# Systemic nutrient signalling: On the road for nitrate.

Sandrine Ruffel, Alain Gojon

► To cite this version:

Sandrine Ruffel, Alain Gojon. Systemic nutrient signalling: On the road for nitrate.. Nature Plants, 2017, 3, pp.17040. 10.1038/nplants.2017.40. hal-01595538

HAL Id: hal-01595538

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Submitted on 22 Aug 2025

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**Strapline**: Systemic Nutrient Signaling

**Title [35c max]:** Glutaredoxins on the road for nitrate

**Authors:** Sandrine Ruffel and Alain Gojon

**Standfirst [around 250c/50w]:**

Whole plant nutrient signaling involves bi-directional exchanges of signal molecules between roots and shoots. For nitrogen, small glutaredoxins are phloem-mobile signals relaying the nitrogen deprivation information to the root system and are key regulators of the nitrate uptake system.

**Main text [800-900w max, here 800]:**

To ensure adequate acquisition of mineral nutrients from the soil, plants need to integrate information on the local external availability of these nutrients and on their own internal nutrient status. This integration relies on the reciprocal exchange of long distance signals between roots and shoots, which ultimately modulates the activity of the root uptake systems to match the nutrient demand of the whole organism. Concerning nitrate, the main nitrogen source for many plant species, part of the whole plant signaling governing its root acquisition has recently been unraveled in *Arabidopsis thaliana*¹. Roots locally starved with nitrate synthesize short mobile peptides (CEPs) that are secreted into the xylem and translocated to the shoot, where they are perceived by two specific CEP-receptor kinases (CEPR1 and CEPR2). This root-to-shoot signaling mechanism in turn triggers a compensatory response in the other roots still fed with nitrate, into which nitrate uptake is stimulated due to enhanced expression of *NRT2.1*, encoding a major nitrate plasmamembrane transporter. This suggested that shoot-to-root mobile signals are involved downstream of the CEPRs to transduce the local nitrate deprivation information to the whole root system.

In this issue of *Nature Plants*, Okhubo *et al.*² report on the identification of these shoot-to-root signals. They show that two glutaredoxins, members of the ROXY family, are induced in the shoot by the CEP-CEPR transduction pathway, and act as phloem mobile signals to trigger the compensatory up-regulation of *NRT2.1* in the roots (Figure 1).

First, Okhubo *et al.*² reinforced their hypothesis that the perception of root-derived CEP peptides by CEPRs activates a shoot-to-root mobile signal by investigating their respective territories of expression/accumulation in leaves. Indeed, reporter-driven localization of *CEPR1* expression and of CEP1 peptide itself revealed that both of them accumulated in phloem cells of the shoot, suggesting that downstream events of CEPR1 activation are associated with phloem transport. Thus, a shoot phloem-specific transcriptomic analysis was performed, allowing the identification of 17 candidate genes for which mRNA accumulation is upregulated by CEP1 peptide supply to roots, and downregulated by the knock-out of *CEPR1*. Given that the perception of CEP1 by CEPR1 is known to mediate *NRT2.1* up-regulation¹, the authors screened overexpressor lines of the 17 genes for enhanced *NRT2.1* expression in conditions usually repressive for this gene; i.e., high nitrogen supply. Among all candidate genes, the overexpression of two close glutaredoxins,
named *CEP Downstream 1 and 2* (CEPD1 and 2) by the authors, lead specifically to an
up-regulation of the *NRT2.1* target.

The second part of the work confirmed that these two polypeptides are nitrogen/CEP-
dependent systemic signals by combining the use of *cepd* mutants with split-root and
grafting experiments. If one part of the root system is nitrogen-deprived, the compensatory
up-regulation of *NRT2.1* in the other roots still supplied with nitrate is lost in the double
*cepd1,2* mutant. Furthermore, despite both mRNA accumulation and promoter activities of
CEPDs were restricted to aerial parts, CEPD1::GFP fusion protein displays CEP1-
dependent accumulation in the root vasculature even in plants with CEPD1::GFP-
expressing shoots grafted on double mutant *cepd1,2* roots. Importantly, two observations
indicate that the CEP-CEPR-CEPD long distance relay is integrated with other nitrogen
signaling pathways in both the shoots and the roots (Figure 1). First, expression of *CEPR1*
in the shoot is reversibly up-regulated by N starvation in a CEP1-independent manner,
suggesting that the N status of the plant may modulate perception of the root-borne CEPs.
Second, even if CEPDs accumulate in the whole root system, *NRT2.1* is specifically up-
regulated in nitrate-supplied roots, indicating combinatorial signaling involving long
distance peptide relay and local nitrate signaling.

The molecular identity of the shoot-to-root signals regulating nitrogen uptake in plants has
been the matter of extensive investigation, and the work of Okhubo et al.² is expected to
have far-reaching consequences, both for fundamental and applied aspects of the nitrogen
nutrition of plants. This finding that proteins like glutaredoxins can act as long distance
nutrient signals was unexpected, as earlier studies have rather suggested that this role is
played by metabolites ³, phytohormones like cytokinins⁴ or miRNAs⁵. However, a link
between glutaredoxins and responses to nitrate was already reported⁶. Furthermore, the
*Arabidopsis* transcription factor HY5 was recently shown to be a shoot-to-root mobile
signal regulating the expression of *NRT2.1* in response to illumination of the shoot⁷.
Therefore, phloem proteins emerge as new key players in long distance nutrient signaling.
Another surprising observation is that the two glutaredoxins apparently specifically
regulate *NRT2.1*, and no other N-responsive genes such as *AMT1.1*, encoding a membrane
transporter for root uptake of ammonium (the other main inorganic nitrogen source). This
contributes to the idea that whole plant nitrogen signaling is probably much more complex
than previously anticipated. It is expected to involve several genetically independent long
distance transduction pathways⁸, possibly specific of each nitrogen source ⁹, and relying
on totally different mobile signal molecules such as glutaredoxins, transcription factors and
cytokinins. Interestingly, the fact that glutaredoxins govern plant responses to nitrate
through interaction with cytokinins⁶ also suggests that although genetically independent,
these transduction pathways are functionally interconnected. Finally, the mechanism by
which glutaredoxins control *NRT2.1* expression remains to be elucidated. Glutaredoxins
generally act in reducing disulfite bonds of target proteins. Therefore, it is of interest to
note that glutaredoxins of the ROXY family may interact with transcription factors of the
TGA family, some members of which were recently reported to regulate *NRT2.1*¹⁰.

S. Ruffel and A. Gojon are at the Biochemistry and Plant Molecular Physiology Research Unit, CNRS, INRA, Montpellier SupAgro, Univ. Montpellier, 2 Place Viala, 34060 Montpellier, France. E-mail: alain.gojon@inra.fr; sandrine.ruffel@inra.fr
1 Tabata, R. et al. Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling. *Science* **346**, 343-346 (2014).

2 Ohkubo, Y., Tanaka, M., Tabata, R., Ogawa-Ohnishi, M. & Matsubayashi, Y. Shoot-to-root mobile polypeptides involved in systemic regulation of nitrogen acquisition. *Nature Plants* **X**, X (2017).

3 Imsande, J. & Touraine, B. N demand and the regulation of nitrate uptake. *Plant Physiol.* **105**, 3-7 (1994).

4 Ruffel, S. et al. Nitrogen economics of root foraging: transitive closure of the nitrate-cytokinin relay and distinct systemic signaling for N supply vs. demand. *Proc Natl Acad Sci U S A* **108**, 18524-18529 (2011).

5 Pant, B. D., Buhtz, A., Kehr, J. & Scheible, W. R. MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. *Plant J* **53**, 731-738 (2007).

6 Patterson, K. et al. Nitrate-Regulated Glutaredoxins Control Arabidopsis Primary Root Growth. *Plant Physiol* **170**, 989-999 (2016).

7 Chen, X. et al. Shoot-to-Root Mobile Transcription Factor HY5 Coordinates Plant Carbon and Nitrogen Acquisition. *Curr Biol* **26**, 640-646 (2016).

8 Ruffel, S. et al. Systemic signaling of the plant nitrogen status triggers specific transcriptome responses depending on the nitrogen source in Medicago truncatula. *Plant Physiol* **146**, 2020-2035 (2008).

9 Gansel, X., Munos, S., Tillard, P. & Gojon, A. Differential regulation of the NO₃⁻ and NH₄⁺ transporter genes *AtNrt2.1* and *AtAmt1.1* in Arabidopsis: relation with long-distance and local controls by N status of the plant. *Plant J* **26**, 143-155 (2001).

10 Alvarez, J. M. et al. Systems approach identifies TGA1 and TGA4 transcription factors as important regulatory components of the nitrate response of Arabidopsis thaliana roots. *The Plant Journal* **80**, 1-13 (2014).
<caption>**Figure 1. Systemic nitrogen-demand signaling involves translocation of peptides and polypeptides between roots and shoots.**</caption>

Whole plant signaling of nitrogen deficiency relies on a root-to-shoot-to-root relay including the following steps: 1- Nitrogen deprivation of one part of the root system induces locally the synthesis of 15 aa CEP peptides, which are translocated to the shoots via the xylem stream. 2- CEP peptides are perceived in the shoots by LRR Receptor kinases (CEPR1 & 2) that are also induced by nitrogen-deprivation at the mRNA level. 3- One molecular event following CEP perception is the induction of two glutaredoxin genes, named *CEPD* (*CEP Downstream1* & 2). 4- CEPD1 and CEPD2 are phloem mobile signals relaying nitrogen-deprivation to the roots for up regulating *NRT2.1*, encoding a major component of the root nitrate transport system. 5- Despite translocation of glutaredoxins downward the whole root system, *NRT2.1* is specifically up regulated in nitrate-supplied roots to trigger a compensatory increase of nitrate uptake. This asymmetric response indicates that CEPD systemic signaling and local nitrate signaling must act in combination to induce *NRT2.1* transporter gene.

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