Periodic Reporting for period 2 - Nitro Systems (Reaching the roots of systemic nitrogen (N) signaling in plants)
Berichtszeitraum: 2017-09-01 bis 2018-08-31
The goal of this project was to develop a molecular understanding of how a multi-organ organism responds to environmental perturbation as an integrated system. Plants especially need to mount a system-wide response to fluctuating signals and are an ideal model to study systemic responses to perturbations. An example of such response is illustrated by a long-distance “root-shoot-root” relay that enables roots to specifically forage for nitrogen (N)-rich patches in a heterogeneous soil environment. Indeed, the level of N is a limiting factor for plant growth and varies by several orders of magnitude in soil. Plants can adapt to this challenge thanks to the ability of roots to sense uneven N concentrations in the soil and to forage via root growth within the N-rich patches. To uncover the underlying regulatory mechanisms, a “split-root” system has been previously studied into the genomic era. Roots of a single plant are split into two halves and exposed to distinct N environments (N-replete vs. N-deplete) (Fig. 1A). It mimics the heterogeneous soil N conditions and allows to distinguish systemic from local N-responses (Fig. 1B). The N-foraging response of roots was shown to be mediated by two distinct systemic N-signals (N-demand and N-supply), dependent on a root-shoot-root signal relay (Fig. 1A). Therefore, the split-root system is a great model to study systemic inter-organ responses to external stimulus in a multi-organ organism.
The goal of Nitro Systems is to decipher the identity of the long-distance signals mediating the systemic signals in Arabidopsis. Our first aim was to generate time-series RNA-seq data from shoots and roots of plants grown on heterogeneous N-environments (Fig.2B left panel and Fig.2C). The following aims were to physically capture inter-organ travelling RNAs by sequencing RNA in phloem cells (Fig.2B right panel and Fig.2C) to identify candidate genes involved in inter-organ signaling by using predictive time-based modeling of RNA-seq data and to validate the highest-rank candidate genes using a genetic approach. The system used is a two compartment Vertical Heterogeneous N-environment (VHN) system (Fig.2A). 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) (Fig.2A). Controls are (i) homogeneous N-depleted (KCl) on each, and (ii) homogeneous N-replete (KNO3) media on each.
After having performed an integrative study of N-foraging response in this VHN system, we concluded that systemic N-demand but not systemic N-supply signaling operates in this system to control root architecture and N-accumulation. Specifically in the older part of the root system, we show for the first time that this systemic N-demand signaling required the activity of the transceptor NRT1.1. Using this system, our last and most interesting conclusion is that in addition to previously known systemic signaling, a « N at tip » signal regulates a part of the molecular responses of the shoots to the nitrate provision allowing the plant to know at a systems level if nitrate is available at the tip of the primary root.
The goal of Nitro Systems is to decipher the identity of the long-distance signals mediating the systemic signals in Arabidopsis. Our first aim was to generate time-series RNA-seq data from shoots and roots of plants grown on heterogeneous N-environments (Fig.2B left panel and Fig.2C). The following aims were to physically capture inter-organ travelling RNAs by sequencing RNA in phloem cells (Fig.2B right panel and Fig.2C) to identify candidate genes involved in inter-organ signaling by using predictive time-based modeling of RNA-seq data and to validate the highest-rank candidate genes using a genetic approach. The system used is a two compartment Vertical Heterogeneous N-environment (VHN) system (Fig.2A). 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) (Fig.2A). Controls are (i) homogeneous N-depleted (KCl) on each, and (ii) homogeneous N-replete (KNO3) media on each.
After having performed an integrative study of N-foraging response in this VHN system, we concluded that systemic N-demand but not systemic N-supply signaling operates in this system to control root architecture and N-accumulation. Specifically in the older part of the root system, we show for the first time that this systemic N-demand signaling required the activity of the transceptor NRT1.1. Using this system, our last and most interesting conclusion is that in addition to previously known systemic signaling, a « N at tip » signal regulates a part of the molecular responses of the shoots to the nitrate provision allowing the plant to know at a systems level if nitrate is available at the tip of the primary root.
The main activity consisted in morphological, physiological and transcriptomics analysis of the root response on the VHN system. The VHN system allows us to detect systemic “N-demand” effect on lateral root (LR) length in both top (R1) and bottom (R2) parts of the root system in the wild-type (Fig.3A). We also showed that the primary root (PR) length responds to “N-demand”. These results indicate that the VHN system is appropriate to study the root foraging response to heterogeneous distribution of N in the growth medium. In a nitrate sensor KO mutant, LR “N-demand” response is affected in the top (R1) part of the root (Fig.3B). This indicates that this sensor is part of the systemic response to heterogeneous N-environment. After only 8h on VHN, shoots accumulate gradually increasing quantities of N depending on the conditions (Ctr+N > N-top > N-tip > Ctr-N) (Fig.4A). The bottom part of the root (R2) is more efficient in N-accumulation than the top (R1) but the later has a higher capacity (Fig.4A to 4C). Nevertheless, after 4 days, plant biomass is similar in Ctr+N and heterogeneous N-conditions (N-top, N-tip) (Fig.4E). These data indicate that plants submitted to heterogeneous N-supply adjust their internal N-status to maintain growth at a level comparable to that of plants grown on homogeneous N-supply. Differential gene expression analysis proved that a systemic N-response is detectable as soon as 2h in both shoots and roots (Fig.5 and 8). However, this systemic response is larger at 8h (Fig.5 and 8) and is largely consistent with response to N in general and heterogeneous N in particular. In roots, we found different dynamics in gene responses to heterogeneous N depending on where N is supplied along the main root axis; systemic N-response is faster in R2 than R1. Moreover, at each time point, the DEG are overall mostly specific to each part of the root but gene expression patterns and biological functions are very similar.
The analysis of the gene expression patterns at 8h in the different tissues converge to indicate that various systemic signals co-exist. We identified 3 root-to-shoot signals (Fig.9 and 11): a N-supply signal, a N-demand signal and a “N at tip” signal coming from the root apex and indicating to the shoot whether N is present of not (Fig. 10). The latter signals is independent of any nutritional consideration and represent a so far unknown and very specific systemic signaling pathway. We showed that “N at tip signal” is dependent on root apex (3mm) and on the nitrate sensor previously mentioned. Besides, we discovered that shoots seem to “sense” how much N has been accumulated in aerial parts and to regulate genes accordingly (Fig. 9 and 11 “N-dose” pattern). In addition, shoots would integrate the “N-status” information with the 3 ascending signals and would send an integrative signal back to the roots (N-repletion, N-depletion, heterogeneous N, etc.) (Fig.7). In roots, those systemic signals would be integrated with local one to adjust gene expression.
The dissemination of these results will be done through the publication of a scientific article in one of the best journal. This article is currently under writing. After the publication, the question of a root tip signal directly transduced to the shoots will be further explored. Notably, we will address the respective role of the root and shoot apical meristems by exploiting the shoot transcriptional responses highlighted in this project.
The analysis of the gene expression patterns at 8h in the different tissues converge to indicate that various systemic signals co-exist. We identified 3 root-to-shoot signals (Fig.9 and 11): a N-supply signal, a N-demand signal and a “N at tip” signal coming from the root apex and indicating to the shoot whether N is present of not (Fig. 10). The latter signals is independent of any nutritional consideration and represent a so far unknown and very specific systemic signaling pathway. We showed that “N at tip signal” is dependent on root apex (3mm) and on the nitrate sensor previously mentioned. Besides, we discovered that shoots seem to “sense” how much N has been accumulated in aerial parts and to regulate genes accordingly (Fig. 9 and 11 “N-dose” pattern). In addition, shoots would integrate the “N-status” information with the 3 ascending signals and would send an integrative signal back to the roots (N-repletion, N-depletion, heterogeneous N, etc.) (Fig.7). In roots, those systemic signals would be integrated with local one to adjust gene expression.
The dissemination of these results will be done through the publication of a scientific article in one of the best journal. This article is currently under writing. After the publication, the question of a root tip signal directly transduced to the shoots will be further explored. Notably, we will address the respective role of the root and shoot apical meristems by exploiting the shoot transcriptional responses highlighted in this project.
This project is the first study to investigate gene response to heterogeneous N-supply depending on where N is applied on roots. Our major findings are that the root apex is able to communicate to the shoot whether N is detected or not through a signaling pathway depending on a major nitrate sensor. This work also constitutes an important community ressource that can feed further works to identify key genes contributing to the different systemic signals that 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.