Project description:Sugars modulate expression of hundreds of genes in plants. Previous studies on sugar signaling, using intact plants or plant tissues, were hampered by tissue heterogeneity, uneven sugar transport and/or inter-conversions of the applied sugars. This, in turn, could obscure the identity of a specific sugar that acts as a signal affecting expression of given gene in a given tissue or cell-type. To bypass those biases, we have developed a novel biological system, based on stem-cell-like Arabidopsis suspension culture. The cells were grown in a hormone-free medium and were sustained on xylose as the only carbon source. The functional genomics approach was used to identify sugar responsive genes, which rapidly (within 1 h) respond specifically to low concentration (1 mM) of glucose, fructose and/or sucrose. A habituated A. thaliana cell culture grown in a hormone free full strength MS media in the dark was adapted to growth on xylose as the only carbon source in the media.The cells were subjected to a 1 hour treatment with 1 mM of either Fru, Glc, Suc or Xyl (control). The experiments were carried out in 3 biological repeats per treatment. Whole genome expression analysis was conducted by hybridization of the extracted RNA to the Affymetrix Arabidopsis ATH1 Genome Array.
Project description:We analyzed the transcriptional response of the actinomycete Rhodococcus aetherivorans I24 to biphenyl and polychlorinated biphenyls (PCBs). This species has not been extensively exposed to PCBs, as it was first isolated from a toluene contaminated aquifer, rather than a site contaminated with polychlorinated hydrocarbons. Using a microarray targeting 3524 genes, we assessed gene expression in minimal medium supplemented with various substrates (e.g. PCBs) and in both PCB-contaminated and non-contaminated sediment slurries. Relative to the reference condition (minimal medium supplemented with glucose), 408 genes were up-regulated in the various treatments. In medium and in sediment, PCBs elicited the up-regulation of a common set of 100 genes, including chaperones (groEL), a superoxide dismutase (sodA), alkyl hydroperoxide reductase protein C (ahpC), and a catalase/peroxidase (katG). Analysis of the R. aetherivorans I24 genome sequence identified orthologs of many of the genes in the canonical biphenyl pathway, but very few of these genes were up-regulated in response to PCBs or biphenyl. This study is one of the first which utilizes microarrays to assess the transcriptional response of a soil bacterium to a pollutant under conditions which more closely resemble the natural environment. Our results indicate that the transcriptional response of R. aetherivorans I24 to PCBs, in both medium and sediment, is primarily directed towards reducing oxidative stress, rather than catabolism. In addition, the identification of numerous genes expressed in contaminated soil specifically may have implications for the development of biosensors. Finally, comparative genomic and transcriptomic analyses suggest that the mere presence of orthologs of the required enzymes may not be sufficient to confer a vigorous biphenyl/PCB metabolism. RNA was isolated from cells incubated in the following: sediment from a PCB-contaminated industrial site, uncontaminated sediment from a comparable site, and defined media supplemented with glucose (3 g/L), glucose and biphenyl (3 g/L, 4.5 μM), or glucose and PCBs (3 g/L, 5 mg/L Aroclor 1254). In all cases, there were 3 biological replicates and 2 technical replicates (repeat hybridizations). A total of 3524 genes are represented on the arrays; of these, 41 and 176 are found on the plasmids pRA2 and pRA3, respectively. On average, there are 3 distinct 24nt probes per gene.
Project description:A custom-made microarray was used for comparative transcriptome analysis of transgenic suspension cell line of Nicotiana tabacum that overexpresses the CaRLK1 gene. Ectopic expression of the CaRLK1 preferentially upregulated many amino acid biosynthetic genes under hypoxia: a 1.7-fold higher steady-state concentration of total free amino acids. Free Ala level was predominantly increased by 3-4 times and reached more than half of total free amino acid content. A significantly and markedly increased pyruvate content was also observed. These accumulations are associated with the glyoxylate cycle positively regulated by the CaRLK1. A total of 12 chips were used for microarray. Total RNAs were extracted from BY-2 suspension cells (control), RLK1ox suspension cells (RLK), auxin-free RLK1ox suspension cells (RA), and 10 uM DPI-treated RLK1ox suspension cells (RD) with triple biological replicates.
Project description:The presence of genetic groups of the entomopathogenic fungus Metarhizium anisopliae in soil is shaped by its adaptability to specific soil and habitat types, and by soil insect populations. Although the entomopathogenic life style of this fungus is well studied, its saprophytic life style has received little consideration. While a set of functionally related genes can be commonly expressed for the adaptability of this fungus to different environments (insect cuticle, insect blood and root exudates), a different subset of genes is also expected for each environment. In order to increase the knowledge of the potential use of M. anisopliae as a rhizosphere competent organism, in this study we evaluated the genetic expression of this fungus while growing on plant root exudates in laboratory conditions during a time course. One fungal strain: Metarhizium anisopliae ARSEF 2575; Five time conditions: 0h, 1h, 4h, 8h, 12h; Five-condition experiment: Time0h vs. Time1h, Time1h vs. Time4h, Time4h vs. Time8h, Time8h vs. Time12h and Time12h vs. Time0h. Two Biological replicates: independently grown and harvested. Three replicates per array. Dye-swap was performed on replicate 2.
Project description:Galactose catabolism in Aspergillus nidulans is regulated by at least two regulators, GalR and GalX. In Aspergillus niger only GalX is present, and its role in D-galactose catabolism in this fungus was investigated. Phenotypic and gene expression analysis of a wild type and a galX disruptant revealed that GalX does not substitute for the absence of GalR in A. niger, it regulates the D-galactose oxido-reductive pathway, but not the Leloir pathway. Four genes, including the recently characterized ladB (galactitol dehydrogenase) were found to have differencial expressions that are highly relevant to GalX , indicating a novel oxido-reductive pathway in A.niger . We aim to discover differentially expressed genes in A.niger wild type strain N402 and M-NM-^TgalX mutant while growing on galactose as carbon source. Biological duplicates were made for both strains. The strains were grown O/N in complete medium with 2% frunctose and mycelium was then washed and transferred to minimal medium with 25 mM D-galactose and incubated for 2 hours. Affymetrix microarray experiments were performed RNA isolated from these samples.
Project description:Transcriptional profiling after inhibition of cellulose synthesis by thaxtomin A and isoxaben in Arabidopsis thaliana suspension cells; Perturbations in the cellulose content of the plant cell wall lead to global modifications in cellular homeostasis, as seen in cellulose synthase mutants or after inhibiting cellulose synthesis. In particular, application of inhibitors of cellulose synthesis such as thaxtomin A (TA) and isoxaben (IXB) initiates a programmed cell death (PCD) in Arabidopsis thaliana suspension cells that is dependent on de novo gene transcription. To further understand how TA and IXB activate PCD, a whole genome microarray analysis was performed on mRNA isolated from Arabidopsis suspension cells exposed to TA and IXB. More than 75% of the genes upregulated by TA were also upregulated by IXB, including genes encoding cell wall-related and calcium-binding proteins, defence/stress-related transcription factors, signalling components and cell death-related proteins. Comparisons with published transcriptional analyses revealed an important subset of genes generally induced in response to various biotic and abiotic stress. Experiment Overall Design: TA, IXB and methanol (control) were added to Arabidopsis thaliana suspension cells three days after sub-culture. Cells were harvested for RNA isolation and frozen in liquid nitrogen after 6 hours of contact with the inhibitors. Samples consisted of four replicates for each condition. A total of 12 Affymetrix GeneChips® were used in this study, which correspond to 12 RNA samples from the four biological replicates for each of the TA, IXB or methanol addition.
Project description:DELLA proteins interact with ARR1 and modulate its activity. We have investigated the effect of DELLA proteins on transcriptional regulation by ARR1 A dominant hyperactive allele of ARR1 was induced with dexamethasone in seedlings preincubated with paclobutrazol (allowing DELLA acummulation) or paclobutrazol+GA3 (promoting DELLA degradation)
Project description:Fungi produce a wide range of enzymes that allow them to grow on diverse plant biomass. Wheat bran is a low-cost substrate with high potential for biotechnological applications. It mainly contains cellulose and (arabino)xylan, with starch, proteins, lipids and lignin as minor components. In this study, we dissected the regulatory network governing wheat bran degradation in Aspergillus niger. Deletion of genes encoding transcription factors involved in (hemi-)cellulose utilization (XlnR, AraR, ClrA and ClrB) individually and in combination significantly reduced production of polysaccharide-degrading enzymes, but retained substantial growth on wheat bran. Proteomic analysis suggested the ability of A. niger to grow on minor carbon components, such as starch, which was confirmed by the additional deletion of the amylolytic regulator AmyR. Growth was further reduced but not impaired, indicating that other minor components provide sufficient energy for residual growth, displaying the flexibility of A. niger, and likely other fungi, in carbon utilization.
Project description:Investigation of whole genome gene expression level changes in trichostatin A (TSA)-treated A. fumigatus Af293 compared to non-treated A. fumigatus Af293. Species: Aspergillus fumigatus; Strain: Af293; Type of array: Eukaryotic Expression (4plex, 2plex for a TSA-treated sample and 2plex for a non-treated sample); Technical replicates: Two per treatment.