Effects of salinity acclimation on the expression and activity of Phase I enzymes (CYP450 and FMOs) in coho salmon (Oncorhynchus kisutch).

Phase I biotransformation enzymes are critically important in the disposition of xenobiotics within biota and are regulated by multiple environmental cues, particularly in anadromous fish species. Given the importance of these enzyme systems in xenobiotic/endogenous chemical bioactivation and detoxification, the current study was designed to better characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch) and the effects of salinity acclimation on those enzymes. Livers, gills, and olfactory tissues were collected from coho salmon (O. kisutch) after they had undergone acclimation from freshwater to various salinity regimes of seawater (8, 16 and 32 g/L). Using immunoblot techniques coupled with testosterone hydroxylase catalytic activities, 4 orthologs of cytochrome P450 (CYP1A, CYP2K1, CYP2M1, and CYP3A27) were measured in each tissue. Also, the expression of 2 transcripts of flavin-containing monooxygenases (FMO A and B) and associated activities were measured. With the exception of CYP1A, which was down-regulated in liver, protein expression of the other 3 enzymes was induced at higher salinity, with the greatest increase observed in CYP2M1 from olfactory tissues. In liver and gills, 6ß- and 16ß-hydroxylation of testosterone was also significantly increased after hypersaline acclimation. Similarly, FMO A was up-regulated in all 3 tissues in a salinity-dependent pattern, whereas FMO B mRNA was down-regulated. FMO-catalyzed benzydamine N-oxygenase and methyl p-tolyl sulfoxidation were significantly induced in liver and gills by hypersalinity, but was either unchanged or not detected in olfactory tissues. These data demonstrate that environmental conditions may significantly alter the toxicity of environmental chemicals in salmon during freshwater/saltwater acclimation.

Detailed analysis of putative genes encoding small proteins in legume genomes.

Diverse plant genome sequencing projects coupled with powerful bioinformatics tools have facilitated massive data analysis to construct specialized databases classified according to cellular function. However, there are still a considerable number of genes encoding proteins whose function has not yet been characterized. Included in this category are small proteins (SPs, 30-150 amino acids) encoded by short open reading frames (sORFs). SPs play important roles in plant physiology, growth, and development. Unfortunately, protocols focused on the genome-wide identification and characterization of sORFs are scarce or remain poorly implemented. As a result, these genes are underrepresented in many genome annotations. In this work, we exploited publicly available genome sequences of Phaseolus vulgaris, Medicago truncatula, Glycine max, and Lotus japonicus to analyze the abundance of annotated SPs in plant legumes. Our strategy to uncover bona fide sORFs at the genome level was centered in bioinformatics analysis of characteristics such as evidence of expression (transcription), presence of known protein regions or domains, and identification of orthologous genes in the genomes explored. We collected 6170, 10,461, 30,521, and 23,599 putative sORFs from P. vulgaris, G. max, M. truncatula, and L. japonicus genomes, respectively. Expressed sequence tags (ESTs) available in the DFCI Gene Index database provided evidence that ~one-third of the predicted legume sORFs are expressed. Most potential SPs have a counterpart in a different plant species and counterpart regions or domains in larger proteins. Potential functional sORFs were also classified according to a reduced set of GO categories, and the expression of 13 of them during P. vulgaris nodule ontogeny was confirmed by qPCR. This analysis provides a collection of sORFs that potentially encode for meaningful SPs, and offers the possibility of their further functional evaluation.

Transcript profiling of common bean nodules subjected to oxidative stress.

Several environmental stresses generate high amounts of reactive oxygen species (ROS) in plant cells, resulting in oxidative stress. Symbiotic nitrogen fixation (SNF) in the legume-rhizobia symbiosis is sensitive to damage from oxidative stress. Active nodules of the common bean (Phaseolus vulgaris) exposed to the herbicide paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride hydrate), which stimulates ROS accumulation, exhibited reduced nitrogenase activity and ureide content. We analyzed the global gene response of nodules subjected to oxidative stress using the Bean Custom Array 90K, which includes probes from 30,000 expressed sequence tags (ESTs). A total of 4280 ESTs were differentially expressed in stressed bean nodules; of these, 2218 were repressed. Based on Gene Ontology analysis, these genes were grouped into 42 different biological process categories. Analysis with the PathExpress bioinformatic tool, adapted for bean, identified five significantly repressed metabolic pathways related to carbon/nitrogen metabolism, which is crucial for nodule function. Quantitative reverse transcription (qRT)-PCR analysis of transcription factor (TF) gene expression showed that 67 TF genes were differentially expressed in nodules exposed to oxidative stress. Putative cis-elements recognized by highly responsive TF were detected in promoter regions of oxidative stress regulated genes. The expression of oxidative stress responsive genes and of genes important for SNF in bacteroids analyzed in stressed nodules revealed that these conditions elicited a transcriptional response.

Common bean (Phaseolus vulgaris L.) PvTIFY orchestrates global changes in transcript profile response to jasmonate and phosphorus deficiency.

BACKGROUND: TIFY is a large plant-specific transcription factor gene family. A subgroup of TIFY genes named JAZ (Jasmonate-ZIM domain) has been identified as repressors of jasmonate (JA)-regulated transcription in Arabidopsis and other plants. JA signaling is involved in many aspects of plant growth/development and in defense responses to biotic and abiotic stresses. Here, we identified the TIFY genes (designated PvTIFY) from the legume common bean (Phaseolus vulgaris) and functionally characterized PvTIFY10C as a transcriptional regulator. RESULTS: Nineteen genes from the PvTIFY gene family were identified through whole-genome sequence analysis. Most of these were induced upon methyl-JA elicitation. We selected PvTIFY10C as a representative JA-responsive PvTIFY gene for further functional analysis. Transcriptome analysis via microarray hybridization using the newly designed Bean Custom Array 90 K was performed on transgenic roots of composite plants with modulated (RNAi-silencing or over-expression) PvTIFY10C gene expression. Data were interpreted using Gene Ontology and MapMan adapted to common bean. Microarray differential gene expression data were validated by real-time qRT-PCR expression analysis. Comparative global gene expression analysis revealed opposite regulatory changes in processes such as RNA and protein regulation, stress responses and metabolism in PvTIFY10C silenced vs. over-expressing roots. These data point to transcript reprogramming (mainly repression) orchestrated by PvTIFY10C. In addition, we found that several PvTIFY genes, as well as genes from the JA biosynthetic pathway, responded to P-deficiency. Relevant P-responsive genes that participate in carbon metabolic pathways, cell wall synthesis, lipid metabolism, transport, DNA, RNA and protein regulation, and signaling were oppositely-regulated in control vs. PvTIFY10C-silenced roots of composite plants under P-stress. These data indicate that PvTIFY10C regulates, directly or indirectly, the expression of some P-responsive genes; this process could be mediated by JA-signaling. CONCLUSION: Our work contributes to the functional characterization of PvTIFY transcriptional regulators in common bean, an agronomically important legume. Members from the large PvTIFY gene family are important global transcriptional regulators that could participate as repressors in the JA signaling pathway. In addition, we propose that the JA-signaling pathway involving PvTIFY genes might play a role in regulating the plant response/adaptation to P-starvation.

Effects of salinity acclimation on the pesticide-metabolizing enzyme flavin-containing monooxygenase (FMO) in rainbow trout (Oncorhynchus mykiss).

Thioether-containing pesticides are more toxic in certain anadromous and catadromous fish species that have undergone acclimation to hypersaline environments. Enhanced toxicity has been shown to be mediated through the bioactivation of these xenobiotics by one or more flavin-containing monooxygenases (FMOs), which are induced by hyperosmotic conditions. To better understand the number of FMO genes that may be regulated by hyperosmotic conditions, rainbow trout (Oncorhynchus mykiss) were maintained and acclimated to freshwater (<0.5 g/L salinity) and to 18 g/L salinity. The expression of 3 different FMO transcripts (A, B and C) and associated enzymatic activities methyl p-tolyl sulfoxidation (MTSO) and benzydamine N-oxigenation (BZNO) were measured in four tissues. In freshwater-acclimated organisms FMO catalytic activities were as follows: liver>kidney>gills=olfactory tissues; in hypersaline-acclimated animals activities were higher in liver>gills>olfactory tissues>kidney. Acclimation to 18 g/L caused a significant induction in the stereoselective formation of R-MTSO in gill. In olfactory tissues, stereoselective (100%) formation of S-MTSO was observed and was unaltered by acclimation to hypersaline water. When specific transcripts were evaluated, salinity-acclimation increased FMO A in liver (up to 2-fold) and kidney (up to 3-fold) but not in olfactory tissues and gills. FMO B mRNA was significantly down-regulated in all tissues, and FMO C was unchanged by hypersaline acclimation. FMO B and C failed to correlate with any FMO catalytic activity, but FMO A mRNA expression linearly correlated to both FMO catalytic activities (MTSO and BZNO) in liver (r(2)=0.92 and r(2)=0.88) and kidney microsomes (r(2)=0.93 and r(2)=90). FMO A only correlated with MTSO activity in gills (r(2)=0.93). These results indicate unique tissue specific expression of FMO genes in salmonids and are consistent with salinity-mediated enhancement of thioether-containing pesticide bioactivation by FMO which may occur in liver or kidney after salinity acclimation.

MicroRNA expression profile in common bean (Phaseolus vulgaris) under nutrient deficiency stresses and manganese toxicity.

*MicroRNAs (miRNAs) play a pivotal role in post-transcriptional regulation of gene expression in plants. Information on miRNAs in legumes is as yet scarce. This work investigates miRNAs in an agronomically important legume, common bean (Phaseolus vulgaris). *A hybridization approach employing miRNA macroarrays - printed with oligonucleotides complementary to 68 known miRNAs - was used to detect miRNAs in the leaves, roots and nodules of control and nutrient-stressed (phosphorus, nitrogen, or iron deficiency; acidic pH; and manganese toxicity) common bean plants. *Thirty-three miRNAs were expressed in control plants and another five were only expressed under stress conditions. The miRNA expression ratios (stress:control) were evaluated using principal component and hierarchical cluster analyses. A group of miRNAs responded to nearly all stresses in the three organs analyzed. Other miRNAs showed organ-specific responses. Most of the nodule-responsive miRNAs showed up-regulation. miRNA blot expression analysis confirmed the macroarray results. Novel miRNA target genes were proposed for common bean and the expression of selected targets was evaluated by quantitative reverse transcriptase-polymerase chain reaction. *In addition to the detection of previously reported stress-responsive miRNAs, we discovered novel common bean stress-responsive miRNAs, for manganese toxicity. Our data provide a foundation for evaluating the individual roles of miRNAs in common bean.

Global changes in the transcript and metabolic profiles during symbiotic nitrogen fixation in phosphorus-stressed common bean plants.

Phosphorus (P) deficiency is widespread in regions where the common bean (Phaseolus vulgaris), the most important legume for human consumption, is produced, and it is perhaps the factor that most limits nitrogen fixation. Global gene expression and metabolome approaches were used to investigate the responses of nodules from common bean plants inoculated with Rhizobium tropici CIAT899 grown under P-deficient and P-sufficient conditions. P-deficient inoculated plants showed drastic reduction in nodulation and nitrogenase activity as determined by acetylene reduction assay. Nodule transcript profiling was performed through hybridization of nylon filter arrays spotted with cDNAs, approximately 4,000 unigene set, from the nodule and P-deficient root library. A total of 459 genes, representing different biological processes according to updated annotation using the UniProt Knowledgebase database, showed significant differential expression in response to P: 59% of these were induced in P-deficient nodules. The expression platform for transcription factor genes based in quantitative reverse transcriptase-polymerase chain reaction revealed that 37 transcription factor genes were differentially expressed in P-deficient nodules and only one gene was repressed. Data from nontargeted metabolic profiles indicated that amino acids and other nitrogen metabolites were decreased, while organic and polyhydroxy acids were accumulated, in P-deficient nodules. Bioinformatics analyses using MapMan and PathExpress software tools, customized to common bean, were utilized for the analysis of global changes in gene expression that affected overall metabolism. Glycolysis and glycerolipid metabolism, and starch and Suc metabolism, were identified among the pathways significantly induced or repressed in P-deficient nodules, respectively.

Species differences and effects of soft coral extracts from Sinnularia maximus on the expression of cytochrome P4501A and 2N in butterflyfishes (Chaetodon spp.).

Cytochrome P450 (CYP) has been shown to confer resistance in numerous terrestrial insects that consume potentially toxic secondary metabolites in plants, but fewer studies have examined the role of critical biotransformation enzymes in allowing marine organisms to consume chemically defended foods. This study examined the expression of CYP1A and CYP2N mRNAs in several butterflyfish species, which can feed on numerous chemically defended soft and hard corals. In addition, the effect of an extract from a soft coral (Sinnularia maxima) on expression of hepatic CYP1A and CYP2 mRNAs was also examined. Fish were fed extracts on days 1, 3 and 5, and expression was examined on day 5. Phylogenetic analyses of the CYP1A cDNA from 12 species of butterflyfish (DNA, amino acid) indicate well-separated groupings according to their feeding strategies. The non-coralline feeding Chaetodon xanthurus exhibited a 7-fold higher basal expression of CYP2N8 relative to the other species studied. Although induction of CYP2N7 expression was observed in C. punctatofasciatus, CYP1A and CYP2N was largely unaffected or diminished by extract treatment in the other species of butterflyfish. These results indicated groupings of feeding strategy with CYP1A phylogeny in Chaetodon, but generally unaltered expression of CYP1A and CYP2N following dietary treatment with an extract from a chemically defended soft coral suggesting an inconclusive role of these isoforms in the detoxification of chemicals in these extracts.

Osmotic regulation of a novel flavin-containing monooxygenase in primary cultured cells from rainbow trout (Oncorhynchus mykiss).

A cDNA encoding a hepatic isoform of flavin-containing monooxygenase (hFMO) (EF063736) containing an open reading frame of 1792 base pairs (bp) and encoding 554 amino acids was cloned and sequenced from liver mRNA of rainbow trout (Oncorhynchus mykiss). The genomic sequence of hFMO was also characterized and was 4.379 kilobases, possessing 10 exons and 9 introns (EU519462). Structural analysis of the promoter region showed several cis-acting elements including putative glucocorticoid and osmoregulatory response elements, which have been reported to be functionally related to induction of flavin-containing monooxygenase (FMO) proteins in vertebrates. The amino acid sequence showed 74% identity to a putative FMO gene from fugu (Takifugu rubripes; Q6ZZY9), 52 to 55% to zebrafish (Brachydanio rerio; Q5RGM6, Q5RGM3, Q6TLD2, Q7T1D7) FMO5, and 54 and 50% to human FMO1 (Q01740), FMO3 (P49326), and FMO5 (P49326). Southern blot analysis using a 180-bp fragment of the hFMO cDNA indicated at least seven potential genes. Treatment of primary trout hepatocytes with cortisol and sodium chloride for 24 h enhanced hFMO expression. Expression of hFMO was not detected in untreated or solute-treated primary cultures of gill epithelial cells, suggesting tissue-specific expression of hFMO. Induction of hFMO is consistent with the occurrence of cis-osmoregulatory and glucocorticoid response elements identified in the 5'-upstream sequence, indicating regulation of hFMO in response to hypersaline conditions and the osmoregulatory hormone cortisol.

Profilin tyrosine phosphorylation in poly-L-proline-binding regions inhibits binding to phosphoinositide 3-kinase in Phaseolus vulgaris.

The profilin family consists of a group of ubiquitous highly conserved 12-15 kDa eukaryotic proteins that bind actin, phosphoinositides, poly-l-proline (PLP) and proteins with proline-rich motifs. Some proteins with proline-rich motifs form complexes that have been implicated in the dynamics of the actin cytoskeleton and processes such as vesicular trafficking. A major unanswered question in the field is how profilin achieves the required specificity to bind such an array of proteins. It is now becoming clear that profilin isoforms are subject to differential regulation and that they may play distinct roles within the cell. Considerable evidence suggests that these isoforms have different functional roles in the sorting of diverse proteins with proline-rich motifs. All profilins contain highly conserved aromatic residues involved in PLP binding which are presumably implicated in the interaction with proline-rich motif proteins. We have previously shown that profilin is phosphorylated on tyrosine residues. Here, we show that profilin can bind directly to Phaseolus vulgaris phosphoinositide 3-kinase (PI3K) type III. We demonstrate that a new region around Y72 of profilin, as well as the N- and C-terminal PLP-binding domain, recognizes and binds PLP and PI3K. In vitro binding assays indicate that PI3K type III forms a complex with profilin in a manner that depends on the tyrosine phosphorylation status within the proline-rich-binding domain in profilin. Profilin-PI3K type III interaction suggests that profilin may be involved in membrane trafficking and in linking the endocytic pathway with actin reorganization dynamics.