Cytoskeleton dynamics control the first asymmetric division in Arabidopsis

Yusuke Kimataa, Takumi Higakib, Tomokazu Kawashimac,d, Daisuke Kuriharaa,e, Yoshikatsu Satof, Tomomi Yamadaa,f, Seiichiro Hasezawab, Frederic Bergerc, Tetsuya Higashiyamaa,e,f, and Minako Uedaa,f,1

aDivision of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan; bDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan; cGregor Mendel Institute, Vienna Biocenter, Austrian Academy of Sciences, 1030 Vienna, Austria; dDepartment of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546; eJapan Science and Technology Agency, Exploratory Research for Advanced Technology Higashiyama Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan; and fInstitute of Transformative Bio-Molecules, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan

Edited by Robert L. Fischer, University of California, Berkeley, CA, and approved November 1, 2016 (received for review August 22, 2016) The asymmetric of the zygote is the initial and crucial correlated to the position of the two adjacent synergid cells (13, developmental step in most multicellular organisms. In flowering 14) (Fig. 1A), suggesting that the preexisting polarity of the egg plants, whether zygote polarity is inherited from the preexisting cell is retained. Conversely, in Papaver rhoeas, the zygote nu- organization in the or reestablished after fertilization has cleus is positioned at the opposite site of its initial location in remained elusive. How dynamically the intracellular organization the egg cell, implying reorientation of the cell polarity after is generated during zygote polarization is also unknown. Here, we fertilization (15). Thus, origins of zygotic polarization have used a live-cell imaging system with Arabidopsis to visu- remained unknown. alize the dynamics of the major elements of the cytoskeleton, In many animal zygotes, cytoskeletal dynamics have already microtubules (MTs), and filaments (F-), during the en- been characterized as a crucial driving force of cell polarization tire process of zygote polarization. By combining image analysis (16–18). By contrast, studies of cytoskeletal behavior in flow- and pharmacological experiments using specific inhibitors of the ering plants have mainly focused on the central cell, which also cytoskeleton, we found features related to zygote polarization. fuses with the sperm cell to generate the -surrounding The preexisting alignment of MTs and F-actin in the egg cell is lost nurse tissue, the endosperm (19, 20) (Fig. 1A). Although mi- on fertilization. Then, MTs organize into a transverse ring defining crotubule(MT)patternwasobservedinfixedzygotes(21),the the zygote subapical region and driving cell outgrowth in the api- cytoskeletal dynamics, such as how intracellular kinetics drive cal direction. F-actin forms an apical cap and longitudinal arrays zygote polarization and how the zygote elongates directionally, and is required to position the nucleus to the apical region of the remained unsolved. Thus, time sequence information on the zygote, setting the plane of the first asymmetrical division. Our zygote polarization steps has been long awaited. findings show that, in flowering plants, the preexisting cytoskel- In this study, we used modified live-imaging markers of cyto- etal patterns in the egg cell are lost on fertilization and that the skeletal dynamics at fertilization (20) combined with a newly zygote reorients the cytoskeletons to perform directional cell elon- developed in vitro ovule cultivation system (3) to perform high- gation and polar nuclear migration. resolution live-cell imaging of MT and actin filament (F-actin) dynamics in the polarizing zygote of Arabidopsis. We found that Arabidopsis thaliana | zygote polarity | microtubule | actin filament | the cytoskeletal organization of the egg cell is lost on fertilization apical–basal axis and that both fibers are reoriented in the zygote in relation to directional cell elongation and apically directed nuclear migration. ody axis formation is one of the first developmental events Our findings provide insights into the intracellular dynamics of Boccurring after fertilization in multicellular eukaryotes. In zygote polarization in flowering plants. PLANT BIOLOGY most flowering plants, the apical–basal (shoot–root) axis is formed along the longitudinal cell polarity of the egg cell and the Significance zygote, marked by the apical position of the nucleus (1, 2) (Fig. 1A). In Arabidopsis thaliana, within 24 h of fertilization, the zy- In animals and plants, the zygote divides unequally, and the gote elongates markedly and becomes polarized with the nucleus daughter cells inherit different developmental fates to form a lying close to the apical region, leading to the asymmetric zygotic proper embryo along the body axis. The cytological events leading division, which produces a small apical cell and a large basal cell – to zygote polarization have remained unknown in flowering plants. (2 4) (Fig. 1A). The apical cell gives rise to the embryo lineage Here, we report that the two essential components of the cyto- that generates most of the plant body, whereas the basal cell skeleton, microtubules and actin filaments, are both disorganized produces the short-lived suspensor lineage and the hypophysis, on fertilization and then, arranged to form a transverse ring leading the most apically located cell, which becomes essential in the directional cell elongation and longitudinal arrays underlying polar organization of the root meristem (5, 6) (Fig. 1A). nuclear migration, respectively. These results provide insights into In most animal zygotes, the unfertilized oocyte has a clear cell the intracellular dynamics of zygote and the specific roles of cyto- polarity, but the sperm entry site changes its direction to set the skeletons on zygote polarizationinfloweringplants. first zygote division plane in many species, such as mouse, – , Xenopus, and bivalve (7 10). Therefore, Author contributions: Y.K., T. Higaki, T.K., D.K., Y.S., S.H., F.B., T. Higashiyama, and M.U. the initial body axis of their is determined by fertiliza- designed research; Y.K., T.Y., and M.U. performed research; Y.K., T. Higaki, and M.U. tion. In flowering plants, the sperm cell enters from the apex of analyzed data; and Y.K., T. Higaki, T.K., D.K., Y.S., F.B., and M.U. wrote the paper. the egg cell, and thus, the apical–basal axis seems unaltered be- The authors declare no conflict of interest. fore and after fertilization (2, 11, 12). Therefore, it has remained This article is a PNAS Direct Submission. unclear whether zygote polarity is inherited from the egg cell or Freely available online through the PNAS open access option. newly generated after fertilization. In vitro fertilization assays of 1To whom correspondence should be addressed. Email: [email protected]. rice gametes showed that the position of the zygote division This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plane is determined independently to the gamete fusion site and 1073/pnas.1613979113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1613979113 PNAS | December 6, 2016 | vol. 113 | no. 49 | 14157–14162 Downloaded by guest on September 28, 2021 Fig. 1. Live-cell imaging and quantification of MT dynamics during zygote polarization. (A) Schematic diagram of the Arabidopsis zygote that develops deep in the flower. (B–J) 2PEM images of the (B) egg cell and (C–J) time-lapse observations of the zygote in in vitro-cultivated ovules expressing the MT/nucleus marker. The images are representative of three time-lapse images. Numbers indicate the time (hours:minutes) from the first frame. The dotted yellow lines show the site where the egg cell and the zygote attach to the maternal tissue. Arrowheads and brackets show the nucleus and the subapical transverse MT ring, respectively. The lengths from the center of the nucleus to the apical edge and the basal end of the cell are shown as A and B, respectively, in C. Maximum intensity projection images generated by serial optical sections are shown. (Scale bars: 10 μm.) (K) Illustrations showing a summary of the respective stages in B–J.(L) Graph of Δθ (the average angle of the fibers against the longitudinal axis) in the indicated cells. The illustrations show the correlation between the values and the cytoskeleton patterns. *P < 0.05. (M) Graph of cell area in the indicated cells. (N) Time course of A and B shown in C until H. (O) Graph of Δθ of MTs in the apical and basal compartments. Error bars represent the SD of 8–10 samples. Significant differences from the values of young zygotes were determined by Dunnett’stestinL, and the letters in M and O indicate significant differences among stages (P < 0.01 by the Tukey–Kramer test). Elong, elongating zygote; ns, not significant; PPB, preprophase band.

14158 | www.pnas.org/cgi/doi/10.1073/pnas.1613979113 Kimata et al. Downloaded by guest on September 28, 2021 Results intensity distribution (a metric for the appearance of bundled MT Arrays Are Disorganized on Fertilization and Form a Subapical cables) (Fig. S3J). In addition, higher Δθ values in the apical Transverse Ring During Zygote Elongation. To perform live-cell region than in the basal area supported the difference between imaging of the cytoskeleton in the polarizing zygote, we developed the subapical transverse MT ring and the basal oblique MT ar- – a set of markers labeling MTs and F-actin with nuclear reporters rays (Fig. 1O and Fig. S3 K N). (MT/nucleus and F-actin/nucleus) (Figs. 1 and 2 and Fig. S1) After the completion of zygote elongation, the subapical trans- Δθ that could be imaged using two-photon excitation microscopy verse MT ring dispersed (Fig. 1F and Fig. S3G), and thus, values (2PEM) (Fig. S2). We captured the dynamic behavior of MTs by in the apical and basal regions no longer significantly differed (Fig. time-lapse imaging until the first zygotic cell division (Fig. 1, Fig. 1O). Before spindle formation (Fig. 1H), another transverse ring of S3, and Movie S1). Before fertilization, the egg cell showed a cortical MTs appeared surrounding the nucleus as a preprophase vertically elongated shape and a clear polar organization with band (Fig. 1G), indicating the site of the first cell division of the mature zygote. Subsequently, mitosis took place, and the phrag- the nucleus at the apical pole (Fig. 1B). The MT arrays aligned moplast formed between the daughter nuclei (Fig. 1I)beforethe longitudinally (Fig. 1 B and K and Fig. S3 A, C, and D), as shown completion of cytokinesis (Fig. 1J). by the small average angle of the fibers against the cell longi- In summary, fertilization triggers a loss of longitudinal MT tudinal axis (Δθ) (Fig. 1L). arrays in the egg cell, and a subapical transverse MT ring appears After fertilization, the cell markedly shrank (Fig. 1 C and M), specifically during zygote elongation. The presence of a trans- as reported in several species of flowering plants (15, 22, 23). verse MT ring in the immature zygote is in agreement with the The nucleus moved to the cell center, and the MT pattern lost its previous observation by immunofluorescence staining of the original longitudinal orientation (Fig. 1 C and N and Fig. S3E)as Δθ nontransgenic plants (21), supporting that our system visualizes indicated by the increased (Fig. 1L) and the reduced paral- the native dynamics of MT. lelness of each fiber (Fig. S3H). Within ∼2 h, transverse MTs accumulated in the subapical region (Fig. 1D) and organized an F-Actin Is Also Rearranged from a Disorganized State and Forms an MT ring, above which a bulge emerged. This bulge rapidly out- Apical Cap and Longitudinal Bundle Along the Apical–Basal Axis. grew above the transverse MT ring, thus elongating in the apical Next, we performed time-lapse imaging and quantification of direction (Fig. 1E and Fig. S3F). During zygote elongation, the F-actin (Fig. 2, Fig. S4, and Movie S2). In the egg cell, we ob- MTs formed spiral oblique cortical arrays in the basal part of the served a meshwork consisting of thick parallel cables (Fig. 2 A cell (Fig. 1E and Fig. S3F), and the nucleus gradually followed to and H and Fig. S4A), as shown by large skewness (Fig. S4D) and the apical region (Fig. 1N). This MT reorganization during zy- parallelness (Fig. 2I) values. After fertilization, F-actin cables gote elongation was quantified by increased values of parallel- were disorganized in the shrunken zygote (Fig. 2B and Fig. S4B), ness (Fig. S3H), fiber density (Fig. S3I), and skewness of the as quantified by lower parallelness values than in the egg cell PLANT BIOLOGY

Fig. 2. Live-cell imaging and quantification of F-actin dynamics during zygote polarization. (A–G) 2PEM images of the (A) egg cell and (B–G) time-lapse observation of the zygote in in vitro-cultivated ovules expressing the F-actin/nucleus marker. The images are representative of five time-lapse images. Numbers indicate the time (hours:minutes) from the first frame. The dotted yellow lines show the site where the egg cell and zygote attach to the maternal tissue. Arrowheads and brackets show the nucleus and apical cap, respectively. Maximum intensity projection images generated by serial optical sections are shown. (Scale bars: 10 μm.) (H) Illustrations showing a summary of the respective stages in A–G.(I and J) Graphs of (I) parallelness and (J) Δθ of each fiber in the indicated cells. Error bars represent the SD of 8–15 samples. Significant differences from the values of young zygotes were determined by Dunnett’s test. ns, Not significant. *P < 0.05; **P < 0.01.

Kimata et al. PNAS | December 6, 2016 | vol. 113 | no. 49 | 14159 Downloaded by guest on September 28, 2021 (Fig. 2I). In addition, the cables became thinner, as shown by imaging system. The MT filament pattern could not be visualized reduced skewness values (Fig. S4D). These data indicated that with this spinning disk confocal system, but it was suitable for high- the F-actin pattern in the egg cell was disrupted on fertilization, throughput measurement of cell shape and nuclear positioning (Fig. as observed for MTs. 3 A–C and Movie S3). Focusing on mature zygotes, we measured At the apical pole in the emerging bulge and elongating tip, we the cell length (Fig. 3D), cell width (Fig. S5G), and nuclear position found F-actin accumulation at the apical end (Fig. 2 C and D), (Fig. 3E). In contrast with the control treatment (Fig. 3A and Movie similar to the apical cap, a typical structure of tip-growing cells, S3), oryzalin treatment resulted in loss of the restriction of zygote which guides vesicle trafficking to promote cell elongation (24). elongation, and thus, the zygotes became shorter and wider than Similar to a report in which the apical cap was detected only in control zygotes (Fig. 3 B and D and Fig. S5G). Although the nucleus the growing tip (25), the cap structure disappeared after zygote properly migrated to the apical region (Fig. 3E), cell division failed elongation was completed (Fig. 2E). In both elongating and in the presence of oryzalin (Fig. 3B), probably because of the in- mature zygotes, F-actin was also aligned longitudinally (Fig. 2 D hibition of the mitotic and cytokinetic apparatus, such as spindle and E and Fig. S4C), as indicated by an increase of parallelness fibers. Treatment with LatB caused distinct effects. It slightly (Fig. 2I) and a reduction of Δθ (Fig. 2J). In addition, F-actin inhibited cell elongation (Fig. 3 C and D and Fig. S5G)butmore cables become thick and dense, as shown by increases in skew- severely blocked nuclear migration (Fig. 3 C and E). LatB did not ness (Fig. S4D) and density (Fig. S4E). During cell division, disturb the completion of nuclear division and cytokinesis, and F-actin accumulated at the phragmoplast (Fig. 2F), as MTs did. thus, cell division was close to symmetrical (Fig. 3C). These results In summary, the preexisting F-actin pattern in the egg cell was indicated that MTs regulate zygote cell elongation, whereas F-actin lost on fertilization. In the elongating zygote, F-actin was rear- promotes polar migration of the nucleus to the apical tip. ranged into an apical cap and a longitudinal array, both of which are typically associated with directionally elongating cells (25). Discussion Our study showed that fertilization triggers the loss of the MTs and F-Actin Regulate Directional Cell Elongation and Polar Nuclear preexisting organization of the cytoskeleton. This disorganiza- Migration, Respectively. To examine the respective roles of MTs tion occurs coincidentally with the dispersion of the vacuole at and F-actin, we analyzed how their inhibitors affected zygote po- thebasalregionoftheeggcell(4,26,27).Becausealarge larization. First, we confirmed that MTs and F-actin in the zygote vacuole occupies much of the egg cell volume in most flowering were specifically disturbed when their respective inhibitors were plants (15, 22), fertilization would trigger vacuole shrinkage to added to the in vitro ovule cultivation media (Fig. S5 A–F). The lose the dominant fiber orientation in the egg cell and also, filamentous pattern of the MT/nucleus marker disappeared after a reduce the cell volume. The disruption of cytoskeletal array 1-h treatment with an MT polymerization inhibitor (oryzalin) (Fig. after fertilization suggests that the polar organization of the egg S5B), whereas the control DMSO and the actin polymerization cell is associated with fertilization. Then, cytoskeletal elements inhibitor [latrunculin B (LatB)] had no detectable effect (Fig. S5 A are redeployed to establish a pattern required to establish the and C). Similarly, the F-actin signal became diffuse only when embryo axis. This idea is supported by the observation that the LatB was applied (Fig. S5 D–F), confirming that these inhibitors central cell consumes the F-actin array for gamete nuclei fusion were specific and efficient in our experimental setup. (20) and by the wrky2 mutant, which generates proper egg cell We examined the effects of these inhibitors on zygote polariza- polarity but fails to repolarize the zygote, resulting in an abnormal tion with the MT/nucleus marker using a multiposition live-cell embryo shape (4).

Fig. 3. Roles of MT and F-actin in zygote polarization. (A–C) Confocal time-lapse observations of the MT/nucleus marker in the presence of (A) control DMSO and polymerization inhibitors for (B)MTs(1μMoryzalin)and(C)actin(1μM LatB). Numbers indicate the time (hours:minutes) from the first frame. The mature zygote was set as one frame before the nuclear division, and the cell shape and nuclear position in the mature zygotes are summarized in column 4. The lengths from the center of the nucleus to the apical edge and the basal end of the cell are shown as A and B, respectively. The width of the zygote is shown as W. Brackets on the images show the lengths of the apical and basal cells after the zygotic division. Note that the oryzalin-treated zygote failed to complete cell division. Maximum intensity projection images generated by serial optical sections are shown. (Scale bars: 10 μm.) (D and E)Graphsof(D)zygotecelllength(A+ B) and (E)asymmetry (A/B). Error bars represent the SD of 13–14 samples, and significant differences from the values of DMSO-treated zygotes were determined by Dunnett’stest.ns, Not significant. *P < 0.05; **P < 0.01. (F) Schematic representation of the patterns and roles of MTs and F-actin in zygote polarization.

14160 | www.pnas.org/cgi/doi/10.1073/pnas.1613979113 Kimata et al. Downloaded by guest on September 28, 2021 Our analysis of cytoskeletal dynamics identified an essential Materials and Methods role for the MT ring in defining the site of cell outgrowth that Detailed materials and methods are described in SI Materials and Methods. sustains unidirectional zygote elongation (Fig. 3F). This role likely explains the absence of zygote elongation in the pilz mu- Strains and Growth Conditions. All Arabidopsis lines were generated in the tant, which is deficient in MT assembly (28). Similar roles for Columbia background. The plants were grown from 18 °C to 22 °C under MT rings have been observed in directionally elongating cells, continuous light or long-day conditions (16-h light/8-h dark). such as fern protonema (29) and Arabidopsis trichome branches (30), suggesting that MT rings play a general role in defining the Plasmid Construction and Generation of Transgenic Plants. The MT/nucleus marker includes EC1p::Clover-TUA6, consisting of the EC1 promoter (43), GFP- zone of apical growth. Because diverse species, including flow- derived Clover, and TUA6 (AT4G14960). The F-actin/nucleus marker contains ering plants and brown algae, have vertically elongated zygotes EC1p::Lifeact-Venus, which was described previously (20). Both markers were (31–33), similar polarization might be a general strategy of plant combined with histone 2B (H2B) reporters ABI4p::H2B-tdTomato and DD22p:: zygotes in contrast to most animal zygotes that undergo cell di- H2B-mCherry. vision in the absence of cell growth (34). We also observed an apical cap and longitudinal bundles of Zygote Imaging and Inhibitor Treatment. The in vitro ovule culture followed a F-actin in the zygote (Fig. 3F), which are general features of tip- previous report with some modifications (3). growing cells in various plant species (25, 35, 36). Because fern An AxioImager A2 (Zeiss) was used for wide-field epifluorescence mi- protonema elongates in tip-growing manner (29) and Arabidopsis croscopy. 2PEM live-cell imaging was performed using an A1R MP (Nikon), and confocal imaging was done with a CV1000 (Yokogawa Electric), except trichome outgrows as polarized diffuse growth (30), it is in- for the comparison of 2PEM and confocal images in Fig. S2. triguing to determine which manner is used in the zygote elon- For inhibitor treatment, 0.1% DMSO and individual inhibitors [0.1% DMSO gation. Because tip growth is an ancient mechanism observed in containing 1 μM oryzalin (Sigma) or 1 μM LatB (Sigma)] were added to the the gametophytes of bryophytes (37) and budding yeasts (38), it media ∼1 h before observation. might be natural for the zygote, the origin of sporophytic cells, to use this machinery for the first polarization step to establish the Quantitative Analysis of Cytoskeletal Patterns. Image processing and measure- body axis. This idea is supported by the fact that the tip growth ments of metrics, including cell area, Δθ, parallelness, density, and skewness of machinery is conserved between gametophytes in bryophytes and the intensity distribution, were performed with the ImageJ software as pre- sporophytes in flowering plants (39) and the analogy between the viously reported (44, 45) (SI Materials and Methods). Arabidopsis budding of yeast and the bulge outgrowth of the ACKNOWLEDGMENTS. We thank Yoko Mizuta for helpful discussion and zygote. However, diffuse growth is a common form of cell Hanae Tsuchiya for technical support. This work was supported by the Institute expansion in land plants (40). In Arabidopsis roots, pericycle of Transformative Bio-Molecules of Nagoya University and the Japan Advanced cells directionally elongate and then divide asymmetrically, Plant Science Network. This work was also supported by Japan Society for the Promotion of Science (JSPS) on Grant-in-Aid for Young Scientists A JP25711017 giving rise to lateral root primordia (41), and epidermal cell (to T. Higaki); Grants-in-Aid for Challenging Exploratory Research JP15K14542 (to elongation is accompanied with the establishment of new po- Y.S.) and JP16K14753 (to M.U.); Grants-in-Aid for Scientific Research on Innova- larity to generate root hair (42). These facts imply some re- tive Areas JP24114007 (to S.H.), JP16H06465 (to T. Higashiyama), JP16H06464 lation between the polarized diffuse growth and the acquisition (to T. Higashiyama), JP16K21727 (to T. Higashiyama), JP24113514 (to M.U.), JP26113710 (to M.U.), JP15H05962 (to M.U.), and JP15H05955 (to M.U.); Grant- of novel cell fate, which would be also important for the zy- in-Aid for Scientific Research B JP16H04802 (to S.H.); Grants-in-Aid for Young gote to initiate embryogenesis. Additional experiments would Scientists B JP24770045 (to M.U.) and JP26840093 (to M.U.); and Japan Sci- identify the growth manner of zygote by determining whether ence and Technology Agency, Exploratory Research for Advanced Technol- the entire cell surface evenly expands (i.e., diffuse growth) or ogy Grant JP25-J-J4216 (to M.U.). T.K. and F.B. were supported by the Gregor Mendel Institute and the European Research Area Network for Coordinating new cell wall materials are preferentially deposited at the apical Action in Plant Sciences (ERA-CAPS) Grant 2163 B16 provided by the Austrian apex (i.e., tip growth). Science Fund (FWF).

1. Juergens G, Mayer U (1994) Arabidopsis. Embryos: Colour Atlas of Development,ed 17. Nicotra A, Schatten G (1990) Propranolol, a beta-adrenergic receptor blocker, affects

Bard J (Wolfe, London), pp 7–21. microfilament organization, but not microtubules, during the first division in sea PLANT BIOLOGY 2. Mansfield SG, Briarty LG (1991) Early embryogenesis in Arabidopsis thaliana. II. The urchin eggs. Cell Motil Cytoskeleton 16(3):182–189. developing embryo. Can J Bot 69:461–476. 18. Wodarz A (2002) Establishing cell polarity in development. Nat Cell Biol 4(2):E39–E44. 3. Gooh K, et al. (2015) Live-cell imaging and optical manipulation of Arabidopsis early 19. Kawashima T, Berger F (2015) The central cell nuclear position at the micropylar end is embryogenesis. Dev Cell 34(2):242–251. maintained by the balance of F-actin dynamics, but dispensable for karyogamy in 4. Ueda M, Zhang Z, Laux T (2011) Transcriptional activation of Arabidopsis axis patterning Arabidopsis. Plant Reprod 28(2):103–110. genes WOX8/9 links zygote polarity to embryo development. Dev Cell 20(2):264–270. 20. Kawashima T, et al. (2014) Dynamic F-actin movement is essential for fertilization in 5. Jürgens G (2001) Apical-basal pattern formation in Arabidopsis embryogenesis. EMBO Arabidopsis thaliana. eLife 3:e04501. J 20(14):3609–3616. 21. Webb MC, Gunning BE (1991) The microtubular cytoskeleton during development of 6. Wendrich JR, Weijers D (2013) The Arabidopsis embryo as a miniature morphogenesis the zygote, proembryo and free-nuclear endosperm in Arabidopsis thaliana (L.) model. New Phytol 199(1):14–25. Heynh. Planta 184(2):187–195. 7. Gerhart J, et al. (1989) Cortical rotation of the Xenopus egg: Consequences for the ante- 22. Ashley T (1972) Zygote shrinkage and subsequent development in some Hibiscus roposterior pattern of embryonic dorsal development. Development 107(Suppl):37–51. hybrids. Planta 108(4):303–317. 8. Goldstein B, Hird SN (1996) Specification of the anteroposterior axis in Caenorhabditis 23. Jensen WA (1968) Cotton embryogenesis: The zygote. Planta 79(4):346–366. elegans. Development 122(5):1467–1474. 24. Fu Y (2015) The cytoskeleton in the pollen tube. Curr Opin Plant Biol 28:111–119. 9. Luetjens CM, Dorresteijn AW (1998) The site of determines dorsoventral 25. Braun M, Baluska F, von Witsch M, Menzel D (1999) Redistribution of actin, profilin polarity but not chirality in the zebra mussel embryo. Zygote 6(2):125–135. and phosphatidylinositol-4, 5-bisphosphate in growing and maturing root hairs. 10. Piotrowska K, Zernicka-Goetz M (2001) Role for sperm in spatial patterning of the Planta 209(4):435–443. early mouse embryo. Nature 409(6819):517–521. 26. Faure JE, Rotman N, Fortuné P, Dumas C (2002) Fertilization in Arabidopsis thaliana 11. Hamamura Y, et al. (2011) Live-cell imaging reveals the dynamics of two sperm cells wild type: Developmental stages and time course. Plant J 30(4):481–488. during double fertilization in Arabidopsis thaliana. Curr Biol 21(6):497–502. 27. Kuroiwa K, Nishimura Y, Higashiyama T, Kuroiwa T (2002) Pelargonium embryo- 12. Mansfield SG, Briarty LG, Erni S (1991) Early embryogenesis in Arabidopsis thaliana.I. genesis: Cytological investigations of organelles in early embryogenesis from the egg The mature embryo sac. Can J Bot 69:447–460. to the two-celled embryo. Sex Plant Reprod 15(1):1–12. 13. Nakajima K, Uchiumi T, Okamoto T (2010) Positional relationship between the gamete 28. Steinborn K, et al. (2002) The Arabidopsis PILZ group genes encode tubulin-folding fusion site and the first division plane in the rice zygote. JExpBot61(11):3101–3105. cofactor orthologs required for cell division but not cell growth. Genes Dev 16(8): 14. Sato A, Toyooka K, Okamoto T (2010) Asymmetric cell division of rice zygotes located 959–971. in embryo sac and produced by in vitro fertilization. Sex Plant Reprod 23(3):211–217. 29. Murata T, Kadota A, Hogetsu T, Wada M (1987) Circular arrangement of cortical mi- 15. Olson AR, Cass DD (1981) Changes in megagametophyte structure in Papaver nudicaule L. crotubules around the subapical part of a tip-growing fern protonema. Protoplasma (Papaveraceae) following in vitro placental pollination. Amer J Bot 68(10):1333–1341. 141(2):135–138. 16. Hiiragi T, Solter D (2004) First plane of the mouse egg is not predetermined but 30. Tian J, et al. (2015) Orchestration of microtubules and the actin cytoskeleton in tri- defined by the topology of the two apposing pronuclei. Nature 430(6997):360–364. chome cell shape determination by a plant-unique kinesin. eLife 4:e09351.

Kimata et al. PNAS | December 6, 2016 | vol. 113 | no. 49 | 14161 Downloaded by guest on September 28, 2021 31. Goodner B, Quatrano RS (1993) Fucus embryogenesis: A model to study the estab- 42. Balcerowicz D, Schoenaers S, Vissenberg K (2015) Cell fate determination and the lishment of polarity. Plant Cell 5(10):1471–1481. switch from diffuse growth to planar polarity in Arabidopsis root epidermal cells. 32. He YC, He YQ, Qu LH, Sun MX, Yang HY (2007) Tobacco zygotic embryogenesis Front Plant Sci 6:1163. in vitro: The original cell wall of the zygote is essential for maintenance of cell po- 43. Sprunck S, et al. (2012) Egg cell-secreted EC1 triggers sperm cell activation during – larity, the apical-basal axis and typical suspensor formation. Plant J 49(3):515 527. double fertilization. Science 338(6110):1093–1097. 33. Pritchard HN (1964) A cytochemical study of embryo development in Stellaria media. 44. Higaki T, Kutsuna N, Sano T, Kondo N, Hasezawa S (2010) Quantification and cluster – Amer J Bot 51(5):472 479. analysis of actin cytoskeletal structures in plant cells: Role of actin bundling in sto- 34. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of em- matal movement during diurnal cycles in Arabidopsis guard cells. Plant J 61(1): bryonic development of the zebrafish. Dev Dyn 203(3):253–310. 156–165. 35. Ketelaar T, et al. (2002) Positioning of nuclei in Arabidopsis root hairs: An actin- 45. Ueda H, et al. (2010) Myosin-dependent endoplasmic reticulum motility and F-actin regulated process of tip growth. Plant Cell 14(11):2941–2955. organization in plant cells. Proc Natl Acad Sci USA 107(15):6894–6899. 36. Vidali L, Rounds CM, Hepler PK, Bezanilla M (2009) Lifeact-mEGFP reveals a dynamic 46. Wang D, et al. (2010) Identification of transcription-factor genes expressed in the apical F-actin network in tip growing plant cells. PLoS One 4(5):e5744. Arabidopsis female gametophyte. BMC Plant Biol 10:110. 37. Rounds CM, Bezanilla M (2013) Growth mechanisms in tip-growing plant cells. Annu 47. Steffen JG, Kang IH, Macfarlane J, Drews GN (2007) Identification of genes expressed Rev Plant Biol 64:243–265. – 38. Martin SG, Arkowitz RA (2014) Cell polarization in budding and fission yeasts. FEMS in the Arabidopsis female gametophyte. Plant J 51(2):281 292. Microbiol Rev 38(2):228–253. 48. Völz R, Heydlauff J, Ripper D, von Lyncker L, Groß-Hardt R (2013) Ethylene signaling is 39. Menand B, et al. (2007) An ancient mechanism controls the development of cells with required for synergid degeneration and the establishment of a pollen tube block. Dev a rooting function in land plants. Science 316(5830):1477–1480. Cell 25(3):310–316. 40. Kropf DL, Bisgrove SR, Hable WE (1998) Cytoskeletal control of polar growth in plant 49. Kroj T, Savino G, Valon C, Giraudat J, Parcy F (2003) Regulation of storage protein cells. Curr Opin Cell Biol 10(1):117–122. gene expression in Arabidopsis. Development 130(24):6065–6073. 41. Dubrovsky JG, Doerner PW, Colón-Carmona A, Rost TL (2000) Pericycle cell pro- 50. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-medi- liferation and lateral root initiation in Arabidopsis. Plant Physiol 124(4):1648–1657. ated transformation of Arabidopsis thaliana. Plant J 16(6):735–743.

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