Fig 1.
MpCLE1 peptide variants mimic Arabidopsis CLE41/TDIF.
(A) A phylogenetic tree of CLE peptides based on their 12 amino-acid CLE peptide sequences. Two subgroups R-type and H-type are indicated. (B) A phylogenetic tree of CLE peptide receptors based on their kinase domain sequences. TDR and CLV1 subclades are indicated. ER(ERECTA) is set as the outgroup. The posterior probabilities of trees are shown at the nodes in (A) and (B). At, Arabidopsis thaliana; Sm, Selaginella moellendorffii; Nsp, Nothoceros sp.; Pp, Physcomitrella patens; Sl, Sphagnum lescurii; Me, Marchantia emarginata; Mp, Marchantia polymorpha; Rn, Ricciocarpos natans; St, Sphaerocarpos texanus. (C) Peptides used in the assays. Blue characters indicate residues different from the MpCLE1 peptide. Residues “O” indicate hydroxyprolines. (D) Effects of 10 μM peptide treatment on stele thickening in the hypocotyls of 10-day-old Arabidopsis. Peptides are indicated below. “Hyp” and “-” indicate the peptides with and without hyrdoxyprolines, respectively. Note that AtCLE41-Hyp is identical to TDIF. Data represent mean values ± s.d. (n = 13–16). Asterisks indicate a significant difference from mock treatment (0 M) in Welch’s t-test, p<0.05. (E) Effects of 10 μM peptide treatment in the leaf vein of 10-day-old Arabidopsis. Red and cyan lines indicate vein and xylem strand, respectively. Scale bars = 100um.
Fig 2.
Gain-of-function phenotypes of MpCLE1.
Morphology of 14-day-old M. polymorpha plants grown from gemmae. (A and B) Overall morphology of (A) wild type (Tak-1) and (B) MpEF1α promoter-driven MpCLE1-overexpression line. (C) Area of thalli (mean ± s.d.). Two independent transgenic lines were used for quantification. Asterisks indicate a significant difference from WT in Weltch’s t-test (p < 0.001, n = 18). (D) Schematic diagram of section planes in apical notch. Longitudinal sections of apical notches in (E) wild type (Tak-1) and (F) the MpCLE1-overexpression line. Arrows, red and black arrowheads, and brackets indicate developing gemmae cups, apical cells, developing air chambers closest to the apical cell, and proliferative region of meristem, respectively. Scale bars = 1cm in (A and B), 200 μm in (E, F).
Fig 3.
Reduction of meristem activity by MpCLE1.
Consecutive transverse sections of apical notches in 14-day-old plants grown from gemmae. WT (A1-A7) and MpCLE1 overexpression (B1-B7) plants are compared. The relative position (μm) is indicated at the right bottom corner of each panel. Note that “0 μm” (A2 and B2) is set at the section in which two flanking lobes merged in the consecutive transverse sectioning as illustrated in (C). Arrows indicate a gemmae cup. Scale bars = 200 μm.
Fig 4.
Loss-of-function phenotypes of MpCLE1 in apical notch.
(A and B) Longitudinal sections of apical notches in 14-day-old M. polymorpha plants grown from gemmae in (A) homologous recombination-based Mpcle1 knock-out line and (B) its complementation line. (C and D) Transverse sections of apical notches at “20 μm” position from the junction of two flanking lobes. Note that consecutive sections including these figures are shown in S6 Fig. Red and black arrowheads, and brackets indicate apical cells, developing air chambers closest to the apical cell, and proliferative region of meristem, respectively. Scale bars = 200 μm.
Fig 5.
Loss-of-function phenotypes of MpCLE1 in antheridiophore.
Comparison of antheridiophore morphology between WT (A, C, E, G and I) and Mpcle1 knock-out line (B, D, F, H and J). (A and B) Overall morphology. (C and D) Stalk cross-sections. (E and F) Antheridial receptacles. (G and H) Hand sections of the antheridial receptacles. (I and J) Antheridia. Scale bars = 2 mm in (A, B and E-H), 1 mm in (I) and (J) and 200 μm in (C) and (D).
Fig 6.
Genetic interaction of MpCLE1 and MpTDR. Mptdr knock-out mutant is insensitive to excess MpCLE1/TDIF.
(A and B) Overall morphology of 21-day-old plants grown from gemmae. (C) Area of thalli in 14-day-old plants (mean ± s.d.). No significant difference was found between Mptdr and Mptdr MpCLE1ox in Weltch’s t-test (p = 0.19, n = 17–18). (D-G) Overall morphology of 17-day-old plants grown from gemmae with or without 10 μM TDIF. (H-K) Archegonial receptacles. Mpcle1 and Mptdr indicate knock-out mutants. MpCLE1ox indicates MpEF1α promoter-driven overexpression. Note that MpCLE1 overexpression plants do not make gametangiophores (S3G Fig). Scale bars = 1 cm in (A,B,D-G) and 2 mm in (H-K).
Fig 7.
Expression patterns of MpCLE1 and MpTDR in apical notch.
(A and B) Promoter-GUS reporter assays for MpCLE1 and MpTDR expression in 5-day-old gemmalings. (C and D) Longitudinal sections of the promoter:GUS lines at the notch in 5-day-old gemmalings. (E) Schematic illustration of promoter activities of CLE (red) and receptor (cyan) genes in the meristem. M. polymorpha (left) genes are contrasted with A. thaliana (right) genes. Scale bars = 200 μm (A and B), 100 μm (C and D).