Systematic Annotation Reveals CEP Function in Tomato Root Development and Abiotic Stress Response

Tomato (Solanum lycopersicum) is one of the most important vegetable crops worldwide; however, environmental stressors severely restrict tomato growth and yield. Therefore, it is of great interest to discover novel regulators to improve tomato growth and environmental stress adaptions. Here, we applied a comprehensive bioinformatics approach to identify putative tomato C-TERMINALLY ENCODED PEPTIDE (CEP) genes and to explore their potential physiological function in tomato root development and abiotic stress responses. A total of 17 tomato CEP genes were identified and grouped into two subgroups based on the similarity of CEP motifs. The public RNA-Seq data revealed that tomato CEP genes displayed a diverse expression pattern in tomato tissues. Additionally, CEP genes expression was differentially regulated by nitrate or ammonium status in roots and shoots, respectively. The differences in expression levels of CEP genes induced by nitrogen indicate a potential involvement of CEPs in tomato nitrogen acquisition. The synthetic CEP peptides promoted tomato primary root growth, which requires nitric oxide (NO) and calcium signaling. Furthermore, we also revealed that CEP peptides improved tomato root resistance to salinity. Overall, our work will contribute to provide novel genetic breeding strategies for tomato cultivation under adverse environments.


Introduction
Cell-to-cell communication is pivotal for plants to coordinate their growth in response to various developmental and environmental cues, and this cellular communication depends on small regulatory peptides [1,2]. Small secretory peptides are classified into two major groups based on their structure differences, the post-translationally modified peptide (PMT) or cysteine-rich polypeptides [3]. The C-TERMINALLY ENCODED PEPTIDE (CEP) peptide belongs to the PMT family, which often undergoes multiple proteolytic modifications. During this process, CEP peptides are cleaved into their bioactive form with approximately 15 amino acids in length [4][5][6]. CEP members have been identified across the plant kingdom; however, the biological function of most CEP genes remains largely untapped due to lack of loss-of-function mutants or genetic redundance [5][6][7][8][9][10][11][12][13][14][15].
In total, 15 CEP genes have been identified in model plant Arabidopsis thaliana. The Arabidopsis CEP genes are differentially expressed in various tissues, and they also respond to environmental stress, suggesting their diverse roles in the regulation of various aspects of Arabidopsis development and adaptions [5,6]. The application of synthesized Arabidopsis CEP1 (AtCEP1) peptide arrests root growth [4]. The synthesized AtCEP3 peptide inhibits primary root growth and lateral root emergence [5]. AtCEP5 is perceived by its putative receptor XYLEM INTERMIXED WITH PHLOEM 1 (XIP1)/CEP RECEPTOR 1 (CEPR1) to inhibit primary root growth and lateral root development [6,16]. These reports indicate crucial roles of CEPs in plant development.
Nitrogen is an essential macronutrient for plant growth and yield. CEP peptides have been reported to mediate nitrogen acquisition [17][18][19][20][21][22]. Under nitrogen starvation conditions, CEPR1 and CEPR2 receptors perceive and transmit the CEP peptides from roots to shoots, thus activating nitrate transporter gene expression, which resulted in nitrogen acquisition [17]. The shoot-derived polypeptides, CEP DOWNSTREAM 1 (CEPD1), CEPD2 and CEPD-LIKE2 (CEPDL2), act as descending long-distance mobile signals downstream of the CEP-CEPR pathway to activate the expression of nitrate transporter genes, thus mediating systemic nitrogen acquisition [18,19]. In Medicago truncatula, MtCEP together with its receptor COMPACT ROOT ARCHITECTURE 2 (MtCRA2) regulates nodulation numbers via modulating the expression of NODULE INCEPTION (NIN) transcription factor and miR2111 [20][21][22], and also via ethylene and auxin hormone signaling [23]. Compared to nitrogen, CEP also regulates sucrose-dependent lateral root development [24]. The synthesized AtCEP3 peptide inhibits primary root growth by decreasing cell division under starvation conditions in a CEPR1-dependent manner [25]. Additionally, AtCEP5 plays roles in Arabidopsis osmotic and drought stress responses via interfering with auxin signaling [26]. These data suggest that CEPs play an essential role in plant environmental adaptions.
Tomato (Solanum lycopersicum) is one of major crop species with high economic value that is cultured world-wide, and there is a growing demand in the fresh fruit market and processed food industries. Notably, environmental stressors severely limit tomato growth and yield. Hence, the generation of tomato cultivars with enhanced growth and stress tolerance is one of the most sustainable approaches for its successful production [27]. The major goals of tomato breeding require identifying novel regulators of tomato development and stress adaptions from available genomic resources [28]. Here, we performed a systematic search of putative CEP genes in the tomato genome and then studied their potential physiological roles in tomato development and abiotic stress responses. We identified 17 tomato CEP genes in total, and they exhibited a diverse expression pattern in tomato tissues. In addition, we revealed that tomato CEP genes differentially responded to nitrogen status in roots and shoots, respectively. Exogenous application of the chemical synthesized tomato CEP peptides promoted tomato root growth, which requires nitric oxide (NO) and Ca 2 + signaling. Furthermore, the exogenous application of synthetic CEP peptides improved tomato root resistance to salinity stress. Taken together, our systematic study of the tomato CEP gene family demonstrates the potential physiological function of CEP peptides in tomato development and abiotic stress responses, and it will provide novel tactics for genetic breeding to improve tomato fitness and to increase the yield under adverse environmental conditions.

Genome-Wide Annotation of Putative Tomato CEP Gene Family
To determine the tomato CEP gene (named as SlCEP) family members, the previously reported 15 Arabidopsis CEP (AtCEP) and six tomato CEP full-length protein sequences were used as query sequences to perform protein BLAST searches against the re-assembly tomato genome (ITAG 4.0) [5][6][7]. As a result, a total of 17 SlCEP genes were identified ( Figure 1A; Table S1). SlCEP proteins shared similar but divergent CEP motifs compared to the AtCEP proteins ( Figure 1B and Figure S1) [5,6]. The SlCEP genes were re-arranged according to their location on the chromosomes (Table S1). The corresponding coding sequence (CDS) of SlCEPs ranged from 204 base pairs (SlCEP13) to 936 base pairs (SlCEP17) with the protein size ranging from 68 (SlCEP13) to 312 (SlCEP17) amino acids in length. The molecular weight and isoelectric point of SlCEP proteins ranged from 7365.5 Da (SlCEP13) to 32878.64 Da (SlCEP17) and from 6.58 (SlCEP9) to 9.92 (SlCEP16), respectively (Table S1).

Motif Analysis, Gene Structure and Chromosome Localization of Slcep Proteins
The AtCEP proteins contain one or more conserved CEP motifs at the C-terminus [5,6]. Therefore, we analyzed the presence and distribution of CEP motifs in the 17 SlCEP proteins. Our analysis showed that SlCEP8 and SlCEP12 contained two CEP domains; SlCEP5 and SlCEP15 displayed three CEP domains; SlCEP16 showed four CEP motifs ( Figure 1A; Table S1). Notably, some SlCEP proteins shared identical CEP motifs ( Figure 1A). The Nterminal signal peptide proteolytic processing is essential for generating mature and functional CEP peptides [29,30]; we next searched for the presence and location of the putative N-terminal signal peptide cleavage sites in each SlCEP proteins. Based on the prediction, it is likely that the cleavage site occurs at a conserved arginine site ( Figure 1C; Table S2), which has been also shown in CEP proteins identified in other plant species [10,12,13]. However, we did not find any cleavage site for SlCEP12; this may be due to the limitations of the software.
Gene structural analysis of the SlCEPs showed that SlCEPs lacked introns ( Figure 2A). Analysis of the chromosomal location showed that 17 SlCEPs were mapped on four chromosomes (Chr1, Chr2, Chr3, Chr7) at different densities ( Figure 2B). For example, SlCEP4, SlCEP5, SlCEP6 and SlCEP7 were organized sequentially in tandem on chromosome 2. A similar cluster was also observed for SlCEP8, SlCEP9, SlCEP10 and SlCEP11 on chromosome 3; and it was observed for SlCEP12, SlCEP13, SlCEP14, SlCEP15, SlCEP16 and SlCEP17 on chromosome 7. Notably, the clustered SlCEP proteins showed low sequence similarity but shared the consensus CEP motifs, suggesting that these genes might arise from recent tandem duplication events.

Phylogenetic Analysis of SlCEP Proteins
To further gain insights into evolutionary relationships among SlCEPs and to group them within the established subfamilies, SlCEP and AtCEP proteins were used to construct an unrooted phylogenetic tree. The SlCEPs were divided into two subgroups based on the CEP motifs ( Figure 2C), and the CEP motifs of the two groups were aligned, resulting in a consensus of sequences supporting the classification of the two groups ( Figure 2C). Notably, the phylogenetic relationship based on the CEP domains was not well supported when the full-length sequences of SlCEP proteins were analyzed, which was due to the amino acids outside the CEP motifs ( Figure S2).
The phylogenetic relationship between AtCEP and SlCEP proteins was also analyzed using either the conversed CEP motifs ( Figure S3) or the full-length protein sequences ( Figure S4). The SlCEP and AtCEP proteins were grouped into several clades with varying degrees (Figures S3 and S4). We further investigated the evolution and origin of the CEP genes of tomato in comparison with Arabidopsis ( Figure 2D). We only identified one pair of the syntenic relationship between Arabidopsis and tomato, where SlCEP9 was linked to AtCEP3; this may suggest a distant evolutionary relationship between these two species. However, the tandem gene duplication events may occur within the tomato genome, as SlCEP12, SlCEP15 and SlCEP16 shared complete identical CEP motifs ( Figure 1A). The divergent evolution of SlCEP genes suggested that SlCEP peptides may play divergent roles in tomato development compared to the well-known AtCEP peptides.

Distinct Expression Pattern of SlCEP Genes in Response to Developmental and Nitrogen Signal
The spatial gene expression patterns are essential for investigating gene function in various developmental processes; hence, we searched the public tomato transcriptome database to explore the SlCEPs expression patterns in tomato tissues. The SlCEP genes' expression levels in 12 tissues were visualized ( Figure S5). The expression patterns of SlCEPs were varied in the examined tissues. For example, SlCEP7, SlCEP8, and SlCEP10 showed a relative high expression level in roots; SlCEP16 was highly expressed in fruit, and SlCEP2 was highly expressed in young flower buds, indicating their potential role in regulating various tomato developmental processes.
Nitrogen regulates AtCEP genes expression; then, CEPR receptors sense the CEP signal to activate nitrogen transporter genes transcriptions, which resulted in nitrogen acquisition [17][18][19]. Plants use either nitrate (NO 3 − ) or ammonium (NH 4 + ) as a nitrogen resource; we then examined the expression profiles of SlCEPs under nitrate or ammonium treatment. The tomato seedlings were treated with low (0.5 mM) and high (5 mM) nitrate or ammonium for 72 h, and the roots and shoots, respectively, were collected for gene expression analysis. Under low nitrate treatment, SlCEP4, SlCEP5, SlCEP6, and SlCEP11 were significantly upregulated, and SlCEP1, SlCEP2, SlCEP3, SlCEP7, SlCEP8, SlCEP9, SlCEP12, SlCEP16 SlCEP17 were downregulated in roots; SlCEP3, SlCEP5, SlCEP6, SlCEP8, SlCEP9, SlCEP10, SlCEP11, SlCEP13, and SlCEP14 were greatly upregulated in shoots. Under high nitrate treatment, SlCEP5, SlCEP6, SlCEP16 were greatly upregulated and SlCEP1 was downregulated in roots; and SlCEP8 and SlCEP16 were prominently downregulated in shoots. Under low ammonium treatment, SlCEP5, SlCEP6, SlCEP13, and SlCEP14 were significantly upregulated and SlCEP15 was downregulated in roots, while SlCEP2 and SlCEP15 were prominently downregulated in shoots. Under high ammonium treatment, SlCEP5, SlCEP6, and SlCEP11 were upregulated and SlCEP1, SlCEP7, SlCEP12, SlCEP15 were downregulated in roots; SlCEP3, SlCEP6, SlCEP8, and SlCEP10 were upregulated and SlCEP15 and SlCEP16 were downregulated in shoots. Our analysis indicated that the expression levels of SlCEPs in roots and shoots varied greatly among nitrate or ammonium treatment, implying crucial but divergent roles of SlCEPs in tomato nitrogen acquisition ( Figure 3). Considering some SlCEPs were not detected under our experimental conditions, this may be due to their spatio-temporal expression patterns or their responses to a certain nitrogen form and status. Overall, our analysis reveals the expression pattern of SlCEPs in response to nitrate or ammonium status, and SlCEPs would play diverse roles in nitrogen acquisition by regulating the corresponding nitrogen transporters in tomato roots and shoots, respectively ( Figure 3) [17][18][19].
6 of 16 their responses to a certain nitrogen form and status. Overall, our analysis reveals the expression pattern of SlCEPs in response to nitrate or ammonium status, and SlCEPs would play diverse roles in nitrogen acquisition by regulating the corresponding nitrogen transporters in tomato roots and shoots, respectively ( Figure 3) [17][18][19]. Figure 3. SlCEP genes expression are differentially regulated by nitrogen status in tomato roots and shoots. Expression levels of SlCEP genes in roots and shoots were quantified under low (0.5 mM) and high (5 mM) nitrate (A) and ammonium (B) treatment for 72 h. Relative expression levels of the genes were normalized to normal nitrogen condition, and the color represents log2 values. The heatmap was generated by TBtools.

NO and Ca 2+ Mediate CEP Peptide to Promote Tomato Root Growth
Next, we synthesized SlCEP9 and SlCEP11 peptides, the most identical CEP peptides to known function of AtCEP ( Figures 2D and S1), as examples to verify the physiological function of SlCEP peptides in tomato root development. After germination, tomato seedlings with a similar primary root length were transferred to new plates supplied with the synthetic SlCEP peptides and were cultured for another 6 days. Under our experimental conditions, we observed that tomato seedlings treated with both SlCEP9 and SlCEP11 peptide showed a significantly longer primary root ( Figure 4). . SlCEP genes expression are differentially regulated by nitrogen status in tomato roots and shoots. Expression levels of SlCEP genes in roots and shoots were quantified under low (0.5 mM) and high (5 mM) nitrate (A) and ammonium (B) treatment for 72 h. Relative expression levels of the genes were normalized to normal nitrogen condition, and the color represents log 2 values. The heatmap was generated by TBtools.

NO and Ca 2+ Mediate CEP Peptide to Promote Tomato Root Growth
Next, we synthesized SlCEP9 and SlCEP11 peptides, the most identical CEP peptides to known function of AtCEP ( Figure 2D and Figure S1), as examples to verify the physiological function of SlCEP peptides in tomato root development. After germination, tomato seedlings with a similar primary root length were transferred to new plates supplied with the synthetic SlCEP peptides and were cultured for another 6 days. Under our experimental conditions, we observed that tomato seedlings treated with both SlCEP9 and SlCEP11 peptide showed a significantly longer primary root ( Figure 4). It has been reported that the inhibition of NO could promote tomato root growth 33]; we then focused on the involvement of NO in SlCEP11-mediated tomato root gro NO can be oxidized to NO2, and superoxide can be detected by NBT staining; hence performed NBT staining as an indirect indication of NO level in SlCEP11 peptide-tre tomato primary roots. Our staining result revealed that SlCEP11 peptide significantl duced the superoxide level compared to control treatment ( Figure 5A,B), suggesting a tential reduced NO level triggered by the SlCEP11 peptide. We next assessed the effe NO inhibitors on SlCEP11 peptide-mediated root growth. In line with previous rep [31][32][33], tomato primary root growth was promoted when NO signaling was inhib and SNP (a NO donor) repressed the primary root growth. However, SlCEP11 and inhibitor exhibited a synergistic effect on primary root growth ( Figure 5C). When ex nous NO was supplied, the synergistic effect was partially abolished, suggesting that is involved in SlCEP11 peptide function ( Figure 5C). Ca 2+ is an important signaling plant development [34]; we then addressed whether Ca 2+ participates in SlCEP11-m ated root growth. When the calcium channel was blocked by LaCl3, root growth pro tion triggered by the SlCEP11 peptide was also counteracted ( Figure 5D), and exogen Ca 2+ partially suppressed the LaCl3 effect. These data indicate an involvement of calc signaling in SlCEP11-mediated root growth. Taken together, these preliminary showed that NO and Ca 2+ were involved in SlCEP peptide function in tomato root de opment. It has been reported that the inhibition of NO could promote tomato root growth [31][32][33]; we then focused on the involvement of NO in SlCEP11-mediated tomato root growth. NO can be oxidized to NO 2 , and superoxide can be detected by NBT staining; hence, we performed NBT staining as an indirect indication of NO level in SlCEP11 peptide-treated tomato primary roots. Our staining result revealed that SlCEP11 peptide significantly reduced the superoxide level compared to control treatment ( Figure 5A,B), suggesting a potential reduced NO level triggered by the SlCEP11 peptide. We next assessed the effect of NO inhibitors on SlCEP11 peptide-mediated root growth. In line with previous reports [31][32][33], tomato primary root growth was promoted when NO signaling was inhibited and SNP (a NO donor) repressed the primary root growth. However, SlCEP11 and NO inhibitor exhibited a synergistic effect on primary root growth ( Figure 5C). When exogenous NO was supplied, the synergistic effect was partially abolished, suggesting that NO is involved in SlCEP11 peptide function ( Figure 5C). Ca 2+ is an important signaling for plant development [34]; we then addressed whether Ca 2+ participates in SlCEP11-mediated root growth. When the calcium channel was blocked by LaCl 3 , root growth promotion triggered by the SlCEP11 peptide was also counteracted ( Figure 5D), and exogenous Ca 2+ partially suppressed the LaCl 3 effect. These data indicate an involvement of calcium signaling in SlCEP11-mediated root growth. Taken together, these preliminary data showed that NO and Ca 2+ were involved in SlCEP peptide function in tomato root development.

SlCEP Peptide Promotes Tomato Root Resistance to Salinity
The CEP peptide has also been suggested to play roles in stress response [5,11,26]. The atcep3 mutant displayed resistance to slat stress [5]; hence, we investigated whether the synthetic SlCEP9 and SlCEP11 peptides play a role in salinity response. After germination, tomato seedlings with a similar primary root length were transferred to new plates supplied with the synthetic SlCEP peptides in the presence of 100 mM NaCl and were cultured for another 4 days. Under our experimental conditions, we observed that salinity stress greatly inhibited tomato primary root growth; however, tomato seedlings treated with synthetic SlCEP peptides displayed longer primary roots, implying that SlCEP pep could improve tomato fitness under salt stress ( Figure 6).

SlCEP Peptide Promotes Tomato Root Resistance to Salinity
The CEP peptide has also been suggested to play roles in stress response [5,11,26 The atcep3 mutant displayed resistance to slat stress [5]; hence, we investigated whethe the synthetic SlCEP9 and SlCEP11 peptides play a role in salinity response. After germi  cultured for another 4 days. Under our experimental conditions, we observed that salinity stress greatly inhibited tomato primary root growth; however, tomato seedlings treated with synthetic SlCEP peptides displayed longer primary roots, implying that SlCEP pep could improve tomato fitness under salt stress ( Figure 6).

Discussion
Tomato is an essential cultural crop; however, the underlying mechanisms for tomato growth and development remain elusive. Numbers of studies have been reported that the CEP peptide family plays crucial roles in a wide range of plant developmental processes [29]. The CEP peptide family has been identified across various plant genomes; however, little is known about this family in tomato. Hence, we performed a genome-wide searching of putative tomato CEP peptide family to explore their potential physiological function in tomato development and stress responses (Figure 7).

Discussion
Tomato is an essential cultural crop; however, the underlying mechanisms for tomato growth and development remain elusive. Numbers of studies have been reported that the CEP peptide family plays crucial roles in a wide range of plant developmental processes [29]. The CEP peptide family has been identified across various plant genomes; however, little is known about this family in tomato. Hence, we performed a genome-wide searching of putative tomato CEP peptide family to explore their potential physiological function in tomato development and stress responses (Figure 7). stress greatly inhibited tomato primary root growth; however, tomato seedlings treated with synthetic SlCEP peptides displayed longer primary roots, implying that SlCEP pep could improve tomato fitness under salt stress ( Figure 6).

Discussion
Tomato is an essential cultural crop; however, the underlying mechanisms for tomato growth and development remain elusive. Numbers of studies have been reported that the CEP peptide family plays crucial roles in a wide range of plant developmental processes [29]. The CEP peptide family has been identified across various plant genomes; however little is known about this family in tomato. Hence, we performed a genome-wide search ing of putative tomato CEP peptide family to explore their potential physiological func tion in tomato development and stress responses (Figure 7).  To address the physiological roles the SlCEP genes, publicly available RNA-seq data were extracted to study the expression patterns of SlCEP genes in tomato tissues ( Figure S5). The SlCEP gene family displayed diverse expression patterns, suggesting the diverse roles of SlCEPs in controlling various aspects of tomato development. Considering that only some of the SlCEPs can be detected in examined tissues, the expression patterns of these undetectable SlCEPs require further investigation in the future.
Exogenous application of synthetic CEP peptides regulates root growth and development, which mimics its endogenous functions [29]. Our data showed that the application synthetic SlCEP peptides promoted tomato primary root growth (Figure 4). Notably, the promotion of tomato root growth triggered by the exogenous application of synthesized SlCEP peptides just suggests their potential physiological roles and by no means limits their function in other developmental processes or rules out other SlCEP peptides as pivotal regulators in tomato growth and stress responses. Compared to the well-known inhibitory roles of the AtCEP peptides [4,5,16,29], the CEP peptides in tomato, cucumber and Brassica rapa exhibited an oppositive effect (Figure 4) [12,13]. Antagonistic peptide technology has been proposed to re-write CLE peptide function [35,36], the promotion of primary root growth triggered by SlCEP peptides (Figure 4), indicating that SlCEP9 and SlCEP11 may be an antagonistic form. However, it definitely requires careful examinations. On the other hand, it is likely that different downstream regulatory networks could be activated by SlCEP peptides in tomato roots. It is also necessary to investigate whether conserved serine (at position 10) and glycine (at position 14) are crucial for SlCEP peptide function, as these amino acid residues are important for apple MdCEP1 function [10]. The loss-of-function or gain-of-function of SlCEP mutants would assist with better elucidating the untapped physiological functions in the tomato life cycle. In addition, the CEP peptide is perceived by membrane localized CEPR receptors to trigger downstream responses [29]. A homology of AtCEPR receptors has been identified in tomato, and it can recognize CEP peptides [37]. It is interesting to test whether the tomato CEPR receptor can transmit the SlCEP signal to regulate tomato root growth and responses to environmental cues. On the other hand, 234 LRR-RLK receptors genes have been identified in tomato [38], suggesting various combination of SlCEPs and SlRLKs to modulate tomato development and environmental adaptions. Screening CRISPR-Cas9 targeted tomato receptor mutants [39] will assist with identifying novel corresponding receptors for SlCEP peptides. Additionally, the usage of 4-azi-dosalicylic acid ([ 125 I] ASA)-labeled SlCEP peptides could assist with screening the RLK library in BY2 cells, and it will also help identify their putative binding proteins [40].
Plants are unable to adjust their growth in the ever-changing environments when reactive oxygen species (ROS) homeostasis is disturbed in the roots, indicating the essential role of ROS in root development [41,42]. We showed that NO is required for SlCEP peptide function in tomato root development ( Figure 5A-C). How SlCEP peptides regulate NO biosynthesis and metabolism or proteins which are main targets of NO-related post-translational modifications [43] requires further investigations. Additionally, it is also necessary to detect endogenous NO levels using a DAF-FM-DA probe [44]. Hydrogen peroxide (H 2 O 2 ) is also involved in CEP-mediated root growth [12,13]; it is intriguing to test the involvement of H 2 O 2 in SlCEP-mediated tomato root growth. Additionally, Ca 2+ was also involved in SlCEP-mediated root growth ( Figure 5D). The genetic mutants related to ROS biosynthesis and signaling mutants, as well as Ca 2+ signaling mutants [45][46][47][48], will help to corroborate the critical roles of ROS and Ca 2+ in the SlCEP peptide signaling pathway.
Abiotic stress negatively affects plant growth and productivity. Therefore, plants have evolved multiple mechanisms such as an increased expression of the stress-associated genes or hormones level to control their adaption to the ever-changing environments [49]. The small peptide family works in parallel with plant hormones to regulate plant stress responses [11,20,26]. Our qRT-PCR analysis showed that SlCEPs expression levels in roots and shoots were differentially regulated by nitrate or ammonium status, suggesting the potential involvement of SlCEPs in modulating tomato adaptions to the nitrogen status ( Figure 3). In the past few decades, the nitrogen signaling regulatory networks have been established, and many genes play crucial roles in modulating nitrogen acquisition [50]; how SlCEP peptides recruit these known or undefined nitrogen regulators in tomato nitrogen adaptions needs to be determined in future investigations. Furthermore, the exogenous application of SlCEP peptides promoted tomato root resistance to salinity ( Figure 6). However, the mechanisms underlying SlCEP-mediated salt stress responses requires more investigations [49]. Additionally, it is also worthwhile to reveal the untapped functions of SlCEPs in other developmental processes as well as abiotic and biotic stress responses.

Genome-Wide Annotation of Tomato CEP Peptide Family
In total, 15 Arabidopsis and 6 previously identified tomato CEP proteins [5][6][7] were used to perform protein BLAST searches against a re-assembled tomato genome (ITAG 4.0) released to Phytozome 13 (https://phytozome-next.jgi.doe.gov/, V13, accessed on 16 March 2022) [51]. Each newly identified protein was subsequently used to conduct protein blast against the tomato genome to avoid any missed SlCEP proteins until no novel proteins were found.

SlCEP Protein Features Analysis
SlCEP protein N-terminal signal peptide prediction was performed by searching

Alignment and Phylogenetic Analysis
ClustalX was applied for multiple alignment analysis [57]; the alignments were then refined and displayed via Jalview [58]. MEGA X software (https://www.megasoftware. net/, Version 10, accessed on 13 May 2022, and the software was downloaded and installed) was used to build the phylogenetic trees [59] using the conserved CEP domains or the full length of CEP proteins by the neighbor-joining method. Bootstrap analysis was conducted with 1000 replicates to verify the significance of nodes.

Gene Duplication Analysis
The Arabidopsis thaliana (TAIR 10) and Solanum lycopersicum (ITAG 4.0) genome and annotation files were downloaded from the Phytozome website. TBtools was used to scan the genome to identify duplicated gene pairs. Finally, the orthologous gene pairs were identified using a Dual synteny plotter in TBtools (https://github.com/CJ-Chen/TBtools/ releases, Version 1.0987663, accessed on 16 May 2022, and the software was downloaded and installed) [60].

SlCEP Gene Expression in Tomato Tissues
A published tomato RNA-seq data in wild species S. pimpinellifolium (LA1589) was used to determine the expression patterns of the SlCEP genes in various tomato tissues (D006, http://ted.bti.cornell.edu/, accessed on 25 May 2022).

Plant Material and Growth Conditions
The cultivar tomato seeds "Dahong" were brought from Shanghai Hongqiao Tianlong Seed Company and were used in this study. All seeds were washed with distilled water. Tomato seeds were sterilized with 2.3% sodium hypochlorite for 5 min. The sterilized seeds were washed with distilled water 5-6 times. The seeds were kept in darkness at 28 • C to induce germination. The seedlings were grown in a plant growth chamber (16 h light: 8 h dark photoperiod, 21 • C, 112 µmol m −2 sec −1 ).

Total RNA Extraction and Gene Expression Analysis
After germination, tomato seedlings with a similar root length were transferred to liquid 1/2 MS solution (normal nitrogen as control) or liquid Hoagland solution without nitrogen (NS10205-NCoolaber, China) supplied with KNO 3 (0.5 and 5 mM) or NH 4 Cl (0.5 and 5 mM) for 72 h, and the roots and shoots parts were collected, respectively. The RNA extraction kit (DP432, Tiangen, China) was used to extract total RNA. A Hifair III cDNA synthesis kit was used to generate first-strand cDNA from 1 µg of total RNA (Cat NO. 11139ES60, Yeasen Biotechnology, Bejing, China). The qRT-PCR was performed using Hieff qPCR SYBR Mix (Cat NO. 11170ES03, Yeasen Biotechnology, China) with an ABI 7500 Real-Time PCR System (Thermo Fisher, Waltham, MA, USA). The primers used for qRT-PCR analysis are listed in Table S3. The average expression level of SlCEP genes was calculated using the ∆∆ CT method via TBtools [60,61]. Three independent experiments were performed.

SlCEP Peptides Treatment
SlCEP9 (DFGPTGPGHSPGIGH) and SlCEP11 (GFSPYGRGHSPGIGH) were synthesized by DGpeptide company. All peptides were dissolved in distilled water to a concentration of 10 mM and were stored at −20 • C. After germination, tomato seedlings with a similar primary root length were transferred to new plates supplied with 1 µM of SlCEP9 and SlCEP11 peptides, respectively. Plates were imaged via the EPSON V370 scanner. Primary root length was quantified via ImageJ software. Three independent experiments were performed.

NBT (Nitroblue Tetrazolium) Staining
After germination, tomato seedlings with a similar root length were transferred to new agar plates supplied with 1 µM of SlCEP11 peptide, and the seedlings were cultured for another 6 days; then, the seedlings were used for NBT staining. The seedlings were incubated in NBT staining buffer (0.5 mg/mL NBT in 50 mM phosphate buffer pH = 7.6) for 5 min in the dark. An inverted BDS 400 microscopy was used to capture the pictures. The relative NBT signal was quantified via ImageJ. Three independent biological repeats were performed.

NO and Ca 2+ Inhibitor Treatment
After germination, tomato seedlings with a similar root length were transferred to new agar plates supplied with NO inhibitors (L-NAME, 25 µM, NO synthase-like enzyme inhibitor and Na 2 WO 4 , 5 µM, nitrate reductase inhibitor), SNP (50 µM, a NO donor), lanthanum chloride (LaCl 3 , 500 µM, Ca 2+ channel blocker), and CaCl 2 (500 µM) in the presence of 1 µM of SlCEP11 peptide for another 6 days. Plates were imaged via the EPSON Cells 2022, 11, 2935 13 of 16 V370 scanner. The root length was quantified via ImageJ. Three independent biological repeats were performed.

Salinity Treatment and Root Growth Quantification
After germination, tomato seedlings with a similar primary root length were transferred to new agar plates supplied with 100 mM NaCl in presence of 1 µM of SlCEP9 and SlCEP11 peptide, respectively, and the seedlings were cultured for another 4 days. Plates were imaged via the EPSON V370 scanner. The root length was quantified via ImageJ. Three independent biological repeats were performed.

Statistical Analysis
All statistical analysis was performed using a one-way ANOVA test with a significant difference via GraphPad Prism 8.0 (* p < 0.05; ** p < 0.01).

Conclusions
We aim to unravel the potential physiological function of a small signaling peptide in tomato development and adaptions to environmental stress, thus providing novel strategies for tomato cultivation and genetic breeding. To this end, we presented the comprehensive overview of putative CEP gene family in tomato, including their gene structure, conserved motifs and expression patterns in tissues. We also revealed that tomato CEP genes were differentially regulated by nitrogen form and status in roots and shoots, respectively. Synthetic tomato CEP peptides significantly promoted tomato primary root elongation via regulating NO and Ca 2+ signaling. Additionally, we showed that the tomato CEP peptide promoted tomato root resistance to salt stress. Overall, our work would provide a very useful reference for future functional analysis CEP function in tomato, and it would also provide novel strategies to improve tomato fitness and to increase yield under adverse environments.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11192935/s1, Table S1: Summary of the identified 17 SlCEP genes. Table S2: Predication of N-terminal signal peptide of SlCEP proteins. Table S3: Primers for qRT-PCR analysis. Figure S1: Alignment of CEP motifs in tomato and Arabidopsis. Figure S2: Phylogenetic tree of full-length SlCEP proteins. Figure S3: Phylogenetic tree AtCEP and SlCEP proteins based on the CEP motif. Figure S4: Phylogenetic tree of full-length AtCEP and SlCEP proteins. Figure S5. Expression patterns of SlCEPs in tomato tissues.  Data Availability Statement: The public transcriptome data were downloaded from Tomato Functional Genomics database, which we mentioned in "Materials and Methods".