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First published online 19 January 2005
doi: 10.1242/dev.01654

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The PRETTY FEW SEEDS2 gene encodes an Arabidopsis homeodomain protein that regulates ovule development

Department of Botany, University of Florida, Gainesville, FL 32611-8526, USA

View larger version (14K): [in a new window]   Fig. 1. The anatomical structures of Arabidopsis ovules were outlined at three developmental stages. (A) Ovule primordia emerge from the placenta and differentiate into three distinct zones: the nucellus (n), chalaza (c), and funiculus (f). (B) The inner integument (ii) and outer integument (oi) initiate from the chalaza. The megaspore mother cell (mmc) undergoes meiosis, forming four megaspores. (C) The largest megaspore undergoes three rounds of cell division and these cells differentiate into the embryo sac (shaded), which contains the antipodals, synergids, egg cell (e), and central cell (cc).

View larger version (64K): [in a new window]   Fig. 2. The phenotypes of pfs2 mutants. (A) Wild-type ovules have an outer integument (oi), inner integument (ii), and embryo sac (es). (B) In pfs2-1 mutants, the embryo sac, or gametophyte, usually does not completely differentiate. In this ovule, the embryo sac failed to differentiate in the nucellus (n). (C) When the embryo sac does form in this mutant, it frequently has fewer than the normal complement of seven cells. This embryo sac has two cells. (D) In 5% of the pfs2 mutant ovules, the embryo sac is indistinguishable from wild type. (E) Wild-type leaves. (F) Wild-type petal. (G) In addition to the aberrant ovule phenotype, the leaves and petals of pfs2 mutants displayed abnormalities. In pfs2 mutants, the leaves curled downward and (H) the petal margins were ragged and wavy. Scale bars: 20 µm.

View larger version (113K): [in a new window]   Fig. 3. Complementation of the pfs2 mutant. (A) Fruit length was reduced in pfs2-1 mutants. In pfs2-1 mutants that were transformed with a wild-type copy of the PFS2 gene (complemented), fruit size and fecundity were identical to wild type (wt). (B) pfs2 mutant ovules. (C) In pfs2 mutants that contained the PFS2 transgene, many ovules were indistinguishable from wild type, but (D) some ovules exhibited subtle changes in outer integument (oi) morphology. (E) Wild-type ovule. Scale bars: 20 µm. f, funiculus; ii, inner integument.

View larger version (76K): [in a new window]   Fig. 4. Molecular lesions in pfs2 alleles and alignment of PFS2 with similar proteins. (A) In the pfs2-1 allele, the T-DNA inserted into the third intron, whereas in pfs2-2 the T-DNA inserted into the second intron. The homeodomain region is encoded by the second exon. (B) Protein alignment is shown for PFS2, PFS2-LIKE, WUS and PRS. Within the homeodomain region (amino acids 59-122), these proteins share 95% amino acid identity. Other conserved regions are similarly boxed. Outside these regions, the homology drops markedly. Conserved amino acids are denoted with a dot, while identical amino acids are marked with an asterisk.

View larger version (116K): [in a new window]   Fig. 5. PFS2 transcripts were present in developing primordia. The localization of PFS2 transcripts was determined by in situ hybridization. (A) Transcripts were present in the suspensor (su), developing embryos (e), and (B) endosperm (en). Note that the brown color in the endothelium (et) derives from a naturally occurring pigment in these cells. (C) In seedlings, transcripts are present in the shoot apical meristem (sam) and leaf primordia (lp), but absent in mature cotyledons (c) and leaves. (D) Similarly, this gene is expressed in floral meristems (fm) and floral primordia (fp). (E) In developing flowers, PFS2 mRNA was most prevalent in the developing anthers (a) and ovule primordia (op). (F,G) In ovules the transcripts localized to the chalaza (ch) and nucellus (n). (H) Extended incubation of slides in substrate reveals that PFS2 is also expressed in carpel walls and petals (p). (I) The anti-sense probe control did not stain. (J) The relative abundance of PFS2 transcripts was measured using RT-PCR and expressed as a fraction of the level found in wild-type inflorescences. RNA for cDNA synthesis was extracted from: (1) wild-type pistils; (2) the outer three whorls (sepals, petals, and stamens); (3) mature leaves; (4) wild-type inflorescences; (5) pfs2 mutant inflorescences. GAPC transcripts were measured to determine if equal amounts of template were used in each PCR reaction. Scale bars in A,B,F: 20 µm in A,B,F; 50 µm in C-E,G-H. f, funiculus; g, gynoecium; s, sepals; tt, transmitting tract.

View larger version (91K): [in a new window]   Fig. 6. Ectopic expression of PFS2 affected organ differentiation. The 35S cauliflower mosaic virus promoter drove PFS2 expression in these transgenic plants. (A) In plants with the highest level of expression, had defects in differentiation of leaf primordia. This four-week-old plant had an enlarged apical meristem (am), but lacked leaf primordia. (B) In a few plants, leaf primordia (lp) emerged, but none of these developed into a mature leaf (l). (C) Other transformants exhibited altered phyllotaxy and reduced leaf development as the plant aged. (D) In plants with lower levels of PFS2 expression, flowers formed. However, these flowers sometimes had carpelloid stamens (cs) with stigmatic papillae (sp) on the tips. (E) In other flowers, the petals were highly reduced in size and carpelloid stamens arose between the second and third floral whorls. (F) In ovules, the outer integument frequently did not undergo asymmetric cell expansion, which is characteristic of wild-type ovules. (G) In the nucellus (n) of these plants, the MMC was absent. In its place were small parenchymatous cells. (H) Relative PFS2 transcript levels were measured using RT-PCR in wild-type flowers (wt) and flowers overexpressing PFS2 (OE). ACTIN11 (ACT11) transcript abundances were used as controls. Scale bars: 20 µm in F,G; 50 µm in D,E. a, anther; c, cotyledon; et, endothelium; f, funiculus; ii, inner integument; oi, outer integument; p, petal; pi, pistil; s, sepals; st, stamens.

View larger version (68K): [in a new window]   Fig. 7. PFS2 activity repressed AG expression. (A) In ag mutant flowers, repeating whorls of sepals and petals develop. In addition, the loss of floral determinacy in ag mutants results in many more floral whorls than the four found in wild-type flowers. (B) Except for undulating petal edges, flowers in the ag pfs2 double mutant appeared similar to ag single mutants. (C) Petals in pfs2 mutants curled downward. (D) In ag pfs2 mutants, the leaf curling phenotype was attenuated, but not eliminated. (E) RNA was extracted from flowers and leaves of pfs2 mutants, wild-type plants, and PFS2 OE plants. Relative AG mRNA levels were measured by RT-PCR and expressed as a fraction of the level found in pfs2 mutants. As an internal control, GAP mRNAs were simultaneously amplified. The OE1 and OE2 plants exhibited phenotypes similar to that shown in Fig. 6C and Fig. 6B, respectively. (F) In wild-type flowers, AG transcripts were present in developing floral primordia (fp), anthers (a) and gynoecia (g). (G) In pfs2 mutants, the expression of AG in floral primordia decreased, but appeared similar to wild type in developing anthers and gynoecia. (H) In pfs2 mutants, AG transcripts were expressed through the nucellus (n). (I) In some of the pfs2 floral primordia, AG transcripts were present in not only the gynoecium and stamen (st) primordia, but also in the petal primordia (arrow). Scale bars: 50 µm in F-I. ch, chalaza; ov, ovule.

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