Genome-Wide Identification and Functional Analysis of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) in Three Populus Species

Abstract

Intercellular communication mediated by CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides and their receptors is crucial for plant development and environmental adaptation. In this study, 45 and 89 CLEs were identified in Populus tomentosa and Populus alba × Populus glandulosa, respectively, and, together with the 52 CLEs in Populus trichocarpa, the chromosome localization, gene and protein characteristics, collinearity and gene duplication events, cis-acting regulatory elements in promoters and evolutionary relationships of CLEs in these three poplar species were analyzed. The CLEs of three poplar species were divided into four subfamilies. Among them, the CLEs in subfamilies I, II and IV were A-type CLEs, while those in subfamily III were B-type CLEs. During the evolutionary process of poplar, the selection pressure faced by whole-genome duplication or segmental duplication was purifying selection, and the duplication events led to the expansion of the CLE family in poplar. The exogenous addition of a certain concentration of poplar CLE13 peptides inhibits the root growth of Arabidopsis thaliana and poplar and simultaneously reduces the expression levels of ARFs and LBDs in the roots. In addition, drought stress induces the expression of PtrCLE13A. The overexpression of preCLE13A significantly enhances the osmotic and drought tolerance in Populus tomentosa. These results have provided valuable information for further research on the molecular mechanisms of CLE peptide signaling pathways in the woody model plant poplar regarding plant growth and stress resistance.

Keywords: Populus alba × Populus glandulosa; Populus tomentosa; Populus trichocarpa; bioinformatic analysis; drought resistance; plant peptides.

Figure 1

The phylogenetic tree was constructed using MEGA7 with the neighbor-joining method. (A) The phylogenetic tree of CLEs among P. trichocarpa, P. tomentosa and P. alba × P. glandulosa. (B) The weblogo represents CLE motifs (12 conserved amino acids) of four groups. (C) The weblogo represents CLE motifs (12 conserved amino acids) of P. trichocarpa, P. tomentosa and P. alba × P. glandulosa. The website of weblogo is https://weblogo.berkeley.edu/logo.cgi (accessed on 4 March 2024).

Figure 2

The collinear relationships of CLE genes within and between species of P. trichocarpa, P. tomentosa and P. alba × P. glandulosa. (A–C) respectively represent the distribution of the intra-genomic collinear pairs of CLE genes within P. trichocarpa, P. tomentosa and P. alba × P. glandulosa on the chromosomes, and the orange boxes represent chromosomes. (D) P. tomentosa and P. trichocarpa; (E) P. alba × P. glandulosa and P. trichocarpa; (F) P. tomentosa and P. alba × P. glandulosa. The gray lines: the collinearity of the whole genome among poplar. The red lines: the collinearity of CLE gene pairs. The collinearity analysis was conducted using TB tools, and the website is https://github.com/CJ-Chen/TBtools (accessed on 16 March 2024).

Figure 3

Analysis of cis-acting regulatory element numbers of CLE genes of P. trichocarpa (A), P. tomentosa (B) and P. alba × P. glandulosa (C). The left-most column of each heatmap shows the gene names. Above the heatmap represent different types of CAREs (the CAREs corresponding to the area below the brown horizontal line are related to stress response; the CAREs corresponding to the area below the green horizontal line are related to hormone regulation; the CAREs corresponding to the area below the orange horizontal line are related to plant development; and the CAREs corresponding to the area below the black horizontal line are related to light response). In the heatmap, the color intensity of the color blocks indicates the number of CAREs (as shown by the color strip on the far right of the entire figure, the number increases from light yellow to dark red). All the heatmaps were generated by GraphPad Prism 8.

Figure 4

The expression patterns of preCLE13 and subcellular localization of pre-propeptide of PtrCLE13A. (A–C) Expression patterns of preCLE13A, preCLE13B and preCLE13C in different tissues of P. trichocarpa. (D) Expression of preCLE13A and NCED3 in response to dehydration treatment. Three-month-old P. trichocarpa with good growth was used as the material for RT-qPCR analysis, and each experiment was based on three biological replicates of each sample and three technical replicates of each biological replicate. (E) The 35Spro:eGFP and 35Spro:preCLE13A-eGFP were transiently infiltrated in N. benthamiana leaves. (F) The 35Spro:preCLE13A-eGFP construct was transiently infiltrated in N. benthamiana leaves with pm-ck CD3-1001 construct as PM (plasma membrane) marker. Microscopic images contain green fluorescence field, chloroplast field, bright field and merged microscope images. Bars = 50 µm. Data are means ± SE.

Figure 4

The expression patterns of preCLE13 and subcellular localization of pre-propeptide of PtrCLE13A. (A–C) Expression patterns of preCLE13A, preCLE13B and preCLE13C in different tissues of P. trichocarpa. (D) Expression of preCLE13A and NCED3 in response to dehydration treatment. Three-month-old P. trichocarpa with good growth was used as the material for RT-qPCR analysis, and each experiment was based on three biological replicates of each sample and three technical replicates of each biological replicate. (E) The 35Spro:eGFP and 35Spro:preCLE13A-eGFP were transiently infiltrated in N. benthamiana leaves. (F) The 35Spro:preCLE13A-eGFP construct was transiently infiltrated in N. benthamiana leaves with pm-ck CD3-1001 construct as PM (plasma membrane) marker. Microscopic images contain green fluorescence field, chloroplast field, bright field and merged microscope images. Bars = 50 µm. Data are means ± SE.

Figure 5

PtrCLE13 and CLE9 peptides inhibit root meristem activity in A. thaliana. (A–D) Inhibitory effects on root growth of Arabidopsis seedlings after 10-day treatment with different concentrations of PtrCLE13 and CLE9 peptides. Bar = 1 cm. (E–G) Length of meristematic zone (MZ) and elongation zone (EZ) of Arabidopsis root tips after 10-day treatment with 0 [(a,e)], 0.1 µM [(b,f)], 1 µM [(c,g)] and 10 µM [(d,h)] PtrCLE13 and CLE9 peptide concentrations, respectively. Bar = 0.125 mm. Data are presented as means ± SD (n = 30 for (C,D,F,G)). Different lowercase letters indicate a significant difference at p < 0.05 based on ANOVA.

Figure 6

PtrCLE13 and CLE9 peptides exert inhibitory effects on root growth in P. tomentosa and A. thaliana by downregulating the expression of ARF and LBD genes. (A,B) Root morphology of P. tomentosa after two-week treatment with 0 (left), 0.1 (middle) and 1 µM (right) PtrCLE13 and CLE9 peptides concentrations. Bar = 1 cm. (C,D) The root length of P. tomentosa was measured after three-week treatment with different concentrations of PtrCLE13 and CLE9 peptides. (E,F) Expression levels of AtARF7, AtLBD16 and AtLBD29 in Arabidopsis roots after 12-day treatment with different concentrations of PtrCLE13 and CLE9 peptides, respectively. (G,H) Expression levels of PtoARF5.2, PtoLBD16.2 and PtoLBD29 in P. tomentosa roots after three-week treatment with different concentrations of PtrCLE13 and CLE9 peptides, respectively. Data are presented as means ± SD (n = 20 for (C,D) or n = 3 for (E–H). Different lowercase letters indicate a significant difference at p < 0.05 based on ANOVA.

Figure 7

PtrCLE13 peptide suppressed the growth of P. tomentosa by inhibiting plant height and leaf development. (A) Whole plants of P. tomentosa untreated (control check, CK) and treated by 10 µM PtrCLE13 [(a) front view; (b) top view]. Bar = 1 cm. (B,C) Plant height (B) and fresh weight (C) of P. tomentosa seedlings in (A). (D), Leaves of P. tomentosa untreated (control check, CK) and treated by 10 µM PtrCLE13. 3rd, 4th and 5th refer to the leaves on the third, fourth and fifth nodes of P. tomentosa seedlings, respectively. Bar = 1 cm. (E–G) Leaf length (E), leaf width (F) and leaf area (G) of P. tomentosa seedlings in (A). Data are presented as means ± SD (n = 9 for B,C,E–G). Different lowercase letters indicate a significant difference at p < 0.05 based on ANOVA.

Figure 8

Overexpression of PtrCLE13A enhances osmotic and drought tolerance in P. tomentosa. (A) Morphological phenotypes of EV (empty vector), OE-6 (overexpression line 6) and OE-3 (overexpression line 3) poplar roots subjected to mannitol treatments at concentrations of 50 mM, 100 mM and 150 mM. Bar = 1 cm. (B–D) The length of adventitious roots of poplar seedlings in (A). Data are presented as means ± SD (n = 12 for (B–D)). (E) Phenotypes of EV, OE-6 and OE-3 poplar seedlings before and after drought stress. (F–L) Net photosynthetic rate (F), stomatal conductance (G), transpiration (H), Fv/Fm (I), Y (Ⅱ) (J), ETR (K) and relative electrolyte leakage (L) of leaves of poplar seedlings in (E). Data are presented as means ± SD (n = 6 for (F–L)). Different lowercase letters indicate a significant difference at p < 0.05 based on ANOVA.