Nitrogen-Activated CLV3/ESR-Related 4 (CLE4) Regulates Shoot, Root, and Stolon Growth in Potato

In potato, high levels of nitrogen (N) can lead to excessive vegetative growth at the expense of tuber development, resulting in lower yield and poor-quality tubers. We found that Solanum tuberosum CLE4 (StCLE4) is expressed most strongly in the roots grown in N-rich media, and it positively regulates potato root growth under N-deficient conditions. We noted that StCLE4 functions as a negative regulator of normal shoot apex development similar to CLV3 in Arabidopsis. Transcriptomic analysis revealed that overexpression of StCLE4 resulted in the repression of the StIT1 gene, a regulator of potato tuber initiation. StCLE4-overexpressing stolons were converted into branches, that were similar to a mild phenotype of the it1 (identity of tuber 1) mutant. We also found that NIN-like proteins, key regulators of nitrate signaling bind to the regulatory sequence of StIT1 in a yeast one-hybrid assay. Taken together, our findings suggest that StCLE4 regulates shoot, root, and stolon growth in potato.


Introduction
The CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are a group of hormones that play a crucial role in plant development and growth.Among them, CLV3 regulates the size of the shoot apical meristem (SAM) by downregulating the expression of the WUSCHEL (WUS) gene encoding a homeodomain transcription factor.Other CLE genes, including CLE1, CLE4, CLE6, and CLE7, can complement the enlarged meristem of the clv3 mutants when they are expressed under the control of the CLV3 promoter [1,2].At the same time, the CLE1, CLE3, CLE4, and CLE7 genes are induced in roots under nitrogen-deficient conditions, and they regulate root growth and branching in response to nitrogen availability [3].CLE genes have been identified in various plant species, including Arabidopsis thaliana, rice, and maize.Previously we have identified CLE genes in potato, Solanum tuberosum L. (StCLE) and found that the expression level of several CLE genes is regulated by nitrogen supply [4].Nitrogen is a macronutrient that is essential for plant growth and development, and it is often a limiting factor in crop production.Adequate nitrogen supply is necessary for the formation of healthy and high-yielding potato tubers.During the early stages of tuber development, nitrogen regulates the formation of stolons, which are stem-like structures that develop from the lower nodes of the potato plant.As tuber development progresses, nitrogen is required for the accumulation of starch and other nutrients in the tubers.Nitrogen deficiency during this stage can result in reduced tuber size and lower yield.However, excessive nitrogen supply can also have negative effects on tuber development.Excessive nitrogen levels can cause an overabundance of vegetative growth, which can have a negative impact on tuber development, ultimately resulting in lower yields and lower quality tubers.Therefore, it is important to maintain a balanced nitrogen supply throughout the growing season to ensure optimal tuber development in potato plants.We previously found that the StCLE4 gene was induced in potato roots under N-rich growth conditions and suggested that StCLE4 could be a negative regulator of tuberization.In this study, we show that the StCLE4 promoter drives GUS reporter activity in the roots grown in N-rich media.Plants with the overexpression of StCLE4 demonstrated a wus-like phenotype, with the arrested growth of the shoot apical meristem (SAM).The overexpression of StCLE4 also resulted in the stimulation of root growth and non-swelling stolons that were converted into branches.The transcriptomic studies of StCLE4-overexpressing plants revealed the repression of the IDENTITY OF TUBER1 (IT1) gene, a regulator of potato tuber initiation.Overall, overexpression phenotype and RNA-Seq expression profiles suggest the roles of StCLE4 in the regulation of shoot, stolon, and root growth.

Promoter Activity of StCLE4 Gene in Potato Roots
The StCLE4 gene belongs to the same clade of CLEs as the nitrate-regulated CLE genes of A. thaliana (AtCLE1, -3, -4, and -7) [4] that are induced upon N deprivation and suppress the initiation of lateral roots [3].Previously, we evaluated the expression of the StCLE4 gene in the roots of potato exposed to different N-availability conditions using RT-qPCR.Opposite to A. thaliana CLE genes, the StCLE4 gene was positively regulated by nitrogen supply [4].In this study, to determine the expression pattern of StCLE4 in potato roots, we generated promCLE4:GUS fusion constructs using 2963 bp of the genomic region upstream of StCLE4.The StCLE4 promoter drove GUS activity in the vascular tissues of the root (Figure 1b), and the reporter gene was expressed most strongly on N-rich media (Figure 1a), confirming that this gene is activated under high nitrogen conditions.
important to maintain a balanced nitrogen supply throughout the growing season to ensure optimal tuber development in potato plants.
We previously found that the StCLE4 gene was induced in potato roots under N-rich growth conditions and suggested that StCLE4 could be a negative regulator of tuberization.In this study, we show that the StCLE4 promoter drives GUS reporter activity in the roots grown in N-rich media.Plants with the overexpression of StCLE4 demonstrated a wus-like phenotype, with the arrested growth of the shoot apical meristem (SAM).The overexpression of StCLE4 also resulted in the stimulation of root growth and non-swelling stolons that were converted into branches.The transcriptomic studies of StCLE4-overexpressing plants revealed the repression of the IDENTITY OF TUBER1 (IT1) gene, a regulator of potato tuber initiation.Overall, overexpression phenotype and RNA-Seq expression profiles suggest the roles of StCLE4 in the regulation of shoot, stolon, and root growth.

Promoter Activity of StCLE4 Gene in Potato Roots
The StCLE4 gene belongs to the same clade of CLEs as the nitrate-regulated CLE genes of A. thaliana (AtCLE1, -3, -4, and -7) [4] that are induced upon N deprivation and suppress the initiation of lateral roots [3].Previously, we evaluated the expression of the StCLE4 gene in the roots of potato exposed to different N-availability conditions using RT-qPCR.Opposite to A. thaliana CLE genes, the StCLE4 gene was positively regulated by nitrogen supply [4].In this study, to determine the expression pattern of StCLE4 in potato roots, we generated promCLE4:GUS fusion constructs using 2963 bp of the genomic region upstream of StCLE4.The StCLE4 promoter drove GUS activity in the vascular tissues of the root (Figure 1b), and the reporter gene was expressed most strongly on N-rich media (Figure 1a), confirming that this gene is activated under high nitrogen conditions.

Effects of Overexpressing StCLE4 and StCLE4G6T Genes on Root, Shoot, and Stolon Growth
To further investigate the functions of StCLE4, we used antagonistic peptide technology [5].Specifically, we created an antagonistic StCLE4 construct by mutating a codon encoding glycine in the CLE motif-coding region (which is known to play a crucial role in the biological activity of CLE) to threonine-coding codon (G6T) (Figure 2a).Vectors carrying the StCLE4 and StCLE4G6T genes under the constitutive 35S promoter were obtained.The overexpression of StCLE4 resulted in the stimulation of root growth in aeroponic nutrient medium without nitrogen (Figure 2b,c) and the reduction in tuber weight (Figure 2d).At the same time, we did not detect any alterations in the growth of StCLE4G6T-overexpressing potato plants, except lower tuber weight (Figures 2, S1 and S2).

Effects of Overexpressing StCLE4 and StCLE4 G6T Genes on Root, Shoot, and Stolon Growth
To further investigate the functions of StCLE4, we used antagonistic peptide technology [5].Specifically, we created an antagonistic StCLE4 construct by mutating a codon encoding glycine in the CLE motif-coding region (which is known to play a crucial role in the biological activity of CLE) to threonine-coding codon (G6T) (Figure 2a).Vectors carrying the StCLE4 and StCLE4 G6T genes under the constitutive 35S promoter were obtained.The overexpression of StCLE4 resulted in the stimulation of root growth in aeroponic nutrient medium without nitrogen (Figure 2b,c) and the reduction in tuber weight (Figure 2d).At the same time, we did not detect any alterations in the growth of StCLE4 G6T -overexpressing potato plants, except lower tuber weight (Figures 2, S1 and S2).

RNA-Sequencing of Transgenic Potato Leaves and Roots with StCLE4 and StCLE4 G6T Overexpression
To study the effect of gene overexpression on the potato transcriptome, we performed RNA sequencing analysis of the leaves and roots of transgenic potato plants overexpressing the StCLE4 and StCLE4 G6T genes.RNA was isolated from the plants grown in both nitrogen-rich and nitrogen-free media.The obtained RNA-seq data were analyzed using bioinformatics tools to identify differentially expressed genes (DEGs) between the transgenic and control plants.A total of 1096 DEGs were detected in the leaves, and 384 DEGs were detected in the roots of control and StCLE4-overexpressing plants grown in nitrogen-free media (Table S1, Figure S3).In total, 1942 DEGs were detected in the leaves and 391 DEGs in the roots of control and StCLE4 G6T -overexpressing plants grown on nitrogen-free media (Table S1).
Gene ontology analysis was performed to identify the biological processes affected by StCLE4 and StCLE4 G6T overexpression.The results showed that the overexpression of the StCLE4 and StCLE4 G6T genes altered the expression of genes involved in regulation of shoot development, response to gibberellin, leaf development, auxin homeostasis, and other processes (Table S1).Based on our RNA-Seq data, we supposed that the long-root phenotype can be caused by the altered expression of auxin response genes, including SMALL AUXIN-UP RNAs (SAURs), Glycoside hydrolase 3 (GH3), and Auxin Response Factor  S1).Among the DEGs in StCLE4 overexpressing leaves, the StIT1 gene was found.StIT1 encodes a TCP transcription factor that determines tuber identity and interacts with the tuberization signal SP6A.The stolons of it1 knockout mutants were converted into aerial shoot branches instead of swelling [8].Based on our RNA-Seq analysis and the observed effects of StCLE4 overexpression, it could be speculated that the StCLE4 peptide may initiate a signaling pathway that results in the inhibition of the StIT1 gene expression.

RNA-Sequencing of Transgenic Potato Leaves and Roots with StCLE4 and StCLE4G6T Overexpression
To study the effect of gene overexpression on the potato transcriptome, we performed RNA sequencing analysis of the leaves and roots of transgenic potato plants overexpressing the StCLE4 and StCLE4G6T genes.RNA was isolated from the plants grown in both nitrogen-rich and nitrogen-free media.The obtained RNA-seq data were analyzed using bioinformatics tools to identify differentially expressed genes (DEGs) between the transgenic and control plants.A total of 1096 DEGs were detected in the leaves, and 384 DEGs were detected in the roots of control and StCLE4-overexpressing plants grown in nitrogen-free media (Table S1, Figure S3).In total, 1942 DEGs were detected in the leaves and 391 DEGs in the roots of control and StCLE4G6T-overexpressing plants grown on nitrogen-free media (Table S1).
Gene ontology analysis was performed to identify the biological processes affected by StCLE4 and StCLE4G6T overexpression.The results showed that the overexpression of the StCLE4 and StCLE4G6T genes altered the expression of genes involved in regulation of shoot development, response to gibberellin, leaf development, auxin homeostasis, and other processes (Table S1).Based on our RNA-Seq data, we supposed that the long-root phenotype can be caused by the altered expression of auxin response genes, including SMALL AUXIN-UP RNAs (SAURs), Glycoside hydrolase 3 (GH3), and Auxin Response Factor (ARF) (Table S1).Among the DEGs in StCLE4 overexpressing leaves, the StIT1 gene was found.StIT1 encodes a TCP transcription factor that determines tuber identity and interacts with the tuberization signal SP6A.The stolons of it1 knockout mutants were We next performed yeast one-hybrid (Y1H) assay to identify the putative regulators of StCLE4.Previously, we found that the crucial regulator of the photoperiod-dependent tuberization StBEL5 did not bind to the DNA sequence corresponding to 500 bp upstream of the start codon of the StCLE4 gene [4].In this study, we performed an analysis of the direct binding of the key nitrate regulators NLPs to the promoter regions of StCLE4.NIN-like proteins (NLPs) are a family of transcription factors that play important roles in plant growth and development, as well as in plant-microbe interactions.The name "NIN-like" comes from the fact that these proteins are structurally similar to the Nodule Inception (NIN) protein, which is involved in the formation of root nodules in legume plants.One of the key functions of NLPs is the regulation of nitrate signaling and metabolism [9,10].In Arabidopsis, the AtNLP6 and AtNLP7 proteins are crucial for the regulation of gene expression in response to nitrate [9][10][11].Using NLP protein sequences from A. thaliana, Medicago truncatula, and Solanum lucopersicum as queries, three StNLP genes homologues to AtNLP6 and AtNLP7 were identified (StNLP3.1 (Soltu.DM.08G004830.1),StNLP3.2(Soltu.DM.08G004830.2) and StNLP5 (Soltu.DM.08G029670.1))(Figure S4).Among the two variants of the StNLP3 gene, StNLP3.1 was selected for further investigation due to its higher expression levels in com-parison to StNLP3.2 (Table S1) (means of transcripts per million (TPM) values are 1722 and 80, respectively).The StNLP5 gene is also highly expressed in all samples (mean of TPM = 2037).
NLPs are known to bind to nitrate-response elements (NREs) and activate the expression of nitrate-regulated genes in response to high nitrate levels [11].We searched for the NREs in the promoter of the StCLE4 gene, and in the promoters of major regulators of tuber development (StBEL5, StSP6A, and StIT1 genes) using the motifs described in the studies by Nishida et al. [12] and Laffont et al. [13] In the promoters of the StIT1, StCLE4, and StBEL5 genes, we found one, two, and three putative NLP-binding sites, respectively (Figure 4a, Table S2).Next, we checked whether these putative NREs motifs could be bound by StNLP3.1 and StNLP5 using yeast one-hybrid assay.We found that StNLP3.1 and StNLP5 bound to the promoter of the StIT1 gene, whereas no interaction was detected for the StCLE4 and StBEL5 promoters (Figure 4b).

Discussion
Nitrogen is a crucial nutrient for the growth and development of potato plants, including tuber development.Previously, we found that the StCLE4 gene was positively regulated by nitrogen supply and suggested that StCLE4 could be a negative regulator of tuberization.In this study, we found that StCLE4-overexpressing plants demonstrated a wus-like shoot phenotype, with the arrested growth of the SAM and subsequent recovery of shoot growth via the activation of axillary buds.This phenotype was similar to that described for overexpression of CLV3, CLE2, -3, -4, -5, -6, -7, -9, -10, -11, and -13 genes in A. thaliana [6].Like Arabidopsis plants with AtCLE1-7 overexpression, potato plants overexpressing the StCLE4 gene resumed growth after the initial SAM arrest.Furthermore, the StCLE4 is close to these AtCLE proteins according to phylogenetic analysis [4].Besides this, StCLE4 overexpression promoted root growth, and a similar effect was also observed for AtCLE2 and AtCLE4-7 overexpression [6].However, opposite to A. thaliana CLE1, -3, -4, and -7 genes, the StCLE4 gene was positively regulated by nitrogen supply, like the AtCLE2 and AtCLE6 genes [3].At the same time AtCLE2 and AtCLE6 promoters drive specific GUS staining in the cells at the junction between the primary root and the lateral roots [14], whereas StCLE4 reporter expression was detected in the vascular tissues of the root.Therefore, StCLE4 is closely related to Arabidopsis AtCLE1-7 peptides according to phylogenetic analysis and functional studies; however, the regulation of

Discussion
Nitrogen is a crucial nutrient for the growth and development of potato plants, including tuber development.Previously, we found that the StCLE4 gene was positively regulated by nitrogen supply and suggested that StCLE4 could be a negative regulator of tuberization.In this study, we found that StCLE4-overexpressing plants demonstrated a wus-like shoot phenotype, with the arrested growth of the SAM and subsequent recovery of shoot growth via the activation of axillary buds.This phenotype was similar to that described for overexpression of CLV3, CLE2, -3, -4, -5, -6, -7, -9, -10, -11, and -13 genes in A. thaliana [6].Like Arabidopsis plants with AtCLE1-7 overexpression, potato plants overexpressing the StCLE4 gene resumed growth after the initial SAM arrest.Furthermore, the StCLE4 is close to these AtCLE proteins according to phylogenetic analysis [4].Besides this, StCLE4 overexpression promoted root growth, and a similar effect was also observed for AtCLE2 and AtCLE4-7 overexpression [6].However, opposite to A. thaliana CLE1, -3, -4, and -7 genes, the StCLE4 gene was positively regulated by nitrogen supply, like the AtCLE2 and AtCLE6 genes [3].At the same time AtCLE2 and AtCLE6 promoters drive specific GUS staining in the cells at the junction between the primary root and the lateral roots [14], whereas StCLE4 reporter expression was detected in the vascular tissues of the root.Therefore, StCLE4 is closely related to Arabidopsis AtCLE1-7 peptides according to phylogenetic analysis and functional studies; however, the regulation of StCLE4 activity in potato is different from that described of the AtCLE1-7 genes.
The overexpression of StCLE4 not only affected shoot apex and root growth, but also promoted stolon conversion into branches.We performed RNA-Seq analysis of the leaves and roots of transgenic and control plants.At the same time, the downregulation of the StIT1 gene was found in the leaves of StCLE4-overexpressing plants grown in N-deficient media.IT1 is a transcription factor that directly interacts with a mobile tuberigen, SP6A, and regulates tuber identity [8].The stolons of it1 knockout mutants were converted into branches instead of swelling [8].Opposite to it1 mutants, StCLE4-overexpressing plants produced tubers, but their weight was lower than that of the control plants.Therefore, we suppose that StCLE4 promotes the growth of stolons as shoots, similar to milder it1 phenotype, and that the StCLE4-overexpressing phenotype could be explained by the downregulation of IT1, which in turn regulates stolon swelling.
Based on the StCLE4-overexpressing phenotype, we supposed that the overexpression of StCLE4 G6T should induce root shortening and increase tuber weight.However, we observed no changes in the growth of potato plants overexpressing StCLE4 G6T , except for a decrease in tuber weight, compared to the control plants.This result indicates that while a Gly at position six is crucial for StCLE4 activity, StCLE4 G6T does not seem to function as an antagonistic peptide.Although Song et al. [5], Xu et al. [15], and Ren et al. [16] have demonstrated the effectiveness of the antagonistic peptide approach in several cases, its universal applicability remains to be determined.This approach was initially used for CLV3, CLE8, CLE19, and CLE22, and recently for the CLE25 peptide, but at the same time, for CLE45, it was shown that the antagonistic peptide acts as a weak native peptide rather than its antagonistic version [17].Moreover, for some CLE peptides, it was demonstrated that the sixth amino acid in the CLE domain is not critical for their function, and its substitution does not produce an antagonistic peptide [17].Our finding suggests that StCLE4 G6T may acts as a weak version of StCLE4 but not as an antagonistic peptide.
The NLPs transcription factors can respond to nitrate signaling, regulate gene expression, and participate in N absorption and utilization.Previously, it was shown that NLP can regulate the expression of the CLE genes [13,18,19], and therefore, we assumed that StNLP3 and StNLP5 can bind to the promoter region of StCLE4.However, we did not detect this interaction in the yeast one-hybrid system.Moreover, previously, we also did not find any interaction of photoperiod-dependent tuberization factor StBEL5 and the promoter region of the StCLE4 gene [4], and therefore, we still do not know which transcription factor regulates StCLE4 gene expression.However, we found that StNLP3 and StNLP5 bound to the promoter of StIT1.Previously, it was shown that N plays an important role in regulating tuberization [20], and based on our data, we speculate that the StCLE4 peptide, as well as the StNLP3 and StNLP5 transcription factors, may act on the N-depended tuberization pathway and regulate the expression of StIT1, however, further investigations are necessary to study the mechanisms of such regulation in more detail.Further research on the mechanisms of CLE genes regulation and their interactions with nitrogen signaling pathways will be crucial for understanding how plants respond to changes in nitrogen availability and for developing strategies to improve crop productivity.

Plant Materials and Growth Conditions
Tubers of the Solanum tuberosum cultivar Desirée was obtained from the stock of N. I. Vavilov All-Russian Institute of Plant Genetic Resources (Saint-Petersburg, Russia).The plants were propagated by two-node stem cuttings and were cultivated in vitro on a Murashige and Skoog (MS) medium containing 0.8% (w/v) agar and 1% sucrose under long-day conditions (16 h light/8 h dark) at 22 • C. N-depleted or N-rich medium was prepared as described in [21].The N-rich medium contains 10 mM NH 4 Cl and 10 mM KNO 3 as sources of nitrogen and potassium, respectively.The other elements are present at half-strength concentrations.The N-depleted medium does not contain NH 4 Cl and KNO 3 but instead has 10 mM KCl added.
For the investigation of the effects of StCLE4 and StCLE4 G6T overexpression on root growth, aeroponic nutrient medium without nitrate was used [22].Seven-day-old stem cuttings were transferred into aeroponic system in a controlled greenhouse (16 h light/8 h dark and 22 • C) and were grown for four weeks.The root length was measured using ImageJ v. 1.52r [23] software, and Tukey's test was used to compare the means of three groups.Stem cuttings were also transferred to pots (9 × 9 × 9 cm) with vermiculite and grown in a growth chamber for three months (16 h light/8 h dark and 22 • C for the first two months; 8 h light/16 h dark and 22 • C for the induction of tuber development).Twice a month plants were supplied with 1  4 MS media without sucrose.Tubers, obtained from plants growing in the vermiculite, were planted in the field (Leningrad Region, Russia) in June 2023 and were grown for 2.5 months.The tuber weight of the plants grown in the field was measured, and Tukey's test was used to compare the means of three groups.Boxplots were generated using boxplot package in R [24].

Constructs
The primers used for PCR are listed in Table S3.Polymerase chain reaction (PCR) with Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA, USA) was used to amplify coding sequences or predicted promoter fragments of genes from the whole-plant DNA of Solanum tuberosum L. cv Desiree.PCR fragments were extracted from an agarose gel using a Cleanup Mini Kit (Evrogen, Moscow, Russia) and cloned into the entry vector pDONR207.Then, coding sequences were cloned into the vector pMDC32 [25] under the CaMV 35S promoter, via Gateway technology, according to the manufacturer's instructions (Invitrogen, Waltham, MA, USA).Promoter sequences for GUS or GFP reporter analysis were cloned into the vector pBGWFS7 [26] or pHm43GW (Invitrogen, USA).For the yeast one-hybrid assay, the promoter fragments of the StCLE4, StSP6A, and StIT1 genes (Table S2) were cloned into the pHISLEU2GW vector (kindly provided by Dr. Rogers from Cardiff University).The coding sequences of StNLP3.1 and StNLP5 were introduced into pDEST22 (Invitrogen, USA).The resulting plasmids were transformed into Top10 Escherichia coli Competent Cells.Plasmid DNA was isolated using a Plasmid Miniprep Kit (Evrogen, Russia).Each plasmid was checked by sequencing.The resulting binary vectors were transferred to the Agrobacterium rhizogenes strain Arqua or Agrobacterium tumefaciens strain AGL1.Agrobacterium cultures were grown overnight on a shaking incubator at 28-30 • C in Luria-Bertani supplemented with antibiotics.

Transformation of Potato
For hairy potato root transformation with the promStCLE4::GUS construct, stems were wounded and inoculated with the A. rhizogenes and then co-cultivated on Murashige and Skoog (MS) media with 10 g/L of sucrose for 2 days.Transgenic hairy roots were grown in MS medium supplemented with cefotaxime (500 mg/L) to prevent Agrobacterium overgrowth.
Transgenic S. tuberosum plants containing prom35S::StCLE4 and prom35S::CLE4 G6T constructs were generated by Agrobacterium tumefaciens-mediated transformation mainly as described previously [27][28][29].The wounded leaf explants were incubated with agrobacteria in liquid MS medium with 16 g/L glucose for 2 days.Then, the explants were moved to solidified MS medium (8 g/L agar) that induces callus growth (16 g/L glucose; 500 mg/L cefotaxime; 3 mg/L hygromycin; 5 mg/L NAA; 0.1 mg/L BAP) and were incubated for 7-8 d under LD at 27 • C. The explants were later transferred to a shoot-regeneration MS medium (16 g/L glucose; 500 mg/L cefotaxime; 3 mg/L hygromycin; 1 mg/L BAP; 0.1 mg/L GA) and were repeatedly transferred to fresh medium with the same composition Plants 2023, 12, 3468 8 of 10 every 10 days until shoots developed on the calli.The resulting 3 cm shoots were then transferred to MS medium (10 g/L sucrose; 500 mg/L cefotaxime) for rooting.

Histochemical Assays
β-Glucuronidase (GUS) staining was performed as described in [30].Incubation periods ranged from 2 to 16 h following vacuum infiltration.Roots were imaged using a ZEISS SteREO Discovery.V12 microscope.

RNA Isolation and RNA-Seq
For RNA-Seq analyses, total RNA was extracted from samples using a RNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA).The enrichment of mRNA was performed using a NEBNext ® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA).The libraries were prepared from three to four biological replicates for each of the transgenic plants and conditions for a total of 30 transcriptome libraries using a NEBNext ® Ultra™ II Directional kit (New England Biolabs, USA).For sequencing, a HiSeq PE Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2 were used.Manufacturer's instructions were used for all procedures.The libraries were sequenced in the Centre for Molecular and Cell Technologies of Saint Petersburg State University Research Park using the Illumina HiSeq 2500 sequencing platform.

Yeast One-Hybrid Assay
The 2.5 kb sequences upstream of the start codon were used for the promoter analysis.Putative NRE motifs were identified using sequences revised in several recent studies [12,13] and MAST algorithms available on the MEME suite server (version 5.1.0,https://meme-suite.org/meme/tools/meme(accessed on 1 September 2023)).The transformation of the Saccharomyces cerevisiae strain Y2HGold (Clontech) was performed as described in [38].A yeast one-hybrid assay was performed as described in [39].

Identification and Phylogenetic Analysis of the NLP proteins
To identify NLP proteins in potato, BLASTP analyses were performed using all the Arabidopsis thaliana NLPs as queries against proteins of the S. tuberosum group Phureja DM1-3 516 R44.The amino acid sequences of the NLP proteins from Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, Oryza sativa, Lotus japonicas, and Solanum tuberosum were aligned using the muscle algorithm in MEGA7 software.A phylogenetic tree with 1000 bootstrap replicates was constructed using the MEGA7 neighbor-joining method with default parameters [40].

Figure 1 .
Figure 1.promStCLE4:GUS expression in potato roots.(a) The promStCLE4-driven GUS reporter is more active in the roots grown in N-rich media (+N).(b) The StCLE4 promoter activity in the vascular tissues of the root.

Figure 1 .
Figure 1.promStCLE4:GUS expression in potato roots.(a) The promStCLE4-driven GUS reporter is more active in the roots grown in N-rich media (+N).(b) The StCLE4 promoter activity in the vascular tissues of the root.

11 Figure 3 .
Figure 3. Effects of StCLE4 overexpression (StCLE4-oe) on apex growth of three-week-old plants (a) and stolon growth of two-month-old plants (b) that were grown in a growth chamber under long days (16 h light/8 h dark and 22 °C).Non-swelling stolons that transformed into branches are marked by a red asterisk.Scale bar in panel (a) = 2 mm.

Figure 3 .
Figure 3. Effects of StCLE4 overexpression (StCLE4-oe) on apex growth of three-week-old plants (a) and stolon growth of two-month-old plants (b) that were grown in a growth chamber under long days (16 h light/8 h dark and 22 • C).Non-swelling stolons that transformed into branches are marked by a red asterisk.Scale bar in panel (a) = 2 mm.2.4.Yeast One-Hybrid Assay of Interaction between StNLPs and the Promoters of the StCLE4, StBEL5, StSP6A, and StIT1 Genes