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# Nitrogen Systemic Signaling: From Symbiotic Nodulation to Root Acquisition

Pierre Gautrat, Carole Laffont, Florian Frugier, Sandrine Ruffel

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## To cite this version:

Pierre Gautrat, Carole Laffont, Florian Frugier, Sandrine Ruffel. Nitrogen Systemic Signaling: From Symbiotic Nodulation to Root Acquisition. Trends in Plant Science, 2021, 26 (4), pp.392-406. 10.1016/j.tplants.2020.11.009. hal-03432519

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Submitted on 23 Nov 2021

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Distributed under a Creative Commons Attribution 4.0 International License
1 Nitrogen Systemic Signaling: from symbiotic nodulation to root acquisition

2

3 Pierre Gautrat¹, Carole Laffont¹, Florian Frugier¹*, Sandrine Ruffel²*

4

5 ¹IPS2 (Institute of Plant Sciences – Paris Saclay), CNRS, INRAe, Université Paris-

6 Diderot, Université d'Evry, Université Paris-Saclay, Bâtiment 630, Gif-sur-Yvette,

7 France

8 ²BPMP, Univ Montpellier, CNRS, INRAe, Montpellier SupAgro, Montpellier, France

9

10 * Correspondence:

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12 sandrine.ruffel@inrae.fr (S. Ruffel)

13 https://www1.montpellier.inra.fr/wp-inra/bpmp/en/research/the-teams/hormones-

14 nutrients-and-development/

15 @SRuffel

16

17 florian.frugier@cnrs.fr (F. Frugier)

18 http://www.ips2.universite-paris-saclay.fr/en/research/dgg-department-

19 developmental-genomics-and-genetics/sileg-signaling-pathways-controlling-legume-

20 root-system-development.html

21

22 Keywords

23 Systemic signaling; nitrogen fixing symbiotic nodule; legume; root development;

24 nitrate transport; carbon

25
Gautrat et al

Highlights

* N nutrition relies on root acquisition of mineral resources, as well as on symbiotic N₂ fixation by soil bacteria in legume plants

* Both processes are regulated by systemic signaling pathways aiming to adjust N acquisition depending on plant N needs and assimilation capacities

* Similar effectors acting in systemic signaling pathways are shared between both N nutrition modes, including hormones such as cytokinins and signaling peptides as well as related NIN-Like Protein (NLP) transcription factors

* Recent advances highlighted that systemic signaling pathways linked to N fixation and acquisition are tightly related to phosphate systemic signaling

* Light and/or CO₂ shoot environmental factors impact N systemic signaling, suggesting mechanisms allowing integration of N and C signaling and metabolism
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**Abstract**

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Plant nutrient acquisition is tightly regulated by resources availability and metabolic needs, implying the existence of communication between roots and shoots to ensure their integration at the whole plant level. Here, we focus on systemic signaling pathways controlling nitrogen (N) nutrition, achieved both by the root import of mineral N and in legume plants through atmospheric N fixation by symbiotic bacteria inside dedicated root nodules. We explore conserved features between systemic pathways repressing or enhancing symbiotic N fixation and the regulation of mineral N acquisition by roots, as well as their integration with other environmental factors, such as phosphate, light and CO₂ availability.
Root-Shoot-Root communication is an essential circuit to regulate plant nitrogen acquisition

Plant metabolism combines light energy capture and carbon (C) fixation by photosynthetic shoots with water and nutrient acquisition by roots. Both organs are challenged with very different local environments, and yet, they need to cooperate to optimize nutrient supply to ensure plant growth. To do so, in addition to the local perception of different nutritional-related cues in the environment, dedicated systemic signaling networks integrating nutrient availability/needs with the growth/developmental status operate at the whole plant level [1]. Plant vascular tissues play an essential role in this long-distance communication between roots and shoots, with a directionality of nutrient and mobile signals exchanges achieved by xylem and phloem vessels [2]. Exhaustive analyses of xylem and phloem saps revealed, beside nutrients and metabolic product of their assimilation, a diversity of molecules ranging from hormones, small and long RNAs, signaling peptides and proteins [3-5]. Understanding the role in systemic root and shoot regulations of signals moving within this complex sap composition, as well as their origins and targets, is a challenging but crucial objective for improving the efficiency of nutrient use and homeostasis at the whole plant level.

Plant nitrogen (N) acquisition by roots is achieved by importing different mineral N sources from the soil as well as, in some specific plants such as legumes, by the fixation of atmospheric N₂ through a symbiotic interaction with soil bacteria, collectively referred to as rhizobia, within a dedicated root lateral organ, the nodule.

Both root and nodule N acquisition is under the control of a long-distance homeostatic signaling, which identification is described in Box 1 for nodules and in Box 2 for roots. These regulatory pathways share similarities as they both aim to regulate the plant N nutrition from different sources (atmospheric N₂, soil mineral NO₃⁻ or NH₄⁺) depending on i) environmental N availability, and ii) plant N needs and assimilation capacities that are notably driven by the C metabolism. Symbiotic N₂ fixation in root nodules is a unique model to study this cost-benefit control of N acquisition by long-distance signals. Indeed, a new organ only dedicated to N acquisition is formed, the nodule, that is non-essential when mineral N is available, in contrast to roots that achieve many other functions in addition to N nutrition. This specificity allowed performing efficient genetic screens to unravel the genetic basis of
71 symbiotic root nodulation [6]. In this review, the systemic pathways regulating N fixing
72 root nodules in legumes will be used as a core to intertwine them with the knowledge
73 gained on N acquisition in roots of the non-nodulating reference plant *Arabidopsis*
74 *thaliana*. We will successively explore i) mechanisms ensuring the homeostatic
75 repression of N acquisition, ii) mechanisms promoting N acquisition when low N is
76 available and plants N needs are high, and iii), how shoot environmental conditions
77 and C metabolism capacities may impact N-systemic signaling.
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79 Repressing root nitrogen fixation and acquisition by long-distance signaling

80 The AON (Autoregulation Of Nodulation) homeostatic systemic signaling pathway
81 restricts the number of N fixing nodules that form on legume roots dependent on
82 rhizobial infections and nodules that were previously formed (Box 1). In nodulated
83 roots, the AON signaling initiates with the production of specific SSP (Small Secreted
84 Peptides) of the CLE (CLAVATA3/EMBRYO-SURROUNDING REGION RELATED)
85 family (Figure 1A). Those 12-amino acid long peptides are conserved among
86 legumes, and their expression is induced when roots interact with rhizobia and by
87 high mineral N availability. These AON-related CLE peptides are encoded by the
88 rhizobium-induced *MtCLE12* and *MtCLE13* genes in *Medicago truncatula* [7, 8]; the
89 rhizobium-induced *LjCLE-RS1* (*CLE-Root Signal1*), *LjCLE-RS2* and *LjCLE-RS3* in
90 *Lotus japonicus*, the two latter genes being also induced by NO₃⁻ [9, 10]; and in
91 soybean the rhizobium-induced *GmRIC1* (*Rhizobia-Induced CLE1*) and *GmRIC2*
92 genes and the NO₃⁻-induced *GmNIC1* (*Nitrate-Induced-CLE1*) gene [11]. SSP activity
93 and/or stability frequently rely on post-translational modifications, and accordingly,
94 some of those CLE peptides were reported to be hydroxyprolinated and tri-
95 arabinosylated in roots, by the MtRDN1 (Root Determined Nodulation1) enzyme for
96 MtCLE12 [12, 13] and by LjPLENTY for LjCLE-RS2 and LjCLE-RS3 [14-16]. Those
97 SSPs can be detected in the xylem sap and are thus proposed to be root-to-shoot
98 mobile signals, perceived in shoots by the CLV1-like (CLAVATA1-like) LRR-RLKs
99 (Leucine Rich Repeats - Receptor Like Kinases) MtSUNN [7, 17], LjHAR1 [15, 18-
100 21], and GmNARK [11, 22] (Figure 1A). A direct binding of LjCLE-RS2 to LjHAR1
101 was demonstrated [15], and several co-receptors (LjKLAVIER, MtCLAVATA2,
102 MtCORYNE) were shown to interact with MtSUNN/LjHAR1 [23-25].
103

103
In Arabidopsis, local functions of CLE peptides in meristems were extensively described, but only few links with nutrient and systemic regulations were reported [26, 27]. A role for the CLE-CLV1 signaling pathway in the regulation of lateral root development by N provision was however shown [28]. Indeed, the *AtCLE1*, *3*, *4* and *7* genes are expressed in pericycle cells and are induced by N-limited conditions. Corresponding peptides would then be translocated into root phloem companion cells to repress the emergence of lateral root primordia in a CLV1-dependent manner [28]. This signaling module may thus also negatively regulate N acquisition locally in roots and would be the target of an unknown N-deficiency shoot-derived signal (Figure 1B). Interestingly in *L. japonicus*, the *har1* mutant, affected in the LRR-RLK most closely related to AtCLV1, has also a root architecture phenotype, consisting in shorter roots and an increased number of lateral roots [18]. The existence of a root-to-shoot CLE-related signaling pathway regulating root architecture and N acquisition remains to be explored further in Arabidopsis, as well as a function in shoots of the AtCLV1 receptor in relation to N-related CLE peptides.

In legumes, the induction of CLE genes expression by $NO_3^-$ and rhizobia relies on transcription factors of the NODULE INCEPTION (NIN) family. Indeed, both in *M. truncatula* and *L. japonicus*, the induction of *MtCLE13* and *LjCLE-RS1/2* expression by rhizobia depends on NIN [8, 29], which binds *LjCLE-RS1/2* promoters [29]. Interestingly, cytokinin (CK) hormones, that are essential for the establishment of nodules, activate NIN and *MtCLE13* or *LjCLE-RS1/2* expression [7, 29-32] (Figure 1A). A subset of NIN-LIKE PROTEIN (NLP) similarly mediates the CLE-dependent $NO_3^-$ inhibition of nodulation both in *L. japonicus* and *M. truncatula* [33, 34] (Figure 1A). Indeed, *Nitrate Unresponsive Symbiosis 1 (NRSYM1)/LjNLP4* binds the *LjCLE-RS2* promoter and activates its expression in response to $NO_3^-$, as well as the NITRITE REDUCTASE 1 (NIR1) encoding gene involved in $NO_3^-$ assimilation but not nodulation, highlighting shared symbiotic and non-symbiotic root functions of this N/NLP/CLE module [33]. In *M. truncatula*, *MtNLP1* and *MtNLP4* redundantly accumulates in the nucleus in response to $NO_3^-$ and interact with NIN to potentially inhibits its function, including hampering the activation of the CK receptor *MtCRE1* expression that is required for nodule initiation [34].
In Arabidopsis, regulators of CLE peptides expression in response to N provision are yet unknown. Given that AtNLP7 is an important hub for the NO₃⁻ root response [35-38], its involvement may be speculated, even though no CLE peptide linked to NO₃⁻ provision was yet identified in Arabidopsis. Transcriptomic analyses however did not identify any NLP7-dependent gene encoding a CLE peptide [37, 39]. It remains to be explored if other NLPs could nevertheless regulate the expression of CLE genes linked to N signaling. In addition, several NLPs interact together via their PB1 (Phox and Bem1) domain [40, 41], as shown for MtNIN and MtNLP1, or with other transcription factors, which may allow coordinating the root response to different N environments. AtNLP7 notably interacts with AtTCP20 (Teosinte branched1/Cycloidea/Proliferating cell factor1-20) to regulate the response of NO₃⁻ responsive genes and root meristem growth depending on N-availability [41]. Interestingly, AtTCP20 is a target in roots of the N-demand systemic signaling [42], indicating that the transcriptional complex AtNLP7/AtTCP20 may integrate local and systemic N signaling pathways (Figure 1B).

Downstream of the AON-related CLE receptor activation in legume shoots, shoot-to-root systemic effectors inhibiting root nodulation were identified. First, as the shoot expression of the *ISOPENTENYLTRANSFERASE3* (*IPT3*) gene increases in response to rhizobium inoculation depending on LjCLE-RS/LjHAR1 signaling pathway, as well as an intermediate form of CKs (*N*⁶-(*Δ*²-isopentenyl) adenine ribosides 5'-phosphates, iPRPs), and as CKs applied to shoots are transported through the phloem towards roots and inhibit nodulation independently of LjHAR1, it was proposed that CK could be a shoot-to-root signal inhibiting root nodulation, in addition to its role as a NIN/CLE activator in roots [43] (Figure 1A). In soybean, a *LjIPT3* ortholog, *GmIPT5*, is also shoot-induced by symbiotic conditions but independently of GmNARK, and CK applications on either shoots or roots similarly promote or inhibit nodulation, depending on the low or high concentration used, respectively [44]. Thus, the negative role of shoot CKs on root nodulation downstream of the AON pathway proposed in *L. japonicus*, implying a potential systemic action of CKs, seems not a general feature of AON in all legumes.

In Arabidopsis, *AtIPT3* expression is triggered by NO₃⁻ [45]. In roots, this ultimately leads to the accumulation of the CK precursor *tZR* (*trans*Zeatin Riboside) and of the
biologically active *tZ*, which are then translocated to the shoots through the ABCG14 transporter [46-48]. Both *tZR* and *tZ* act as systemic signals to promote shoot growth in response to NO₃⁻ [49-52]. The shoot apical meristem fate is notably likely modulated by the influence of *tZR* on WUSCHEL expression [50, 51] (Figure 1B), WUSCHEL being directly activated by a subset of type B Arabidopsis Response Regulators (see [53] for an overview). Moreover, in split-root experiments mimicking NO₃⁻ heterogeneity, the *tZ/tZR* integration in shoots is also required to enhance lateral root development and NO₃⁻ transport in NO₃⁻-supplied roots, compensating for the partial N deficiency [54-56] (Figure 1B). As shown in legumes, CK are proposed to act at several levels of N-signaling since it also regulates locally the NO₃⁻-dependent primary root growth [57], lateral root development and NO₃⁻ transport [58, 59].

In legumes, another shoot-to-root signal conserved between *L. japonicus* and *M. truncatula* was more recently proposed, corresponding to the microRNA miR2111, which expression is shoot-specific and repressed by rhizobium inoculation, dependent on LjHAR1/MtSUNN [60, 61] (Figure 1A). This miRNA regulates post-transcriptionally the accumulation of *TML* (*Too Much Love*) genes (LjTML in *L. japonicus* [60, 62, 63] and MtTML1/MtTML2 in *M. truncatula* [61, 64]), which encode E3 ubiquitin ligases acting in roots to inhibit nodulation. Although TMLs targets remain unknown, one could anticipate that they regulate the stability of symbiotic regulators, and notably of NIN that is a homeostatic target of the NIN/CLE/AON pathway [29]. Overall, whereas the initial AON model was proposing a nodulation "Shoot-Derived Inhibitor" (SDI) signal, as could be CKs in *L. japonicus* (see above), the miR2111 shoot-to-root systemic effector behaves as a nodulation "Shoot-Derived Activator" (SDA) signal that is repressed by the AON pathway (Figure 1A).

In Arabidopsis, the miR2111/TML regulatory module is evolutionary conserved, suggesting a possible recruitment in the regulation of root responses to N availability beyond nodulation. Indeed, one of the two TML homologs (At3g27150) encoding an uncharacterized E3 ubiquitin ligase, is targeted by mir2111, which was detected in Arabidopsis phloem saps in agreement with a function as a systemic shoot-to-root effector [65]. However, miR2111 accumulation is induced under phosphate (Pi)-limited conditions and not by N-limitation [65], in contrast to legumes (Figure 1B).
Interestingly, others F-box proteins that are also regulated by systemic miRNAs control nutrient homeostasis depending on combined P and N availability. Indeed, the E3 ubiquitin ligase NLA (NITROGEN LIMITATION ADAPTATION), and the E2 ubiquitin conjugase PHO2 (PHOSPHATE2), are targeted by the shoot-to-root mobile mir827 and mir399 miRNAs, respectively, in Pi-limiting conditions to inhibit their activity and to favor Pi acquisition [66-68]. Furthermore, the phosphate starvation response (PSR) is conditional to the NO₃⁻ provision [69-71] and this feature depends on the reciprocal regulation of the transceptor NPF6.3/NRT1.1 and of PHO2 [69] (Figure 1B). This is achieved by integrating local and systemic signals, suggesting that the furniture of a N signal to forage for P acquisition is mandatory even at long-distance [69].

## Promoting root nitrogen acquisition and fixation by long-distance signaling

As a mirror of the negative AON systemic pathway, a positive systemic pathway promoting nodulation was more recently evidenced in *M. truncatula*, involving another class of SSP, the CEP (C-terminally Encoded Peptide) family, which expression is generally enhanced in N-deficient roots [72] (Figure 1A). CEP peptides increase the number of nodules formed [72] through a systemic pathway involving the activity in shoots of the MtCRA2 (Compact Root Architecture2) LRR-RLK receptor [73, 74]. Under N deficiency conditions, this systemic MtCEP/MtCRA2 pathway upregulates in shoots the expression of the miR2111 shoot-to-root systemic effector, promoting in roots the cleavage of *MtTML* transcripts that encode an inhibitor of nodulation (see above) [61]. The MtCEP/MtCRA2 pathway thus actively maintains roots competent for nodulation in N deficiency conditions (Figure 1A). Interestingly, the previously described rhizobium- and N-induced MtSUNN AON pathway and the MtCRA2 pathways act independently [75] but modulate antagonistically the same mir2111/TML regulatory module, which is thus a central hub to regulate dynamically nodule number depending on environmental N-availability and the plant N-metabolic status [61]. Interestingly, the MtCEP/MtCRA2 pathway also regulates locally the root system architecture, inhibiting lateral root development and being required for primary root growth depending on a YUCCA-dependent local auxin biosynthesis pathway [72, 73, 75-77] (Figure 1A). *cra2* mutant roots perceive changes in N environments but have altered responses to these conditions [77]. Indeed, instead of repressing root growth and increasing lateral root
239 density, NO₃⁻ promotes *cra2* mutant root growth and represses its lateral root density.
240 In addition, this pathway regulates systemically from shoots the gravitropic set-point
241 angle of lateral roots by limiting shoot auxin transport to roots, ultimately allowing a
242 wider foraging area of the root system [78] (Figure 1A).
243

244 In Arabidopsis, the first CEP peptide, AtCEP1, was identified in *silico* and then by LC-MS (Liquid Chromatography-Mass Spectrometry) as a 15 amino acid long
245 hydroxyprolined peptide. A subset of AtCEP peptides, including notably AtCEP3, 5
246 and 9 accumulates in N-deficient roots and inhibit primary root growth (AtCEP3 and
247 5), with a reduction of meristematic cell number and of the size of the meristem, as
248 well as the emergence of lateral roots (AtCEP3 and 5), and/or the gravitropic set-
249 point angle of lateral roots (AtCEP3) [78-84] (Figure 1B). In addition, AtCEP1
250 upregulates systemically the expression of the *AtNRT2.1* high affinity NO₃⁻
251 transporter depending on the LRR-RLKs AtCEPR1/AtCEPR2 (CEP Receptor 1 and
252 2) [85], closely related to MtCRA2 in *M. truncatula* (Figure 1B). Noteworthy, the
253 binding of the AtCEP1 peptide to these two CEPR receptors was demonstrated [85].
254 A shoot-to-root systemic signal upregulating *AtNRT2.1* expression in roots
255 downstream of the AtCEP/CEPR1 pathway was identified as belonging to the class
256 III glutaredoxins family, and so called AtCEPD1 (CEP Downstream1) and AtCEPD2.
257 Split-root experiments revealed that these shoot-produced AtCEPDs are translocated
258 through the phloem in the root system to upregulate *AtNRT2.1* expression only in
259 roots locally supplied with NO₃⁻, through a yet unknown mechanism implying an
260 interaction with local N conditions [86] (Figure 1B). Interestingly, it was previously
261 shown that the Class III glutaredoxins AtGRX3/4/5/8, homologous to CEPDs, were
262 regulated by NO₃⁻ and CK, and negatively controlled primary root growth [87],
263 suggesting a complex involvement of GRXs in the NO₃⁻-dependent root plasticity.
264 Overall, the CEP/CEPR/CEPD pathway is a root-shoot-root circuit allowing plants to
265 adapt to heterogeneous NO₃⁻ supply in soils.
266

267
268 In Medicago, CEPDs homologs were recently identified as upregulated by N
269 deficiency conditions depending on the MtCRA2 receptor, both in shoots and in
270 roots, in agreement with the role of MtCRA2 in shoots or roots to regulate nodule or
271 lateral root development, respectively [61, 73, 75], but it remains to be established
272 whether *MtNRT2.1* homologs as well as nodulation are targeted by a
273 MtCRA2/MtCEPD pathway. Conversely, a relationship between the Arabidopsis
274 CEP/CEPR pathway and the conserved miR2111/TML-like module remains to be
275 explored.

276
277 Finally, in Arabidopsis, another member of the class III GRX family expressed
278 predominantly in shoots under N deficiency, and denominated AtCEDPlike2
279 (AtCEPDL2), was recently shown to regulate root NO3- uptake and root-to-shoot
280 transport [88] (Figure 1B). AtCEPDL2 acts independently of AtCEPR1/2 but
281 complements cepd1/2 mutations for the NO3- uptake [88]. Interestingly, the maximal
282 induction of AtCEPDL2 expression in shoots requires tZ accumulation [88] (Figure
283 1B). As described in the previous part, tZ/tZR is a N-supply root-borne signal whose
284 translocation in shoots has a positive effect on NO3- uptake, including AtNRT2.1
285 upregulation, and on lateral root growth [55]. Altogether, these results show that
286 multiple systemic signals coexist to positively regulate NO3- uptake in plants. This
287 would allow integrating local N-deprivation pathways (i.e., CEP/CEPR/CEPD) with
288 global N-deprivation pathways (i.e., CEPDL) and local NO3- supply (i.e., tZ/tZR) to
289 finely modulate N acquisition in NO3- heterogeneous environment (Figure 1B).

290
291 Influence of shoot environment on N-related long-distance signaling

292 The systemic regulation of root N acquisition and fixation relies on the perception of
293 root-borne signals modulated by local environmental soil conditions, and by their
294 integration in shoots. This suggests that the N acquisition may be modulated
295 depending on the nutrient status of shoots, i.e. the N status but also C resources that
296 can be provided to sustain root and nodule development as well as N assimilation,
297 and thus on plant C fixation capacities and C allocation to roots. Aboveground
298 atmospheric environmental conditions, including CO2 levels as well as light quality
299 and quantity, are thus anticipated to impact systemic signaling pathways targeting N
300 acquisition. The increase of atmospheric CO2 concentrations associated to the global
301 climate change has various consequences depending on plant species (Box 3),
302 notably impacting C allocation to roots and the different modes of N mineral vs
303 symbiotic acquisition. Below, we expose emerging data indicating that C provision
304 likely impacts N-systemic signaling effectors and pathways regulating N acquisition.
305
In *M. truncatula*, additionally to their transcriptional upregulation in response to low NO₃⁻, the expression of most *CEP* genes is cumulatively induced by low N and high CO₂ (800 ppm) conditions [72]. In Arabidopsis, the root architecture phenotype of the *cep3* mutant is observed not only under low N but also under low light conditions [80]. In addition, a subset of *AtCEP* genes (*AtCEP5-AtCEP9*) is upregulated by a treatment with metabolizable sugars, such as sucrose [89]. AtCEP5 notably restricts the promotion of lateral root growth in response to sucrose depending on an AtCEPR1 activity in both roots and shoots. An upregulation of sucrose responsive genes is detected in the *Atcepr1* transcriptome, suggesting a potential interaction with the SnRK1 (Sucrose non-fermenting 1-Related protein Kinase 1) C-signaling pathway [89]. This indicates that the Arabidopsis CEP/CEPR1 pathway, and thus likely by homology the *M. truncatula* CEP/CRA2 pathway, integrates N and C signals to modulate root system architecture and growth. Whether this coordination implies a regulation by C sources of already known systemic effectors (e.g. CEPDs and/or the miR2111), and/or of novel shoot-to-root signals, remain to be discovered (Figure 1B).

In addition, the role of the AtCEPR1/MtCRA2 pathway into the regulation by C provision of root NO₃⁻ transport, assimilation, and corresponding genes should be investigated. Indeed, photosynthesis products stimulate NO₃⁻ uptake and NO₃⁻ transporter gene expression [90, 91]. To date, the regulation of *AtNRT2.1* and *AtNPF6.3/NRT1.1* seems not dependent on the SnRK1 pathway [92] but rather linked to the oxidative pentose phosphate pathway (OPPP) [93, 94]. In addition, glucose itself promotes NRT2.1 protein accumulation [94]. Interestingly, the redox metabolism, including the OPPP output, may actually regulate NO₃⁻ transport depending on N and C signaling (see [95] for a review). On this line, the CEPDs/CEPDLs shoot-born mobile signals, being class III GRX, may thus participate to such redox integration of C and N regulations.

In Arabidopsis, a systemic shoot-to-root function of the transcription factor AtHY5 (Elongated Hypocotyl5) was also proposed to control N-acquisition and shoot/root development, depending on the C status of shoots [96]. AtHY5 was initially described as a positive regulator of photomorphogenesis, modulating hypocotyl growth depending on light availability, but was also linked to various aspects of root development and to redox regulations [97-101]. Recently, under light illumination, AtHY5 was shown to up-regulate the expression of sucrose biosynthesis and efflux
340 genes in shoots (*AtTPS1* for Trehalose-6-Phosphate Synthase; *SWEET11* and *SWEET12*), and *AtHY5* proteins was shown to be translocated from shoots-to-roots 341 through the phloem [96]. In roots, *AtHY5* binds its own promoter to generate an 342 amplification loop, as well as the *AtNRT2.1* promoter to enhance its expression and 343 root NO₃⁻ uptake [96] (Figure 1B). In *L. japonicus*, *LjHY5/LjBzf* (BZip ring-Finger) 344 inhibits nodulation but not LR development [102]. However, it is not known if this 345 occurs through a local and/or a systemic regulation. 346

347

348 Finally, hormones are anticipated to mediate the integration of shoot local 349 environmental C-related signals with N-systemic signaling. For instance, CK is a 350 positive regulator of chlorophyll synthesis as well as of chloroplast development [103, 351], and might potentially favor C allocation to roots and thus N acquisition. 352 Conversely in Arabidopsis, photosynthesis-related sugars, which synthesis is favored 353 by elevated CO₂ environments, induce the expression of the IPT3, CYP735A2 and 354 ABCG14 CK biosynthesis enzymes and transporter, and consequently CK 355 accumulation, promoting shoot growth [105]. By promoting CK biosynthesis in 356 shoots, elevated CO₂ may thus interfere with the CK-dependent systemic signaling 357 controlling NO₃⁻ acquisition in roots [55, 88] (Figure 1B). The regulation of CK 358 biosynthesis may depend directly on sugars but also on derived metabolic signals 359 [105], such as glutamine for example, which is also known to influence CK 360 biosynthesis [106]. 361

361 Interestingly, in Arabidopsis shoots, an overrepresented number of genes encoding 362 enzymes involved in the glutamine metabolism show a differential expression 363 depending on NO₃⁻ availability, and this regulation relies on tZ/tZR translocated from 364 roots [55]. The role of amino acids as a nutrient status reporter of the whole plant 365 level, and in particular of glutamine that is the first amino acid issued from C and N 366 assimilation, was a long-standing hypothesis that could however never be validated 367 experimentally [107]. 368

368 Altogether, these studies shed light that CK-glutamine interaction in shoots may thus 369 be a potential hub in systemic signaling controlling root responses and integrating C 370 and N availability. 371

371

372 **Concluding remarks and Future Perspectives**
In legumes, a mechanistic framework of the N-related systemic control of nodulation recently emerged, involving antagonistic CEP/CRA2 and CLE/SUNN-HAR1 pathways differentially activated depending on N availability and/or the whole plant N status to regulate nodulation, and likely more globally root system architecture and N acquisition. This new knowledge allowed reconsidering the historical AON model, which is not only activated by previously established nodules but also by NO₃⁻, indicating that rhizobium seems considered as an N source by the plant. In addition, the initially proposed shoot-to-root systemic signal inhibiting nodulation, which may involve CKs in some legume plants, also comprises the repression of a positive regulator of nodulation produced only in shoots, the miR2111 miRNA. Strikingly, the production of this miRNA is actively maintained under low N and/or N deficit conditions by the CEP/CRA2 pathway (Figure 1A). In addition to these two antagonistic systemic pathways regulating rhizobial infections at the onset of nodule initiation, AON-independent systemic signaling pathways also exist to regulate later nodulation stages including N fixation and assimilation (Box 1).

Whether it is through N₂ fixation in symbiotic nodules of legume plants, or NO₃⁻ import from the soil at the root surface of all plants, N acquisition is constantly modulated by a combination of local and long-distance systemic signals. The parallel made between the legume and Arabidopsis knowledge that is at the basis of this review allowed to highlight similarities and overlaps between N-related systemic pathways regulating N fixing nodules and root N acquisition. The respective role of the mir2111, CEPDs/CEPDLs, CK and HY5 shoot-to-root signals remains however to be better analyzed in parallel between Arabidopsis and legume plants, as well as the potential role of NLP transcription factors to regulate CLE and/or CEP signaling peptides related to N systemic pathways (see "Outstanding Questions"). Some differences exist, such as the regulation of root development by the CEP pathway, both systemically from shoots and locally from roots in Arabidopsis, at least in response to sucrose, or only locally in roots in *M. truncatula*, or such as the LjHAR1/MtSUNN and AtCLV1 CLE peptide receptor functions that, despite being the most closely related proteins, apparently diverged functionally regarding their functions in shoot and floral meristems or N related systemic signaling [108].
Regulatory mechanisms regulating inorganic N acquisition exist in all plants, and are thus expected to be likely conserved in legumes, including the regulation of NO₃⁻ transport (Box 2). The acquisition of nodulation, implying the co-option of local and systemic N- (or P-) related regulatory circuits, thus required an integration of the regulation of N-fixing nodules with root N-acquisition through related pathways. This may also allow ensuring a robustness of repressive mechanisms against detrimental effects of exaggerated root and nodule development resulting from an over C allocation to nodules, as exemplified by the phenotypes of peptide receptors mutants – either “supernodulating” as *sunn/har1* or “superrooting” as *cra2* (see “Outstanding Questions”).

This review also highlights that we know few about interactions between these different systemic signaling pathways and how they integrate the multiplicity of signals that plants continuously perceive in their different organs (e.g., rhizobium, N-limitation, NO₃⁻ provision in roots) (see “Outstanding Questions”). For example, in Arabidopsis, the study of the potential interactions between CK (tZ/tZR)- and CEPD/CEPDL-pathways in shoots remains in its infancy (Figure 1B). Interestingly, results obtained on the antagonistic regulation of the miR2111 by CRA2 and SUNN illustrates the integration of the balance between permissive and repressive nodulation states according to N provision (i.e., N-limitation and rhizobium) [61] (Figure 1A). Indeed, a specific *M. truncatula* CEP gene, *MtCEP7*, systemically promoting nodulation through the MtCRA2 receptor, was recently shown to be induced by rhizobium and CKs, depending on the MtCRE1 CK receptor and on the MtNIN transcription factor [109] (Figure 1A). Interestingly, MtNIN binds both *MtCLE13* and *MtCEP7* promoters and activate their expression through NIN binding sites required for their activation in response to rhizobium [29, 109]. MtNIN thus coordinates the regulation of specific SSPs from two different families regulating antagonistically nodulation, potentially to allow switching from a negative to a positive regulation of nodulation depending on changes in N availability and of the N plant status [109]. In addition to the co-regulation of specific *CLE* and *CEP* genes by a single transcription factor, SSP may also regulate each other’s expression. Indeed, AtCEP3 up-regulates the expression of *AtCLE3*, *4*, *7*, suggesting that the AtCEP3/AtCEPR1-dependent low N response may promote the low N-dependent AtCLV1/CLE signaling pathway [81] (Figure 1B). Finally, different types of CKs have
440 various roles in shoots vs roots, and locally vs systemically, but how these
441 specificities are decoded depending on the N availability/whole plant N status
442 remains to be explored.

443
444 It is likely critical that local and systemic signaling associated to the heterogeneous
445 distribution of other nutrients than N also intersects with N signaling (see
446 "Outstanding questions"). Herein, we covered interactions between C availability and
447 N-systemic signaling, but other nutrients also have an influence. An implication of N
448 in Pi-limitation signaling was reported [69-71], and one can anticipate a large
449 integration between these two major nutrient signaling pathways. Interestingly, the
450 low Pi regulations of nodulation and of the arbuscular endomycorrhiza symbiosis also
451 require the CLE/SUNN systemic pathway, thus mirroring the AON pathway and so-
452 called AOM for Autoregulation Of Mycorrhization [110-112]. The integration of these
453 different systemic pathways ensuring an integrated plant nutrition through the root
454 acquisition of various mineral nutrients and through mutualistic symbioses
455 establishment is key to understand how plants adapt to fluctuating and
456 heterogeneous environments (See "Outstanding Questions").

457
458 Acknowledgments

459 We thank Ambre Miassod (IPS2) for plant illustrations used in Figure 1, and Pascal

460 Ratet (IPS2) and Benjamin Péret (B&PMP) for critical reading of the manuscript.

461 P.G., C.L. and F.F. work is supported by the Agence Nationale de la Recherche

462 (ANR) PSYCHE project and "Saclay Plant Sciences" (SPS) Graduate School of

463 Research. P.G. received a PhD fellowship from the University of Paris-Saclay. S.R.

464 work is supported by the National Science Foundation (IOS 1339362-NutriNet).

465
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736 125. Gazzarrini, S. et al. (1999) Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. *Plant Cell* 11, 937-48.

738 126. Ganeteg, U. et al. (2017) Amino acid transporter mutants of Arabidopsis provides evidence that a non-mycorrhizal plant acquires organic nitrogen from agricultural soil. *Plant Cell Environ.* 40, 413-423.

741 127. Leran, S. et al. (2014) A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. *Trends Plant Sci.* 19, 5-9.

743 128. Tegeder, M. and Masclaux-Daubresse, C. (2018) Source and sink mechanisms of nitrogen transport and use. *New Phytol.* 217, 35-53.

745 129. Vidal, E.A. et al. (2020) Nitrate 2020: Thirty years from transport to signaling networks. *Plant Cell*, 10.1105/tpc.19.00748.

747 130. Jia, Z. and von Wiren, N. (2020) Signaling pathways underlying nitrogen-dependent changes in root system architecture: from model to crop species. *J Exp Bot*, 10.1093/jxb/eraa033.

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751 132. Lima, J.E. et al. (2010) Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1;3-dependent manner. *Plant Cell* 22, 3621-33.

753 133. Ho, C.H. et al. (2009) CHL1 functions as a nitrate sensor in plants. *Cell* 138, 1184-94.

754 134. Alvarez, J.M. et al. (2012) Integration of local and systemic signaling pathways for plant N responses. *Curr. Opin. Plant Biol.* 15, 185-91.

756 135. Li, Y. et al. (2014) Finding a nitrogen niche: a systems integration of local and systemic nitrogen signalling in plants. *J. Exp. Bot.* 65, 5601-10.

758 136. Drew, M.C. and Saker, L.R. (1975) Nutrient supply and the growth of the seminal root system in barley. II. Localized compensatory increases in lateral root growth and rates of nitrate uptake when nitrate is restricted to only part of the root system. *J. Exp. Bot.* 26, 79-90.

761 137. Gile, P.L. and Carrero, J.O. (1917) Absorption of nutrients as affected by the number of roots supplied with the nutrient. *J. of Agri. Res.* 9, 73-95.
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764 AtNrt2.1 and AtAmt1.1 in Arabidopsis: relation with long-distance and local controls by N status of the

765 plant. *Plant Journal* 26, 143-55.

766 139. Zhang, H. et al. (1999) Dual pathways for regulation of root branching by nitrate. *Proc. Natl.*

767 Acad. Sci. U.S.A. 96, 6529-34.

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769 nutrients. *J. of Exp. Bot.* 53, 39-43.

770 141. Okamoto, Y. et al. (2018) Shoot nitrate underlies a perception of nitrogen satiety to trigger

771 local and systemic signaling cascades in Arabidopsis thaliana. *Soil Sci. and Plant Nutrition* 65, 56-64.

772 142. Girin, T. et al. (2010) Identification of Arabidopsis mutants impaired in the systemic regulation

773 of root nitrate uptake by the nitrogen status of the plant. *Plant Physiol.* 153, 1250-60.

774 143. Nazoa, P. et al. (2003) Regulation of the nitrate transporter gene AtNRT2.1 in *Arabidopsis*

775 thaliana: responses to nitrate, amino acids and developmental stage. *Plant Mol. Biol.* 52, 689-703.

776 144. Muller, B. and Touraine, B. (1992) Inhibition of NO3- uptake by various phloem-translocated

777 amino acids in soybean seedlings. *J. Exp. Bot.* 43, 617-623.

778 145. Leakey, A.D.B. et al. (2009) Elevated CO2 effects on plant carbon, nitrogen, and water

779 relations: six important lessons from FACE. *J. of Exp. Bot.* 60, 2859-2876.

780 146. Li, P. et al. (2006) Response diversity of Arabidopsis thaliana ecotypes in elevated [CO2] in

781 the field. *Plant Mol. Biol.* 62, 593-609.

782 147. Myers, S.S. et al. (2014) Increasing CO2 threatens human nutrition. *Nature* 510, 139-42.

783 148. Andrews, M. et al. (2020) Will rising atmospheric CO2 concentration inhibit nitrate assimilation

784 in shoots but enhance it in roots of C3 plants? *Physiol. Plant.*, 10.1111/ppl.13096.

785 149. Bloom, A.J. et al. (2020) Rising atmospheric CO2 concentration inhibits nitrate assimilation in

786 shoots but enhances it in roots of C3 plants. *Physiol. Plant* 168, 963-972.

787 150. Bloom, A.J. et al. (2010) Carbon dioxide enrichment inhibits nitrate assimilation in wheat and

788 Arabidopsis. *Science* 328, 899-903.

789 151. Asensio, J.S. et al. (2015) Responses of Arabidopsis and wheat to rising CO2 depend on

790 nitrogen source and nighttime CO2 levels. *Plant Physiol.* 168, 156-63.

791 152. Andrews, M. et al. (2019) Elevated CO2 effects on nitrogen assimilation and growth of C3

792 vascular plants are similar regardless of N-form assimilated. *J. Exp. Bot.* 70, 683-690.

793 153. Parvin, S. et al. (2020) Carbon sink strength of nodules but not other organs modulates

794 photosynthesis of faba bean (Vicia faba) grown under elevated [CO2] and different water supply. *New*

795 *Phytol.*, 10.1111/nph.16520.

796

797
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Figure 1.</figcaption></figure>
**Figure 1. Regulation of Nitrogen Fixation and Acquisition by Systemic Signaling Pathways in Legumes and in Arabidopsis.**

(A) Systemic signaling repressing (left) and promoting (right) nodulation in legumes.

In legumes, the CLE (CLAVATA3/EMBRYO-SURROUNDING REGION RELATED)-dependent long-distance signaling represses the N fixing nodulation (Blue arrow).

Symbiotic rhizobia infection triggers this systemic signaling partially through the CK (Cytokinin) signaling pathway involving the CK receptor CRE1 and the NIN (Nodule Inception) transcription factor activity that are also required for nodulation (+). Nitrate ($NO_3^-$) also triggers this CLE-systemic signaling through the activity of NLP (NIN-like Protein) transcription factors. CLE peptide signals are perceived in shoots by a CLV1 (CLAVATA1)-like receptor (LRR-RLK, Leucine Rich Repeats - Receptor Like Kinases; SUNN in *M. truncatula*). This induces downstream the expression of *IPT3* (*ISOPENTENYLTRANSFERASE 3*) and thus CK biosynthesis, a putative nodulation "Shoot-Derived Inhibitor" (SDI) signal.

In the *M. truncatula* legume, the CEP (C-Terminally Encoded Peptide)-dependent long-distance signaling promotes N fixing nodulation in low N conditions (Green arrow). CEP peptide signals are perceived in shoots by a LRR-RLK receptor, named CRA2 (Compact Root Architecture2). The MtCEP/MtCRA2 pathway regulates also locally in roots negatively LR (Lateral Root) development and promotes PR (Primary Root) growth. This pathway also systemically regulates LR GSA (Gravitropic Set Angle) by inhibiting shoot-root auxin transport.

These CLE and CEP-systemic signaling pathways respectively repress and induce the expression of the mobile microRNA mir2111, a nodulation "Shoot-Derived Activator" (SDA) signal, that targets the expression of the E3 Ubiquitin Ligase TML (Too Much Love) inhibiting nodulation. Thickness of arrows and lines represents the strength of the signaling pathway.

In addition, rhizobia promote through CK/CRE1 and NIN the expression of MtCEP7, promoting nodulation depending on MtCRA2, thus potentially allowing switching from a negative to a positive regulation of nodulation depending on changes in N availability and of the N plant status.

(B) N provision (left) and N deficiency (right) systemic signaling in Arabidopsis.

On the left (with the light-purple background), $NO_3^-$ signaling requires the NRT1.1/NPF6.3 transceptor and NLP7 in roots. The TCP20 transcription factor (Teosinte branched1/Cycloidea/Proliferating cell factor1-20) interacts with NLP7 and
833 intersects with N-systemic signaling to control the PNR (Primary Nitrate Response)
834 and LR development. In roots, NO₃⁻ induces IPT3 expression and thus the
835 biosynthesis of CK precursor (tZR, trans-Zeatin Riboside) and active (tZ, trans-
836 Zeatin) forms, which is followed by their translocation into shoots. Their perception in
837 shoots promotes shoot growth (tZ) and Shoot Apical Meristem development (SAM
838 Dvt) (tZR), through the induction of the expression of the transcription factor WUS
839 (WUSCHEL) related to the local CLV3/CLV1 peptide signaling. tZ and tZR shoot
840 integration controls a shoot-to-root signaling ultimately enhancing PNR, LR
841 development, and NO₃⁻ transport, also modulating in shoots the expression of
842 glutamine (Gln) metabolism-related genes, which may have a role in N-systemic
843 signaling.

844 On the right (with the light-green background), low N triggers a CEP/LRR-RLK
845 pathway, similarly as in M. truncatula. The CEPR1 receptor homologous to MtCRA2
846 represses LR and PR development dependent on its activity in roots and in shoots.
847 Repression of LR emergence is also controlled by a root CLE/CLV1 signaling
848 pathway induced by a low N (shoot) status, and maybe also by CEP peptides. Low N
849 availability also triggers in shoots the expression of class III Glutaredoxins, named
850 CEPD1&2 (CEP-Downstream) acting downstream of the CEP/CEPR1 pathway, and
851 of CEPD-like2 depending on the shoot low N status. These CEPD shoot-to-root
852 mobile signals enhance the expression of the AtNRT2.1 NO3- transport gene, and
853 thus NO3- uptake. CEPDL enhances also AtNRT1.5 expression and thus NO3- root-
854 to-shoot transport (R→S). tZ/tZR being required for CEPD-like2 expression, the
855 interaction in shoots between the "NO3-/CK" and the "CEP/CEPD/CEPD-like"
856 systemic signaling may finely tune N acquisition depending on root NO3- availability
857 and N-demand.

858 At the extreme left (with the yellow background), the integration between phosphate
859 (P/Pi) or carbon (C) signaling with the N-systemic signaling network is depicted, via i)
860 potentially the conserved miR2111/TML pathway, paralleling the mir399/PHO2
861 pathway known to control Pi acquisition (Acqu.) in relation to the regulation of
862 NRT1.1/NPF6.3; ii) the shoot-to-root HY5 (Elongated Hypocotyl5) transcription factor
863 controlling its own expression, AtNRT2.1 and NO3- uptake, and iii) a possible direct
864 or indirect regulation of the expression of the different shoot systemic effectors (arrow
865 pointing on the grey dotted frame).

866
867 **Box 1. Systemic control of the N₂ fixing nodule symbiosis in legume plants**

868 Nodule organogenesis and maintenance are energetically costly, thus legumes must
869 tightly control their number and activity to balance nutrient gain and loss, thanks to a
870 dedicated network of signaling pathways [113]. First evidences of a systemic control
871 of nodulation, in the early 50's, came from nodule excision, varieties or mutant
872 grafting, and split-root experiments in soybean, which highlighted that mechanisms of
873 host plants limiting the nodule number integrates shoot and root cues [114-116]. This
874 systemic negative regulation of nodulation was named Autoregulation Of Nodulation
875 (AON) [117], as it originally corresponded to a restriction of the nodule number
876 following a first wave of rhizobial infection events. Later, the observation that the
877 nodulation of super/hypernodulating mutants was also NO₃⁻ resistant expanded the
878 delineation of AON to nodulation repressive conditions mediated by high NO₃⁻, which
879 for the plant is a cheaper source of N than N fixing rhizobia are [17]. More recent
880 results showed that the systemic AON restricts rhizobial infections, likely by impairing
881 the perception of rhizobial signals [60, 64, 118]. N-repression involves not only
882 "AON"-dependent mechanisms, but also independent local and/or systemic pathways
883 controlling nodule number and rhizobial infections as well as later nodulation stages,
884 including N fixation and assimilation in nodules [33, 119-121].

885 N-limitation is a mandatory factor for promoting root nodule symbiosis. Split-root
886 experiments showed that new nodule formation and nodule expansion, rather than
887 an increase in N-fixation activity in existing nodules, which seem already at their
888 maximum capacity, is systemically compensated in distal non N-limited area [120,
889 122]. The stimulation of mature nodule metabolism and expansion would act through
890 an independent pathway and rely on a rapid re-allocation of sucrose to sustain
891 nodule metabolism along with a repression of nodule senescence and plant defense
892 markers [123].

893
894

Box 2. Systemic control of nitrogen acquisition in roots

895

896

897

898

899

900

901

902

903

904

905

906

Plants can acquire N from a range of mineral sources, NO3- and NH4+, and from organic compounds (e.g., amino acids) [124-126]. The acquisition of mineral N relies on root transporters belonging to three main gene families: NRT2s and NPFs/NRT1s for NO3-, and AMTs for NH4+ [127, 128]. Their characterization advanced our understanding of N acquisition regulation by environmental cues, including the fluctuating availability of the ions themselves [129, 130]. Moreover, an intricate relationship has been established with a role of some transporters in the control of root development, independently of their transport activity [131-133]. The architecture of N acquisition (i.e., root transporters and development) is tightly regulated by a network of local but also systemic signaling, integrating N external availability with the global plant N need and in coordination with other nutrient resource pools [134, 135].

907

908

909

910

911

912

913

914

915

916

917

918

919

Early works using split-root experiments in rice, corn and barley revealed that a systemic compensation response to a N-deprivation exists in the distant root system consisting in an increased NO3- uptake and root development [136] [137]. Going along with these physiological responses, NO3- supplied roots display a rapid increase in the expression of genes involved in NO3- transport and assimilation, in split-root experiments performed on Arabidopsis and Medicago [54, 121, 138]. These physiological and molecular responses are controlled by a systemic N-demand signaling that likely includes several signaling pathways related to the heterogeneous NO3- availability at the root system level and/or to the NO3-/N-limited status of shoots [54, 85, 90, 138-141]. Local NH4+ availability is not compensated by an increase of NH4+ uptake [121, 138], but rather in Arabidopsis by an AMT1.3-dependent enhanced proliferation of lateral roots [132], indicating some specificity of the N-systemic signaling with respect to the N source.

920

921

922

923

924

On the other hand, all these N acquisition components are repressed by a systemic N supply [54, 121, 142], suggesting a lower specificity for the repressive mechanisms. For instance, as amino acid supply represses N acquisition, and as they can circulate inside plants, their role as a systemic inhibitory signal for N acquisition was proposed [143, 144].

925
926 **Box 3. Differential plant N acquisition efficiencies to elevated CO₂**

927 The "anthropocene" era is synonymous of increased greenhouse gas emissions. In 928 the 1750-2011 period, CO₂ concentrations increased from 280 to 380 ppm, with a 929 dramatic acceleration of emissions these last 40 years (+2ppm/year). Consequences 930 of associated climate changes on plant responses have been evaluated these last 931 years, notably with the FACE system (Free-Air CO₂ Enrichment) that allowed 932 simulating the effect of CO₂-enriched atmosphere up to a whole ecosystem for 933 several years [145]. Increasing CO₂ concentrations in the atmosphere is expected to 934 increase plant biomass and yield due to a "CO₂ fertilization effect" increasing the 935 photosynthesis rate, but several studies revealed a limited beneficial effect on the 936 long-term in some plants, such as C3 grains (wheat, rice) and legumes (field pea, 937 soybean), leading to lower yields, protein contents, and even nutritional food quality, 938 because of a CO₂/photosynthesis acclimation [145-147]. Whether elevated CO₂ 939 consequences on plant growth and protein content are also different depending on 940 the N inorganic form remains under debate [148, 149]. Some studies point to an 941 inhibition of photorespiration and consequently of NO₃⁻ assimilation [150, 151], 942 whereas other studies report no differences in N assimilation and growth in C3 plants 943 supplied with NO₃⁻ or NH₄⁺ [152].

944 CO₂/photosynthesis acclimation also strongly relies on the sink organ strength to 945 consume photosynthesis C products, since a reduced sink strength negatively 946 feedback on photosynthesis [145]. The N₂ fixing symbiosis is particularly interesting 947 in this context since nodules constitute an important C sink, and since the nodule N₂ 948 fixation capacity depends on C allocation. Recent studies suggest that the nodule N₂ 949 fixation activity may allow avoidance of the negative feedback on photosynthesis 950 provoked by elevated CO₂ conditions [153]. This way, symbiosis in N fixing legumes 951 is anticipated to gain higher yields and biomass productivity under elevated CO₂, a 952 condition that will be the future agricultural context to consider. 953

953
Gautrat et al.

OUTSTANDING QUESTIONS

* What are the respective role and relevance of evolutionary conserved regulatory modules, including the mir2111, CEPDs/CEPDLs, CK and HY5 mobile signals, in different plants? Combining evolutionary and functional approaches is needed to understand the origin of these systemic signaling pathways and how they have evolved in different plants to ensure a robust regulation of the different N acquisition modes.

* What are the roles and target overlaps of N-related NLP transcription factors, notably regarding the regulation of CLE and/or CEP signaling peptides?

* The co-existence of various systemic signals, such as peptide and hormones, questions how these molecular effectors interact and impact N-systemic signaling.

* How the various roles of different types of CKs, locally in shoots vs roots, and systemically, are decoded depending on the N availability/whole plant N status?

* How low N / N deficiency signals and high N / N satiety signals, as well as local and systemic regulations, integrate?

* How are integrated the multiplicity of N availability signals that plants continuously perceive in different parts of their body together with the whole plant metabolic status as well as in relation to other nutrients than N (e.g., C and P)? How shoot systemic effectors (mir2111, CEPDs/CEPDLs, CK...) are regulated by these other nutrient inputs? Such questions will require the use of integrated analyses.

* Some systemic signals, such as CKs, are known to regulate the vascular system development. This questions the possibility that systemic signals may by themselves modulate vascular channeling and thus long-distance communication.