SC I ENCE ADVANCES | R E S EARCH ART I C L E

PLANT SC I ENCES

1Precursory Research for Embryonic Science and Technology (PRESTO) Kasahara Saki-gake Project, Japan Science and Technology Agency (JST), Furo, Chikusa, Nagoya, Aichi464-0814, Japan. 2Institute of Transformative Bio-Molecules, Nagoya University, Furo,Chikusa, Nagoya, Aichi 464-0814, Japan. 3Exploratory Research for Advanced Technol-ogy (ERATO) Higashiyama Live-Holonics Project, JST, Furo, Chikusa, Nagoya, Aichi 464-0814, Japan. 4Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya,Aichi 464-0814, Japan. 5PRESTO, JST, Furo, Chikusa, Nagoya, Aichi 464-0814, Japan.6College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho,Kasugai, Aichi 487-8501, Japan. 7Kihara Institute for Biological Research, YokohamaCityUniversity, 641-12 Maioka-cho, Totsuka Ward, Yokohama 244-0813, Japan.*Corresponding author. Email: kasahara@bio.nagoya-u.ac.jp†These authors contributed equally to this work.

Kasahara et al. Sci. Adv. 2016;2 : e1600554 28 October 2016

2016 © The Authors,

some rights reserved;

exclusive licensee

American Association

for the Advancement

of Science. Distributed

under a Creative

Commons Attribution

NonCommercial

License 4.0 (CC BY-NC).

Pollen tube contents initiate ovule enlargement andenhance seed coat development without fertilizationRyushiro D. Kasahara,1,2*† Michitaka Notaguchi,3,4,5† Shiori Nagahara,2,4 Takamasa Suzuki,3,4,6

Daichi Susaki,4,7 Yujiro Honma,1,2 Daisuke Maruyama,2,4,7 Tetsuya Higashiyama2,3,4

In angiosperms, pollen tubes carry two sperm cells toward the egg and central cells to complete double fertilization.In animals, not only sperm but also seminal plasma is required for proper fertilization. However, little is knownregarding the function of pollen tube content (PTC), which is analogous to seminal plasma. We report that thePTC plays a vital role in the prefertilization state and causes an enlargement of ovules without fertilization. Wetermed this phenomenon as pollen tube–dependent ovule enlargement morphology and placed it between pollentube guidance and double fertilization. Additionally, PTC increases endosperm nuclei without fertilization whencombined with autonomous endospermmutants. This finding could be applied in agriculture, particularly in enhancingseed formation without fertilization in important crops.

http://advancesD

ownloaded from

INTRODUCTIONIn animals, numerous sperm cells swim toward the egg, and completefertilization is supported by seminal plasma (1, 2). For example, the SVS2(seminal vesicle secretion 2) protein localized only in the seminal plasmaprotects sperms from uterine attack and is required for sperm capacitationin mice (3). In higher plants, the pollen tube content (PTC) also carriestwo sperm cells toward the embryo sac, where fertilization takes place(4). However, except for being a sperm cell carrier, nothing is knownregarding PTC.Here, we identified the PTC functions that are significantfor seed development initiation, including endosperm proliferation with-out fertilization.

on May 9, 2020

.sciencemag.org/

RESULTSAs shown in animal reproduction (3), we first considered the function ofthe PTC, analogous to the seminal plasma. To investigate how PTCaffects male-female interaction in plants, we analyzed gene expressionprofiles in ovules (seed primordia) in bulk.We performed transcriptomeanalysis on wild-type (WT) ovules without pollination or crossed withWT or gcs1/gcs1 pollen, which are defective in fertilization of sperm cells,resulting in a PTC donor without fertilization (fig. S1 and table S1) (5, 6).As Nagahara et al. (6) reported previously, gcs1/gcs1 sperm cells rarelyfertilize egg or central cells; however, the ratio for this has been unknown.Therefore, we confirmed the fertilization ratio by crossingWTovules andgcs1/gcs1 pollen. As a result, 91.1 ± 3.2% (mean ± SD; n = 11 pistils) ofgcs1/gcs1 spermcells failed fertilization, 4.0 ± 3.2% single-fertilized the eggcell, 4.2 ± 3.7% single-fertilized the central cell, and 0.7 ± 1.5% fertilizedboth the egg and central cells. Thus, using gcs1/gcs1, we could block fer-tilization genetically and show the effect triggered by PTConly. First, weconfirmed that the expression of embryo and endosperm markers,

WOX9 (7) andAGL62 (8), was observed inWT pollination at 12 hoursafter pollination (HAP), soon after fertilization, but not in gcs1/gcs1 pol-lination because there was no fertilization. Second, using genome-wideanalysis, at 12 HAP, we identified 24 up-regulated genes from each dataset of the ovules crossed withWT or gcs1/gcs1 pollen, as compared withthe data set of the ovules without pollination. Although most ovuleswere fertilized at this time in WT pollination, but not in gcs1/gcs1pollination, 21 of the 24 identified genes overlapped in these samplefractions (Fig. 1A and table S2), suggesting that only PTC release, butnot fertilization, accounted for the gene up-regulation. At 24 HAP orlater, which is the stage accompanied by embryogenesis, these expres-sion profiles showed increasing differences (Fig. 1A and table S2). Thetranscriptional similarity of early events at 12 and 24HAP after crossingwith WT and gcs1/gcs1 pollen was also indicated by clustering analysis(Fig. 1B). Furthermore, some of the genes showed similar expressionpatterns of up-regulation at early times after PTC release in ovules polli-natedwithWTor gcs1/gcs1 pollen (Fig. 1, C andD, and tables S3 and S4).To avoid the possibility of contamination through unexpected fertiliza-tion by gcs1/gcs1 pollen, we performed another transcriptome analysisusing ovules that retained two sperm cells from gcs1/gcs1 pollen, indicat-ing that these ovules failed to fertilize (5, 6). As shown in table S5, thesecond transcriptome profile shared genes with the first profile, sug-gesting that the first and second transcriptome profiles were not due tofertilization events. Many of these were identified as the genes that areexpressed in typical spatial manners in fertilized ovules (table S6). Allthese data suggest that the PTC release itself triggers typical gene ex-pression soon after pollination. Notably, multiple genes associated withcell expansion, cell division, or seed coat development were significantlyexpressed even by gcs1/gcs1 pollination at 24 and 48 HAP (Fig. 1E andtable S7). Hence, we hypothesized that PTC could affect ovule shapewithout fertilization through gene induction.

To investigate this, we first observed ovule size by crossingWTovulesand hap2-1/+ (9) (allelic to gcs1) pollen. We observed three classes ofovules of different sizes in the sameWT silique 3 days after pollination(DAP) (Fig. 2, A andB). The largest ovules (66.5 ± 6.4%,mean± SD; n=10 pistils) corresponded to fertilized ovules that received pollen tube(s);the smallest (5.1 ± 1.8%)were virgin ovules that were not inserted witha pollen tube. The remaining unfertilized ovules, previously shown tohave the hap2-1 pollen tube insertion(s) (28.4 ± 5.2%) (10), weresmaller than fertilized ovules but obviously larger than virgin ones.To investigate whether ovule enlargement resulted from cell expansion,

1 of 7

SC I ENCE ADVANCES | R E S EARCH ART I C L E

Kasahara et al. Sci. Adv. 2016;2 : e1600554 28 October 2016

on May 9, 2020

http://advances.sciencemag.org/

Dow

nloaded from

we observed ovule integument cells using a plasma membrane markerline (Fig. 2, C to E, and fig. S2) (11). Cells were expanded when an ovuleaccepted aWT pollen tube (Fig. 2C), but not in a virgin ovule (Fig. 2D).However, when ovules accepted a gcs1/gcs1 pollen tube, the cells werestill expanded (Fig. 2E), indicating that the ovule enlargement resultedfrom cell expansion. To determine whether cell division in ovule integ-ument contributes to the enlargement, we counted the divided cells aftercrossingM-phasemarker pistils (12) andWTor gcs1/gcs1 pollen (Fig. 2,F to I, and fig. S3). The ovule cell division ratio was high with WT pol-lination (Fig. 2F) but low without pollination (Fig. 2G). However, thecell division ratio with gcs1/gcs1 pollinationwas higher (Fig. 2H) than invirgin ovules (Fig. 2G), indicating that ovules underwent cell divisionwithout fertilization. As reported previously (13), ovules at 0 DAPhadmitotic activity (Fig. 2I). However, at 1 to 3DAP, the division ratesof nonpollinated ovule cells decreased steadily. Ovules with WT pol-lination showed mitotic activity until 3 DAP to produce seeds. Ovuleswith gcs1/gcs1 pollination showed mitotic activity until 1 DAP, sug-gesting that ovules continued cell division as though they had under-gone fertilization.

Because cell expansion and division are initiated without fertilization,we speculated that PTC could initiate seed coat development (13). Toinvestigate this, we stained ovules with vanillin to detect proanthocya-nidin, a seed coat marker (14). The largest ovule was stained, but thesmallest virgin ovule was not (Fig. 2, J and K). Intermediate ovules withgcs1/gcs1 pollination were partly stained without fertilization (Fig. 2Land fig. S4). These three phenotypes—cell expansion, cell division,and seed coat formation without fertilization—are completely reflectedin our transcriptome data, indicating thatwe have identified a newplantphenomenon from transcriptome analysis. We call this phenomenonpollen tube–dependent ovule enlargementmorphology (POEM) becauseenlargement is observed only when an ovule has accepted one or twopollen tubes.

To confirm that PTC is the trigger of POEM, we first compared theratio of PTC release with that of POEM. We analyzed lre/lre mutants(15–17), which are defective in synergid cells, by blocking pollen tubebursts. The lre/lremutants sometimes burst PTCs, but the ratio for thiswas not reported (16, 17). To understand the PTC release ratio, wecrossed PTC marker pollen (18) with lre/lre pistils; 49.5 ± 10.8% (n =12 pistils) of pollen tubes did not release PTCs, whereas 50.5 ± 10.8%of pollen tubes did (Fig. 3, A and B). To assess the POEM ratio, wecrossed WT or gcs1/gcs1 pollen with lre/lre mutant pistils; 53.1 ± 11.8%(n = 13 pistils) of ovules showed POEM, but 46.9 ± 11.8% of ovules didnot (Fig. 3, C to E), suggesting that the ratios of POEM and PTC releaseare synchronized. Another possible POEMtrigger is synergid cell contentbecause this includes important intercellular substances, such as LUREpeptides (19) or calcium ions (20). We previously showed that a pollentube entering the embryo sac ruptures one of the two synergid cells (10). Ifsynergid cell content triggers POEM, then synergid cell breakdownshould facilitate ovule enlargement. To break synergid cells using pollentubes, we crossedWT or gcs1/gcs1 pollen and lre/lre pistils with FGR8.0(a synergid cell nucleus marker) (21) background. When we crossedgcs1/gcs1 pollen in 97.1 ± 2.6% (n = 10 pistils) of ovules, at least onesynergid cell was lost during pollen tube attack (Fig. 3, F to H), sug-gesting that almost all ovules have synergid cell content inside the embryosac. These results strongly suggest that the POEMratio corresponds to thePTC release ratio but not to the synergid cell disruption ratio (Fig. 3I).Thus, we concluded that the PTC is the trigger for POEM(Fig. 3, J andK).

After confirming that PTC induces POEM, we further investigatedthe PTC functions inside ovules, including egg and central cells. We

Fig. 1. Transcriptome analysis for ovules with or without fertilization. (A) Up-regulated genes in ovules crossed with WT (WT) and gcs1/gcs1 (gcs1) pollen com-pared with those in ovules without pollination (NPT) at 12, 24, and 48 HAP. Numberof up-regulated genes for each sample fraction and that of common up-regulatedgenes in WT (red circle; WT > NPT) and gcs1 (green circle; gcs1 > NPT) are indicated.Numbers of overlapping up-regulated genes are indicated in orange. (B) Cluster anal-ysis of the tested sample fractions. Bar indicates the height of branches. gcs1_12HAPand WT_12HAP are highlighted in pink, and gcs1_24HAP and WT_24HAP are high-lighted in blue, showing the similarity between clusters. (C) Early-response genestriggered by PTC release were screened in the two classes indicated and pooledfor further expression analyses in (D) (see the Supplementary Materials for details).(D) Temporal expression of the identified genes in NPT, WT, and gcs1. Expressionpeaks were at 12 HAP in WT and gcs1. (E) Temporal expression of the genes for cellexpansion (expansin), cell division (cyclin), and seed coat development in NPT, WT,and gcs1.

2 of 7

SC I ENCE ADVANCES | R E S EARCH ART I C L E

http://advances.sciencD

ownloaded from

Fig. 2. Discovery of POEM. (A and B) POEM after crossing WT pistils (♀) with hap2-1/+ pollen (♂). (a) Largest fertilized ovule with a pollen tube (pt). (b) Smallest ovulewithout a pollen tube. (c) Intermediate ovule with a pollen tube. (C to E) Ovule integument cells observed at 2 DAP using an RPS5Apro::tdTomato-LTI6b marker line. (C)Largest cells (WT) after crossing +/+ pollen, indicating that all the ovules were fertilized. (D) Small cells without pollen tube (Pt–). (E) Intermediate cells with gcs1/gcs1pollen tube(s) (gcs1), indicating that almost all the ovules were unfertilized. (F to H) Yellow fluorescent protein (YFP) spots using a CycB1;2pro:CycB1;2::NLS-YFP celldivision marker line after crossing with WT (F), no pollen (G), and gcs1/gcs1 (H). (I) Number of spots (±SD) observed: 0 DAP, 11.2 ± 1.7 (all); 1 DAP: 25.9 ± 2.9 (W, wildtype), 12.5 ± 2.2 (g, gcs1), and 5.3 ± 2.3 (N, no pollen); 2 DAP: 10.1 ± 2.7 (W), 0.9 ± 0.9 (g), and 3.1 ± 1.7 (N); 3 DAP: 5.4 ± 1.7 (W), 0.1 ± 0.2 (g), and 1.1 ± 1.2 (N). (J to L)Ovules at 3 DAP, stained by vanillin in WT (J); unstained virgin ovules (K); partially stained ovules, which were fertilized with gcs1/gcs1 pollen (L). v, vanillin-stained zone.Scale bars, 100 mm (A and J to L); 60 mm (C to E); 40 mm (F to H).

on May 9, 2020

emag.org/

Fig. 3. PTC triggers POEM. (A and B) PTC release ratio at 1 DAP following crossing with lre/lre pistil and LAT52pro::GFP pollen (PTC marker): (A) 49.5 ± 10.8% (n = 12) ofovules without and (B) 50.5 ± 10.8% of ovules with PTC release. (C to E) Confocal microscopy images after aniline blue staining and the POEM ratio in each ovule at 2 DAPfollowing crossing lre/lre ovules with gcs1/gcs1 pollen. (C) No pollen tube control. (D) Ovule with pollen tube(s) but no POEM (46.9 ± 11.8%; n = 13). (E) Ovule with pollen tube(s)and POEM (53.1 ± 11.8%; n = 13). (F to H) Survival ratio of synergid cell(s) at 2 DAP following crossing with lre/lre pistil with FGR8.0 background and gcs1/gcs1 pollen. (F) Bothsynergid cells (84.6%) broken. (G) One synergid cell survived (12.5%). (H) Both synergid cells survived (3.0%) in each ovule. (I) Bar graph indicating ratios with pollen tube burst(50.5%), POEM (gcs1/gcs1; 53.1%), POEM (WT; 49.1%), and synergid cell (SY) death (97.1%). (J and K) Schematic diagrams of POEM. Pollen tube entry into an ovule is notsufficient to trigger POEM (J), but PTC release triggers POEM (K). Scale bars, 50 mm (A to H).

Kasahara et al. Sci. Adv. 2016;2 : e1600554 28 October 2016 3 of 7

SC I ENCE ADVANCES | R E S EARCH ART I C L E

on May 9, 2020

http://advances.sciencemag.org/

Dow

nloaded from

observed egg and central cells after gcs1/gcs1 pollination (Fig. 4).For WT, some ovules had increased numbers of central cell nuclei, butnot of endosperm nuclei because of lack of AGL62 expression (fig. S5)without fertilization (Fig. 4, A to D). To investigate whether mea/meamutants, which develop endosperm nuclei without fertilization (22, 23),could increase autonomous endosperm proportion, we crossedmea/meaovules with gcs1/gcs1 pollen.mea/mea ovules markedly increased theendosperm amountwithout fertilization at 2 and 3DAP (Fig. 4, E toH).Inmea/mea, 19.0% of ovules at 2 DAP and 45.7% of ovules at 3 DAP

Kasahara et al. Sci. Adv. 2016;2 : e1600554 28 October 2016

increased the endosperm nuclei number (two or more nuclei) withoutfertilization (Fig. 4I). We confirmed that the vanillin staining resultscorrespond to increased numbers of endosperm nuclei at 3 DAP(Fig. 4, J and K) after crossing WT or mea/mea ovules with gcs1/gcs1pollen. After mea/mea pistils were crossed with gcs1/gcs1 pollen,not only all ovules but also the pistils were enlarged (Fig. 4L). Overall,we conclude that PTC can trigger gene induction, resulting in ovuleenlargement and seed coat development without fertilization, and canmarkedly increase endosperm nuclei number without fertilization in

Fig. 4. PTCs increase the number of central cell/autonomous endosperm nuclei. (A to D) WT (C24) virgin ovules with a central cell at 3 DAE (days after emascu-lation) (100%) (A) and 4 DAE (97.7%) (B). gcs1/gcs1-pollinated ovules with a central cell nucleus (92.2%) (C) and with two nuclei (6.8%) (D) at 2 DAP. (E to H) A central cellnucleus (96.4%) (E) and two nuclei (3.6%) (F) in a mea/mea ovule at 3 DAE. gcs1/gcs1-pollinated mea/mea ovule with two nuclei (16.4%) (G) and four nuclei (6.3%) (H) at2 DAP. (I) Ratios of the number of central cell/endosperm nuclei. Pollen tube insertion (P+) increased the nuclei ratios (≥2) for no insertion (P−) from 0 to 7.8% (WT, 2 DAP),2.3 to 12.8% (WT, 3 DAP), 3.6 to 22.6% (mea/mea, 2 DAP), and 6.0 to 51.7% (mea/mea, 3 DAP). Colors indicate number of nuclei. (J and K) Vanillin staining of gcs1/gcs1-pollinated WT (J) and mea/mea (K) pistils at 3 DAP. (J) Few ovules stained. (K) About 50% stained (right) but not without pollination (left). (L) Nonpollinated (left) andgcs1/gcs1-pollinated (right) mea/mea pistils at 3 DAP. (M) PTC induces ovule enlargement, seed coat development, and central cell/endosperm proliferation. Scale bars,50 mm (A to H); 1 mm (J to L).

4 of 7

SC I ENCE ADVANCES | R E S EARCH ART I C L E

the previously identified mutants, which are capable of autonomousendosperm formation (Fig. 4M).

http://advances.sciencD

ownloaded from

DISCUSSIONIn plants, seed formation without fertilization is called apomixisand is valuable for agriculture because the important genetic traitscan be easily fixed in apomictic crops, which then propagate withoutinterference from unfavorable environmental conditions (24). POEMcould contribute to creating apomictic crops when combined with au-tonomous endosperm mutants because PTC can produce enlargedovules and seed coat initiation without fertilization, although it cannotproduce autonomous endosperm or embryo by itself.

In 1910, Bataillon (25, 26) showed that in frogs, physical stimulicould induce segmentation without fertilization. Although not byphysical stimuli alone, PTC promotes endosperm division withoutfertilization, suggesting a function parallel to that in animals whereingerm cells may divide independently of fertilization owing to externalstimuli.

We have identified a new plant phenomenon, POEM, by transcrip-tome analysis and have revealed that POEM is a new step in plant re-production that occurs between pollen tube guidance and doublefertilization. We also note that transcriptome analysis is a powerfultool not only for identifying important genes but also for discoveringnew biological phenomena. PTC plays a crucial role in enlargingovules, initiating seed coat formation, and increasing autonomous en-dosperm without fertilization when combined with autonomous en-dosperm mutants and could therefore be applied to apomixis inmajor crops. Further studies on POEMmight not only lead to agricul-tural improvements but also reveal points common to plant and animalreproduction.

on May 9, 2020

emag.org/

MATERIALS AND METHODSPlant materials and growth conditionsArabidopsis thaliana ecotypes Columbia (Col-0) and C24 were used asWT plants. Testcross experiments were conducted in gcs1/gcs1 (5, 6),hap2-1/+ (9), lre/lre (15–17), and mea/mea (22, 23) and in WT plants.TheRPS5Apro::tdTomato-LTI6b (11) andCycB1;2pro:CycB1;2::NLS-YFP(12) lines were used for POEM assays. Seeds were sterilized with 5%sodium hypochlorite containing 0.5% Triton X-100 and germinatedon plates containing 0.5× Murashige and Skoog salts (pH 5.7) (WakoPure Chemical), 2% sucrose, Gamborg’s B5 vitamin solution (Sigma),and 0.3%Gelrite (Wako Pure Chemical) in a growth chamber at 21.5°Cunder 24 hours of light after cold treatment (4°C) for 2 days. Ten-day-oldseedlings were transferred to Metro-Mix 350 soil (Sun Gro) and grownat 21.5°C under 24 hours of light.

Phenotypic analysesFor staining of silique tissue, WT flowers were emasculated at stage 12c(27) and pollinated with hap2-1/+ gcs1/gcs1 pollen grains. Siliques werecollected at 0, 1, 2, and 3 DAP. After the samples were dissected andviewed with differential interference contrast microscopy, they wererinsed with Milli-Q (Millipore)–purified water and softened with 1 MNaOH for about 16 hours. The samples were directly stained with an-iline blue solution [0.1% (w/v) aniline blue and 0.1 M K3PO4] for morethan 3 hours. For the analysis of embryo crossed by kpl/kpl mutantpollen (28) and other fluorescence markers, including FGR8.0 (21),we followed the protocols as previously described (29). For gcs1/gcs1

Kasahara et al. Sci. Adv. 2016;2 : e1600554 28 October 2016

crossing experiment, we did not count accidentally fertilized ovules aspreviously described (6).

Detection of cell expansion and cell divisionFor both detection of cell expansion and cell division, ovules with polli-nation ofWTand gcs1/gcs1 andwithout pollinationwere analyzed at 1, 2,and 3DAP.Ovuleswere also analyzed at the experimental starting point(0 DAP). To visualize cell shapes, an RPS5Apro::tdTomato-LTI6b plantline ubiquitously expressing a plasma membrane–localized tdTomatowas imaged by a two-photon microscopy system (11). For detectionof cell division, a CycB1;2pro:CycB1;2::NLS-YFP plant line was used(12). A half part of the ovule was z-scanned, from surface to mediansection, and all the YFP spots were counted for each ovule. Each samplefraction included 15 to 41 ovules. Confocal/two-photon images wereacquired using a laser scanning inverted microscope (LSM780-DUO-NLO, Zeiss). The images were processed using the ZEN 2010 software(Zeiss) to create maximum-intensity projection images.

RNA extraction from ovulesTotal RNA for RNA sequencing (RNA-Seq) and quantitative reversetranscription polymerase chain reaction was extracted from ovules surgi-cally isolated from pistils, which were pollinated with WT and gcs1/gcs1pollen or were without pollination, using a pin and a needle. Only ~20ovules at the upper region (around the top one-third of pistil) weresampled from a pistil. Isolated ovules were pooled into a droplet of5 ml of RNAlater on the tip of the pestle. After the collection of 150 to200 ovules within 30 to 60 min, excess RNAlater was removed. Thepestle was inserted into a precooled 1.5-ml tube, and tissues were groundwith liquid nitrogen. Two hundred microliters of lysis buffer for theRNAqueous-Micro Total RNA Isolation Kit (Life Technologies) and175 ml of lysis buffer + 25 ml of Plant RNA Isolation Aid (Ambion) wereadded into the tube, and the samples were stored at −80°C until sub-sequent RNA extraction procedures. Total RNA extraction was per-formed according to the manufacturer’s instructions, where totalRNAwas eluted in 24 ml of elution solution. The RNAwas quantifiedusing theQuant-iT RiboGreen RNAAssay Kit (Life Technologies) withthe EnSpireMultimodePlate Reader (PerkinElmer) and qualified by theRNA integrity number analysis using the Agilent RNA 6000 Nano Kit(Agilent Technologies).

RNA-Seq analysisTotal RNA was treated with RNase-Free DNase I (Life Technologies),according to the manufacturer’s instructions. The TruSeq RNA SamplePreparation Kit (Illumina) was used to construct complementary DNA(cDNA) libraries, according to the manufacturer’s instructions. Thesingle ends of cDNA libraries were sequenced for 36 nucleotides fromsamples using the IlluminaGenomeAnalyzer IIx (Illumina). The result-ing sequence data were deposited in the DDBJ Sequence Read Archiveat the DNA Data Bank of Japan (www.ddbj.nig.ac.jp/) under accessionnos. DRA003909 and DRA004932. The reads were mapped to the Ar-abidopsis reference (TAIR10) using Bowtie (30) with the following op-tions, “-v 3 -m 1 –all –best –strata,” and the number of readsmapped toeach reference was counted. The cluster tree illustrated in Fig. 1B wasbased on Pearson’s correlation matrix among samples. The correlationbetween two samples was derived as follows. First, genes without ex-pression in any of the two samples were removed. Second, the logarith-mic tag count of geneswas used as input for the cor function of R (https://r-project.org/). The cluster tree was plotted by the hclust function of R.Early-response genes in the prefertilization event were collected by

5 of 7

SC I ENCE ADVANCES | R E S EARCH ART I C L E

two categorizations, as shown in Fig. 1C; the value of Zero_HAP, areference, was 0 or >0. For the latter case, the genes of which the valueof WT_12HAP divided by the value of ZeroHAP was >3 were furtherscreened. For each case, the genes that fulfilled all the following require-ments were screened; the value of WT_24HAP divided by the value ofWT_12HAPwas <0.5, the value of gcs1_24HAP divided by the value ofgcs1_12HAP was <0.5, and the value of gcs1_12HAP divided by thevalue ofWT_12HAPwas >0.5. The genes associated with cell division,cyclins, cell expansion, expansins, and seed coat development weremanually picked up and analyzed.

http://advancD

ownloaded from

SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/10/e1600554/DC1fig. S1. Steps to identify PTC-induced genes.fig. S2. Expansion of ovule integument cells by pollen tube insertion.fig. S3. Enlargement of ovules by pollen tube insertion.fig. S4. Fertilization is not required for POEM.fig. S5. Expression of the embryo- and endosperm-specific marker genes was absent in theenlarged ovules after fertilization failure.table S1. All RNA-Seq analysis data on the ovules pollinated with NPT, WT, and gcs1/gcs1.table S2. List of genes in Fig. 1A.table S3. Data for early-response genes in cases of Zero_HAP = 0.table S4. Data for early-response genes in cases of Zero_HAP > 0.table S5. Confirmation data for early-response genes using unfertilized ovules.table S6. Spatial expression patterns in the ovules for the early-response genes listed in tablesS3 and S4.table S7. Data for the genes associated with embryo and endosperm development, cellexpansion, cell division, and seed coat development as well as internal references.References (31–34)

on May 9, 2020

es.sciencemag.org/

REFERENCES AND NOTES1. T. Sasanami, S. Izumi, N. Sakurai, T. Hirata, S. Mizushima, M. Matsuzaki, G. Hiyama,

E. Yorinaga, T. Yoshimura, K. Ukena, K. Tsutsui, A unique mechanism of successfulfertilization in a domestic bird. Sci. Rep. 9, 7700 (2015).

2. T. Sasanami, K. Sugiura, T. Tokumoto, N. Yoshizaki, H. Dohra, S. Nishio, S. Mizushima,G. Hiyama, T. Matsuda, Sperm proteasome degrades egg envelope glycoprotein ZP1 duringfertilization of Japanese quail (Coturnix japonica). Reproduction 144, 423–431 (2012).

3. N. Kawano, N. Araki, K. Yoshida, T. Hibino, N. Ohnami, M. Makino, S. Kanai, H. Hasuwa,M. Yoshida, K. Miyado, A. Umezawa, Seminal vesicle protein SVS2 is required for spermsurvival in the uterus. Proc. Natl. Acad. Sci. U.S.A. 18, 4145–4150 (2014).

4. Y. Hamamura, C. Saito, C. Awai, D. Kurihara, A. Miyawaki, T. Nakagawa, M. M. Kanaoka,N. Sasaki, A. Nakano, F. Berger, T. Higashiyama, Live-cell imaging reveals the dynamics oftwo sperm cells during double fertilization in Arabidopsis thaliana. Curr. Biol. 22, 497–502(2011).

5. T. Mori, H. Kuroiwa, T. Higashiyama, T. Kuroiwa, GENERATIVE CELL SPECIFIC 1 is essentialfor angiosperm fertilization. Nat. Cell Biol. 8, 64–71 (2006).

6. S. Nagahara, H. Takeuchi, T. Higashiyama, Generation of a homozygous fertilization-defective gcs1 mutant by heat-inducible removal of a rescue gene. Plant Reprod. 28,33–46 (2015).

7. A. Haecker, R. Groß-Hardt, B. Geiges, A. Sarkar, H. Breuninger, M. Herrmann, T. Laux,Expression dynamics of WOX genes mark cell fate decisions during early embryonicpatterning in Arabidopsis thaliana. Development 131, 657–668 (2003).

8. I.-H. Kang, J. G. Steffen, M. F. Portereiko, A. Lloyd, G. N. Drews, The AGL62 MADS domainprotein regulates cellularization during endosperm development in Arabidopsis. Plant Cell20, 635–647 (2008).

9. K. von Besser, A. C. Frank, M. A. Johnson, D. Preuss, Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization. Development 133,4761–4769 (2006).

10. R. D. Kasahara, D. Maruyama, Y. Hamamura, T. Sakakibara, D. Twell, T. Higashiyama,Fertilization recovery after defective sperm cell release in Arabidopsis. Curr. Biol. 22,1084–1089 (2012).

11. Y. Mizuta, D. Kurihara, T. Higashiyama, Two-photon imaging with longer wavelengthexcitation in intact Arabidopsis tissues. Protoplasma 252, 1231–1240 (2015).

12. E. Iwata, S. Ikeda, S. Matsunaga, M. Kurata, Y. Yoshioka, M.-C. Criqui, P. Genschik, M. Ito,GIGAS CELL1, a novel negative regulator of the anaphase-promoting complex/

Kasahara et al. Sci. Adv. 2016;2 : e1600554 28 October 2016

cyclosome, is required for proper mitotic progression and cell fate determination inArabidopsis. Plant Cell 23, 4382–4393 (2011).

13. M. Ingouff, P. E. Jullien, F. Berger, The female gametophyte and the endosperm controlcell proliferation and differentiation of the seed coat in Arabidopsis. Plant Cell 18,3491–3501 (2006).

14. P. Roszak, C. Köhler, Polycomb group proteins are required to couple seed coat initiationto fertilization. Proc. Natl. Acad. Sci. U.S.A. 108, 20826–20831 (2011).

15. A. Capron, M. Gourgues, L. S. Neiva, J.-E. Faure, F. Berger, G. Pagnussat, A. Krishnan,C. Alvarez-Mejia, J.-P. Vielle-Calzada, Y.-R. Lee, B. Liu, V. Sundaresan, Maternal control ofmale-gamete delivery in Arabidopsis involves a putative GPI-anchored protein encodedby the LORELEI gene. Plant Cell 20, 3038–3049 (2008).

16. T. Tsukamoto, Y. Qin, Y. Huang, D. Dunatunga, R. Palanivelu, A role for LORELEI, a putativeglycosylphosphatidylinositol-anchored protein, in Arabidopsis thaliana doublefertilization and early seed development. Plant J. 62, 571–588 (2010).

17. T. Tsukamoto, R. Palanivelu, Loss of LORELEI function in the pistil delays initiation butdoes not affect embryo development in Arabidopsis thaliana. Plant Signal. Behav. 5,1487–1490 (2010).

18. D. Twell, J. Yamaguchi, R. A. Wing, J. Ushiba, S. McCormick, Promoter analysis ofgenes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes Dev. 5, 496–507(1991).

19. S. Okuda, H. Tsutsui, K. Shiina, S. Sprunck, H. Takeuchi, R. Yui, R. D. Kasahara, Y. Hamamura,A. Mizukami, D. Susaki, N. Kawano, T. Sakakibara, S. Namiki, K. Itoh, K. Otsuka, M. Matsuzaki,H. Nozaki, T. Kuroiwa, A. Nakano, M. M. Kanaoka, T. Dresselhaus, N. Sasaki, T. Higashiyama,Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells.Nature 458, 357–361 (2009).

20. Y. Hamamura, M. Nishimaki, H. Takeuchi, A. Geitmann, D. Kurihara, T. Higashiyama, Liveimaging of calcium spikes during double fertilization in Arabidopsis. Nat. Commun. 5,4722 (2013).

21. R. Völz, J. Heydlauff, D. Ripper, L. von Lyncker, R. Groß-Hardt, Ethylene signaling isrequired for synergid degeneration and the establishment of a pollen tube block. Dev.Cell 25, 310–316 (2013).

22. U. Grossniklaus, J.-P. Vielle-Calzada, M. A. Hoeppner, W. B. Gagliano, Maternal control ofembryogenesis by MEDEA, a polycomb group gene in Arabidopsis. Science 280, 446–450(1998).

23. M. Luo, P. Bilodeau, A. Koltunow, E. S. Dennis, W. J. Peacock, A. M. Chaudhury, Genescontrolling fertilization-independent seed development in Arabidopsis thaliana. Proc.Natl. Acad. Sci. U.S.A. 96, 296–301 (1999).

24. A. M. Koltunow, U. Grossniklaus, APOMIXIS: A developmental perspective. Annu. Rev.Plant Biol. 54, 547–574 (2003).

25. E. Bataillon, L’embryogénèse complète provoquèe chez les Amphibiens par figurede l’œuf vierge, larves parthénogénétiques de Rana fusca. C.R. Acad. Sci. Paris 150(1910).

26. E. Bataillon, La parthénogénèse experimentale chez Bufo vulgaria. C.R. Acad. Sci. Paris 152(1911).

27. C. A. Christensen, E. J. King, J. R. Jordan, G. N. Drews, Megagametogenesis in Arabidopsiswild type and the Gf mutant. Sex. Plant Reprod. 10, 49–64 (1997).

28. M. Ron, M. Alandete Saez, L. Eshed Williams, J. C. Fletcher, S. McCormick, Properregulation of a sperm-specific cis-nat-siRNA is essential for double fertilization inArabidopsis. Genes Dev. 15, 1010–1021 (2010).

29. D. Maruyama, Y. Hamamura, H. Takeuchi, D. Susaki, M. Nishimaki, D. Kurihara,R. D. Kasahara, T. Higashiyama, Independent control by each female gamete prevents theattraction of multiple pollen tubes. Dev. Cell 13, 317–323 (2013).

30. B. Langmead, C. Trapnell, M. Pop, S. L. Salzberg, Ultrafast and memory-efficient alignmentof short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

31. M. F. Belmonte, R. C. Kirkbride, S. L. Stone, J. M. Pelletier, A. Q. Bui, E. C. Yeung,M. Hashimoto, J. Fei, C. M. Harada, M. D. Munoz, B. H. Le, G. N. Drews, S. M. Brady,R. B. Goldberg, J. J. Harada, Comprehensive developmental profiles of gene activity inregions and subregions of the Arabidopsis seed. Proc. Natl. Acad. Sci. U.S.A. 110,E435–E444 (2013).

32. D. J. Cosgrove, Loosening of plant cell walls by expansins. Nature 407, 321–326 (2000).33. A. Hemerly, C. Bergounioux, M. Van Montagu, D. Inzé, P. Ferreira, Genes regulating the

plant cell cycle: Isolation of a mitotic-like cyclin from Arabidopsis thaliana. Proc. Natl. Acad.Sci. U.S.A. 89, 3295–3299 (1992).

34. T. Czechowski, M. Stitt, T. Altmann, M. K. Udvardi, W.-R. Scheible, Genome–wideidentification and testing of superior reference genes for transcript normalization inArabidopsis. Plant Physiol. 139, 5–17 (2005).

Acknowledgments: We thank E. Matsumoto, N. Furuichi, and N. Ono for technical assistance.We thank D. Kurihara, M. Ito, and R. Groß-Hardt for the gifts of RPS5Apro::tdTomato-LTI6b,CycB1;2pro:CycB1;2::NLS-YFP, and FGR8.0 marker lines. We thank G. N. Drews for critical

6 of 7

SC I ENCE ADVANCES | R E S EARCH ART I C L E

discussion for this project. Funding: This work was supported by the Precursory Research forEmbryonic Science and Technology (grant 13416724 to R.D.K.; Kasahara Sakigake Project),Japan Science and Technology Agency. This work was also supported by a grant-in-aid(25840106 to R.D.K.) from the Japanese Society for the Promotion of Science (JSPS). S.N. wassupported by grant 25009285 from the JSPS Fellowship. A part of this work was supported bythe Japan Advanced Plant Science Network and JSPS KAKENHI (grant 16H06465 to T.H.) andthe Japan Science and Technology Agency (START no. 15657559 and PRESTO no. 15665754 toM.N.). Author contributions: R.D.K. discovered the POEM phenomenon. R.D.K. and S.N.discovered the enhancement of endosperm division by PTCs. R.D.K., M.N., Y.H., and D.M.performed phenotypic analysis for the POEM phenomenon. R.D.K., M.N., S.N., D.S., Y.H., andD.M. performed RNA extraction from ovules for the new-generation sequencer. M.N. andT.S. conducted the transcriptome analysis. R.D.K. and M.N. designed the experiments. R.D.K.,M.N., and T.H. wrote the paper from the input of all authors’ experimental results. Competing

Kasahara et al. Sci. Adv. 2016;2 : e1600554 28 October 2016

interests: The authors declare that they have no competing interests. Data and materialsavailability: All data needed to evaluate the conclusions in the paper are present in the paperand/or the Supplementary Materials. Additional data related to this paper may be requestedfrom the authors.

Submitted 15 March 2016Accepted 27 September 2016Published 28 October 201610.1126/sciadv.1600554

Citation: R. D. Kasahara, M. Notaguchi, S. Nagahara, T. Suzuki, D. Susaki, Y. Honma,D. Maruyama, T. Higashiyama, Pollen tube contents initiate ovule enlargement and enhanceseed coat development without fertilization. Sci. Adv. 2, e1600554 (2016).

7 of 7

on May 9, 2020

http://advances.sciencemag.org/

Dow

nloaded from

fertilizationPollen tube contents initiate ovule enlargement and enhance seed coat development without

Maruyama and Tetsuya HigashiyamaRyushiro D. Kasahara, Michitaka Notaguchi, Shiori Nagahara, Takamasa Suzuki, Daichi Susaki, Yujiro Honma, Daisuke

DOI: 10.1126/sciadv.1600554 (10), e1600554.2Sci Adv 

ARTICLE TOOLS http://advances.sciencemag.org/content/2/10/e1600554

MATERIALSSUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2016/10/24/2.10.e1600554.DC1

REFERENCES

http://advances.sciencemag.org/content/2/10/e1600554#BIBLThis article cites 32 articles, 13 of which you can access for free

PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions

Terms of ServiceUse of this article is subject to the

is a registered trademark of AAAS.Science AdvancesYork Avenue NW, Washington, DC 20005. The title (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 NewScience Advances

Copyright © 2016, The Authors

on May 9, 2020

http://advances.sciencemag.org/

Dow

nloaded from