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Reaching the roots of systemic nitrogen (N) signaling in plants

Periodic Reporting for period 1 - Nitro Systems (Reaching the roots of systemic nitrogen (N) signaling in plants)

Reporting period: 2015-09-01 to 2017-08-31

The major goal of this project is to develop a molecular understanding of how a multi-cellular, multi-organ organism responds to environmental perturbation as an integrated system. As sessile organisms, plants especially need to mount a system-wide response to fluctuating environmental signals hence they serve as an ideal model to study systemic responses to external perturbations. A good example of system-wide response is illustrated by a long-distance, inter-organ “root-shoot-root” relay system that enables plant roots to specifically forage for nitrogen (N)-rich patches in a heterogeneous nutrient soil environment. Indeed, the level of soil nitrogen (N) is a critical limiting factor for optimal plant growth and varies by several orders of magnitude in soil (1). Plants can adapt to this challenge by sensing through their roots uneven N concentrations in the soil, and by actively foraging via root growth within the N-rich patches. To uncover the regulatory mechanisms under root N-foraging, a split-root system has been previously inducted from the physiological archives into the genomic era (7). In this system, roots of a single plant are split into two halves and exposed to distinct N-nutrient environments (N-replete vs. -deplete) (Fig1A). This system hence mimics the heterogeneous N conditions in nature and agricultural contexts. Importantly, it allows to distinguish systemic N-responses from local N-responses (Fig1B) which are undistinguishable from each other in standard homogenous N set-ups (6). Importantly, the N-foraging response was shown to be mediated by two distinct systemic N-signals (N-demand and N-supply), dependent on a root-shoot-root signal relay (7) (Fig1A). Therefore, the split-root system is a great model to study systemic responses to external stimulus in a multi-organ organism. The specific goal of the Nitro systems program is to decipher the molecular identity of the long-distance signals that mediate the two systemic N-signals in Arabidopsis. Two hypotheses concerning systemic N-signaling are tested. 1) That a systemic N-signal emanating from roots affects the expression of specific genes in shoots which are required to induce root foraging in response. 2) That specific long-distance signals (e.g. hormones) trafficking via phloem - the “information highway”- are involved in systemic N-signaling. This Nitro systems program adapt the original split-root system to achieve four aims. Aim 1 is to generate time-series RNA-seq data from shoots and roots of plants grown on heterogeneous N-environments (Fig2B, left panel and Fig2C). Aim 2 is to physically capture inter-organ travelling RNAs by sequencing RNA in phloem cells in plants exposed to a heterogeneous N-environment (Fig2B, right panel and Fig2C). Aim 3 is to identify candidate genes involved in inter-organ signaling by using predictive modeling of RNA-seq data and to rank them. Aim 4 is to test the highest-rank candidate genes for validation using transgenics and shoot/root grafting. The system used here is a two compartment Vertical Heterogeneous N-environment (VHN) system (Fig2A). The VHN system is a variant of split-root that provides sufficient amounts of material to isolate “traveling” RNA from isolated phloem cells, and also allow to identify genes responding to systemic N-signaling. In detail, the growth media on each plate is divided into 2 sections (top/bottom) of medium with one section containing KNO3 (N-replete) and the other section an equivalent concentration of KCl (N-depleted) (Fig2A). Controls are homogeneous N-depleted (KCl) on each and homogeneous N-replete (KNO3) media on each. This project is the first to integrate N-signaling across roots and shoots to study systemic N-signaling in plants.
1 Alvarez et al. 2012 Current Opinion in Plant Biology 15:185-191
2 Canales et al. 2014a TIPS 5
3 Canales et al. 2014b Frontiers in plant science 5
4 Gifford et al. 1999 The Plant Cell 11:309-322
5 Lee et al. 2006 Proceedings of t
Aim 1
Setup and optimization of the VHN system
Caracterization of the root system architecture after 4 days on VHN for a WT and a KO mutant of an important nitrate sensor, NRT1.1
Performance of 4 sets of experiments (3 reps each) on VHN system at 2h and 8h
RNA extraction, cDNA library preparation and sequencing for all sets
Pre-processing of the RNAseq raw data for all sets
Further bioinformatics analysis more or less advanced
The main activity consisted in developmental (A) and transcriptome (B) data analysis of the root response on VHN system.
A. Root system response on VHN after 4 days in WT and in nrt1.1 mutant: We found that the VHN system allows to detect the systemic N-demand in both the top and the tip parts of the root system in WT (Fig3A). The N-supply which is always more difficult to catch on the split-root system is not detected on the VHN system. These results indicate that VHN system is appropriate to study the root N-foraging response but it also has some differences with the original split-root system. In the nrt1.1 mutant, the N-demand is affected in the top part of the root (Fig.3B) indicating that this gene is part of the foraging systemic response.
B. Gene expression in shoots and roots after 8h on VHN (Fig2B, left panel and Fig2C): Gene expression analysis proved that there is a systemic response after 8h on VHN (assessed by the large shoot response), the response is consistent with response to N in general and heterogeneous N in particular. Moreover, the analysis of the gene expression patterns in the different tissues converge to indicate that various systemic signals co-exist: N-supply signal, N-deficiency signal and heterogeneous N-supply signal. A major finding of this work is that some shoot genes specifically respond to the presence of N at the main root tip. This response is independent of any nutritional consideration and represent a novel and very specific systemic signaling pathway.
Our results led to this model: The N-supply and the N-deficiency signals generated in roots are integrated into a heterogeneous N-supply signal in shoots that regulates roots in feedback. In roots, all the different systemic signals overlap and interact with local signals to generate an appropriate root response.
Aim 2
The protocol for isolation of GFP-marked phloem cells (protoplasts) from roots was tuned as well as FACS cell-sorting and RNA extraction. Another important part of the work was to select and amplify a good phloem-specific GFP-marked line.
This project represents the first inter-organ, systems-view of plant adaptation to a changing environment of N-supply and -demand. It will identify genes contributing to these systemic signals to allow the plant to respond to environmental cues as an integrated system. This knowledge will have potential to enhance N-foraging hence to improve nitrogen-use efficiency (NUE) in agriculture by genetic alterations, to reduce energy costs and environmental contamination associated with N-fertilizers.
For Dr. Eléonore Bouguyon, this project has already been the opportunity to diversify her individual competence in terms of skill acquisition through advanced training. Indeed she learnt new techniques and she learnt about basic programming, bioinformatics and statistics.