Egg Cell–Secreted EC1 Triggers Sperm Cell Activation During Double Fertilization

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Science  23 Nov 2012:
Vol. 338, Issue 6110, pp. 1093-1097
DOI: 10.1126/science.1223944


Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple–sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions.

Sexual reproduction depends on the successful union of two gametes of opposite sex at fertilization. In flowering plants such as Arabidopsis thaliana, sexual reproduction is distinct in that two gamete fusion events take place in a coordinated manner, a phenomenon termed “double fertilization” (1). Two nonmotile sperm cells are delivered by a pollen tube into the female gametophyte (embryo sac) that harbors two dimorphic female gametes (Fig. 1A). One sperm fuses with the egg cell, giving rise to the embryo, whereas the second sperm cell fuses with the central cell to develop the triploid endosperm. Although this distinct mode of reproduction was discovered more than a century ago (2, 3) and recent live-cell imaging has shed some light on the behavior of Arabidopsis sperm nuclei during double fertilization (4), almost nothing is known about the molecules mediating gamete interactions. To date, the evolutionary conserved HAP2 (HAPLESS 2)/GCS1 (GENERATIVE CELL SPECIFIC 1) is the only sperm protein known to be essential at a late step in gamete interactions (5, 6), and its role as a fusogen is strongly supported by the observation that HAP2-deficient gametes of Chlamydomonas and Plasmodium berghei are able to adhere but fail to fuse (7).

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.

Based on a transcriptomics approach that uses isolated egg cells of wheat (8), we discovered a family of Arabidopsis genes with sequence similarity to the largest wheat egg cell–specific transcript cluster, termed EC-1 (Egg Cell 1) (see supplementary materials and methods). We found transcripts of the five Arabidopsis EC1-like genes (EC1.1, EC1.2, EC1.3, EC1.4, and EC1.5) only in female reproductive tissues (Fig. 1B). Egg cell–specificity of EC1 was shown by expressing the β-glucuronidase (GUS) reporter under control of individual EC1 promoters (Fig. 1, C and D) and by in situ hybridization to tissue sections. Transcripts of EC1 genes are specifically present in the egg cell (Fig. 1, E to G) but are not detectable early after fertilization (Fig. 1H and fig. S1B), whereas GUS remains active in zygotes and early embryos (fig. S1, D to F).

EC1 proteins belong to the large and unexplored group of ECA1 (Early Culture Abundant 1) gametogenesis-related cysteine-rich proteins characterized by their conserved cysteine-spacing signature (9). Within 118 ECA1 proteins of Arabidopsis, the EC1 family forms a distinct subclade (fig. S2A). Notably, we identified EC1-like genes or transcripts only in flowering plant species, including the basal angiosperm Amborella trichopoda, and not in gymnosperms, ferns (Adiantum sp.), Bryophytes (Physcomitrella patens), or green algae (Volvox sp.; Chlamydomonas sp.). Protein-sequence analyses revealed common features of EC1 proteins, such as a predicted N-terminal signal peptide for secretion and a similar predicted intramolecular disulfide bond arrangement (Fig. 2A). Two conserved signature sequences, termed S1 and S2, were identified by multiple sequence alignments with representatives from monocots, dicots, and basal angiosperms (fig. S2B).

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.

To investigate the subcellular localization of EC1, we stably expressed a translational fusion of EC1.1 and the green fluorescent protein (GFP) under control of the EC1.1 promoter. The calculated molecular mass of the EC1.1-GFP fusion, excluding the leader peptide, corresponds to its size in anti-GFP Western blots (Fig. 2B), arguing against posttranslational proteolytic processing of EC1. We found EC1-GFP to accumulate in spherical vesicle-like structures within the unfertilized egg cell, but never as extracellular signal (Fig. 2, C to E, and fig. S3). To study EC1-GFP localization during the short-lived event of double fertilization, we used a red fluorescent marker for sperm nuclei (10). Upon sperm cell arrival in the female gametophyte and during double fertilization, EC1-GFP signals are detected extracellularly, especially in the apical region of the degenerating synergid cell (Fig. 2G), which is where gamete fusions will take place (4, 11). Quantification of GFP fluorescence in the egg cell and the flanking synergid cell during gamete interaction and fusion revealed that signal intensities are gradually increasing toward the egg cell membrane (Fig. 2H and fig. S4), indicating regulated exocytosis of EC1-GFP by the egg cell during gamete interactions. By contrast, extracellular GFP signals were never observed in control egg cells expressing a GFP fusion of ARO1 (ARMADILLO REPEAT-ONLY 1) (Fig. 2K and fig. S4), a cytoplasmic and endomembrane-associated gametophyte-specific Armadillo repeat protein found to be essential for polar pollen tube growth (12).

Recent live-cell imaging revealed that the sperm cells remain in the boundary region between the egg and the central cell for only 7.4 ± 3.3 min before they fuse (4). We therefore proposed the narrow time slot of EC1 secretion to be associated with sperm-egg recognition or adhesion, with egg cell signaling, or with a polyspermy block on the egg cell. However, the five EC1 genes appear to be functionally redundant, as we did not find negative effects on fertility in single, double, or triple knockout mutants (fig. S5). EC1.2 and EC1.3 are tandemly arranged on chromosome 2 and, thus, are not suitable to generate double mutants. We used the strong EC1.1 promoter for simultaneous knockdown of EC1.2 and EC1.3 expression in the egg cell via RNA interference (RNAi), but we did not find any effect on fertility (fig. S5). We only observed reduced seed set when we down-regulated EC1.2/3 in egg cells of the triple knockout ec1.1/4/5 (Fig. 3B and fig. S5; referred to as ec1-RNAi). Reciprocal backcrosses and segregation ratios of self-fertilized ec1-RNAi lines revealed the expected female gametophytic effect on transfer DNA transmission, resulting in only heterozygous or wild-type (WT) offspring (tables S1 and S2). Despite the lower transcript abundance of EC1.2 and EC1.3 in heterozygous ec1-RNAi pistils (fig. S6), both embryo sac development (fig. S7) and pollen tube reception (fig. S8) were comparable to the same processes in WT ovules.

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.

To study the EC1 knockdown phenotype in more detail, we pollinated pistils of the wild type and four independent ec1-RNAi lines with the sperm marker HTR10-mRFP1 (10). The evaluation of ovules at 30 to 40 hours after pollination (hap) revealed that ec1-RNAi ovules allow sperm release but block gamete fusion (table S3). We found that 43.5 to 46.7% of ec1-RNAi embryo sacs had two, or even four, unfused sperm cells (Fig. 3, D to F), whereas in the wild type, both sperm cell delivery and fusion are completed within 6 to 10 hap (Fig. 3C) (13). Notably, multiple–sperm cell delivery in ec1-RNAi ovules involves both synergids (fig. S9). We conclude that, due to failed gamete fusion, the second synergid of an ec1-RNAi ovule continues to secrete pollen tube attractants, even after the first pollen tube has successfully discharged its sperm pair into the receptive synergid cell. This suggests a gamete fusion–based molecular mechanism preventing multiple–pollen tube attraction and is in accordance with recent observations of mutants with defective sperm cells that fail to fuse (14, 15).

It is vital to ensure that both gamete fusion events take place in a timely and efficient manner, because typically only one pair of sperm cells is delivered into the embryo sac, and both female gametes need to be fertilized for reproductive success. The two sperm cells of flowering plants are known to be physically associated (arrowhead in Fig. 4A) (16), and this connection may ensure the simultaneous delivery of both sperm cells to the gamete fusion sites. To address the question of whether EC1 secretion serves as a precisely timed signal for sperm cell separation, we generated transgenes expressing a GFP-labeled membrane protein (TET9-GFP) in sperm cells to visualize the sperm membranes during double fertilization. Both the intercellular sperm connection and the cytoplasmic projection that is known to connect the front sperm (sc1) with the vegetative nucleus (16) are visible (Fig. 4, A and B). Notably, the physical link between the two sperm cells appears to be membrane-enriched (arrowhead in Fig. 4B). This membranous link remains between the unfused sperm cells in ec1-RNAi ovules (Fig. 4, C and D). However, we also detect the physical association at the time point of sperm-egg fusion in WT embryo sacs (Fig. 4, E and F), arguing against a role for EC1 in sperm separation. After plasmogamy, remnants of the membranous link are visible at the apical edge of the degenerating synergid (Fig. 4, G and H), where they stay attached, even when the synergid collapses. At that stage, we also find traces of GFP-labeled sperm membranes at the surface of the fertilized egg cell, most likely representing the sperm-egg fusion site (fig. S10).

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.

Gamete-interaction studies in other organisms revealed that gamete adhesion and/or binding initiates a signaling cascade that leads to the activation of the male gamete and to the exposure of fusogenic membrane regions (1721). So far, the only known flowering plant candidate gamete fusogen is HAP2/GCS1, a sperm-expressed single-pass transmembrane domain protein (5, 6). Notably, fluorescent signals of HAP2–yellow fluorescent protein (YFP) in sperm cells of semi–in vivo grown Arabidopsis pollen tubes are endomembrane-associated and not located at the sperm surface (Fig. 4, I and J) (22), implying the need for membrane remodeling to acquire full fertilization competence when the sperm cells reach their fusion sites. However, the weak fluorescence of HAP2-YFP prevented us from detecting any redistribution of HAP2/GCS1 within the ovule. To investigate the effect of exogenously applied EC1 on HAP2-YFP localization, we developed a bioassay with sperm cells freshly released from semi–in vivo grown pollen tubes. Because all attempts to express recombinant EC1 or to generate synthetic EC1 proteins failed, we tested the effect of two synthetic EC1.1 peptides, comprising the signature motifs S1 and S2 (Fig. 2A). Neither peptide alone showed an obvious effect on sperm cells, but when we treated the sperm cells with an equimolar peptide mixture [EC1(pep)], the HAP2-YFP signal significantly shifted toward the sperm plasma membrane, compared with controls treated with a mixture of two randomized peptides based on the same amino acids or a peptide-free solution (Fig. 4, K to Q).

Taken together, our results strongly suggest that EC1 is a protein factor controlling Arabidopsis gamete fusion by mediating sperm activation. We propose that the regulated secretion of EC1 by the egg cell upon sperm-egg interaction ensures the appropriate localization of the cell-fusion machinery in distinct sperm membrane domains to accomplish gamete fusion. Although both sperm cells stay close together after being delivered at the site where the two fusion events take place, we show that they remain spatially separated from one another, which may be a strategy to avoid polyspermy and to guarantee reliable and efficient double fertilization after achieving full fertilization competence. The observed mutual gamete activation furthermore suggests a complex mode of intercellular communication between the flowering plant egg and sperm cell before fusion. This may include the on-time delivery of other, yet undiscovered fertilization molecules to the gamete surfaces.

Although the reproductive strategy of flowering plants is considerably different from those of other sexual reproducing organisms, we report here on some essential common processes and principles. Our results indicate that, like the male gametes of other species, Arabidopsis sperm cells must be activated to acquire fertilization competence. However, animal sperm become activated after interacting with the outer vestment of the egg, whereas the egg responds by undergoing the cortical reaction only after fusion (1719, 23). Thus, the mutual activation of Arabidopsis gametes before fusion points to an evolutionary link between flowering plant fertilization and fertilization in unicellular sexual reproducing organisms such as yeast and Chlamydomonas (20, 21), where both mating-type gametes become activated before fusion.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S10

Tables S1 to S3

References (2446)

Movies S1 and S2

References and Notes

  1. The Amborella EC1-like1 protein sequence was identified by a tblastn search in the raw data set of the Amborella RNASeq Trinity Assembly (posted date: Jan 12, 2012), using EC1.1 as protein query. The sequence data were produced by the Amborella Genome Project (, in collaboration with the user community.
  2. Acknowledgments: Seeds were kindly provided by M. Johnson (HAP2p:HAP2-YFP) and F. Berger (HTR10p:HTR10-mRFP1). We thank M. Gebert for her help in in situ hybridization, M. Gahrtz for providing plasmids, M. Kammerer and B. Bellmann for technical assistance, and F. Sprenger for his support in spinning disc microscopy. This work was supported by the Deutsche Forschungsgemeinschaft (grants SP 686/1-2 and SFB924 to S.S. and DR 334/5 to T.D.), by a graduate scholarship of Universität Bayern e.V. (to S.R.), and, in part, by the Australian Grains Research Development Corporation (to T.D.) and the Swiss National Science Foundation (grant 3100AO-112489 to U.G.). S.S. and T.D. are inventors on a patent (WO 2007/092992) related to the EC1 promoter sequences. Accession numbers and author contributions are listed in the supplementary materials.
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