**3. Discussion**

MiRNAs have been considered for a long time as non-coding RNAs. However, a few years ago, it was shown that pri-miRNAs can encode regulatory peptides, which were named miPEPs. These miPEPs activate the transcription of their associated miRNA and thus downregulate the expression of their target genes [5]. Among miPEPs, miPEP165a induces the accumulation of mature miR165a, known to repress the expression of all five class III homeodomain-leucine zipper (HD-ZIP III) transcription factors, i.e., *REV*, *PHB*, *PHV*, *CORONA* (*CAN*/*AtHB15*), and *AtHB8* [5,37]. In *Arabidopsis*, the overexpression of all HD-ZIP III results in plants with shorter roots whereas *phb, phv* double mutants and *phv-11* mutants display longer roots as well as an increase in the number of meristem cells compared to wild-type plants [38,39]. Moreover, the overexpression of miR166, differing by only

one nucleotide from miR165 and targeting the expression of three HD-ZIP III genes, also promotes primary root growth in *Arabidopsis* [39]. These results can be correlated with those of the present study, since we showed that miPEP165a promotes primary root growth by increasing cell division in the root apical meristem (Figure 1). Moreover, misexpression of the HD-ZIP III genes by making them resistant to miR165/166 and a reduction in the expression of HD-ZIP IIIs by overexpression of miR165/166 induces prolonged activity of floral stem cells [30]. Here, we observed that miPEP165a accelerates the appearance of the inflorescence stem and the flowering time of *Arabidopsis* wild-type plants (Figure 2).

Since some small peptides were considered as long-distance signaling molecules, we wondered whether miPEP165a was involved in root/shoot communication [40–42]. By tracking the FAM-labelled miPEP165a across all layers of *Arabidopsis* roots, we showed that the labelled peptide entered into the epidermis and migrated up to the pericycle but did not reach the root vessels (Figure 4). Moreover, the acceleration of flowering observed in response to the miPEP165a treatment of the shoot apical meristem was not observed after watering *Arabidopsis* roots with miPEP165a (Figure 2). Taken together, these results indicate that miPEP165a is not a root-to-shoot mobile signal molecule.

Consequently, in order to have a better understanding of miPEP uptake into plants, we investigated the mobility of FAM-labelled miPEP165a in *Arabidopsis* roots. Clathrin-mediated endocytosis is the major and the most studied route of entry in plants [8]. A recent study showed that this endocytic pathway is necessary for the internalization of the elicitor peptide *At*pep1 and its receptor, leading to *At*pep1-induced responses [16]. Here, we showed that the entry of miPEP165a could also be dependent on clathrin since miPEP165a uptake was significantly decreased in the primary roots of *chc1-1* and strongly reduced in the mature zone in the three mutants *chc1-1*, *chc2-1*, and *ap2*σ*2* (Figure 5). These results were confirmed by the fact that the increase of the root length by miPEP165a was not observed in the *chc1-1* mutant or after treatment with TyrA23 (Figure 6A, Figure 8A), the most commonly used CME inhibitor [8,32,36]. Similarly, the acceleration of the flowering time induced by miPEP165a in wild-type plants was not observed in the *chc1-1* mutant (Figure 7).

Besides clathrin-mediated endocytosis, membrane microdomain-associated endocytosis has been described in plants as an alternative route of entry pathway [8]. This endocytosis pathway is sensitive to sterol depletion and consequently to the sterol-depleting agen<sup>t</sup> MβCD [8,17,18]. In the present study, we showed that MβCD prevented miPEP165a-FAM entry and correlatively the increase of root length induced by miPEP165a (Figure 8B, Figure S4). Collectively, our results indicate that both clathrin-dependent pathways and microdomain-associated events may cooperate in peptide entry into *Arabidopsis* roots. Previous results have demonstrated that internalization of the aquaporin PIP2;1 and RbohD involved both dependent and independent clathrin-mediated endocytosis, the latter being stimulated in saline stress conditions [17,22]. Stimulation of the endocytic pathway under salt stress requires the simultaneous action of both clathrin-dependent and membrane microdomain-associated endocytosis [17,22]. In addition, Baral and his colleagues have shown that clathrin-mediated endocytosis allows the internalization of transmembrane proteins in all cell root layers whereas a sterol-sensitive clathrin-independent pathway internalizes lipid-anchored cargoes only in the epidermal cell layer [18]. Moreover, these authors showed that salt stress activates an additional clathrin-independent endocytosis pathway across all cell root layers that takes up both molecule types [18]. Considering membrane microdomain-associated endocytosis, it is known that proteins assemble into clusters in lipid rafts [8]. Among these proteins, remorins are considered as markers of membrane microdomains [35]. In *Medicago truncatula*, the symbiotic remorin 1 forms clusters and interacts with symbiotic receptors at the plasma membrane, playing a key role in bacterial signal perception [21]. Here, we showed that remorins 1-2 and 1-3, which are among the 10% of the most highly expressed genes in *Arabidopsis* [43], were also involved in miPEP165a entry into *Arabidopsis* roots (Figure 6B, Figure 7). Indeed, miPEP165a-FAM failed to enter the differentiation zone of *Arabidopsis* roots in *rem1-2* and *rem1-3* mutants. Moreover, root length and flowering acceleration induced by miPEP165a were perturbed in both remorin mutants (Figure 6B, Figure 7).

#### *Int. J. Mol. Sci.* **2020**, *21*, 2266

To conclude, we showed that endocytic pathways participate in miPEP uptake in plants. Thus, clathrin-mediated endocytosis as well as membrane microdomain-associated pathways seem to cooperate, allowing miPEPs to regulate their corresponding miRNAs and consequently modulate the plant phenotype, such as flowering and root development. Due to the simplicity of the mode of administration of miPEPs, a better understanding of miPEP uptake into plants is a first step towards the possible agronomic application of peptides.

#### **4. Materials and methods**

### *4.1. Peptide Synthesis*

miPEP165a (MRVKLFQLRGMLSGSRIL), miPEP165a fused to fluorescein (miPEP165a-FAM), scrambled miPEP165a (LMGRQGLKISSLVFRMLR), PEP1 (KSNKTRVNFPS), PEP2 (MCFSFPDL), and PEP3 (MASAAKVYMA) were synthetized by Smart Bioscience (https://www.smart-bioscience. com/). They were dissolved in water (control) as a 10 mM stock solution (except for PEP2, which was dissolved in 50% acetonitrile as a 2 mM stock solution), aliquoted, and conserved at −80 ◦C until use.

### *4.2. Plant Materials*

Di fferent *Arabidopsis thaliana* plant lines (Columbia Col-0 ecotype) were used: the *chc1-1* (At3g11130), *chc2-1* (At3g08530), *ap2*σ*2* (At1g47830), *rem1-2* (At2g45820), and *rem1-3* (At3g61260) *Arabidopsis* mutants.

#### *4.3. Peptide Treatment of Arabidopsis Roots*

Surface-sterilized *Arabidopsis* seeds were sown on the surface of cellophane membrane placed on 1 2 MS solid medium and stratified for one day at 4 ◦C in the dark. Seeds were vertically grown in controlled environmental chambers at 22/20 ◦C, with a photoperiod of 16h light/8h dark, an irradiance of ~ 97.5 μmol photons.m−2.s−1, and a relative humidity of 40%. Three days after sowing, seedlings were treated daily for 4 days either with water, 2.5% acetonitrile, 100 μM scrambled miPEP165a, 100 μM irrelevant peptides (PEP1, PEP2, PEP3), or fluorescein (control conditions) or with 100 μM miPEP165a or miPEP165a-FAM (treated conditions). Twenty-four hours after the last treatment, seedlings were scanned in order to measure primary root lengths using NeuronJ plugin of ImageJ.

#### *4.4. Peptide Uptake in Arabidopsis Roots*

Surface-sterilized wild-type and mutant *Arabidopsis* seeds were grown onto 1 2 MS solid medium in the same conditions as those described in the previous section. Three days after germination, three seedlings were transferred to each well of a 48-well plate containing 200 μL of 1 2 MS liquid medium. One day later, medium was replaced by 10 μM miPEP165a-FAM diluted in 1 2 MS liquid medium until confocal microscopy observations. FAM fluorescence was analyzed with a confocal laser scanning microscope (Leica TCS SP2-AOBS using a 40 X water immersion objective lens (numerical aperture 0.80; HCX APO). FAM fluorescence was excited with the 488-nm ray line of the argon laser and recorded in the 511–551-nm emission range.

For quantification of miPEP165a-FAM entry into wild-type and mutant *Arabidopsis* roots, the fluorescence intensity was determined per surface unit in the di fferent root zones using ImageJ software.

### *4.5. Inhibitor Treatment*

TyrA23 was dissolved in dimethyl sulfoxide to yield a 50 mM stock solution and MβCD was prepared in deionized water at a final concentration of 38 mM. For each experiment, 3-day-old seedlings germinated on 1 2 MS solid medium + 1% sucrose (wt/vol) were pre-treated with 50 μM TyrA23 or 10 mM MβCD for 30 min [17]. Seedlings were then treated with the inhibitors supplemented with 100 μM miPEP165a. Treatments were performed daily for an additional 3 days and plates were scanned for analysis of the primary root length with NeuronJ, an Image J plugin [29,44].

### *4.6. Flowering Phenotype*

*Arabidopsis* seeds were grown on Ji ffy® under a 16 h light/8 h dark cycle (22/20 ◦C), with a relative humidity of 80%. Fifteen days after seed sowing, either a 2-μL droplet of 100 μM miPEP165a was put on the shoot apical meristem or seedlings were watered with 500 μL of 10 μM miPEP165a three times a week. Analyses of the aerial parts were performed 24 days after sowing.

#### *4.7. Propidium Iodide Staining*

Wild-type seeds were grown for 3 days on 1 2 MS solid medium + 1% sucrose (wt/vol) in the same growth conditions as described above. Seedlings were then treated with water or 100 μM miPEP165a daily for 3 additional days and placed in the growth chamber at the same settings. Seedlings were then stained with 10 μg/mL propidium iodide for 20 min and *Arabidopsis* cell roots were analyzed with a laser scanning confocal microscope (Leica TCS SP8-AOBS) with a ×25 water immersion objective lens (numeral aperture 0.95; Fluotar Visir). The excitation and emission wavelengths of propidium iodide were 561 and 570–640 nm, respectively.

The meristematic zone for the cortex cells was defined as the region between quiescent center cells and the first elongating cell that was twice the length compared to its distal neighbor [20,26]. The meristematic cell length and cell number were determined with the software tool Cell-o-Tape, an open source ImageJ/Fiji macro [27–29]. At least 20 roots were analyzed for each treatment.

#### *4.8. Immunoblots and RT-qPCR*

Seven-week-old *Arabidopsis* seedlings were treated with 100 μM miPEP165a or its corresponding control for 24 h, and then the expression of pri-miR165a was evaluated by RT-qPCR according to Lauressergues et al. [5].

To evaluate miPEP165a stability, 5 nanomoles of miPEP165a were subjected to several freeze/thaw cycles and its degradation was detected by immunoblotting with an anti-miPEP165a antibody as previously described [5].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/21/7/2266/s1, Figure S1. Effect of miPEP165a and importance of its stability. Figure S2. Physiochemical properties of miPEP165a. Figure S3. Quantification of miPEP165a-FAM uptake in Arabidopsis roots. Figure S4. MβCD impairs the miPEP165a-FAM entry in the Arabidopsis root cap/meristematic zone.

**Author Contributions:** Conceptualization, M.O., S.P. and J.-P.C.; Methodology, M.O., A.L.R., S.P. and J.-P.C.; Validation, M.O., A.L.R. and J.-P.C; Formal Analysis, M.O., J.-P.C; Investigation, M.O., A.L.R., C.D. and J.-P.C; Resources, H.J.; Writing—Original Draft Preparation, M.O., S.P. and J.-P.C.; Writing—Review and Editing, M.O., H.J., P.T., S.P. and J.-P.C.; Project Administration, S.P. and J.-P.C.; Funding Acquisition, S.P. and J.-P.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the *Laboratoire d'Excellence* entitled TULIP (ANR-10-LABX-41), and National Institute of Health (R01 GM093008) to H.J.

**Acknowledgments:** We thank Martina Beck (Micropep Technologies, France), Jean-Malo Couzigou (LRSV, France) and Nathalie Leborgne-Castel (Université Bourgogne Franche-Comté, Dijon, France) for their helpful advices.

**Conflicts of Interest:** The authors declare no conflict of interest. The sponsors had no role in the design, execution, interpretation, or writing of the study.

*Int. J. Mol. Sci.* **2020**, *21*, 2266
