《种子学》课程教学资源（文献资料）Double fertilization on the move

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 immo￾tile 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 mech￾anism 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 repro￾duction [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 unexpect￾edly 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 move￾ment of sperm cells was rapid and continuous until reach￾ing 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 reach￾ing 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 micro￾pylar 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 ima￾ging 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 pos￾ition 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 syner￾gid cells always lack the membrane when observed by electron microscopy [8]. Visualizing the endocytic mem￾brane 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 double￾fertilization 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 mem￾brane fusion. Another possibility might be that male and/ or female gametes require complex cell-to-cell communi￾cation 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 homo￾logs of a molecular chaperone Hsp70 in the endoplasmic reticulum [28] were shown to be involved. GCS1/HAPLESS2 (HAP2) is a sperm cell transmem￾brane protein necessary for gamete fusion [29,30] that is conserved in the plant kingdom and other lower eukar￾yotes 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 glycosylphosphati￾dylinositol-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 ferti￾lize 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 histone￾fused 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 supernu￾merary 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 ferti￾lization 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 fertili￾zation targets are predetermined has long been debated [38]. Two sperm cells, which are enclosed by the endo￾cytic 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 pre￾ferentially 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 pre￾ferential fertilization. For example, two egg cells in an eostre and rbr1 mutant female gametophyte can be ferti￾lized 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, mono￾meric 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 con￾ceivably assume that isomorphic sperm cells of flowering plants are functionally identical and can be involved in both embryogenesis and endosperm formation. Prefer￾ence 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 embryo￾genesis 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 function￾ally 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 Ara￾bidopsis 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 power￾ful tools to gain higher temporal and spatial-resolution information. Additionally, novel approaches including visible screening using fluorescent marker lines of game￾tophytic 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

Double fertilization Hamamura.Nagahara and Higashivama 75 2.Kle shen in pollen de ery in 3. M- n ML:Usir 23. 2 ing th ception and f Me0eTaKenR4aniedtgs0otheEmbnologyolAngospems 2009.1913271新 25. n-D 9 o3 26. 2。 d time-la and 28 it of sp clei.By 29. 12epmpna8sa路en8m296atonn 15.Wetor2t5107S118 16 ntntTor o t2010137.i4731481. de enabled the zygote to avoid 18 ver GP, 3. 5e1595 by G s1. nsab as the N- ativ ntial eful f the mo te Pe Wong JL.Leydon AR.Joh MA: To ide on.theN www.sciencedirect.com Current Opinion in Plant:70-77

2. Kessler SA, Grossniklaus U: She’s the boss: signaling in pollen tube reception. Curr Opin Plant Biol 2011, 14:622-627. 3. Dresselhaus T, Lausser A, Marton ML: Using maize as a model to study pollen tube growth and guidance, cross-incompatibility and sperm delivery in grasses. Ann Bot 2011, 108:727-737. 4. Takeuchi H, Higashiyama T: Attraction of tip-growing pollen tubes by the female gametophyte. Curr Opin Plant Biol 2011, 14:614-621. 5. Kinoshita T: Reproductive barrier and genomic imprinting in the endosperm of flowering plants. Genes Genet Syst 2007, 82:177-186. 6. Feng S, Jacobsen SE, Reik W: Epigenetic reprogramming in plant and animal development. Science 2010, 330:622-627. 7. Maheshwari P: An Introduction to the Embryology of Angiosperms. New York: McGraw-Hill; 1950. 8. Russell SD: Double fertilization. Int Rev Cytol: Surv Cell Biol 1992, 140:357-387. 9. Berger F, Hamamura Y, Ingouff M, Higashiyama T: Double fertilization – caught in the act. Trends Plant Sci 2008, 13:437-443. 10. Berger F: Imaging fertilization in flowering plants, not so abominable after all. J Exp Bot 2011, 62:1651-1658. 11. Hamamura Y, Saito C, Awai C, Kurihara D, Miyawaki A, Nakagawa T, Kanaoka MM, Sasaki N, Nakano A, Berger F et al.: Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana. Curr Biol 2011, 21:497-502. The authors performed time-lapse imaging of double fertilization and found three behavioral steps of the two sperm cells: rapid movement from the ruptured pollen tube, a stationary phase between the two female gametes, and gamete fusion leading to movement of sperm cell nuclei toward the target female nuclei. By labeling the two sperm cells differently with a photo-convertible fluorescent protein, they found no preference in fertilization targets between the front and back sperm cells of the male germ unit. 12. Twell D: Male gametogenesis and germline specification in flowering plants. Sex Plant Reprod 2011, 24:149-160. 13. Chevalier E, Loubert-Hudon A, Zimmerman EL, Matton DP: Cell￾cell communication and signalling pathways within the ovule: from its inception to fertilization. New Phytol 2011, 192:13-28. 14. Tian HQ, Yuan T, Russell SD: Relationship between double fertilization and the cell cycle in male and female gametes of tobacco. Sex Plant Reprod 2005, 17:243-252. 15. Weterings K, Russell SD: Experimental analysis of the fertilization process. Plant Cell 2004, 16(Suppl.):S107-S118. 16. Higashiyama T, Kuroiwa H, Kawano S, Kuroiwa T: Explosive discharge of pollen tube contents in Torenia fournieri. Plant Physiol 2000, 122:11-14. 17. Faure JE, Rotman N, Fortune P, Dumas C: Fertilization in Arabidopsis thaliana wild type: developmental stages and time course. Plant J 2002, 30:481-488. 18. Sandaklie-Nikolova L, Palanivelu R, King EJ, Copenhaver GP, Drews GN: Synergid cell death in Arabidopsis is triggered following direct interaction with the pollen tube. Plant Physiol 2007, 144:1753-1762. 19. Huck N, Moore JM, Federer M, Grossniklaus U: The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception. Development 2003, 130:2149-2159. 20. Rotman N, Rozier F, Boavida L, Dumas C, Berger F, Faure JE: Female control of male gamete delivery during fertilization in Arabidopsis thaliana. Curr Biol 2003, 13:432-436. 21. Escobar-Restrepo JM, Huck N, Kessler S, Gagliardini V, Gheyselinck J, Yang WC, Grossniklaus U: The FERONIA receptor-like kinase mediates male-female interactions during pollen tube reception. Science 2007, 317:656-660. 22. Capron A, Gourgues M, Neiva LS, Faure JE, Berger F, Pagnussat G, Krishnan A, Alvarez-Mejia C, Vielle-Calzada JP, Lee YR et al.: Maternal control of male-gamete delivery in Arabidopsis involves a putative GPI-anchored protein encoded by the LORELEI gene. Plant Cell 2008, 20:3038-3049. 23. Kessler SA, Shimosato-Asano H, Keinath NF, Wuest SE, Ingram G, Panstruga R, Grossniklaus U: Conserved molecular components for pollen tube reception and fungal invasion. Science 2010, 330:968-971. In this study, nortia (nta) was identified as a female gametophytic mutant displaying pollen tube overgrowth in the female gametophyte, such as feronia ( fer). The NTA gene encodes a member of the Mildew Resistance Locus O (MLO) family, suggesting that pollen tube reception and fungal invasion share common components. Using fer/fer mutant plants, the authors also showed that FER was involved in powdery mildew infection as well as pollen tube reception. 24. Miyazaki S, Murata T, Sakurai-Ozato N, Kubo M, Demura T, Fukuda H, Hasebe M: ANXUR1 and 2, sister genes to FERONIA/ SIRENE, are male factors for coordinated fertilization. Curr Biol 2009, 19:1327-1331. 25. Boisson-Dernier A, Roy S, Kritsas K, Grobei MA, Jaciubek M, Schroeder JI, Grossniklaus U: Disruption of the pollen￾expressed FERONIA homologs ANXUR1 and ANXUR2 triggers pollen tube discharge. Development 2009, 136:3279-3288. 26. Antoine AF, Faure JE, Cordeiro S, Dumas C, Rougier M, Feijo JA: A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants. Proc Natl Acad Sci USA 2000, 97:10643-10648. 27. Portereiko MF, Sandaklie-Nikolova L, Lloyd A, Dever CA, Otsuga D, Drews GN: NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization. Plant Physiol 2006, 141:957-965. 28. Maruyama D, Endo T, Nishikawa S: BiP-mediated polar nuclei fusion is essential for the regulation of endosperm nuclei proliferation in Arabidopsis thaliana. Proc Natl Acad Sci USA 2010, 107:1684-1689. 29. Mori T, Kuroiwa H, Higashiyama T, Kuroiwa T: GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization. Nat Cell Biol 2006, 8:64-71. 30. von Besser K, Frank AC, Johnson MA, Preuss D: Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization. Development 2006, 133:4761-4769. 31. Hirai M, Arai M, Mori T, Miyagishima SY, Kawai S, Kita K, Kuroiwa T, Terenius O, Matsuoka H: Male fertility of malaria parasites is determined by GCS1, a plant-type reproduction factor. Curr Biol 2008, 18:607-613. 32. Liu Y, Misamore MJ, Snell WJ: Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy in Chlamydomonas. Development 2010, 137:1473-1481. Both FUS1 and HAP2 (GCS1) of the green alga Chlamydomonas are expressed in the plus and the minus gametes, respectively, and are rapidly degradated after gamete fusion. The authors concluded that this rapid degradation of essential proteins enabled the zygote to avoid polygamy in Chlamydomonas. 33. Mori T, Hirai M, Kuroiwa T, Miyagishima SY: The functional domain of GCS1-based gamete fusion resides in the amino terminus in plant and parasite species. PLoS ONE 2010, 5:e15957. The authors performed an Arabidopsis gcs1 mutant complementation assay by transforming plants with modified versions of GCS1, of which various domains were disrupted, separated, or exchanged by GFP insertions. Expression and localization of transformed genes was visua￾lized by GFP fluorescence. The C-terminus of GCS1, a putative intracel￾lular region, was dispensable, whereas the N-terminus domain, a putative extracellular region, was essential for successful fertilization. Further￾more, an in vitro fertilization assay of the mouse malaria parasite Plas￾modium berghei possessing modified versions of GCS1 also represented less contribution of the C-terminus to gamete fusion in P. berghei. 34. Wong JL, Leydon AR, Johnson MA: HAP2(GCS1)-dependent gamete fusion requires a positively charged carboxy-terminal domain. PLoS Genet 2010, 6:e1000882. To identify the HAP2 (GCS1) functional region, the N-terminus and C-terminus of Arabidopsis HAP2 was exchanged with those of other Double fertilization Hamamura, Nagahara and Higashiyama 75 www.sciencedirect.com Current Opinion in Plant Biology 2012, 15:70–77

76 Growth and Development in Arabidopis thallana. 35出微2a地88bae。 HAP d ceser the d two s ete.Ho the ca d in the rotcrationo ell was a ntry,bu of m 2337 37. “二声空 ization 3a眼2含8pR湍88是72s63品。 39.umGadFertiocalclum a key sper 40. he c rather than the e 7 vas iden R19 h “ M 5oganond ssP】 46. 8 ns Panta 2011. of male g with the P ago zeylanica IPT ger xpresed ini的& 。 ion of 50. 61. ant Chen TannoMrrChromin Current Opinion in Plant Biology 2012.15:70-7 www.scie edirect.com

species, and a complementation assay was performed. The N-terminus was functional when exchanged with that of closely related species, such as Sisymbrium irio, whereas it was nonfunctional when exchanged with that of distantly related species such as Oryza sativa. The positive charge of the histidines in the C-terminus contributed to the full function of HAP2. 35. Liu Y, Tewari R, Ning J, Blagborough AM, Garbom S, Pei J, Grishin NV, Steele RE, Sinden RE, Snell WJ et al.: The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes. Genes Dev 2008, 22:1051-1068. 36. Yu F, Shi J, Zhou J, Gu J, Chen Q, Li J, Cheng W, Mao D, Tian L, Buchanan BB et al.: ANK6, a mitochondrial ankyrin repeat protein, is required for male-female gamete recognition in Arabidopsis thaliana. Proc Natl Acad Sci USA 2010, 107:22332- 22337. The mitochondrial ankyrin repeat protein ANK6 was abundantly expressed in the male and female gametophyte, both of which were unexpectedly involved in gamete recognition for fusion. 37. Tsukamoto T, Qin Y, Huang Y, Dunatunga D, Palanivelu R: A role for LORELEI, a putative glycosylphosphatidylinositol￾anchored protein, in Arabidopsis thaliana double fertilization and early seed development. Plant J 2010, 62:571-588. 38. Knox RB, Zee SY, Blomstedt C, Singh MB: Male gametes and fertilization in angiosperms. New Phytologist 1993, 125:679-694. 39. Dumas C, Gaude T: Fertilization in plants: is calcium a key player? Semin Cell Dev Biol 2006, 17:244-253. 40. Spielman M, Scott RJ: Polyspermy barriers in plants: from preventing to promoting fertilization. Sex Plant Reprod 2008, 21:53-65. 41. Ebel C, Mariconti L, Gruissem W: Plant retinoblastoma homologues control nuclear proliferation in the female gametophyte. Nature 2004, 429:776-780. 42. Ingouff M, Jullien PE, Berger F: The female gametophyte and the endosperm control cell proliferation and differentiation of the seed coat in Arabidopsis. Plant Cell 2006, 18:3491-3501. 43. Ingouff M, Sakata T, Li J, Sprunck S, Dresselhaus T, Berger F: The two male gametes share equal ability to fertilize the egg cell in Arabidopsis thaliana. Curr Biol 2009, 19:R19-R20. The Arabidopsis thaliana mutant of RETINOBLASTOMA RELATED1 (rbr1) contains an additional egg cell and a sterile central cell. The two egg cells in the rbr1 ovule fused with sperm cells to intiate normal embryogenesis, indicating that both sperm cells in a single pollen tube have equal ability to fertilize an egg cell. 44. Kranz E, Vonwiegen P, Lorz H: Early cytological events after induction of cell-division in dgg cells and zygote development following in-vitro fertilization with angiosperm gametes. Plant J 1995, 8:9-23. 45. Mogensen HL: The male germ unit: concept, composition and significance. Int Rev Cytol: Surv Cell Biol 1992, 140:129-147. 46. Russell SD, Cass DD: Ultrastructure of the sperms of Plumbago zeylanica. 1. Cytology and association with the vegetative nucleus. Protoplasma 1981, 107:85-107. 47. Sodmergen, Chen GH, Hu ZM, Guo FL, Guan XL: Male gametophyte development in plumbago-zeylanica – cytoplasm localization and cell determination in the early generative cell. Protoplasma 1995, 186:79-86. 48. Saito C, Nagata N, Sakai A, Mori K, Kuroiwa H, Kuroiwa T: Angiosperm species that produce sperm cell pairs or generative cells with polarized distribution of DNA-containing organelles. Sex Plant Reprod 2002, 15:167-178. 49. Russell SD: Preferential fertilization in Plumbago: Ultrastructural evidence for gamete-level recognition in an angiosperm. Proc Natl Acad Sci USA 1985, 82:6129-6132. 50. Pagnussat GC, Yu HJ, Sundaresan V: Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1. Plant Cell 2007, 19:3578-3592. 51. Chen Z, Tan JL, Ingouff M, Sundaresan V, Berger F: Chromatin assembly factor 1 regulates the cell cycle but not cell fate during male gametogenesis in Arabidopsis thaliana. Development 2008, 135:65-73. 52. Aw SJ, Hamamura Y, Chen Z, Schnittger A, Berger F: Sperm entry is sufficient to trigger division of the central cell but the paternal genome is required for endosperm development in Arabidopsis. Development 2010, 137:2683-2690. Loss-of-function mutations in Arabidopsis CYCLIN-DEPENDENT KINASE A1 (CDKA;1) cause the production of a single sperm cell. In this study, the authors showed that cdka;1 single spermcells fertilizedeitherthe eggcell or the central cell in Arabidopsis. They also revealed that a significant propor￾tion of cdka;1 pollen tubes contained two sperm cells, which appeared to fuse with each female gamete. However, the karyogamy of the cdka;1 sperm nucleus failed in the fertilized central cell. The following autonomous proliferation of the central cell was activated by cdka;1 sperm entry, but incorporation of the paternal genome (i.e. karyogamy of male and female nuclei in the fertilized central cell) was required for early development of the endosperm. 53. Kim HJ, Oh SA, Brownfield L, Hong SH, Ryu H, Hwang I, Twell D, Nam HG: Control of plant germline proliferation by SCF(FBL17) degradation of cell cycle inhibitors. Nature 2008, 455: 1134-U1114. 54. Frank AC, Johnson MA: Expressing the diphtheria toxin A subunit from the HAP2(GCS1) promoter blocks sperm maturation and produces single sperm-like cells capable of fertilization. Plant Physiol 2009, 151:1390-1400. Expression of the translation inhibitor diphtheria toxin A subunit (DTA) under the control of the male germ line-specific HAP2 (GCS1) promoter led to the production of a single sperm-like cell rather than two sperm cells. The single sperm-like cell preferentially fused with the central cell rather than the egg cell, suggesting the possibility of a differential gene expression program between the two sperm cells in directing preference to the targeted female gametes. 55. Ron M, Saez MA, Williams LE, Fletcher JC, McCormick S: Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis. Genes Dev 2010, 24:1010- 1021. A male gametophytic mutant of Arabidopsis, kokopelli (kpl), was identi- fied, which showed single-fertilization events and resulted in the reduc￾tion of seed set. The KPL and inversely transcribed ARIADNE14 (ARI14; putatively encoding a ubiquitin E3 ligase) genes generated cis-nat-siRNA that targeted ARI14 specifically in sperm cells. ARI14 overexpression in sperm cells also induced the reduced seed set phenotype, indicating that cis-nat-siRNA played a key role in successful double fertilization of plants by regulating the ARI14 transcript level. 56. Bayer M, Nawy T, Giglione C, Galli M, Meinnel T, Lukowitz W: Paternal control of embryonic patterning in Arabidopsis thaliana. Science 2009, 323:1485-1488. Mutations in the IRAK/Pelle-like kinase SHORT SUSPENSOR (SSP) gene resulted in the suppression of suspensor formation such as yoda (yda) mitogen-activated protein kinase pathway mutants. SSP transcripts existed in sperm cells, but the protein was undetectable. Alternatively, SSP protein accumulated in the zygote and the endosperm transiently after fertilization, suggesting the presence of paternal effects over zygote growth. Because the ectopically expressed SSP in seedlings solely activated YDA-dependent signaling, the authors concluded that SSP plays an important role upon fertilization via the YDA-dependent signaling cascade. 57. Ge L, Gou X, Yuan T, Strout GW, Nakashima J, Blancaflor EB, Tian HQ, Russell SD: Migration of sperm cells during pollen tube elongation in Arabidopsis thaliana: behavior during transport, maturation and upon dissociation of male germ unit associations. Planta 2011, 233:325-332. A GFP reporter gene fused with the Plumbago zeylanica IPT gene promoter of the sperm associated with the vegetative nucleus was expressed in both sperm cells at an equal level in Arabidopsis. The authors used Arabidopsis plants transformed with this pPzIPT:GFP con￾struct to examine movement of sperm cells during pollen tube elongation and to show that the male germ unit is an essential assemblage for transmission of sperm cells. 58. Ueda M, Zhang Z, Laux T: Transcriptional activation of Arabidopsis axis patterning genes WOX8/9 links zygote polarity to embryo development. Dev Cell 2011, 20:264-270. The plant specific zinc-finger transcription factor WRKY2 directly activates the transcription of WUSCHEL-RELATED HOMEOBOX (WOX) 8 and its redundant gene WOX9 in the Arabidopsis zygote. In wrky2-1 mutant plants, the zygote failed to reestablish polar distribution of organelles, and the zygote underwent abnormal cell division. The WRKY2-WOX8/9 76 Growth and Development Current Opinion in Plant Biology 2012, 15:70–77 www.sciencedirect.com

Double fertilization Hamamura.Nagahara and Higashivama 77 deveopment byut- sspp1agreaeciopPn8ece20o218e2988mic 59. 98 65 “之 61 67.M 62. 63 68. M omin contining Current Opinion in Plant Biology 2012.15:70-77

transcription cascade played a key role in zygote development by regulat￾ing zygote polarity and embryo patterning. 59. Ingouff M, Hamamura Y, Gourgue SM, Higashiyama T, Berger F: Distinct dynamics of HISTONE3 variants between the two fertilization products in plants. Curr Biol 2007, 17:1032-1037. 60. Slotkin RK, Vaughn M, Borges F, Tanurdzic M, Becker JD, Feijo JA, Martienssen RA: Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 2009, 136:461-472. 61. Autran D, Baroux C, Raissig MT, Lenormand T, Wittig M, Grob S, Steimer A, Barann M, Klostermeier UC, Leblanc O et al.: Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis. Cell 2011, 145:707-719. 62. Baroux C, Raissig MT, Grossniklaus U: Epigenetic regulation and reprogramming during gamete formation in plants. Curr Opin Genet Dev 2011, 21:124-133. 63. Wolff P, Weinhofer I, Seguin J, Roszak P, Beisel C, Donoghue MT, Spillane C, Nordborg M, Rehmsmeier M, Kohler C: High￾resolution analysis of parent-of-origin allelic expression in the Arabidopsis Endosperm. PLoS Genet 2011, 7:e1002126. 64. Ikeda Y, Kinoshita Y, Susaki D, Iwano M, Takayama S, Higashiyama T, Kakutani T, Kinoshita T: HMG domain containing SSRP1 is required for DNA demethylation and genomic imprinting in Arabidopsis. Dev Cell 2011, 21:589-596. 65. Borges F, Gomes G, Gardner R, Moreno N, McCormick S, Feijo JA, Becker JD: Comparative transcriptomics of Arabidopsis sperm cells. Plant Physiol 2008, 148:1168-1181. 66. Qin Y, Leydon AR, Manziello A, Pandey R, Mount D, Denic S, Vasic B, Johnson MA, Palanivelu R: Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil. PLoS Genet 2009, 5:e1000621. 67. Wuest SE, Vijverberg K, Schmidt A, Weiss M, Gheyselinck J, Lohr M, Wellmer F, Rahnenfuhrer J, von Mering C, Grossniklaus U: Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes. Curr Biol 2010, 20:506-512. 68. Wang D, Zhang C, Hearn DJ, Kang IH, Punwani JA, Skaggs MI, Drews GN, Schumaker KS, Yadegari R: Identification of transcription-factor genes expressed in the Arabidopsis female gametophyte. BMC Plant Biol 2010, 10:110. 69. Scott RJ, Armstrong SJ, Doughty J, Spielman M: Double fertilization inArabidopsis thaliana involves a polyspermy block on the egg but not the central cell. Mol Plant 2008, 1:611-619. Double fertilization Hamamura, Nagahara and Higashiyama 77 www.sciencedirect.com Current Opinion in Plant Biology 2012, 15:70–77