Transcription profiling of A. glutinosa roots growing with indirect contact with Frankia in nitrogen-free culture medium against A. glutinosa roots alone in nitrogen-free culture medium during 2 days
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ABSTRACT: Experiment with 2 conditions: - Contactless : A. glutinosa alone in nitrogen-free culture medium during 2 days (reference condition). - Indirect : A. glutinosa in nitrogen-free culture medium containing the Frankia bacterium confined in a dialysis tube during 2 days.
Project description:Experiment with 2 conditions: - BAP-PCM : A. glutinosa alone in nitrogen-free culture medium plus frankialess culture medium during 2 days (reference condition). - CN BAP-PCM : A. glutinosa in nitrogen-free culture medium with Frankia culture supernatant during 2 days.
Project description:Alnus glutinosa belongs to a family of angiosperms called actinorhizal plants because they can develop nitrogen-fixing nodules in association with the soil bacteria Frankia. The aim of this transcriptomic study was to get a global view of the plant symbiotic genetic program and to identify new key plant genes that control nodulation during symbiosis in A. glutinosa. Symbiosis between A. glutinosa and Frankia was obtained after inoculation of young plant with a concentrated culture of the bacteria. Inoculation was performed in a medium depleted in nitrogen which favors the induction of nitrogen fixing symbiosis. For this study we considered two stages of symbiosis: - an early stage where inoculated roots were harvested 7 days after inoculation with the bacteria and compared to two controls (non-inoculated roots grown with or without nitrogen and harvested at the same time) - a late stage where nodules (nitrogen-fixing specific organs) were harvested 21 days after inoculation and compared to non-inoculated roots harvested on the day of inoculation (which is our reference time 0d). Three biological replicates were used for each condition. Microarrays were designed by Imaxio (Clermont Ferrand, France ; http://www.imaxio.com/index.php) which has been accredited by Agilent Technologies (Palo Alto, CA, USA; http://www.home.agilent.com/agilent/home.jspx) as a certified service provider for microarray technologies. Based on 14327 annotated unigenes for A. glutinosa, 60mers probes were designed using eArray software (1 probe per unigene) and custom 8 x 15K Oligo Microarrays were manufactured by Agilent
Project description:Trees establish a symbiotic relationship with specialized soil fungi, called ectomycorrhizae, which is essential for nutrition, growth and health of temperate forest ecosystems. Understanding the mechanisms governing the establishment and functioning of ectomycorrhiza is important because of the role of forests in sequestering CO2 and also to develop ways to optimize tree productivity and sustainability. Here, we investigated the response of an oak species to ectomycorrhiza formation using a two dimensional differential in gel electrophoresis (2D-DIGE) and MALDI-TOF/TOF mass spectrometry proteomics approach. At the root level, changes in the abundance of 34 unique oak proteins were detected and revealed proteins involved in carbon and energy metabolism, protein processing and degradation, response to oxidative stress, lipid metabolism/transport, nitrogen and phosphorous assimilation and cell wall modification. Proteins supporting the importance of the secretory pathway functioning, in particular of the endoplasmic reticulum, during ectomycorrhiza functioning were identified. These proteins were identified as components of the endoplasmic reticulum folding/chaperoning machinery and proteins involved in the ER quality control system. This study constitutes an important contribution for the understanding of the mechanisms underlying the response of plants to ectomycorrhizal symbiosis establishment.
Project description:In this study, roots of Rehmannia glutinosa were used as experimental material, and three tuber roots of Rehmannia glutinosa in extension stage (I), expansion stage (E) and mature stage (M) were selected as samples, The iTRAQ quantitative proteomic technology combined with two-dimensional liquid chromatography and tandem mass spectrometry (2D-LC-MSMS) technology was used to construct the root tuber proteome database of Rehmannia glutinosa, the genes related to the growth and development of Rehmannia glutinosa root tuber were found.
Project description:The actinobacteria Frankia alni is able to induce the formation of nodules on the root of a large spectrum of actinorhizal plants, where it converts dinitrogen to ammonia in exchange for plant photosynthates. In the present study, transcriptional analyses were performed on nitrogen-replete free-living cells and on Alnus glutinosa nodule bacteria, using whole genome microarrays. Distribution of nodule-induced genes on the genome was found to be mostly over regions with high synteny between three Frankia genomes, while nodule-repressed genes, which were mostly hypothetical and not conserved, were spread around the genome. Genes known to be related to symbiosis were highly induced: nif (nitrogenase), hup2 (hydrogenase uptake), suf (sulfur-iron cluster) and shc (hopanoids synthesis). The expression of genes involved in ammonium assimilation and transport was strongly modified suggesting that bacteria ammonium assimilation was limited. Genes involved in particular in transcriptional regulation, signalling processes, protein drug export, protein secretion, lipopolysaccharide and peptidoglycan biosynthesis that may play a role in symbiosis were also identified. We showed that this nodule transcriptome of Frankia was highly similar among phylogenetically distant plant families. To address gene expression changes of Frankia alni ACN in the symbiotic state, we compared transcript levels between young nodules formed on 4 species of trees (Alnus glutinosa, Alnus nepalensis, Myrica gale and Myrica rubra) and free-living cells grown in nitrogen-replete minimal medium. For A. glutinosa nodule and free-living cells, two sets of experiments (A and B) were performed in two different laboratories. Three biological replicates were preformed for each condition.
Project description:To better understand the molecular mechanism underlying the rice root response to low nitrogen and high nitrogen, comparative proteomic analysis was performed using tandem mass tag (TMT)-based proteomics, and related proteins were further characterized.
Project description:We study the effect of nitrogen limitation on the growth and development of poplar roots. We used microarrays to detail the global program of gene expression underlying morphological and developmental changes driven by low nitrogen in the growth media. We report the effect of nitrogen limitation on the growth and development of poplar roots. Low nitrogen concentration led to increased root elongation followed by lateral root proliferation and finally increased root biomass. These morphological responses correlated with high and specific activation of genes encoding regulators of cell cycle and enzymes involved in cell wall biogenesis, growth and remodeling. Comparative analysis of poplar and Arabidopsis root transcriptomes under nitrogen deficiency indicated many similarities and diversification in the response in the two species. A reconstruction of genetic regulatory network (GRN) analysis revealed a sub-network centered on a PtaNAC1-like transcription factor. Consistent with the GRN predictions, root-specific upregulation of PtaNAC1 in transgenic poplar plants increased root biomass and led to significant changes in the expression of the connected genes specifically under low nitrogen. PtaNAC1 and its regulatory miR164 showed inverse expression profiles during response to LN, suggesting of a micro RNA mediated attenuation of PtaNAC1 transcript abundance in response to nitrogen deprivation. Poplar roots from low nitrogen treated and untreated from in vitro condition was selected for RNA extraction and hybridization on Affymetrix microarrays. Roots were sampled at 6, 12, 24, 48, 96 and 504h after transfer to control and low nitrogen media and RNA was extacted.
Project description:Rice seedlings grown on a hydroponic set-up depleted of nitrogen were supplemented with 5mM of nitrate, 5mM of ammonium, 2.5mM of ammonium-nitrate or with a negative control (potassium nitrate). Samples were harvested immediately after treatment and over an 8 points time-course up to 48h after treatment. Roots and shoots were harvested separately.
Project description:Over the past decade, growing demand from many domains (research, cosmetics, pharmaceutical industries, etc.) has given rise to significant expansion of the number of in vitro cell cultures. Despite the widespread use of fetal bovine serum, many issues remain. Among them, the whole constitution of most serums remains unknown and is subject to significant variations. Furthermore, the presence of potential contamination and xenogeny elements is challenging for clinical applications, while limited production is an obstacle to the growing demand. To circumvent these issues, a Serum-Free Medium (SFM) has been developed to culture dermal and vesical fibroblasts and their corresponding epithelial cells, namely, keratinocytes and urothelial cells. To assess the impact of SFM on these cells, proliferation, clonogenic and metabolic assays have been compared over three passages to conditions associated with the use of a classic Fetal Bovine Serum-Containing Medium (FBSCM). The results showed that the SFM enabled fibroblast and epithelial cell proliferation while maintaining a morphology, cell size and metabolism similar to those of FBSCM. SFM has repeatedly been found to be better suited for epithelial cell proliferation and clonogenicity. Fibroblasts and epithelial cells also showed more significant mitochondrial metabolism in the SFM compared to the FBSCM condition. However, the SFM may need further optimization to improve fibroblast proliferation.
Project description:Legumes, in interaction with resistant rhizobia, combined both moderate tolerance and accumulation of metal(loids) in roots, with the ability to grow without nitrogen supply (Pajuelo et al., 2011). This quality has attracted attention for phytostabilisation of polluted soils (Reichman, 2007). Physiological studies suggest that low arsenite concentrations lead to a decrease of nodulation process (Dary et al., 2010; Pajuelo et al., 2008). Moreover, Lafuente et al. (2010) described a reduction in the expression patterns of nodulins genes in the presence of arsenite. Nevertheless, a global transcriptomic analysis has never been approached. In order to decipher the genetic regulation underlying the arsenite effect on the model symbiotic interaction Medicago-Sinorhizobium, we have performed a meta-analysis of three different hybridizations. These compare transcriptomic profiles of roots cultivated under different treatments (M-125 M-5M arsenite, M-1rhizobia).