**2. Results**

#### *2.1. MiPEP165a Promotes Cell Division in the Meristematic Zone to Increase Primary Root Length and Acts on Flowering Time in Arabidopsis*

It has been previously shown that *A. thaliana* miPEP165a, as well as miR165a, is expressed in endodermis cells [5,23]. Exogenous treatment of *A. thaliana* seedlings with synthetic miPEP165a is sufficient to increase the primary root length [5]. However, the precise mechanisms (spatial and temporal) involved in the peptide uptake remained unknown. To study the entry of miPEPs, especially miPEP165a, we first defined the best experimental conditions to obtain a significant effect of miPEP165a on plant development. We first observed that watering plants with 100 μM of peptide was much more efficient at increasing the primary root length than treatments performed with only 10 μM of peptide, the concentration used in the previous study [5] (Figure 1A). In addition, similar to the concentration of 10 μM previously used [5], applying miPEP165a at 100 μM also induced the activation of the pri-miRNA from which it originates (Figure S1A). In addition, during the initial stages of the study, when the effect of miPEP165a on primary root length was studied, whatever the control used, i.e., scrambled miPEP165a, irrelevant peptides, or their corresponding solvents (acetonitrile or water), no response was observed compared to miPEP165a treatments (Figure S1B). Similarly, water and scrambled miPEP165a had no effect on the expression of pri-miR165a compared to miPEP165a (Figure S1A). Finally, we observed that several freeze/thaw cycles of the peptide were detrimental to its activity on the length of primary roots (Figure S1C). For these reasons, we used aliquots of unfrozen peptides only once and kept water as a reference in all the following experiments.

The increase in root length upon treatment may be a consequence of higher cell elongation or increased cell proliferation. To address this point, we analyzed the effect of miPEP165a at the cellular level on the meristematic zone since root growth was often determined by meristematic activity [24]. We revealed that more cells were present in the meristematic zone when roots were treated with miPEP165a (Figure 1B–E). Therefore, these experiments sugges<sup>t</sup> that the increase in root length induced by the miPEP165a treatment is likely due to the stimulation of cellular proliferation rather than an increase in cell length.

**Figure 1.** MiPEP165a promotes root growth by enhancing cell division. (**A**) Effect of miPEP165a on primary root length. *Arabidopsis thaliana* seedlings were treated with various concentrations of miPEP165a. Seedlings were treated daily with the peptide for 4 days, with the exception of those that received only one treatment. Peptides were thawed once, except those that underwent five freeze/thaw cycles. Two-way analysis of variance (ANOVA) significance levels were based on Tukey's post-test (1-way ANOVA), (a–c, *p* < 0.05, *n* = 70). At least three biological replicates were performed (**B**–**E**). Three-day-old seedlings were treated daily with water or 100 μM miPEP165a for a further 3 days and stained with 10 μg/mL propidium iodide for 20 min. (**B**) Confocal images showing the meristematic zone for the cortex cells, defined as the region between quiescent center cells and the first elongating cell that was twice the length compared to its distal neighbor (distance between white arrows) [25,26]. Meristematic cell number (**C**) and cell length (**D**) were determined with the software tool Cell-o-Tape, an open source ImageJ/Fiji macro [27–29]. (**E**) Quantification of root apical meristem length. (**B**–**E**) Four biological replicates were performed with at least 20 seedlings. Errors bars represent SEM. Asterisks indicate a significant difference at *p* < 0.01 (\*) according to the t-test. Scale bar = 25 μm. Water was used as a control.

MiR165a and its target genes, *REVOLUTA* (*REV*), *PHABULOSA* (*PHB*), and *PHAVOLUTA* (*PHV*), are also known to be involved in flowering [30]. To investigate whether miPEP165a could have an effect on flowering, we treated the shoot apical meristem with a droplet of 100 μM miPEP165a three times a week during plant development. Treatments with miPEP165a accelerated plant development as illustrated by the decrease of the flowering day (Figure 2A,B) and the increase of the length of the

inflorescence stem (Figure 2C,D). Interestingly, watering the roots with 10 μM peptide had no effect on the flowering, suggesting that peptides cannot migrate throughout the plant (Figure 2).

**Figure 2.** Flowering phenotypes of *Arabidopsis* plants in response to miPEP165a treatment. *Arabidopsis* plants were treated with either water (control) or a droplet of 100 μM miPEP165a placed on the shoot apical meristem or by watering with 10 μM miPEP165a three times a week until analyses. Flowering time measurements were determined using the number of days to obtain an inflorescence stem of 1 cm (**A**) and the number of days to obtain the first flowers (**B**). (**C**) The length of the *Arabidopsis* inflorescence stem was determined 24 days after sowing. Error bars indicate SEM. Statistical analysis was performed using a *t*-test (*p* < 0.01). (**D**) Representative pictures showing the flowering phenotype according to the miPEP165a treatment. Experiments were performed at least 4 independent times (*n* > 78 plants). Bar = 1 cm.

#### *2.2. MiPEP165a Entry Involves both Passive Di*ff*usion at the Root Apex and Endocytic Pathways in the Di*ff*erentiation and Mature Zones*

To document this observation, we used the miPEP165a labelled with FAM, a fluorescent dye derived from fluorescein. As illustrated in Figure S2, the physiochemical properties of the miPEP165a-FAM are similar to those of the non-modified peptide. Although slightly less active, the labelled peptide was still able to increase the primary root length (Figure 3).

Interestingly, while the labelled peptide penetrated rapidly (~2 h) into the root cap and the meristematic zone, it took longer to penetrate the other parts of the root (Figure 4). Twenty-four hours after the application of the labelled peptide, the latter was present in most external parts of the roots. The central cylinder was never labelled by the peptide, which seemed to be blocked by the pericycle (Figure 4).

**Figure 3.** MiPEP165a-FAM is biologically active. Seedlings were treated with water (control), 100 μM miPEP165a, miPEP165a-FAM, or fluorescein. At least 70 seedlings were used to determine the normalized *Arabidopsis* root length. Data are given as ± SEM and statistical analysis was performed using a t-test (a–c, *p* < 0.01).

**Figure 4.** Kinetics of miPEP165a uptake into *Arabidopsis* roots. The mobility of miPEP165a-FAM was followed at the indicated time in different zones of *Arabidopsis* roots, as defined by [31]. Confocal images are representative of four independent experiments, with at least 6 seedlings for each condition. Bar = 50 μm (root cap/meristematic zone) or 25 μm (differentiation and mature zones). The different cell layers are indicated in the differentiation zone image at 48 h as follows: cc, central cylinder; p, pericycle; en, endodermis; co, cortex; ep, epidermis.

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

The entry of peptides into plants might occur passively, by di ffusion, or actively, via specific transporters or by endocytosis. Because of the huge diversity of miPEPs in a plant and the lack of conservation between species [5], we hypothesized that specific transporters for each peptide are unlikely to exist and, more likely, the miPEPs might be internalized by generic internalization machinery or, more simply, by passive di ffusion. To decipher the mechanisms involved in the entry of peptides into cells, we used *A. thaliana* mutants impaired on genes encoding proteins associated to the clathrin pathway (*chc1-1*, *chc2-1*, *ap2*σ*2*) [12,16,32] or to the membrane microdomain (*rem1-2*, *rem1-3*) [33–35]. Internalization of miPEP165a was not a ffected in most of the mutants tested, except in the root cap/meristematic zone of the *chc1-1* mutant and in the di fferentiation zone of the *chc1-1* and *rem1-2* mutants, suggesting that uptake in these parts was mainly passive (Figure 5, Figure S3). Conversely, the entry of the peptide into the mature zone of all mutants was strongly impaired (Figure 5, Figure S3). These data sugges<sup>t</sup> that peptide entry in plants involves, in addition to passive di ffusion, both clathrin and membrane microdomain-mediated pathways.

**Figure 5.** Internalization of miPEP165a is clathrin and remorin dependent. Representative confocal images showing the uptake of miPEP165-FAM 48 h after treatment in wild-type seedlings and *chc1-1*, *chc2-1*, *ap2*σ*2*, *rem1-2*, and *rem1-3* mutants. A significant fluorescence decrease for each condition is indicated in each panel by asterisks. Quantifications of the fluorescence intensity from more than 15 seedlings are shown in Figure S3. Bar = 50 μm (root cap/meristematic zone) or 25 μm (differentiation and mature zones).

In order to determine how and to what extent a defect in the peptide entry affects its biological effect on plant development, we treated the roots of *chc1-1*, *rem1-2*, and *rem1-3* mutants with the peptide in parallel with the wild-type roots. While the mutants showed a longer primary root in the control conditions compared to the wild type plants, they were unable to respond to the peptide by increasing their primary root length (Figure 6). Indeed, the *rem1-2* mutant, which was strongly affected in the peptide uptake, was unable to respond to miPEP165a.

**Figure 6.** MiPEP165a-mediated root growth induction involves clathrin and remorin proteins. Measurement of the primary root length in *chc1-1* (**A**) and remorin (*rem1-2* and *rem1-3*) mutants (**B**) after water (control) or miPEP165a (100 μM) treatment. The error bars indicate SEM of at least three biological replicates (n > 110 seedlings) and statistical analyses were performed using a t-test (a–c, *p* < 0.01).

We next treated the aerial parts of the mutants with miPEP165a, and we observed similar results on the flowering time (Figure 7). These results sugges<sup>t</sup> that the mechanisms of miPEP165a uptake into roots and aerial parts could be similar.

**Figure 7.** Flowering time depends on clathrin- and membrane microdomain-associated pathways. The number of days to obtain a 1-cm inflorescence stem (**A**) and the number of days to observe the first flowers (**B**) were determined for wild-type plants as well as for *chc1-1*, *rem1-2*, and *rem1-3* mutant plants. (**C**) Measurement of the inflorescence stem length was determined 24 days after sowing for wild-type and mutant plants. Data are representative of the average of at least four independent experiments with at least 10 plants per condition, for each experiment. Error bars represent SEM and statistical analyses were performed using a t-test (\*, *p* < 0.01). (**D**) Representative images comparing wild-type and mutant plants treated with water (control) or miPEP165a. Bar = 1 cm.

Finally, we used TyrA23, a chemical inhibitor known to affect clathrin-mediated endocytosis [22,32,36], and MβCD, a cholesterol-depleting agent, which have been suggested to block microdomain-dependent endocytosis [17,18,22]. Interestingly, both molecules were able to inhibit the miPEP165a-activated root length phenotype, suggesting that peptide entry in plant involves clathrin-mediated endocytosis and membrane microdomain-dependent pathways (Figure 8).

**Figure 8.** Disruption of endocytic pathways prevents miPEP165a-induced root growth. Normalized primary root growth analysis after treatment with miPEP165a and TyrA23 (**A**) or miPEP165a and MβCD (**B**). Three biological replicates were performed by using at least 100 seedlings for each condition and root lengths were statistically analyzed using a *t*-test (*p* < 0.01, \*). The data represent the mean value ± SEM. Water was used as a control for the miPEP165a treatment.

Altogether, our results showed that miPEP165a entry used passive diffusion at the root apex followed by endocytosis in the differentiation or mature zone of plant roots. All pathways are required to mediate full peptide uptake (and activity).
