**1. Introduction**

Gene expression is the consequence of the transcription of an RNA molecule from a gene—modulated by transcription factors and modifications of the chromatin structure—and post-transcriptional mechanisms acting on the RNA stability of translation or on the protein it encodes. One of the best-known mechanisms of post-transcriptional regulation of gene expression is gene silencing induced by microRNAs (miRNAs). MiRNAs are small, regulatory RNA molecules (21–24 nucleotides) first discovered in the worm *Caenorhabditis elegans* and later in plants and humans [1–4]. Each miRNA regulates the expression of specific target gene(s) either by cleaving the mRNA transcribed from it or by inhibiting its translation. Target genes of miRNAs are often key regulatory genes encoding, for example, transcription factors or hormone receptors. MiRNAs are therefore required for the correct regulation of most developmental processes in plants and animals, and dysregulation of miRNA expression is a feature of many human pathologies.

MiRNAs are themselves encoded by genes and are transcribed in the form of long primary transcripts (pri-miRNAs). One of the first steps in the maturation of pri-miRNAs involves a nuclear protein complex containing an enzyme called dicer-like 1 (DCL1), which cleaves pri-miRNAs to form precursor miRNAs (pre-miRNAs). A second cleavage step then forms mature miRNAs. In the cytoplasm, the mature miRNA anneals by homology with the mRNA of its target gene(s). This heteroduplex molecule is recognized by a protein complex called RISC, containing the enzyme Argonaute (AGO1), which either cleaves the targeted mRNA or inhibits its translation. Because the main role of miRNAs is to act as regulatory small RNAs and not in the direct translation of proteins, miRNAs have always been thought to be non-coding RNAs.

Surprisingly, the characterization of plant pri-miRNAs revealed that they encode small regulatory peptides, which were called miPEPs for miRNA-encoded peptides [5]. MiPEPs are involved in a positive autoregulatory feedback loop. They specifically activate transcription of their primary transcript and consequently enhance the synthesis of the mature miRNA, thus turning down the expression of specific genes. Interestingly, the application of exogenous synthetic miPEPs to plants is su fficient to stimulate the synthesis of their corresponding miRNAs and to modify plant development accordingly [6,7]. Given their e fficiency simply by an external application on plants, miPEPs are promising molecules for many agronomic applications. In particular, they o ffer a new way of modulating plant development, stimulating plant symbioses, or increasing plant fitness, to name a few potential uses. Moreover, as natural and endogenous peptides, they are likely to be much less harmful to the environment than chemical treatments and more acceptable to the general public than genetically modified organisms.

Endocytosis plays a crucial role in the internalization of extracellular molecules and plasma membrane proteins into eukaryotic cells [8]. Clathrin-mediated endocytosis (CME) remains the most extensively studied and characterized endocytosis and constitutes the major route of entry and pathway in eukaryotes [8,9]. Clathrin is a triskelion-shaped sca ffold protein composed of three clathrin light chains (CLCs) and three clathrin heavy chains (CHCs). The formation of clathrin-coated vesicle at the plasma membrane requires adaptor proteins, including AP2 complex [10]. In plants, CME is involved in multiple important biological processes, including growth, development, nutrient uptake, and biotic and abiotic stress responses [8,10–15]. For instance, clathrin is required for plasma membrane-located receptor endocytosis upon peptide perception, leading to peptide-mediated responses and thus to plant immunity [15,16]. Moreover, recent studies have also reported the existence of sterol-sensitive clathrin-independent pathways in plants, although this alternative endocytosis pathway is far less understood [17,18]. The best-studied clathrin-independent pathway in plants corresponds to flotillin-1-mediated endocytosis, a membrane microdomain-associated protein involved in plant development and promoted by flg22, a flagellin-derived 22-amino acid peptide [19,20]. Alternatively, proteins can assemble into clusters in membrane microdomains [8]. For instance, remorins form clusters at the plasma membrane and interact with a symbiotic receptor that allows bacterial infection in *Medicago truncatula* [21]. Finally, both clathrin-dependent and -independent pathways can be constitutive or di fferentially regulated in response to stimuli [17,18,22]. In summary, di fferent endocytosis pathways have been reported to be involved in many biological outcomes.

Due to their capacity to modulate plant development, miPEPs are of interest in agronomy as an alternative to chemicals to stimulate plant development. Nevertheless, to achieve this goal, a better understanding of their mode of action at the molecular level, including the mechanisms of their entry into plants, is required. In this study, we investigated how miPEPs enter into plants. We first reported in detail the phenotypes observed after treatment of *Arabidopsis thaliana* with miPEP165a, previously used to decipher the mode of action of miPEPs [5]. By using this miPEP labelled with a fluorescent dye, we followed the internalization of the peptide into plants. The peptide entered rapidly into the root cap and the meristematic zone and it took longer to penetrate the other parts of the root. Using mutants potentially altered in endocytic pathways or chemical inhibitors a ffecting endocytosis, we identified two mechanisms of miPEP165a entry into roots, passive di ffusion followed by an endocytosis process.
