Constitutive expression of Medicago truncatula NRT1 PTR FAMILY 1.7 enhances growth in Arabidopsis thaliana
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ABSTRACT: To gain insight into the molecular mechanisms underlying the increased growth of Col-0:MtNPF1.7 CE plants compared to WT, leaves from plants grown for 21 days on defined media were subjected to transcript profiling using the Affymetrix Arabidopsis ATH1 microarray. We tested gene expression from plants grown at 0.1 mM nitrate and at 10 mM nitrate. The 0.1 mM nitrate concentration is below MtNPF1.7’s Km and at that nitrate concentration, large growth differences between Col-0:MtNPF1.7 CE and wild-type Col-0 plants were observed, while 10 mM nitrate is well above MtNPF1.7’s Km, where fewer differences in growth were seen. Three independent biological replicates were collected.
Project description:Plants aquire nitrogen from the soil, most commonly in the form of either nitrate or ammonium. Unlike ammonium, nitrate must be reduced (with NADH and ferredoxin as electron donors) prior to assimilation. Thus, nitrate nutrition imposes a substantially greater energetic cost than ammonium nutrition. Our goal was to compare the transcriptomes of nitrate-supplied and ammonium-supplied plants, with a particular interest in characterizing the differences in redox metabolism elicited by different forms of inorganic nitrogen. We used microarrays to compare the short-term transcriptional response to either nitrogen supply or ammonium supply in Arabidopsis roots. Genes upregulated or downregulated by nitrate only, ammonium only, or both ammonium and nitrate were identified and analyzed. Arabidopsis thaliana (Col-0) plants were grown hydroponically until they reached growth stage 5.10. They were then transferred to a nitrogen-free medium for 26 hr and then supplied with 1 mM nitrate or 1 mM ammonium. RNA isolation (and subsequent microarray analysis) was performed on root tissue isolated just before nitrogen supply (time 0) and at 1.5 hr and 8 hr after nitrogen supply (1.5 hr nitrate, 8 hr nitrate, 1.5 hr ammonium, 8 hr ammonium).
Project description:Nitrate and other nitrogen metabolites can act as signals that regulate global gene expression in plants. Adaptive changes in plant morphology and physiology triggered by changes in nitrate availability are partly explained by these changes in gene expression. Despite several genome-wide efforts to identify nitrate-regulated genes, no comprehensive study of the Arabidopsis root transcriptome under contrasting nitrate conditions has been carried out. In this work, we employed the Illumina high throughput sequencing technology to perform an integrated analysis of the poly-A+ enriched and the small RNA fractions of the Arabidopsis thaliana root transcriptome in response to nitrate treatments. Our sequencing strategy identified new nitrate-regulated genes including 40 genes not represented in the ATH1 Affymetrix GeneChip, a novel nitrate-responsive antisense transcript and a new nitrate responsive miRNA/TARGET module consisting of a novel microRNA, miR5640 and its target, AtPPC3. This nitrate-responsive miRNA/target module might be involved in controlling carbon flux to assimilate nitrate into amino acids, suggesting that microRNAs can have both developmental and metabolic functions in the nitrate response of Arabidopsis roots. Arabidopsis thaliana wild-type Col-0 plants were grown in hydroponic nitrate-free medium with 0.5 mM ammonium succinate as the only N-source for two weeks and were treated with 5 mM KNO3, or 5 mM KCl as control, for 2 hours. Total RNA from two independent sets of plants (biological replicates) was extracted from roots, and poly-A+ enriched and sRNA fractions were used to construct libraries for Illumina sequencing.
Project description:Transcriptional profile of leaves from poplar plants transformed with a pine GS1a gene under two different nitrate nutrition level (50mM and 10mM). Two conditions: nitrate nutrition (50mM and 10 mM) and genotype (GS1a transgenic poplar vs WT poplar)
Project description:In this project, we treated Arabidopsis wild type (Col-0) with low nitrate (0.2 mM KNO3) and high nitrate (5 mM KNO3) after nitrate starvation. Next we harvested root, and then we performed memebrane fraction and cytosolic fraction isolation for trypsin digestion and phosphopeptide enrichment.
Project description:Freshly harvested Arabidopsis seeds (ecotype Col0 obtained under 10 mM nitrate nutrition, referred to as C10) are dormant and do not germinate when sown on an agarose-based medium. This experiment was performed to analyze the genes that are differentially expressed in freshly harvested C10 seeds sown on agarose (dormant) versus freshly harvested C10 dry seeds (dormant) to analyze differential expression of genes during the first 6 hrs of imbibition of dormant seeds. Reference: Alboresi et al. Plant Cell Environ (2005) 28 : 500-512 - 16 DAP siliques were harvested from Col 0 plants grown on 10 or 50 mM nitrate or from G4 3 mutant grown on 10 mM nitrate in growth chambers. Keywords: treated vs untreated comparison
Project description:Nitrate regulates plant growth and development and acts as a potent signal to control gene expression in Arabidopsis. Using an integrative bioinformatics approach we identified TGA1 and TGA4 as putative regulatory factors that mediate nitrate responses in Arabidopsis thaliana roots. We showed that both TGA1 and TGA4 mRNAs accumulate strongly and quickly after nitrate treatments in root organs in a tissue-specific manner. Phenotypic analysis of tga1/tga4 double mutant plants indicated that TGA1 and TGA4 are necessary for nitrate modulation of both primary and lateral root growth. Global gene expression analysis revealed that 97% of the genes with altered expression in tga1/tga4 double mutant plants are regulated by nitrate treatments indicating these transcription factors have a specific role in nitrate responses in Arabidopsis root organs. Among the nitrate-responsive genes that depend on TGA1/TGA4 for normal regulation of gene expression, we found nitrate transporters NRT2.1, NRT2.2 and nitrite reductase (NIR) genes. Specific binding of TGA1 to its cognate DNA sequence on the target gene promoters was confirmed by chromatin immunoprecipitation assays. These results identify TGA1 and TGA4 as important regulatory factors of the nitrate response in Arabidopsis roots. Arabidopsis seedlings of the Col-0 and tga1/tga4 genotypes were grown on hydroponic medium containing 1X MS salts without Nitrogen, supplemented with 0.5 mM ammonium succinate as Nitrogen source and 3 mM sucrose on a Percival chamber under a photoperiod of 16 hours of light (100 ?E/m2/sec) and 8 hours of dark at 22°C for 14 days. The plants were treated at the onset of the light cycle with 5 mM KNO3 or 5 mM KCl as control for 2 hours. Whole roots were cut from seedlings and frozen on liquid Nitrogen. Total RNA was extracted using the TriZol reagent. 3 independent biological replicates were performed.
Project description:We used Arabidopsis thaliana Col-0 for all experiments. The plants were grown hydroponically on nutrient solution as described previously [Plant J 1999, 18(5):509-519]. Briefly, plants were grown on sand, placed in custom-designed styrofoam rafts, in a growth chamber (EGC, Chagrin Falls, OH, USA) at 22°C with 60 mmol photons m-2s-1 light intensity and 8 h/16 h light/dark cycles. The seeds were initially germinated in tap water. After one week, the water was replaced with a complete nutrient solution [Plant J 1999, 18(5):509-519]. All the experiments were performed with 6 week old plants. Nutrient solutions were renewed weekly and on the day before the experiments. For treatments, individual rafts were transferred to containers with 300mL of nutrient solution supplemented with various concentrations of nitrate (as a mix of 2/1 KNO3/Ca(NO3)2) and/or sucrose. The N-free nutrient solutions contained 0.25 mM K2SO4 and 0.25 mM CaCl2 instead of KNO3 and Ca(NO3)2. Plants were transferred to treatments media at the beginning of the light period and were harvested 8h afterwards. Roots and leaves were harvested separately and quickly frozen in liquid N2.
Project description:Freshly harvested Arabidopsis seeds (ecotype Col0 obtained under 10 mM nitrate nutrition, referred to as C10 seeds) are dormant and do not germinate when sown on an agarose-based medium. However, when C10 seeds are sown on 10mM nitrate or when Col0 mother plants are grown on 50 mM nitrate (referred to as C50 seeds), the produced seeds are non dormant and germinate readily on agarose. The G4-3 arabidopsis mutant which is impaired in nitrate assimilation and accumulates nitrate produces likewise non dormant seeds when grown on 10mM nitrate (referred to as G4-3 seeds). The goals of these experiments are to better understand the effects of nitrate feeding of mother plants on the produced seed dormancy.
Project description:Because nitrogen (N) nutrition is a key determinant of plant growth, we explored the role of N availability in grafted grapevine development. Vitis vinifera cv. Cabernet Sauvignon was grafted on two rootstock genotypes known to confer high (1103 Paulsen, 1103P) and low (Riparia Gloire de Montpellier, RGM) vigour. One-year-old plants were cultivated in sand-filled pots in a greenhouse and irrigated with the control nutrient solution for 15 days of acclimation (1.6 mM N). At the end of the acclimation period (0 days post treatment (dpt)), the plants were divided in two groups of 5 plants per combination and irrigated with nutrient solutions varying only in their nitrate concentration (0.8 mM (Nitrate -) and 2.45 mM (Nitrate +)). Roots were harvested at 15 and 60 dpt. Gene expression profiling was done using the Nimblegen whole genome array with 3 biological replicates per condition to analyze the combined effect of N treatment and rootstock genotype on gene expression.
Project description:ra05-12_nar2 - atnar2.1-1 - Does the ko of AtNAR2.1 leads to a differential expression in comparison to the WT when grown under high nitrate condition and transfered onto low nitrate concentration? - Plants were grown for 41 days in hydroponics in media containing 6 mM nitrate and then transferred to 6 or 0.2 mM nitrate media for 24h ( short days, irradiation 150 uE). Plants were harvested on day 42 during the first 2 hours of light. Keywords: dose response,gene knock out