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