Non-cell-autonomous control of vascular stem cell fate by a CLE peptide/receptor system

Abstract

Land plants evolved a long-distance transport system of water and nutrients composed of the xylem and phloem, both of which are generated from the procambium- and cambium-comprising vascular stem cells. However, little is known about the molecular mechanism of cell communication governing xylem-phloem patterning. Here, we show that a dodecapeptide (HEVHypSGHypNPISN; Hyp, 4-hydroxyproline), TDIF (tracheary element differentiation inhibitory factor), is secreted from the phloem and suppresses the differentiation of vascular stem cells into xylem cells through a leucine-rich repeat receptor-like kinase (LRR-RLK). TDIF binds in vitro specifically to the LRR-RLK, designated TDR (putative TDIF receptor), whose expression is restricted to procambial cells. However, the combined analysis of TDIF with a specific antibody and the expression profiles of the promoters of two genes encoding TDIF revealed that TDIF is synthesized mainly in, and secreted from, the phloem and its neighboring cells. The observation that TDIF is capable of promoting proliferation of procambial cells while suppressing xylem differentiation suggests that this small peptide functions as a phloem-derived, non-cell-autonomous signal that controls stem cell fate in the procambium. Our results indicate that we have discovered a cell communication system governing phloem-xylem cross-talk.

Fig. 1.

Vascular phenotypes caused by excess TDIF. (A–H) TDIF suppresses xylem vessel differentiation in the leaf vein. Pc, procambium; Xy, xylem vessel; dXy, differentiating Xy. (A and G) No peptide application. (B–E) Application of 1 μM TDIF. (H) Application of 10 μM TDIF. (B and C) Discontinuous xylem vessel. (D) Sieve elements (SEs) visualized by aniline blue in the same position as C. (E) Proliferated procambium cells in the region in which TE formation was suppressed. (F) Discontinuous xylem vessel formation in leaves of a CLE44-overexpressing plant; the yellow arrows show the non-TE region in the veins. Veins are outlined in red, and xylem vessels are outlined in blue. (G and H) Sections of the vein; xylem vessels are not often formed in veins of TDIF-treated seedlings. Arrowheads show SEs. (I) Dose-dependent suppression of TE formation in the veins by TDIF. (Right) A schematic illustration of the quantification method. The presence (+Xy) or absence (−Xy) of the xylem vessels was examined at the position where arbitrary transverse lines and veins of a high order crossed. (Left) Based on the method, veins with discontinuous xylem vessel strands were quantified in the presence of TDIF or P9A, an inactive derivative of TDIF. Blue boxes indicate veins with xylem vessels; red boxes, veins without xylem vessels. (J) TDIF enlarges the stele of hypocotyls. (K) The number of procambial cells but not phloem or xylem cells is increased by TDIF application. White boxes indicate P9A application; black boxes, TDIF application. (L) Procambium marker genes (AtHB8 and VH1) were up-regulated by TDIF application. (M–U) TDIF promotes procambial cell proliferation in the hypocotyl. (M, O, Q, and S) Application of 1 μM P9A. (N, P, R, and T) Application of 1 μM TDIF. (M–R) Toluidine blue-stained transverse sections of hypocotyls of 7-day-old Arabidopsis seedlings. (O and P) Magnification of the stele. (Q and R) Magnification of the boxed area in O and P. Arrowheads indicate the division plane of newly divided cells. (S–U) Hypocotyls of 10-day-old seedlings that were treated with P9A and TDIF and that were overexpressing CLE44. TDIF enhanced random division of procambium cells. (Scale bars: A–D, F, M, N, and S–U, 100 μm; E, G, H, Q, and R, 20 μm; O and P, 50 μm.) Error bars indicate SEM, n = 6 (I), 18 (J), and 7 (K). Asterisks indicate significant differences from mock/P9A application by Student's t test (P < 0.01).

Fig. 2.

Identification of a putative TDIF receptor. (A) Each of three homozygous alleles in an LRR-RLK gene (tdr) resulted in xylem vessel formation that was insensitive to exogenous TDIF. (B) Xylem vessel formation in clv1 and clv2 was TDIF sensitive. Error bars indicate SEM (n = 6). Asterisks indicate that the ratios of the number of non-TE positions to all examined positions were significantly different compared with wild type (WT) treated with 1 μM TDIF by Student's t test (P < 0.01). (C) Predicted TDR protein structure and positions of T-DNA insertions. (D) Levels of TDR mRNA in tdr-1, tdr-2, and tdr-3 measured with RT-PCR. (E) A 5-week-old tdr plant. (F) A transverse section of the hypocotyl of a 7-day-old tdr seedling grown in the presence of P9A or TDIF peptide. Note that phloem cells (Ph) are located close to xylem vessel cells (Xy) and, in an extreme case, are adjacent to a xylem vessel cell (asterisk). (Scale bars: E, 10 cm; F, 50 μm.)

Fig. 3.

Direct interaction between TDR and TDIF. (A) Subcellular localization of TDR. (B) Suppression of TE differentiation in Zinnia cell culture. TDIF, ASA-TDIF, and the nonfunctional peptide P9A are shown. (C) Visualization of TDR-ΔKD-HT in transgenic tobacco BY-2 cells overexpressing TDR-ΔKD-HT. (D) Photoaffinity labeling of TDR-ΔKD-HT by ¹²⁵I-ASA-TDIF in the absence (−) or presence (+) of excess unlabeled TDIF in microsomal fractions from TDR-ΔKD-HT-overexpressing cells (TDR-OX) or nontransformed tobacco BY2 cells (BY2-WT). (E) Immunoprecipitation with an anti-HaloTag antibody for the photoaffinity-labeled samples used in D. (F) Photoaffinity labeling of TDR-ΔKD-HT by ¹²⁵I-ASA-TDIF in the absence (−) or presence of excess unlabeled TDIF, CLV3, CLE2, CLE9, CLE19, or CLE46 peptide.

Fig. 4.

Vascular cell-specific localization of TDIF and TDR. (A–F) Distinctive vascular cell-specific expression of the CLE41, CLE44, and TDR genes in roots (A, C, and E) and hypocotyls (B, D, and F). (A and B) pCLE41::GUS. (C and D) pCLE44::GUS. (E and F) pTDR::GUS. Ph, phloem; Pc, procambium; Pe, pericycle; En, endodermis. (G–K) Immunohistochemical localization of the TDIF peptide in the root tip (G and H) and hypocotyls (I–K). (J) Magnification of the box in I. Note that the signal is detected around the phloem cells. (L and M) Positional (L) and functional (M) models of the TDIF/TDR signaling system, which regulates the fate of procambial cells in a non-cell-autonomous manner. (Scale bars: A–F, 20 μm; G and H, 10 μm; I and K, 50 μm; J, 10 μm.)