ARABIDOPSIS THALIANA HOMEOBOX GENE1 establishes the basal boundaries of shoot organs and controls stem growth

Abstract

Apical meristems play a central role in plant development. Self-renewing cells in the central region of the shoot meristem replenish the cell population in the peripheral region, where organ primordia emerge in a predictable pattern, and in the underlying rib meristem, where new stem tissue is formed. While much is known about how organ primordia are initiated and their lateral boundaries established, development at the interface between the stem and the meristem or the lateral organs is poorly understood. Here, we show that the BELL-type ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1) is required for proper development of the boundary between the stem and both vegetative and reproductive organs and that this role partially overlaps with that of CUP-SHAPED COTYLEDON genes. During the vegetative phase, ATH1 also functions redundantly with light-activated genes to inhibit growth of the region below the shoot meristem. Consistent with a role in inhibiting stem growth, ATH1 is downregulated at the start of inflorescence development and ectopic ATH1 expression prevents growth of the inflorescence stem by reducing cell proliferation. Thus, ATH1 modulates growth at the interface between the stem, meristem, and organ primordia and contributes to the compressed vegetative habit of Arabidopsis thaliana.

Figure 1.

ATH1 Is Required to Form the Basal Boundaries of Shoot Organs. (A) and (B) Inflorescences of wild-type (A) and ath1-3 (B) plants, showing that stamens remain attached to the developing fruits of ath1-3 (arrows). (C) and (D) Sections through the base of flowers, with arrows indicating the dehiscence zone at the base of a wild-type stamen (C) and the corresponding region in ath1-3 (D). (E) and (F) Closeups of the base of mature stamens in the wild type (E) and in ath1-3, which has partially fused stamens in this region (F). (G) and (H) Scanning electron micrographs of stage 11 wild-type (G) and ath1-3 (H) flowers; the arrow shows the partial fusion at the base of stamens in ath1-3. (I) and (J) Scanning electron micrographs of wild-type (I) and ath1-3 (J) floral buds, with arrowheads indicating the boundary between sepals and pedicel. (K) and (L) Whole-mount staining of BP:GUS in wild-type (K) and ath1-3 (L) backgrounds. (M) and (N) Scanning electron micrographs of the base of cauline leaves of the wild type (M) and ath1-3 (N); arrows indicate the boundary between the leaf and inflorescence stem. (O) and (P) Base of the rosette of 2-week-old wild-type (O) and ath1-3 (P) plants; h and c mark the hypocotyls and the petioles of cotyledons, respectively, and the arrowheads show the boundary at the base of leaf petioles. Bars = 1 cm in (A) and (B), 1 mm in (K), (L), (O), and (P), and 100 μm in (C) to (J), (M), and (N).

Figure 2.

Interaction between ath1-3 and cuc Genes. (A) and (B) One-week-old homozygous cuc1 cuc2 (A) and ath1-3 cuc1 cuc2 (B) seedlings. (C) to (F) Scanning electron micrographs of mature (stage 13) flowers of the wild type (C), ath1-3 (D), cuc2/cuc2 cuc1/+ (E), and ath1-3/ath1-3 cuc2/cuc2 cuc1/+ (F); the arrowheads show the point where sepals become separate. (G) to (J) Scanning electron micrographs of the base of flowers comparable to (C) to (F), respectively; note that the partial loss of the basal boundaries in (H) is enhanced in (J). (K) to (M) RNA in situ hybridization showing expression of CUC1 at the developing organ boundaries of a stage 3 bud (arrows) but absent from the basal region of organs at later stages (arrowheads) in the wild type (K), ath1-3 (L), and 35S:ATH1 (M). Bars = 1 mm in (A) and (B) and 100 μm in (C) to (M).

Figure 3.

ATH1 Expression Pattern. (A) and (B) In situ hybridization showing the expression pattern of ATH1 in the inflorescence tip. (A) shows a section through a stage 6 floral bud, showing ATH1 expression in developing stamens (st), carpels (ca), and the base of sepals (se); im indicates the inflorescence meristem. (B) shows a section through buds at different stages (numbers marked on the floral pedicels); arrows indicate expression at the base of sepals and stamens at stages 10 to 12. (C) and (D) Whole-mount staining of ATH1:ATH1-GUS inflorescences. (C) shows a lateral inflorescence showing expression in young buds, in the carpels and stamens of older buds, and at the base of a cauline leaf (arrow). (D) shows an inflorescence with most buds removed, showing expression at different stages (numbered); arrows indicate expression at the base of stage 10 to 12 buds. (E) and (F) Sections through ATH1:ATH1-GUS at an early stage ([E]; stage 3), showing expression in the floral meristem (fm), and at a late stage ([F]; stage 10 to 11), showing expression in carpels (ca) and at the bases of sepals (se) and stamens (st). (G) and (H) Sections through seedling apices, hybridized with ATH1 antisense probe (G) and sense control (H); m marks the shoot apical meristem, and lp indicates leaf primordia. (I) and (J) Staining of ATH1:ATH1-GUS seedlings, showing expression in the shoot apex and at the base of developing leaves. (I) shows whole-mount staining. (J) shows a section through a seedling comparable to that in (I), showing GUS expression throughout the meristem and leaf primordia. Bars = 50 μm in (A), (E), (G), (H), and (J), 100 μm in (B) and (F), or 1 mm in (C), (D), and (I).

Figure 4.

ATH1 Functions Redundantly with Light-Activated Genes to Inhibit Growth of the Subapical Region of the Shoot. (A) and (B) Expression of ATH1:ATH1-GUS in the shoot apex of seedlings grown in the dark for 1 week without sucrose (A) or with 1% sucrose (B). (C) and (D) Shoot apices of wild-type (C) and ath1-3 (D) seedlings after 2 weeks of growth in the dark with 1% sucrose; the arrow in (D) indicates the displaced meristem and leaf primordia in ath1-3. (E) A 2-week-old ath1-3 seedling grown in standard light conditions; note that the leaf primordia emerged from the normal position at the junction of the cotyledon petioles. (F) to (K) Sections through the shoot apex of wild-type ([F], [H], and [J]) or ath1-3 ([G], [I], and [K]) seedlings grown in the dark at different times after germination on medium with 1% sucrose ([F] and [G], 0 weeks; [H] and [I], 1 week; [J] and [K], 2 weeks); arrows indicate the shoot meristem, and st indicates stipules. (L) and (M) Expression of the stipule marker P_FCA:GUS (Laurie, 2003) in wild-type (L) and ath1-3 (M) seedlings after 2 weeks of growth in the dark with 1% sucrose. (N) and (O) BP:GUS expression in wild-type (N) and ath1-3 (O) seedlings grown for 2 weeks in the dark on medium with 1% sucrose; the arrow in (O) indicates the extended BP:GUS-expressing region below the meristem of ath1-3. Bars = 0.5 mm in (A), (B), and (E), 20 μm in (F) and (G), and 100 μm in (C), (D), and (H) to (O).

Figure 5.

ATH1 Inhibits Cell Proliferation in the Subapical Region of the Shoot. (A) to (D) Whole-mount staining of vascular strands of wild-type ([A] and [C]) or ath1-3 ([B] and [D]) seedlings grown in standard light ([A] and [B]) or in low light ([C] and [D]); the arrowheads mark the distance between the base of the shoot meristem and the point where the vascular strands converge at the top of the hypocotyls. hy, hypocotyl; pe, petiole. (E) to (H) Sections through the shoot apices of seedlings comparable to those shown in (A) to (D), respectively. (I) Cell length in the region indicated between arrowheads in (A) to (D), measured in sections comparable to those shown in (E) to (H). Bars represent averages ± sd (n = 16 to 18 for each treatment). (J) Total length of the region between arrowheads in (A) to (D), measured in sections equivalent to those in (E) to (H). Bars represent averages ± sd (n = 4 for each treatment). Numbers over each bar are ratios between total length and average cell length. Bars = 1 mm in (A) to (D) and 200 μm in (E) to (H).

Figure 6.

ATH1 Is Downregulated at the Transition to Bolting. (A) and (B) Expression of ATH1:ATH1-GUS in the shoot apex of three different plants grown for 33 d in short days (A) or for 30 d in short days followed by 3 d in long days to induce flowering (B). Bars = 1 mm. (C) Expression of ATH1 measured by quantitative RT-PCR in plants grown for 33 d in short days (SD), 32 d in short days followed by 1 long day (LD1), or 30 short days followed by 3 long days (LD3). The vertical axis shows expression levels relative to the average of the SD treatment; the bars show averages ± sd for three independent RNA extractions (each from three to four whole rosettes).

Figure 7.

Ectopic Expression of ATH1 Inhibits Cell Proliferation in the Stem. (A) Inflorescence of a 35S:ATH1 plant. Note the maturing siliques (arrows) in spite of very little stem elongation. (B) and (C) Closeups of the inflorescence stem of a 35S:ATH1 plant (B) and a wild-type control (C). Note that the internodes that separate developing siliques are much shorter in 35S:ATH1. (D) to (I) Sections through the inflorescence apex ([D] and [E]), through the internode between siliques at positions 9 and 10 (Bleecker and Patterson, 1997) ([F] and [G]), or through the base of the inflorescence stem ([H] and [I]) of wild-type ([D], [F], and [H]) and 35S:ATH1 (line nr.3) ([E], [G], and [I]) plants. (J) Length of the inflorescence stem measured at the same stage of development in the wild type and three independent 35S:ATH1 lines (nr.1, nr.2, and nr.3). The bars show averages ± sd (n = 11 for the wild type, n = 5 for each 35S:ATH1 line). (K) Length of pith cells near the apical region (100 to 500 μm from the meristem), internode (positions 9 and 10), and basal region of the stem of wild-type or 35S:ATH1 plants. The bars represent averages ± sd (n = 13 to 17). Bars = 1 mm in (A) to (C) and 100 μm in (D) to (I).