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Link to original content: https://pubmed.ncbi.nlm.nih.gov/18171480
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. 2008 Jan 3:8:1.
doi: 10.1186/1471-2229-8-1.

Bioinformatic analysis of the CLE signaling peptide family

Karsten Oelkers  1 , Nicolas Goffard, Georg F Weiller, Peter M Gresshoff, Ulrike Mathesius, Tancred Frickey
Affiliations

Bioinformatic analysis of the CLE signaling peptide family

Karsten Oelkers et al. BMC Plant Biol. .

Erratum in

  • BMC Plant Biol. 2009;9:17

Abstract

Background: Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases.

Results: We identified 114 new members of the CLE protein family from various plant species, as well as five protein sequences containing multiple CLE domains. We were able to cluster the CLE domain proteins into 13 distinct groups based on their pairwise similarities in the primary CLE motif. In addition, we identified secondary motifs that coincide with our sequence clusters. The groupings based on the CLE motifs correlate with known biological functions of CLE signaling peptides and are analogous to groupings based on phylogenetic analysis and ectopic overexpression studies. We tested the biological function of two of the predicted CLE signaling peptides in the legume Medicago truncatula. These peptides inhibit the activity of the root apical and lateral root meristems in a manner consistent with our functional predictions based on other CLE signaling peptides clustering in the same groups.

Conclusion: Our analysis provides an identification and classification of a large number of novel potential CLE signaling peptides. The additional motifs we found could lead to future discovery of recognition sites for processing peptidases as well as predictions for receptor binding specificity.

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Figures

Figure 1
Figure 1
Multidomain CLE sequences. The potential multidomain CLE signaling peptides CLE75, CLE76, CLE68, CLE31 and CLE30 are represented. The figure is a scaled representation of the domain organization. The relative positions of the first amino acid of the motifs are specified.
Figure 2
Figure 2
Analysis of sequence similarity in the CLE domain. CLANS clustering of 174 sequences based on their sequence similarity in the CLE domain. Sequences are represented by dots and the various groups are highlighted by ovals. Sequences of the same group are assigned the same color. Lines connecting the dots correspond to BLASTP values better than 1.2E-7. Characterized CLE members HgCLE (CLE47), TDIF (CLE49) and ZmESR (CLE143–CLE147), as well as the known orthologs CLV3/FON4 and CLE19/BnCLE19 (CLE162) are highlighted with red stars. The single CLE member found from Physcomitrella patens (moss, CLE170), which clusters into group 11, is highlighted with a grey star. A putative CLE sequence from Chlamydomonas reinhardtii (alga, CLE177) is also marked with a grey star but does not cluster close to any group. The grouping established upon cluster analysis is analogous to previous classifications [8, 12, 24]. Group 2 contains CLE1–CLE7, which were previously shown to have no effect on RAM growth or on vascular cell differentiation in peptide assays and which led to wus-like dwarf growth only at 21 days after germination when ectopically overexpressed. CLE9–CLE13 can be found in group 7. These CLE members had an effect on the RAM but not on vascular cell differentiation in peptide assyas and wus-like dwarf growth could be observed at 14 and 21 days after germination in overexpression studies. The CLE family members CLE41, CLE42, CLE44, which had no effect on RAM but on vascular cell differentiation in peptide assays, and had a shrub-like overexpression phenotype are located in group 5.
Figure 3
Figure 3
Weblogo representation of the conservation pattern of residues in each group and for the entire protein family. The previously described main CLE motif of 12 amino acid length is marked with a black frame. Group specific residues are marked in black in the various groups. Invariant residues are marked in black in the bottommost logo. Conserved residues are marked grey. The size of the letter symbolizes the frequency of that residue in the group and at that position. A secondary motif was identified at around 50 amino acids upstream of the primary CLE motif in groups 1, 2, 8 and 13. Extensions of the motif are recognizable at both the C- and N-terminus. Bracketed figures indicate the number of sequences assigned to the respective group.
Figure 4
Figure 4
Biological activity of CLE peptides in Medicago truncatula. Confirmation of the biological activity of synthetic CLE peptides corresponding to 14 amino acids of the conserved domain of predicted CLE signaling peptides in a plate assay using M. truncatula. Peptides were added at a concentration of 10 μM as growth media additives. The top row (A-C) shows plant growth in the absence of peptide, the middle row (D-F) in the presence of peptide 1 (SKRKVPSCPDPLHN), and the bottom row (G-I) in the presence of peptide 2 (SKRRVPNGPDPIHN). Plant growth is shown on day 6 after treatment (left column; A, D, G), on day 20 after treatment (middle column; B, E, H) and on day 20 of recovery, whereby seedlings were treated for 6 days and then transferred to plates without peptide for the remaining 14 days (right column; C, F, I). Bar on the bottom of each column indicates 2 cm.
Figure 5
Figure 5
Sequence specificity of CLE peptide activity. Root length of Medicago truncatula plants at 6 days after treatment with different peptides. Control plates did not contain peptide, peptide 1 (SKRKVPSCPDPLHN) and peptide 2 (SKRRVPNGPDPIHN) resemble the CLE motif, peptide 3 (randomized version of peptide 1, DHKSKPPVLRPNSC) and peptide 4 (randomized version of peptide 2, PVHPKGNRNDISPR) do not resemble the CLE motif. Bars with different letters differ significantly at p < 0.0001 (N = 27; one-way ANOVA). Both CLE peptides are significantly different from the no-peptide control and the control peptides with randomized amino acid sequence.
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