Article Figures & Data
Figures
- Figure 1.
Phenotypes of ap2-1 and 28-5 ap2-1 Mutant Flowers.
(A) ap2-1 mutant flower.
(B) and (C) 28-5 ap2-1 mutant flowers. The pistils of 28-5 ap2-1 mutant flowers are three to five times wider laterally than those of ap2-1 (B), and the enlarged region is empty (C).
(D) Scanning electron micrograph (SEM) of a 28-5 ap2-1 flower viewed from above.
(E) to (I) SEM of ap2-1 pistil.
(J) to (N) SEM of 28-5 ap2-1 pistil.
(E) and (J) View of stage 16 pistil, showing lateral expansion of the mutant ovary. (F) and (K) Close-up view toward the top of the pistils. (G) and (L) Close-up view near the centers of the pistils. Re, replum; Va, valve. (H) and (M) Close-up view of the edges of the pistils. The carpel cells of 28-5 ap2-1 are distorted and larger than those of ap2-1. (I) and (N) Higher magnification of the cells at the edge of (H) and (M), respectively.
Bars in (A), (B), and (C) = 1 mm; bars in (D), (E), (F), (J), and (K) = 500 μm; bars in (G), (H), (L), and (M) = 200 μm; bars in (I) and (N) = 50 μm.
- Figure 2.
Phenotypes of Wild-Type and 28-5 Single Mutant Flowers.
(A) and (B) Wild-type (Landsberg erecta) (left) and 28-5 single mutant flowers (right). The length of the sepals and petals of 28-5 flowers did not differ from those of the wild type. However, stamens of 28-5 mutants were ∼50% shorter than those of the wild type. The 28-5 single mutant pistils were longer and wider than those of the wild type.
(C) Wild-type silique ∼14 days after emasculation (left), 28-5 single mutant siliques ∼3 days (middle), and 5 days (right) after anthesis. The siliques of 28-5 mutants continued to elongate without fertilization. Stigmatic papillae of elongating 28-5 siliques were still intact.
(D) Unpollinated dried 28-5 single mutant silique. The silique of the 28-5 single mutant showed a parthenocarpic phenotype. One carpel was removed to view the inside (right). No seeds were produced.
(E) SEM of 28-5 single mutant pistil. Part of one carpel was removed to view the ovules inside.
(F) to (H) Close-up view of the ovules. Most of the ovules were shriveled around the region where the embryo sac would be in the wild type (G). However, a few ovules showed normal morphology (H).
(I) Self-pollinated wild-type silique (left) and pollinated 28-5 single mutant silique with wild-type pollen (middle). Pollinated 28-5 silique elongated to as much as 18 mm in one extreme case (right).
Bars in (C), (D), and (I) = 1 mm; bar in (E) = 500 μm; bar in (F) = 200 μm; bars in (G) and (H) = 50 μm.
- Figure 3.
Physical Map of the 28-5 Locus and Overexpression of 28-5 Gene 1 in the 28-5 Mutant.
(A) Schematic map of the T-DNA activation-tagged 28-5 gene. The regions of the T-DNA activation tagging vector pSKI015 (http://biosun.salk.edu/LABS/pbio-w/) are shown: Bastar, bar gene; pB KS, pBluescript KS+; triangles, cauliflower mosaic virus 35S enhancers. Recognition sites for restriction endonucleases BamHI (B), EcoRI (E), KpnI (K), and XhoI (X) are shown on the map. Rescued plasmids, designated by lines, are shown below. 28-5 genomic DNA was digested with EcoRI, XhoI, and KpnI to clone the sequences adjacent to the right border and with BamHI and SpeI to clone the sequences adjacent to the left border. The right border clones revealed two genes at that locus (shown in schematic). 28-5 gene 1 was identified ∼2 kb from the right border of the T-DNA insertion site. 28-5 gene 2, for which transcription starts in the opposite orientation to gene 1, was found 8 kb from the right border.
(B) DNA gel blot genomic DNA hybridization analysis of the wild type (Wt) and the 28-5 mutant. Genomic DNA was digested with EcoRI, separated by electrophoresis, and blotted. The blot was probed with the EcoRI-rescued plasmid. Arrows indicate DNA size markers in kilobases.
(C) RNA gel blot hybridization analysis of wild-type and 28-5 mutant callus tissue. Ten micrograms of total RNA was hybridized with the EcoRI fragment shown in (A) as a probe. The result indicated that the 28-5 gene 1 was overexpressed in the 28-5 callus and that the transcribed RNA was 1.9 kb long.
- Figure 4.
Overexpression of 28-5 Gene 1 Reproduced the 28-5 Mutant Phenotypes.
(A) Diagram of the transformation construct. The KpnI fragment, containing four tandemly arrayed cauliflower mosaic virus 35S enhancer and the 28-5 gene 1 (CYP78A9), was cloned into Agrobacterium binary vector pPZP211, and the construct was transformed into ap2-1 or wild-type plants by Agrobacterium-mediated transformation. Bastar, bar gene; LB, left border; RB, right border; pB KS, pBluescript KS+.
(B) Amplification by RT-PCR of 28-5 gene 1 cDNA with primers 28-5-1F and 28-5-1R. Total RNA was isolated from inflorescences of wild-type plants (Wt), 28-5 single mutants (28-5), a transgenic plant showing wild-type phenotypes (T.G.–), and a transgenic plant showing strong mutant phenotypes (T.G.+). The arrow indicates the 580-bp PCR product amplified from 28-5 gene 1. As a control, ubiquitin extension protein (UBQ5) (Callis et al., 1990) was amplified.
(C) Transgenic plant in the wild-type background. A pistil continued to elongate without fertilization. Stamens were very short and were not visible before removal of the outer floral organs.
(D) Silique of a transgenic plant in the wild-type background showing parthenocarpy. One carpel was removed to view the ovules inside.
(E) Flower of a transgenic plant in the ap2-1 mutant background. A very wide pistil was observed. Some sepals and petals were removed to show the stamens, which were very short.
(F) ap2-1 flower at anthesis.
(G) Capsella bursa-pastoris flower at anthesis.
(H) C. bursa-pastoris fruit.
Bars in (C) to (H) = 1 mm.
- Figure 5.
In Situ Hybridization of CYP78A9 to Arabidopsis Flowers.
(A) Longitudinal section of the flowers at stage 6 and stage 12. In the wild-type Arabidopsis flower, the strong localization of the CYP78A9 transcript was not detected in the floral buds up to stage 13.
(B) Cross-section of the pistil of a stage 14 flower. The CYP78A9 gene was expressed in the funiculi (stalks of the developing ovules) in stage 14 flowers (arrowheads).
(C) Longitudinal section of the pistil of a stage 14 flower. Arrowheads show funiculi.
(D) Longitudinal medial section of the 28-5 ap2-1 mutant pistil. The CYP78A9 RNA is ectopically overexpressed in the carpel valves, especially in the inner side of the carpels.
Bars in (A) to (D) = 200 μm.
Tables
- Table 1.
Dimensions of Fully Elongated Siliques of Emasculated ap2-1, 28-5 ap2-1 Mutant, Emasculated Wild Type, 28-5 Single Mutant, Self-Pollinated Wild Type, and Pollinated 28-5 Single Mutant with Wild-Type Pollen or 28-5 Pollen
Genotype Length (mm) Width (mm) Length/Width Emasculated ap2-1
(n = 10)a2.7 ± 0.6b 0.7 ± 0.1 3.9 28-5 ap2-1 (n = 31) 5.2 ± 0.5 2.2 ± 0.2 2.4 Emasculated wild type
(n = 10)3.1 ± 0.2 0.6 ± 0.1 5.2 28-5 single (n = 45) 7.8 ± 1.2 1.0 ± 0.1 7.8 Wild type (self-
pollinated) (n = 16)12.0 ± 1.0 1.0 ± 0.0 12.0 Pollinated 28-5 single
with wild-type
pollen (n = 16)14.0 ± 0.3 1.8 ± 0.4 7.7 Pollinated 28-5 single
with 28-5 pollen
(n = 8)9.8 ± 0.2 1.7 ± 0.1 5.8
