Project description:Although urea is the most used nitrogen fertilizer worldwide, little is known on the capacity of crop plants to use urea per se as a nitrogen source for development and growth. To date, the molecular and physiological bases of its transport have been investigated only in a limited number of species. In particular, up to date only one study reported the transcriptomic modulation induced by urea treatment in the model plant Arabidopsis (Mérigout et al., 2008 doi: 10.1104/pp.108.119339). In maize, one of crops using huge amount of urea, only a physiological characterization of uptake and assimilation of the N-source has been conducted. General aim of the present work was the comprehension of the molecular basis of urea uptake and assimilation in maize plants, using a transcriptomic approach. In addition, the work focused on the possible interactions between the two main N-sources, conceivably occurring concomitantly in the soil, urea and nitrate.
Project description:Although urea is the most used nitrogen fertilizer worldwide, little is known on the capacity of crop plants to use urea per se as a nitrogen source for development and growth. To date, the molecular and physiological bases of its transport have been investigated only in a limited number of species. In particular, up to date only one study reported the transcriptomic modulation induced by urea treatment in the model plant Arabidopsis (MM-CM-)rigout et al., 2008 doi: 10.1104/pp.108.119339). In maize, one of crops using huge amount of urea, only a physiological characterization of uptake and assimilation of the N-source has been conducted. General aim of the present work was the comprehension of the molecular basis of urea uptake and assimilation in maize plants, using a transcriptomic approach. In addition, the work focused on the possible interactions between the two main N-sources, conceivably occurring concomitantly in the soil, urea and nitrate. 5 dd-old maize plants were treated for 8 hours with nutrient solution containing nitrogen in form of urea; nitrate; urea and nitrate; or not exposed to any form of nitrogen. Three different biological replicates were used for each sample repeating the experiment three times. All samples were obtained pooling roots of six plants.
Project description:Cryptococcus neoformans causes meningoencephalitis and is an increasing human health threat. C. neoformans is neurotropic, and persists in the cerebrospinal fluid (CSF) of the mammalian host during infection. In order to survive in the host, pathogenic fungi must procure nutrients such as carbon and nitrogen. To enhance our understanding of nutrient acquisition during infection by Cryptococcus species, we examined utilization of nitrogen sources available in CSF. We screened for growth and capsule production of 817 global environmental and clinical isolates on various sources of nitrogen. Capsule production was assessed using ammonium and urea in the presence or absence of benomyl to determine the relationship of urea exposure to capsule production. Since urea is metabolized to ammonia and CO2 (a known signal for capsule induction), we examined urea metabolism mutants for their response to urea regarding capsule production. Non-preferred nitrogen sources were found to greatly affect capsule production in pathogenic species of Cryptococcus. Urea induced the greatest magnitude of capsule production. Capsule induction by urea was greater in Cryptococcus gattii strains than in C. neoformans strains. In addition, both environmental and clinical strains grew robustly on uric acid, casamino acids, creatinine, and asparagine as sole nitrogen sources. While substantial growth on nitrate was not apparent at day 3, growth was apparent by day 6 for all serotypes. In this study, transcription profiles of urea pathway mutants (ure1 and amt1/2) and WT Cryptococcus neoformans strains were compared in a dye-swap experiment following 1hr exposure to proline or proline + urea (.25g/L).
Project description:Cryptococcus neoformans causes meningoencephalitis and is an increasing human health threat. C. neoformans is neurotropic, and persists in the cerebrospinal fluid (CSF) of the mammalian host during infection. In order to survive in the host, pathogenic fungi must procure nutrients such as carbon and nitrogen. To enhance our understanding of nutrient acquisition during infection by Cryptococcus species, we examined utilization of nitrogen sources available in CSF. We screened for growth and capsule production of 817 global environmental and clinical isolates on various sources of nitrogen. Capsule production was assessed using ammonium and urea in the presence or absence of benomyl to determine the relationship of urea exposure to capsule production. Since urea is metabolized to ammonia and CO2 (a known signal for capsule induction), we examined urea metabolism mutants for their response to urea regarding capsule production. Non-preferred nitrogen sources were found to greatly affect capsule production in pathogenic species of Cryptococcus. Urea induced the greatest magnitude of capsule production. Capsule induction by urea was greater in Cryptococcus gattii strains than in C. neoformans strains. In addition, both environmental and clinical strains grew robustly on uric acid, casamino acids, creatinine, and asparagine as sole nitrogen sources. While substantial growth on nitrate was not apparent at day 3, growth was apparent by day 6 for all serotypes.
Project description:Purpose: RNA-seq technology was used to profile the transcriptome of a Chinese strain of A. anophagefferens that was grown on urea, nitrate, and a mixture of urea and nitrate, and that was under N-replete, limited and recovery conditions to understand the molecular mechanisms that underlie nitrate and urea utilization. Methods: mRNA profiles of Aureococcus anophagefferens that was grown on urea, nitrate, and a mixture of urea and nitrate, and that was under N-replete, limited and recovery conditions were generated by deep sequencing using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed by aligning to reference genome and transcript using SOAPaligner/SOAP2 (version 2.21). Results: Deconvolution and filtering of raw reads yielded a mean of 6,938,798 reads (range: 6,539,842 to 7,242,083 reads) per individual RNA-seq library (Table 1). Subsequent alignment of the clean reads to the A. anophagefferens reference genome yielded a mean of 5,852,408 reads (84.3%) for each sample that mapped to at least one location in the A. anophagefferens genome. However, of these mapped reads, only 47.4 to 50.9% of the total reads were mapped to the reference transcript for each sample (Table 2). In A. anophagefferens grown on three different N sources, 9148 to 9526 genes were detected for each sample. A comparison of gene expression among the three N sources was performed. 322 differentially expressed genes were detected between nitrate-grown and urea-grown cells, 237 between nitrate-grown and mixture N-grown cells and 29 between urea-grown and mixture N-grown cells. Fewer differentially expressed genes between urea-grown and mixture N-grown cells were identified, which further suggested the preferred utilization of cells for urea in media with mixture N. For nitrogen-limited and recovery experiments, 707 genes were up-regulated significantly, and 766 were down-regulated significantly in N-depleted cells relative to N-replete cells. N-depleted cells exhibited a broad transcriptional response to nitrogen re-addition, with 681 genes up-regulated and 874 genes down-regulated in urea recovery cells, and 312 genes up-regulated and 688 genes down-regulated in nitrate recovery cells. Conclusions: We noted that transcripts upregulated by nitrate and N-limitation included those encoding proteins involved in amino acid, nucleotide and aminosugar transport, degradation of amides and cyanates, and nitrate assimilation pathway. The data suggest that A. anophagefferens possesses an ability to utilize a variety of dissolved organic nitrogen. Moreover, transcripts for synthesis of proteins, glutamate-derived amino acids, spermines and sterols were upregulated by urea. Transcripts encoding key enzymes that are involved in the ornithine-urea and TCA cycles were differentially regulated by urea and nitrogen concentration, which suggests that the OUC may be linked to the TCA cycle and involved in reallocation of intracellular carbon and nitrogen. These genes regulated by urea may be crucial for the rapid proliferation of A. anophagefferens when urea is provided as the N source. Seven mRNA samples for Aureococcus anophagefferens were sequencd using Illumina HiSeqTM 2000, including three samples that was grown on urea, nitrate, and a mixture of urea and nitrate, and four samples that was under N-replete, limited and recovery conditions
Project description:Purpose: RNA-seq technology was used to profile the transcriptome of a Chinese strain of A. anophagefferens that was grown on urea, nitrate, and a mixture of urea and nitrate, and that was under N-replete, limited and recovery conditions to understand the molecular mechanisms that underlie nitrate and urea utilization. Methods: mRNA profiles of Aureococcus anophagefferens that was grown on urea, nitrate, and a mixture of urea and nitrate, and that was under N-replete, limited and recovery conditions were generated by deep sequencing using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed by aligning to reference genome and transcript using SOAPaligner/SOAP2 (version 2.21). Results: Deconvolution and filtering of raw reads yielded a mean of 6,938,798 reads (range: 6,539,842 to 7,242,083 reads) per individual RNA-seq library (Table 1). Subsequent alignment of the clean reads to the A. anophagefferens reference genome yielded a mean of 5,852,408 reads (84.3%) for each sample that mapped to at least one location in the A. anophagefferens genome. However, of these mapped reads, only 47.4 to 50.9% of the total reads were mapped to the reference transcript for each sample (Table 2). In A. anophagefferens grown on three different N sources, 9148 to 9526 genes were detected for each sample. A comparison of gene expression among the three N sources was performed. 322 differentially expressed genes were detected between nitrate-grown and urea-grown cells, 237 between nitrate-grown and mixture N-grown cells and 29 between urea-grown and mixture N-grown cells. Fewer differentially expressed genes between urea-grown and mixture N-grown cells were identified, which further suggested the preferred utilization of cells for urea in media with mixture N. For nitrogen-limited and recovery experiments, 707 genes were up-regulated significantly, and 766 were down-regulated significantly in N-depleted cells relative to N-replete cells. N-depleted cells exhibited a broad transcriptional response to nitrogen re-addition, with 681 genes up-regulated and 874 genes down-regulated in urea recovery cells, and 312 genes up-regulated and 688 genes down-regulated in nitrate recovery cells. Conclusions: We noted that transcripts upregulated by nitrate and N-limitation included those encoding proteins involved in amino acid, nucleotide and aminosugar transport, degradation of amides and cyanates, and nitrate assimilation pathway. The data suggest that A. anophagefferens possesses an ability to utilize a variety of dissolved organic nitrogen. Moreover, transcripts for synthesis of proteins, glutamate-derived amino acids, spermines and sterols were upregulated by urea. Transcripts encoding key enzymes that are involved in the ornithine-urea and TCA cycles were differentially regulated by urea and nitrogen concentration, which suggests that the OUC may be linked to the TCA cycle and involved in reallocation of intracellular carbon and nitrogen. These genes regulated by urea may be crucial for the rapid proliferation of A. anophagefferens when urea is provided as the N source.
Project description:We developed an experimental model to elevate BUN during diestrus. There were both urea and control treatments (7 mares/treatment), done in a crossover design. Urea treatment consisted of a loading dose of urea (0.03 g/kg of urea) and urea injections over 6 hours (0.03 g/kg/hr). Control mares received the same volume of saline solution. Blood samples were collected to measure BUN. Uterine and vaginal pH were evaluated after the last intravenous infusion, then endometrial biopsies were collected for RNA-sequencing