Project description:We investigated the morphological roots decisions of Arabidopsis in a NO3- heterogeneous medium. To do so, we used the Split-Root System which is an experimental set up to assess root decisions in nutrient heterogeneous medium. Split-root plants have been subjected to three different treatments. ‘Control KNO3’ plants received KNO3 on both sides of the root system (C.NO3) and ‘Control KCl’ plants received KCl on both sides (C.KCl) as a nitrogen deprivation treatment. 'Split' plants received KNO3 on one side (Sp.NO3) and KCl on the other side (Sp.KCl) of the root system to assess the root decision-making in a heterogeneous environment. We observed that the total lateral roots length in the Sp.NO3 and C.KCl compartments is induced as compared to C.NO3 and Sp.KCl compartments. This corresponds to a root proliferation response in strategic territories to compensate the nitrogen deprivation. To decipher the molecular basis of this morphological root response on day 4 after the beginning of the split-root treatment, we used a transcriptomic approach on roots at 2hours, 8 hours and 2 days after the beginning of the treatment. From our microarrays data, we have identified a global set of 150 genes for which the expression pattern match with the lateral roots responses. Among them, we selected 8 early marker genes of the root decisions, which allowed us to show that the shoots and the NO3- itself are essential for the decision. Finally, we tested the role of the cytokinins phytohormones as a NO3--derived systemic signal in the root decision. Interestingly, we have demonstrated that the systemic cytokinins are involved into the decision of inducing maker genes expression and making lateral roots in the Sp.NO3 compartment specifically. 36 samples were analyzed. They correspond to three biological repeats of the C.NO3, Sp.NO3, Sp.KCl and C.KCl root samples (12 samples) that we have collected at 2hours, 8 hours and 2 days (12 samples x 3 time points) after the beginning of the split-root treatment. We analyzed the normalized microarrays data by using a three way ANOVA. The three factors of the ANOVA are 1) presence/absence of NO3-, 2) split/control and 3) time. The measures of the significance of each probe were done by the Q-value method (q<0.2, panova<0.001). The genes differentially expressed between the C.NO3 and Sp.NO3 samples, and the C.KCl and Sp.KCl samples were determined by the post-hoc Tukey test (p<0.05).

Project description:affy_nitrogen_medicago - affy_nitrogen_medicago - Experiment has been designed to characterize the molecular expression patterns associated to a contrasted modification of the nitrogen status of the whole plant. The systemic effects of nitrogen status modifications are investigated and compared on non nodulated plant supplied with NO3, NH4 or nodulated plants (Sinorhizobium meliloti 2011) supplied with air. The root systems were separated in two compartments of unequal sizes (split root system). Two treatments were applied on the larger compartment in order to modulate the nitrogen status of the plant: for the S treatment, roots are supplied with nutrient solution containing 10 mM NH4NO3,, whereas for the C treatment, roots are supplied with nitrogen free medium. In the case of N2 fixing plants, N limitation was obtained by replacing air by a mixture of Ar and O2 80 per cent and 20 per cent. The effects of these treatments were investigated on roots of the minor compartment supplied continuously with either NO3 1 mM, NH4 1 mM or air (N2) and on the shoots. We were also interested in the molecular expression patterns associated to the roots deprived of N.-The root system of non-nodulated (NO3- and NH4+) or nodulated (N2) plants is split into two unequal parts and each one is installed in a separate compartment. For the S treatement, the major root part is supplied with NH4NO3 10 mM whereas the minor part is supplied with either NO3- 1mM, NH4+ 1mM or N2. For the C treatement, the major root part is supplied with nitrogen-free nutrient solution whereas the minor part is supplied with either NO3- 1mM, NH4plus 1mM or N2. Each treatement is four days long. Samples of roots of six biological types (NO3S, NO3C, NH4S, NH4C, N2S and N2C) were collected. Two biological repeats per biological types have been analyzed. The effect of the S and C treatments were investigated for each N sources by comparing Affymetrix transcriptomes (NO3C vs NO3S, NH4C vs NH4S, N2C vs N2S). Keywords: treatement (nitrogen-sufficient) vs treatement (nitrogen-limited)

Project description:affy_nitrogen_medicago - affy_nitrogen_medicago - Experiment has been designed to characterize the molecular expression patterns associated to a contrasted modification of the nitrogen status of the whole plant. The systemic effects of nitrogen status modifications are investigated and compared on non nodulated plant supplied with NO3, NH4 or nodulated plants (Sinorhizobium meliloti 2011) supplied with air. The root systems were separated in two compartments of unequal sizes (split root system). Two treatments were applied on the larger compartment in order to modulate the nitrogen status of the plant: for the S treatment, roots are supplied with nutrient solution containing 10 mM NH4NO3,, whereas for the C treatment, roots are supplied with nitrogen free medium. In the case of N2 fixing plants, N limitation was obtained by replacing air by a mixture of Ar and O2 80 per cent and 20 per cent. The effects of these treatments were investigated on roots of the minor compartment supplied continuously with either NO3 1 mM, NH4 1 mM or air (N2) and on the shoots. We were also interested in the molecular expression patterns associated to the roots deprived of N.-The root system of non-nodulated (NO3- and NH4+) or nodulated (N2) plants is split into two unequal parts and each one is installed in a separate compartment. For the S treatement, the major root part is supplied with NH4NO3 10 mM whereas the minor part is supplied with either NO3- 1mM, NH4+ 1mM or N2. For the C treatement, the major root part is supplied with nitrogen-free nutrient solution whereas the minor part is supplied with either NO3- 1mM, NH4plus 1mM or N2. Each treatement is four days long. Samples of roots of six biological types (NO3S, NO3C, NH4S, NH4C, N2S and N2C) were collected. Two biological repeats per biological types have been analyzed. The effect of the S and C treatments were investigated for each N sources by comparing Affymetrix transcriptomes (NO3C vs NO3S, NH4C vs NH4S, N2C vs N2S). Experiment Overall Design: 26 arrays - medicago

Project description:Arable soils are extremely heterogeneous in spatial nutrient distribution. It is well documented that lateral roots (LRs) proliferate in nitrate-rich patch. Yet less information is available as to which genes are involved in this process, in particular in cereals. To understand the molecular mechanism for local nitrate induced LR growth in maize (Zea mays L.), we analyzed the gene expression profiling in maize root in early response (1 hour) to local nitrate stimulation by using Maize Oligonucleotide Array (http://www.maizearray.org) and a split-root system. Selected differentially expressed genes were further confirmed by semi-quantitative RT-PCR. The results showed that reception and/or transduction of local NO3- signal involve some important protein kinases and protein phosphatases (histidine kinases, serine/threonine kinases, protein phosphatase 2A etc.) and transcription factors (F-box, Zinc finger, Myb and bZIP transcription factors and response regulator). Increasing expression of genes encoding auxin response factor 7b, ethylene receptor, and cytokinin oxidase suggests strong interaction among hormonal pathways and local NO3- signaling pathways. Genes involving NO3- uptake and assimilation (NRT2.1, NR, etc.), sugar transport (a sugar transporter) and utilization (a sucrose synthase) were enhanced in the N-fed root. Furthermore, local NO3- induces rapid expression of genes related to cell division and expansion such as alpha-expansin, celluose synthase, kinesin, plasma membrane and tonoplast aquaporins. Based on the differentially expressed genes, a putative model which combines both the 'NO3- signal' and 'metabolic sink' theories is proposed to explain the molecular mechanism controlling local nitrate induced LR elongation in maize. plants were pre-cultured in solution containing 0.5 mmol L-1 NO3- for 6 days and then transferred to N-free solution to grow for another 2 days. Then these plants were transferred to a two-compartment split-root system with only half root supplied with 1.0 mmol L-1 NO3-. Supply of Ca2+ in the N-free half was compensated by adding 1.0 mmol L-1 CaCl2. One hour after the local nitrate treatment, root segments (15cm from root tip to lateral root elongation zone) were sampled for RNA extraction. Two biological replicates were performed for each treatment.

Project description:The organs of multicellular species are comprised of cell types that must function together to perform specific tasks. One critical organ function is responding to internal or external change but little is known about how responses are tailored to specific cell types or coordinated among them on a global level. Here we use cellular profiling of five Arabidopsis root cell types in response to a limiting resource, nitrogen, to uncover a vast and predominantly cell-specific response that was largely undetectable using traditional methods. These methods reveal a new class of cell-specific nitrogen responses. As a proof-of-principle, we dissected one cell-specific response circuit that mediates nitrogen-induced changes in root branching from pericycle cells. Thus, cellular response profiling links gene modules to discrete functions in specific cell types. Experiment Overall Design: The whole experiment was carried out in triplicate with 84 chips in total (28 experiments). The nitrogen response in 5 FACS-sorted root cell types (LRC, lateral root cap; EpiC, epidermis and cortex; EndoP, endodermis and pericycle; Peri, pericycle; Stele, stele), protoplasts and Col-0 whole roots was assayed using microarrays. 5mM KNO3 was used as a nitrogen treatment for 2 hours. 5mM KCl was used as a control treatment for the same length of time. 1mM MSX in combination with 5mM KNO3 (or with 5mM KCl as a control) was used to block the assimilation of KNO3, and 5mM Gln added to restore it in order to separate KNO3 effects from assimilated-nitrogen effects. 5mM KNO3 was either maintained in the protoplast-generating solution (continuous treat) or not (transitory treat) to test how to best preserve the cellular nitrogen-response during the 1 hr protoplast generating procedure. As controls we used protoplasts, whole roots frozen directly after the 2hr treatment or 3.5hr treatment, or whole roots incubated in protoplast-generating solution (minus enzymes; transitory or continuous treated) and then frozen. After FACS-sorting or mock FACS-sorting cells were frozen.

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A systemic view of coordinated root responses to NO3- heterogeneous environment in Arabidopsis

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