (API). In general, mutations of both genes increased exopolysaccharide (EPS) synthesis and did not affect swimming or surface motility; mutations also delayed nodule formation, but increased competitiveness. We found that the indole-3-acetamide (IAM) pathway was active in CIAT 899 and not affected by the mutations, and-noteworthy-that API was required to activate the tryptamine (TAM) and the indol-3-pyruvic acid (IPyA) pathways in all strains, particularly in the mutants. High up-regulation of y4wF and tidC genes was observed in both the wild-type and the mutant strains in the presence of API. The results obtained revealed an intriguing relationship between IAA metabolism and nod-gene-inducing activity in R. tropici CIAT 899. We discuss the IAA pathways, and, based on our results, we attribute functions to the y4wF and tidC genes of R. tropici.

Quorum sensing communication: Bradyrhizobium-Azospirillum interaction via N-acyl-homoserine lactones in the promotion of soybean symbiosis.

Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth.

Antioxidant activity and induction of mechanisms of resistance to stresses related to the inoculation with Azospirillum brasilense.

We investigated the effects of Azospirillum brasilense strains Ab-V5 and Ab-V6 in the induction of mechanisms of systemic acquired resistance (SAR) and induced system resistance (ISR) on maize (Zea mays L.) plants. Under normal growth conditions, the treatments consisted of the standard inoculation of cells at sowing, and leaf spray of cells or their metabolites at the V2.5 growth stage; under saline stress (170 mM NaCl), the treatment consisted of standard single and co-inoculation of A. brasilense and Rhizobium tropici. The main compounds in the Azospirillum metabolites were identified as indole-3-acetic acid (IAA) and salicylic acid (SA). Under normal conditions, A. brasilense cells applied at sowing or by leaf spray increased the activities of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) in leaves, and of ascorbate peroxidase (APX) in roots; however, interestingly, in general the highest activities were observed by leaf spray of metabolites. Under normal conditions, the highest levels of salicylic acid (SA) and jasmonic acid (JA) were achieved in leaves by leaf spray of metabolites, of SA in roots by leaf spray of cells, and of JA in roots by standard inoculation and leaf spray of metabolites. Under saline stress, plant protection occurred via SA and abscisic acid (ABA), but not JA. In general, inoculation resulted in further increases in SA in leaves and roots, and ABA in leaves. We hypothesize that A. brasilense confers protection to maize plants by simultaneous induction of JA and SA pathways, and, under saline stressing conditions, by SA and ABA pathways.

Revealing strategies of quorum sensing in Azospirillum brasilense strains Ab-V5 and Ab-V6.

Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

Promoter architecture and transcriptional regulation of musculoskeletal embryonic nuclear protein 1b (mustn1b) gene in zebrafish.

BACKGROUND: Mustn1 is a specific musculoskeletal protein that plays a critical role in myogenesis and chondrogenesis in vertebrates. Whole-mount in situ hybridization revealed that mustn1b mRNAs are specifically expressed in skeletal and cardiac muscles in Zebrafish embryos. However, the precise function and the regulatory elements required for its muscle-specific expression are largely unknown. RESULTS: The purpose of this study was to explore and uncover the target genomic regions that regulate mustn1b gene expression by in vivo functional characterization of the mustn1b promoter. We report here stable expression analyses of eGFP from fluorescent transgenic reporter Zebrafish line containing a 0.8kb_mustn1b-Tol2-eGFP construct. eGFP expression was specifically found in the skeletal and cardiac muscle tissues. We show that reporter Zebrafish lines generated replicate the endogenous mustn1b expression pattern in early Zebrafish embryos. Specific site directed-mutagenesis analysis revealed that promoter activity resides in two annotated genomic regulatory regions, each one corresponding to a specific functional transcription factor binding site. CONCLUSIONS: Our data indicate that mustn1b is specifically expressed in skeletal and cardiac muscle tissues and its muscle specificity is controlled by the 0.2-kb promoter and flanking sequences and in vivo regulated by the action of two sequence-specific families of transcription factors. Developmental Dynamics 246:992-1000, 2017. © 2017 Wiley Periodicals, Inc.

BAC Recombineering of the Agouti Loci from Spotted Gar and Zebrafish Reveals the Evolutionary Ancestry of Dorsal-Ventral Pigment Asymmetry in Fish.

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts.

RNA-seq analysis of the Rhizobium tropici CIAT 899 transcriptome shows similarities in the activation patterns of symbiotic genes in the presence of apigenin and salt.

BACKGROUND: Rhizobium tropici strain CIAT 899 establishes effective symbioses with several legume species, including Phaseolus vulgaris and Leucaena leucocephala. This bacterium synthesizes a large variety of nodulation factors in response to nod-gene inducing flavonoids and, surprisingly, also under salt stress conditions. The aim of this study was to identify differentially expressed genes in the presence of both inducer molecules, and analyze the promoter regions located upstream of these genes. RESULTS: Results obtained by RNA-seq analyses of CIAT 899 induced with apigenin, a nod gene-inducing flavonoid for this strain, or salt allowed the identification of 19 and 790 differentially expressed genes, respectively. Fifteen of these genes were up-regulated in both conditions and were involved in the synthesis of both Nod factors and indole-3-acetic acid. Transcription of these genes was presumably activated through binding of at least one of the five NodD proteins present in this strain to specific nod box promoter sequences when the bacterium was induced by both apigenin and salt. Finally, under saline conditions, many other transcriptional responses were detected, including an increase in the transcription of genes involved in trehalose catabolism, chemotaxis and protein secretion, as well as ribosomal genes, and a decrease in the transcription of genes involved in transmembrane transport. CONCLUSIONS: To our knowledge this is the first time that a transcriptomic study shows that salt stress induces the expression of nodulation genes in the absence of flavonoids. Thus, in the presence of both nodulation inducer molecules, apigenin and salt, R. tropici CIAT 899 up-regulated the same set of symbiotic genes. It could be possible that the increases in the transcription levels of several genes related to nodulation under saline conditions could represent a strategy to establish symbiosis under abiotic stressing conditions.

Genome of Rhizobium leucaenae strains CFN 299(T) and CPAO 29.8: searching for genes related to a successful symbiotic performance under stressful conditions.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici "group" are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian "Cerrados" soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299(T) and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions. RESULTS: The genomes of R. leucaenae strains CFN 299(T) and CPAO 29.8 were estimated at 6.7-6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299(T) is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different. CONCLUSION: A detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899(T) and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.

Opening the "black box" of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899.

BACKGROUND: Transcription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes--five--and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants. METHODS: The three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.). RESULTS: Phenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions. CONCLUSIONS: We report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene-nodD4-might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresses.

Regulatory nodD1 and nodD2 genes of Rhizobium tropici strain CIAT 899 and their roles in the early stages of molecular signaling and host-legume nodulation.

BACKGROUND: Nodulation and symbiotic nitrogen fixation are mediated by several genes, both of the host legume and of the bacterium. The rhizobial regulatory nodD gene plays a critical role, orchestrating the transcription of the other nodulation genes. Rhizobium tropici strain CIAT 899 is an effective symbiont of several legumes-with an emphasis on common bean (Phaseolus vulgaris)-and is unusual in carrying multiple copies of nodD, the roles of which remain to be elucidated. RESULTS: Phenotypes, Nod factors and gene expression of nodD1 and nodD2 mutants of CIAT 899 were compared with those of the wild type strain, both in the presence and in the absence of the nod-gene-inducing molecules apigenin and salt (NaCl). Differences between the wild type and mutants were observed in swimming motility and IAA (indole acetic acid) synthesis. In the presence of both apigenin and salt, large numbers of Nod factors were detected in CIAT 899, with fewer detected in the mutants. nodC expression was lower in both mutants; differences in nodD1 and nodD2 expression were observed between the wild type and the mutants, with variation according to the inducing molecule, and with a major role of apigenin with nodD1 and of salt with nodD2. In the nodD1 mutant, nodulation was markedly reduced in common bean and abolished in leucaena (Leucaena leucocephala) and siratro (Macroptilium atropurpureum), whereas a mutation in nodD2 reduced nodulation in common bean, but not in the other two legumes. CONCLUSION: Our proposed model considers that full nodulation of common bean by R. tropici requires both nodD1 and nodD2, whereas, in other legume species that might represent the original host, nodD1 plays the major role. In general, nodD2 is an activator of nod-gene transcription, but, in specific conditions, it can slightly repress nodD1. nodD1 and nodD2 play other roles beyond nodulation, such as swimming motility and IAA synthesis.

Epigenetic regulation of sex ratios may explain natural variation in self-fertilization rates.

Self-fertilization (selfing) favours reproductive success when mate availability is low, but renders populations more vulnerable to environmental change by reducing genetic variability. A mixed-breeding strategy (alternating selfing and outcrossing) may allow species to balance these needs, but requires a system for regulating sexual identity. We explored the role of DNA methylation as a regulatory system for sex-ratio modulation in the mixed-mating fish Kryptolebias marmoratus. We found a significant interaction between sexual identity (male or hermaphrodite), temperature and methylation patterns when two selfing lines were exposed to different temperatures during development. We also identified several genes differentially methylated in males and hermaphrodites that represent candidates for the temperature-mediated sex regulation in K. marmoratus. We conclude that an epigenetic mechanism regulated by temperature modulates sexual identity in this selfing species, providing a potentially widespread mechanism by which environmental change may influence selfing rates. We also suggest that K. marmoratus, with naturally inbred populations, represents a good vertebrate model for epigenetic studies.

Direct retino-iridal projections and intrinsic iris contraction mediate the pupillary light reflex in early vertebrates.

The pupillary light reflex (PLR) adapts the amount of light reaching the retina, protecting it and improving image formation. Two PLR mechanisms have been described in vertebrates. First, the pretectum receives retinal inputs and projects to the Edinger-Westphal nucleus (EWN), which targets the ciliary ganglion through the oculomotor nerve (nIII). Postganglionic fibers enter the eye-globe, traveling to the iris sphincter muscle. Additionally, some vertebrates exhibit an iris-intrinsic PLR mechanism mediated by sphincter muscle cells that express melanopsin inducing muscle contraction. Given the high degree of conservation of the lamprey visual system, we investigated the mechanisms underlying the PLR to shed light onto their evolutionary origins. Recently, a PLR mediated by melanopsin was demonstrated in lampreys, suggested to be brain mediated. Remarkably, we found that PLR is instead mediated by direct retino-iridal cholinergic projections. This retina-mediated PLR acts synergistically with an iris-intrinsic mechanism that, as in other vertebrates, is mediated by melanopsin and has contribution of gap junctions between muscle fibers. In contrast, we show that lampreys lack the brain-mediated PLR. Our results suggest that two eye-intrinsic PLR mechanisms were present in early vertebrate evolution, whereas the brain-mediated PLR has a more recent origin.

Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.).

BACKGROUND: Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. RESULTS: Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. CONCLUSIONS: Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.

Neuromeric Distribution of Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase Activity in the Adult Lamprey Brain.

This study reports for the first time the distribution and morphological characterization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d; a reliable marker of nitric oxide synthase activity) positive elements in the central nervous system of the adult river lamprey (Lampetra fluviatilis) on the framework of the neuromeric model and compares their cytoarchitectonic organization with that of gnathostomes. Both NADPH-d exhibiting cells and fibers were observed in all major divisions of the lamprey brain as well as in the spinal cord. In the secondary prosencephalon, NADPH-d positive cells were observed in the mitral cell layer of the olfactory bulb, evaginated pallium, amygdala, dorsal striatum, septum, lateral preoptic nucleus, caudal paraventricular area, posterior entopeduncular nucleus, nucleus of the stria medullaris, hypothalamic periventricular organ and mamillary region sensu lato. In the lamprey diencephalon, NADPH-d labeled cells were observed in several nuclei of the prethalamus, epithalamus, pretectum, and the basal plate. Especially remarkable was the staining observed in the right habenula and several pretectal nuclei. NADPH-d positive cells were also observed in the following mesencephalic areas: optic tectum (two populations), torus semicircularis, nucleus M5 of Schöber, and a ventral tegmental periventricular nucleus. Five different cell populations were observed in the isthmic region, whereas the large sensory dorsal cells, some cells located in the interpeduncular nucleus, the motor nuclei of most cranial nerves, the solitary tract nucleus, some cells of the reticular nuclei, and small cerebrospinal fluid-contacting (CSF-c) cells were the most evident stained cells of the rhombencephalon proper. Finally, several NADPH-d positive cells were observed in the rostral part of the spinal cord, including the large sensory dorsal cells, numerous CSF-c cells, and some dorsal and lateral interneurons. NADPH-d positive fibers were observed in the olfactory pathways (primary olfactory fibers and stria medullaris), the fasciculus retroflexus, and the dorsal column tract. Our results on the distribution of NADPH-d positive elements in the brain of the adult lamprey L. fluviatilis are significantly different from those previously reported in larval lampreys and demonstrated that these animals possess a complex nitrergic system readily comparable to those of other vertebrates, although important specific differences also exist.

Comparison of vertebrate skin structure at class level: A review.

The skin is a barrier between the internal and external environment of an organism. Depending on the species, it participates in multiple functions. The skin is the organ that holds the body together, covers and protects it, and provides communication with its environment. It is also the body's primary line of defense, especially for anamniotes. All vertebrates have multilayered skin composed of three main layers: the epidermis, the dermis, and the hypodermis. The vital mission of the integument in aquatic vertebrates is mucus secretion. Cornification began in apmhibians, improved in reptilians, and endured in avian and mammalian epidermis. The feather, the most ostentatious and functional structure of avian skin, evolved in the Mesozoic period. After the extinction of the dinosaurs, birds continued to diversify, followed by the enlargement, expansion, and diversification of mammals, which brings us to the most complicated skin organization of mammals with differing glands, cells, physiological pathways, and the evolution of hair. Throughout these radical changes, some features were preserved among classes such as basic dermal structure, pigment cell types, basic coloration genetics, and similar sensory features, which enable us to track the evolutionary path. The structural and physiological properties of the skin in all classes of vertebrates are presented. The purpose of this review is to go all the way back to the agnathans and follow the path step by step up to mammals to provide a comparative large and updated survey about vertebrate skin in terms of morphology, physiology, genetics, ecology, and immunology.

Priming of defense-related genes in Brassica oleracea var. capitata using concentrated metabolites produced by Rhizobium tropici CIAT 899.

To verify the potential of metabolites extracted from Rhizobium tropici to trigger the priming of defense responses in cruciferous plants, we analyzed the expression of defense-related genes by qRT-PCR. Brassica oleracea var. capitata, susceptible to Xanthomonas campestris pv. campestris, were grown in greenhouse conditions. At 18 days after sowing, plants were inoculated with 1 mL of 1% concentrated metabolites produced by R. tropici (CM-RT) in the root. In a second experiment, leaves were sprayed with 1 mL of a solution containing 1% CM-RT. Aerial and root tissue were collected separately at 0 (non-treated control condition), 24, and 48 h after application, submitted to RNA extraction and gene expression analysis by qRT-PCR. The results showed that, after root treatment with CM-RT, most evaluated genes were upregulated at 24 h after application and downregulated at 48 h after application in roots, while in leaves, genes were downregulated both at 24 and 48 h after application. On the other hand, leaf treatment with CM-RT showed that most evaluated genes in leaves and roots were upregulated at 24 and 48 h after application. These results indicate that the effect of CM-RT applied in roots seems restricted to the applied region and is not sustained, while the application in leaves results in a more systemic response and maintenance of the effect of CM-RT for a longer period. The results obtained in this study emphasize the biotechnological potential of using metabolites of R. tropici as an elicitor of active defense responses in plants.

The nodulation of alfalfa by the acid-tolerant Rhizobium sp. strain LPU83 does not require sulfated forms of lipochitooligosaccharide nodulation signals.

The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of the nod gene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod gene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfa-nodulating rhizobia, Sinorhizobium meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partial nod sequences of Rhizobium mongolense USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic analysis of the intergenic region nodEG positions strain LPU83 and the type strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation.

Brief history of biofertilizers in Brazil: from conventional approaches to new biotechnological solutions.

Brazil has a long history of research with rhizobia and plant growth-promoting rhizobacteria (PGPR). Currently, the use of bio-based products in Brazil, containing microorganisms that are effective in promoting plant growth through various mechanisms, is already a consolidated reality for the cultivation of several crops of agricultural interest. This is due to the excellent results obtained over many years of research, which contributed to reinforce the use of rhizobia and PGPR by farmers. The high quality of the products offered, containing elite strains, allows the reduction and prevention in the use of mineral fertilization, contributing to low-cost and sustainable agriculture. Currently, research has turned its efforts in the search for new products that further increase the efficiency of those already available on the market and for new formulations or inoculation strategies that contribute to greater productivity and efficiency of these products. In this review, the history of biological products for main crops of agricultural interest and the new biotechnologies and research available in the agricultural market are discussed.

The nodC, nodG, and glgX genes of Rhizobium tropici strain PRF 81.

Rhizobium tropici is a diazotrophic microsymbiont of common bean (Phaseolus vulgaris L.) that encompasses important but still poorly studied tropical strains, and a recent significant contribution to the knowledge of the species was the publication of a genomic draft of strain PRF 81, which revealed several novel genes [Pinto et al. Funct Int Gen 9:263-270, 2009]. In this study, we investigated the transcription of nodC, nodG, and glgX genes, located in the nod operon of PRF 81 strain, by reverse-transcription quantitative PCR. All three genes showed low levels of transcription when the cells were grown until exponential growth phase in the presence of common-bean-seed exudates or of the root nod-gene inducer naringenin. However, when cells at the exponential phase of growth were incubated with seed exudates, transcription occurred after only 5 min, and nodC, nodG, and glgX were transcribed 121.97-, 14.86-, and 50.29-fold more than the control, respectively, followed by a rapid decrease in gene transcription. Much lower levels of transcription were observed in the presence of naringenin; furthermore, maximum transcription required 8 h of incubation for all three genes. In light of these results, the mechanisms of induction of the nodulation genes by flavonoids are discussed.