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. 2024 Apr 29;13(5):549.
doi: 10.3390/antiox13050549.

The Truncated Peptide AtPEP1(9-23) Has the Same Function as AtPEP1(1-23) in Inhibiting Primary Root Growth and Triggering of ROS Burst

Junmei Cui  1 , Ermei Sa  1   2 , Jiaping Wei  1 , Yan Fang  1 , Guoqiang Zheng  1   2 , Ying Wang  1   2 , Xiaoxia Wang  1   2 , Yongjie Gong  1   2 , Zefeng Wu  1 , Panfeng Yao  1 , Zigang Liu  1   2
Affiliations

The Truncated Peptide AtPEP1(9-23) Has the Same Function as AtPEP1(1-23) in Inhibiting Primary Root Growth and Triggering of ROS Burst

Junmei Cui et al. Antioxidants (Basel). .

Abstract

Currently, the widely used active form of plant elicitor peptide 1 (PEP1) from Arabidopsis thaliana is composed of 23 amino acids, hereafter AtPEP1(1-23), serving as an immune elicitor. The relatively less conserved N-terminal region in AtPEP family indicates that the amino acids in this region may be unrelated to the function and activity of AtPEP peptides. Consequently, we conducted an investigation to determine the necessity of the nonconserved amino acids in AtPEP1(1-23) peptide for its functional properties. By assessing the primary root growth and the burst of reactive oxygen species (ROS), we discovered that the first eight N-terminal amino acids of AtPEP1(1-23) are not crucial for its functionality, whereas the conserved C-terminal aspartic acid plays a significant role in its functionality. In this study, we identified a truncated peptide, AtPEP1(9-23), which exhibits comparable activity to AtPEP1(1-23) in inhibiting primary root growth and inducing ROS burst. Additionally, the truncated peptide AtPEP1(13-23) shows similar ability to induce ROS burst as AtPEP1(1-23), but its inhibitory effect on primary roots is significantly reduced. These findings are significant as they provide a novel approach to explore and understand the functionality of the AtPEP1(1-23) peptide. Moreover, exogenous application of AtPEP1(13-23) may enhance plant resistance to pathogens without affecting their growth and development. Therefore, AtPEP1(13-23) holds promise for development as a potentially applicable biopesticides.

Keywords: plant elicitor peptide 1; primary root; reactive oxygen species burst; root tip.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Sequence information of AtPEP family peptides. (a) Alignment of AtPEP family peptides, the darker the color, the higher the similarity; (b) Peptide sequence information used in this study.
Figure 2
Figure 2
Phenotypes and data of primary roots after treatment with AtPEP1(1–23) (a), AtPEP1(5–23) (b), AtPEP1(7–23) (c), AtPEP1(9–23) (d), AtPEP1(11–23) (e), AtPEP1(13–23) (f), and AtPEP1(9–22) (g). Five-day-old seedlings were transferred from 1/2 MS to culture media with different concentrations of peptides. The left side shows the phenotypes of the primary roots, while the right side presents the data on primary root length. Results are presented as mean ± standard deviation (SD). “ns” represents no significant difference compared to the control treatment (0 nM). *, **, **** indicate significant differences at p < 0.05, p < 0.01, and p < 0.0001 levels, respectively, compared to the control treatment (two-way ANOVA, n = 20).
Figure 3
Figure 3
Root tip phenotypes. Five-day-old seedlings were treated with peptide AtPEP1(1–23) (a), AtPEP1(5–23) (b), AtPEP1(7–23) (c), AtPEP1(9–23) (d), AtPEP1(11–23) (e), AtPEP1(13–23) (f), and AtPEP1(9–22) (g) for 4 days, followed by observation of root tip phenotypes (n = 20). The magnification of the eyepiece and objective lenses of a microscope is both 10 times.
Figure 4
Figure 4
Root phenotypes and root length of transgenic lines DR5::GFP (a) and WOX5::GFP (b). The left panel shows the primary root phenotypes on day 0 and day 4 after transferring 5-day-old seedlings to medium containing 500 nM peptide. The right panel presents the primary root length data after transfer. Results are presented as mean ± SD. “ns” represents no significant difference compared to the control treatment (mock); **** indicates significant differences at p < 0.0001 level compared to the control treatment (two-way ANOVA, n = 20).
Figure 5
Figure 5
The GFP expression in root tips of transgenic lines DR5::GFP and WOX5::GFP. Five-day-old seedlings were treated with 500 nM peptide for four days, followed by observation of DR5::GFP (a) and WOX5::GFP (c) using confocal microscopy. The scale was 50 μm. (b,d) The GFP fluorescence regions were measured as indicated, and the results are presented as mean ± SD. “ns” represents no significant difference compared to the control treatment (mock); **, **** indicate significant differences compared to the control treatment (n = 20), with p < 0.01 and p < 0.0001, respectively. One-way ANOVA was conducted for (b), and two-way ANOVA for (d).
Figure 6
Figure 6
Phenotype and root length of double mutant pepr1/2 primary roots. On the left side, the primary root phenotypes were observed on Day 0 and Day 4 after transferring 5-day-old seedlings to culture media containing 500 nM peptide. On the right side, the data for primary root length were recorded for the seedlings transferred to culture media containing 500 nM peptide. Results are expressed as mean ± SD; “ns” represents no significant difference compared to the mock treatment (two-way ANOVA, n = 20).
Figure 7
Figure 7
TMHMM analysis of AtPEPR1 and AtPEPR2. (a) Topology model and probability of the target proteins; (b) distribution of amino acids in signal peptide (SP), extracellular domain (outside), transmembrane domain (TM), and intracellular domain (inside).
Figure 8
Figure 8
Molecular docking of ligand peptides with receptor proteins. (a) Optimal docking poses between ligand peptides and receptor proteins. Receptor proteins AtPEPR1 and AtPEPR2 are represented by blue and green, respectively. Ligand peptides AtPEP1(1–23), AtPEP1(9–23), and AtPEP1(13–23) are represented by pink, orange, and purple, respectively. (b) Complexes in terms of contact sites, energy, and binding properties. The possible contact sites of peptides are highlighted in red. (c) Binding interactions between peptide ligands and AtPEPR1 and AtPEPR2. The blue ball, nitrogen atom; red ball, oxygen atom; black ball, carbon atom. The dotted black line above represents the peptide ligand, while the line below represents the receptor.
Figure 9
Figure 9
Dose-dependent ROS burst. Dynamic curves of ROS burst and total ROS within 15 min in Arabidopsis leaves induced by different doses of peptides AtPEP1(1–23) (a), AtPEP1(5–23) (b), AtPEP1(7–23) (c), AtPEP1(9–23) (d), AtPEP1(11–23) (e), AtPEP1(13–23) (f), and AtPEP1(9–22) (g). Results are expressed as mean ± SD; “ns” indicates no significant difference compared to mock treatment. “*”, “**”, “***”, and “****” indicate significant differences compared to the control group at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 levels, respectively (one-way ANOVA, n = 4). (h) The occurrence of ROS burst. The symbol “N” represents no occurrence of ROS, while the symbol “Y” signifies the presence of ROS.
Figure 10
Figure 10
Dynamic curves of ROS burst and total ROS of pepr1/2 mutant and wild-type Col-0. Panels (a,c,e) show the ROS results of pepr1/2 mutant treated with peptides at concentrations of 500 nM, 1000 nM, and 2000 nM, respectively. Panels (b,d,f) show the ROS results of Col-0 treated with peptides at concentrations of 500 nM, 1000 nM, and 2000 nM, respectively. Total ROS represents the cumulative amount of active ROS generated within 15 min. Results are expressed as mean ± SD (n = 4); “ns” indicates no significant difference compared to mock; “*” indicates a significant difference to mock at p < 0.05 level.
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