ECS1 and ECS2 suppress polyspermy and the formation of haploid plants by promoting double fertilization

  1. Yanbo Mao
  2. Thomas Nakel
  3. Isil Erbasol Serbes
  4. Saurabh Joshi
  5. Dawit G Tekleyohans
  6. Thomas Baum
  7. Rita Groß-Hardt  Is a corresponding author
  1. University of Bremen, Centre for Biomolecular Interactions, Germany

Abstract

The current pace of crop plant optimization is insufficient to meet future demands and there is an urgent need for novel breeding strategies. It was previously shown that plants tolerate the generation of triparental polyspermy-derived plants and that polyspermy can bypass hybridization barriers. Polyspermy thus has the potential to harness previously incompatible climate-adapted wild varieties for plant breeding. However, factors that influence polyspermy frequencies were not previously known. The endopeptidases ECS1 and ECS2 have been reported to prevent the attraction of supernumerary pollen tubes by cleaving the pollen tube attractant LURE1. Here, we show that these genes have an earlier function that is manifested by incomplete double fertilization in plants defective for both genes. In addition to supernumerary pollen tube attraction, ecs1 ecs2 mutants exhibit a delay in synergid disintegration, are susceptible to heterofertilization, and segregate haploid plants that lack a paternal genome contribution. Our results thus uncover ECS1 and ECS2 as the first female factors triggering the induction of maternal haploids. Capitalizing on a high-throughput polyspermy assay, we in addition show that the double mutant exhibits an increase in polyspermy frequencies. As both haploid induction and polyspermy are valuable breeding aims, our results open new avenues for accelerated generation of climate-adapted cultivars.

Editor's evaluation

This important work convincingly shows that the endopeptidases ECS1 and ECS2 repress the formation of polyspermy-derived triparental offspring and haploid induction by promoting double fertilization. While the underlying mechanisms remain to be further elucidated, the data presented in this study represent a valuable foundation for understanding the regulation of offspring genome size. This study will be of particular interest to the large community of scientists who are interested in plant reproduction and breeding.

https://doi.org/10.7554/eLife.85832.sa0

Introduction

Sexually reproducing organisms have evolved a plethora of ingenious tools to make gamete fusion a safe event (Iwao et al., 2020; Tekleyohans and Groß-Hardt, 2020; Toda and Okamoto, 2020). The production of supernumerary sperm is commonly found in both animals and plants, but this strategy comes with the risk of polyspermy. While polyspermy is lethal in most animals, molecular and genetic evidence for supernumerary sperm fusion in plant egg cells has long been lacking, and so has knowledge of the fate of a polyspermy-derived egg cell. In 2008 Rod Scott et al. made elegant use of Arabidopsis thaliana tes mutants, which form pollen with supernumerary sperm, to show that, similar to animal systems, polyspermy in the egg cell is typically prevented by an efficient polyspermy block (Scott et al., 2008). However, Toda et al., 2016 were later able to artificially bypass the polyspermy block using delicate electrofusion experiments and combined two sperms with a single egg to generate polyploid plants.

In 2017, we provided molecular and genetic evidence of egg polyspermy in planta capitalizing on a high-throughput polyspermy detection (HIPOD) assay, which was established in the Landsberg erecta accession (Nakel et al., 2017). The assay consists of two pollen donors, that contain the individual elements of the yeast GAL4-UAS two-component system. The elements control expression of a herbicide resistance conferring gene, that consequently only gets activated if both paternal constructs are combined in a single egg cell. This enables the generation and identification of plants with three parents, one mother and two fathers (Nakel et al., 2017). Using a modified HIPOD assay that allows detection of embryos from three parents based on seed color, we also showed that polyspermy can specifically affect the egg cell, such that DNA of the second father bypasses DNA control mechanisms of the endosperm (Mao et al., 2020). This has significant implications for plant breeding, where the introduction of beneficial traits, for example from heat or drought-adapted wild species, is often prevented by hybridization barriers of the endosperm.

Plants can also be exposed to an insufficient number of fertilization-competent sperm (Aw et al., 2010; Cyprys et al., 2019; Mori et al., 2006; Ron et al., 2010; Takahashi et al., 2018; von Besser et al., 2006) in which case they activate a fertilization recovery program: The mechanism is triggered when fertilization in one of the female gametes stalls at the level of plasmo - or karyogamy and includes a transient halt of second synergid disintegration and concomitant attraction of supernumerary pollen tubes (Beale et al., 2012; Kasahara et al., 2012; Maruyama et al., 2013). This recovery program allows for recruitment of further sperm transporting pollen tubes and hence constitutes an important back-up mechanism.

Various proteases have previously been associated with the cortical reaction in animals, a fertilization-induced exocytosis of secretary granules that mediates a biochemical renovation of the egg coat to prevent further sperm entry (Burkart et al., 2012; Haley and Wessel, 1999). Based on the analysis of more than 450,000 seedlings, we here and in a preprint (Mao et al., 2022) show that the aspartic endopeptidases ECS1 and ECS2 repress polyspermy. The defect is associated with a transient delay in synergid disintegration, supernumerary pollen tube attraction, and heterofertilization which are characteristic features of the fertilization recovery program (Beale et al., 2012; Kasahara et al., 2012; Maruyama et al., 2013). In fact, we show that a fraction of ecs1 ecs2 double mutants exhibit defects in gamete fusion and segregate seeds lacking paternally contributed fluorescence marker either in embryo or endosperm indicative of single fertilization events. These results are in line with a recent report by Jiang et al., 2022 concordantly showing that ECS1 and ECS2 are required for gamete attachment. Furthermore, we show that the double mutants segregate haploid plants, as later confirmed by Zhang et al., 2023. ECS1 and ECS2 have previously been demonstrated to prevent the attraction of supernumerary pollen tubes by cleaving the pollen tube attractant LURE1 (Yu et al., 2021), indicating that polytubey is prevented by at least two different functions of ECS1 and ECS2.

Results and discussion

The aspartic proteases ECS1 and ECS2 negatively regulate polyspermy and the formation of triparental offspring

ECS1 and ECS2 were identified in a candidate gene approach for factors regulating polyspermy: T-DNA insertion lines for egg cell-expressed proteases, that is, ecs1, ecs2-1, ecs2-2 single mutants, and ecs1 ecs2-1 double mutants were subjected to HIPOD by synchronously applying the pollen of two fathers onto the stigmatic surface. One father contained a construct providing constitutive expression of the yeast transcription factor mGAL4-VP16 while the second father was engineered to express a herbicide conferring YFP-tagged BAR gene under the GAL4 responsive promoter UAS (Figure 1A). The resulting seedlings were subjected to herbicide selection (Figure 1B) and resistant plants were inspected under the fluorescence microscope for YFP expression (Figure 1C). The fusion of one egg cell with two sperm cells is expected to yield triploid rather than diploid offspring, which was demonstrated by flow cytometry (Figure 1D). Finally, we confirmed the triparental status of the seedlings by PCR, which revealed inheritance of GAL4 and UAS from the two genetically distinct fathers (Figure 1E). For wild-type Columbia plants, we recovered 19 triparental seedlings out of 92,680 seeds, corresponding to a polyspermy frequency of 0.041%. This is significantly higher than the polyspermy frequency previously recovered for the Landsberg accession (Nakel et al., 2017), and experimentally re-assessed in this study (Figure 1F). By comparison, we detected an up to threefold increase in polyspermy frequencies in ecs1 ecs2-1 double mutants among the 77,441 seedlings analyzed (Figure 1G). ECS1 and ECS2 are hence the first molecular factors demonstrated to affect egg polyspermy and three parent formation in plants.

Figure 1 with 1 supplement see all
ecs1 ecs2 double mutants segregate polyspermy-derived triparental offspring.

(A) Illustration of the high-throughput polyspermy detection (HIPOD) assay. The two components of the yeast GAL4-UAS system are provided by distinct pollen donors (pollen donor 1, yellow and pollen donor 2, blue) such that herbicide resistance (green seedling) is only conferred if the mGAL4 transcription factor and the UAS-driven BAR_YFP protein are combined in a single egg (grey). (B–D) Herbicide treatment (B), YFP fluorescence (C), ploidy analysis (D) of triparental triploid plants (TP), and biparental diploid plants (BP). (E) PCR targeting pUAS::BAR-YFP (blue) and pRPS5A::mGAL4-VP16 (yellow) in a herbicide-resistant plant recovered from HIPOD (1), pUAS::BAR-YFP/ + (2), pRPS5A::mGAL4-VP16/ +  (3), wild-type control (4), water control (5). (F) Polyspermy frequency (Psf) following crosses of either wild-type Ler (pollen acceptor) or wild-type Col-0 (pollen acceptor) with Ler pollen donor 1 and Ler pollen donor 2. (G) Polyspermy frequency in ecs1 ecs2 mutants compared to wild type and a rescue line harboring ecs1 ecs2 pECS1::gECS1-YFP. (H) ECS1-YFP and ECS2-YFP localization before fertilization in the corresponding transgenic plants of pECS1::ECS1-YFP and pECS2::ECS2-YFP. Arrow indicates egg cell. (I) Promoter activity analysis using NLS-tdTomato expression driven by a promoter fragment upstream of either ECS1 or ECS2. The arrow points to the egg cell nucleus. Scale bars, 5 mm (B), 50 μm (C) and (I), 20 μm (H).

We confirmed that this phenomenon was causally linked to ECS1 by introducing a pECS1::gECS1-YFP rescue construct, which restored triparental frequencies to wild-type levels (Figure 1G). We next asked whether the polyspermy defect was of gametophytic origin. To answer this, we performed the polyspermy assay in ecs1/ecs1, ecs2-1/ECS2 background, where each gynoecium is expected to segregate a 1:1 ratio of double and single mutant gametophytes. We first assessed ecs2-1 segregation in the reference offspring population that was not herbicide treated by genotyping PCR and found an ECS2:ecs2-1 ratio of 109:97, in line with the expected 1:1 segregation ratio. We furthermore reasoned that the segregation of the ecs2-1 mutant allele would be enhanced in the triparental offspring if the absence of ECS2 in the female gametophyte positively affects polyspermy. Following herbicide selection of 78,338 seedlings, we recovered 24 triparental plants (Figure 1—figure supplement 1A), of which 18 plants exhibited an ecs2-1 tDNA-specific PCR product (Figure 1—figure supplement 1B), corresponding to an ECS2:ecs2-1 ratio of 6:18. This finding fits well with the threefold increase in polyspermy frequency found in ecs1 ecs2-1 double mutants. In addition, analysis of YFP-tagged ECS1 and ECS2 fusion proteins, as well as pECS1::NLS-tdTomato and pECS2::NLS-tdTomato promoter reporter constructs revealed that ECS1 and ECS2 are exclusively expressed in the egg cell prior to fertilization (Figure 1H and I). Together, these results indicate a gametophytic origin of the polyspermy defect.

ecs1 ecs2 mutants are defective in gamete fusion and activate a fertilization recovery program

Making use of aniline blue staining, we found the reproductive defect to be associated with an 8.4% increase in supernumerary pollen tube attraction (Figure 2A). Polytubey in ecs1 ecs2-1 double mutants was also reported by Yu et al., 2021, who provide a compelling set of experiments to show that the endopeptidases cleave specific variants of LURE1. In contrast to Yu et al., 2021, however, we observed reproductive defects, reflected by 13% abnormal seeds (Figure 2B). To investigate whether the phenotype was causally linked to ECS1 and ECS2, we first generated a second double mutant using the independent ecs2-2 allele, which exhibited similar defects in seed development (Figure 2B and Figure 2—figure supplement 1A). Second, we introduced pECS1:gECS1-YFP and pECS2:gECS2-YFP into the ecs1 ecs2 double mutants and demonstrated that each of the constructs restored fertility (Figure 2B and Figure 2—figure supplement 1A). Our results thus indicate that the seed defect in ecs1 ecs2 double mutants is caused by loss of ECS1 and ECS2. To determine whether the defect was due to the role of ECS1 and ECS2 in the male or female reproductive tissue, we conducted reciprocal crosses between the double mutant and wild type. While the introduction of the mutant alleles from the female crossing partner recapitulated the defect, the reciprocal cross yielded fertile seeds, suggesting that the defect was of female origin (Figure 2—figure supplement 1B).

Figure 2 with 1 supplement see all
ecs1 ecs2 double mutants exhibit reproductive defects.

(A) Occurrence of polytubey in ecs1 ecs2 mutants 20 hr after pollination (HAP) (n=1039/835/1070 for wild type/ecs1 ecs2-1/ecs1 ecs2-2, respectively). Circles represent data recovered from individual pistils. The images indicate one pollen tube (left) or two pollen tubes (right) targeting an embryo sac. Pollen tube is highlighted by a yellow arrow. (B) Silique of wild-type and ecs1 ecs2 mutants with quantification of seed abortion in different genotypes (n>800 for each genotype). Circles represent data recovered from individual siliques. Arrowheads, aborted seeds. Different letters show significant difference; p<0.0001, F=106.0, by one-way ANOVA with a Tukey multiple comparison test. (C) Cleared whole mount and quantitative analysis of ecs1 ecs2 mutant seed categories 3 days after pollination (DAP): In comparison to wild-type like seeds containing embryo and endosperm, ecs1 ecs2 mutants segregate seeds without embryo (I) and seeds containing no or retarded endosperm (II) (n=655/540/522 for wild type/ecs1 ecs2-1/ecs1 ecs2-2, respectively). Circles represent data recovered from individual pistils. Embryo and unfertilized egg cells were false colored in yellow (wild type) and orange (mutant). Asterisk in image; unfertilized egg nucleus; arrowhead, endosperm nucleus. Similar results for (B) and (C) were obtained in independent experiments by a different scientist (data shown in Figure 2—figure supplement 1A and Figure 2-figure supplement 1D). Data in (A–C) are represented in box and whisker plot, center line, median; bottom and up, 25th and 75th percentiles. Whisker, the minimum and maximum. Two-tailed Mann-Whitney comparison test between wild type and mutants. Scale bars, 50 μm (A) and (C), 500 μm (B). **p<0.01; ***p<0.001; ****p<0.0001.

To further narrow down the reproductive defect, we performed a comprehensive analysis of cleared whole mounts of mutant and wild-type seeds at different developmental stages. Prior to fertilization, ecs1 ecs2 female gametophytes exhibited a morphology similar to that of wild type (Figure 2—figure supplement 1C). Three days after pollination, however, we observed 8.1% of seeds that failed to develop an embryo and 7.1% of seeds that exhibited defects in endosperm development (Figure 2C and Figure 2—figure supplement 1D).

Together, the above-described results suggest a defect after or during fertilization. The latter scenario, that is, a defect in gamete fusion, has been shown to activate a fertilization recovery program, which delays synergid disintegration in order to attract additional pollen tubes (Beale et al., 2012; Kasahara et al., 2012; Maruyama et al., 2013). When comparing young ecs1 ecs2 seeds with wild type, we indeed found that supernumerary pollen tube attraction correlates with a significant increase of synergid-containing seeds at the two-nucleate endosperm stage (Figure 3A). Overall progression of seed development between mutant and wild type was comparable (Figure 3—figure supplement 1A) and ecs1 ecs2 synergid nuclei eventually degenerated (Figure 3A).

Figure 3 with 2 supplements see all
ecs1 ecs2 double mutants segregate embryos that lack a paternal genome contribution.

(A) Frequency of seeds containing a synergid nucleus at different endosperm stages (n for 2nES = 67/72, 4nES = 143/158, and 8nES = 179/141 for wild type/ecs1 ecs2-1, respectively). The image shows the second synergid nucleus at the two-nucleate endosperm stage. (B) Quantitative analyses of single fertilizations 8-10 HAP in ecs1 ecs2 (n=90) and wild-type ovules (n=89) using HTR10-mRFP. Successful double fertilization (DF) is visualized by two dispersed sperm nuclei signals (arrow heads). In the middle and right panel, only one dispersed sperm cell nuclear signal (arrow heads) can be observed, while an additional sperm nuclear signal (arrows) is condensed suggesting the unfertilized state. The positions of the dispersed sperm signals correspond to the position of central cell (middle) and egg cell (right), respectively, and thus suggest different single fertilization patterns (CC-F and EC-F). (C–E) Analyses of seed development 3 days after dual pollination. (C) Diagram of the dual pollination experiment. (D) Successful double fertilization by a single pollen tube from either pRPS5A::H2B_tdTomato (left panel) or pRPS5A::H2B_GFP (middle panel). Heterofertilization involving the content of two genetically distinct pollen tubes is depicted in the right panel. Endosperm nuclear signal (arrow heads), embryo nuclear signal (arrows). (E) Percentage of heterofertilization events in wild type (n=276) and ecs1 ecs2-1 (n=284) ovules. (F) Fluorescence signal in 6-day-old progenies of ecs1ecs2-1 female plants upon paternal introduction of pRPS5A::H2B-tdTomato. WT: wild-type like seeds, showing paternal marker expression in both embryo and endosperm. Only EN (I): paternal marker expression only in endosperm. Only EM (II): paternal marker expression only in embryo. Dotted line; embryo, yellow arrowhead; sperm nuclei. (G) Percentage of 6-day-old progenies of ecs1ecs2-1 or wild-type female plants crossed with pRPS5A::H2B-tdTomato, showing paternal marker expression in different fertilized tissues. (H) Assessment of paternally introduced marker expression in endosperm (EN) and embryo (EM) of 6-day-old siliques in ecs1 ecs2-1 or wild type. The ecs1 ecs2-1 seeds were divided into a normally looking and an abnormal seed fraction and analyzed individually. EM+EN; expression in both embryo and endosperm. (G) and (H), n=349/332 for wild type/ecs1 ecs2-1, respectively. Brightness was manually enhanced with Adobe Photoshop in (A–C). Data in (A), (B), (G), and (H) indicate mean ± SD. Two-tailed Mann-Whitney comparison test between wild type and mutants, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Scale bars, 20 μm (A), 25 μm (B), 75 µm (D), 50 μm (F).

Our observations suggest a defect at the level of egg-sperm fusion in line with Jiang et al. However, they conflict with work by Yu et al., 2021 and Zhang et al., 2023 which reported that the defect manifests only during early embryo development. To gain a more detailed understanding of the mutant defect, we capitalized on an observation described by Takahashi et al. according to which the condensation status of sperm nuclei relaxes upon gamete fusion. Upon fertilization of ecs1 ecs2 with HTR10-RFP (Ingouff et al., 2007) labeled sperm cells, we observed a pair containing two decondensed sperm nuclei signals in the wild type, indicating successful double fertilization (Figure 3B and Figure 3—figure supplement 2). In the ecs1 ecs2 mutants, we frequently detected a pair of sperm nuclei that differed in the condensation status analogous to the single fertilization pattern described by Takahashi et al., 2018. In one category the more dispersed signal corresponded to egg cell, (EC-F) in the other to central cell (CC-F) position indicating single fertilizations of either central (CC-F) or egg cell (EC-F) (Takahashi et al., 2018; Figure 3B and Figure 3—figure supplement 2). This is in line with later observations using the same experimental setup, where we detect an increase of unfused sperm nuclei in ecs1 ecs2 double mutants over wild type (Figure 3—figure supplement 1B).

In a complementary experiment we confirmed the fertilization defect and an active fertilization recovery program by genetic evidence: It was previously shown that egg and central cell can be fertilized by sperm from different pollen, making this so called heterofertilization a hallmark of single fertilization and an active recovery program (Maruyama et al., 2013): To visualize heterofertilization we pollinated ecs1 ecs2 pistils with pollen from two distinct fathers carrying either pRPS5A::H2B-GFP or pRPS5A::H2B-tdTomato (Figure 3C–E). While we did not detect heterofertilization in wild-type (n=276), ecs1 ecs2 mutants (n=284) exhibited a small fraction of seeds where the two fertilization products (egg and central cell) had inherited different fluorophores provided by the two different pollen donors (1.8%) (Figure 3D–E). This frequency is comparable to the one described by Maruyama et al., which observed heterofertilization using gamete fusion defective kokopelli sperm (Maruyama et al., 2013).

ecs1 ecs2 segregate maternal haploids

The early fertilization defect implies that the inheritance of paternal DNA is compromised in ecs1 ecs2 mutants. To characterize this further we analyzed transmission of a paternally introduced pRPS5a::H2B_tdTomato marker (Figure 3F). While the majority of wild-type seeds showed a fluorescence signal in both endosperm and embryo, a significant number of ecs1 ecs2 mutants contained seeds without paternal marker expression in the embryo (10.9%) or the endosperm (9.7%) (Figure 3G). When comparing marker distribution between aborted and normal seeds, we detected a relative enrichment of seeds without paternal contribution in the endosperm in the aborted seed fraction (Figure 3H), stressing the importance of a paternal endosperm contribution for seed development. Intriguingly, among the normal looking seeds we detected a fraction that lacked a paternal contribution in the embryo (Figure 3H).

This observation prompted us to trace the fate of such seeds. We inspected in total 200 ecs1 ecs2 double mutant-derived plants and identified seven plants exhibiting short siliques and small flowers (Figure 4A). Flow cytometry revealed that all seven plants exhibited a haploid-characteristic signature, indicating that only one set of chromosomes had been inherited (Figure 4B and Figure 4—figure supplement 1). To substantiate this result, we performed a complementary approach and determined the chromosome number by chromosome spread. A. thaliana contains five different chromosomes and in the diploid Col-0 accession used in this study they exist in two copies. Notably, in nuclei of plants with short siliques and small flowers, we detected 5 instead of 10 chromosomes (Figure 4C), confirming the haploid nature of these plants. To determine whether these haploids were of maternal origin, we conducted inter-accession crosses combining diploid ecs1 ecs2 in the Col-0 background with wild-type Ler accession. We recovered eight haploid plants out of 934 F1 seedlings (Figure 4—figure supplement 2) and profiled accession-dependent restriction fragment length polymorphisms (RFLPs) for each of the five chromosomes. We only detected polymorphisms specific for Col-0 on all chromosomes in all eight haploids (Figure 4D and Figure 4—figure supplement 3), indicating that only the maternal genome had been inherited. Such maternal induction of haploidy has only been reported in a recent preprint for the gynoecium-expressed phospholipase AII (pPLAIIγ) (Jang et al., 2022) and these examples differ conceptionally from earlier reported haploid inducers, which operate through selective elimination of aberrant paternal chromosomes (Gilles et al., 2017; Karimi-Ashtiyani et al., 2015; Kelliher et al., 2017; Khanday et al., 2019; Li et al., 2021; Liu et al., 2017; Ravi and Chan, 2010; Underwood et al., 2022; Zhong et al., 2020; Zhong et al., 2019).

Figure 4 with 3 supplements see all
ecs1 ecs2 double mutants segregate haploid offspring.

(A–C) Inflorescence (A), flow cytometry (B), and DAPI stained chromosome spreads (C) of diploid (2n) wild-type and haploid (1n) ecs1 ecs2 plant (n=200, see also Figure 4—figure supplement 1). Brightness was manually enhanced with Adobe Photoshop in (A). The arrowhead and arrow point at a small flower and an undeveloped silique, respectively. (D) Accession-dependent restriction fragment length polymorphisms (RFLPs) in the hybrid progeny from a cross of ecs1 ecs2 mutant in Col-0 background (green) with Ler wild type pollen (blue). Scale bars, 5 mm (A), 2 μm (C). (E) Schematic model illustrating causes and consequences of an active fertilization recovery program in ecs1 ecs2. Left panel: Double fertilization of both, egg and central cell, induces synergid disintegration in wild type. Right panel: Defects in double fertilization in ecs1 ecs2 (egg cell or central cell only) result in delayed synergid disintegration and concomitant attraction of supernumerary pollen tubes increasing the likelihood of polyspermy (blue arrows). In addition, the fertilization defect contributes to seed abortion and can infrequently result in the generation of haploid plants (orange arrows). Dotted lines indicate incomplete penetrance of the respective phenotype. m and p indicate the segregation of maternal and paternal genomes, respectively. Note that according to Yu et al., 2021 the attraction of supernumerary pollen tubes is additionally controlled by ECS1/2-meditated LURE1 cleavage after fertilization.

The segregation of haploid plants in ecs1 ecs2 mutants has similarly been observed by Zhang et al., 2023. However, in contrast to our work and the report by Jiang et al., 2022, Zhang et al. describe that the defect of ecs1 ecs2 double mutants is triggered by semigamy post fertilization. According to previous work, semigamy scenarios would be expected to infrequently result in inheritance of paternal signatures (Gerlach-Cruse, 1970; Lanaud, 1988; Rao and Narayana, 1980) which was, thus far, neither observed by Zhang et al. nor us. Such a scenario would thus require the existence of an additional, previously undiscovered mechanism for the selective elimination of paternal wild-type DNA.

Several factors regulating gamete fusion have been described (Aw et al., 2010; Cyprys et al., 2019; Mori et al., 2014; Mori et al., 2006; Rademacher and Sprunck, 2013; Ron et al., 2010; Sprunck et al., 2012; Takahashi et al., 2018; Tian et al., 2018; von Besser et al., 2006). Among these, the sperm-specific AtDMP8 and AtDMP9 and the pollen-specific NLD have been implicated in haploid induction (Gilles et al., 2017; Kelliher et al., 2017; Liu et al., 2017; Zhong et al., 2020). It will be an interesting task for the future to determine whether other gamete fusion defective mutants similarly give rise to haploids or whether this phenotype uncovers an additional function of ECS1 and ECS2. In addition, it will be interesting to determine whether the activation of the fertilization recovery program is typically associated with increased polyspermy rates.

In summary, our and previous data suggest that ECS1 and ECS2 function at the level of gamete fusion (Jiang and Qu, 2023; Jiang et al., 2022; Mao et al., 2022) and support the coordination of double fertilization. Thus, single fertilization of egg or central cell can be observed in ecs1 ecs2 double mutants that contributes to seed abortion and can infrequently result in the generation of haploid plants. It also leads to delayed synergid disintegration and concomitant attraction of supernumerary pollen tubes. The supply of additional sperm cells can either lead to successful fertilization recovery or the induction of polyspermy (Figure 4E). It was recently reported that following successful double fertilization ECS1/2 are secreted and play a role in degradation of LURE peptides (Yu et al., 2021). To what extent this process in addition contributes to supernumerary pollen tube attraction is currently unclear.

Taken together, we have shown that ECS1 and ECS2 regulate offspring genome size via haploid and polyspermy induction, two agriculturally highly relevant mechanisms. This is particularly important, as the current pace of crop plant optimization is insufficient to meet future demand (Hickey et al., 2019). ECS1 and ECS2 provide molecular clues opening up new avenues for the introduction and/or combination of agricultural relevant traits.

Materials and methods

Plant material and growth conditions

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All experiments were performed with A. thaliana L., ecotype Col-0 or Ler. The seeds were sown on soil. Two days after stratification at 4°C, they were transferred and germinated in a Conviron MTPS growth chamber under long-day conditions (16 hr light/8 hr dark) at 23°C. Plants were transferred into 18°C after bolting. T-DNA insertion mutants SALK_021086 (ecs1), SALK_090795 (ecs2-1), SALK_036333 (ecs2-2) were obtained from European Arabidopsis Stock Center (NASC) (Nottingham, UK).

Plasmid construction and plant transformation

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The promoter and gene fragments of ECS1 and ECS2 were amplified from Arabidopsis Col-0 by using YM76s/as and YM78s/as and introduced into pUAS::BASTA-YFP (Nakel et al., 2017) using AscI/NotI restriction sites. The resulting constructs were then transformed into ecs1 ecs2 double mutants by floral dip (Zhang et al., 2006). To generate pECS1/2::NLS-tdTomato transgenic plants, the promoter region of ECS1 and ECS2 was individually amplified from Col-0 by the respective primer pair YM76s/86as and YM78s/87as. The resulting fragment was then subcloned into DR13 plasmid (pAt5g40260::NLS-tdTomato) (Völz et al., 2013) through AscI/PacI restriction sites. Primer sequences used for genotyping and plasmid construction are listed in Supplementary file 1.

Histology and microscopy

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For the analysis of mature female gametophytes, the oldest closed flower buds were emasculated and harvested 2 days later. For the analysis of seed development, the emasculated flowers were manually pollinated and collected as specified in the figure legends. For whole-mount clearings, samples were treated with acetic acid/ethanol (vol/vol=1:9) overnight, washed in a 80%, 70%, and 50% ethanol series and mounted in chloral hydrate:glycerol:water (8:2:1; wt:vol:vol)(Gross-Hardt et al., 2007). For each experiment, more than five emasculated flowers or pollinated pistils were analyzed. With regard to the determination of persistent synergid cell, more than 10 pistils 19–21 hr after pollination (HAP) were scored and the data from the pistils containing less than 10 fertilized ovules were excluded. Cleared whole-mount ovules and seeds were analyzed under Zeiss Axioscope (Zeiss, Oberkochen, Germany) and the images were captured by Canon PowerShot G10 camera. Mature siliques were dissected and observed under Leica S6E or S8apo stereomicroscope (Leica, Germany).

For aniline blue staining, pistils were collected 20 HAP and processed as described previously (Mori et al., 2006). This experiment was analyzed for each genotype with two technical replicates. Supernumerary pollen tube attraction was examined using an epifluorescence inverted microscope (Leica DMI6000b) with DAPI filter.

Triparental plant screening

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To determine the occurrence of polyspermy-induced triparental plants, HIPOD screening was applied as previously described (Nakel et al., 2017). Two to three closed flower buds per inflorescence were emasculated. Two to three days after emasculation, pollen grains collected from plants of pRPS5A::mGAL4-VP16/+ (pollen donor 1, PD1) and pUAS::BAR-YFP /+ (pollen donor 2, PD2) were applied onto the stigmatic surface. Landsberg erecta accession was used for PD1 and PD2. The resulting mature seeds were sown on soil and the plants were subjected to herbicide treatment. The triparental status of herbicide-resistant plants was confirmed by flow cytometry, fluorescence microscopy inspection, and PCR targeting of the GAL4 and UAS loci.

Chromosome spread

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To detect the exact chromosome number in the diploid and haploid plants, chromosome spreads from pollen mother cells were prepared according to published protocols (Ross et al., 1996).

Ploidy analysis

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Leaves of ecs1 ecs2 plants or herbicide-resistant plants from HIPOD screenings were subjected to flow cytometry as previously described (Nakel et al., 2017).

Statistical analysis

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Data sets were processed in Microsoft Excel. Bar charts, box and whisker plots, as well as all statistical tests were generated using Microsoft Excel or GraphPad Prism.

Data availability

All data generated or analysed during this study are included in the manuscript and supplementary files.

References

  1. Book
    1. Gerlach-Cruse D
    (1970)
    Induction of Semigamy in Arabidopsis Thaliana
    Heynh.

Decision letter

  1. Hao Yu
    Reviewing Editor; National University of Singapore & Temasek Life Sciences Laboratory, Singapore
  2. Jürgen Kleine-Vehn
    Senior Editor; University of Freiburg, Germany
  3. Sharon A Kessler
    Reviewer; Purdue University, United States
  4. Tomoko Igawa
    Reviewer; Chiba University, Japan

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "ECS1 and ECS2 suppress polyspermy and the formation of haploid plants by promoting double fertilization" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by the Reviewing Editor, Hao Yu, and Jürgen Kleine-Vehn as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Sharon A Kessler (Reviewer #1); Tomoko Igawa (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. As the single fertilization events seem to be the most prominent phenotype in this study, additional evidence of single fertilization is necessary to determine whether the defects in ECS1 and ECS2 indeed influence gamete fusion.

2. This manuscript should be improved by providing clearer data analysis and descriptions (e.g. Figure 3), images with higher resolution or better representation (e.g. Figures 2B and 3B), and extra image data (e.g. the DIC image of the synergid nucleus in the embryo-only and endosperm-only ecs1 ecs2 ovules).

3. More discussions should be provided in the context of several recent studies reporting ECS1/2, and regarding the potential roles of ECS1/2 peptidases in regulating gamete fusion and/or repressing parthenogenesis.

Please also take into consideration the other specific comments from the reviewers below to revise the manuscript.

Reviewer #1 (Recommendations for the authors):

Very interesting story, but the presentation of the data in the text was a little confusing since it starts out with triploids, moves on to single fertilization, and ends with haploids but there is not a clear connection made for how all of these phenotypes relate to problems regulating gamete fusion. I'd suggest adding another paragraph at the end that brings these concepts together a little more clearly. It might be helpful to note that the single fertilization events seem to be the most prominent phenotype while the haploid induction and triparental triploids are quite rare. What does this tell us about the wild-type function of ECS1/2?

Reviewer #2 (Recommendations for the authors):

Although Zhang et al. have published similar data on haploid formation, the authors had released bioRxiv in advance, so I think there's no problem with novelty. The data quality is enough for a short report; however, the present descriptions are confusing and hard to understand soon. Please try to use uniform expressions in the description of each data. In addition, as I suggest in a public view, additional evidence of single fertilization would be critical to determine the ecs1ecs2 influence on gamete fusion. I encourage authors to try this or a similar alternative experiment.

Reviewer #3 (Recommendations for the authors):

1. The production of embryo-only seeds in ecs1 ecs2 mutants probably needs more explanation. They claimed that the embryo-only seeds could be observed in ecs1 ecs2 siliques. However, the embryo-only seeds in ecs1 ecs2 siliques are not reported in Jiang et al., (2022) and Zhang et al., (2023). Although the authors provided the reasons that this phenotype might result from the different growth conditions and developmental stages, the explanation is not satisfactory to me. I'm actually curious about one thing, that is, whether the percentage of the ovules with only embryo and arrested sperm cell nuclei is comparable with the percentage of embryo-only seeds (7.1%) in line 143. If they were comparable, the authors probably need to have a deeper discussion on how the egg cell- specifically expressed proteases only affect the interaction between the central cell and sperm cell in those embryo-only seeds. If they were not comparable, I probably have another explanation for these embryo-only seeds, that is, there is a possibility that both of the two sperm cells delivered by one pollen tube (in less pollinated condition) fertilize the egg cell simultaneously due to the polyspermy block defect in ecs1 ecs2 ovules. These ovules were not targeted by the second pollen tube, therefore the central cell did not fertilize in the end. Since the phenotype of having embryo-only seeds was completely not observed in the other two published papers, I would suggest the authors explain the cause of the embryo-only seeds more clearly.

2. In Figure 2B, the images of ecs1 ecs2 mutant siliques are not representative. The authors reported that there were two types of aborted seeds in siliques of ecs1 ecs2 mutants, including embryo-only seeds and endosperm-only seeds (Figure 2C). According to Xiong et al., (2021) and Li et al., (2022), embryo-only seeds do not enlarge and are small dot-like, which are significantly smaller than endosperm-only seeds and normal seeds. I notice that in Figure 2—figure supplement 1B, they showed small dot-like embryo-only seeds and white-but-smaller endosperm-only seeds in the siliques of ecs1 ecs2-1×ecs1 ecs2-1. Thus, I would suggest the authors provide representative images of siliques of ecs1 and ecs2 which simultaneously show the two types of aborted seeds and mark them respectively in Figure 2B.

3. In Figure 3—figure supplement 1B, I notice that the frequency of unfused sperm cells in ecs1 ecs2 ovules is approximately 15% (in my own calculation), but this ratio is significantly higher than the percentage of polytubey in ecs1 ecs2 mutants (Figure 2A). Therefore, I think that the phenotype of unfused sperm cells can not be fully attributed to and explained by the mechanism that is reported by Yu et al., (2021).

4. The logic about how ECS1 and ECS2 participate in persistent synergid cell degeneration is a little bit confusing. The authors think that the defect of persistent synergid cell degeneration is caused by the defect of gamete fusion. However, in Figure 3A, they provided the persistent synergid cell nucleus in ovules undergoing successful double fertilization. The conclusion and the evidence are two different things. I suggest that the authors show ovules with gamete fusion defect and polytubey to support that persistent synergid cell degeneration is caused by the defect of gamete fusion. For example, provide the DIC image of the synergid nucleus in the embryo-only and endosperm-only ecs1 ecs2 ovules. I also suggest the authors discuss the relationship between ECS1/2 and EIN3/EIL1/EIN2 since the phenotype in figure 3A is very similar to those of ein3 eil1 and ein2 mutants. I'm very much curious about whether the EIN3-dependent ethylene-response cascade that is activated by fertilization of the egg cell is affected in ecs1 ecs2 ovules. Whether the affected cleavage of pollen tube attractant AtLURE1 is related to persistent synergid cell death/nucleus disintegration in ecs1 ecs2 ovules?

In Figure 3B, what do the "unfertilized ovules" indicate? (Based on my understanding, this type of seed has neither embryo nor endosperm). The authors should explain this type of ovule in detail here.

https://doi.org/10.7554/eLife.85832.sa1

Author response

Essential revisions:

1. As the single fertilization events seem to be the most prominent phenotype in this study, additional evidence of single fertilization is necessary to determine whether the defects in ECS1 and ECS2 indeed influence gamete fusion.

We here provide two additional data sets that clearly show that ecs1 ecs2 mutant plants indeed exhibit single fertilization, leading to fertilization recovery.

First, we used HTR10_RFP marker as described in Takahashi et al. and suggested by Reviewer 2 to analyze the decondensation of sperm cell nuclei 8-10 HAP. We indeed found an increase in single fertilization events as evidenced by condensed sperm cells either at the micropylar position (egg cell) or at the chalazal position (central cell) (Figure 3B).

Second, we performed a dual pollination experiment using pRPS5A:H2B_GFP and pRPS5A:H2B_tdTomato as male pollen donors and analyzed the fluorescence signal in the seeds 3-6 DAP. In ecs1 ecs2 mutants we found 5 seeds that exhibited differential paternal contribution in embryo and endosperm, while we did not encounter a single such heterofertilization event in wild type. This experiment thus gives additional evidence, that after single fertilization of either egg or central cell, the fertilization recovery mechanism was activated and a second pollen tube (different color) was attracted and fertilized the hitherto unfertilized gamete. Please note that we found fertilization recovery at a frequency of 1.8 %, which is even slightly higher than the frequency reported by Maruyama et al. 2013 when using pollen tubes that contain a single sperm cell (1.2 %).

2. This manuscript should be improved by providing clearer data analysis and descriptions (e.g. Figure 3), images with higher resolution or better representation (e.g. Figures 2B and 3B), and extra image data (e.g. the DIC image of the synergid nucleus in the embryo-only and endosperm-only ecs1 ecs2 ovules).

Thank you for your suggestion. We added images for the silique analysis (Figure 1—figure supplement 1A) and now provide evidence for the different categories as suggested by Reviewer 2. In addition, we provide high resolution pictures throughout the manuscript for all figures. Finally, and in response to the comments by Reviewer 1, we exchanged the original Figure 3B with newly generated Figure 3B-E, to determine fertilization failure by checking the decondensation HTR10-RFP labelled sperm nuclei 8-10 HAP and the frequency of heterofertilization (see above).

3. More discussions should be provided in the context of several recent studies reporting ECS1/2, and regarding the potential roles of ECS1/2 peptidases in regulating gamete fusion and/or repressing parthenogenesis.

We added a model figure (Figure 4E) and included additional discussion to the manuscript: Line 207 to 210 and Line 223 to 232

Reviewer #1 (Recommendations for the authors):

Very interesting story, but the presentation of the data in the text was a little confusing since it starts out with triploids, moves on to single fertilization, and ends with haploids but there is not a clear connection made for how all of these phenotypes relate to problems regulating gamete fusion. I'd suggest adding another paragraph at the end that brings these concepts together a little more clearly. It might be helpful to note that the single fertilization events seem to be the most prominent phenotype while the haploid induction and triparental triploids are quite rare. What does this tell us about the wild-type function of ECS1/2?

Thank you for your suggestions. We created a model figure (Figure 4E) to facilitate the understanding. In addition, we also added a paragraph in the end that summarizes the results and brings the concepts clearer together (Line 223 to 232)

Reviewer #2 (Recommendations for the authors):

Although Zhang et al. have published similar data on haploid formation, the authors had released bioRxiv in advance, so I think there's no problem with novelty. The data quality is enough for a short report; however, the present descriptions are confusing and hard to understand soon. Please try to use uniform expressions in the description of each data. In addition, as I suggest in a public view, additional evidence of single fertilization would be critical to determine the ecs1ecs2 influence on gamete fusion. I encourage authors to try this or a similar alternative experiment.

Thank you for your suggestion. We rephrased the descriptions in the figure legends to make them easier to understand. In addition, we added the experiments suggested by the reviewer (see answers above).

Reviewer #3 (Recommendations for the authors):

1. The production of embryo-only seeds in ecs1 ecs2 mutants probably needs more explanation. They claimed that the embryo-only seeds could be observed in ecs1 ecs2 siliques. However, the embryo-only seeds in ecs1 ecs2 siliques are not reported in Jiang et al., (2022) and Zhang et al., (2023). Although the authors provided the reasons that this phenotype might result from the different growth conditions and developmental stages, the explanation is not satisfactory to me. I'm actually curious about one thing, that is, whether the percentage of the ovules with only embryo and arrested sperm cell nuclei is comparable with the percentage of embryo-only seeds (7.1%) in line 143. If they were comparable, the authors probably need to have a deeper discussion on how the egg cell- specifically expressed proteases only affect the interaction between the central cell and sperm cell in those embryo-only seeds. If they were not comparable, I probably have another explanation for these embryo-only seeds, that is, there is a possibility that both of the two sperm cells delivered by one pollen tube (in less pollinated condition) fertilize the egg cell simultaneously due to the polyspermy block defect in ecs1 ecs2 ovules. These ovules were not targeted by the second pollen tube, therefore the central cell did not fertilize in the end. Since the phenotype of having embryo-only seeds was completely not observed in the other two published papers, I would suggest the authors explain the cause of the embryo-only seeds more clearly.

Thank you for your suggestion. We cannot exclude the possibility that the first pollen tube delivers both sperm cells to the egg cell. However, we now provide an extra data set that shows that we do encounter single egg cell fertilization (EC only– Figure 3B).

While secretion of ECS1 could not be observed (Susaki et al., 2021) before fertilization, it is not unlikely that these molecules are secreted directly at the initiation of fertilization and thus likewise influence the correct fertilization of the central cell. In addition it was previously shown that aspartyl proteases of the same class contain small peptides that can be cleaved off and act as mobile signal transducers (Xia et al., 2004).

2. In Figure 2B, the images of ecs1 ecs2 mutant siliques are not representative. The authors reported that there were two types of aborted seeds in siliques of ecs1 ecs2 mutants, including embryo-only seeds and endosperm-only seeds (Figure 2C). According to Xiong et al., (2021) and Li et al., (2022), embryo-only seeds do not enlarge and are small dot-like, which are significantly smaller than endosperm-only seeds and normal seeds. I notice that in Figure 2—figure supplement 1B, they showed small dot-like embryo-only seeds and white-but-smaller endosperm-only seeds in the siliques of ecs1 ecs2-1×ecs1 ecs2-1. Thus, I would suggest the authors provide representative images of siliques of ecs1 and ecs2 which simultaneously show the two types of aborted seeds and mark them respectively in Figure 2B.

We added Figure 2—figure supplement 1 containing also the class of embryo only seeds.

3. In Figure 3—figure supplement 1B, I notice that the frequency of unfused sperm cells in ecs1 ecs2 ovules is approximately 15% (in my own calculation), but this ratio is significantly higher than the percentage of polytubey in ecs1 ecs2 mutants (Figure 2A). Therefore, I think that the phenotype of unfused sperm cells can not be fully attributed to and explained by the mechanism that is reported by Yu et al., (2021).

Thank you for your observation. We agree with the reviewer’s assessment. We, however, believe that it is difficult to directly compare two differently conducted experiments using two different methods. We determined the fertilization failure by checking the decondensation HTR10-RFP labelled sperm nuclei 8-10 HAP (Figure 3B) and the frequency of heterofertilization through dual pollination experiment (Figure 3C-E) see above.

4. The logic about how ECS1 and ECS2 participate in persistent synergid cell degeneration is a little bit confusing. The authors think that the defect of persistent synergid cell degeneration is caused by the defect of gamete fusion. However, in Figure 3A, they provided the persistent synergid cell nucleus in ovules undergoing successful double fertilization. The conclusion and the evidence are two different things. I suggest that the authors show ovules with gamete fusion defect and polytubey to support that persistent synergid cell degeneration is caused by the defect of gamete fusion. For example, provide the DIC image of the synergid nucleus in the embryo-only and endosperm-only ecs1 ecs2 ovules.

Thank you very much for your comment. In the context of this particular experimental setup, we can only deduce that fertilization has occurred based on the division of the endosperm nuclei. At the same time, at this early stage the oocyte/zygote has not yet divided and therefore we cannot confidently distinguish between double fertilization or single fertilization of the central cell. Analysis of later time points requires introgression of fluorescent reporters which is beyond the time frame of this work but will be possible subsequently. We are sorry for the confusion and changed the figure caption accordingly (Figure 3A). In addition, we have included two experiments that clearly show single fertilization events (Figure 3B and Figure 3C-E). For further explanation of new experiments see above.

I also suggest the authors discuss the relationship between ECS1/2 and EIN3/EIL1/EIN2 since the phenotype in figure 3A is very similar to those of ein3 eil1 and ein2 mutants. I'm very much curious about whether the EIN3-dependent ethylene-response cascade that is activated by fertilization of the egg cell is affected in ecs1 ecs2 ovules. Whether the affected cleavage of pollen tube attractant AtLURE1 is related to persistent synergid cell death/nucleus disintegration in ecs1 ecs2 ovules?

We thank the reviewer for this observation. It was actually established earlier that in light of the fertilization-compensation system, all defects that interfere with gamete fusion are expected to extend the life-span of the second synergid (Beale, Leydon, & Johnson, 2012; Volz, Heydlauff, Ripper, von Lyncker, & Gross-Hardt, 2013). At the same time, we think that the involvement of the ethylene response cascade exceeds the scope of this report.

In Figure 3B, what do the "unfertilized ovules" indicate? (Based on my understanding, this type of seed has neither embryo nor endosperm). The authors should explain this type of ovule in detail here.

We removed the figure and exchanged with new data (see above).

https://doi.org/10.7554/eLife.85832.sa2

Article and author information

Author details

  1. Yanbo Mao

    University of Bremen, Centre for Biomolecular Interactions, Bremen, Germany
    Present address
    School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China
    Contribution
    Conceptualization, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Contributed equally with
    Thomas Nakel
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5520-8202
  2. Thomas Nakel

    University of Bremen, Centre for Biomolecular Interactions, Bremen, Germany
    Contribution
    Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing
    Contributed equally with
    Yanbo Mao
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9033-5987
  3. Isil Erbasol Serbes

    University of Bremen, Centre for Biomolecular Interactions, Bremen, Germany
    Contribution
    Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7854-1243
  4. Saurabh Joshi

    University of Bremen, Centre for Biomolecular Interactions, Bremen, Germany
    Contribution
    Data curation, Investigation, Visualization, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4354-6762
  5. Dawit G Tekleyohans

    University of Bremen, Centre for Biomolecular Interactions, Bremen, Germany
    Present address
    Wageningen Plant Research, Bioscience, Wageningen, Netherlands
    Contribution
    Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7383-5971
  6. Thomas Baum

    University of Bremen, Centre for Biomolecular Interactions, Bremen, Germany
    Contribution
    Formal analysis, Investigation, Visualization
    Competing interests
    No competing interests declared
  7. Rita Groß-Hardt

    University of Bremen, Centre for Biomolecular Interactions, Bremen, Germany
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing, Writing – original draft
    For correspondence
    gross-hardt@uni-bremen.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1998-0507

Funding

European Research Council (646644)

  • Rita Groß-Hardt

European Innovation Council (101057189)

  • Rita Groß-Hardt

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Prof. Dr. Tetsuya Higashiyama (Nagoya University, Japan) for providing the pRPS5A::H2B-tdTomato and pRPS5A::H2B-GFP seeds. We thank Prof. Dr. Frederic Berger (Gregor Mendel Institute of Molecular Plant Biology, Austria) for providing transgenic plants expressing sperm marker pHTR10::HTR10-mRFP. We thank members of the Groß-Hardt laboratory for valuable comments on the manuscript. This work is supported by grants of the European Research Council (ERC Consolidator Grant 'bi-BLOCK' ID. 646644) and the European Innovation Council (EIC Transition '3P-Tec' 101057189) to RG.

Senior Editor

  1. Jürgen Kleine-Vehn, University of Freiburg, Germany

Reviewing Editor

  1. Hao Yu, National University of Singapore & Temasek Life Sciences Laboratory, Singapore

Reviewers

  1. Sharon A Kessler, Purdue University, United States
  2. Tomoko Igawa, Chiba University, Japan

Version history

  1. Preprint posted: January 22, 2022 (view preprint)
  2. Received: December 28, 2022
  3. Accepted: July 24, 2023
  4. Accepted Manuscript published: July 25, 2023 (version 1)
  5. Version of Record published: August 11, 2023 (version 2)

Copyright

© 2023, Mao, Nakel et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Yanbo Mao
  2. Thomas Nakel
  3. Isil Erbasol Serbes
  4. Saurabh Joshi
  5. Dawit G Tekleyohans
  6. Thomas Baum
  7. Rita Groß-Hardt
(2023)
ECS1 and ECS2 suppress polyspermy and the formation of haploid plants by promoting double fertilization
eLife 12:e85832.
https://doi.org/10.7554/eLife.85832

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https://doi.org/10.7554/eLife.85832

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