TDIF peptide signaling regulates vascular stem cell proliferation via the WOX4 homeobox gene in Arabidopsis

Abstract

The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell-cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth.

Figure 1.

Identification and Expression Analyses of TDIF Signaling Target Genes. (A) Effects of TDIF treatment on relative expression levels of WOX genes in the top part of 7-d-old seedlings. For each WOX gene, the rate of expression level of TDIF treatment per P9A treatment as measured by qRT-PCR is shown. The asterisk indicates significant difference between TDIF and P9A treatment. Peptides were treated for 7 d. (B) Relative WOX4 expression level measured by qRT-PCR in Col-0 and tdr-1 plants that were either mock treated or treated with TDIF or P9A for the indicated amounts of time. Enhancement of WOX4 expression compared with mock treatment was observed only in the samples treated with TDIF for 1 h or more. The values of expression level are normalized by both the mock treatment in Col-0 and TUA4. (C) Relative expression levels of procambium marker genes (ATHB8, ATHB15, and TDR) and the TDIF genes (CLE41 and CLE44). Expression levels of the procambium marker genes but not the TDIF genes were significantly upregulated compared with mock treatment only when plants were treated with TDIF for 7 d. The values of expression level are normalized by both the mock treatment and TUA4. Error bars indicate sd, n = 3; *Student’s t test significance at P < 0.05 for different means.

Figure 2.

Expression Patterns of WOX4. WOX4 promoter activity was observed using WOX4_pro:GUS plants. (A) to (D) The 7-d-old cotyledon (A), root (B), and hypocotyl (C) and 14-d-old first leaf (D). (E) and (F) In the SAM (E) and RAM (F), the GUS signal was below the detectable level. (G) to (J) Comparison of WOX4_pro:GUS ([G] and [I]) and TDR_pro:GUS ([H] and [J]) expression in young leaves. Each pair of (G) and (I), and (H) and (J) shows nearly the same stages in leaf development. (K) and (L) Procambium/cambium-specific WOX4_pro:GUS staining in transverse sections of 7-d-old (K) and 4-week-old (L) hypocotyls. Weak staining was also observed in the phloem and pericycle cells in the 7-d-old hypocotyl. (M) to (R) Treatment with 1 μM P9A ([M], [O], and [Q]) or TDIF ([N], [P], and [R]) did not change the pattern of GUS staining in the hypocotyl ([M] and [N]), root ([O] and [P]), and cotyledon ([Q] and [R]) of 7-d-old plants. The arrow in (E) indicates the SAM. The arrowhead in (F) indicates the position of the cortex transition zone of the root meristem. Arrows in (K) indicate the phloem cells. In (L), ph and xy indicate phloem and xylem tissues, respectively. Bars = 500 μm in (A), (Q), and (R), 200 μm in (B) and (C), 1 mm in (D), 50 μm in (E) and (F), 10 μm in (G) and (H), 20 μm in (I) to (L), and 100 μm in (M) to (P).

Figure 3.

TDIF-Sensitive Xylem Formation in wox4 Mutants. (A) Effects of 1 μM TDIF on 10-d-old leaf veins of different strains. TDIF caused fragmentation of xylem strands (indicated by white arrowheads) in wox4-1 and in the wild type but did not in tdr-1 or the tdr-1 wox4-1double mutant. Veins are outlined in red, and xylem strands are outlined in blue. (B) Quantification of the effects of TDIF. The blue and white boxes indicate the frequencies of the xylem-absent and xylem-present veins, respectively. Bars = 100 μm in (A). Error bars indicate se, n = 8; *Student’s t test significance at P < 0.01 for different means of the frequency of xylem-absent veins in (B).

Figure 4.

Differences and Similarities between TDR and WOX4 Functions in Determining Procambial Cell Behavior. (A) to (H) Magnification of the procambium region in hypocotyls of seedlings grown for 7 d with 1 μM P9A ([A], [C], [E], and [G]) or TDIF ([B], [D], [F], and [H]). tdr ([C] and [D]) and tdr wox4-1 ([G] and [H]), but not the wild type ([A] and [B]) or wox4-1 ([E] and [F]), often had adjoining phloem and xylem cells. Panels at the right show the arrangement of vascular cells. Red, blue, and yellow areas show phloem, xylem, and procambial cells, respectively. (I) and (J) Number of procambial cells per section (I) and stele width (J) in 7-d-old plants grown with P9A (white) or TDIF (blue). The ± indicates heterozygous mutants. Bars = 10 μm in (A) to (H). Error bars indicate se, n = 9 to 12; *Student’s t test significance at P < 0.01 for different means in (I) and (J).

Figure 5.

Effects of WOX4 Overexpression on Vascular Development and Root Growth. (A) Relative expression level of WOX4 per TUA4 in the 35S_pro:WOX4 lines measured by qRT-PCR. The wild-type expression level is set to 1. (B) Stele width of 7-d-old hypocotyls. No significant differences were found in the four different 35S_pro:WOX4 lines when compared with the wild type (Col-0). (C) and (D) Transverse sections of 7-d-old hypocotyls. Cellular arrangement in the stele was similar between the wild type (C) and 35S_pro:WOX4 (D). (E) Relative expression of TDIF genes (CLE41 and CLE44) per TUA4 in 35Spro:WOX4 lines (#6 and #18). The wild-type expression level is set to 1. (F) Root growth of 7-d-old plants. (G) Tissue anatomy in the RAM of 7-d-old plants. Bars = 20 μm in (C) and (D), 1 cm in (F), and 100 μm in (G). Error bars indicate sd (n = 3, *Student’s t test significance at P < 0.05 for different means compared with the wild-type expression levels) in (A) and (E) and se (n = 9 to 10, *Student’s t test significance at P < 0.01 for different means compared with the wild type) in (B).

Figure 6.

Phenotypes of cle41 Mutants. (A) Stele widths of the 7-d-old hypocotyls treated with P9A (white) or TDIF (blue). (B) Magnification of the procambial region in 5-d-old (top) and 7-d-old (bottom) hypocotyls. Only the 7-d-old cle41-1 mutants had adjoining phloem and xylem cells. Scale bars = 10 μm in (B). Error bars indicate se, n = 9 to 10; *Student’s t test significance at P < 0.01 for different means in (A).

Figure 7.

Exhaustion of Vascular Stem Cells Caused Premature Termination of Secondary Growth. (A) to (D) Overall radial structure of the 4-week-old hypocotyls. (E) to (H) Magnifications of the cambial region located between the secondary phloem and xylem tissues. In contrast with the wild-type (E) and wox4-1 (G) plants, tdr (F) and tdr wox4 (H) plants had cavities in the outline of xylem tissues and the continuous cambium ring was disrupted. Some sieve elements (white arrowheads) were found at the bottom of the cavities ([F] and [H]). Numerals at left bottom show the number of cavities per the number of plants tested in (E) to (H). (I) to (L) Central regions of the vascular cylinder in the wild type ([I] and [K]) and the most severely affected tdr-1 wox4-1 plant ([J] and [L]). In (K) and (L), the most central region of the vascular cylinder was magnified. Black arrows and arrowheads in (L) indicate the axis of primary xylem and primary phloem cells respectively. Stained by safranin-O and aniline blue, cells in xylem, phloem, and phellem (cork) show orange, green, and blue fluorescence, respectively, in (A) to (D), (I), and (J). Bars = 100 μm in (A) to (D), (I), and (J), 50 μm in (E) to (H), and 20 μm in (K) and (L).