<i>LIS</i> Is Strongly Expressed in Gametic Cells

<div><p>(A) RT-PCR analysis of <i>LIS</i> expression in leaves (1), roots (2), flower buds (3), open flowers (4), inflorescences (5), siliques (6), and stem (7) (upper panel). <i>ACTIN 2</i> was used as control (lower panel).</p> <p>(B–E), Expression of <i>pLIS::NLS_GUS</i> in wild-type ovules during female gametophyte development. (B) Arrowhead points at the functional megaspore in which <i>GUS</i> expression is detected in the nucleus. (C) Two-nucleate embryo sac showing <i>GUS</i> expression in both nuclei (arrowheads). (D) Young eight-nucleate gametophyte. Antipodal nuclei are not visible. Inset shows area with GUS-positive nuclei at higher magnification. White arrowheads point to the synergid nuclei; star points to the egg cell nucleus; and black arrowheads point to the unfused polar nuclei. (E) Mature gametophyte showing strong GUS signal in both the egg cell (star) and fused central cell nucleus (arrowhead). Expression in synergid cells is hardly detectable.</p> <p>(F) Proposed model for <i>LIS</i> function. Expression of <i>LIS</i> in gametic cells (egg cell [ec] and central cell [cc]) is necessary for the generation of a lateral inhibition signal that prevents the adjacent accessory cells (synergids [s] and antipodal cells [a]) from adopting gametic cell fate.</p></div

<i>LIS</i> Codes for a WD40 Repeat Protein

<div><p>(A) Gene (upper panel) and protein (lower panel) structure of <i>LIS</i>. The localization of the <i>lis-1</i> and <i>lis-2</i> mutations and the seven WD40 repeats are indicated.</p> <p>(B and C) <i>LIS</i> cDNA driven by a 2.6-kb upstream promoter complements the gametophytic (B) as well as possible sporophytic defects of <i>lis-1</i>/<i>lis-1</i> mutants, allowing for the generation of homozygous plants, as demonstrated by PCR-based genotyping (C).</p></div

Successive Recruitment of Cells as Gametic Cells in <i>lis-1</i> Gametophytes

<div><p>(A and B) Ectopic expression of the egg cell marker ET1119 (A) and the central cell marker <i>pMEA::GUS</i> (B) in wild-type and <i>lis-1/LIS</i> plants. Dark bars represent wild-type, light bars represent <i>lis-1</i>/<i>LIS</i> plants. The <i>y</i>-axis shows the percentage of ectopic expression of total GUS-staining ovules. Ectopic expression was scored zero (0d), one (1d), and two days (2d) after emasculation. Total number of ovules counted was greater than 250.</p> <p>(C–G) Development of five morphological features in <i>lis-1/LIS</i> plants as compared to wild type, zero (0d), one (1d), and two days (2d) after emasculation. The <i>y</i>-axis shows percent deviation from wild type (data from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050047#pbio-0050047-st001" target="_blank">Table S1</a>). (C) Synergid nuclei smaller than egg cell nucleus. (D) Different polarity of synergids and egg cell with respect to position of nucleus. (E) Polar nuclei unfused. (F) Ectopic cellularization. (G) Protruded antipodal cells.</p></div

Functional Analysis of Synergids and Central Cells in <i>lis-1/LIS</i> Plants

<div><p>(A–C) GUS staining in synergids after fertilization of wild-type and <i>lis-1/LIS</i> plants with pollen from the ET434G pollen-tube marker line. (A) Ovule with GUS-stained synergids. The arrowhead points at pollen tube. (B) Ovule in which no GUS staining was detected in synergids. (C) Frequencies of GUS negative synergids. Dark bars represent wild-type, light bars represent <i>lis-1</i>/<i>LIS</i> plants. The <i>y</i>-axis shows the percentage of the scored phenotype (<i>lis-1/LIS:</i> 50.8%, <i>n</i> = 789; wild-type: 21.5%, <i>n</i> = 287).</p> <p>(D–F) Endosperm development after fertilization of wild-type and <i>lis-1/LIS</i> plants with wild-type pollen. (D) Ovule with developing embryo (star) and endosperm (arrowhead). (E) Ovule with a developing embryo (star), but no endosperm. The undeveloped central cell nucleus is visible (arrowhead). (F) Frequencies of ovules with a developing embryo, but no endosperm. Dark bars represent wild-type, light bars represent <i>lis-1/LIS</i> plants. The <i>y</i>-axis shows the percentage of the scored phenotype (<i>lis-1/LIS:</i> 11.2%, <i>n</i> = 267; wild type: 0.5%, <i>n</i> = 191).</p></div

Accessory Cells in <i>lis-1</i> Gametophytes Morphologically Resemble Gametic Cells

<div><p>(A–D) Schematic representation of wild-type female gametophyte development. Sporophytic structures are shown in grey; gametophytic structures are colored. (A) After meiosis, the haploid functional megaspore is formed. (B) A series of three mitotic divisions results in the formation of an eight-nucleate syncytium. (C) After nuclear migration and cellularization, a seven-celled gametophyte is formed containing two synergids at the micropylar end (dark green), one egg cell (red), one central cell (orange) with two polar nuclei, and three antipodal cells at the chalazal pole (light green). (D) Prior to fertilization, the two polar nuclei fuse to form one large central cell nucleus, and the antipodal cells degenerate.</p> <p>(E) Wild-type silique showing full seed set.</p> <p>(F) Silique of <i>lis-1/LIS</i> plants containing aborted ovules (arrowheads).</p> <p>(G–I) Mature wild-type gametophyte. (G) At the micropylar end, the two small synergid nuclei are detected (stars). The larger egg cell nucleus (arrowhead) is oriented towards the adjacent central cell. (H) A large central cell nucleus (arrowhead) resulting from the fusion of the two polar nuclei can be detected. (I) The antipodal cells at the chalazal end degenerate (star).</p> <p>(J–N) <i>lis-1</i> gametophytes. (J) The synergid nuclei are enlarged and mis-polarized (star). As a consequence, synergid and egg cell become indistinguishable when lying in a similar position (arrowheads). (K) Polar nuclei are unfused (arrowheads) and occasionally ectopically cellularized (arrowheads in M). (L and M) Antipodal cells do not degenerate, but enlarge and protrude towards the center (stars). (N) Disintegration of antipodal cells and fused antipodal nuclei (star).</p></div