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Article

Characterization of the CLE Family in Three Nicotiana Species and Potential Roles of CLE Peptides in Osmotic and Salt Stress Responses

by
Yumeng Chu
1,†,
Xiaoming Gao
2,†,
Lichao Wen
2,
Zhichao Deng
2,
Tao Liu
2 and
Yongfeng Guo
2,*
1
Qingdao Agricultural University, Qingdao 266109, China
2
Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(6), 1480; https://doi.org/10.3390/agronomy13061480
Submission received: 1 April 2023 / Revised: 3 May 2023 / Accepted: 24 May 2023 / Published: 27 May 2023
(This article belongs to the Special Issue Emerging Topics in Tobacco Genomics)
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Abstract

:
The CLE family (CLAVATA3/embryo surrounding region-related), a class of small secreted proteins, play important roles in plant development and stress responses. Members of the CLE family have been characterized in a number of plant species, including Arabidopsis and rice. However, limited information is available about CLE peptides in tobacco (Nicotiana tabacum) and related Nicotiana species. Here we report the identification of 84 CLE family members in three Nicotiana species based on sequence similarity. The newly identified CLE members, including 41 from N. tabacum, 19 from N. sylvestris, and 24 from N. tomentosiformis, together with 32 CLEs from Arabidopsis and 52 CLEs from tomato, formed 9 subgroups in a phylogenic tree. The unbalanced distribution of the Nicotiana CLEs in the subgroups suggested potential preferential gene family expansion during evolution. Expression of the NtCLE genes was analyzed and a number of the NtCLEs showed induced expression upon abiotic stress treatments. Synthetic peptides of several NtCLEs, when applied to detached tobacco leaf discs, were able to increase plants’ tolerance to osmotic and salinity stresses, suggesting potential roles of CLE peptides in the stress responses of tobacco.

1. Introduction

Plant hormones are involved in the regulation of plant growth, development, senescence, stress responses, and other physiological processes. Traditional phytohormones are endogenous small molecules derived from secondary metabolism which are often present at very low concentrations. Small peptides derived from gene-encoded proproteins have also been characterized as signaling molecules similar to phytohormones [1]. Plant signaling peptide was first reported in tomato (Lycopersicon esculentum) in 1991 when systemin was identified as a hormonal peptide [2]. A large number of signaling peptides (or peptide hormones) from various plant species have been identified and characterized since then [3,4,5,6,7]. It is generally believed that plant peptide hormones are a class of mature peptide molecules with a length of fewer than 100 amino acids and special functions, and their concentrations are usually very low in plant cells [8,9].
The CLAVATA3/embryo surrounding region-related (CLE) gene family is one of the largest families encoding plant signaling peptides. CLE peptides are also the most studied group of plant peptides in the past decade. The first CLE gene was reported to be an endosperm-specific gene in maize in 1997, and the name was taken from two genes in the same family, CLAVATA3 (CLV3) from Arabidopsis [10] and embryo surrounding region (ESR) from maize [11]. CLE family members have been reported to participate in a variety of physiological and developmental processes in plants, and play an important role in regulating cell-to-cell communication [12,13].
Including CLV3, in Arabidopsis there are 32 CLE genes encoding CLE precursor proteins. In addition, 52 SlCLE genes from tomato [14], 41 CLE genes from potato (Solanum phureja DM-1-3 516R44) [15], 45 BnCLE genes from Brassica napus [16], 50 PtCLEs from Populus trichocarpa [17], 93 GhCLE genes from cotton [18], 26 CLE genes from cucumber [19], and 104 TaCLE genes from wheat [20] have been identified at the genome level. The CLE proproteins have a similar structure, typically with fewer than 100 amino acids, consisting of an N-terminal signal peptide (SP) of 15–69 amino acids, followed by a variable domain of 40–99 amino acids with significant sequence diversity, and a conserved C-terminal CLE domain with 12–14 amino acids [9,21,22,23]. A few CLE precursor proteins have multiple CLE domains, and some CLE precursor proteins contain a C-terminal extension region composed of 1–150 amino acids [23,24]. The minimum length of a mature CLE peptide has been reported to be 12 amino acids, and the functional CLE motif was suggested to be released from the CLE precursor protein via the function of a peptidase [9,25,26]. Fiers et al. showed that the deletion of most flanking sequences of the conserved CLE domain did not affect the function of the CLE peptide [27]. Domain exchange and deletion experiments with CLV3 showed that the signal peptide and CLE domain play a key role in the function of CLE proteins [28,29].
CLE peptide ligands assist in different processes by interacting with different receptor kinases. In Arabidopsis, CLV3 is the most widely studied CLE peptide; it regulates the homoeostasis of stem cell division and differentiation in the shoot apical meristem (SAM) by suppressing the expression of transcription factor WUSCHEL (WUS) [10,30]. CLV3 acts as a ligand to activate the CLV1/CLV2 receptor complex to transmit CLV3 signals [30,31,32]. Similarly, CLE40 functions in maintaining the root apical meristem (RAM) via binding to the receptor ACR4 and suppressing the expression of WUSCHEL-related homeobox5 (WOX5) [33,34]. In vascular tissues, the tracheary element differentiation factor (TDIF) peptides encoded by CLE41, CLE42, and CLE44 regulate the expression of WOX4 by binding to the TDR/PXY receptor, stimulating cell proliferation and suppressing the differentiation of vascular elements [35,36].
In addition to stem cell division and differentiation, CLE peptides have also been characterized as being involved in several other processes. Zhang et al. reported that CLE14 peptides assist in the suppression of leaf senescence by regulating ROS homeostasis in Arabidopsis [37]. Thereafter, CLE42 has also been characterized as regulating senescence by communicating with the ethylene pathway [38]. A number of studies have indicated the regulatory role of CLE peptides in plants’ responses to abiotic stresses. The Arabidopsis CLE45-SKM1/SKM2 signaling pathway plays an important role in ensuring the normal mutual recognition of pollen and stigma at high temperature [39]. CLE25 peptides can be produced in roots under water deficit conditions, and the peptides move from roots to leaves through vascular bundles, regulating the accumulation of abscisic acid and inducing stomatal closure by binding to the BAM receptors in leaves [40]. Another study also showed that CLE9 peptides play a regulatory role in inducing stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 led to stomatal closure and enhanced osmotic stress tolerance, whereas CLE9 loss-of-function mutants were sensitive to osmotic stress [41]. Under the condition of N deficiency, the transcription level of CLE1/3/4/7 in roots increases. The receptor kinase CLV1 located in the phloem senses the CLE3 signal and inhibits the development of lateral roots [42]. Under the condition of sulfur (S) deficiency, the transcription levels of CLE2 and CLE3 in roots are repressed, resulting in a reduction in lateral root density [43]. CLE14 mediates low phosphorus stress signals in roots, triggering RAM differentiation through the CLV2/PEPR2 receptors, inhibiting primary root growth [44].
Tobacco (Nicotiana tabacum L.) is an important cash crop widely grown in the world. It also plays an important role in plant molecular biology as a model plant and can be used as a bioreactor for vaccine production [45,46]. In addition, due to the huge biomass of tobacco, it has been studied as a potential bioenergy plant [47,48]. Although CLE peptides have been studied in a large number of plant species, such as Arabidopsis, rice, tomato, and poplar, the CLE gene family in tobacco has not been reported. Tobacco is an allotetraploid (2n = 48) species originating through hybridization and chromosome doubling of N. sylvestris (2n = 24) and N. tomentosiformis (2n = 24), with a 4.5 Gb genome and more than 70% repetitive sequences [49,50]. In this study, the CLE genes of N. tabacum, N. sylvestris, and N. tomentosiformis were identified by comparing the published CLE peptide sequences of Arabidopsis and tomato, and the bioinformatics analysis of their gene structure, expression pattern, and protein characteristics were carried out. In addition, the CLE peptides (12 amino acids) identified in tobacco were synthesized, and their roles in osmotic stress and salinity stress of tobacco were determined through exogenous application. Several CLE peptides that can improve tolerance to osmotic stress and salinity stress in tobacco were identified.

2. Materials and Methods

2.1. Plant Materials

Common tobacco (N. tabacum) variety K326 was used for all analyses in this study. Tobacco plants were grown in a growth room at 23 °C with 16 h of light and 8 h of dark in a daily cycle.

2.2. Identification of Tobacco CLE Proteins and Protein Feature Analysis

The genome sequences of N. tabacum, N. sylvestris, and N. tomentosiformis were searched via BLASTP and TBLASTN in the China Tobacco Genome Database (http://218.28.140.17/, accessed on 1 January 2020), the database is not publicly available) using the protein sequences of 32 AtCLEs and 52 SlCLEs [14,51] as queries. Genes with E values ≤ 10−10 were selected as candidate CLE genes. The protein sequences of obtained tobacco CLE candidate genes were used as new queries to search the databases repeatedly until no new candidates were found. Finally, genomic sequences, CDS sequences, and protein sequences of the candidate genes were obtained.
The N-terminal signal peptide prediction of tobacco CLEs was performed using the online prediction tool SignaIP4.1 Sever (https://services.healthtech.dtu.dk/services/SignalP-4.1/, accessed on 1 January 2020). The conserved CLE domain was analyzed via the online software MEME (https://meme-suite.org/, accessed on 1 January 2020). The maximum number of discoveries was set to five, and the other parameters were the default values. Physical and chemical characteristics of the tobacco CLEs were analyzed using the ExPASy Proteomics Server software (http://www.expasy.org/, accessed on 1 January 2020). The online prediction tool GSDSv2.0 (http://gsds.cbi.pku.edu.cn/, accessed on 1 January 2020) was used to analyze and map the gene structure of the tobacco CLE genes.

2.3. Multiple Sequence Alignment and Phylogenetic Analysis

Multiple sequence alignment of tobacco CLE protein sequences was performed using the MEGA7.0 software [52]. A phylogenetic tree was constructed by MEGA7.0 using the sequences of the CLE motifs. The maximum-likelihood algorithm was used, and the bootstrap replications were set to 500.

2.4. Expression Analysis of Tobacco CLE Genes

For gene expression analysis in different tissues, total RNA was extracted from whole plants at the seedling stage (4–5 true leaves) and from roots, stems, leaves, flowers, and fruits of mature plants (60 d old).
For gene expression analysis under stressed conditions, plants with 4 true leaves were subject to liquid culture treatments containing MS for the control, 300 mM mannitol in MS for osmotic stress, or 150 mM NaCl in MS for salinity stress. Samples were harvested 0, 1, 3, and 6 h after treatments. Leaf samples from 9 plants in each treatment group were used for RNA extraction.
Total RNA was extracted using the TRIzol reagent (Invitrogen, Shanghai, China) and first-strand cDNA was synthesized from 1 μg of total RNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Q312, Nanking, China) according to the manufacture’s instructions. qRT-PCR was carried out on a Thermal Cycler Block 7500 (ABI) using the ChamQ SYBR® qPCR Master Mix (Vazyme, Q311). The primers were designed according to the CDS sequence of CLE genes in N. tabacum. The means of three biological and technical replicates were analyzed. The tobacco actin gene Ntab0037910 was used as an internal control in qPCR. The primer sequences are listed in Table S1 (Supplementary Materials). Relative expression levels were normalized using the housekeeping gene actin. The results were analyzed via the 2−ΔΔCt method. IBM SPSS Statistics 25 was used as the statistical analysis software, and the ANOVA test was used to determine the significance of difference. Multiple comparisons were performed using the Waller–Duncan and LSD tests.

2.5. Exogenous Application of Synthetic NtCLE Peptides under Osmotic Stress and Salinity Stress Conditions

NtCLE peptides with a purity of >90% were synthesized by the Biology Corporation of GenScript, Nanjing, China, and dissolved in sterilized ddH2O before use. Leaf discs were prepared from middle leaves of 60 d old tobacco plants grown in a greenhouse. Detached leaves were cut into small discs with diameters of 1.5 cm and incubated on filter paper in Petri dishes with ddH2O, 300 mM mannitol, or 150 mM NaCl. NtCLE peptides were added to the treatments at a concentration of 1 µM to test their effects on plants’ stress tolerance. Pictures were taken and tissues were harvested for analysis 4 d and 8 d after treatments. The chlorophyll content and Fv/Fm ratio were determined as described previously [53]. Three biological replicates were done for each treatment.

3. Results

3.1. Identification and Sequence Analysis of CLE Proteins in Three Nicotiana Species

Using the CLE protein sequences in Arabidopsis and tomato, eighty-four CLE genes were identified in the three Nicotiana species studied, including forty-one CLE genes from N. tabacum, nineteen CLE genes from N. sylvestris, and twenty-four CLE genes from N. tomentosiformis, named NtCLE1~NtCLE41, NsyCLE1~NsyCLE19, and NtomCLE1~NtomCLE24, respectively (Table 1). The physical and chemical characteristics of the Nicotiana CLEs were analyzed (Table 1). The average length of the CLE proteins is 94.41463 amino acids, with the longest NtCLE6 and the shortest NtCLE5 consisting of 176 and 51 amino acids, respectively. The maximum and minimum molecular weights are 19,906.78 and 5321.6 Da, respectively. NtCLE41 has the lowest isoelectric point of 5.66 and NtCLE18 has the highest isoelectric point of 12.13, with an average isoelectric point of 9.89 between all the Nicotiana CLEs, indicating that these CLE proteins are alkaline. Signal peptide prediction using SignalP revealed that 28 NtCLEs, 10 NsyCLEs, and 12 NtomCLEs contain a signal peptide structure, suggesting that most of them are secreted proteins. The hydrophilicity of all the Nicotiana CLE proteins is negative, indicating that they are hydrophilic proteins. Instability index analysis indicated that the majority of the Nicotiana CLE proteins are unstable (index > 40). The aliphatic index of the CLE family proteins was 79.82 on average, ranging from 38.87 for NsylCLE3 to 106.95 for NtCLE36, suggesting large variations in thermo-stability among Nicotiana CLE proteins (Table 1). The coding sequences in eleven of the forty-one NtCLEs are interrupted by one or two introns (Figure 1).
The sequence characteristics of the 84 Nicotiana CLE proteins were analyzed using MEME online software, and 4 conserved motifs were identified (Figure 2A–C). Among them, Motif1, which contains the core functional sequence of the CLE peptide, is present at the C-termini of all 84 Nicotiana CLE proteins. By comparing the protein sequences of AtCLEs, SICLEs, and NtCLEs, the twelve conserved amino acids of CLE proteins in common tobacco, representing the CLE domain, were identified (Figure 2A). A sequence logo was built for tobacco CLE motifs to visualize the conserved residues, among which 5 residues including R (1), V (3), P (4), G (6), and P (9) are almost invariant over the whole NtCLE family. Residues at the other positions in the CLE domain are relatively variable, including positions 2, 5, 8, and 10 (Figure 2B). Some of the signature amino acids of CLE motifs in other plant species are also conserved in NtCLEs [9,23].

3.2. Phylogenetic Analysis and Categorization of Nicotiana CLE Proteins

A phylogenetic tree of 32 CLEs from Arabidopsis, 52 CLEs from tomato, and 84 CLEs from the 3 Nicotiana species was constructed through the neighbor-joining method using the MEGA7.0 software (Figure 3). These CLE proteins were divided into 9 sub-groups, namely, Groups A, B, C, D, E, F, G, H, and I. Some subgroups contain more CLE members, such as Group B (28 CLEs), Group E (28 CLEs), Group G (45 CLEs), and Group I (35 CLES), while Groups C, D, and H are relatively small subgroups, with only 2, 2, and 3 CLEs, respectively. Some of the subgroups contain more CLE members from the Nicotiana species. For example, there are 25 Nicotiana CLEs out of the 45 Group G members and 25 out of 35 CLEs are from Nicotiana species in Group I. It’s interesting to note that the NtomCLEs showed an unbalanced distribution among the subgroups. For example, Group A contains 5 NtomCLEs out of the 9 subgroup members, while Groups C and D do not contain any NtomCLEs. CLEs from N. sylvestris are mainly found in Groups B, G and H, containing 4, 6, and 4 NsylCLEs, respectively. Groups A, C, D and E did not contain NsylCLE genes. For N. tabacum, Group B (9), Group G (12), and Group I (10) contain more NtCLEs, while Groups A and F do not have any NtCLEs (Figure 3). As ancestor species of allotetraploid N. tabacum, diploid species N. sylvestris and N. tomentosiformis showed unbalanced distributions of CLEs among the subgroups, suggesting potential preferential gene family expansion during evolution. A number of the CLE genes from Arabidopsis and tomato have been characterized as being involved in plant development and stress response [1,13]. Biological functions of the Nicotiana CLEs can be partially predicted based on their sequence similarity to known CLE peptides.

3.3. Expression of Tobacco CLE Genes in Different Tissues

Since the function of NtCLEs is often dependent on precise expression in specific locations, the expression of the 41 NtCLE genes in various tissues, including roots (from 20-day-old seedlings), stems (at the bolting stage), leaves (from 20-day-old seedlings), flowers, 14-day-old fruits, and whole plants at the seedling stage, were analyzed using qRT-PCR. Expression of the NtCLE genes showed significant differences in different parts of tobacco plants (Figure 4). Among the NtCLE genes, expression of NtCLE2, NtCLE16, NtCLE24, NtCLE26, and NtCLE27 was detected in all tested tissues. NtCLE11 seemed to be expressed specifically in leaves, but not in the other tested tissues. Expression of NtCLE11, NtCLE21, NtCLE25, and NtCLE32 was not detectable in roots. Expression of NtCLE1, NtCLE11, NtCLE12, and NtCLE32 was not detected in stems. Expression of NtCLE4, NtCLE17, and NtCLE22 was not detected in leaves. Expression of NtCLE11 and NtCLE31 was not detected in flowers and fruits (Figure 4).

3.4. Expression of Tobacco CLE Genes in Response to Stress Treatments

We further analyzed the expression levels of the NtCLE genes under different stress treatments via qRT-PCR. The results showed that several NtCLE genes showed differential expression in response to stress treatments (Figure 5). The expression levels of NtCLE3, NtCLE12, NtCLE16, and NtCLE29 continuously increased under osmotic stress, reaching the highest level 6 h after treatments. On the other hand, the expression of NtCLE15, NtCLE18 and NtCLE27 decreased continuously under osmotic stress treatments (Figure 5A). The expression of NtCLE12 and NtCLE41 increased gradually under salinity stress treatments, and NtCLE37 was down-regulated by salinity treatments (Figure 5B). Under salinity stress, NtCLE18 was upregulated 1 h after treatments and down-regulated afterward (Figure 5B). The NtCLEs showing differential expression after stress treatments are likely to be involved in stress responses of tobacco.

3.5. Function of NtCLE Peptides in Response to Osmotic Stress and Salinity Stress in Tobacco

To mimic mature plants grown under stress conditions in the field, we used leaf disc treatments in evaluating the stress responses of tobacco plants. Suitable concentrations of NaCl and mannitol were determined in pre-experiments mimicking salinity and osmotic stresses. To test the effects of CLE peptides on stress tolerance, synthetic peptides were added into Petri dishes with 1.5 cm-diameter leaf discs incubated in 15 mL of 150 mM NaCl or 300 mM mannitol for stress treatments (Figure S1).
The results showed that after 4 days’ mannitol treatments, in comparison with mock treatments, the leaf discs started showing chlorophyll loss, and more profound leaf yellowing was observed 8 days after osmotic stress treatments (Figure 6). The treamtns co-inoculating with NtCLE29 or NtCLE12 peptides were able to significantly delay the osmotic stress-induced yellowing process of the leaf discs (Figure 6A,B). Exogenous addition of NtCLE4 peptides, however, did not have significant effects on the osmotic stress responses of the leaf discs (Figure 6C). The leaf-yellowing phenotypes of different treatments were well reflected by chlorophyll contents and photosynthetic rates represented by the Fv/Fm ratio.
For salinity stress treatments, significant leaf yellowing was observed 4 d after treatments. Among the three peptides tested, NtCLE12 showed a significant effect in delaying salt-induced chlorophyll loss in leaf discs (Figure 7).

4. Discussion

Compared to peptide signals in animals, research in plant peptides is lagging. However, in the past twenty years, with the further development of genetics and molecular biology, plant peptides have drawn much attention, and significant progression has been made in the identification and characterization of plant peptides and in related signal transduction [1,13]. CLE peptides are the largest family of peptide signals identified in plants by far [1,12]. CLE family members have been characterized as being involved in a large number of developmental and stress response processes through mediating intercellular signal transduction [12,13].
In this study, 84 CLE genes were identified in three Nicotiana species, including forty-one from N. tabacum, nineeteen from N. sylvestris, and twenty-four from N. tomentosiformis. The Nicotiana CLEs were compared with CLE proteins from Arabidopsis and tomato through sequence alignment and phylogenetic analysis, and all the CLE proteins were classified into 9 subgroups. Gene expansion in certain subgroups was revealed by the unbalanced distribution of Nicotiana CLEs in different subgroups (Figure 3).
The CLE domain of CLE family members in N. tabacum was found to be highly conserved (Figure 2). Fiers et al. showed that most of the flanking sequences of the CLE domain in the CLV3 gene were deleted without affecting the function of CLV3 [27]. Further studies showed that the treatment of Arabidopsis seedlings with synthetic CLE peptides reduced the root apical meristem and inhibited root growth, showing a phenotype similar to the overexpression of CLE genes [25,26]. In addition, studies have found that ectopic expression of CLE19 and CLE40 in Arabidopsis roots can promote the differentiation of root apical meristem stem cells, and the in vitro application of CLE19 and CLE40 peptides has the same effects [54,55]. These studies have shown that the core sequence of twelve amino acids in the CLE domain is the true active form of the CLE proteins and the smallest structural form required for their functions [56,57,58]. In this study, by comparing the twelve amino acid core domain of tobacco CLEs with other species, it was found that eight residues within the CLE motif sequences were more conservative (Figure 2B), which is similar to CLE domain sequences from other plant species [9,23].
Osmotic stress and salinity are abiotic stress factors affecting plant growth and crop yield [59]. Studying the mechanisms of plants’ responses to osmotic stress and salinity for the improvement of agricultural production and ecological harmony is of great significance. At present, many genes related to osmotic stress tolerance and salinity stress tolerance in plants have been cloned and used in transgenic technology for increasing stress tolerance in crops [59]. As important regulatory signals in stress response, synthetic peptides can be applied to plants to improve stress tolerance without genetic modification of the plants.
Several plant peptide signals have been characterized as being involved in stress responses [7,13,60]. In addition to CLE members CLE25, CLE9, and CLE14, which function as regulators of stress tolerance in Arabidopsis [37,40,41], members in the CEP family [61], RALFL family [62], Pep family [63], CAPE family [64], PIP family [65], and SCREW family [66] have been reported to be involved in plants’ responses to abiotic stresses. Chemically synthesized peptides with regulatory roles in stress responses can be potentially used as plant protection agents to improve stress tolerance. In a recent study, StPep1 peptides were synthesized in Bacillus subtilis, and the application of the biosynthesized peptides promoted potato resistance against root-knot nematodes [67]. The biosynthetic approach of producing peptides significantly reduced costs, and this could greatly foster the use of peptide agents in agriculture. In this study, the exogenous application of CLE peptides enhanced tobacco’s tolerance to osmotic stress and salinity (Figure 6 and Figure 7). These peptides can be potentially used in tobacco production for agricultural improvement.

5. Conclusions

Through genome-wide analysis, we identified 41 NtCLE genes, 19 NsylCLE genes, and 24 NtomCLE genes from three Nicotiana species. Phylogenic analysis showed that the Nicotiana CLEs, together with CLEs from Arabidopsis and tomato, could be divided into 9 subgroups. A number of NtCLEs showed differential expression upon osmotic stress and salinity treatments. Synthetic peptides of several NtCLEs improved stress tolerance of tobacco leaf discs when exogenously applied.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13061480/s1, Figure S1: Screen of suitable NaCl and mannitol concentrations; Table S1: Primers for qRT-PCR.

Author Contributions

Conceptualization, X.G. and Y.G.; Data curation, Y.C., X.G. and L.W., Z.D. and T.L.; Formal analysis, Y.C.; Funding acquisition, Y.G.; Methodology, Y.C.; Writing—original draft, Y.C. and X.G.; Writing—review & editing, Y.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Agricultural Science and Technology Innovation Program, grant number ASTIP-TRIC02 and by National Natural Science Foundation of China, grant number 32270332.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author Y.G. upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Olsson, V.; Joos, L.; Zhu, S.; Gevaert, K.; Butenko, M.A.; De Smet, I. Look Closely, the Beautiful May Be Small: Precursor-Derived Peptides in Plants. Annu. Rev. Plant Biol. 2019, 70, 153–186. [Google Scholar] [CrossRef] [Green Version]
  2. Pearce, G.; Strydom, D.; Johnson, S.; Ryan, C.A. A Polypeptide from Tomato Leaves Induces Wound-Inducible Proteinase Inhibitor Proteins. Science 1991, 253, 895–897. [Google Scholar] [CrossRef] [PubMed]
  3. Lease, K.A.; Walker, J.C. The Arabidopsis unannotated secreted peptide database, a resource for plant peptidomics. Plant Physiol. 2006, 142, 831–838. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Boschiero, C.; Dai, X.; Lundquist, P.K.; Roy, S.; Christian de Bang, T.; Zhang, S.; Zhuang, Z.; Torres-Jerez, I.; Udvardi, M.K.; Scheible, W.R.; et al. MtSSPdb: The Medicago truncatula Small Secreted Peptide Database. Plant Physiol. 2020, 183, 399–413. [Google Scholar] [CrossRef] [Green Version]
  5. Pan, B.; Sheng, J.; Sun, W.; Zhao, Y.; Hao, P.; Li, X. OrysPSSP: A comparative platform for small secreted proteins from rice and other plants. Nucleic Acids Res. 2013, 41, D1192–D1198. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Li, Y.L.; Dai, X.R.; Yue, X.; Gao, X.-Q.; Zhang, X.S. Identification of small secreted peptides (SSPs) in maize and expression analysis of partial SSP genes in reproductive tissues. Planta 2014, 240, 713–728. [Google Scholar] [CrossRef]
  7. Tian, D.; Xie, Q.; Deng, Z.; Xue, J.; Li, W.; Zhang, Z.; Dai, Y.; Zheng, B.; Lu, T.; De Smet, I.; et al. Small secreted peptides encoded on the wheat (Triticum aestivum L.) genome and their potential roles in stress responses. Front. Plant Sci. 2022, 13, 1000297. [Google Scholar] [CrossRef]
  8. Murphy, E.; Smith, S.; De Smet, I. Small Signaling Peptides in Arabidopsis Development: How Cells Communicate Over a Short Distance. Plant Cell 2012, 24, 3198–3217. [Google Scholar] [CrossRef] [Green Version]
  9. Gao, X.; Guo, Y. CLE peptides in plants: Proteolytic processing, structure-activity relationship, and ligand-receptor interaction. J. Integr. Plant Biol. 2012, 54, 738–745. [Google Scholar] [CrossRef]
  10. Fletcher, L.C.; Brand, U.; Running, M.P.; Simon, R.; Meyerowitz, E.M. Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. Science 1999, 283, 1911–1914. [Google Scholar] [CrossRef]
  11. Opsahl-Ferstad, H.G.; Le Deunff, E.; Dumas, C.; Rogowsky, P.M. ZmEsr, a novel endosperm-specific gene expressed in a restricted region around the maize embryo. Plant J. Cell Mol. Biol. 1997, 12, 235–246. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Willoughby, A.C.; Nimchuk, Z.L. WOX going on: CLE peptides in plant development. Curr. Opin. Plant Biol. 2021, 63, 102056. [Google Scholar] [CrossRef]
  13. Xie, H.; Zhao, W.; Li, W.; Zhang, Y.; Hajný, J.; Han, H. Small signaling peptides mediate plant adaptions to abiotic environmental stress. Planta 2022, 255, 72. [Google Scholar] [CrossRef]
  14. Carbonnel, S.; Falquet, L.; Hazak, O. Deeper genomic insights into tomato CLE genes repertoire identify new active peptides. BMC Genom. 2022, 23, 756. [Google Scholar] [CrossRef]
  15. Gancheva, M.S.; Losev, M.R.; Gurina, A.A.; Poliushkevich, L.O.; Dodueva, I.E.; Lutova, L.A. Polymorphism of CLE gene sequences in potato. Vavilovskii Zh Genet. 2021, 25, 746–753. [Google Scholar] [CrossRef] [PubMed]
  16. Han, S.; Khan, M.H.U.; Yang, Y.; Zhu, K.; Li, H.; Zhu, M.; Amoo, O.; Khan, S.U.; Fan, C.; Zhou, Y. Identification and comprehensive analysis of the CLV3/ESR-related (CLE) gene family in Brassica napus L. Plant Biol. 2020, 22, 709–721. [Google Scholar] [CrossRef] [PubMed]
  17. Han, H.; Zhang, G.; Wu, M.; Wang, G. Identification and characterization of the Populus trichocarpa CLE family. BMC Genom. 2016, 17, 174. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Wan, K.; Lu, K.; Gao, M.; Zhao, T.; He, Y.; Yang, D.L.; Tao, X.; Xiong, G.; Guan, X. Functional analysis of the cotton CLE polypeptide signaling gene family in plant growth and development. Sci. Rep. 2021, 11, 5060. [Google Scholar] [CrossRef] [PubMed]
  19. Qin, N.; Gao, Y.; Cheng, X.; Yang, Y.; Wu, J.; Wang, J.; Li, S.; Xing, G. Genome-wide identification of CLE gene family and their potential roles in bolting and fruit bearing in cucumber (Cucumis sativus L.). BMC Plant Biol. 2021, 21, 143. [Google Scholar] [CrossRef]
  20. Li, Z.; Liu, D.; Xia, Y.; Li, Z.; Niu, N.; Ma, S.; Wang, J.; Song, Y.; Zhang, G. Identification and Functional Analysis of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Wheat. Int. J. Mol. Sci. 2019, 20, 4319. [Google Scholar] [CrossRef] [Green Version]
  21. Cock, J.M.; McCormick, S. A large family of genes that share homology with CLAVATA3. Plant Physiol. 2001, 126, 939–942. [Google Scholar] [CrossRef] [Green Version]
  22. Strabala, T.J.; O’Donnell, P.J.; Smit, A.M.; Ampomah-Dwamena, C.; Martin, E.J.; Netzler, N.; Nieuwenhuizen, N.J.; Quinn, B.D.; Foote, H.C.C.; Hudson, K.R. Gain-of-function phenotypes of many CLAVATA3/ESR genes, including four new family members, correlate with tandem variations in the conserved CLAVATA3/ESR domain. Plant Physiol. 2006, 140, 1331–1344. [Google Scholar] [CrossRef] [Green Version]
  23. Oelkers, K.; Goffard, N.; Weiller, G.F.; Gresshoff, P.M.; Mathesius, U.; Frickey, T. Bioinformatic analysis of the CLE signaling peptide family. BMC Plant Biol. 2008, 8, 1–15. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Kinoshita, A.; Nakamura, Y.; Sasaki, E.; Kyozuka, J.; Fukuda, H.; Sawa, S. Gain-of-function phenotypes of chemically synthetic CLAVATA3/ESR-related (CLE) peptides in Arabidopsis thaliana and Oryza sativa. Plant Cell Physiol. 2007, 48, 1821–1825. [Google Scholar] [CrossRef] [PubMed]
  25. Djordjevic, M.A.; Oakes, M.; Wong, C.E.; Singh, M.; Bhalla, P.; Kusumawati, L.; Imin, N. Border sequences of Medicago truncatula CLE36 are specifically cleaved by endoproteases common to the extracellular fluids of Medicago and soybean. J. Exp. Bot. 2011, 62, 4649–4659. [Google Scholar] [CrossRef]
  26. Ni, J.; Guo, Y.; Jin, H.; Hartsell, J.; Clark, S.E. Characterization of a CLE processing activity. Plant Mol. Biol. 2011, 75, 67–75. [Google Scholar] [CrossRef] [PubMed]
  27. Fiers, M.; Golemiec, E.; van der Schors, R.; van der Geest, L.; Li, K.W.; Stiekema, W.J.; Liu, C.M. The CLAVATA3/ESR motif of CLAVATA3 is functionally independent from the nonconserved flanking sequences. Plant Physiol. 2006, 141, 1284–1292. [Google Scholar] [CrossRef] [Green Version]
  28. Rojo, E.; Sharma, V.K.; Kovaleva, V.; Raikhel, N.V.; Fletcher, J.C. CLV3 Is Localized to the Extracellular Space, Where It Activates the Arabidopsis CLAVATA Stem Cell Signaling Pathway. Plant Cell 2002, 14, 969–977. [Google Scholar] [CrossRef] [Green Version]
  29. Song, X.F.; Guo, P.; Ren, S.C.; Xu, T.T.; Liu, C.M. Antagonistic Peptide Technology for Functional Dissection of CLV3/ESR Genes in Arabidopsis. Plant Physiol. 2013, 161, 1076–1085. [Google Scholar] [CrossRef] [Green Version]
  30. Clark, S.E.; Running, M.P.; Meyerowitz, E.M. Clavata3 Is a Specific Regulator of Shoot and Floral Meristem Development Affecting the Same Processes as Clavata1. Development 1995, 121, 2057–2067. [Google Scholar] [CrossRef]
  31. Clark, S.E. Cell signalling at the shoot meristem. Nat. Rev. Mol. Cell Biol. 2001, 2, 276–284. [Google Scholar] [CrossRef] [PubMed]
  32. Guo, Y.; Han, L.; Hymes, M.; Denver, R.; Clark, S.E. CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification. Plant J. 2010, 63, 889–900. [Google Scholar] [CrossRef] [Green Version]
  33. Sarkar, A.K.; Luijten, M.; Miyashima, S.; Lenhard, M.; Hashimoto, T.; Nakajima, K.; Scheres, B.; Heidstra, R.; Laux, T. Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature 2007, 446, 811–814. [Google Scholar] [CrossRef] [PubMed]
  34. Stahl, Y.; Wink, R.H.; Ingram, G.C.; Simon, R. A Signaling Module Controlling the Stem Cell Niche in Arabidopsis Root Meristems. Curr. Biol. 2009, 19, 909–914. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Ito, Y.; Nakanomyo, I.; Motose, H.; Iwamoto, K.; Sawa, S.; Dohmae, N.; Fukuda, H. Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science 2006, 313, 842–845. [Google Scholar] [CrossRef] [PubMed]
  36. Hirakawa, Y.; Kondo, Y.; Fukuda, H. TDIF Peptide Signaling Regulates Vascular Stem Cell Proliferation via the WOX4 Homeobox Gene in Arabidopsis. Plant Cell 2010, 22, 2618–2629. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Zhang, Z.; Liu, C.; Li, K.; Li, X.; Xu, M.; Guo, Y. CLE14 functions as a “brake signal” to suppress age-dependent and stress-induced leaf senescence by promoting JUB1-mediated ROS scavenging in Arabidopsis. Mol. Plant 2022, 15, 179–188. [Google Scholar] [CrossRef]
  38. Zhang, Y.; Tan, S.; Gao, Y.; Kan, C.; Wang, H.L.; Yang, Q.; Xia, X.; Ishida, T.; Sawa, S.; Guo, H.; et al. CLE42 delays leaf senescence by antagonizing ethylene pathway in Arabidopsis. New Phytol. 2022, 235, 550–562. [Google Scholar] [CrossRef] [PubMed]
  39. Endo, S.; Shinohara, H.; Matsubayashi, Y.; Fukuda, H. A novel pollen-pistil interaction conferring high-temperature tolerance during reproduction via CLE45 signaling. Curr. Biol. 2013, 23, 1670–1676. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  40. Takahashi, F.; Suzuki, T.; Osakabe, Y.; Betsuyaku, S.; Kondo, Y.; Dohmae, N.; Fukuda, H.; Yamaguchi-Shinozaki, K.; Shinozaki, K. A small peptide modulates stomatal control via abscisic acid in long-distance signalling. Nature 2018, 556, 235–238. [Google Scholar] [CrossRef]
  41. Zhang, L.S.; Shi, X.; Zhang, Y.T.; Wang, J.J.; Yang, J.W.; Ishida, T.; Jiang, W.Q.; Han, X.Y.; Kang, J.K.; Wang, X.N.; et al. CLE9 peptide-induced stomatal closure is mediated by abscisic acid, hydrogen peroxide, and nitric oxide in Arabidopsis thaliana. Plant Cell Environ. 2019, 42, 1033–1044. [Google Scholar] [CrossRef] [PubMed]
  42. Araya, T.; Miyamoto, M.; Wibowo, J.; Suzuki, A.; Kojima, S.; Tsuchiya, Y.N.; Sawa, S.; Fukuda, H.; von Wiren, N.; Takahashi, H. CLE-CLAVATA1 peptide-receptor signaling module regulates the expansion of plant root systems in a nitrogen-dependent manner. Proc. Natl. Acad. Sci. USA 2014, 111, 2029–2034. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Dong, W.; Wang, Y.H.; Takahashi, H. CLE-CLAVATA1 Signaling Pathway Modulates Lateral Root Development under Sulfur Deficiency. Plants 2019, 8, 103. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Gutierrez-Alanis, D.; Yong-Villalobos, L.; Jimenez-Sandoval, P.; Alatorre-Cobos, F.; Oropeza-Aburto, A.; Mora-Macias, J.; Sanchez-Rodriguez, F.; Cruz-Ramirez, A.; Herrera-Estrella, L. Phosphate Starvation-Dependent Iron Mobilization Induces CLE14 Expression to Trigger Root Meristem Differentiation through CLV2/PEPR2 Signaling. Dev. Cell 2017, 41, 555. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Monreal-Escalante, E.; Ramos-Vega, A.A.; Salazar-Gonzalez, J.A.; Banuelos-Hernandez, B.; Angulo, C.; Rosales-Mendoza, S. Expression of the VP40 antigen from the Zaire ebolavirus in tobacco plants. Planta 2017, 246, 123–132. [Google Scholar] [CrossRef]
  46. Budzianowski, J. Tobacco against Ebola virus disease. Prz. Lek. 2015, 72, 567–571. [Google Scholar]
  47. Andrianov, V.; Borisjuk, N.; Pogrebnyak, N.; Brinker, A.; Dixon, J.; Spitsin, S.; Flynn, J.; Matyszczuk, P.; Andryszak, K.; Laurelli, M.; et al. Tobacco as a production platform for biofuel: Overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass. Plant Biotechnol. J. 2010, 8, 277–287. [Google Scholar] [CrossRef]
  48. Vanhercke, T.; El Tahchy, A.; Liu, Q.; Zhou, X.R.; Shrestha, P.; Divi, U.K.; Ral, J.P.; Mansour, M.P.; Nichols, P.D.; James, C.N.; et al. Metabolic engineering of biomass for high energy density: Oilseed-like triacylglycerol yields from plant leaves. Plant Biotechnol. J. 2014, 12, 231–239. [Google Scholar] [CrossRef] [PubMed]
  49. Leitch, I.J.; Hanson, L.; Lim, K.Y.; Kovarik, A.; Chase, M.W.; Clarkson, J.J.; Leitch, A.R. The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae). Ann. Bot. 2008, 101, 805–814. [Google Scholar] [CrossRef] [Green Version]
  50. Renny-Byfield, S.; Chester, M.; Kovarik, A.; Le Comber, S.C.; Grandbastien, M.A.; Deloger, M.; Nichols, R.A.; Macas, J.; Novak, P.; Chase, M.W.; et al. Next generation sequencing reveals genome downsizing in allotetraploid Nicotiana tabacum, predominantly through the elimination of paternally derived repetitive DNAs. Mol. Biol. Evol. 2011, 28, 2843–2854. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Zhang, Y.; Yang, S.; Song, Y.; Wang, J. Genome-wide characterization, expression and functional analysis of CLV3/ESR gene family in tomato. BMC Genom. 2014, 15, 827. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Guo, Y.F.; Gan, S.S. AtNAP, a NAC family transcription factor, has an important role in leaf senescence. Plant J. Cell Mol. Biol. 2006, 46, 601–612. [Google Scholar] [CrossRef] [PubMed]
  54. Fiers, M.; Hause, G.; Boutilier, K.; Casamitjana-Martinez, E.; Weijers, D.; Offringa, R.; van der Geest, L.; van Lookeren Campagne, M.; Liu, C.M. Mis-expression of the CLV3/ESR-like gene CLE19 in Arabidopsis leads to a consumption of root meristem. Gene 2004, 327, 37–49. [Google Scholar] [CrossRef]
  55. Fiers, M.; Golemiec, E.; Xu, J.; Geest, L.; Heidstra, R.; Stiekema, W.; Liu, C.M. The 14–Amino Acid CLV3, CLE19, and CLE40 Peptides Trigger Consumption of the Root Meristem in Arabidopsis through a CLAVATA2-Dependent Pathway. Am. Soc. Plant Biol. 2005, 17, 2542–2553. [Google Scholar]
  56. Song, X.F.; Yu, D.L.; Xu, T.T.; Ren, S.C.; Guo, P.; Liu, C.M. Contributions of Individual Amino Acid Residues to the Endogenous CLV3 Function in Shoot Apical Meristem Maintenance in Arabidopsis. Mol. Plant 2012, 5, 9. [Google Scholar] [CrossRef] [Green Version]
  57. Kondo, T.; Sawa, S.; Kinoshita, A.; Mizuno, S.; Kakimoto, T.; Fukuda, H.; Sakagami, Y. A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis. Science 2006, 313, 845–848. [Google Scholar] [CrossRef] [PubMed]
  58. Ohyama, K.; Shinohara, H.; Ogawa-Ohnishi, M.; Matsubayashi, Y. A glycopeptide regulating stem cell fate in Arabidopsis thaliana. Nat. Chem. Biol. 2009, 5, 578–580. [Google Scholar] [CrossRef] [PubMed]
  59. Parmar, N.; Singh, K.H.; Sharma, D.; Singh, L.; Kumar, P.; Nanjundan, J.; Khan, Y.J.; Chauhan, D.K.; Thakur, A.K. Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: A comprehensive review. 3 Biotech 2017, 7, 239. [Google Scholar] [CrossRef]
  60. Chen, Y.L.; Fan, K.T.; Hung, S.C.; Chen, Y.R. The role of peptides cleaved from protein precursors in eliciting plant stress reactions. New Phytol. 2020, 225, 2267–2282. [Google Scholar] [CrossRef] [Green Version]
  61. Smith, S.; Zhu, S.S.; Joos, L.; Roberts, I.; Nikonorova, N.; Vu, L.D.; Stes, E.; Cho, H.; Larrieu, A.; Xuan, W.; et al. The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis. Mol. Cell Proteom. 2020, 19, 1248–1262. [Google Scholar] [CrossRef]
  62. Atkinson, N.J.; Lilley, C.J.; Urwin, P.E. Identification of Genes Involved in the Response of Arabidopsis to Simultaneous Biotic and Abiotic Stresses. Plant Physiol. 2013, 162, 2028–2041. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Nakaminami, K.; Okamoto, M.; Higuchi-Takeuchi, M.; Yoshizumi, T.; Yamaguchi, Y.; Fukao, Y.; Shimizu, M.; Ohashi, C.; Tanaka, M.; Matsui, M.; et al. AtPep3 is a hormone-like peptide that plays a role in the salinity stress tolerance of plants. Proc. Natl. Acad. Sci. USA 2018, 115, 5810–5815. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Chien, P.S.; Nam, H.G.; Chen, Y.R. A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis. J. Exp. Bot. 2015, 66, 5301–5313. [Google Scholar] [CrossRef] [Green Version]
  65. Zhou, H.P.; Xiao, F.; Zheng, Y.; Liu, G.Y.; Zhuang, Y.F.; Wang, Z.Y.; Zhang, Y.Y.; He, J.X.; Fu, C.X.; Lin, H.H. PAMP-INDUCED SECRETED PEPTIDE 3 modulates salt tolerance through RECEPTOR-LIKE KINASE 7 in plants. Plant Cell 2022, 34, 927–944. [Google Scholar] [CrossRef]
  66. Liu, Z.Y.; Hou, S.G.; Rodrigues, O.; Wang, P.; Luo, D.X.; Munemasa, S.; Lei, J.X.; Liu, J.; Ortiz-Morea, F.A.; Wang, X.; et al. Phytocytokine signalling reopens stomata in plant immunity and water loss. Nature 2022, 605, 332. [Google Scholar] [CrossRef]
  67. Zhang, L.; Gleason, C. Enhancing potato resistance against root-knot nematodes using a plant-defence elicitor delivered by bacteria. Nat. Plants 2020, 6, 625–629. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Schematic diagram of the genomic structures of common tobacco CLE family members (exons are shown as green boxes and introns are shown as grey lines).
Figure 1. Schematic diagram of the genomic structures of common tobacco CLE family members (exons are shown as green boxes and introns are shown as grey lines).
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Figure 2. Characteristics of the tobacco CLEs. (A) Multiple sequence alignment of tobacco CLE proteins. The conserved CLE domains are marked in the red box. (B) Weblogo of conserved motifs identified in tobacco CLEs. The height of the bars indicates the number of identical residues per position. (C) Conserved motifs of tobacco CLEs.
Figure 2. Characteristics of the tobacco CLEs. (A) Multiple sequence alignment of tobacco CLE proteins. The conserved CLE domains are marked in the red box. (B) Weblogo of conserved motifs identified in tobacco CLEs. The height of the bars indicates the number of identical residues per position. (C) Conserved motifs of tobacco CLEs.
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Figure 3. Phylogenetic analysis of Nicotiana CLEs, AtCLEs, and SICLEs. The tree was generated from the aligment of the CLE motifs of all the CLE proteins from Arabidopsis, tomato, and three Nicotiana species. The 9 distinct identity subgroups are shown by colored branches. Homoeologous genes consistently cluster together with high confidence (indicated by high bootstrap values). The 9 subgroups (Group A–I) were assigned based on clustering in the tree, in addition to sequence similarity. The tree is shown with bootstrap confidence values expressed as a percentage from 500 bootstrap replications.
Figure 3. Phylogenetic analysis of Nicotiana CLEs, AtCLEs, and SICLEs. The tree was generated from the aligment of the CLE motifs of all the CLE proteins from Arabidopsis, tomato, and three Nicotiana species. The 9 distinct identity subgroups are shown by colored branches. Homoeologous genes consistently cluster together with high confidence (indicated by high bootstrap values). The 9 subgroups (Group A–I) were assigned based on clustering in the tree, in addition to sequence similarity. The tree is shown with bootstrap confidence values expressed as a percentage from 500 bootstrap replications.
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Figure 4. Expression of representative NtCLEs in different tissues. WP is an abbreviation of “whole plant at the seedling stage”. Different lowercase letters indicate significant differences at the level of p = 0.05.
Figure 4. Expression of representative NtCLEs in different tissues. WP is an abbreviation of “whole plant at the seedling stage”. Different lowercase letters indicate significant differences at the level of p = 0.05.
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Figure 5. qRT-PCR analysis of NtCLEs under different stress treatments. (A) Osmotic stress treatments. (B) Salinity treatments. Different lowercase letters indicate significant differences at the level of p = 0.05.
Figure 5. qRT-PCR analysis of NtCLEs under different stress treatments. (A) Osmotic stress treatments. (B) Salinity treatments. Different lowercase letters indicate significant differences at the level of p = 0.05.
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Figure 6. The role of CLE peptides in osmotic stress responses. Tobacco leaf discs were used in tesing whether the synthesized CLE peptides have an effect on tobacco osmotic stress tolerance. Pictures in the left column (A1,E1,I1) (A2,E2,I2) (A3,E3,I3) represent the results of adding ddH2O as a control. Pictures in the second column (B1,F1,J1) (B2,F2,J2) (B3,F3,J3) represent the results of adding 300 mM mannitol only, and pictures in the third column (C1,G1,K1) (C2,G2,K2) (C3,G3,K3) represent the results of adding 300 mM mannitol plus 1 µM of peptide. Pictures in the fourth column (D1,H1,L1) (D2,H2,L2) (D3,H3,L3) represent the results of adding 1 µM of peptide only. Photographs were taken 0 days (A1–D1) (A2–D2) (A3–D3), 4 days (E1–H1) (E2–H2) (E3–H3), and 8 days after treatments (I1–L1) (I2–L2) (I3–L3). NtCLE29 (HVVPGGPDPLHN). NtCLE12 (RLVPTGPNPLHH). NtCLE4 (RRVPTGPNAIHN). Different lowercase letters indicate significant differences at the level of p = 0.05.
Figure 6. The role of CLE peptides in osmotic stress responses. Tobacco leaf discs were used in tesing whether the synthesized CLE peptides have an effect on tobacco osmotic stress tolerance. Pictures in the left column (A1,E1,I1) (A2,E2,I2) (A3,E3,I3) represent the results of adding ddH2O as a control. Pictures in the second column (B1,F1,J1) (B2,F2,J2) (B3,F3,J3) represent the results of adding 300 mM mannitol only, and pictures in the third column (C1,G1,K1) (C2,G2,K2) (C3,G3,K3) represent the results of adding 300 mM mannitol plus 1 µM of peptide. Pictures in the fourth column (D1,H1,L1) (D2,H2,L2) (D3,H3,L3) represent the results of adding 1 µM of peptide only. Photographs were taken 0 days (A1–D1) (A2–D2) (A3–D3), 4 days (E1–H1) (E2–H2) (E3–H3), and 8 days after treatments (I1–L1) (I2–L2) (I3–L3). NtCLE29 (HVVPGGPDPLHN). NtCLE12 (RLVPTGPNPLHH). NtCLE4 (RRVPTGPNAIHN). Different lowercase letters indicate significant differences at the level of p = 0.05.
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Figure 7. The role of CLE peptides in salinity stress responses. Tobacco leaf discs were used in tesing whether the synthesized CLE peptides have an effect on tobacco salinity stress tolerance. Pictures in the left column (A1,E1) (A2,E2) (A3,E3) represent the results of adding ddH2O as a control. Pictures in the second column (B1,F1) (B2,F2) (B3,F3) represent the results of adding 150 mM NaCl only, and pictures in the third column (C1,G1) (C2,G2) (C3,G3) represent the results of adding 150 mM NaCl plus 1 µM of peptide. Pictures in the fourth column (D1,H1) (D2,H2) (D3,H3) represent the results of adding 1 µM of peptide only. Photographs were taken 0 days (A1–D1) (A2,D2) (A3,D3) and 4 days after treatments (E1–H1) (E2,H2) (E3.H3). NtCLE29 (HVVPGGPDPLHN). NtCLE12 (RLVPTGPNPLHH). NtCLE4 (RRVPTGPNAIHN). Different lowercase letters indicate significant difference at the level of p = 0.05.
Figure 7. The role of CLE peptides in salinity stress responses. Tobacco leaf discs were used in tesing whether the synthesized CLE peptides have an effect on tobacco salinity stress tolerance. Pictures in the left column (A1,E1) (A2,E2) (A3,E3) represent the results of adding ddH2O as a control. Pictures in the second column (B1,F1) (B2,F2) (B3,F3) represent the results of adding 150 mM NaCl only, and pictures in the third column (C1,G1) (C2,G2) (C3,G3) represent the results of adding 150 mM NaCl plus 1 µM of peptide. Pictures in the fourth column (D1,H1) (D2,H2) (D3,H3) represent the results of adding 1 µM of peptide only. Photographs were taken 0 days (A1–D1) (A2,D2) (A3,D3) and 4 days after treatments (E1–H1) (E2,H2) (E3.H3). NtCLE29 (HVVPGGPDPLHN). NtCLE12 (RLVPTGPNPLHH). NtCLE4 (RRVPTGPNAIHN). Different lowercase letters indicate significant difference at the level of p = 0.05.
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Table
Table 1. Detailed characteristics of tobacco CLEs.
Table 1. Detailed characteristics of tobacco CLEs.
Gene NumberProtein IDNumber of Amino AcidsMolecular WeightTheoretical pISignal PeptideGrand Average of HydropathicityInstability IndexAliphatic Index
NtCLE1Ntab0794960.19410,569.3911Yes−0.25165.295.43
NtCLE2Ntab0439970.19410,514.3610.7Yes−0.2257.6198.51
NtCLE3Ntab0614790.19410,575.469.78Yes−0.02989.44101.7
NtCLE4Ntab0869460.1839544.9610.38No−0.84628.3775.3
NtCLE5Ntab0636720.1515408.039.96No−0.83532.4451.57
NtCLE6Ntab0578590.117619,906.7810.42No−0.22635.3198.64
NtCLE7Ntab0047380.115817,155.339.27No−0.83343.259.87
NtCLE8Ntab0059110.110611,922.7610.65Yes−0.28351.8487.45
NtCLE9Ntab0437050.110712,226.1911.67No−0.38847.9780.84
NtCLE10Ntab0468120.19010,261.6910.28No−0.90640.7168.33
NtCLE11Ntab0806210.110912,760.839.84No−0.41342.4284.86
NtCLE12Ntab0572710.11111292410No−0.45643.7785.14
NtCLE13Ntab0797810.110511,621.3210.03Yes−0.31934.7278
NtCLE14Ntab0422310.110511,621.3210.03Yes−0.31934.7278
NtCLE15Ntab0788230.112113,530.589.08Yes−0.40178.9370.99
NtCLE16Ntab0955370.1818988.416.25Yes−0.12559.9775.8
NtCLE17Ntab0954490.18610,045.6710.28Yes−0.65569.6379.3
NtCLE18Ntab0467670.18610,045.6710.28Yes−0.65569.6379.3
NtCLE19Ntab0710930.19310,708.611.49No−0.5959.2879.57
NtCLE20Ntab0916110.19310,716.7111.67No−0.42767.7787.96
NtCLE21Ntab0110010.1718186.5810.17Yes−0.36279.0476.9
NtCLE22Ntab0776730.1718439.0410.28Yes−0.30671.1985.21
NtCLE23Ntab0923360.1717813.1212.31Yes−0.13264.4389.3
NtCLE24Ntab0197240.1728023.3512.01Yes−0.07272.1885.42
NtCLE25Ntab0962310.1788993.4111.64Yes−0.39756.8565.13
NtCLE26Ntab0934300.1788929.4312.13Yes−0.25353.3765.49
NtCLE27Ntab0086070.1839162.9212.02Yes−0.08960.3376.39
NtCLE28Ntab0219740.19510,837.5810.42Yes−0.65344.2968.95
NtCLE29Ntab0218060.1889559.119.87No039.0795.45
NtCLE30Ntab0643500.1798823.016.23Yes−0.22779.7392.53
NtCLE31Ntab0293510.1859889.429.69Yes−0.30762.7582.59
NtCLE32Ntab0933210.19310,837.7110.06Yes−0.32828.7185.91
NtCLE33Ntab0701160.110611,238.628.14Yes−0.17930.573.77
NtCLE34Ntab0316120.19010,020.588.89Yes−0.00464.1981.33
NtCLE35Ntab0123790.1879726.218.82Yes−0.11765.3387.47
NtCLE36Ntab0302810.1829093.59.03Yes−0.10245.68106.95
NtCLE37Ntab0489230.19410,398.410.24No−0.55562.0156.06
NtCLE38Ntab0151960.19110,195.5310.44No−0.5342.7162.2
NtCLE39Ntab0586090.110411,392.028.66Yes−0.12845.7886.35
NtCLE40Ntab0934130.1788929.4312.13Yes−0.25353.3783.71
NtCLE41Ntab0359830.110511,338.785.66Yes−0.18739.8883.71
NsylCLE1Nsyl0128110.110411,402.058.66Yes−0.13646.386.35
NsylCLE2Nsyl0355150.110411,436.068.66Yes−0.14543.4182.6
NsylCLE3Nsyl0143890.1535321.65.35No−0.69835.1338.87
NsylCLE4Nsyl0228420.1505608.4411.41Yes−1.11272.9487.75
NsylCLE5Nsyl0213440.1535321.65.35No−0.69835.1386.11
NsylCLE6Nsyl0219800.18910,061.529.25Yes−0.6441.9468.73
NsylCLE7Nsyl0072460.19510,728.5511.32No−0.30671.5185.19
NsylCLE8Nsyl0151300.110211,565.949.25No−0.57154.7165.63
NsylCLE9Nsyl0153010.1798762.896.02Yes−0.26687.1984.72
NsylCLE10Nsyl0087640.111913,332.349.36Yes−0.4575.1584.86
NsylCLE11Nsyl0348610.110812,989.919.65No−0.64148.7184.39
NsylCLE12Nsyl0173530.110912,730.819.84No−0.40938.3974.18
NsylCLE13Nsyl0440460.1829341.929.73Yes−0.1726.9984.39
NsylCLE14Nsyl0324930.112213,358.866.65No−0.79963.3874.18
NsylCLE15Nsyl0295080.19410,672.399.35No−0.28654.186.06
NsylCLE16Nsyl0050760.1657215.2511.04No−1.11743.1368.92
NsylCLE17Nsyl0368090.1778443.619.36Yes−0.42113.4871.04
NsylCLE18Nsyl0196480.117619,938.810.02Yes−0.66133.2573.75
NsylCLE19Nsyl0085490.19410,672.399.35Yes−0.28654.186.06
NtomCLE1Ntom0043790.110011,325.179.51Yes−0.0946.2691.6
NtomCLE2Ntom0180900.112714,069.2910.67Yes−0.4838.0486.05
NtomCLE3Ntom0272530.112112,573.139.6Yes−0.1640.168.68
NtomCLE4Ntom0132020.110511,437.926.55Yes−0.22639.9783.71
NtomCLE5Ntom0078650.19811,064.959.56Yes−0.38471.9186.53
NtomCLE6Ntom0236520.112113,529.599.25Yes−0.40178.9370.99
NtomCLE7Ntom0348130.111412,974.869.74Yes−0.45558.2182.98
NtomCLE8Ntom0005610.1718303.729.78Yes−0.3969.0282.39
NtomCLE9Ntom0065030.19410,575.469.78Yes−0.02989.44101.7
NtomCLE10Ntom0321940.19410,569.3911Yes−0.25165.295.43
NtomCLE11Ntom0106270.110211,589.089.73Yes−0.55454.2570.69
NtomCLE12Ntom0221410.117519,473.935.33No−0.65958.1881.43
NtomCLE13Ntom0272700.19010,187.879.42No−0.29162.3892
NtomCLE14Ntom0158810.113114,541.14.64No−0.73965.3459.39
NtomCLE15Ntom0275520.115016,466.254.57No−0.50971.3379.13
NtomCLE16Ntom0113520.115016,446.264.76No−0.58967.9276.53
NtomCLE17Ntom0144670.112413,505.954.34No−0.37269.3577.66
NtomCLE18Ntom0117310.116317,665.474.33No−0.58768.7166.26
NtomCLE19Ntom0019740.113114,765.424.58No−0.4358.2177.94
NtomCLE20Ntom0324220.19710,470.294.07No−0.65764.461.13
NtomCLE21Ntom0005610.1718303.729.78Yes−0.3969.0282.39
NtomCLE22Ntom0144670.112413,505.954.34No−0.37269.3577.66
NtomCLE23Ntom0272700.115016,466.254.57No−0.50971.3392
NtomCLE24Ntom0275520.115016,466.254.57No−0.50971.3379.13
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MDPI and ACS Style

Chu, Y.; Gao, X.; Wen, L.; Deng, Z.; Liu, T.; Guo, Y. Characterization of the CLE Family in Three Nicotiana Species and Potential Roles of CLE Peptides in Osmotic and Salt Stress Responses. Agronomy 2023, 13, 1480. https://doi.org/10.3390/agronomy13061480

AMA Style

Chu Y, Gao X, Wen L, Deng Z, Liu T, Guo Y. Characterization of the CLE Family in Three Nicotiana Species and Potential Roles of CLE Peptides in Osmotic and Salt Stress Responses. Agronomy. 2023; 13(6):1480. https://doi.org/10.3390/agronomy13061480

Chicago/Turabian Style

Chu, Yumeng, Xiaoming Gao, Lichao Wen, Zhichao Deng, Tao Liu, and Yongfeng Guo. 2023. "Characterization of the CLE Family in Three Nicotiana Species and Potential Roles of CLE Peptides in Osmotic and Salt Stress Responses" Agronomy 13, no. 6: 1480. https://doi.org/10.3390/agronomy13061480

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