CLAVATA3/EMBRYO SURROUNDING REGION ( CLE ) Gene Family in Potato ( Solanum tuberosum L.): Identiﬁcation and Expression Analysis

: CLE genes encode a group of small secretory peptides, which regulate cell proliferation and differentiation in plants. CLE genes have been studied in many plants; however, little is known about this gene family in potato. In this study, we characterized members of the CLE gene family in potato Solanum tuberosum ( StCLE ) and comprehensively analyzed their phylogenetic relationships, structure, and expression patterns. Using available transcriptomic data, we found a relative high expression level of StCLE8 , StCLE12 , and StCLE13 in stolons and tubers. Real-time PCR analysis showed that the StCLE23 gene was upregulated by water deﬁciency, whereas the expression of StCLE4 and StCLE10 was induced by nitrogen supply. Besides that, using data from transcriptomic studies obtained previously for plants with the induction the StBEL5 gene, a positive regulator of tuber development, we found that StCLE4 was among genes upregulated in response to StBEL5 induction, suggesting that StCLE4 could be a target of StBEL5 transcription factor. However, we did not reveal a direct binding of StBEL5 to the regulatory sequences of StCLE4 using yeast one-hybrid assay. Taken together, our data provide basic information for future functional studies of CLE peptides in potato growth and tuberization and in response to various environmental stimuli.


Introduction
CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are a group of post-translationally modified peptide hormones that act as mediators of cell-to-cell communication. CLE peptides are produced from larger polypeptide precursors, which contain an N-terminal signal peptide, a variable domain, and conserved CLE domain near the C-terminus [1,2]. Then, CLE domain undergoes proteolytic processing and some posttranslational modifications to yield the functional CLE peptide. In Arabidopsis, CLE peptides control cell divisions of stem cell in different types of plant meristems including the shoot apical meristem (AtCLV3) [3], the root apical meristem (AtCLE40 and AtCLE19) [4,5], and the vascular meristem (tracheary element differentiation inhibitory factor (TDIF) peptides, which are encoded by AtCLE41 and AtCLE44) [6]. CLE peptides also play roles in symbiosis, parasitism, and responses to abiotic cues (reviewed in [7]). Although the CLE gene family has been studied in many plants, there is still a lack of research on the phylogenetic relationship and expression of the CLE gene family in potato Solanum tuberosum L. (StCLE). Potato, a perennial herbaceous plant that forms edible tubers, is one of the world's most important food crops. In the present study, we annotated 41 StCLE genes and analyzed expression levels of the StCLE genes in different tissues and conditions. Tuber development is associated with active cell divisions that led to thickening of the stolon. TDIF peptides in other plants were shown to participate in regulation of secondary thickening in the root and shoot [8,9]. Using transcriptomic data, we found relative high expression level of potato TDIF-like genes in stolons and tubers, suggesting these CLE peptides could be involved in tuber growth regulation. Tuberization is regulated by environmental factors such as nitrogen supply, day length, water availability, and temperature. Here, we identified candidate CLE peptides that could mediate signaling from these environmental factors. We found that the StCLE10 and StCLE4 were induced in potato roots under N-rich growth conditions. Furthermore, we found that StCLE23 expression was induced in roots under dehydration stress condition. In addition to this, based on available transcriptomic data we found that StCLE4 could be regulated by StBEL5 transcription factor, which was previously characterized as a short-day activated inductor of tuberization. Therefore, in this study we identified CLE peptides that could be potentially involved in response to water stress, nitrate-induced reactions, and regulation of tuberization.

Plant Materials and Growth Conditions
The study was performed with the widely used Solanum tuberosum L. cv. Désirée. Plants were propagated by two-node stem cuttings and grown in vitro on Murashige-Skoog (MS) medium with 0.8% (w/v) agar and 1% sucrose under long-day conditions (16 h light: 8 h dark) at 22 • C. Induction of dehydration stress was performed as described in [10] using 14 days-old plantlets. MS medium for different N-availability conditions was modified as described in [11]. Two-node stem cuttings were kept on N-depleted or N-rich medium for 14 days (shoots were transferred to the fresh medium of the same composition every 7 d) until roots were formed on the shoots.

Identification and Phylogenetic Analysis of CLE Genes
To identify CLE genes in potato, BLASTN and TBLASTN analyses with all the Arabidopsis thaliana and known Solanum lycopersicum CLEs as queries were used against genomic sequences of S. tuberosum group Phureja DM1-3 516 R44 available at https://www.ncbi. nlm.nih.gov/genome/?term=txid4113[orgn] (accessed on 13 May 2021). Alignment of CLE amino acid (AA) sequences (Table S1) was generated by MEGA7 software using the muscle algorithm [12]. A phylogenetic tree with 1,000 bootstrap replicates was constructed using MEGA7 neighbor-joining method with default parameters [12].

Quantitative RT-PCR Analysis
Total RNA was isolated using a Trizol reagent (Invitrogen, Waltham, MA, USA). First-strand cDNA was synthesized from 500 µg of total RNA using oligodT primers and RevertAid reverse transcriptase (Thermo Scientific, Waltham, MA, USA). Quantitative RT-PCR was performed with EvaGreen intercalating dye (Syntol, Novosibirsk, Russia) using CFX96 (BioRad, Hercules, CA, USA). Relative expression was normalized against the ubiquitin gene (PGSC0003DMT400011939). Primers used are listed in Table S2. Each sample was tested in three technical repeats. At least three biological replicates were used in each experiment. The statistical significance was evaluated by Student's t-test.

Yeast One-Hybrid Assay
Transformation of Saccharomyces cerevisiae (strain Y2H Gold (Clontech)) was performed as described in [17]. The promoter fragments of StCLE4 and StSP6A genes (Table S3) were cloned into the pHISLEU2GW (kindly provided by Dr Rogers from Cardiff University) destination vector. Coding sequences of StBEL5 was introduced into pDEST22 (Invitrogen, Waltham, MA, USA). A yeast one-hybrid assay was performed as described in [18]. The primers used for cloning are listed in Table S2.

Identification and Phylogenetic Analysis of the CLE Family Genes in Solanum tuberosum
Previously, Goad et al. [19] analyzed genomic data from 57 plant species, and as a result, 21 CLE-like sequences were identified in potato genome database. However, no comprehensive analysis of these sequences was carried out, and other CLE genes may remain unidentified in potato. In this study, we identified previously unknown CLE genes in potato database and analyzed their sequences. To achieve this, the sequences of all previously reported CLE genes from Arabidopsis thaliana and Solanum lycopersicum (AtCLEs and SlCLEs, respectively) were used as queries to perform BLASTN and TBLASTN searches against potato genome at SpudDB (http://solanaceae.plantbiology.msu.edu (accessed on 13 May 2021)) and NCBI (S. tuberosum group Phureja DM1-3 516 R44). As a result, we identified 41 CLE genes in potato (Table S4), including 20 new genes that have not been identified by Goad et al. [19]. Eighteen genes (StCLE24- 41) have not been annotated previously as coding sequences possibly due to fact that StCLE genes are often missed by many standard annotation pipelines [19].
Analysis of chromosomal location showed that the StCLE genes were distributed throughout all 12 potato chromosomes except for chromosome 6, which does not contain CLE genes ( Figure 1). The CLE genes StCLE26 and StCLE27 are located very close to each other (approximately 4 kb apart) and have similar sequences (88% identity), suggesting that they possibly emerged from tandem duplications.
Most of the StCLE genes lacked introns, with the exception of five genes (StCLE14, StCLE15, StCLE19, StCLE29, and StCLE30). For the StCLE14 gene, two protein isoforms with different length (164 AA for isoform 1 and 144 AA for isoform 2) were found in NCBI database due to the presence of second intron in transcript variant X2. The StCLE proteins contain 66 (StCLE31) to 163 (StCLE14) AA. Like the CLE proteins in other plants [1], potato CLEs also have a signal peptide at the N-terminus, a central variable domain, and a 12 AA CLE domain at the C-terminus. Similar to SlCLEs and AtCLEs, all identified StCLE proteins contain only one CLE domain. The online MEME software was used to visualize the conserved residues within 12 AA StCLE domains ( Figure 2a). Moreover, we also searched for CLE homologs in the Solanum lycopersicum genome and found 16 previously unidentified CLE genes in addition to previously characterized 15 CLE genes (SlCLE1-15) in the tomato genome [20] (Table S5). To do this, BLASTN search against Solanum lycopersicum cv. "Heinz-1706" genome using potato and Arabidopsis CLE genes as queries were performed. The sequence consensus over the whole StCLE family was slightly different from one generated for AtCLEs and SlCLEs ( Figure 2a); however, six residues Agronomy 2021, 11, 984 4 of 11 (R 1 , P 4 , G 6 , P 7 , P 9 , and H 11 ) within CLE domain were found to be highly conserved in Arabidopsis, tomato, and potato CLEs (Figure 2a). Most of the StCLE genes lacked introns, with the exception of five genes (StCLE14, StCLE15, StCLE19, StCLE29, and StCLE30). For the StCLE14 gene, two protein isoforms with different length (164 AA for isoform 1 and 144 AA for isoform 2) were found in NCBI database due to the presence of second intron in transcript variant X2. The StCLE proteins contain 66 (StCLE31) to 163 (StCLE14) AA. Like the CLE proteins in other plants [1], potato CLEs also have a signal peptide at the N-terminus, a central variable domain, and a 12 AA CLE domain at the C-terminus. Similar to SlCLEs and AtCLEs, all identified StCLE proteins contain only one CLE domain. The online MEME software was used to visualize the conserved residues within 12 AA StCLE domains ( Figure 2a). Moreover, we also searched for CLE homologs in the Solanum lycopersicum genome and found 16 previously unidentified CLE genes in addition to previously characterized 15 CLE genes (SlCLE1-15) in the tomato genome [20] (Table S5). To do this, BLASTN search against Solanum lycopersicum cv. "Heinz-1706" genome using potato and Arabidopsis CLE genes as queries were performed. The sequence consensus over the whole StCLE family was slightly different from one generated for AtCLEs and SlCLEs ( Figure 2a); however, six residues (R1, P4, G6, P7, P9, and H11) within CLE domain were found to be highly conserved in Arabidopsis, tomato, and potato CLEs (Figure 2a). To study the phylogenetic relationship of the StCLE family members to Arabidopsis and tomato CLEs, an unrooted neighbor-joining phylogenetic tree, based on multiple alignments of the AA sequences of the CLEs, was created (Figure 2b, Figure S1). As expected, a perfect match was observed for potato CLEs with tomato CLEs (for example, StCLE7 and SlCLE7). Phylogenetic analysis also showed that there were some closely related homologous CLEs between potato and Arabidopsis. For example, StCLE7 and StCLE21 demonstrated high similarity with AtCLE45 that is involved in regulation of To study the phylogenetic relationship of the StCLE family members to Arabidopsis and tomato CLEs, an unrooted neighbor-joining phylogenetic tree, based on multiple alignments of the AA sequences of the CLEs, was created (Figure 2b, Figure S1). As expected, a perfect match was observed for potato CLEs with tomato CLEs (for example, StCLE7 and SlCLE7). Phylogenetic analysis also showed that there were some closely related homologous CLEs between potato and Arabidopsis. For example, StCLE7 and StCLE21 demonstrated high similarity with AtCLE45 that is involved in regulation of pollen tube growth and protophloem differentiation [21,22]. StCLE12 and StCLE8 had identical CLE domain to AtCLE41 and AtCLE44, which play crucial roles in vascular meristem maintenance [23]. AtCLE46 and StCLE30 grouped together, but little is known about functions of AtCLE46. The StCLE4, StCLE5, StCLE10, StCLE11, StCLE24, StCLE25, StCLE34, and StCLE36 group is close to nitrate-regulated CLE peptides of A. thaliana (AtCLE1,-3,-4,-7) [24,25]. For other proteins, no clear homology with AtCLEs was found.

Expression Profiles of StCLE Genes in Different Tissues According to Transcriptomic Data
To provide some clues on the roles of the StCLE genes in potato growth, the expression profiles of the StCLE genes in shoot apex, flower, leaf, petiole, stem, stolon (modified shoot, which gives rise to potato tuber), tuber (young and mature), and root were analyzed using transcriptomic data from NCBI database ( Figure 3). Reads were filtered and counted with kallisto [16].

Expression of StCLEs in Response to Dehydration Stress
In Arabidopsis and other plants, several CLE peptides were found to be involved in responses to abiotic cues. Specifically, the AtCLE25 gene expression is induced in the root in response to dehydration stress, and AtCLE25 systemically activates signaling pathway that leads to stomatal closure thereby enhancing the resistance to dehydration stress [10]. Interestingly, AtCLE25 is also involved in protophloem cell differentiation [26]. In potato, StCLE6 and StCLE19 are grouped together with AtCLE25 according to phylogenetic analysis (Figure 2b). According to expression data collected previously by [27], StCLE19 was expressed in phloem, as well as StCLE12, which showed a closer relationship to Arabidopsis CLE41/44 peptides involved in regulation of cambium cell proliferation and vascular development [21,28]. In our experiment after dehydration for four hours, we found no activation of StCLE6 and StCLE19 in roots in response to dehydration stress. However, the expression of StCLE23 was increased in response to four hours of dehydration ( Figure 4). StCLE23 does not demonstrate any clear homology with known Arabidopsis CLE peptides, and its possible role in dehydration stress remains to be investigated in more details.

Expression of StCLEs under Low and High Nitrogen Conditions
In Arabidopsis, the expression levels of AtCLE1, AtCLE3, AtCLE4, and AtCLE7 genes were found to be induced in roots under nitrogen (N) deficient conditions [24,25]. To identify genes specifically induced by N deficiency in potato, we evaluated the expression of StCLE genes (which were grouped with nitrate-regulated CLE peptides of A. thaliana according to phylogenetic analysis) in the roots of plants exposed to different N-availability conditions. To do this, we cultivated plants on N-rich medium for two weeks, after which control plants were kept on the same media (N-rich, 10 mM nitrogen), whereas others plants were transferred to N-depleted medium without a nitrogen source (0 mM nitrogen). After 48 h, we estimated the expression levels of StCLE genes in the roots of control and N-depleted plants. No upregulation of StCLE genes was detected in the roots of plants cultivated on N-depleted medium. However, we observed downregulation of StCLE4 and StCLE10 expression levels under N-depleted conditions ( Figure 5).

Expression of StCLEs under Low and High Nitrogen Conditions
In Arabidopsis, the expression levels of AtCLE1, AtCLE3, AtCLE4, and AtCLE7 genes were found to be induced in roots under nitrogen (N) deficient conditions [24,25]. To identify genes specifically induced by N deficiency in potato, we evaluated the expression of StCLE genes (which were grouped with nitrate-regulated CLE peptides of A. thaliana according to phylogenetic analysis) in the roots of plants exposed to different N-availability conditions. To do this, we cultivated plants on N-rich medium for two weeks, after which control plants were kept on the same media (N-rich, 10 mM nitrogen), whereas others plants were transferred to N-depleted medium without a nitrogen source (0 mM nitrogen). After 48 h, we estimated the expression levels of StCLE genes in the roots of control and N-depleted plants. No upregulation of StCLE genes was detected in the roots of plants cultivated on N-depleted medium. However, we observed downregulation of StCLE4 and StCLE10 expression levels under N-depleted conditions ( Figure 5).
N-availability conditions. To do this, we cultivated plants on N-rich medium for two weeks, after which control plants were kept on the same media (N-rich, 10 mM nitrogen), whereas others plants were transferred to N-depleted medium without a nitrogen source (0 mM nitrogen). After 48 h, we estimated the expression levels of StCLE genes in the roots of control and N-depleted plants. No upregulation of StCLE genes was detected in the roots of plants cultivated on N-depleted medium. However, we observed downregulation of StCLE4 and StCLE10 expression levels under N-depleted conditions ( Figure 5). To check the hypothesis that StCLE4 and StCLE10 gene expression is activated by high nitrogen content, we first cultivated plants on N-depleted media and then exposed them to the N-rich or N-depleted media for 48 h. We detected upregulation of StCLE4, StCLE5, and StCLE10 expression levels ( Figure 6) on N-rich media, suggesting that these genes are activated under high nitrogen conditions. In legume plants, the CLE genes have been described that are activated in response to high concentration of nitrate (10 mM) and mediate nitrate-dependent inhibition of symbiotic nodulation [29][30][31][32][33]. These nodulation-specific CLE genes belong to the same clade as nitrate-regulated CLE peptides of A. thaliana (AtCLE1, -3, -4, and -7) [24,25]. Therefore, we suggest that StCLE4, StCLE5, and StCLE10 genes, which also belong to the same clade of nitrate-regulated genes, upregulated by high N condition could mediate nitrate-dependent processes in potato. To check the hypothesis that StCLE4 and StCLE10 gene expression is activated by high nitrogen content, we first cultivated plants on N-depleted media and then exposed them to the N-rich or N-depleted media for 48 h. We detected upregulation of StCLE4, StCLE5, and StCLE10 expression levels ( Figure 6) on N-rich media, suggesting that these genes are activated under high nitrogen conditions. In legume plants, the CLE genes have been described that are activated in response to high concentration of nitrate (10 mM) and mediate nitrate-dependent inhibition of symbiotic nodulation [29][30][31][32][33]. These nodulationspecific CLE genes belong to the same clade as nitrate-regulated CLE peptides of A. thaliana (AtCLE1, -3, -4, and -7) [24,25]. Therefore, we suggest that StCLE4, StCLE5, and StCLE10 genes, which also belong to the same clade of nitrate-regulated genes, upregulated by high N condition could mediate nitrate-dependent processes in potato. y 2021, 11, x FOR PEER REVIEW 8 of 12 Figure 6. StCLE4, StCLE5, and StCLE10 are expressed in roots exposed to the N-rich medium (10 mM NO3 − and 10 mM NH4 + ). NS., Not Significant; *, p-value < 0.05.

StBEL5 Transcription Factor as a Possible Regulator of the StCLE4 Gene
StBEL5 acts as a signal mRNA in potato, which moves from leaves to stolons and mediates the activation of tuberization [34]. The SP6A protein, also known as tuberigen, is another mobile regulator of tuber initiation that moves from shoots to stolons and induces tuberization. Expression of both StBEL5 and SP6A genes is activated by short-day conditions, which are inductive for tuberization in potato (reviewed in [35]). Sharma et al. [36] performed transcriptomic studies of plants with elevated StBEL5 expression levels obtained in ethanol-inducible system and identified approximately 10,000 potential target genes, including genes involved in the biosynthesis or bioactivity of phytohormones that regulate tuber development [36]. Interestingly, according to the list of differentially expressed genes from this study, transcription factor StBEL5 upregulates the expression of StCLE4 ( Figure S2). StBEL5 is known to interact with the tandem TGAC motif in the promoters of target genes [36]. We found that there are three TGAC motifs in a genomic DNA sequence corresponding to 500 bp upstream of the start codon of the StCLE4 gene (Table S3). To check if these motifs could be bound by StBEL5 transcription factor, we performed yeast-one-hybrid screening. According to our data, StBEL5 did not bind to regulatory sequences of the StCLE4 gene (Figure 7). At the same time, in our yeast-one-hybrid screening StBEL5 demonstrated binding with regulatory sequences Figure 6. StCLE4, StCLE5, and StCLE10 are expressed in roots exposed to the N-rich medium (10 mM NO 3 − and 10 mM NH 4 + ). NS., Not Significant; *, p-value < 0.05, **, p-value < 0.01.

StBEL5 Transcription Factor as a Possible Regulator of the StCLE4 Gene
StBEL5 acts as a signal mRNA in potato, which moves from leaves to stolons and mediates the activation of tuberization [34]. The SP6A protein, also known as tuberigen, is another mobile regulator of tuber initiation that moves from shoots to stolons and induces tuberization. Expression of both StBEL5 and SP6A genes is activated by short-day conditions, which are inductive for tuberization in potato (reviewed in [35]). Sharma et al. [36] performed transcriptomic studies of plants with elevated StBEL5 expression levels obtained in ethanol-inducible system and identified approximately 10,000 potential target genes, including genes involved in the biosynthesis or bioactivity of phytohormones that regulate tuber development [36]. Interestingly, according to the list of differentially expressed genes from this study, transcription factor StBEL5 upregulates the expression of StCLE4 ( Figure S2). StBEL5 is known to interact with the tandem TGAC motif in the promoters of target genes [36]. We found that there are three TGAC motifs in a genomic DNA sequence corresponding to 500 bp upstream of the start codon of the StCLE4 gene (Table S3). To check if these motifs could be bound by StBEL5 transcription factor, we performed yeast-one-hybrid screening. According to our data, StBEL5 did not bind to regulatory sequences of the StCLE4 gene (Figure 7). At the same time, in our yeastone-hybrid screening StBEL5 demonstrated binding with regulatory sequences from the promoter of the SP6A gene (Figure 7), which was previously shown to be the direct target of StBEL5 according to the results of electrophoretic mobility shift assay [36].
conditions, which are inductive for tuberization in potato (reviewed in [35]). Sharma et al. [36] performed transcriptomic studies of plants with elevated StBEL5 expression levels obtained in ethanol-inducible system and identified approximately 10,000 potential target genes, including genes involved in the biosynthesis or bioactivity of phytohormones that regulate tuber development [36]. Interestingly, according to the list of differentially expressed genes from this study, transcription factor StBEL5 upregulates the expression of StCLE4 ( Figure S2). StBEL5 is known to interact with the tandem TGAC motif in the promoters of target genes [36]. We found that there are three TGAC motifs in a genomic DNA sequence corresponding to 500 bp upstream of the start codon of the StCLE4 gene (Table S3). To check if these motifs could be bound by StBEL5 transcription factor, we performed yeast-one-hybrid screening. According to our data, StBEL5 did not bind to regulatory sequences of the StCLE4 gene (Figure 7). At the same time, in our yeast-one-hybrid screening StBEL5 demonstrated binding with regulatory sequences from the promoter of the SP6A gene (Figure 7), which was previously shown to be the direct target of StBEL5 according to the results of electrophoretic mobility shift assay [36].

Discussions and Conclusions
In this study, we annotated 41 CLE genes in S. tuberosum and analyzed their structures, phylogenetic relationships, and expression profiles in different tissues of potato and in responses to abiotic cues.
Expression profiles for StCLEs in different organs suggest that corresponding CLE peptides could play various roles in potato growth. Specifically, StCLE8 and StCLE12 are expressed at high levels in stolon and tubers. According to phylogenetic analysis, StCLE8 and StCLE12 peptides showed a closer relationship to Arabidopsis AtCLE41 and AtCLE44 peptides, which promote cambium cell proliferation and inhibit xylem cell differentiation (TDIF) [21,28] (Figure 2b). It is known that TDIF homologs stimulate radial growth of shoots and roots in various plants [8,9]. However, during tuberization, active cell divisions resulting in thickening of stolons occur in the perimedullary region of the stolon, but not in the cambium [37,38]. The molecular mechanisms that control the changes in cell growth and proliferation during tuberization have not yet been studied in details, and the increased expression of TDIF-related StCLE8 and StCLE12 genes in potato stolons and tubers may suggest that these genes could be possible regulators of tuberization in the potato.
CLE peptides are known as regulators of plant responses to different environmental factors, such as nutrient availability and dehydration stress (reviewed in [39]). The potato is very sensitive to water stress: drought could decrease the photosynthetic rate, leaf area, tuber number, and weight [40]. We found that expression of the StCLE23 gene was activated under water stress. In Arabidopsis, the AtCLE25 peptide acts as a water-deficiency signal and mediates stomatal control [10]. However, StCLE23 does not show high similarity to AtCLE25. StCLE23 clustered together with AtCLE10 that was reported to regulate protoxylem vessel formation in roots [41]. However, in comparison with AtCLE10, the StCLE23 peptide has two substitutions in the CLE domain sequence (R 2 -to-L 2 and T 5 -to-S 5 ), that might confer novel functions to this peptide in the potato. Single AA substitutions in the CLE domain are known to essentially affect function of the corresponding CLE Agronomy 2021, 11, 984 9 of 11 peptide. For example, Arabidopsis AtCLE25 is involved in dehydration response, but its close homologue, AtCLE26, which differs from CLE25 only by one AA substitution, is not involved in the dehydration stress response [10]. Future studies should elucidate whether the StCLE23 peptide is involved in the dehydration response pathway similar to AtCLE25-activated one, or it acts through other signaling pathways.
In addition to dehydration stress, CLE peptides were also shown to be involved in the N-nutritional response. In Arabidopsis, AtCLE1, -3, -4, and -7 peptides locally regulate lateral root development under N-limited growth condition [25]. In legume plants, CLE peptides were found that mediate systemic response to nitrate and transmit signal about N availability from the root to the shoot [42]. In our study we found that expression of StCLE4, StCLE5, and StCLE10 was activated in the roots, which were exposed to the N-rich medium. These genes are closely related to nitrate-regulated CLE peptides of A. thaliana, suggesting that they could be involved in nitrogen-depended regulation of potato development and a possible negative regulation of tuberization, the latter is known to be inhibited by a high nitrogen amount. The low amount of nitrogen is one of the signals indicating the end of the growing season, and it induces tuberization in the potato. We can speculate that downregulation of StCLE4, StCLE5, and StCLE10 under N-depleted conditions could have stimulating effect on tuberization; however, further experiments are required to check this hypothesis.
Other environmental factors that control tuberization include day length and temperature. Tuberization is inhibited by long days and high temperatures and is stimulated by short days and low positive temperatures [43,44]. Therefore, in a changing environmental condition, different kinds of mobile signals can mediate organ-to-organ communication regulating tuber formation. Two mobile signals, which are produced in the leaves under short-days condition and transported to the stolons to induce tuber formation, are known. The first one represents the StSP6A protein, a member of flowering locus T (FT)like protein family, involved in photoperiodic regulation of flowering and some other developmental processes (reviewed in [39]). It is known that heat-mediated inhibition of tuber growth correlated with downregulation of StSP6A expression [45]. Another mobile signal transported from leaves to stolons and stimulating tuberization represents mRNA of homeodomain transcription factor StBEL5 [36]. Using transcriptomic data, we found that the StCLE4 expression level was increased upon StBEL5 activation, suggesting that StCLE4 could be a potential target of StBEL5 transcription factors. Importantly, StCLE4 is also upregulated at high-nitrogen conditions according to our data; therefore, this gene can combine regulatory signals derived from different environmental regulators, including photoperiod-dependent StBEL5-mediated pathway and nitrogen-induced one. To check the possibility that the StCLE4 gene could be directly regulated by StBEL5 transcription factor, we performed yeast-one-hybrid assay. However, the direct interaction of StBEL5 with the regulatory sequences of the StCLE4 gene was not found in our assay. Therefore, either StBEL5 transcription factor activates StCLE4 by binding other regulatory sequences within its promoter that were not included in the 500 bp-upstream region used in this assay, or StBEL5 indirectly regulates StCLE4 expression. Functional analysis of the StCLE4 gene and its possible regulation by different environmental cues is of great interest and should be addressed in future studies.
Taken together, the present study provides comprehensive analysis of the CLE genes in the potato that allowed selecting candidate genes for further functional analysis in potato tuber development and response to environmental cues.  Table S1: AA sequences of CLE proteins used for phylogenetic tree construction, Table S2: List of primers, Table S3: Promoter regions of StSP6A and StCLE4 used for Yeast One-Hybrid Assay, Table S4: Potato CLE genes, Table S5: Tomato CLE genes. Funding: This work was funded by Ministry of Science and Higher Education of the Russian Federation in accordance with agreement № 075-15-2020-922 date 16.11.2020 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center "Agrotechnologies for the Future".