# Rhizobium symbiotic efficiency meets CEP signaling peptides

Carole Laffont, Florian Frugier

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## To cite this version:

Carole Laffont, Florian Frugier. Rhizobium symbiotic efficiency meets CEP signaling peptides. New Phytologist, 2024, 241 (1), pp.24-27. 10.1111/nph.19367. hal-04800564

HAL Id: hal-04800564

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Submitted on 24 Nov 2024

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Rhizobium symbiotic efficiency
meets CEP signaling peptides

Summary

C-terminally encoded peptides (CEP) signaling peptides are drivers of systemic pathways regulating nitrogen (N) acquisition in different plants, from Arabidopsis to legumes, depending on mineral N availability (e.g. nitrate) and on the whole plant N demand. Recent studies in the *Medicago truncatula* model legume revealed how root-produced CEP peptides control the root competence for endosymbiosis with N fixing rhizobia soil bacteria through the activity of the Compact Root Architecture 2 (CRA2) CEP receptor in shoots. Among *CEP* genes, *MtCEP7* was shown to be tightly linked to nodulation, and the dynamic temporal regulation of its expression reflects the plant ability to maintain a different symbiotic root competence window depending on the symbiotic efficiency of the rhizobium strain, as well as to reinitiate a new window of root competence for nodulation.

The developmental plasticity of the root system is crucial for plant
adaptation to fluctuating soil environments. Nutrient availability,
such as N, controls root growth and differentiation as well as the
beneficial root-microorganism symbiotic association with N-
fixing soil bacteria collectively referred to as rhizobia in legumes
(Gautrat et al., 2021). Rhizobia colonize host roots depending on
local signaling pathways allowing first a strain-specific recognition
through the perception of bacterial lipo-chito-oligosaccharidic
signaling molecules, so-called Nod factors, and second the
perception of a mineral N deficit that promotes nodulation.

Nodules hosting differentiated rhizobia then provide an alternative N source originating from the atmosphere through bacterial N fixation and further assimilation into organic molecules by host plant cells.

Because sustaining nodule organogenesis, bacterial metabolism,
and ammonium assimilation is highly energetically costly for the
host plants, the symbiotic interaction is tightly regulated depending
on mineral N availability. It is thus not surprising that evolutionary
conserved mechanisms related to N perception and signaling were
co-opted to regulate atmospheric N fixing nodulation, considering
that it is a substitute for the acquisition of mineral N from the soil.
One initial key symbiotic factor identified was the NIN
transcription factor, required for ‘nodule inception’, which turned
to be the founding member of the highly evolutionary conserved
NIN-like protein family, including notably AtNLP7 in Arabidop-
sis that was recently proposed to act as a nitrate sensor (Liu &
Bisseling, 2020; Liu et al., 2022; Yan & Nambara, 2023). Another
component of the N signaling pathways that was more recently
recognized as key for legume root competence to nodulate are the
C-terminally encoded peptides (CEP) signaling peptides (Gautrat
et al., 2021).

CEP signaling peptides at the onset of the N deficit-
driven root competence for nodulation

Following the initial discovery of CEP signaling peptides in
Arabidopsis, their link with N availability was rapidly identified
based on their highly induced expression under N deficit conditions
(Ohyama et al., 2008; Delay et al., 2013), a regulatory feature that is
evolutionarily conserved in the *M. truncatula* legume (Imin
et al., 2013). Identification of CEP receptors (CEPR) in Arabidopsis
further linked the CEP/CEPR pathway to the long distance, termed
systemic, activation of mineral N acquisition, notably through the
induction of the NRT2.1 high affinity nitrate transporter expression

**Fig. 1** C-terminally encoded peptides (CEP) systemic pathways regulate in legumes nitrogen acquisition in relation to its availability and to rhizobium strain symbiotic efficiency. (a, b) Scheme of the nitrogen (N) deficit induced CEP signaling peptide systemic pathway in the *Arabidopsis thaliana* model (a), and in the *Medicago truncatula* model legume (b) for the CEP1 pathway (left), the CEP7 pathway (middle), and the CLE13 pathway (right). CEPD, CEP Downstream; CEPR, CEP receptor; CRA, compact root architecture; miR2111, microRNA 2111; NRT, nitrate transporter; SUNN, SuperNumerary Nodules; TML, Too Much Love. Blue connectors represent positive interactions, red connectors negative interactions, and dotted grey connectors indicate inhibited interactions. (c) Representative images of the overall plant growth and nodulation (inset) phenotypes of *Medicago truncatula* Jemalong A17 plants grown on low N medium and inoculated with the efficient N fixing strain (WSM419; Terpolilli et al., 2008), the weak N fixing strain (Sm1021; Terpolilli et al., 2008), or the non-N fixing strain (T173; Bromfield et al., 2023). Pink elongated nodules accumulate leghemoglobin that is essential for N fixation, whereas white round nodules do not. (d) Quantification of the number of pink vs white nodules 20 d post-rhizobium inoculation in plants shown in (b). A Fisher's exact test was used to assess significant differences, indicated with asterisks (*P* < 0.05; *n* > 19 plants per condition). (e) Kinetic of the number of nodule initiations induced by the efficient N fixing strain (WSM419) or the weak N fixing strain (Sm1021) between 0 and 8 d after rhizobium inoculation (left graph); or by the WSM419 and the non-N fixing strain (T173) between 0 and 20 d after rhizobium inoculation (right graph). Error bars represent the standard deviation, and a Kruskal–Wallis test was used to assess significant differences, indicated with letters (α < 0.05, *n* > 46 plants per condition). ns, not significant. (f) Expression of the *MtCLE13* (upper graphs) or of the *MtCEP7* (lower graphs) gene in the nodulation kinetics described in (e). Data were normalized relative to the non-inoculated condition for each rhizobium strain, and three biological (pools of 6 plants) and two technical (independent cDNA synthesis) replicates were performed. Error bars represent the standard deviation. In (c–f), plant growth conditions, rhizobium inoculation, and qRT-PCR experiments were performed as described in Laffont *et al.* (2020). The same WSM419-inoculated kinetic was used in left and right graphs.
(Tabata *et al.*, 2014; Fig. 1a). A combination of grafting, split-root assays aiming to separate local and systemic regulations, and xylem sap peptidomic analyses, revealed that CEP peptides produced in roots move through the xylem vasculature to activate CEPR receptors

in shoots, promoting the downstream production of glutaredoxin-
like CEPD (CEP Downstream) shoot-to-root signals trafficking
through the phloem (Tabata *et al.*, 2014; Ohkubo *et al.*, 2017;
Fig. 1a). An orthologous receptor to CEPR, called Compact Root

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Architecture 2 (CRA2), antagonistically regulating lateral root and symbiotic nodulation, was simultaneously identified in the *M. truncatula* legume and rapidly shown to be required for CEP peptide action (Huault *et al.*, 2014; Mohd-Radzman *et al.*, 2016; Fig. 1b, left scheme). Grafting revealed that, whereas the lateral root number phenotype required the CRA2 receptor function in roots, the regulation of root nodule number was systemic from shoots (Huault *et al.*, 2014). Downstream of this legume CEP/CEPR pathway, a shoot-to-root systemic effector microRNA was identified, the miRNA2111 (miR2111), targeting the transcript accumulation of the Too Much Love (TML) F-box protein negative regulators of nodulation that act locally in roots (Gautrat *et al.*, 2020; Fig. 1b, left scheme). This illustrates that different downstream effectors of the N-deficit-induced systemic pathway involving CEP signaling peptides were recruited to regulate in legumes root mineral N acquisition or symbiotic N fixation.

*MtCEP7*, an atypical *CEP* gene upregulated by rhizobium, exhibits an expression correlated with the strain-specific root competence window to initiate nodules

In the course of characterizing how *CEP* gene expression was regulated by N availability and rhizobia environmental cues in the *M. truncatula* legume, a first regulatory pathway was identified as directly repressing the expression of several *CEP* genes in high N conditions through the MtNLP1 transcription factor (Luo *et al.*, 2022). However, it remains an open question whether another pathway would additionally activate *CEP* gene expression in N deficit. In response to a short-term treatment with rhizobium or its NF signaling molecules, a unique *CEP* gene emerged as upregulated, *MtCEP7* (Laffont *et al.*, 2020; Fig. 1b, middle scheme). This additional regulatory pathway involves a direct regulation of *MtCEP7* expression through the MtNIN transcription factor, allowing thus to draw a parallel with the previously mentioned role of MtNLP1 in the high N repression of *CEP* gene expression. The expression of *MtCEP7* is spatially linked to early rhizobial infection events in root epidermal and cortical cells, and is additionally transiently temporally upregulated in a window corresponding to the onset of nodule initiation (Laffont *et al.*, 2020; Ivanovici *et al.*, 2023). Another systemic pathway antagonizes the CEP nodulation-promoting pathway through the action of another family of signaling peptides, the CLE (CLAVATA3/Embryo surrounded) peptides, such as MtCLE13 in *M. truncatula* (Gautrat *et al.*, 2021, and references therein; Fig. 1b, right scheme). These CLE peptides inhibit nodulation through the SUNN (Supernumerary Nodules) receptor acting in shoots and are induced by rhizobium and/or high mineral N to repress the miR2111 systemic effector, thus favoring the accumulation of TML inhibitors in roots (Fig. 1b, right scheme). Consequently, nodule initiation is inhibited when a sufficient number of nodules is already formed (autoregulation of nodulation pathway, AON) or when soil mineral N is available as an alternative to atmospheric N (nitrate inhibition of nodulation, NON). Increasing the length of the temporal window of *MtCEP7* expression by exchanging its promoter with the *MtCLE13*

promoter mitigated the CLE13-dependent negative AON regula-
tion and thus led to an increased nodulation-promoting activity
(Laffont *et al.*, 2020). The length of the temporal window of root
nodulation competence to rhizobium is affected by environmental
conditions and notably by the ability of rhizobial strains to fix N
(Laguerre *et al.*, 2012; Kassaw *et al.*, 2015). We thus tested how
*MtCEP7* symbiotic induction was regulated in response to rhizobia
strains that were either an efficient N fixer in the *M. truncatula*
genotype Jemalong A17, for example *Sinorhizobium/Ensifer
medicae* WSM419 strain (Terpolilli *et al.*, 2008), a weak N fixer,
for example the *S./E. meliloti* 1021 strain (Terpolilli *et al.*, 2008), or
a non-N fixer (so-called *fix*-) strain, for example the *E. adhaerens*
T173 strain (Bromfield *et al.*, 2023; Fig. 1c-e). The contrasted
outputs of these symbiotic interactions can be easily observed
through the overall aspect of plants, that is, green healthy-growing
plants with WSM419 compared with yellowing chlorotic plants
with T173 (Fig. 1c). Similarly at the nodule level (Fig. 1c, insets),
WSM419 nodules efficiently fixing N were elongated and pink due
to the accumulation of leghemoglobin pigments required for an
efficient N fixation, whereas T173 *fix*- nodules remained round-
shaped and white, and the less symbiotically efficient Sm1021
nodules showed a mixture of pink and white nodules, as quantified
in the Fig. 1(d). A key behavioral difference between the WSM419
highly efficient and the Sm1021 weakly efficient strains is the rate of
nodule initiation, which rapidly drops with the WSM419 strain
due to the fast activation of the negative AON systemic pathway, as
attested by the strong and rapid activation of *CLE13* gene
expression (Fig. 1e,f, upper left graph). The expression of the
*CEP7* nodulation-promoting gene is transiently upregulated in a
tight temporal window, between 1 and 3 d after inoculation with
WSM419 (Fig. 1f, lower left graph), as previously reported
with another efficient strain (Ivanovici *et al.*, 2023). By contrast, the
weakly efficient Sm1021 strain shows a longer *CEP7* temporal
window correlating with the maintenance over time of nodule
initiations, which is associated with a weaker induction of the
*MtCLE13* gene compared with the high efficient WSM419 strain
(Fig. 1f, left graphs). Finally, the T173 *fix*- strain shows a similar
initial rate of nodule initiation as the WSM419 highly efficient
strain, but is then characterized by a drop in initiation events 8 d
after inoculation, followed by the set-up of a new nodulation wave
(Fig. 1e, right graph). This re-initiation of root competence for
nodulation, starting at day 11, again tightly correlates with a higher
level of *CEP7* expression and a lower level of *CLE13* expression
(Fig. 1f, right graphs). Overall, this indicates that the temporal
window of the induction of the nodule-promoting CEP7 signaling
peptide expression by rhizobium is correlated with the strain
symbiotic efficiency.

Concluding remarks and perspectives

The dynamic and coordinated regulation of the expression of CEP
and CLE signaling peptides appears as a mechanism used by legume
plants to adapt their root competence nodulation window not only
to the availability of mineral N but also depending on the output of
the symbiotic interaction, namely the nutritional benefit gained
from the interaction with a more or less efficient rhizobial strain.
Analyzing the expression pattern of such CLE and CEP genes in response to a mix of different rhizobia strains with various symbiotic efficiencies should help to better understand the function of these signaling peptide systemic pathways in natural environments. In addition, by modulating the expression pattern of CLE or CEP peptide encoding genes promoting or inhibiting nodulation, for example by exchanging their promoters to modify the symbiotic root competence temporal window (Laffont et al., 2020), it remains to be evaluated whether these systemic pathways contribute to conditional systemic sanctioning mechanisms that favor strains with higher efficiencies over poor mutualist interactions (Westhoek et al., 2021). Finally, gaining a mechanistic knowledge on the transcriptional regulations of these signaling peptide-encoding genes, beyond NIN and NLP transcription factors, will allow better understanding, and therefore manipulating, the CEP temporal expression dynamics and thus the legume root symbiotic potential.

## Acknowledgements

Work in the FF laboratory was supported by the CNRS, the 'PSYCHE' ANR project, and the 'Ecole Universitaire de Recherche' (EUR) 'Saclay Plant Science' (SPS). We thank Kevin Magne (IPS2) for suggesting using the T173 rhizobium strain and for discussions.

## Competing interests

None declared.

## Author contributions

CL performed the experiments. FF wrote the manuscript with contributions from CL.

## ORCID

Florian Frugier ID https://orcid.org/0000-0002-9783-7418

Carole Laffont ID https://orcid.org/0000-0002-8447-1736

## Data availability

All data generated for this study are found in Fig. 1.

Carole Laffont ID and Florian Frugier* ID

Institute of Plant Sciences Paris Saclay (IPS2), CNRS, Paris-Saclay University, Paris-Cité University, INRAE, Univ d'Evry, Bat. 630, Avenue des Sciences, Gif-sur-Yvette, 91190, France
(*Author for correspondence: email florian.frugier@universite-paris-saclay.fr)

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**Key words:** Compact Root Architecture 2 receptor, C-terminally encoded peptides, nitrogen deficit, nitrogen fixation, rhizobia bacteria, root nodule symbiosis.

Received, 12 August 2023; accepted, 13 October 2023.