An Egg Apparatus-Specific Enhancer of Arabidopsis, Identified by Enhancer Detection

Despite a central role in angiosperm reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer-detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis (Arabidopsis thaliana). The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5′ and 3′ deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp. Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This EASE works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE. The potential application of the EASE element and similar control elements as part of an open-source biotechnology toolkit for apomixis is discussed

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

<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

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