Figure 1.
Sequence analysis of AtPV42a and AtPV42b.
(A) Gene structures of AtPV42a and AtPV42b. A triangle indicates the T-DNA insertion site in atpv42b-1 (CS823876). Black boxes, grey boxes, and lines represent exons, introns, and untranslated regions, respectively. (B) Alignment of CBS-domain containing proteins from plants including Arabidopsis (At), Phaseolus vulgaris (Pv), and Medicago truncatula (Mt), and yeast (Saccharomyces cerevisiae). Identical residues are marked with asterisks. Conserved and semi-conserved substitutions are denoted by ‘:’ and ‘.’, respectively. The overlined CBS domains were predicted using Pfam in the following website (http://www.sanger.ac.uk/Users/agb/CBS/CBS.html). A triangle indicates the position of the T-DNA insertion in atpv42b-1.
Figure 2.
Expression of AtPV42a and AtPV42b in wild-type and transgenic plants.
(A) Transcript levels of AtPV42a and AtPV42b in various tissues from 28-day-old adult plants and dry seeds of Col wild-type. R, root; RL, rosette leaf; St, stem; CL, cauline leaf; Bud, unopen floral bud; OF, open flower; Si, silique; DS, dry seed. (B) Transcript levels of AtPV42a and AtPV42b in the seedlings 3, 8, and 14 days after germination. (C-J) In situ hybridization of AtPV42a and AtPV42b in wild-type developing flowers. (C, F) Transverse section of a stage 9 flower hybridized with the antisense AtPV42a (C) or AtPV42b (F) probe. Arrows indicate the labelled septum inside the gynoecia, while arrowheads indicate some labelled microspore mother cells in the locules. Bars, 100 µm. (D, E, G, H) Transverse section of a gynoecium from a stage 11 flower hybridized with the antisense (D) or sense probe (E) of AtPV42a or the antisense (G) or sense probe (H) of AtPV42b. f, funiculus; o, ovule. Bars, 50 µm. (I, J) Longitudinal section of a gynoecium from a stage 13 flower hybridized with the antisense AtPV42a (I) or AtPV42b (J) probe. Bars, 70 µm. (K) Transcript levels of AtPV42a and AtPV42b in rosette leaves of 7 selected amiR-atpv42a independent transgenic lines at the T1 generation. (L) Transcript levels of AtPV42a and AtPV42b in rosette leaves of 10 selected amiR-atpv42b-1 independent transgenic lines at the T1 generation. Asterisks indicate the transgenic lines showing the low fertility phenotype as shown in Figure 3A, C. Transcript levels in (A, B, K, L) were determined by real-time PCR and are shown relative to TUB2 expression. Values are the mean ± standard deviation from three replicates.
Figure 3.
Phenotypes of siliques and developing flowers in wild-type and amiR-atpv42b-1(line 10).
(A) Comparison of fully grown siliques from wild-type and amiR-atpv42b-1 plants. (B) A wild-type silique with full seed set. (C) An amiR-atpv42b-1 silique with reduced seed set and undeveloped ovules, some of which are indicated by arrows. (D-G) Wild-type flowers at early stage 12 (D), late stage 12 (E), stage 13 (F) and stage 14 (G). (H-K) amiR-atpv42b-1 flowers at early stage 12 (H), late stage 12 (I), stage 13 (J) and stage 14 (K). Flowers at stage 14 show delayed filament elongation and significantly reduced production of pollen grains. Bars in (A–C), 1 mm; Bars in (D, E, H, and I), 150 µm; Bars in (F, G, J, and K), 200 µm.
Table 1.
Phenotypic analysis of wild-type, amiR-atpv42b-1, and amiR-atpv42b-2 seeds.
Figure 4.
Pollen grain development in wild-type and amiR-atpv42b-1.
(A) Scanning electron micrograph (SEM) of mature pollen grains collected from wild-type flowers at stages 13–14. (B) SEM of pollen grains collected from amiR-atpv42b-1 flowers at stages 13–14. The majority of the pollen grains are shrunken and exhibit a collapsed morphology (arrowheads). (C) Transverse sections of wild-type and amiR-atpv42b-1 anthers at anther stages 9, 10, 11, and 12. Arrowheads indicate defective pollen grains in amiR-atpv42b-1 anthers. Bars, 50 µm.
Figure 5.
Transmission electron microscopy of wild-type and amiR-atpv42b-1 pollen grains.
Wild-type and amiR-atpv42b-1 pollen grains at anther stages 9 and 12 are shown. In amiR-atpv42b-1, during plasmolysis occurring at anther stage 9, the plasma membrane (arrows) of the pollen grain is withdrawn from the cell wall, while at anther stage 12, the cytoplasm is almost invisible in the severely shrunken pollen grain. Bars, 2 µm.
Figure 6.
Phenotypic analyses of reciprocal crosses between wild-type and amiR-atpv42b-1 plants.
(A) SEM of wild-type ovules at flower stage 12 prior to fertilization. (B, C) SEMs of developing seeds 2 days after pollination from the following reciprocal crosses: Col ♀×Col ♂ (B) and Col ♀× amiR-atpv42b-1 ♂ (C). (D) SEM of amiR-atpv42b-1 ovules at flower stage 12 prior to fertilization. (E, F) SEMs of developing seeds 2 days after pollination from the following reciprocal crosses: amiR-atpv42b-1 ♀×Col ♂ (E) and amiR-atpv42b-1 ♀× amiR-atpv42b-1 ♂ (F). Arrows indicate the unfertilized ovules. (G–J) Aniline blue staining of pollen tube growth inside pistils collected 20 hours after pollination from the following reciprocal crosses: Col ♀×Col ♂ (G), Col ♀×amiR-atpv42b-1 ♂ (H), amiR-atpv42b-1 ♀×Col ♂ (I), and amiR-atpv42b-1 ♀× amiR-atpv42b-1 ♂ (J). Arrows indicate the lateral growth of pollen tubes. Bars in (A-F), 100 µm.
Table 2.
Phenotypic analysis of reciprocal crosses between wild-type and amiR-atpv42b-1 plants.