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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.
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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.

Mentions: M. truncatula seedlings were grown with the peptide as growth media additive [22]. A termination of root growth was clearly observable six days after treatment in all of the seedlings treated with peptides 1 and 2 compared to control plants in the absence of either peptide and compared to the randomized peptides (Figure 4, Figure 5). After six days of treatment, root growth of the plants treated with peptide 1 and peptide 2 was significantly (p < 0.0001, one-way analysis of variance) reduced compared to the no-peptide and the random peptide controls. After 20 days, almost no further root growth was observed in seedlings treated with peptide 1 or 2. We noted an increased formation of lateral roots in both peptide treatments. Similar to the RAM, the newly formed meristems of the lateral roots terminated their growth shortly after lateral root emergence. We tested the reversibility of the peptide treatment by transferring half of the plants to a fresh plate not containing peptides. The RAM recovered within two weeks. In some cases the main root terminated its growth, and a lateral root elongated instead. We also observed that the main root could recover its growth after release from the peptide-containing medium. In this experiment, shoot growth was not noticeably affected by the presence of peptide in the agar, although shoots were not in direct contact with the agar.

Bioinformatic analysis of the CLE signaling peptide family

Oelkers K, Goffard N, Weiller GF, Gresshoff PM, Mathesius U, Frickey T - BMC Plant Biol. (2008)

Bottom Line: 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.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.

Affiliation: School of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT, Australia. karsten.oelkers@anu.edu.au

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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