Plant CLE peptides from two distinct functional classes synergistically induce division of vascular cells

Abstract

The Clavata3 (CLV3)/endosperm surrounding region (CLE) signaling peptides are encoded in large plant gene families. CLV3 and the other A-type CLE peptides promote cell differentiation in root and shoot apical meristems, whereas the B-type peptides (CLE41-CLE44) do not. Instead, CLE41 inhibits the differentiation of Zinnia elegans tracheary elements. To test whether CLE genes might code for antagonistic or synergistic functions, peptides from both types were combined through overexpression within or application onto Arabidopsis thaliana seedlings. The CLE41 peptide (CLE41p) promoted proliferation of vascular cells, although delaying differentiation into phloem and xylem cell lineages. Application of CLE41p or overexpression of CLE41 did not suppress the terminal differentiation of the root and shoot apices triggered by A-type CLE peptides. However, in combination, A-type peptides enhanced all of the phenotypes associated with CLE41 gain-of-function, leading to massive proliferation of vascular cells. This proliferation relied on auxin signaling because it was enhanced by exogenous application of a synthetic auxin, decreased by an auxin polar transport inhibitor, and abolished by a mutation in the Monopteros auxin response factor. These findings highlight that vascular patterning is a process controlled in time and space by different CLE peptides in conjunction with hormonal signaling.

Fig. 1.

Combined effect of CLE6p (A-type) and CLE41p (B-type). All CYCB1,1_pro:GUS plantlets were transferred to media supplemented with the indicated peptide(s), 3 dag (n = 10 for each treatment). (A) Root growth rate after transfer. (B) GUS expression patterns in mature primary root tissue (7 dat). (C) First true leaves (10 dat; Top), cotyledons (7 dat; Middle), and hypocotyls (7 dat; Bottom). (D) Hypocotyl sections showing vascular GUS expression (7 dat in liquid medium) and counterstained with ruthenium red. In C and D, initial concentration of each added peptide was 10 μM. [Scale bars: 100 μm in B, C (Bottom), and D; 1 mm in C (Top and Middle).]

Fig. 2.

Radial enlargement of the hypocotyl stele after CLE6p and CLE41p treatments. (A) Hypocotyl stele width of the wild type (Col-0) transferred 3 dag and grown for 15 days in liquid medium supplemented with the indicated peptide(s) (n = 15). (B) Representative hypocotyls. Samples are aligned according to labels of abscissa in A. Arrows indicate the measured width. (Scale bar, 100 μm.) (C) Hypocotyl stele width of wild-type (Ler), clv1–1, and clv2–1 seedlings transferred 3 dag and grown for 10 days in liquid medium with or without combined CLE6p/CLE41p (n = 10). The asterisk indicates samples significantly different from control without peptide. (Student t test P < 0.05.)

Fig. 3.

Cellular details of vascular tissues in hypocotyls treated with CLE peptides. Hypocotyls were sectioned directly above the collet and stained with ruthenium red. Seedlings were transferred 3 dag and grown for 15 days in liquid medium supplemented with (A) no peptide, (B) CLE6p, (C) CLE6p and CLE41p, and (D) CLE41p (n = 10). (A, B, and D) Black arrows indicate phloem poles. Initial concentration of each added peptide was 10 μM. (Scale bars, 50 μM.)

Fig. 4.

Defects in vascular development after CLE peptide treatment. GUS expression in (A) APL_pro:GUS and (B) ATHB8_pro:GUS hypocotyls. Seedlings were transferred 3 dag and grown for 15 days in liquid medium supplemented with the peptide(s) indicated, each at an initial concentration of 10 μM (n = 10). (Scale bars, 50 μm.) (Inset) Longitudinal section through a hypocotyl stele showing xylem (X) and phloem strands (PS). (Scale bar, 10 μm.)

Fig. 5.

Interaction between CLE peptide and hormone signaling. (A) Hypocotyl stele width of whole or decapitated wild-type seedlings transferred at 3 and 5 dag, respectively, and for 10 days into liquid medium supplemented with the indicated peptide(s) (n = 15). (B) Treatments combining CLE peptides, auxin, and auxin transport inhibitor. Ten seedlings were transferred 3 dag and grown for 10 days either with NAA (1 mM) or NPA (1 μM), in combination with no peptide, CLE6p, CLE41p, or CLE6p/CLE41p. Initial concentration of each added peptide was 10 μM in both experiments. The asterisk marks samples significantly different from mock-treated controls. (Student t test P < 0.05.)