Next Article in Journal
Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses
Next Article in Special Issue
Melatonin-Mediated Molecular Responses in Plants: Enhancing Stress Tolerance and Mitigating Environmental Challenges in Cereal Crop Production
Previous Article in Journal
Perineuronal Net Microscopy: From Brain Pathology to Artificial Intelligence
Previous Article in Special Issue
Comparative Physiological and Transcriptome Analyses of Tolerant and Susceptible Cultivars Reveal the Molecular Mechanism of Cold Tolerance in Anthurium andraeanum
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 8
10.3390/ijms25084231
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

A Genome-Wide Analysis of the CEP Gene Family in Cotton and a Functional Study of GhCEP46-D05 in Plant Development

by
Zhenyu Mei
1,†,
Bei Li
1,†,
Shouhong Zhu
1,
Yan Li
1,
Jinbo Yao
1,
Jingwen Pan
1,
Yongshan Zhang
1,2,* and
Wei Chen
1,*
1
National Key Laboratory of Cotton Bio-Breeding and Integrated Utilization, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang 455000, China
2
Zhengzhou Research Base, National Key Laboratory of Cotton Bio-Breeding and Integrated Utilization, School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, China
*
Authors to whom correspondence should be addressed.
†
These authors equally contributed to this work.
Int. J. Mol. Sci. 2024, 25(8), 4231; https://doi.org/10.3390/ijms25084231
Submission received: 14 March 2024 / Revised: 7 April 2024 / Accepted: 8 April 2024 / Published: 11 April 2024
(This article belongs to the Special Issue Molecular Mechanisms of Plant Abiotic Stress Tolerance)
Browse Figures
Versions Notes

Abstract

:
C-TERMINALLY ENCODED PEPTIDEs (CEPs) are a class of peptide hormones that have been shown in previous studies to play an important role in regulating the development and response to abiotic stress in model plants. However, their role in cotton is not well understood. In this study, we identified 54, 59, 34, and 35 CEP genes from Gossypium hirsutum (2n = 4x = 52, AD1), G. barbadense (AD2), G. arboreum (2n = 2X = 26, A2), and G. raimondii (2n = 2X = 26, D5), respectively. Sequence alignment and phylogenetic analyses indicate that cotton CEP proteins can be categorized into two subgroups based on the differentiation of their CEP domain. Chromosomal distribution and collinearity analyses show that most of the cotton CEP genes are situated in gene clusters, suggesting that segmental duplication may be a critical factor in CEP gene expansion. Expression pattern analyses showed that cotton CEP genes are widely expressed throughout the plant, with some genes exhibiting specific expression patterns. Ectopic expression of GhCEP46-D05 in Arabidopsis led to a significant reduction in both root length and seed size, resulting in a dwarf phenotype. Similarly, overexpression of GhCEP46-D05 in cotton resulted in reduced internode length and plant height. These findings provide a foundation for further investigation into the function of cotton CEP genes and their potential role in cotton breeding.

1. Introduction

Phytopeptide hormones play an essential role in regulating plant growth, development and adaptation in a non-cell-autonomous manner through intercellular communication [1,2,3]. The C-TERMINALLY ENCODED PEPTIDE (CEP) family is one of the most universal peptide classes [4]. The first CEP gene identified was AtCEP1, which was discovered using an in silico approach in Arabidopsis thaliana [5]. CEP genes are abundant in seed plants, but not in ancient land plant lineages that lack true branching roots or root vasculature [6]. To date, 16 members of the CEP family have been identified in Arabidopsis thaliana [6,7]. Each CEP consists of a signal peptide at the N-terminus and a certain number of conserved CEP domains at the C-terminus. In Arabidopsis, members of the CEP family have been shown to have distinct expression patterns during development, and the expression of some CEP genes could be regulated by environmental cues such as exogenous hormones, nutrients and stress.
Recent evidence has implicated CEPs in the regulation of plant development and response to abiotic stress in model plants [8]. For example, constitutive expression of AtCEP1 in Arabidopsis suppressed primary root growth and delayed lateral root elongation by inhibiting meristematic cell division and expansion [5,9]. In AtCEP3-overexpressing plants, primary root length was significantly reduced but shoots were enlarged, fewer rosette leaves of reduced size were differentiated, and flowering time was delayed. In addition, leaf morphology was altered (more flattened and rounded). AtCEP3 could be induced by nitrogen-limiting or high-salt conditions, and cep3 knockout lines showed increased root system size under these abiotic stress conditions [7,10]. Overexpression of OsCEP6.1, a CEP gene highly expressed in reproductive tissues in rice (Oryza sativa), causes multiple phenotypic variations, such as reduced primary root length and seedling height, fewer tillers, shorter panicle lengths and smaller flag leaves [11]. In addition, several rice CEP genes were found to be reduced by various abiotic stresses, and abiotic stress-responsive elements were enriched in their promoters [12]. ZmCEP1, a homolog of OsCEP6.1 in maize, is preferentially expressed in reproductive organs such as young ears and tassels. Overexpression of ZmCEP1 reduces plant height, spikelet size and kernel size. Kernel length and width are significantly increased in ZmCEP1 knockout lines, and plant and ear height are also slightly increased [13]. In Setaria italica, 14 CEP genes have been identified whose expression can be induced by various abiotic stresses (such as salt and drought) and phytohormones [such as abscisic acid (ABA)]. One of them, SiCEP3, was highly expressed in panicles. Overexpression of SiCEP3 inhibits plant growth, and exogenous synthetic SiCEP3 promotes ABA signalling by increasing ABA uptake [4,14]. Post-translational modification of CEPs also affects their activities. For example, in Medicago truncatula, overexpression of MtCEP1 inhibits lateral root formation but promotes nodulation. MtCEP1 with different hydroxylation patterns was found to exhibit differential inhibition of lateral root formation and altered patterns of auxin response [15].
Several components of the CEP signalling cascades have been identified, some of which are also involved in the signalling of other hormones. In Arabidopsis, two leucine-rich repeat receptor kinase (LRR-RK) genes have been identified as CEP receptors (CEPR1 and CEPR2). The CEP genes are induced by nitrogen starvation, and CEP peptides secreted in the root may act as long-distance root-to-shoot signals received by these two receptors [16]. CEPR2 can interact with and phosphorylate NITRATE TRANSPORTER 1.2 (NRT1.2), an ABA transporter with ABA import activity that positively regulates the ABA response [17]. In addition, CEPR2 interacts with and phosphorylates some members of the pyrabactin resistance 1 (PYR1)/PYR1-like (PYL) abscisic acid (ABA) receptors [18]. The cepr1 knockout mutants showed a reduction in vegetative and reproductive growth, and subsequently, seed yield and seed size were significantly reduced [19]. In Medicago truncatula, COMPACT ROOT ARCHITECTURE2 (CRA2), a homologue of CEPR1, is a receptor of MtCEP1. CRA2 interacts with MtCEP1 to regulate ethylene signalling [20]. Thus, CEP signalling usually shows crosstalk with other hormone signals.
Cotton is one of the most important fibre crops in China and the world. The improvement of cotton yield and quality is highly desired for cotton breeding [21]. Although CEPs have been shown to be important phytopeptide hormones that modulate plant or stress resistance in model plants, the role and function of CEP genes in cotton are still poorly understood. Identification and analysis of the phylogenetic relationships of CEP genes in cotton, their chromosomal locations, domain structures, collinearity, expression patterns and functions in cotton development would benefit the understanding of CEP genes and provide important gene resources for yield improvement in cotton. In this study, we performed a comprehensive analysis of CEP genes in four cotton (Gossypium) species (G. hirsutum, G. barbadense, G. raimondii and G. arboretum) and a functional study of GhCEP46-D05 was conducted to explore the potential role of cotton CEP genes in plant development.

2. Results

2.1. Genome-Wide Identification of CEP Genes

Using the amino acid sequences of 16 reported CEP proteins from Arabidopsis as queries in BLASTP, 46, 44, 57, and 72 candidate CEP genes were identified from G. arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. After filtering for the presence of signal peptides and CEP domains, 34, 35, 54 and 59 CEP genes were retained, respectively. In each genome, the identified CEP genes were renamed according to their chromosomal locations, with suffixes added to represent the chromosomes (GaCEP1-01~GaCEP34-14, GrCEP1-02~GrCEP35-13, GhCEP1-A01~GhCEP54-D13 and GbCEP1-A01~GbCEP59-D13).
The basic characteristics of the cotton CEP genes, such as gene length, exon number, molecular weight and isoelectric point of the protein, are listed in Table S1. Most of the cotton CEP genes have only one exon, and the others contain 2–5 exons. Most of the cotton CEPs encode small proteins (77–214 amino acids; 7.09–21.4 kDa), except for GbCEP2-A01 (546aa), GbCEP37-D03 (391aa) and GhCEP37-D03 (410aa). The theoretical isoelectric points and charges of the cotton CEP genes show a wide range of variation, with theoretical isoelectric points ranging from 4.45 to 11.48 and charges from −8.5 to 17.5.

2.2. CEP Gene Structure and Phylogenetic Tree Analysis

A phylogenetic analysis was performed using the complete protein sequences of Arabidopsis and cotton CEPs to construct a tree. Consistent with previous research, the resulting phylogenetic tree was separated into two distinct groups: Group I and Group II (see Figure 1 for details). Each group could be further subdivided into three subgroups. In Group I, all Arabidopsis CEP genes (AtCEP1–AtCEP12) were located in subgroup I-2, and subgroups I-1 and I-3 contained only cotton CEPs. Group II contained four Arabidopsis CEP genes (AtCEP13–AtCEP16); subgroup II-1 and subgroup II-3 each contained one Arabidopsis CEP gene (AtCEP16 and AtCEP15, respectively).
WebLogo plots showed that the CEP domains of the cotton CEP genes had a high amino acid similarity between two groups and that a core motif (SPG [V/A/I] G [H/N] in Group I and PSPG [V/A/I] G [H/N] in Group II) was present in both groups. Another motif (PT [T/A] PGH) was also found in front of the core motif in Group I (Figure 2). Thus, the CEP domains contain three prolines in Group I and two prolines in Group II. Previous studies have shown that hydroxyprolylation can affect the biological activity and hydrophilicity of CEP peptides [5,15].

2.3. Chromosomal Location and Collinearity Analysis of the CEP Gene in Cotton

The distribution of CEP genes is relatively scattered across the genome, with most of the chromosomes in the four cotton species harbouring CEPs (Figure 3). In G. arboreum, 34 CEP genes are located on 11 chromosomes, and the rest are located on two scaffolds. In the G. raimondii genome, a total of 35 CEP genes are found on all chromosomes, except Chr01 and Chr06. In G. hirsutum and G. barbadense, the distribution of CEP genes spans 20 and 21 chromosomes, respectively. It is noteworthy that more than half of the cotton CEP genes are located within gene clusters, which are defined as regions containing more than three genes in close proximity. For example, in G. barbadense, there are six gene clusters (containing 32 genes) located at the ends of three pairs of homologous chromosomes: A01/D01, A02/D03 and A05/D05.
The possible ancestors of G. hirsutum and G. barbadense are widely considered to be the two extant diploid cotton species, G. arboretum and G. raimondii [22]. To explore the evolutionary history of the CEP gene family in Gossypium, synteny analyses were performed to identify directly homologous CEP genes in the four cotton species (Figure 4). Collinearity analyses identified 20 directly homologous gene pairs between the D subgenome of G. hirsutum and G. raimondii, 21 directly homologous gene pairs between the A subgenome of G. hirsutum and G. arboreum, 11 directly homologous gene pairs between G. raimondii and G. arboreum, and 16 directly homologous gene pairs between the A and D subgenomes of G. hirsutum. These results indicate that the CEP family genes have been highly conserved during the evolution of cotton.

2.4. Analysis of the Expression Pattern of CEP Gene in G. hirsutum

The public RNA-seq data in G. hirsutum show that CEP genes in the same gene cluster tend to have the same expression pattern (Figure 5A). For example, the genes in gene clusters on A02/D03 (GhCEP08-A02~GhCEP14-A02 and GhCEP38-D03~GHCEP43-D03) are almost all preferentially expressed in the root. The genes in another pair of gene clusters on A01/D01 (GhCEP5-A01~GhCEP7-A01 and GhCEP32-D01~GHCEP35-D01) are preferentially expressed in the pistil. In addition, several CEP genes show high expression in the stem (GhCEP15-A03, GhCEP26-A11, GhCEP27-A11, GhCEP46-D05 and GhCEP47-D06). No CEP gene is preferentially expressed in fiber except for CEP51-D11.
Quantitative RT-PCR was used to further analyse the expression pattern of nine CEP genes. (Figure 5B). Consistent with the RNA-seq data, GhCEP08-A02 and GhCEP14-A02 were found to be preferentially expressed in roots, and GhCEP19-A05 was highly expressed in floral tissues, such as bract, sepal and anther. GhCEP46-D05 showed a high level of expression in the leaf and stem. Some discrepancies were found between RNA-seq and quantitative RT-PCR for some tissues or CEP genes, which could be due to experimental conditions or differences in the tissues collected.
GhCEP46-D05 was selected for further analysis of the expression pattern using promoter::GUS reporter lines (Figure 5C). Its expression was found in the shoot apex, leaf veins, leaf tips and stems.

2.5. Function of Cotton CEPs in Plant Development

To investigate the function of cotton CEP genes in plant growth and development, the GhCEP46-D05 gene was isolated and subsequently inserted into a construct downstream of the 35S promoter for overexpression. (p35S::GhCEP46-D05) in Arabidopsis Col-0. When grown on standard medium, a strong reduction in primary root length was observed in p35S::GhCEP46-D05 lines. The final plant height of these overexpression lines was also significantly reduced when compared with Col-0 (Figure 6A–D). These lines also showed an earlier bolting and flowering time (Figure 6E). In addition, both the length and width of mature seeds were reduced in the overexpression lines (Figure 6F–H).
To further investigate the function of GhCEP46-D05 in cotton, p35S::GhCEP46-D05 was also transformed into the cotton line Jin668. Similar to the phenotype in Arabidopsis, the overexpression lines showed a reduced plant height resulting from a reduced internode length of the main shoot (Figure 7A–C). In addition, a shorter fiber length was observed in the overexpression lines (Figure 7D,E), suggesting that GhCEP46-D05 inhibits cell expansion, considering that each fiber is a single cell.
Thus, the above results indicate that the overexpression of GhCEP46-D05 has an effect on the development of roots, shoots and seeds in plants.

3. Discussion

Previous studies have identified over 900 putative CEP genes in plant genomes [4,14,23,24]. Despite this, a thorough investigation of the CEP gene family in cotton species has not been undertaken. In our research, we carried out a comprehensive investigation of the cotton CEP genes, covering aspects such as their evolutionary relationships, structural domains, genomic locations, gene synteny and expression patterns.
The two allotetraploid cotton species, G. hirsutum and G. barbadense, are thought to be derived from natural interspecific hybridization events between their ancestral species, usually speculated to be G. raimondii and G. arboreum [25]. It would therefore be expected that the total number of CEPs in these two allotetraploid cotton species would be equal to the combined CEPs of G. raimondii and G. arboreum. However, the CEPs identified in G. hirsutum (54) and G. barbadense (59) were fewer than the combined CEPs found in the two allotetraploid progenitors, G. arboreum (34) and G. raimondii (35). This discrepancy suggests that the process of polyploidisation, characterised by chromosome duplication and rapid genome rearrangement, may lead to different levels of gene loss or pseudogenization [26].
A characteristic feature of the CEP genes found in the four cotton species is that many of these genes are located in gene clusters. In Arabidopsis, AtCEP5, AtCEP6, AtCEP7, and AtCEP8 also appear to be tandem repeats on chromosome 5 [7,8]. This clustered distribution may therefore be a typical feature of the CEP gene family. Compared to Arabidopsis, the discovery of a greater number of gene clusters in cotton suggests that tandem gene duplication may be a driving force for the expansion of the cotton CEP gene family. In addition, in this study, we found that the CEP genes in the same cluster tend to have similar expression patterns, and this phenomenon has been found in many previous studies, demonstrating an efficient mechanism for gene replication and expression regulation [27,28].
Phylogenetic tree analysis showed that cotton CEP genes could be separated into two distinct groups (Group I and Group II), which is consistent with the phylogenetic relationships found in Arabidopsis and other plants [7,8,12,14,22]. In addition, within each group, the present study found three subgroups that have not been reported in other plants. In Group 1, there are 12 Arabidopsis CEPs (AtCEP1–AtCEP12), but they are only present in subgroup I-2. This result suggests that the differentiation of novel CEP genes (paralogs) in cotton is another driving force for the expansion of the cotton CEP gene family. Considering that the same conserved CEP domain is shared between these subgroups in each group, it can be hypothesized that the differentiation of non-conserved segments in CEPs is responsible for the formation of subgroups. These subgroups also suggest that there may be functional differentiation within each group of cotton CEPs.
In Arabidopsis, CEP gene expression is detected in different tissues at different stages of the plant’s life cycle. Some of these expressions are triggered by different environmental cues, such as abiotic stress, nutrient deficiency, and unfavourable temperature fluctuations. Our results indicate that the expression of cotton CEP genes is widespread throughout the cotton plant, although a subset of these genes have distinct expression profiles. For example, certain genes, such as GhCEP08-A02 and GhCEP14-A02, are more highly expressed in roots, whereas others, such as GhCEP19-A05 and GhCEP22-A06, are highly expressed in flower tissues. These findings suggest that cotton CEP genes play a significant role in the growth and development of cotton plants.
Previous studies have highlighted the important role of CEP genes in various plant growth and developmental processes [5,6,15,29]. In the case of Arabidopsis, the enhanced expression of AtCEP1 or AtCEP3 has been shown to suppress primary root growth and lateral root emergence [30]. Similarly, in Medicago truncatula, overexpression of MtCEP1 reduces the number of lateral roots and increases the number of nodules [20]. In rice, the upregulation of OsCEP6.1 resulted in pleiotropic effects on panicle and grain development, such as dwarfism, smaller leaves, fewer tillers, reduced grain size, and fewer grains per panicle. In this study, we found that overexpression of GhCEP46-D05 in Arabidopsis inhibited primary root elongation and reduced plant height and seed size. In cotton, overexpression of GhCEP46-D05 also leads to a reduction in internode length and fiber length. This suggests that GhCEP46-D05 may play a role in the regulation of cotton plant growth. It is possible that GhCEP46-D05 inhibits internode elongation and fiber growth by decreasing cell elongation. Based on our results, it can be inferred that cotton CEP genes play a crucial role in cotton plant development. This is supported by the observation that GhCEP46-D05 has similar effects on plant development as observed in other plants such as Arabidopsis and rice. Therefore, the present results suggest that manipulation of CEP genes could be an effective way to improve the yield or quality of cotton.
Recently, the CEP signalling pathway has attracted a lot of research attention, and many key factors have been identified, such as the CEP receptors CEPR1, CEPR2 and CRA2 [16,19,20]. Crosstalk between CEP signalling and other hormones, such as ABA and ethylene, has also been observed [17,18,20]. Future studies are needed to identify the CEP receptor genes and other factors involved in CEP signalling and to analyse their functions in plant development or stress resistance in cotton.

4. Materials and Methods

4.1. Identification of CEP Protein Family Members

The complete genome sequence data of four cotton species, Gossypium arboreum (A2) (CRI v1), G. raimondii (D5) (JGI v2), G. hirsutum (AD1) (ZJU v2.1) and G. barbadense (AD2) (ZJU v1.1), were retrieved from the COTTONGEN database (https://www.cottongen.org/, accessed on 15 July 2022) [31]. The complete amino acid sequences of CEPs from Arabidopsis thaliana were downloaded from TAIR (https://www.arabidopsis.org/, accessed on 18 July 2022). The AtCEP protein sequences were used as queries and a genome-wide similarity search was performed using BLASTP to identify the cotton CEP gene family with an E-value cut-off of 1. SignalP 5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0, accessed on 20 July 2022) was used to search for signal peptides. Only sequences containing both the signal peptides and the conserved 15-amino acid peptide (CEP) domains were retained for further analysis [32]. The physicochemical properties of CEP proteins, such as length, molecular weight, and isoelectric point, were predicted using the ExPASy suite of tools (https://www.expasy.org/, accessed on 25 July 2022) [33].

4.2. Sequence Alignment and Phylogenetic Tree Construction

The Clustal W 1.81 program (http://www.clustal.org/clustal2/, accessed on 12 May 2020) with default parameters was used to align the amino acid sequences of all CEP proteins. The consensus CEP domains of the G. hirsutum CEP genes were represented using Weblogo (http://weblogo.berkeley.edu/logo.cgi, accessed on 2 August 2022). The NJ tree with bootstrap tests of 1000 replicates was constructed by aligning the amino acid sequences of the CEP proteins using MEGA X (https://www.megasoftware.net/, accessed on 28 July 2022) [34].

4.3. Chromosomes Locations and Collinearity of CEPs in Cotton

The physical location data for the CEP genes were retrieved from the Gossypium genomic databases using gff3 files. The local database contained protein sequences from four cotton species, which were searched using the Basic Local Alignment Search Tool (BLAST). The blastp results were analysed using the Dual Systeny Plotter of TBtools and MCscanX to examine the collinearity of the CEP genes [35,36].

4.4. Analysis of Expression Pattern of CEPs

To characterize the gene expression patterns of the CEP gene family in different tissues, we downloaded public RNA-seq data from the Cotton Omics Database project website (http://cotton.zju.edu.cn/, accessed on 10 August 2022) [37,38], and the heat map of the tissue expression of the CEP gene family was constructed using the Fragments Per Kilobase of exon model per Million mapped reads (FPKM) values.

4.5. RNA Extraction and Quantitative RT-PCR

The Fastpure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China) was used to extract total RNA from various samples (Vazyme, Nanjing, China). First-strand cDNA was synthesized using the PrimeScript RT reagent kit with the gDNA Eraser (Takara, Dalian, China). Quantitative reverse transcription PCR (qRT-PCR) was conducted on a BIO-RAD CFX96 Connect Real-Time PCR System using a TB Green Premix Ex Taq (Tli RnaseH Plus) Kit (Takara, Dalian, China). Relative gene expression level was calculated using the 2−ΔCt method with cotton ACTIN14 (GeneBank accession number: AY305733) as the internal reference. Three independent biological replicates and four technical replicates were performed for each sample. Primers used are listed in Table S2.

4.6. Promoter Activity Analysis

The 2000-bp upstream fragment of GhCEP46-D05 was cloned and inserted upstream of the GUS reporter gene in plasmid pBI121. The resulting construct was then introduced into Agrobacterium tumefaciens strain GV3101. Arabidopsis ecotype Col-0 was used as the transgenic receptor. Promoter activity analysis was detected by GUS histochemical staining.

4.7. Over-Expression Constructs and Plant Transformation

The full-length coding sequence of GhCEP46-D05 was PCR amplified from the cDNA of TM-1 and then put downstream of the 35S promoter of plasmid pBI121. The resulting construct was then transformed into the Agrobacterium tumefaciens strain GV3101. Arabidopsis thaliana was transformed via Agrobacterium-mediated transformation using the floral-dip method [39]. Jin668, an upland cotton cultivar with a normal growth habit, was used as the receptor for cotton transformation. The Agrobacterium-mediated transformation was carried out according to a previous study [40]. For both Arabidopsis and cotton, positive transformants were selected by kanamycin resistance and PCR analysis. The expression of GhCEP46-D05 was detected by qRT-PCR in the leaves of the transgenic plants.

4.8. Phenotypic Analysis of GhCEP46-D05 Transgenic Plants

For Arabidopsis, seeds from Col-0 (wild-type, WT) and transgenic plants (T3 generation) overexpressing GhCEP46-D05 were germinated on Murashige and Skoog (MS) medium containing 3% sucrose and 0.7% agar (pH 5.8), and Petri dishes were placed vertically to allow the roots of the plants to grow downwards. After vernalization, the seeds were incubated at 21 °C with a 16:8 h light/dark photoperiod. After seven days, 30 plants from each line were randomly selected for the measurement of primary root length. After 15 days, the seedlings were transferred to soil and grown in a growth chamber (at 21 °C with a 16:8 h light/dark photoperiod). The bolting date was defined as the number of days between seed germination and the time when flower buds could be distinguished from leaves. Plant height was measured 40 days after sowing. Seed length and width were measured using ImageJ software (https://imagej.net/software/imagej/, accessed on 8 March 2019). For cotton, seeds were germinated at 28 °C with a 16 h light/8 h dark cycle on 1/2X MS medium, and then seedlings were transplanted into the soil to grow to maturity under normal growing conditions. For each line, 20 plants were randomly selected for measurement of plant height and main shoot internode length at maturity. Mature fibers (15 g/each sample) collected from the bolls at similar positions on the plants (at least 15 plants per line) were used for fiber length measurements using an HVI900 instrument (USTER, Knoxville, TN, USA) at the Center of Cotton Fiber Quality Inspection and Testing, Chinese Ministry of Agriculture (Anyang, China). Statistically significant differences between groups were determined using the Student’s t-test with GraphPad Prism 8.0 software.

5. Conclusions

CEPs are a group of widely distributed peptide hormones in plants. Although numerous CEP genes have been identified in different plants, their functions remain largely unknown, particularly in cotton. In this study, we performed a comprehensive analysis of CEP gene families in four cotton species for the first time. A total of 182 CEP genes were identified on the basis of their conserved CEP domains and the presence of a signal peptide. The cotton CEPs were divided into two groups, each with three subgroups. Further analysis was conducted on the conserved motifs, chromosomal locations, gene collinearity, and expression patterns. The results suggest that segmental duplications may have a significant impact on the expansion of cotton CEPs, with more than half of them located in gene clusters. The CEP domains in both groups showed a high degree of similarity, with a core motif present in both. Expression pattern analysis revealed that CEP genes within the same gene cluster tended to exhibit the same pattern. Expression analyses showed that some CEP genes are expressed exclusively in certain tissues, such as roots and pistils. An additional functional analysis was performed on GhCEP46-D05. Its overexpression inhibits root, shoot and fiber elongation, probably by repressing cell expansion. The results show that the CEP genes in cotton play a key role in plant development. This study provides a solid basis for further investigation of CEP gene functions in cotton.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25084231/s1.

Author Contributions

Conceptualization and methodology, Z.M. and B.L.; validation, Z.M. and S.Z.; formal analysis, Z.M., B.L. and Y.L.; investigation, Z.M., B.L., J.Y. and J.P.; writing—original draft preparation, Z.M. and B.L.; writing—review and editing, Y.Z. and W.C.; supervision, project administration, and funding acquisition Y.Z. and W.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was by supported by the Natural Science Foundation of Henan (242300421340).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article or Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interests.

References

  1. Hirakawa, Y.; Sawa, S. Diverse function of plant peptide hormones in local signaling and development. Curr. Opin. Plant Biol. 2019, 51, 81–87. [Google Scholar] [CrossRef]
  2. Takahashi, F.; Hanada, K.; Kondo, T.; Shinozaki, K. Hormone-like peptides and small coding genes in plant stress signaling and development. Curr. Opin. Plant Biol. 2019, 51, 88–95. [Google Scholar] [CrossRef] [PubMed]
  3. Kim, J.S.; Jeon, B.W.; Kim, J. Signaling peptides regulating abiotic stress responses in plants. Front. Plant Sci. 2021, 12, 704490. [Google Scholar] [CrossRef]
  4. Zhang, L.; Ren, Y.; Xu, Q.; Wan, Y.; Zhang, S.; Yang, G.; Huang, J.; Yan, K.; Zheng, C.; Wu, C. SiCEP3, a C-terminally encoded peptide from Setaria italica, promotes ABA import and signaling. J. Exp. Bot. 2021, 72, 6260–6273. [Google Scholar] [CrossRef] [PubMed]
  5. Ohyama, K.; Ogawa, M.; Matsubayashi, Y. Identification of a biologically active, small, secreted peptide in Arabidopsis by in silico gene screening, followed by LC-MS-based structure analysis. Plant J. 2008, 55, 152–160. [Google Scholar] [CrossRef] [PubMed]
  6. Delay, C.; Imin, N.; Djordjevic, M.A. Regulation of Arabidopsis root development by small signaling peptides. Front. Plant Sci. 2013, 4, 352. [Google Scholar] [CrossRef]
  7. Roberts, I.; Smith, S.; De Rybel, B.; Van Den Broeke, J.; Smet, W.; De Cokere, S.; Mispelaere, M.; De Smet, I.; Beeckman, T. The CEP family in land plants: Evolutionary analyses, expression studies, and role in Arabidopsis shoot development. J. Exp. Bot. 2013, 64, 5371–5381. [Google Scholar] [CrossRef]
  8. Taleski, M.; Imin, N.; Djordjevic, M.A. CEP peptide hormones: Key players in orchestrating nitrogen-demand signalling, root nodulation, and lateral root development. J. Exp. Bot. 2018, 69, 1829–1836. [Google Scholar] [CrossRef]
  9. Song, W.; Liu, L.; Wang, J.; Wu, Z.; Zhang, H.; Tang, J.; Lin, G.; Wang, Y.; Wen, X.; Li, W.; et al. Signature motif-guided identification of receptors for peptide hormones essential for root meristem growth. Cell Res. 2016, 26, 674–685. [Google Scholar] [CrossRef]
  10. Brachi, B.; Faure, N.; Horton, M.; Flahauw, E.; Vazquez, A.; Nordborg, M.; Bergelson, J.; Cuguen, J.; Roux, F. Linkage and association mapping of Arabidopsis thaliana flowering time in nature. PLoS Genet. 2010, 6, e1000940. [Google Scholar] [CrossRef]
  11. Sui, Z.; Wang, T.; Li, H.; Zhang, M.; Li, Y.; Xu, R.; Xing, G.; Ni, Z.; Xin, M. Overexpression of Peptide-Encoding OsCEP6.1 Results in Pleiotropic Effects on Growth in Rice (O. sativa). Front. Plant Sci. 2016, 7, 228. [Google Scholar] [CrossRef] [PubMed]
  12. Aggarwal, S.; Kumar, A.; Jain, M.; Sudan, J.; Singh, K.; Kumari, S.; Mustafiz, A. C-terminally encoded peptides (CEPs) are potential mediators of abiotic stress response in plants. Physiol. Mol. Biol. Plants 2020, 26, 2019–2033. [Google Scholar] [CrossRef] [PubMed]
  13. Xu, R.; Li, Y.; Sui, Z.; Lan, T.; Song, W.; Zhang, M.; Zhang, Y.; Xing, J. A C-Terminal Encoded Peptide, ZmCEP1, is essential for kernel development in maize (Zea mays L.). J. Exp. Bot. 2021, 72, 5390–5406. [Google Scholar] [CrossRef] [PubMed]
  14. Imin, N.; Mohd-Radzman, N.A.; Ogilvie, H.A.; Djordjevic, M.A. The peptide-encoding CEP1 gene modulates lateral root and nodule numbers in Medicago truncatula. J. Exp. Bot. 2013, 64, 5395–5409. [Google Scholar] [CrossRef] [PubMed]
  15. Mohd-Radzman, N.A.; Binos, S.; Truong, T.T.; Imin, N.; Mariani, M.; Djordjevic, M.A. Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula. J. Exp. Bot. 2015, 66, 5289–5300. [Google Scholar] [CrossRef] [PubMed]
  16. Tabata, R.; Sumida, K.; Yoshii, T.; Ohyama, K.; Shinohara, H.; Matsubayashi, Y. Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling. Science 2014, 346, 343–346. [Google Scholar] [CrossRef] [PubMed]
  17. Zhang, L.; Yu, Z.; Xu, Y.; Yu, M.; Ren, Y.; Zhang, S.; Yang, G.; Huang, J.; Yan, K.; Zheng, C.; et al. Regulation of the stability and ABA import activity of NRT1.2/NPF4.6 by CEPR2-mediated phosphorylation in Arabidopsis. Mol. Plant 2022, 15, 1635. [Google Scholar] [CrossRef] [PubMed]
  18. Yu, Z.; Zhang, D.; Xu, Y.; Jin, S.; Zhang, L.; Zhang, S.; Yang, G.; Huang, J.; Yan, K.; Wu, C.; et al. CEPR2 phosphorylates and accelerates the degradation of PYR/PYLs in Arabidopsis. J. Exp. Bot. 2019, 70, 5457–5469. [Google Scholar] [CrossRef] [PubMed]
  19. Taleski, M.; Chapman, K.; Imin, N.; Djordjevic, M.A.; Groszmann, M. The Peptide Hormone Receptor CEPR1 Functions in the Reproductive Tissue to Control Seed Size and Yield. Plant Physiol. 2020, 183, 620–636. [Google Scholar] [CrossRef]
  20. Mohd-Radzman, N.A.; Laffont, C.; Ivanovici, A.; Patel, N.; Reid, D.; Stougaard, J.; Frugier, F.; Imin, N.; Djordjevic, M.A. Different Pathways Act Downstream of the CEP Peptide Receptor CRA2 to Regulate Lateral Root and Nodule Development. Plant Physiol. 2016, 171, 2536–2548. [Google Scholar] [CrossRef]
  21. Yu, J.; Zhang, K.; Li, S.; Yu, S.; Zhai, H.; Wu, M.; Li, X.; Fan, S.; Song, M.; Yang, D.; et al. Mapping quantitative trait loci for lint yield and fiber quality across environments in a Gossypium hirsutum × Gossypium barbadense backcross inbred line population. Theor. Appl. Genet. 2013, 126, 275–287. [Google Scholar] [CrossRef] [PubMed]
  22. Paterson, A.H.; Wendel, J.F.; Gundlach, H.; Guo, H.; Jenkins, J.; Jin, D.; Llewellyn, D.; Showmaker, K.C.; Shu, S.; Udall, J.; et al. Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres. Nature 2012, 492, 423–427. [Google Scholar] [CrossRef] [PubMed]
  23. Ogilvie, H.A.; Imin, N.; Djordjevic, M.A. Diversification of the C-TERMINALLY ENCODED PEPTIDE (CEP) gene family in angiosperms, and evolution of plant-family specific CEP genes. BMC Genom. 2014, 15, 870. [Google Scholar] [CrossRef] [PubMed]
  24. Qiu, Z.; Zhuang, K.; Liu, Y.; Ge, X.; Chen, C.; Hu, S.; Han, H. Functional characterization of C-TERMINALLY ENCODED PEPTIDE (CEP) family in Brassica rapa L. Plant. Signal. Behav. 2022, 17, 2021365. [Google Scholar] [CrossRef] [PubMed]
  25. Wendel, J.F.; Brubaker, C.; Alvarez, I.; Cronn, R.; Stewart, J.M. Evolution and natural history of the cotton genus. In Genetics and Genomics of Cotton; Paterson, A.H., Ed.; Springer: New York, NY, USA, 2009; Volume 3. [Google Scholar]
  26. Paterson, A.H.; Bowers, J.E.; Chapman, B.A. Ancient polyploidization predating divergence of the cereals, and its consequences for comparative genomics. Proc. Natl. Acad. Sci. USA 2004, 101, 9903–9908. [Google Scholar] [CrossRef] [PubMed]
  27. Hurst, L.D.; Pál, C.; Lercher, M.J. The evolutionary dynamics of eukaryotic gene order. Nat. Rev. Genet. 2004, 5, 299–310. [Google Scholar] [CrossRef]
  28. Kustatscher, G.; Grabowski, P.; Rappsilber, J. Pervasive coexpression of spatially proximal genes is buffered at the protein level. Mol. Syst. Biol. 2017, 13, 937. [Google Scholar] [CrossRef] [PubMed]
  29. Roberts, I.; Smith, S.; Stes, E.; De Rybel, B.; Staes, A.; Van De Cotte, B.; Njo, M.F.; Dedeyne, L.; Demol, H.; Lavenus, J.; et al. CEP5 and XIP1/CEPR1 regulate lateral root initiation in Arabidopsis. J. Exp. Bot. 2016, 67, 4889–4899. [Google Scholar] [CrossRef] [PubMed]
  30. Chapman, K.; Taleski, M.; Ogilvie, H.A.; Imin, N.; Djordjevic, M.A. CEP–CEPR1 signalling inhibits the sucrose-dependent enhancement of lateral root growth. J. Exp. Bot. 2019, 70, 3955–3967. [Google Scholar] [CrossRef]
  31. Zhu, T.; Liang, C.; Meng, Z.; Sun, G.; Meng, Z.; Guo, S.; Zhang, R. CottonFGD: An integrated functional genomics database for cotton. BMC Plant Biol. 2017, 17, 101. [Google Scholar] [CrossRef]
  32. Bendtsen, J.D.; Nielsen, H.; Von Heijne, G.; Brunak, S. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 2004, 340, 783–795. [Google Scholar] [CrossRef]
  33. Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R.D.; Bairoch, A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003, 31, 3784–3788. [Google Scholar] [CrossRef] [PubMed]
  34. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
  35. Wang, Y.; Tang, H.; Debarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.-H.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef] [PubMed]
  36. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  37. Zhang, T.; Hu, Y.; Jiang, W.; Fang, L.; Guan, X.; Chen, J.; Zhang, J.; Saski, C.A.; Scheffler, B.E.; Stelly, D.M.; et al. Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement. Nat. Biotechnol. 2015, 33, 531–537. [Google Scholar] [CrossRef]
  38. Hu, Y.; Chen, J.; Fang, L.; Zhang, Z.; Ma, W.; Niu, Y.; Ju, L.; Deng, J.; Zhao, T.; Lian, J.; et al. Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton. Nat. Genet. 2019, 51, 739–748. [Google Scholar] [CrossRef] [PubMed]
  39. Clough, S.J.; Bent, A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998, 16, 735–743. [Google Scholar] [CrossRef]
  40. Jin, S.; Zhang, X.; Nie, Y.; Guo, X.; Liang, S.; Zhu, H. Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton. Biol. Plant. 2006, 50, 519–524. [Google Scholar] [CrossRef]
Ijms 25 04231 g001
Figure 1. Phylogenetic relationships of CEP genes in G. arboreum (Ga), G. raimondii (Gr), G. hirsutum (Gh), G. barbadense (Gb), and Arabidopsis (At). A neighbour-joining (NJ) phylogenetic tree was constructed in MEGA X based on full-length protein sequences of CEP genes.
Figure 1. Phylogenetic relationships of CEP genes in G. arboreum (Ga), G. raimondii (Gr), G. hirsutum (Gh), G. barbadense (Gb), and Arabidopsis (At). A neighbour-joining (NJ) phylogenetic tree was constructed in MEGA X based on full-length protein sequences of CEP genes.
Ijms 25 04231 g001
Ijms 25 04231 g002
Figure 2. CEP domains of the cotton CEP genes. (A) Alignment of the CEP domains of G. hirsutum CEP genes. (B) Sequence logos of CEP domains in G. hirsutum.
Figure 2. CEP domains of the cotton CEP genes. (A) Alignment of the CEP domains of G. hirsutum CEP genes. (B) Sequence logos of CEP domains in G. hirsutum.
Ijms 25 04231 g002
Ijms 25 04231 g003
Figure 3. Distribution of cotton CEP genes in: (A) Gh. (B) Gb. (C) Ga. (D) Gr.
Figure 3. Distribution of cotton CEP genes in: (A) Gh. (B) Gb. (C) Ga. (D) Gr.
Ijms 25 04231 g003
Ijms 25 04231 g004
Figure 4. Collinear correlations between CEP genes from Ga, Gr, Gh and Gb. Homologous relationships of CEP genes are indicated with colour lines.
Figure 4. Collinear correlations between CEP genes from Ga, Gr, Gh and Gb. Homologous relationships of CEP genes are indicated with colour lines.
Ijms 25 04231 g004
Ijms 25 04231 g005
Figure 5. The tissue-specific expression patterns of the CEP family genes in upland cotton (Gh). (A) Heat map of expression profiles of G. hirsutum CEP genes in public RNA-seq data. (B) Expression levels of nine CEP genes in quantitative RT-PCR. (C) Expression of GhCEP46-D05 in Arabidopsis detected by GUS histochemical staining. DPA, days after anthesis.
Figure 5. The tissue-specific expression patterns of the CEP family genes in upland cotton (Gh). (A) Heat map of expression profiles of G. hirsutum CEP genes in public RNA-seq data. (B) Expression levels of nine CEP genes in quantitative RT-PCR. (C) Expression of GhCEP46-D05 in Arabidopsis detected by GUS histochemical staining. DPA, days after anthesis.
Ijms 25 04231 g005
Ijms 25 04231 g006
Figure 6. Overexpression of GhCEP46-D05 in Arabidopsis. (A) Phenotypes of root. (B) Phenotypes of shoot. (C–E) Quantification of primary root length, plant height and bolting time. (F) Phenotypes of seed. (G,H) Quantification of the seed length and seed width. Data are displayed as mean ± standard deviation; p-values are determined by means of Student’s t-test (** p < 0.01). Scale bars: 1 cm in A and B; 100 µm in F.
Figure 6. Overexpression of GhCEP46-D05 in Arabidopsis. (A) Phenotypes of root. (B) Phenotypes of shoot. (C–E) Quantification of primary root length, plant height and bolting time. (F) Phenotypes of seed. (G,H) Quantification of the seed length and seed width. Data are displayed as mean ± standard deviation; p-values are determined by means of Student’s t-test (** p < 0.01). Scale bars: 1 cm in A and B; 100 µm in F.
Ijms 25 04231 g006
Ijms 25 04231 g007
Figure 7. Overexpression of GhCEP46-D05 in cotton. (A) Representative plants of Jin668 and overexpression lines. (B,C) Quantification of plant height and internode length. (D) Phenotypes of fiber. (E) Quantification of fiber length. Data are displayed as mean ± standard deviation; p-values are determined by means of Student’s t-test (** p < 0.01, * p < 0.05). Scale bars: 10 cm in (A) and 10 mm in (D).
Figure 7. Overexpression of GhCEP46-D05 in cotton. (A) Representative plants of Jin668 and overexpression lines. (B,C) Quantification of plant height and internode length. (D) Phenotypes of fiber. (E) Quantification of fiber length. Data are displayed as mean ± standard deviation; p-values are determined by means of Student’s t-test (** p < 0.01, * p < 0.05). Scale bars: 10 cm in (A) and 10 mm in (D).
Ijms 25 04231 g007
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Mei, Z.; Li, B.; Zhu, S.; Li, Y.; Yao, J.; Pan, J.; Zhang, Y.; Chen, W. A Genome-Wide Analysis of the CEP Gene Family in Cotton and a Functional Study of GhCEP46-D05 in Plant Development. Int. J. Mol. Sci. 2024, 25, 4231. https://doi.org/10.3390/ijms25084231

AMA Style

Mei Z, Li B, Zhu S, Li Y, Yao J, Pan J, Zhang Y, Chen W. A Genome-Wide Analysis of the CEP Gene Family in Cotton and a Functional Study of GhCEP46-D05 in Plant Development. International Journal of Molecular Sciences. 2024; 25(8):4231. https://doi.org/10.3390/ijms25084231

Chicago/Turabian Style

Mei, Zhenyu, Bei Li, Shouhong Zhu, Yan Li, Jinbo Yao, Jingwen Pan, Yongshan Zhang, and Wei Chen. 2024. "A Genome-Wide Analysis of the CEP Gene Family in Cotton and a Functional Study of GhCEP46-D05 in Plant Development" International Journal of Molecular Sciences 25, no. 8: 4231. https://doi.org/10.3390/ijms25084231

APA Style

Mei, Z., Li, B., Zhu, S., Li, Y., Yao, J., Pan, J., Zhang, Y., & Chen, W. (2024). A Genome-Wide Analysis of the CEP Gene Family in Cotton and a Functional Study of GhCEP46-D05 in Plant Development. International Journal of Molecular Sciences, 25(8), 4231. https://doi.org/10.3390/ijms25084231

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop