The Importance of Conserved Serine for C-Terminally Encoded Peptides Function Exertion in Apple

Abstract

Background: The C-terminally encoded peptide (CEP) family has been shown to play vital roles in plant growth. Although a genome-wide analysis of this family has been performed in Arabidopsis, little is known regarding CEPs in apple (Malus domestica).

Methods: Here, a comprehensive bioinformatics approach was applied to identify MdCEPs in apple, and 12 MdCEP genes were identified and distributed on 6 chromosomes.

Results: MdCEP1 peptide had an inhibitory effect on root growth of apple seedlings, indicating that MdCEP1 played a negative role in root development. In addition, the serine and glycine residues remained conserved within the CEP domains, and MdCEP1 lost its function after mutation of these two key amino acids, suggesting that Ser¹⁰ and Gly¹⁴ residues are crucial for MdCEPs-mediated root growth of apple. Encouragingly, multiple sequence alignment of 273 CEP domains showed that Ser¹⁰ residue was evolutionarily conserved in monocot and eudicot plants. MdCEP derivative (Ser to Cys) lost the ability to inhibit the root growth of Nicotiana benthamiana, Setaria italic, Samolous parviflorus, and Raphanus sativus L. and up-regulate the NO_3^- importer gene NRT2.1.

Conclusion: Taken together, Ser¹⁰ residue is crucial for CEP function exertion in higher land plants, at least in apple.

Keywords: C-terminally encoded peptide; Malus domestica; bioinformatics; phylogenetic analysis; root growth; secreted peptides.

Figure 1

Identification and phylogenetic analysis of MdCEPs in apple. (A) The phylogenetic relationship of 15 AtCEPs and 12 MdCEPs. The phylogenetic tree based on CEP domain is created with MEGA7 by the neighbor-joining method and the bootstrap tests are indicated on the tree. CEP, C-terminally encoded peptide. (B) The chromosome location of MdCEPs in apple. The unanchored contigs are marked in the open squares. MdCEP11 and MdCEP12 are situated on unanchored contigs (Apple’s whole genome sequencing is incomplete). (C) The gene structures of MdCEPs in apple.

Figure 2

Signal peptide cleavage sites prediction and promoter analysis of MdCEP members in apple. (A) Signal peptide of MdCEPs deduced with SignalP 4.1 website. The red boxes represent signal peptides and the scissors are predicted signal peptide cleavage sites. (B) The predicted signal peptide cleavage site. A WebLogo representation of the cleavage site is shown in the right image. (C) Analysis of various stress response cis elements in the promoter regions upstream of MdCEPs via the PlantCARE website. Eleven cis elements are displayed in different patterns.

Figure 3

The tissue expression pattern of MdCEPs in apple. The expression levels of MdCEP1–MdCEP12 (A–L) examined by qRT-PCR in various tissue and stages in apple. The 18s rRNA was performed as an internal control. Relative abundance of MdCEP1–MdCEP12 transcripts from various tissue and stages represented normalized against 18s rRNA. Error bars indicate SEM (n = 3), p < 0.05. One-way ANOVA Duncan’s test is used for statistical analysis. These experiments were repeated three times with similar results. rRNA, ribosomal RNA.

Figure 4

The expression pattern of MdCEPs under stress treatments in apple. Fourteen-day-old seedlings of Malus hupehesis (Pamp.) Rehd supplied with different treatments and the aboveground parts and roots are collected for RNA extraction and qRT-PCR analysis. (A–L) Representation of the transcription levels of MdCEP1–MdCEP12. Relative abundance of MdCEP1–MdCEP12 transcripts from various treatments represented as fold change relative to mock-treated values and normalized against 18s rRNA. Error bars indicate SEM (n = 3). * p < 0.05; ** p < 0.01 (Student’s t-test). These experiments were repeated three times with similar results.

Figure 5

MdCEPs are divided into 2 subgroups according to the number of CEP domains. (A) Schematic representation of MdCEP family members. Group 1 contains members with one CEP domain; Group 2 contains five-seven CEP domains. The dark grey boxes represent CEP domains. (B) A WebLogo representation of the sequence before CEP domains (Group 1) and adjoining sequence between CEP domains (Group 2). (C) Three-dimensional structures of MdCEPs predicted by I-TASSER website. The CEP domains in Group 2 are pointed by arrows.

Figure 6

The conserved serine and glycine are critical for MdCEPs in root growth. (A) Multiple alignment of the CEP domains from 12 MdCEP members. Overall conserved amino acids are in black. (B) Conserved amino acids analysis of all MdCEPs and AtCEPs. Different substitution derivatives are obtained by changing the conserved amino acids indicate in red. (C) Inhibitory effects of exogenous AtCEP1 and MdCEP1 peptide on root growth of apple seedlings. Apple seeds were plated on MS medium with or without different CEP peptides and grown for 2 weeks. (D) Inhibitory effects of exogenous substitution derivatives of MdCEP1 on root growth of apple seedlings. Apple seeds were plated on MS medium with or without different CEP peptides and grown for 2 weeks. (E,F) The relative primary root length and lateral root number of various plants in (C,D). Error bars indicate SEM (N = 3, n = 24). * p < 0.05; ns, no significance. (Student’s t-test).

Figure 7

MdCEP overexpression leads to a retarded growth in apple callus. (A) Ten-day-old apple callus with similar size were cultured on different concentrations of CEPs (AtCEP1, MdCEP1 and substitution derivatives of MdCEP1). Subsequently, these callus were grown for another 18 d under continuous dark conditions. (B) The fresh weight of various callus in (A). Error bars indicate SEM (N = 3, n > 30). * p < 0.05; ** p < 0.01 (Student’s t-test). Statistical analysis is based on three independent biological repeats. ns, no significance. (C) The phenotype of transgenic and non-transgenic apple callus (empty vector, control) grown on MS medium for 18 d. And the expression levels of AtCEP1, MdCEP1, MdCEP^S, MdCEP^G, and MdCEP^SG were detected by RT-PCR. (D) The fresh weight of transgenic and non-transgenic apple callus in (C). Error bars indicate SEM (N = 3, n > 45), p < 0.05. One-way ANOVA Duncan’s test was used for statistical analysis. Statistical differences are indicated by lowercase letters and different letters represent different significance. These experiments were repeated three times with similar results.

Figure 8

The serine is evolutionarily conserved in 32 higher land plants. (A) The evolutionarily conserved amino acid of CEPs in higher land plants. A WebLogo representation of the sequence of 273 CEP domains in 32 plant species. (B) Inhibitory effects of exogenous MdCEP1 and MdCEP^S on growth of Arabidopsis thaliana, Nicotiana benthamiana, Raphanus sativus L., Gramineae (Setaria italica) and Setaria italica (Brassica campestris L.). These sterile seeds were plated on MS medium with MdCEP1 or MdCEP^S and grown for 2 weeks. Arabidopsis thaliana was used as the positive control to detect the activity of the synthesized MdCEP1. (C) Relative primary root length of the higher land plants shown in (B). Error bars indicate SEM (N = 3, n =30). * p < 0.05; ** p < 0.01 (Student’s t-test). ns, no significance. (D) The relative expression levels of MdCEP1 in 14-day-old apple seedlings treated with 0.5 mM nitrates. And the relative expression levels of MdNRT2.1 in 14-day-old apple seedlings treated with AtCEP1, MdCEP1 or MdCEP^S. Error bars indicate SEM (N = 3). * p < 0.05 (Student’s t-test). ns, no significance.