# MiPEPs and cPEPs as tools to monitor plant gene expression and develop alternative strategies in agriculture

Patrice Thuleau, Mélanie Ormancey, Serge Plaza, Jean-Philippe Combier

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Patrice Thuleau, Mélanie Ormancey, Serge Plaza, Jean-Philippe Combier. MiPEPs and cPEPs as tools to monitor plant gene expression and develop alternative strategies in agriculture. Journal of Experimental Botany, 2024, pp.erae501. 10.1093/jxb/erae501. hal-05343745

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# MiPEPs and cPEPs as tools to monitor plant gene expression and develop alternative strategies in agriculture

Patrice Thuleau¹, Mélanie Ormancey¹,², Serge Plaza¹, Jean-Philippe Combier¹,³*

¹ Laboratoire de Recherche en Sciences Végétales, CNRS/UT3/INPT, Auzeville-Tolosane, France.

² Present address: Epigenetics and Plant Development, Centre for Research in Agricultural Genomics (CRAG), UAB, Cerdanyola del Vallès, 08193 Barcelona, Spain

³ Micropep Technologies, Auzeville- Tolosane, France

*Correspondence: jean-philippe.combier@univ-tlse3.fr

Mélanie Ormancey: melanie.ormancey@cragenomica.es

Serge Plaza: serge.plaza@univ-tlse3.fr

Jean-Philippe Combier: jean-philippe.combier@univ-tlse3.fr

Patrice Thuleau: patrice.thuleau@univ-tlse3.fr

## 1 Abstract

In order to develop a sustainable agriculture respecting the environment and to reduce chemical inputs, a new strategy has emerged in recent years, based on the use of products targeting plants' natural defense and growth mechanisms. In this context, a few years ago we demonstrated the existence in plants of regulatory peptides called miPEPs for "microRNA-encoded peptides". MicroRNAs (miRNAs) are small RNAs that down-regulate the expression of numerous genes in eukaryotes. MiPEPs increase the amount of their cognate miRNA, resulting in an even greater decrease in the expression of genes targeted by these miRNAs. Thus, the application of an artificial miPEP to a plant causes a phenotype associated with a change in the genes targeted by the corresponding miRNA. More recently we also identified new regulatory peptides, namely cPEPs for "complementary peptides", capable of enhancing the activity of a given protein, independently of any increase in transcriptional activity. Moreover, as for the miPEPs, the external application of these new peptides makes it possible to regulate the phenotypes associated with the targeted proteins. This review highlights the most recent advances in the potential use of miPEPs and cPEPs in agronomy to improve plant development and stress tolerance.

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## 17 Highlight

One of the main challenges facing agriculture is the control of plant development without using chemical compounds harmful to the environment. Here, we present natural regulatory peptides as promising alternatives.

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## 22 Keywords

peptide; miRNA; miPEP; microRNA-encoded peptide; cPEP, complementary peptide; gene regulation;

biostimulant; biotechnology; plant.
25 **Introduction**

For many years, phytosanitary products have been widely used by farmers. Currently the global market for phytosanitary products amounts to approximately 70 billion euros per year, of which almost half is devoted to herbicides alone (https://www.statista.com/statistics/272493/revenue-of-the-plant-protection-market-worldwide-since-2007/). For example, one of the main problems in agriculture today is the difficulty in controlling the development of weeds, which requires the massive use of weedkillers such as glyphosate, but their use in the field remains a major problem because of the environmental risks on flora and fauna. However, except genetically modified organisms (GMOs), no current tools or chemicals are available to specifically target weeds without inhibiting crop growth. Therefore, in order to develop a sustainable agriculture, the identification of natural molecules to control plant development remains a major challenge.

In recent years, in order to effectively protect crops, innovative strategies have been developed based on the use of products that take advantage of natural mechanisms that plants display to combat their aggressors. Among these strategies, some relate to the mechanisms of RNA interference by siRNA gene silencing and more particularly in plants by the use of microRNAs (miRNAs) (Zhou and Luo, 2013; Das and Sherif, 2020; He et al., 2022;). MiRNAs are small single stranded regulatory RNA molecules of 20-22 nt in length which negatively regulate, in plants or animal, the expression of many fundamental genes at the post-transcriptional level by binding to the messenger RNA (mRNA) in order to inhibit translation or cleaving the mRNA. Because their target genes are mainly key regulatory genes, such as genes encoding transcription factors or hormone receptors, miRNAs are involved at the crossroads of several biological processes.

Since miRNAs have the property of controlling gene expression and therefore plant growth and development or stress responses, without resorting to transgenesis and GMOs, these last years, attempts to use them in the field have been multiplied. However, these attempts have been hampered by the difficulty for miRNAs to enter plants (Dalakouras et al., 2018). Nevertheless, recent studies have demonstrated that miRNA-derived peptides could be interesting tools for agronomic applications.

Here, we summarize the most recent advances on the use of these and other more recently discovered regulatory peptides as effective molecules for improving traits of interest in plant development and responses to biotic and abiotic stresses.

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**miPEPs, newcomers in regulation of gene expression**

MiRNAs are transcribed as long primary transcripts (pri-miRNAs) by RNA Polymerase II, and are
capped and polyadenylated, like the genes encoding proteins. This gave the idea, albeit initially
thought as non-coding RNAs, that pri-miRNAs might be translatable as previously shown for other
non-coding RNAs. Several studies have now revealed that a number of plant pri-miRNAs encode
regulatory peptides called miPEPs for miRNA-encoded peptides.

So far, miPEPs have been defined as encoded by the first short open reading frame (sORF) present
in the 5' region after the transcription start site of the pri-miRNA. However, it is possible that
64 downstream sORFs might be functional. Interestingly, miPEPs are not specific to plants. Some miPEPs
65 have been recently identified in humans, enlarging the concept of coding pri-miRNAs from plants to
66 animals (Dozier and Plaza, 2022; Ormancey et al., 2023a). Initially characterized in *Arabidopsis*
67 *thaliana*, *Medicago truncatula* and *Glycine max* (Lauressergues et al., 2015; Couzigou et al., 2016;
68 Couzigou et al., 2017), more recent studies revealed the presence of miPEPs in other plant species,
69 suggesting that the number of miPEPs has been largely underestimated so far, and that presence of
70 miPEPs might be a common feature of plant pri-miRNAs (Ormancey et al., 2023a) (Box1; Box 2).
71

Until now, all identified miPEPs have the property of enhancing the transcription of their cognate
71 pri-miRNA, which increases the amounts of mature miRNAs, thus leading, through a positive
72 regulatory loop, to an even greater decrease in the expression of mRNAs targeted by these miRNAs
73 (Box 3; Box 4).
74

75
76 The molecular mechanisms by which plant miPEPs are able to specifically increase the
77 transcription of their corresponding pri-miRNAs are beginning to be elucidated. First, it has been
78 shown that miPEPs enter plants by both passive diffusion and endocytosis (Ormancey et al., 2020;
79 Badola et al., 2022). Second, miPEPs have recently been found to be in close association with the pri-
80 miRNAs from which they are derived (Lauressergues et al., 2022) (Box 3; Box 4). More importantly,
81 on pri-miRNA, the nucleotide sequence encoding the miPEP (called miORF) appears to be required
82 for activation of pri-miRNA expression by the corresponding miPEP. It likely acts on the
83 transcriptional machinery via its interaction with the pri-miRNA and the protein transcriptional
84 complex. Indeed, if the miORF is deleted, miPEP activation is lost (Box 3). Similarly, when this
85 sequence is exchanged with another sequence encoding a different peptide, activation of expression
86 is restored, but only by the latter peptide (Lauressergues et al., 2022).
87

87
88 Besides the interaction between miPEPs and RNA, a second hypothesis that could explain the
89 ability of miPEPs to activate the transcription of pri-miRNAs is that the peptides may also interact
90 with proteins involved in the transcription and processing of miRNAs (Box 4).
91

91
92 The different steps of miRNA transcription and processing and the proteins involved are now well
93 characterized and form, together with the transcription complex, the so-called DICER complex. Since
94 the expression of mature miRNAs is also increased by miPEPs, each of the proteins of these
95 complexes could be a potential interactor (Box 4). We are currently investigating these different
96 hypotheses.
97

97 miPEPs as potential tools for agronomy

98
99 The application of a chemically synthesized miPEP to a plant, either by spraying or watering, led to
100 a developmental response consistent with the role of the targeted miRNAs. This includes growth
101 control, increased resistance to biotic and abiotic stresses. This makes miPEPs particularly attractive
102 tools for sustainable and environmentally friendly agronomy by reducing chemical inputs or to
respond to various agronomic issues (Yadav et al., 2021; Chen et al., 2023) (Box 1; Box 2).
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Since the discovery of miPEPs (Lauressergues et al., 2015), several teams have been interested in these new regulatory peptides.

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For example, an external application of At-miPEP858a increases the transcription of miRNA858a in *A. thaliana*, which in turn decreases the amount of the transcription factor MYB12, ultimately leading to inhibition of the enzymes responsible for the anthocyanin synthesis and an increase in root length (Sharma *et al.*, 2020). Also in *A. thaliana*, miR408 and its encoded peptide At-miPEP408, have been shown to control sulfur assimilation and arsenic stress (Kumar *et al.*, 2023). Similarly, it has been shown that the external application of Vvi-miPEP171d1 from grapevine (*Vitis vinifera*) increased the formation of adventitious roots in this plant, specifically for this species (Chen *et al.*, 2020). More recently, the same team identified two other miPEPs, Vvi-miPEP172b and Vvi-miPEP3635b which are able to increase cold tolerance of grapevine by regulating their corresponding *MIR* gene (Chen *et al.*, 2022).

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By studying somatic embryogenesis of cell cultures of *Dimocarpus longan*, a plant widely used in traditional Chinese medicine, a functional analysis of miR166 family members, suspected of regulating embryogenesis in response to plant hormones, was performed (Zhang *et al.*, 2020). First, an *in-silico* approach revealed the presence of sORFs in the 5' region of the pri-miR166 studied. External application of a synthetic peptide potentially encoded by one of these sORFs to somatic embryo led to a modulation of miR166 expression and to an inhibition of the expression of a target gene of the miR166 (*ATHB15*). Whether this miPEP has a biological function remains to be determined.

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In the literature, examples relating the importance of miPEPs for agronomic application have been described (Box 2). In 2016, Couzigou and colleagues showed that soybean miPEP172c was able to increase nodulation (Couzigou et al., 2016). We can imagine that a field treatment with this miPEP would improve yields by increasing nitrogen fixation.

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Very recently, a panel of 87 *A. thaliana* miPEPs were tested on this plant for their ability to positively modulate the expression of the *PR1* gene, a molecular marker of defense reactions whose expression is induced by many pathogens. These miPEPs had been deduced from bioinformatics analyses aimed at identifying miRNAs conserved between plant species. Among the miPEPs tested, 3 were found to be active, namely miPEP169c, miPEP169h and miPEP396b. Based on these data, we wondered whether these miPEPs could be used to improve plant resistance to pathogens. To answer this question, following inoculation of *A. thaliana* leaves with the necrotrophic fungus *Botrytis cinerea*, we showed that exogenous application of the peptides to these leaves reduced the infection rate, without any toxic effect on the fungus spores. To investigate whether the activity of these peptides was conserved in plants, all miPEPs from the miR169 and miR396 families in three plants of agronomic interest, tomato, bean and strawberry, were tested for their effect on resistance to *B. cinerea*. Some members of the miR169 and miR396 families were found to significantly improve resistance to the fungus, while others increased susceptibility. Interestingly, tomato *S/miPEP169d* which had the greatest effect on improving resistance to *B. cinerea*, was also shown to improve tomato resistance to *Alternaria solani*, a fungus responsible for downy mildew, and to *Pseudomonas*
syringae pv. tomato, which is responsible for bacterial speck. Similarly, this miPEP is capable of improving tomato resistance in the field (Ordancey et al., 2024a)

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With the aim of identifying miPEPs able to control weed development without interfering with that of crops, a similar approach was taken to determine those that modulate *A. thaliana* root growth (Ordancey et al., 2021). Thus, among the same panel of 87 previous miPEPs, 23 were found to have an effect on both the length of the main root and the number and length of lateral roots, most of them having a negative effect on this growth. As before, in order to see whether these results could be transposed to plants of agronomic interest, the study focused on two peptides in particular, miPEP164b and miPEP397a, which respectively had the greatest negative and positive effect on root development in *A. thaliana*. As these miPEPs are conserved in Brassicaceae, their homologues were sought in plants of agronomic interest belonging to this family, namely *Brassica oleracea* (*BomiPEP397a*) and *Barbarea vulgaris* (*BvmiPEP164b*), a weed that can contaminate seeds (MacDonald and Cavers, 1991). Interestingly, exogenous application of *BomiPEP397a* increased cabbage root growth, while *BvmiPEP164b* decreased that of Barbarea vulgaris. Consistently, cabbages sprayed with *BomiPEP397a* were more developed than those sprayed with a control peptide, while *B. vulgaris* sprayed with *BvmiPEP164b* were smaller than those treated with a control peptide (Ordancey et al., 2021).

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Similarly, a recent *in silico* analysis of the genome of *Orobanche cumuna*, a parasitic plant of sunflower roots, identified 39 miRNAs potentially involved in the germination of this weeds. From these miRNAs, miPEPs were chemically generated and tested for their ability to modulate *O. cumuna* germination *in vitro*. A total of 19 miPEPs were found to inhibit germination. The most active was miPEP319a, which was shown to specifically increase pri-miR319a expression, while miPEP319b had no effect. Since *O. cumuna* occurs at very high densities, outcompetes sunflower and significantly reduces crop yield if not controlled, miPEPs could provide sustainable phytosanitary molecules to fight this parasitic plant (Tourneur et al., 2024).

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In conclusion, from an applied point of view, it now seems clear that miPEPs constitute an innovative tool that can offer interesting prospects in terms of plant improvement for agronomic purposes. Indeed, as mentioned before, we now know that many physiological processes can be modulated simply by spraying plants with appropriate miPEPs. These peptides, like many others encoded by sORFs present on non-coding RNAs, are not conserved and can therefore be species-specific. Furthermore, since miRNAs are found in all plant lineages, miPEPs can also be expected to be present in many, if not all, plant species. This makes them power tools for large-scale application.

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## What about cPEPs?

Very recently, a new class of peptides capable of targeting and regulating plant protein translation was identified in *A. thaliana* (Ordancey et al., 2023b). Based on the results described above showing that miPEPs were able to interact with their pri-miRNAs, it was investigated whether other peptides
183 could interact with messenger RNAs. Thus, peptides derived from any protein were shown to bind
184 the mRNA encoding that protein and, more interestingly, to enhance its activity, independently of
185 any transcriptional activity, since no increase in the corresponding mRNA was observed (Box 3).
186 These peptides were named cPEPs for complementary peptides. In fact, cPEPs enhance translation of
187 target proteins by promoting recruitment of the ribosomal machinery (Box 4). As with miPEPs,
188 external application of cPEPs was effective in regulating phenotypes associated to the targeted
189 proteins both in the model plant A. thaliana and in plants of agronomic interest or weeds (Box 1; Box
190 2). Moreover, unlike miPEPs which are highly species-specific (Laressergues et al., 2022), cPEPs can
191 be designed to specifically target one protein in one species, or to target homologous proteins in
192 different species (Ormancey et al., 2024b). This property could potentially be used to target and
193 enhance the translation of multiple members of protein families at once, thus providing exciting new
194 tools for the study of entire protein families.

195 Confirming this property, a recent study focusing on key proteins of the phenylpropranoid
196 pathway, namely two transcription factors MYB12 and HY5, and chalcone synthase (CHS) a pivotal
197 enzyme of this pathway, showed that treatment of A. thaliana, tobacco as well as tomato with cPEPs
198 targeting these proteins induced a significant increase in their quantity without increasing the
199 transcription of their encoding gene. Consistently, cPEPMYB12 enhance flavonol accumulation in
200 Arabidopsis and a subsequent reduction in root length (Sharma et al., 2024).
201

## 202 Conclusion - Future prospects

203 At the fundamental level, the identification of miPEPs and cPEPs provides a new level of gene
204 regulation and represents an exciting tool to easily control the functions of miRNAs and protein-
205 coding genes. However, a number of questions remain unanswered to better understand their
206 activity (see Box 4):

207 1. How do miPEPs activate the transcription of their nascent pri-miRNA? By interacting with other proteins, such as those involved in the DICER complex?

209 2. Are there any negative feedback mechanisms that could control miPEP and cPEP activities?

210 3. What are the molecular bases of cPEP/RNA/ribosome interactions?

211 4. Do natural cPEPs exist in plant cells? Indeed, recent evidence has shown that sORFs in 5' and 3' untranslated regions or present in either intergenic sequences or in the main coding sequences can encode peptides (Fesenko et al., 2019; Wang et al., 2020; Brunet et al., 2021).
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213 In addition, other peptides may be derived from the proteolytic degradation of proteins. It
214 remains to be investigated whether all these peptides, potentially natural cPEPs, increase the
215 expression of their target protein.
216

217 5. MiPEPs increase transcription of their nascent pri-miRNA whereas cPEPs activate translation
218 of their targeted protein, although both are capable of interacting with their "nascent" RNA.
219 Is this difference solely due to their cellular localization, i.e. nuclear for miPEPs, and
220 cytoplasmic for cPEPs?
221

6. Do miPEPs underlie other biological functions? Indeed, several human miPEPs cannot increase the transcription of their nascent pri-miRNA but have other biological functions (Ordancey et al., 2023a)

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From an applied point of view, it now seems clear that miPEPs and cPEPs constitute a fantastic innovative tool that can offer interesting prospects in terms of plant improvement for agronomic purposes. Indeed, as mentioned before, we now know that many physiological processes, like development, immunity, stress response, can be modulated simply by spraying plants with appropriate peptides. For the first time, this opens the door to identifying credible alternatives to chemicals in agriculture.

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## Acknowledgements -Fundings

233

This work was funded by the French ANR projects BiomiPEP (ANR-16-CE12-0018-01) and DeepPEP (ANR-23-CE20-0020). The boxes 1 and 4 were created using BioRender.com.

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## Boxes

**Box 1: miPEPs and cPEPs playing a role in development and response to stress in model plants.**

miPEPs are represented in green and targets of cPEPs are represented in blue.

References: (1) Badola *et al.*, 2022; (2) Couzigou *et al.*, 2017; (3) Kumar *et al.*, 2022; (4) Lauressergues *et al.*, 2015; (5) Ormancey *et al.*, 2020; (6) Ormancey *et al.*, 2021; (7) Ormancey *et al.*, 2023b; (8) Ormancey *et al.*, 2024a; (9) Ormancey *et al.*, 2024b; (10) Sharma *et al.*, 2020; (11) Sharma *et al.*, 2024.

**Box 2: miPEPs and cPEPs playing a role in development and response to stress in plants of agronomic interest.**

miPEPs are represented in green and targets of cPEPs are represented in blue.

**Box 3: Nucleotide sequences encoding miPEPs or the target of cPEPs are required for the mRNA/peptide interaction and the activity of miPEPs and cPEPs.**

a, c: Schematic representation of constructs used, with wild type construct (left) and constructs in which the ORF (for miPEP) (a) or the target sequence of cPEP (c) is deleted (right).
b, d: Left panel: Interaction assay, using FRET-FLIM analysis between fluorescent peptides and mRNA labelled or not with Sytox. Right panel: Activity of peptides on expression of pri-miR171b (b) and NSP1 (d). Adapted from Lauressergues *et al.*, 2022 and Ormancey *et al.*, 2023b.

**Box 4: Global overview of the mode of action of miPEPs and cPEPs.**

In red, remaining questions (see corresponding numbers in the "Conclusion – Future prospects" section of the manuscript). Adapted from Lauressergues *et al.*, 2022 and Ormancey *et al.*, 2023b.
**Box 1: miPEPs and cPEPs playing a role in development and response to stress in model plants.** miPEPs are represented in **green** and targets of cPEPs are represented in **blue**.

References: (1) Badola *et al.*, 2022; (2) Couzigou *et al.*, 2017; (3) Kumar *et al.*, 2022; (4) Lauressergues *et al.*, 2015; (5) Ormancey *et al.*, 2020; (6) Ormancey *et al.*, 2021; (7) Ormancey *et al.*, 2023b; (8) Ormancey *et al.*, 2024a; (9) Ormancey *et al.*, 2024b; (10) Sharma *et al.*, 2020; (11) Sharma *et al.*, 2024.

## Arabidopsis thaliana

### Response to stress

**Sensitivity to arsenic and low sulfur**
miPEP408
(3)

**Heat tolerance**
HSC70, HSP101, SHOT1
(7, 9)

**PR1 activity**
miPEP156c, 159a, 160b, 161, 164a, b, 166d, 167a, b, d, 169c, h, j, m, n, 172d, 390a, 391, 394b, 396b
(8)

**plant defense**
miPEP169c, h
miPEP396b
ABCG34, AGB1, COI1, CPK3, JAR1, MAPKKK3, 5, RING1
(7, 9)

### Development

**Growth**
miPEP165a
BAK1, BRI1, EIN2, GA2OX7, HAG5, MRB1, PHYB, SHR, SHY2, SPT, TAP46, WUS
(5, 7)

**Seedlings growth**
miPEP408
(3)

**Chlorophyl content**
ABI5, SGR1
(7)

**Root Development**
miPEP157b, c, 159b, c, 163, 164b, c, 165a, 166a, g, 169a, 171c, 390a, 391, 393b, 394b, 395a, c, f, 396a, 397a, 398c, 399d, 858a
DCL1, EIN2
(1, 4, 5, 6, 7, 9, 10)

<figure><img src="image_2.png" /></figure>

## Medicago truncatula

### Response to stress

**Arbuscular mycorrhizal symbiosis**
miPEP171a-f
(2)

**Plant defense**
RH10
(7)

**Nodulation**
miPEP169g
SKL
(7, 9)

### Development

**Root Development**
miPEP171b
NSP1, SKL
(4, 7, 9)

<figure><img src="image_3.png" /></figure>
<table><caption>Box 2: miPEPs and cPEPs playing a role in development and response to stress in plants of agronomic interest. miPEPs are represented in green and target of cPEPs are represented in blue.</caption><thead><tr><th>Plants</th><th>Target (pri-miRNA or protein)</th><th>Phenotype</th><th>Reference</th></tr></thead><tbody><tr><td>Amaranthus hypochondriacus</td><td>Mix EIN2-BRI1-BAK1-WUS</td><td>Plant growth</td><td>Ormancey et al., 2023b</td></tr><tr><td rowspan="2">Barbarea vulgaris</td><td>Pri-miR164b</td><td>Root development</td><td>Ormancey et al., 2021</td></tr><tr><td>Mix EIN2-BRI1-BAK1-WUS</td><td>Plant growth</td><td>Ormancey et al., 2023b</td></tr><tr><td>Brassica oleacera</td><td>Pri-miR397a</td><td>Root development</td><td>Ormancey et al., 2021</td></tr><tr><td>Brassica rapa</td><td>Pri-miR156a</td><td>Root development</td><td>Erokhina et al., 2021</td></tr><tr><td>Dimocarus longan</td><td>Pri-miR166</td><td>Embryogenesis</td><td>Zhang et al., 2020</td></tr><tr><td>Fragaria vesca</td><td>Pri-miR169b-j, l<br>Pri-miR396a-d, f</td><td>Plant defense</td><td>Ormancey et al., 2024a</td></tr><tr><td rowspan="4">Glycine max</td><td>Pri-miR172c</td><td>Nodulation</td><td>Couzigou & Combier, 2016</td></tr><tr><td>Pri-miR167c</td><td>Chlorophyl content</td><td>Ormancey et al., 2024b</td></tr><tr><td>HSP101</td><td>Heat tolerance</td><td>Ormancey et al., 2023b</td></tr><tr><td>Mix MRB1-SHY2-SGR1</td><td>Plant growth</td><td>Ormancey et al., 2023b</td></tr><tr><td>Hordeum vulgare</td><td>Pri-miR444c</td><td>Root and shoot development</td><td>Chojnacka et al., 2023</td></tr><tr><td>Lotus japonicus</td><td>Pri-miR171b</td><td>Arbuscular mycorrhizal symbiosis</td><td>Couzigou et al., 2017</td></tr><tr><td>Orobanche cumana</td><td>Pri-miR156d, 159, 160a-d, 164, 167b, 169a, b, 172a, b, 319a, 393a, b, 395, 396a-c</td><td>Seed germination</td><td>Tourneur et al., 2024</td></tr><tr><td rowspan="2">Oryza sativa</td><td>Pri-miR171i</td><td>Arbuscular mycorrhizal symbiosis</td><td>Couzigou et al., 2017</td></tr><tr><td>Pri-miR156e</td><td>Cadmium tolerance</td><td>Lu et al., 2024</td></tr><tr><td>Petunia hybrida</td><td>Pri-miR169</td><td>Flowering</td><td>Ormancey et al., 2024b</td></tr><tr><td>Phaseolus vulgaris</td><td>Pri-miR169b, h-l, o, p<br>pri-miR396</td><td>Plant defense</td><td>Ormancey et al., 2024a</td></tr><tr><td>Rosa chinensis</td><td>Mix MYB12-CHS-HY5</td><td>Anthocyanin content</td><td>Sharma et al., 2024</td></tr><tr><td rowspan="3">Solanum lycopersicum</td><td>Pri-miR171e</td><td>Arbuscular mycorrhizal symbiosis</td><td>Couzigou et al., 2017</td></tr><tr><td>Pri-miR169e, i, j, l, n, o-q<br>pri-miR396a, b, d<br>JAR1</td><td>Plant defense</td><td>Ormancey et al., 2023b, 2024a</td></tr><tr><td>Pri-miR396a</td><td>Root development</td><td>Wang et al., 2024</td></tr><tr><td rowspan="3">Vitis vinifera</td><td>Mix MYB12-CHS-HY5</td><td>Anthocyanin content</td><td>Sharma et al., 2024</td></tr><tr><td>Pri-miR171d</td><td>Root development</td><td>Chen et al., 2020</td></tr><tr><td>Pri-miR164c, 166c</td><td>Anthocyanin synthesis/ accumulation</td><td>Valle et al., 2021, 2024</td></tr><tr><td></td><td>Pri-miR172b, 3635b</td><td>Cold tolerance</td><td>Chen et al., 2022</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Box 3: Nucleotide sequences encoding miPEPs or the target of cPEPs are required for the mRNA/peptide interaction and the activity of miPEPs and cPEPs.** a, c: Schematic representation of constructs used, with wild type construct (left) and constructs in which the ORF (for miPEP) (a) or the target sequence of cPEP (c) is deleted (right). b, d: Left panel: Interaction assay, using FRET-FLIM analysis between fluorescent peptides and mRNA labelled or not with Sytox. Right panel: Activity of peptides on expression of pri-miR171b (b) and NSP1 (d). Adapted from Lauressergues *et al.*, 2022 and Ormancey *et al.*, 2023b.</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>**Box 4: Global overview of the mode of action of miPEPs and cPEPs.** In red, remaining questions (see corresponding numbers in the “Conclusion – Future prospects” section of the manuscript). Adapted from Lauressergues *et al.*, 2022 and Ormancey *et al.*, 2023b.</figcaption></figure>