Available online at www.sciencedirect.com Current opinion in SciVerse ScienceDirect Plant Biology ELSEVIER Double fertilization on the move Yuki Hamamura',Shiori Nagahara'and Tetsuya Higashiyama1.2 Double fertilization isaflo ng plant mechanism wher by twc nut ent tissue. ofheteopscesierzateodere embryo and the other fertilizes the central ce o pro ce the and agncult Sin ation by owing to difficulties as investigated this phenomenon using sections of fixed emale gametes in the matemal tissue.How matena a owing to the difficulti techniques have beaun to clarify the actual behavior of the s of the wo nyte 、ells and the sperm cells,which is different from that described by previous anism of double fertilization e remained largely own.n this decade,cons cells in Arabidoosis. mutants defective in fertilization processe Addresso aided by advan cesn technicalive-cell the behavior of hiyama,Tetsuya Live-cell imaging of double fertilization in Arabidopsis revealed three erm cell behavioral steps after the start of pollen tube charge (Figure 1) .During the f in Plant.15:70-7 t iveret（step) This reviey es from a themed issue on minut Available online 5th December 2011 steps.R ted review have focused more on duction [13. entire process of repro D0110.1016/.pbi201.1.001 Step 1:Pollen tube disc h arge phase netophyte (Figures 1 and 2).The maximum flowering plants that enables rapid sced formaion this a88±5.5s1.This wer g plants do not produc edly faster than previous estimates.Previous observation dynein on fixed or dis daferm of othe plant 2000)Instead,polen tube two imr :141i tile sperm cells to the female gametophyte,which the degenerated synergid cel ll,possibly tracking rails o cn cab t from the tube is attracted to the female gametophyte and enters However,direct observation ggesting that the move the le nale gam etophyte, ment of sperm 141 d and discharge is the onset of double-fertilization ga sibly hased on plasmon vsis-like flow of the pollen One of the two released sperm cells fertiliz Current Opinion in Plant Biology 2012.15:70-7 .com
Double fertilization on the move Yuki Hamamura1 , Shiori Nagahara1 and Tetsuya Higashiyama1,2 Double fertilization is a flowering plant mechanism whereby two immotile sperm cells fertilize two different female gametes. One of the two sperm cells fertilizes the egg cell to produce the embryo and the other fertilizes the central cell to produce the endosperm. Despite the biological and agricultural significance of double fertilization, the mechanism remains largely unknown owing to difficulties associated with the embedded structure of female gametes in the maternal tissue. However, molecular genetic approaches combined with novel live-cell imaging techniques have begun to clarify the actual behavior of the sperm cells, which is different from that described by previous hypotheses. In this review article, we discuss the mechanism of double fertilization based on the dynamics of the two sperm cells in Arabidopsis. Addresses 1 Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Aichi, Japan 2 JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Aichi, Japan Corresponding author: Higashiyama, Tetsuya (higashi@bio.nagoya-u.ac.jp) Current Opinion in Plant Biology 2012, 15:70–77 This review comes from a themed issue on Growth and Development Edited by Xuemei Chen and Thomas Laux Available online 5th December 2011 1369-5266/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2011.11.001 Introduction Double fertilization is a unique reproductive system in flowering plants that enables rapid seed formation even under dry conditions. Flowering plants do not produce motile sperm with flagella (flagellar dynein genes are lacking in the genome; Arabidopsis Genome Initiative, 2000). Instead, a pollen tube rapidly conveys two immotile sperm cells to the female gametophyte, which includes two synergid cells, an egg cell, and a central cell. Through interactions with synergid cells, the pollen tube is attracted to the female gametophyte and enters the female gametophyte, finally discharging contents from an aperture at the tip [1–4]. This pollen tube discharge is the onset of double-fertilization gametic interactions. One of the two released sperm cells fertilizes the egg cell to form the embryo and the other sperm cell fertilizes the central cell to form the endosperm, the seed nutrient tissue. Endosperm formation is appropriately accelerated by the male genome delivered by double fertilization [5,6]. Since the discovery of double fertilization by Nawashin (1898) and Guignard (1899), many plant biologists have investigated this phenomenon using sections of fixed material owing to the difficulties in accessing the embedded female gametophyte [7,8]. However, the dynamics of the two immotile sperm cells and the mechanism of double fertilization have remained largely unknown. In this decade, considerable progress has been achieved using molecular genetic analyses of Arabidopsis thaliana mutants defective in fertilization processes, aided by advances in technical live-cell imaging [9,10]. In thisreview article, we discussthe mechanism of double fertilization, focusing particularly on the behavior of Arabidopsis sperm cells. Live-cell imaging of double fertilization in Arabidopsis revealed three sperm cell behavioral steps after the start of pollen tube discharge (Figure 1) [11]. During the first step, sperm cells are discharged and delivered into the female gametophyte rapidly in several seconds (step 1). Sperm cells remain in the female gametophyte for several minutes without further migration (step 2). The sperm begin to fuse with each target cell (step 3). Below, we discuss the mechanism of double fertilization using these steps. Related review articles have focused more on sperm cell formation [12] and the entire process of reproduction [13]. Step 1: Pollen tube discharge phase The first step is pollen tube discharge, wherein two sperm cells are rapidly transported from the pollen tube into the female gametophyte (Figures 1 and 2). The maximum velocity of sperm cells was 10 mm/s, and the duration of this movement was 8.8 5.5 s [11]. This was unexpectedly faster than previous estimates. Previous observations on fixed or dissected materials of other plants after in vivo pollination suggested that the two sperm cells migrate gradually (e.g. several hours in Nicotiana tabacum; [14]) in the degenerated synergid cell, possibly tracking rails of actin cables called ‘corona’ due to the actomyosin-based movement from the micropylar part to the apical part [15]. However, direct observations suggesting that the movement of sperm cells was rapid and continuous until reaching a position between the egg and central cell were possibly based on plasmoptysis-like flow of the pollen tube cytoplasm as observed in Torenia fournieri, which is a unique plant with a protruding embryo sac [16]. Two sperm cells always overtake the vegetative nucleus of the Available online at www.sciencedirect.com Current Opinion in Plant Biology 2012, 15:70–77 www.sciencedirect.com
Double fertilization Hamamura.Nagahara and Higashivama 71 Figure 1 e and concluded that breakdown of the synergid cell occurs just afterolenowfth dischar In T. fe theopn Molecular genetic analyses in Arabidopsis also support the erated r-like 2l for mber of the m二com nich are pre at the micro charge [19- zation or plasma membrane of the pollen tube tip and are nccessary Sperm cells ning pllen tubethupre Step 2:Immobility phase of the two sperm t of the oollen tube long period,approximately 7.4 min [11".Two-photon cell be havior during double in ana alysis enab high-re ametophyte (Figure 3a).Figure 3b is an illustration ase d on a two-photor nthat shows the pos dclivered to the apical cdge of the svnerid tep 2 is th ng the apical edge of the egg cel and the central This area pthe I ma th fe he cent pollen tube a rm c d b membrane is believed to rupture during pollen tube ccept the pollen tube arrival and synergid cell de neration has clectron microscop Visu the ate for many years [ idopst recep the a s are actually re preciscly after am a drasti www.sciencedirect.com Current Opinion in Plant Biology 0.1:70-77
pollen tube after pollen tube discharge but before reaching the position between the egg and central cell (Figure 1) [11]. One of the two synergid cells degenerates to accept the pollen tube contents. However, the timing between pollen tube arrival and synergid cell degeneration has been an issue of debate for many years [1]. In Arabidopsis, degeneration of the receptive synergid cell has been suggested to begin upon pollen tube arrival [17] or more precisely after pollen tube arrival but before pollen tube discharge [18]. In higher time-resolution, Hamamura et al. [11] observed a drastic change in morphology of the green fluorescent protein (GFP)-labeled synergid cell nucleus just after the start of pollen tube discharge and concluded that breakdown of the synergid cell occurs just after pollen tube discharge (Figure 2). In T. fournieri, breakdown of the synergid cell (including rupture of the plasma membrane) also occurs just after the start of pollen tube discharge [16]. Molecular genetic analyses in Arabidopsis also support the idea that the receptive synergid cell is not degenerated before the pollen tube arrives (see [2] for a detailed review). For example, a receptor-like kinase, FERONIA, and a member of the mildew resistance locus o family NORTIA, which are predominantly localized at the micropylar end of the synergid cell upon pollen tube arrival, are involved in a direct interaction between the synergid cell and the pollen tube to trigger pollen tube discharge [19– 22,23 ]. A change in the localization of NORTIA from the entire synergid cell to the micropylar end was observed in both synergid cells. How the receptive synergid cell is determined remains unknown. Interestingly, homologs of FERONIA, ANXUR1, and ANXUR2 are expressed in the plasma membrane of the pollen tube tip and are necessary for maintaining pollen tube growth without rupture [24,25]. Step 2: Immobility phase of the two sperm cells After rapid transport of the sperm cells by pollen tube discharge, sperm cells remain immobile for a relatively long period, approximately 7.4 min [11]. Two-photon microscopy analysis enabled high-resolution deep imaging of the positioning of sperm cells in the female gametophyte (Figure 3a). Figure 3b is an illustration based on a two-photon observation that shows the position of two sperm cells in this phase. Sperm cells are delivered to the apical edge of the degenerated synergid cell facing the apical edge of the egg cell and the central cell [11]. This area between the egg and central cell may be the region where the sperm cells fuse with female gametes [8]. In the second step, sperm cells remain in this boundary region, keeping contact with the egg cell and the central cell. Notably, the two sperm cells are always in close vicinity during this phase [11]. The two sperm cells in the pollen tube are enclosed by an endocytic membrane. This membrane is believed to rupture during pollen tube discharge because sperm cells in the degenerated synergid cells always lack the membrane when observed by electron microscopy [8]. Visualizing the endocytic membrane during the fertilization process would be of interest to address when two sperm cells are actually released to initiate the gametic interaction. What is the meaning of this immobility? An in vitro fertilization assay using isolated egg and sperm cells of Double fertilization Hamamura, Nagahara and Higashiyama 71 Figure 1 1 2 3 Central cell Sperm cells Ovule Vegetative nucleus Synergid cells pollen tube Egg cell Current Opinion in Plant Biology Schematic representation of sperm cell behavior during double fertilization in Arabidopsis. Sperm cell behavior during double fertilization is divided into three steps. Step 1 is the pollen tube discharge phase. Sperm cells are delivered rapidly (8.8 5.5 s; [11]) from the pollen tube into the female gametophyte. The receptive synergid cell is likely to breakdown just after the start of pollen tube discharge. Step 2 is the immobility phase. After delivery, sperm cells remain at the boundary region between the egg cell and the central cell for 7.4 3.3 min [11]. Step 3 is the doublefertilization phase. After the immobility phase, sperm cells fuse with each target (plasmogamy) and their nuclei reinitiate movement toward the target gamete nuclei. The time lag between the first and second fertilization is 2.5 1.7 min [11]. www.sciencedirect.com Current Opinion in Plant Biology 2012, 15:70–77
72 Growth and Development Figure2 20m ent Opinion in Plan arge rom the egg ce SYN,ynergid cll nuceus Scale,20m een two flagella of the minus-gamete [35]and is rapidly degraded upon membrane 32 probably to 3d36 Step 3:Double-fertilization phase the egg cell (37). ci (Figre c)This behavior of nuclei was no ofthedoube-mehnism On Two-photon mic oscopy sugges 40.Observation of retinoblastom fusion of gametes(plasmogamy)to karyogamy with target ferie nacland the clcan be by pemumerary sperm cells in a pollen tube always ferti- GCSI/HAPLESS2 (HAP2) ver. sed that not only the egg cel Current Opinion in Plant Biology 201.1570-7 www. edirect.com
Zea mays (maize) may shed light on the meaning. A maize in vitro fertilization assay revealed that adhesion of gametes before fertilization lasted 3.8 1.8 min [26]. This immobile state may be important to establish adhesion (anchoring) with target cells, leading to membrane fusion. Another possibility might be that male and/ or female gametes require complex cell-to-cell communication including a change in physiological condition to accomplish double fertilization. Step 3: Double-fertilization phase After the 7.4 min immobility phase, sperm cell nuclei suddenly reinitiate movement toward the female gamete nuclei (Figure 3c) [11]. This behavior of the sperm cell nuclei was not observed in generative cell specific 1 (gcs1) mutant sperm cells defective in fertilization (Figure 3d). Two-photon microscopy suggested that reinitiation of sperm cell nuclei movement indicates gamete fusion of both the egg and central cell [11]. Thus, the third step can be defined as the double-fertilization phase from the fusion of gametes (plasmogamy) to karyogamy with target nuclei. Karyogamy events, including fusion of polar nuclei of the central cell before fertilization, has been studied by molecular genetic approaches and a putative mitochondrial 50S ribosomal subunit L21 [27] and homologs of a molecular chaperone Hsp70 in the endoplasmic reticulum [28] were shown to be involved. GCS1/HAPLESS2 (HAP2) is a sperm cell transmembrane protein necessary for gamete fusion [29,30] that is conserved in the plant kingdom and other lower eukaryotes including some protists and invertebrates [31,32 ]. A GCS1 domain analysis revealed that the N-terminus region is possibly an extracellular domain important for gamete fusion [33 ,34 ]. In Chlamydomonas reinhardtii, GCS1/HAP2 is exclusively localized at the fusion site between two flagella of the minus-gamete [35] and is rapidly degraded upon membrane fusion, probably to prevent polygamy with other gametes [32]. Other genetic analyses in Arabidopsis found that the mitochondrial ankyrin repeat protein ANK6, expressed in male and female gametes, is necessary for fertilization recognition (Figure 3d) [36 ] and that a putative glycosylphosphatidylinositol-anchored protein, LORELEI, might be involved in not only pollen tube reception by the synergid cell, as FERONIA and NORTIA, but also fertilization of the egg cell [37]. One of the double-fertilization mechanism models is based on blocking polyspermy, wherein the remaining sperm cell is obligated to fertilize the remaining female gamete [38–40]. Observation of retinoblastoma related 1 (rbr1; [41,42]) mutants suggest a polyspermy block in the fertilized egg cell: the rbr1 mutant ovule has two egg cells and a non-fertile central cell, and the two egg cells can be fertilized with wild-type sperm cells one by one (Figure 3e) [43]. Spielman and Scott [40] observed that supernumerary sperm cells in a pollen tube always fertilize the central cell, resulting in polyspermy in a tetraspore mutant (Figure 3f). This observation proposes a stricter polyspermy block in fertilized egg cells, suggesting that the egg cell might be fertilized earlier than the central cell. However, live-cell imaging showed no particular order of the egg and central cells for fertilization [11]. Hamamura et al. [11] proposed that not only the egg cell but the central cell might rapidly block polyspermy to ensure double fertilization (Figure 3c). Rapid and local cell wall formation at the fertilization point of the egg cell 72 Growth and Development Figure 2 -1 min 0 min 1 min 5 min 20 min CCN ECN SYN SCN Current Opinion in Plant Biology Live-cell imaging of the entire double-fertilization process. Sperm cell nuclei (SCN; arrowheads) were labeled with histone-fused mRFP. Female gametophyte nuclei and the ovules were labeled with histonefused green fluorescent protein (GFP). The asterisk represents the nucleus of a broken-down synergid cell. Note that two sperm cells are rapidly transported from the pollen tube to the boundary of the egg and central cell, where gamete fusion occurs. Time indicates min after pollen tube discharge. Modified after [11]. CCN, central cell nucleus; ECN, egg cell nucleus; SYN, synergid cell nucleus. Scale, 20 mm. Current Opinion in Plant Biology 2012, 15:70–77 www.sciencedirect.com
Double fertilization Hamamura.Nagahara and Higashivama 73 Figure 3 (a) (d) CC acs ion in Piant Bio at th nt cel e of cdka:1 a y (h)The the egg and central cell [55"].Female gametophytic gene might be involved in fertilization of the egg cell [37]. sted during in fertilizati maiz 441.The cenual celis than the sel thus.it After doub The time difference between the two fertilization events n In time-lap imagin 38.Two sperm cells,which are enclosed by the endo tion and Current Opinion in Plant:70-77
has been suggested during in vitro fertilization of maize [44]. The central cell is larger than the egg cell; thus, it might be less apt to block polyspermy when supernumerary sperm cells arrive. The time difference between the two fertilization events was only 2.5 min on average [11]. In time-lapse imaging with 30-s and 1-min intervals, two sperm cells sometimes fertilized the egg and central cell simultaneously. Additionally, a correlation occurred between the time of fertilization of the egg and central cell. Double fertilization may not simply depend on blocking polyspermy but may also be determined by an complex cell-to-cell communication between the two male gametes and the two female gametes during the immobility phase. Are the two isomorphic sperm cells of Arabidopsis functionally equivalent? Two sperm cells follow considerably different fates depending on their fertilization targets. After double fertilization, one sperm cell is involved in producing an embryo, which survives to the next generation, but the other sperm cell is involved in producing the endosperm, which supplies nutrition to the embryo. Whether fertilization targets are predetermined has long been debated [38]. Two sperm cells, which are enclosed by the endocytic membrane, associate with the vegetative nucleus to form the male germ unit (Figure 1) [45]. In some plants such as Plumbago zeylanica, two dimorphic sperm cells are always arranged in the same alignment [46–48]. The smaller sperm cell of Plumbago, which is not associated Double fertilization Hamamura, Nagahara and Higashiyama 73 Figure 3 (a) (b) (c) (d) (e) (f) (g) (h) (i) CC CC CC EC EC SC EC ? F:B=1:1 F:B=1:1 EC CC SC SY SC DSY gcs1 rbr1 tes cdka;1, kpl diftheria toxin or Current Opinion in Plant Biology or Sperm cell behavior at the double fertilization phase in wild-type and mutant plants. (a) Two-photon microscopy observation of sperm cells at the immobility phase, namely, just before fusions. Two sperm cells (SC) are at the boundary of the egg cell (EC; mitochondria are labeled) and the central cell (CC; the nucleus and cytosol are labeled). Scale, 10 mm. (b) Illustration of the sperm cells based on a two photon microscopy observation. Sperm cells appear to maintain contact with the egg cell and central cell before fusions with them. (c) Schematic representation of the mechanism of double fertilization in wild type. The front (F) and back (B) sperm cells of the male germ unit fertilize the egg and the central cell at an equal frequency, suggesting that the two sperm cells might be identical in their functions. Rapid and local blocking might ensure double fertilization between two identical sperm cells. (d)–(h) Summary of observations in mutant plants. Among the following mutants, the behavior of the sperm cell has been visually examined in gcs1 [11,29], ank6 [36 ], cdka;1 [52], and rbr1 [43]. The behavior of sperm cells in the other mutants is suggestions from observations of the embryogenesis and the endosperm development. Nuclei of the pollen tube cell and the synergid cell are not shown. (d) Two mutant cells do not fuse with female gametes in gcs1/hap2, ank6, duo pollen 1 and 3 [12]. (e) Two wild-type sperm cells fertilize two egg cells in an embryo sac of an rbr1 mutant, on which the central cell is sterile, suggesting that polyspermy block might be working in the fertilized egg cell and that no specific sperm cell fertilizes the central cell. Fertilization of a pair of two egg cells by two sperm cells has also been suggested in eostre [50]. (f) Supernumerary sperm cells of tetraspore (tes) caused polyspermy only in the central cell, suggesting a stricter block in the egg cell [69]. We propose that the central cell is bigger than the egg cell and is less apt to block polyspermy of supernumerary sperm cells. (g) The single fertile sperm cell of cdka;1 and msi1 [51] fertilizes both female gametes at an equal frequency. (h) The single fertile sperm cell produced by diphtheria toxin preferentially fertilizes the central cell [54]. (i) In kpl mutant, defects in sperm-specific cis-nat-siRNA caused single fertilization in both the egg and central cell [55]. Female gametophytic gene LORELEI might be involved in fertilization of the egg cell [37]. www.sciencedirect.com Current Opinion in Plant Biology 2012, 15:70–77
74 Growth and Developmen with the vegetative nucleus.contains plastids and pre both embrvogenesis and endost r formation.Prefer ferentially fertilizes the egg cell(16/17)49.As the male ence in fertilization targets would not be critical in thi uni s generally in flowering plants cas plants with isomorphic sperm cells such has zinc-finger transeription factor WRKY2 1581.An increase been an issue of debate. ferential he sperm cells have been inte lized with each wild-type sperm cell.suggesting that n cells has been reported.Knowing whether a sperm cel A pem-like sc has appropriare for both embryo D)mutant The twa ntral easons why the functio developmental programs after fusion with each targer and its T-DNA mutant gonclhusionsln advances have enabled us to same manner 53.Ho a single sperm-l efpnihomal nhi tha A gu live-cell imaging and molecular genetic analysis in A (Figure 3h) the 0 the approach the n sm between the two male gametes and the two female Furthe Additionally the fert cells 6n cen or gam the front (associated with the leading vegetative nuleus ion analyses am ong each gametophytic cel and back sperm cell in the male gemm uni had the s eidentifying new genes involved in egg cel will clucidate the lasti target was observed betwe een the front and back sperm cells accurately fertilize different partners. cells of Arabidopsis This result was consistent with no preference for fusion. Acknowledgements which was observed in most previous mutant analyse RPENSOR.which cells ar essed equally of ere 1. 8S尚器aown Current Opinion in Plant Biology 012.15:70-77 irect.com
with the vegetative nucleus, contains plastids and preferentially fertilizes the egg cell (16/17) [49]. As the male germ unit is generally observed in flowering plants, whether such a preferential fertilization occurs in usual plants with isomorphic sperm cells such as Arabidopsis has been an issue of debate. Emerging data based on an analysis of A. thaliana mutants are complicated and have not clarified the issue of preferential fertilization. For example, two egg cells in an eostre and rbr1 mutant female gametophyte can be fertilized with each wild-type sperm cell, suggesting that no sperm cell fertilizes only the central cell (Figure 3e) [43,50]. A single sperm-like cell of multicopy suppressor of ira1 and cyclin-dependent kinase a1 (cdka;1) mutants showssingle fertilization with either the egg or the central cell at an equal frequency (Figure 3g) [51,52]. An F-box protein of Arabidopsis, F-box-like 17 (FBL17), targets degradation of cyclin-dependent kinase A1 inhibitors, specifically in male germ cells, and its T-DNA mutant shows a similar impaired seed formation phenotype as that of cdka;1, possibly due to single fertilization in the same manner [53]. However, a single sperm-like cell formed by translational inhibition of sperm cells that express the diphtheria toxin A subunit is likely to cause single fertilization preferentially with the central cell (Figure 3h) [54]. Defects in the regulation of a sperm-specific cis-nat-siRNA of kokopelli (kpl) mutant also result in single fertilization, wherein one of two sperm cells fertilizes either the egg or central cell (Figure 3i) [55]. These discussions about the fertilization capacity of two sperm cells were based on mutant analyses, and therefore, a wild-type analysis is expected. Using a photo-convertible fluorescent protein, monomeric Kikume Green-Red, nuclei of two isomorphic Arabidopsis sperm cells were differentially labeled in the pollen tube [11]. Double fertilization of these differently colored wild-type sperm cells revealed that the front (associated with the leading vegetative nucleus) and back sperm cells in the male germ unit had the same opportunity to fertilize the egg cell and the central cell (Figure 3c). That is, no preference in the fertilization target was observed between the front and back sperm cells of Arabidopsis. This result was consistent with no preference for fusion, which was observed in most previous mutant analyses, and no gene was identified as preferentially expressed in one of the two Arabidopsis sperm cells. For example, SHORT SUSPENSOR, which is necessary for regulating unequal divisions of the zygote, is transcribed in both sperm cells [56 ]. Homologs of genes distributed unequally in Plumbago sperm cells are expressed equally in both sperm cells of Arabidopsis [57 ]. One may conceivably assume that isomorphic sperm cells of flowering plants are functionally identical and can be involved in both embryogenesis and endosperm formation. Preference in fertilization targets would not be critical in this case. Another gene expressed in the male gametophyte to establish polarity in the zygote has been identified as the zinc-finger transcription factor WRKY2 [58 ]. An increase in the number of such genes will provide insights into the capacity of the two sperm cells. Additionally, epigenetic regulation and reprogramming of the sperm cells have been intensively studied [6,59–63]. No epigenetic regulation specific to one of the two sperm cells has been reported. Knowing whether a sperm cell has appropriate epigenetic regulation for both embryogenesis and endosperm formation would be of interest. The two male chromatins undergo distinct epigenetic reprogramming (chromatin remodeling) after fertilization [59]. This might be one of the reasons why the functionally identical sperm cells might contribute to different developmental programs after fusion with each target. Conclusions Recent live-cell imaging advances have enabled us to visualize male and female gamete interactions during double fertilization. The three-step behavior of sperm cells during double fertilization is now apparent. Both live-cell imaging and molecular genetic analysis in Arabidopsis support the idea that the two sperm cells have an equal ability to fertilize each female gamete. Rapid blocking in both the egg and central cell might be critical to avoid mis-targeting of two identical sperm cells. To approach the mechanism of double fertilization, further insights are required into the cell-to-cell communications between the two male gametes and the two female gametes during the 7.4-min immobility phase. Further development of imaging techniques will provide powerful tools to gain higher temporal and spatial-resolution information. Additionally, novel approaches including visible screening using fluorescent marker lines of gametophytic cells [64] and large-scale transcriptome and gene-expression analyses among each gametophytic cell [65–68] will accelerate identifying new genes involved in double fertilization. In the near future, these new insights will elucidate the long-lasting issue of how two sperm cells accurately fertilize different partners. Acknowledgements We thank Taeko Sasaki for illustration of Figure 3b. Y.H. was supported by a grant (number 9138) from the Japan Society for the Promotion of Science Fellowships and by GCOE program (Nagoya Univeristy). References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Higashiyama T: The synergid cell: attractor and acceptor of the pollen tube for double fertilization. J Plant Res 2002, 115:149-160. 74 Growth and Development Current Opinion in Plant Biology 2012, 15:70–77 www.sciencedirect.com