The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway

Abstract

CLAVATA3 (CLV3), CLV3/ESR19 (CLE19), and CLE40 belong to a family of 26 genes in Arabidopsis thaliana that encode putative peptide ligands with unknown identity. It has been shown previously that ectopic expression of any of these three genes leads to a consumption of the root meristem. Here, we show that in vitro application of synthetic 14-amino acid peptides, CLV3p, CLE19p, and CLE40p, corresponding to the conserved CLE motif, mimics the overexpression phenotype. The same result was observed when CLE19 protein was applied externally. Interestingly, clv2 failed to respond to the peptide treatment, suggesting that CLV2 is involved in the CLE peptide signaling. Crossing of the CLE19 overexpression line with clv mutants confirms the involvement of CLV2. Analyses using tissue-specific marker lines revealed that the peptide treatments led to a premature differentiation of the ground tissue daughter cells and misspecification of cell identity in the pericycle and endodermis layers. We propose that these 14-amino acid peptides represent the major active domain of the corresponding CLE proteins, which interact with or saturate an unknown cell identity-maintaining CLV2 receptor complex in roots, leading to consumption of the root meristem.

Figure 1.

The CLE Motif and the Peptides Used in the Root Assays. (A) Alignment of CLV3, CLE5, CLE19, Bn CLE19, and CLE40. The signal sequences are underlined, and the CLE motif is framed. (B) Chemically synthesized peptides used in the root assays. Identical amino acids in five or more proteins/peptides are shaded in black, and amino acids that are similar or identical in at least three proteins are highlighted in gray.

Figure 2.

Treatment with CLE Peptides Gave a Short Root Phenotype in Arabidopsis. (A) Effect of different concentrations of CLE19p and AGp (control) on wild-type (Col-0) root development. The lengths of the main roots were measured after 7 d of growth on peptide-containing media (n = 8 for each treatment). (B) Effects of different peptides on the root lengths of Arabidopsis (Ler). The roots were measured after 14 d of growth on media with 10 μM various peptides. Note that CLE5p, CLV3m, CLV3t, and CLE19t did not inhibit root growth (n = 10 for each treatment). Data and error bars represent mean ± sd.

Figure 3.

Effect of CLE Peptides on Root Meristems. (A) to (G) The morphology of the primary roots of Arabidopsis (Ler) 14 d after treatments with different peptides. Note the consumption of the root meristem ([E] to [G]) after treatments with CLE peptides. The bar in (G) = 50 μm for (A) to (G). (H) Number of meristematic cells in roots of Ler seedlings after 14 d of treatment with different peptides. Data and error bars represent mean ± sd (n = 10).

Figure 4.

Effect of Peptides on the Root Growth in Different Mutants. All roots were measured after 2 weeks growing on media containing different peptides. (A) The length of the primary roots of clv1-1, clv2-1, and clv3-2 grown on media with different peptides. Note that the CLE peptides did not give a significant inhibition in clv2-1 (n = 10). (B) Number of meristematic cells in roots of clv1-1, clv2-1, and clv3-2 grown on media with different peptides (n = 10). Data and error bars in (A) and (B) represent mean ± sd. (C) to (F) The morphology of roots of Ler and clv mutants treated with CLE19p, showing that clv2-1 is not sensitive to the peptide. Bar in (C) = 50 μm for (C) to (F).

Figure 5.

Heterologous Production of CLE19 Protein and Its Effect on Root Development. (A) SDS-PAGE gel of thrombin-cleaved GST-Bn CLE19 and GST protein produced in E. coli with a mass of ∼5 and 24 kD, respectively. The molecular mass (in kD) is shown at the right. (B) Effect of purified the Bn CLE19 or GST proteins on root development. Wild-type (Col-0) seedlings were grown for 10 d on plates containing 10 μM Bn CLE19 or GST. Data and error bars represent mean ± sd (n = 8).

Figure 6.

Effects of CLE Peptides on the Cell Identity of Roots. The ground tissue initial cells are marked by arrows, and the ground tissue daughter cells, including cells at this position, are labeled with arrowheads. Abnormal expression of GFP or YFP in other cell layers is marked by asterisks. (A) to (D) Confocal analysis after 4-d treatment of the roots of J0571. Note that treatments with CLV3p (C) or CLE19p (D) lead to a delayed separation of cortex and endodermis and expression of GFP in pericycle cells. No peptide (A), 10 μM CLVm (B), CLV3p (C), and CLE19p (D). (E) to (H) Confocal analyses of P_SCR:GFP seedlings after 4-d treatments. No peptide (E), 10 μM CLV3s (F), 10 μM CLV3p (G), and 10 μM CLE19p (H). The inset in (G) shows the delayed separation of the cortex and endodermis cells (with GFP expression). (I) to (M) P_CO2:YFP-H2B marker line treated with different peptides. Note the YFP expression in the ground tissue daughter cells and the pericycle cells, but not in ground tissue initial cells. (I) No peptide, 4 d after germination. The inset shows the absence of YFP expression in the QC, ground tissue initial, and ground daughter cells. (J) 10 μM CLE19p, 4 d after treatment. (K) 10 μM CLE40p, 5 d after treatment. (L) and (M) 10 μM CLE19p, 5 d after treatment. Bars = 30 μm.

Figure 7.

Penetration of Fluorescence-Labeled CLE19p in Roots. The roots of Arabidopsis (Col-0) were incubated for 1 min ([A] to [C]) or 4 min ([D] to [F]) with 10 μM R-CLE19p ([A] and [D]), 10 μM lissamine rhodamine ([B] and [E]), or 10 μM PI ([C] and [F]). Bar in (F) = 30 μm for (A) to (F).