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Science 338 (6110): 1093-1097
Copyright © 2012 by the American Association for the Advancement of Science
Egg Cell–Secreted EC1 Triggers Sperm Cell Activation During Double Fertilization
Stefanie Sprunck1,*,
Svenja Rademacher1, ,
Frank Vogler1,
Jacqueline Gheyselinck2, ,
Ueli Grossniklaus2, and
Thomas Dresselhaus1
1 Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
2 Institute of Plant Biology and Zürich-Basel Plant Science Center, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland.
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Fig. 1. The EC1 gene family is specifically expressed in the egg cell. (A) Schematic of an Arabidopsis ovule harboring the haploid generation, termed "female gametophyte" (dashed line), which comprises two female gametes (egg cell and central cell) and two accessory cell types (synergids and antipodal cells). (B) Reverse transcription polymerase chain reaction (RT-PCR) detects EC1 transcripts only in female reproductive tissues. (C and D) Egg cell–specific β-glucuronidase (GUS) reporter activity, driven by the EC1.1 (C) and EC1.2 (D) promoters. (E to H) Expression pattern of EC1 as detected by in situ hybridization on ovules. EC1 transcripts are present only in egg cells (E to G) but not in zygotes (H). ap, antipodal cells; as, antisense RNA; cc, central cell; ec, egg cell; fert., fertilized; fg, female gametophyte; syn, synergid cell; zyg, zygote; -, water control; +, genomic DNA. Scale bars, 20 μm.
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Fig. 2. Triggered secretion of small cysteine-rich EC1 proteins. (A) Schematic of EC1 precursor protein. Predicted N-terminal signal peptide cleavage site (arrow). Prolines at the C terminus (P-box), two signature motifs (S1 and S2), and a conserved cysteine-spacing signature (C1 to C6) forming three predicted disulfide bonds (black lines) are shown. (B) Western blot showing the 42.4-kD EC1-GFP fusion in a pistil protein extract, compared with endoplasmic reticulum–localized GFP (29 kD) from leaves. (C to H) EC1-GFP is secreted upon sperm cell arrival. Merged bright-field and fluorescence images at a single z plane (C, F, and I) and corresponding fluorescence images (D, G, and J) are shown together with plot profiles of relative signal intensities (E, H, and K) detected along a line drawn across one synergid cell and the egg cell (red arrows in C, F, and I). x axis, distances (in micrometers) along the drawn line; y axis, relative signal intensities of GFP (green) and reciprocal grayscale values [1/bright field; gray]. Dashed lines denote the position of cell borders. (C to E) EC1-GFP signals are visible as vesicle-like structures (solid arrowheads) in the cytoplasm of unfertilized egg cells but not outside the egg cell. (F to H) EC1-GFP is detected extracellularly when the sperm cells (red nuclei) reach the gamete fusion sites (open arrows). Insets in (F) and (G) represent different focal planes. One sperm nucleus is out of focus. (I to K) Control. Egg cell–expressed cytoplasmic ARO1-GFP is not detected extracellularly during gamete interactions. dsyn, degenerating synergid; sc, sperm cell. Dotted lines delimit egg cell borders; open arrowheads point at synergid cell borders. Scale bars, 10 μm. See figs. S3 and S4 and movies S1 and S2 for additional data.
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Fig. 3. The EC1 gene family is essential for gamete fusion and for blocking supernumerary sperm cell delivery. (A) ec1.1/4/5 is a triple knockout, shown by RT-PCR. (B) The reduced seed set was observed only in siliques of the quintuple knockdown ec1-RNAi. (C to E) Confocal images of sperm nuclei (red) in the wild type and in ec1-RNAi ovules. (C) Sperm cells (arrowheads) fusing with WT female gametes at 7 hap. (D and E) ec1-RNAi ovules with two (D) or four (E) unfused sperm cells (arrowheads). (F) Quantitative assessment of ec1-RNAi phenotypes at 30 to 40 hap. Phenotypes of ovules targeted by a pollen tube were classified as indicated. Mean values ± SEM (error bars) are shown [n = numbers of siliques counted (number of ovules)]. Scale bar in (C), 20 μm.
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Fig. 4. EC1 peptides activate the sperm endomembrane system. (A and B) Confocal images of sperm cell marker line simultaneously labeling sperm nuclei (red) and sperm membranes (green). Note the membranous connection between the sperm cells (solid arrowheads) and the cytoplasmic projection associated with the vegetative nucleus (dashed arrows). (C to F) Sperm membranes stay physically connected (solid arrowheads) during gamete interaction, in both ec1-RNAi (C and D) and WT ovules (E and F). (G and H) After plasmogamy, the membranous connection (solid arrowheads) is visible at the apical edge of the degenerating synergid. (I to P) Spinning disc images of sperm cells expressing HAP2-YFP. (I and J) HAP2-YFP localization in the endomembrane system of sperm cells within a semi–in vivo growing pollen tube. Solid arrowheads point at the position of the membranous intercellular connection. (K and L) Sperm cell pair released in peptide-free control solution, showing endomembrane-associated HAP2-YFP (asterisks). (M and N) HAP2-YFP localization at the plasma membrane (arrowheads) of sperm cells treated with EC1.1 peptide mix [EC1(pep)]. (O) Single sperm cell in a peptide-free control solution. (P) HAP2-YFP at the plasma membrane (arrowheads) of EC1(pep)-treated single sperm cell. (Q) Quantitative assessment of HAP2-YFP localization at plasma membranes of EC1(pep)-treated sperm cells, compared with randomized control peptides and a peptide-free solution. Mean values (±SEM) of at least three independent experiments with  20 sperm cells each are shown. Asterisks indicate statistically significant difference from the controls (*P < 0.05), according to Student’s t test. vn, vegetative nucleus. Scale bars, 5 μm.
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