Cytoskeleton dynamics control the first asymmetric celldivision in Arabidopsis zygoteYusuke 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 ofIntegrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan; cGregor MendelInstitute, 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 cell division of the zygote is the initial and crucialdevelopmental step in most multicellular organisms. In floweringplants, whether zygote polarity is inherited from the preexistingorganization in the egg cell or reestablished after fertilization hasremained elusive. How dynamically the intracellular organizationis generated during zygote polarization is also unknown. Here, weused a live-cell imaging system with Arabidopsis zygotes to visu-alize the dynamics of the major elements of the cytoskeleton,microtubules (MTs), and actin filaments (F-actins), during the en-tire process of zygote polarization. By combining image analysisand pharmacological experiments using specific inhibitors of thecytoskeleton, we found features related to zygote polarization.The preexisting alignment of MTs and F-actin in the egg cell is loston fertilization. Then, MTs organize into a transverse ring definingthe zygote subapical region and driving cell outgrowth in the api-cal direction. F-actin forms an apical cap and longitudinal arraysand is required to position the nucleus to the apical region of thezygote, setting the plane of the first asymmetrical division. Ourfindings show that, in flowering plants, the preexisting cytoskel-etal patterns in the egg cell are lost on fertilization and that thezygote reorients the cytoskeletons to perform directional cell elon-gation and polar nuclear migration.

Arabidopsis thaliana | zygote polarity | microtubule | actin filament |apical–basal axis

Body axis formation is one of the first developmental eventsoccurring after fertilization in multicellular eukaryotes. In

most flowering plants, the apical–basal (shoot–root) axis isformed along the longitudinal cell polarity of the egg cell and thezygote, marked by the apical position of the nucleus (1, 2) (Fig.1A). In Arabidopsis thaliana, within 24 h of fertilization, the zy-gote elongates markedly and becomes polarized with the nucleuslying close to the apical region, leading to the asymmetric zygoticdivision, which produces a small apical cell and a large basal cell(2–4) (Fig. 1A). The apical cell gives rise to the embryo lineagethat generates most of the plant body, whereas the basal cellproduces the short-lived suspensor lineage and the hypophysis,the most apically located cell, which becomes essential in theorganization of the root meristem (5, 6) (Fig. 1A).In most animal zygotes, the unfertilized oocyte has a clear cell

polarity, but the sperm entry site changes its direction to set thefirst zygote division plane in many species, such as mouse,Caenorhabditis elegans, Xenopus, and bivalve (7–10). Therefore,the initial body axis of their embryos is determined by fertiliza-tion. In flowering plants, the sperm cell enters from the apex ofthe egg cell, and thus, the apical–basal axis seems unaltered be-fore and after fertilization (2, 11, 12). Therefore, it has remainedunclear whether zygote polarity is inherited from the egg cell ornewly generated after fertilization. In vitro fertilization assays ofrice gametes showed that the position of the zygote divisionplane is determined independently to the gamete fusion site and

correlated to the position of the two adjacent synergid cells (13,14) (Fig. 1A), suggesting that the preexisting polarity of the eggcell is retained. Conversely, in Papaver rhoeas, the zygote nu-cleus is positioned at the opposite site of its initial location inthe egg cell, implying reorientation of the cell polarity afterfertilization (15). Thus, origins of zygotic polarization haveremained unknown.In many animal zygotes, cytoskeletal dynamics have already

been characterized as a crucial driving force of cell polarization(16–18). By contrast, studies of cytoskeletal behavior in flow-ering plants have mainly focused on the central cell, which alsofuses with the sperm cell to generate the embryo-surroundingnurse tissue, the endosperm (19, 20) (Fig. 1A). Although mi-crotubule (MT) pattern was observed in fixed zygotes (21), thecytoskeletal dynamics, such as how intracellular kinetics drivezygote polarization and how the zygote elongates directionally,remained unsolved. Thus, time sequence information on thezygote polarization steps has been long awaited.In this study, we used modified live-imaging markers of cyto-

skeletal dynamics at fertilization (20) combined with a newlydeveloped in vitro ovule cultivation system (3) to perform high-resolution live-cell imaging of MT and actin filament (F-actin)dynamics in the polarizing zygote of Arabidopsis. We found thatthe cytoskeletal organization of the egg cell is lost on fertilizationand that both fibers are reoriented in the zygote in relation todirectional cell elongation and apically directed nuclear migration.Our findings provide insights into the intracellular dynamics ofzygote polarization in flowering plants.

Significance

In animals and plants, the zygote divides unequally, and thedaughter cells inherit different developmental fates to form aproper embryo along the body axis. The cytological events leadingto zygote polarization have remained unknown in flowering plants.Here, we report that the two essential components of the cyto-skeleton, microtubules and actin filaments, are both disorganizedon fertilization and then, arranged to form a transverse ring leadingdirectional cell elongation and longitudinal arrays underlying polarnuclear migration, respectively. These results provide insights intothe intracellular dynamics of zygote and the specific roles of cyto-skeletons on zygote polarization in flowering plants.

Author contributions: Y.K., T. Higaki, T.K., D.K., Y.S., S.H., F.B., T. Higashiyama, and M.U.designed research; Y.K., T.Y., and M.U. performed research; Y.K., T. Higaki, and M.U.analyzed data; and Y.K., T. Higaki, T.K., D.K., Y.S., F.B., and M.U. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. Email: m-ueda@itbm.nagoya-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1613979113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1613979113 PNAS | December 6, 2016 | vol. 113 | no. 49 | 14157–14162

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

021

Fig. 1. Live-cell imaging and quantification of MT dynamics during zygote polarization. (A) Schematic diagram of the Arabidopsis zygote that develops deepin 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/nucleusmarker. The images are representative of three time-lapse images. Numbers indicate the time (hours:minutes) from the first frame. The dotted yellow linesshow the site where the egg cell and the zygote attach to the maternal tissue. Arrowheads and brackets show the nucleus and the subapical transverse MTring, 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 respectivestages 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 correlationbetween 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 youngzygotes were determined by Dunnett’s test in L, 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.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

021

ResultsMT Arrays Are Disorganized on Fertilization and Form a SubapicalTransverse Ring During Zygote Elongation. To perform live-cellimaging of the cytoskeleton in the polarizing zygote, we developeda set of markers labeling MTs and F-actin with nuclear reporters(MT/nucleus and F-actin/nucleus) (Figs. 1 and 2 and Fig. S1)that could be imaged using two-photon excitation microscopy(2PEM) (Fig. S2). We captured the dynamic behavior of MTs bytime-lapse imaging until the first zygotic cell division (Fig. 1, Fig.S3, and Movie S1). Before fertilization, the egg cell showed avertically elongated shape and a clear polar organization withthe nucleus at the apical pole (Fig. 1B). The MT arrays alignedlongitudinally (Fig. 1 B and K and Fig. S3 A, C, and D), as shownby the small average angle of the fibers against the cell longi-tudinal axis (Δθ) (Fig. 1L).After fertilization, the cell markedly shrank (Fig. 1 C and M),

as reported in several species of flowering plants (15, 22, 23).The nucleus moved to the cell center, and the MT pattern lost itsoriginal longitudinal orientation (Fig. 1 C and N and Fig. S3E) asindicated by the increased Δθ (Fig. 1L) and the reduced paral-lelness of each fiber (Fig. S3H). Within ∼2 h, transverse MTsaccumulated in the subapical region (Fig. 1D) and organized anMT ring, above which a bulge emerged. This bulge rapidly out-grew above the transverse MT ring, thus elongating in the apicaldirection (Fig. 1E and Fig. S3F). During zygote elongation, theMTs formed spiral oblique cortical arrays in the basal part of thecell (Fig. 1E and Fig. S3F), and the nucleus gradually followed tothe apical region (Fig. 1N). This MT reorganization during zy-gote elongation was quantified by increased values of parallel-ness (Fig. S3H), fiber density (Fig. S3I), and skewness of the

intensity distribution (a metric for the appearance of bundledcables) (Fig. S3J). In addition, higher Δθ values in the apicalregion than in the basal area supported the difference betweenthe subapical transverse MT ring and the basal oblique MT ar-rays (Fig. 1O and Fig. S3 K–N).After the completion of zygote elongation, the subapical trans-

verse MT ring dispersed (Fig. 1F and Fig. S3G), and thus, Δθ valuesin the apical and basal regions no longer significantly differed (Fig.1O). Before spindle formation (Fig. 1H), another transverse ring ofcortical MTs appeared surrounding the nucleus as a preprophaseband (Fig. 1G), indicating the site of the first cell division of themature zygote. Subsequently, mitosis took place, and the phrag-moplast formed between the daughter nuclei (Fig. 1I) before thecompletion of cytokinesis (Fig. 1J).In summary, fertilization triggers a loss of longitudinal MT

arrays in the egg cell, and a subapical transverse MT ring appearsspecifically during zygote elongation. The presence of a trans-verse MT ring in the immature zygote is in agreement with theprevious observation by immunofluorescence staining of thenontransgenic plants (21), supporting that our system visualizesthe native dynamics of MT.

F-Actin Is Also Rearranged from a Disorganized State and Forms anApical Cap and Longitudinal Bundle Along the Apical–Basal Axis.Next, we performed time-lapse imaging and quantification ofF-actin (Fig. 2, Fig. S4, and Movie S2). In the egg cell, we ob-served a meshwork consisting of thick parallel cables (Fig. 2 Aand H and Fig. S4A), as shown by large skewness (Fig. S4D) andparallelness (Fig. 2I) values. After fertilization, F-actin cableswere disorganized in the shrunken zygote (Fig. 2B and Fig. S4B),as quantified by lower parallelness values than in the egg cell

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-lapseobservation 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 maternaltissue. Arrowheads and brackets show the nucleus and apical cap, respectively. Maximum intensity projection images generated by serial optical sections areshown. (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 theindicated 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

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

021

(Fig. 2I). In addition, the cables became thinner, as shown byreduced skewness values (Fig. S4D). These data indicated thatthe F-actin pattern in the egg cell was disrupted on fertilization,as observed for MTs.At the apical pole in the emerging bulge and elongating tip, we

found F-actin accumulation at the apical end (Fig. 2 C and D),similar to the apical cap, a typical structure of tip-growing cells,which guides vesicle trafficking to promote cell elongation (24).Similar to a report in which the apical cap was detected only inthe growing tip (25), the cap structure disappeared after zygoteelongation was completed (Fig. 2E). In both elongating andmature zygotes, F-actin was also aligned longitudinally (Fig. 2 Dand E and Fig. S4C), as indicated by an increase of parallelness(Fig. 2I) and a reduction of Δθ (Fig. 2J). In addition, F-actincables become thick and dense, as shown by increases in skew-ness (Fig. S4D) and density (Fig. S4E). During cell division,F-actin accumulated at the phragmoplast (Fig. 2F), as MTs did.In summary, the preexisting F-actin pattern in the egg cell was

lost on fertilization. In the elongating zygote, F-actin was rear-ranged into an apical cap and a longitudinal array, both of whichare typically associated with directionally elongating cells (25).

MTs and F-Actin Regulate Directional Cell Elongation and Polar NuclearMigration, Respectively. To examine the respective roles of MTsand F-actin, we analyzed how their inhibitors affected zygote po-larization. First, we confirmed that MTs and F-actin in the zygotewere specifically disturbed when their respective inhibitors wereadded to the in vitro ovule cultivation media (Fig. S5 A–F). Thefilamentous pattern of the MT/nucleus marker disappeared after a1-h treatment with an MT polymerization inhibitor (oryzalin) (Fig.S5B), whereas the control DMSO and the actin polymerizationinhibitor [latrunculin B (LatB)] had no detectable effect (Fig. S5 Aand C). Similarly, the F-actin signal became diffuse only whenLatB was applied (Fig. S5 D–F), confirming that these inhibitorswere specific and efficient in our experimental setup.We examined the effects of these inhibitors on zygote polariza-

tion with the MT/nucleus marker using a multiposition live-cell

imaging system. The MT filament pattern could not be visualizedwith this spinning disk confocal system, but it was suitable for high-throughput measurement of cell shape and nuclear positioning (Fig.3 A–C and Movie S3). Focusing on mature zygotes, we measuredthe cell length (Fig. 3D), cell width (Fig. S5G), and nuclear position(Fig. 3E). In contrast with the control treatment (Fig. 3A and MovieS3), oryzalin treatment resulted in loss of the restriction of zygoteelongation, and thus, the zygotes became shorter and wider thancontrol zygotes (Fig. 3 B andD and Fig. S5G). Although the nucleusproperly migrated to the apical region (Fig. 3E), cell division failedin the presence of oryzalin (Fig. 3B), probably because of the in-hibition of the mitotic and cytokinetic apparatus, such as spindlefibers. Treatment with LatB caused distinct effects. It slightlyinhibited cell elongation (Fig. 3 C and D and Fig. S5G) but moreseverely blocked nuclear migration (Fig. 3 C and E). LatB did notdisturb the completion of nuclear division and cytokinesis, andthus, cell division was close to symmetrical (Fig. 3C). These resultsindicated that MTs regulate zygote cell elongation, whereas F-actinpromotes polar migration of the nucleus to the apical tip.

DiscussionOur study showed that fertilization triggers the loss of thepreexisting organization of the cytoskeleton. This disorganiza-tion occurs coincidentally with the dispersion of the vacuole atthe basal region of the egg cell (4, 26, 27). Because a largevacuole occupies much of the egg cell volume in most floweringplants (15, 22), fertilization would trigger vacuole shrinkage tolose the dominant fiber orientation in the egg cell and also,reduce the cell volume. The disruption of cytoskeletal arrayafter fertilization suggests that the polar organization of the eggcell is associated with fertilization. Then, cytoskeletal elementsare redeployed to establish a pattern required to establish theembryo axis. This idea is supported by the observation that thecentral cell consumes the F-actin array for gamete nuclei fusion(20) and by the wrky2 mutant, which generates proper egg cellpolarity but fails to repolarize the zygote, resulting in an abnormalembryo shape (4).

Fig. 3. Roles of MT and F-actin in zygote polarization. (A–C) Confocal time-lapse observations of theMT/nucleus marker in the presence of (A) control DMSO andpolymerization inhibitors for (B) MTs (1 μMoryzalin) and (C) actin (1 μM LatB). Numbers indicate the time (hours:minutes) from the first frame. The mature zygotewas 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 thecenter 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 theimages show the lengths of the apical and basal cells after the zygotic division. Note that the oryzalin-treated zygote failed to complete cell division. Maximumintensity projection images generated by serial optical sections are shown. (Scale bars: 10 μm.) (D and E) Graphs of (D) zygote cell length (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’s test. 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.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

021

Our analysis of cytoskeletal dynamics identified an essentialrole for the MT ring in defining the site of cell outgrowth thatsustains unidirectional zygote elongation (Fig. 3F). This rolelikely explains the absence of zygote elongation in the pilz mu-tant, which is deficient in MT assembly (28). Similar roles forMT rings have been observed in directionally elongating cells,such as fern protonema (29) and Arabidopsis trichome branches(30), suggesting that MT rings play a general role in defining thezone of apical growth. Because diverse species, including flow-ering plants and brown algae, have vertically elongated zygotes(31–33), similar polarization might be a general strategy of plantzygotes in contrast to most animal zygotes that undergo cell di-vision in the absence of cell growth (34).We also observed an apical cap and longitudinal bundles of

F-actin in the zygote (Fig. 3F), which are general features of tip-growing cells in various plant species (25, 35, 36). Because fernprotonema elongates in tip-growing manner (29) and Arabidopsistrichome outgrows as polarized diffuse growth (30), it is in-triguing to determine which manner is used in the zygote elon-gation. Because tip growth is an ancient mechanism observed inthe gametophytes of bryophytes (37) and budding yeasts (38), itmight be natural for the zygote, the origin of sporophytic cells, touse this machinery for the first polarization step to establish thebody axis. This idea is supported by the fact that the tip growthmachinery is conserved between gametophytes in bryophytes andsporophytes in flowering plants (39) and the analogy between thebudding of yeast and the bulge outgrowth of the Arabidopsiszygote. However, diffuse growth is a common form of cellexpansion in land plants (40). In Arabidopsis roots, pericyclecells directionally elongate and then divide asymmetrically,giving rise to lateral root primordia (41), and epidermal cellelongation is accompanied with the establishment of new po-larity to generate root hair (42). These facts imply some re-lation between the polarized diffuse growth and the acquisitionof novel cell fate, which would be also important for the zy-gote to initiate embryogenesis. Additional experiments wouldidentify the growth manner of zygote by determining whetherthe entire cell surface evenly expands (i.e., diffuse growth) ornew cell wall materials are preferentially deposited at the apicalapex (i.e., tip growth).

Materials and MethodsDetailed materials and methods are described in SI Materials and Methods.

Strains and Growth Conditions. All Arabidopsis lines were generated in theColumbia background. The plants were grown from 18 °C to 22 °C undercontinuous light or long-day conditions (16-h light/8-h dark).

Plasmid Construction and Generation of Transgenic Plants. The MT/nucleusmarker includes EC1p::Clover-TUA6, consisting of the EC1 promoter (43), GFP-derived Clover, and TUA6 (AT4G14960). The F-actin/nucleus marker containsEC1p::Lifeact-Venus, which was described previously (20). Both markers werecombined with histone 2B (H2B) reporters ABI4p::H2B-tdTomato and DD22p::H2B-mCherry.

Zygote Imaging and Inhibitor Treatment. The in vitro ovule culture followed aprevious report with some modifications (3).

An AxioImager A2 (Zeiss) was used for wide-field epifluorescence mi-croscopy. 2PEM live-cell imaging was performed using an A1R MP (Nikon),and confocal imaging was done with a CV1000 (Yokogawa Electric), exceptfor the comparison of 2PEM and confocal images in Fig. S2.

For inhibitor treatment, 0.1%DMSO and individual inhibitors [0.1%DMSOcontaining 1 μM oryzalin (Sigma) or 1 μM LatB (Sigma)] were added to themedia ∼1 h before observation.

Quantitative Analysis of Cytoskeletal Patterns. Image processing and measure-ments of metrics, including cell area, Δθ, parallelness, density, and skewness ofthe intensity distribution, were performed with the ImageJ software as pre-viously reported (44, 45) (SI Materials and Methods).

ACKNOWLEDGMENTS. We thank Yoko Mizuta for helpful discussion andHanae Tsuchiya for technical support. This work was supported by the Instituteof Transformative Bio-Molecules of Nagoya University and the Japan AdvancedPlant Science Network. This work was also supported by Japan Society for thePromotion of Science (JSPS) on Grant-in-Aid for Young Scientists A JP25711017(to T. Higaki); Grants-in-Aid for Challenging Exploratory Research JP15K14542 (toY.S.) and JP16K14753 (to M.U.); Grants-in-Aid for Scientific Research on Innova-tive Areas JP24114007 (to S.H.), JP16H06465 (to T. Higashiyama), JP16H06464(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-in-Aid for Scientific Research B JP16H04802 (to S.H.); Grants-in-Aid for YoungScientists B JP24770045 (to M.U.) and JP26840093 (to M.U.); and Japan Sci-ence and Technology Agency, Exploratory Research for Advanced Technol-ogy Grant JP25-J-J4216 (to M.U.). T.K. and F.B. were supported by the GregorMendel Institute and the European Research Area Network for CoordinatingAction in Plant Sciences (ERA-CAPS) Grant 2163 B16 provided by the AustrianScience Fund (FWF).

1. Juergens G, Mayer U (1994) Arabidopsis. Embryos: Colour Atlas of Development, edBard J (Wolfe, London), pp 7–21.

2. Mansfield SG, Briarty LG (1991) Early embryogenesis in Arabidopsis thaliana. II. Thedeveloping embryo. Can J Bot 69:461–476.

3. Gooh K, et al. (2015) Live-cell imaging and optical manipulation of Arabidopsis earlyembryogenesis. Dev Cell 34(2):242–251.

4. Ueda M, Zhang Z, Laux T (2011) Transcriptional activation of Arabidopsis axis patterninggenes WOX8/9 links zygote polarity to embryo development. Dev Cell 20(2):264–270.

5. Jürgens G (2001) Apical-basal pattern formation in Arabidopsis embryogenesis. EMBOJ 20(14):3609–3616.

6. Wendrich JR, Weijers D (2013) The Arabidopsis embryo as a miniature morphogenesismodel. New Phytol 199(1):14–25.

7. Gerhart J, et al. (1989) Cortical rotation of the Xenopus egg: Consequences for the ante-roposterior pattern of embryonic dorsal development. Development 107(Suppl):37–51.

8. Goldstein B, Hird SN (1996) Specification of the anteroposterior axis in Caenorhabditiselegans. Development 122(5):1467–1474.

9. Luetjens CM, Dorresteijn AW (1998) The site of fertilisation determines dorsoventralpolarity but not chirality in the zebra mussel embryo. Zygote 6(2):125–135.

10. Piotrowska K, Zernicka-Goetz M (2001) Role for sperm in spatial patterning of theearly mouse embryo. Nature 409(6819):517–521.

11. Hamamura Y, et al. (2011) Live-cell imaging reveals the dynamics of two sperm cellsduring double fertilization in Arabidopsis thaliana. Curr Biol 21(6):497–502.

12. Mansfield SG, Briarty LG, Erni S (1991) Early embryogenesis in Arabidopsis thaliana. I.The mature embryo sac. Can J Bot 69:447–460.

13. Nakajima K, Uchiumi T, Okamoto T (2010) Positional relationship between the gametefusion site and the first division plane in the rice zygote. J Exp Bot 61(11):3101–3105.

14. Sato A, Toyooka K, Okamoto T (2010) Asymmetric cell division of rice zygotes locatedin embryo sac and produced by in vitro fertilization. Sex Plant Reprod 23(3):211–217.

15. Olson AR, Cass DD (1981) Changes in megagametophyte structure in Papaver nudicaule L.(Papaveraceae) following in vitro placental pollination. Amer J Bot 68(10):1333–1341.

16. Hiiragi T, Solter D (2004) First cleavage plane of themouse egg is not predetermined butdefined by the topology of the two apposing pronuclei. Nature 430(6997):360–364.

17. Nicotra A, Schatten G (1990) Propranolol, a beta-adrenergic receptor blocker, affectsmicrofilament organization, but not microtubules, during the first division in seaurchin eggs. Cell Motil Cytoskeleton 16(3):182–189.

18. Wodarz A (2002) Establishing cell polarity in development. Nat Cell Biol 4(2):E39–E44.19. Kawashima T, Berger F (2015) The central cell nuclear position at the micropylar end is

maintained by the balance of F-actin dynamics, but dispensable for karyogamy inArabidopsis. Plant Reprod 28(2):103–110.

20. Kawashima T, et al. (2014) Dynamic F-actin movement is essential for fertilization inArabidopsis thaliana. eLife 3:e04501.

21. Webb MC, Gunning BE (1991) The microtubular cytoskeleton during development ofthe zygote, proembryo and free-nuclear endosperm in Arabidopsis thaliana (L.)Heynh. Planta 184(2):187–195.

22. Ashley T (1972) Zygote shrinkage and subsequent development in some Hibiscushybrids. Planta 108(4):303–317.

23. Jensen WA (1968) Cotton embryogenesis: The zygote. Planta 79(4):346–366.24. Fu Y (2015) The cytoskeleton in the pollen tube. Curr Opin Plant Biol 28:111–119.25. Braun M, Baluska F, von Witsch M, Menzel D (1999) Redistribution of actin, profilin

and phosphatidylinositol-4, 5-bisphosphate in growing and maturing root hairs.Planta 209(4):435–443.

26. Faure JE, Rotman N, Fortuné P, Dumas C (2002) Fertilization in Arabidopsis thalianawild type: Developmental stages and time course. Plant J 30(4):481–488.

27. Kuroiwa K, Nishimura Y, Higashiyama T, Kuroiwa T (2002) Pelargonium embryo-genesis: Cytological investigations of organelles in early embryogenesis from the eggto the two-celled embryo. Sex Plant Reprod 15(1):1–12.

28. Steinborn K, et al. (2002) The Arabidopsis PILZ group genes encode tubulin-foldingcofactor orthologs required for cell division but not cell growth. Genes Dev 16(8):959–971.

29. Murata T, Kadota A, Hogetsu T, Wada M (1987) Circular arrangement of cortical mi-crotubules around the subapical part of a tip-growing fern protonema. Protoplasma141(2):135–138.

30. Tian J, et al. (2015) Orchestration of microtubules and the actin cytoskeleton in tri-chome cell shape determination by a plant-unique kinesin. eLife 4:e09351.

Kimata et al. PNAS | December 6, 2016 | vol. 113 | no. 49 | 14161

PLANTBIOLO

GY

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

021

31. Goodner B, Quatrano RS (1993) Fucus embryogenesis: A model to study the estab-lishment of polarity. Plant Cell 5(10):1471–1481.

32. He YC, He YQ, Qu LH, Sun MX, Yang HY (2007) Tobacco zygotic embryogenesisin vitro: The original cell wall of the zygote is essential for maintenance of cell po-larity, the apical-basal axis and typical suspensor formation. Plant J 49(3):515–527.

33. Pritchard HN (1964) A cytochemical study of embryo development in Stellaria media.Amer J Bot 51(5):472–479.

34. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of em-bryonic development of the zebrafish. Dev Dyn 203(3):253–310.

35. Ketelaar T, et al. (2002) Positioning of nuclei in Arabidopsis root hairs: An actin-regulated process of tip growth. Plant Cell 14(11):2941–2955.

36. Vidali L, Rounds CM, Hepler PK, Bezanilla M (2009) Lifeact-mEGFP reveals a dynamicapical F-actin network in tip growing plant cells. PLoS One 4(5):e5744.

37. Rounds CM, Bezanilla M (2013) Growth mechanisms in tip-growing plant cells. AnnuRev Plant Biol 64:243–265.

38. Martin SG, Arkowitz RA (2014) Cell polarization in budding and fission yeasts. FEMSMicrobiol Rev 38(2):228–253.

39. Menand B, et al. (2007) An ancient mechanism controls the development of cells witha rooting function in land plants. Science 316(5830):1477–1480.

40. Kropf DL, Bisgrove SR, Hable WE (1998) Cytoskeletal control of polar growth in plantcells. Curr Opin Cell Biol 10(1):117–122.

41. Dubrovsky JG, Doerner PW, Colón-Carmona A, Rost TL (2000) Pericycle cell pro-liferation and lateral root initiation in Arabidopsis. Plant Physiol 124(4):1648–1657.

42. Balcerowicz D, Schoenaers S, Vissenberg K (2015) Cell fate determination and the

switch from diffuse growth to planar polarity in Arabidopsis root epidermal cells.

Front Plant Sci 6:1163.43. Sprunck S, et al. (2012) Egg cell-secreted EC1 triggers sperm cell activation during

double fertilization. Science 338(6110):1093–1097.44. Higaki T, Kutsuna N, Sano T, Kondo N, Hasezawa S (2010) Quantification and cluster

analysis of actin cytoskeletal structures in plant cells: Role of actin bundling in sto-

matal movement during diurnal cycles in Arabidopsis guard cells. Plant J 61(1):

156–165.45. Ueda H, et al. (2010) Myosin-dependent endoplasmic reticulum motility and F-actin

organization in plant cells. Proc Natl Acad Sci USA 107(15):6894–6899.46. Wang D, et al. (2010) Identification of transcription-factor genes expressed in the

Arabidopsis female gametophyte. BMC Plant Biol 10:110.47. Steffen JG, Kang IH, Macfarlane J, Drews GN (2007) Identification of genes expressed

in the Arabidopsis female gametophyte. Plant J 51(2):281–292.48. Völz R, Heydlauff J, Ripper D, von Lyncker L, Groß-Hardt R (2013) Ethylene signaling is

required for synergid degeneration and the establishment of a pollen tube block. Dev

Cell 25(3):310–316.49. Kroj T, Savino G, Valon C, Giraudat J, Parcy F (2003) Regulation of storage protein

gene expression in Arabidopsis. Development 130(24):6065–6073.50. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacterium-medi-

ated transformation of Arabidopsis thaliana. Plant J 16(6):735–743.

14162 | www.pnas.org/cgi/doi/10.1073/pnas.1613979113 Kimata et al.

Dow

nloa

ded

by g

uest

on

Sep

tem

ber

7, 2

021