Article type : Original Article

Accepted Article
Ethylene signaling is critical for synergid cell functional specification and pollen tube
attraction

Cheng Zhang#, 1, Xiao-Dong Teng#, 1, Quan-Quan Zheng1 Yan-Yun Zhao1, Jie-Yang Lu1,
Yichuan Wang2, Hongwei Guo*, 2, Zhong-Nan Yang*, 1, 3

1
College of Life and Environmental Sciences, Shanghai Normal University, 100
Guilin Road, Shanghai 200234, China
2
Institute of Plant and Food Science, Department of Biology, Southern University
of Science and Technology, Shenzhen, Guangdong 508055, China
3
CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant
Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200233, China

# These authors contributed equally to this work

* For correspondence: (znyang@shnu.edu.cn, guohw@sustc.edu.cn)

Tel: 86-21- 64324650
Fax: 86-21-64322143

RUNNING TITLE
Ethylene signal controls pollen tube attraction

KEYWORDS
Ethylene signaling, EBF1, EBF2, EIN3, Pollen tube attraction, Synergid cell, SAG29

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/tpj.14027

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 SUMMARY

ETHYLENE INSENSITIVE 3 (EIN3) is a key regulator of ethylene signaling, and EIN3-

Accepted Article
BINDING F-BOX1 (EBF1) and EBF2 are responsible for EIN3 degradation. Previous report
showed that the ebf1 ebf2 double homozygous mutant cannot be identified. In this work, the
genetic analysis revealed that the ebf1 ebf2 female gametophyte is defective. The pollination
experiment showed that ebf1 ebf2 ovules failed to attract pollen tubes. In female
gametophyte/ovule, the synergid cell is responsible for pollen tube attraction. Observation of
the pEIN3::EIN3-GFP transgenic lines showed that EIN3 signal was over-accumulated at the
micropylar end of ebf1 ebf2 female gametophyte. The overexpression of stabilized EIN3 in
synergid cell led to the defect of pollen tube guidance. These results suggest that the over-
accumulated EIN3 in ebf1 ebf2 synergid cell blocks its pollen tube attraction which leads to
the failure of ebf1 ebf2 homozygous plant. We identified that EIN3 directly activated the
expression of a sugar transporter, SENESCENCE-ASSOCIATED GENE29
(SAG29/SWEET15). Overexpression of SAG29 in synergid cells blocked pollen tube
attraction, suggesting SAG29 might play a role in ethylene signaling to repel pollen tube
entry. Taken together, our study reveals that strict control of ethylene signaling is critical for
the synergid cell function during plant reproduction.

INTRODUCTION

Ethylene is a gaseous phytohormone that plays crucial roles in various biological processes,
including fruit ripening, abscission, stress response, cell expansion inhibition and senescence
(Abeles et al., 1988; Bleecker and Kende, 2000). Ethylene signaling initiates with the
perception of ethylene by ER-located receptors, which interact with a Raf-like protein kinase,
CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), to form a negative regulator complex
(Bleecker et al., 1988; Clark et al., 1998; Hua and Meyerowitz, 1998). This complex inhibits
the essential positive regulator of ethylene signaling, ETHYLENE INSENSITIVE 2 (EIN2),
until ethylene is present. Once ethylene signaling is activated, EIN2 activates two master
transcription factors, ETHYLENE INSENSITIVE 3 (EIN3) and its homologue EIN3-LIKE 1
(EIL1), in the nucleus, and these transcription factors regulate multiple ethylene responses
(Binder et al., 2004; Ju et al., 2012; Qiao et al., 2012; Chang et al., 2013; Wen et al., 2012; Li
et al., 2015).

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 In the ethylene signaling pathway, EIN3 and EIL1 regulate the vast majority of downstream
target genes (Li et al., 2013; Kim et al., 2014; Qiu et al., 2015). The amounts of these two
transcription factors are strictly governed by two critical F-box proteins, EIN3-BINDING F-
Accepted Article
BOX1 (EBF1) and EBF2, which have been reported to degrade EIN3 and EIL1 through the
ubiquitin/26S proteasome pathway (Guo and Ecker, 2003; Potuschak et al., 2003; An et al.,
2010). The simultaneous loss of EBF1 and EBF2 results in the over-accumulation of EIN3
and EIL1 proteins, which, in turn, leads to a constitutive ethylene response (Guo and Ecker,
2003; An et al., 2010). The ebf1 and ebf2 single mutants show normal vegetative and
reproductive growth (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004, Figure
S1). However, the homozygous double mutant ebf1 ebf2 is difficult to obtain from
heterozygous parents (An et al., 2010). Interestingly, knockout of EIN3 in ebf1 ebf2 double
mutants results in a significant restoration of fertility, although the mutant still shows
numerous ethylene response phenotypes in vegetative growth (An et al., 2010). These
observations indicate that endogenous control of EIN3 protein abundance by EBF1/2
signaling is crucial for plant reproduction, but the underlying mechanism is still unknown.

Sexual reproduction in flowering plants requires the production of two gametophytes: the
pollen (male gametophyte) and embryo sac (female gametophyte). Pollen is produced by the
anthers, which are initiated from the stamen primordia (Sanders et al., 1999). Each pollen
grain contains one vegetative cell and two sperm cells. In contrast, the embryo sac is located
inside the ovule, which includes the outer integuments; the nucellus, which is controlled by
the sporophyte, and the inner embryo sac, which is controlled by the gametophyte. The
embryo sac is composed of seven cells in Arabidopsis. One egg cell and two synergid cells
are located at the micropylar end of the ovule. One central cell is in the middle of the embryo
sac, and three antipodal cells are at the chalazal end of the ovule (Drews and Yadegari, 2002;
Yang et al., 2010). Synergid cells are responsible for attracting the pollen tube, but only one
pollen tube is attracted into each ovule. The disappearance of the persistent synergid cell after
double fertilization ensures that the attraction of the second pollen tube is blocked. Ethylene
signaling activation triggered by egg cell fertilization has been demonstrated to be largely
responsible for the programmed cell death of the persistent synergid cell (Volz et al., 2013;
Maruyama et al., 2015). On the other hand, over-activation of ethylene signaling, such as that
in the ctr1 mutant, also leads to abnormal synergid degradation (Volz et al., 2013). These
observations collectively implicate ethylene signaling as an important regulator of synergid
cell lifespan and pollen tube attraction.

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 Here, we discovered that EBF1 and EBF2 are essential for synergid functioning for pollen
tube attraction. Their loss in ebf1 ebf2 synergid cells impairs pollen tube attraction, which can
be largely restored in ebf1 ebf2 ein3 triple mutants. EIN3 is over-accumulated in ebf1 ebf2
Accepted Article
synergid cells, impairing pollen tube attraction. SAG29 was identified to be one of the direct
targets of EIN3, and its overexpression was sufficient to impair pollen tube attraction. Our
results indicate that ethylene signaling mediated by EIN3 and its direct target SAG29 should
be strictly controlled by EBF1 and EBF2 to ensure normal synergid function and embryonic
development.

RESULTS
ebf1 ebf2 is defective in female gametophytic and embryonic development
Previous studies have reported that it is difficult to identify homozygous ebf1-/-ebf2-/- plants
and that the fertility of ebf1-1-/-ebf2-1+/- is reduced (Guo and Ecker, 2003). We obtained the
ebf1-1-/-ebf2-1+/- allele used in the previous study and observed the vegetative growth and
seed set of ebf1-/-ebf2+/-. The plant is slightly small and weak in vegetative growth and
displayed reduced fertility. A number of aborted seeds could be observed in its siliques
(Figure 1a). In the wild type, approximately 95.53% of seeds were normal, while only
42.38% of seeds were normal in ebf1-/-ebf2+/- siliques (Figure 1b). Seed abortion can result
from developmental defects of the male sporophyte and gametophyte, female sporophyte and
gametophyte or embryo. To understand the functions of EBF1 and EBF2 in plant
reproduction in detail, a genetic analysis was performed. In the selfed progeny of ebf1-/-
ebf2+/- plants, no ebf1-/-ebf2-/- was identified, and the segregation ratio of ebf1-/-ebf2+/+ to
ebf1-/-ebf2+/- was approximately 1:1.2 (n=462, Table 1). This result indicates that the
gametophytic or embryonic development of ebf1 ebf2 was defective. When wild-type plants
were pollinated with pollen grains from ebf1-/-ebf2+/- plants, the ratio of ebf1+/-ebf2+/+ and
ebf1+/-ebf2+/- in the progeny was 1:1 (n=92; Table 1). This result suggests that ebf1 ebf2
pollen grains (male gametophyte) are normal. When ebf1-/-ebf2+/- stigmas were pollinated
with wild-type pollen grains, the ratio of ebf1+/-ebf2+/+and ebf1+/-ebf2+/- in F1 progenies was
approximately 5:1 (n=108; Table 1), which deviated from the expected ratio of 1:1. This
result indicates that only 33.3% of ebf1 ebf2 female gametophytes could accept the male
gametophyte for double fertilization, while the majority of them (66.7%) were defective.
Although genetic analysis revealed that all ebf1 ebf2 pollen grains and about 33.3% of ebf1
ebf2 female gametophytes had transmission ability, no ebf1-/-ebf2-/- plant has been identified.
This result implies that ebf1-/-ebf2-/- embryonic development might also be defective.

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 To confirm whether these defects were caused by the knockout of EBF1 and EBF2, we
constructed PEBF1::EBF1-GFP and PEBF2::EBF2-GFP vectors and introduced them into ebf1-
/-
ebf2+/- and ebf1+/-ebf2-/- plants, respectively. We successfully obtained ebf1-/-ebf2-/- plants
Accepted Article
with the complemented PEBF1::EBF1 genomic-GFP, and these plants showed normal fertility
(Figure 1a). This result indicates that PEBF1::EBF1-GFP could complement the defective
female gametophytic and embryonic development of ebf1-/-ebf2+/-. We also obtained
PEBF2::EBF2-GFP transgenic plants with an ebf1-/-ebf2-/- background, but these plants were
arrested in the early vegetative stage. They gradually turned yellow and failed to bolt (Figure
S2). This result indicates that EBF2 is sufficient to complement the reproductive defect in the
ebf1-/-ebf2+/- plant. However, EBF2 alone is not sufficient to complement its vegetative
growth of the ebf1-/-ebf2-/- plant.

Pollen tube attraction is impaired in the ebf1 ebf2 female gametophyte

Since the reproductive defect of ebf1-/-ebf2+/- plants is of female gametophytic origin, we next
used confocal laser scanning microscopy to examine whether ovule development is normal in
ebf1-/-ebf2+/- pistils (Christensen et al., 1998). The development of the female gametophyte in
ebf1-/-ebf2+/- and wild type was indistinguishable during the coenocytic and later
differentiation stages (Figure S3a-S3b), indicating that female gametophytic development in
ebf1 ebf2 is normal.

Pollen tube attraction is one of the most important functions of the female gametophyte. To
test whether the female gametophytic defects of ebf1 ebf2 are associated with impaired pollen
tube attraction, we pollinated the stigmas of both ebf1-/-ebf2+/- and wild type with wild-type
pollen and examined the pollen tube growth. Sixteen hours after pollination, 96.57% of the
ovules of wild-type plants had attracted one pollen tube (n=423; Figure 2a, b and f). In
contrast, only 45.97% of the ovules of ebf1-/-ebf2+/- plants attracted pollen tubes (n=442;
Figure 2c, d and f), indicating that ebf1 ebf2 ovules are defective in attracting pollen tubes.

In the canonical ethylene signaling pathway, EBF1 and EBF2 are responsible for the
degradation of EIN3 (Guo and Ecker, 2003). To test whether EIN3 accumulation in the ebf1
ebf2 female gametophyte is responsible for the defective pollen tube attraction, we pollinated
the stigma of the ebf1-/-ebf2-/-ein3-/- triple mutant with wild-type pollen. Sixteen hours after
pollination, more than 70% of pollen tubes have entered the ovule of the ebf1 ebf2 ein3 triple

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 mutant at the micropylar end (n=343, Figure 2e and f). Therefore, the removal of EIN3 from
ebf1 ebf2 restored its pollen tube attraction, suggesting that EIN3 accumulation leads to the
pollen tube attraction defect in ebf1 ebf2 female gametophyte.
Accepted Article
Accumulated EIN3 at the micropylar end of ebf1 ebf2 ovule leads to synergid cell
dysfunction
Previous studies have reported that the protein level of EIN3 was elevated in ebf1 ebf2
seedlings and 2-week-old light-grown plants (Guo and Ecker, 2003; Potuschak et al., 2003;
Gagne et al., 2004). To determine EIN3 localization in the female gametophyte of ebf1 ebf2,
we made a PEIN3::EIN3-GFP construct and introduced it into wild-type and ebf1-/-ebf2+/-
plants. GFP signals in the ovules of these transgenic lines were examined before pollination.
In wild-type PEIN3::EIN3-GFP transgenic lines, no GFP fluorescent signal could be detected (Figure
3a). However, in the transgenic lines in the ebf1-/-ebf2+/- background, the GFP fluorescent
signal was observed in 17.87% of ovules (n=1080; Figure 3a). The signal was over-
accumulated at the micropylar ends of ebf1 ebf2 ovules. These results suggest that over-
accumulation of EIN3 at the micropylar end was harmful to pollen tube attraction.

The synergid cells, located at the micropylar end, are crucial for pollen tube attraction (Yang
et al., 2010; Higashiyama et al., 2002). To investigate whether synergid cell function was
abnormal in ebf1 ebf2, we introduced the synergid-specific marker PDD31::GFP (Steffen et al.,
2007) into ebf1-/-ebf2+/- plants to examine synergid cell identity, with ebf1 as a control. In the
transgenic ebf1 lines, GFP signals were detected in more than 80% of female gametophytes
(n=554; Figure 3b). However, in the ebf1-/-ebf2+/- transgenic lines, GFP signals were detected
in only 44.76% of female gametophytes (n=791; Figure 3b). The ebf1-/-ebf2+/- plant is
expected to produce female gametophytes of ebf1 ebf2 and ebf1 EBF2 at a ratio of 1:1. These
observations suggest that the identity of ebf1 ebf2 synergid cells was severely disrupted,
which may further affect their function. Therefore, the EIN3 over-accumulation at the
micropylar end of the ovule may affect synergid cell function.

Overexpression of stabilized EIN3 in synergid cells leads to defective pollen tube

attraction
To investigate whether EIN3 would affect synergid cell function or pollen tube attraction, we
ectopicly expressed EIN3 in synergid cells by DD31 promoter. Because EBF1 and EBF2 can
directly interact with EIN3, and the C-terminus of EIN3 is required for this interaction (Guo

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 and Ecker, 2003; An et al., 2010), we made EIN3 constructs with different C-terminal length.
These constructs were named PDD31::ΔEIN31~550, PDD31::ΔEIN31~450 and PDD31::EIN3,
respectively (Figure 4a). A total of 12 and 15 transgenic plants were obtained for
Accepted Article
PDD31::ΔEIN31~550 and PDD31::ΔEIN31~450, respectively. The transgenic lines of PDD31::EIN3
and PDD31::ΔEIN31~550 showed normal siliques and ovules (Figure 4d and g; Figure 4e and h;
n=3). However, among the 15 PDD31::ΔEIN31~450 transgenic lines, 12 plants showed
obviously reduced fertility. The siliques of PDD31::ΔEIN31~450 transgenic lines showed
numerous aborted ovules that failed to develop into seeds (Figure 4f). Statistical analysis
showed that, in the PDD31::ΔEIN31~450 transgenic lines, approximately 40% of seeds were
aborted (Figure 4i), and approximately 30% of ovules failed to attract pollen tubes (Figure 4b
and c). Collectively, these results demonstrate that the activation of ethylene signaling in
synergid cells is sufficient to induce defects in their pollen tube attraction.

EIN3 directly activates SAG29/SWEET15

EIN3 regulates the expression of the vast majority of ethylene-related genes in Arabidopsis
(Chao et al., 1997; Solano et al., 1998). It has also been reported that an EIN3-dependent
ethylene response is activated after double fertilization, and this response is largely
responsible for persistent synergid cell degradation and the consequent second pollen tube
block (Maruyama et al., 2015). However, the downstream gene targets of EIN3 in synergid
cells that are related to synergid degradation and the pollen tube block are still unknown.
Because synergid cell degeneration can be regarded as a process of senescence. we searched
for candidate genes in the Leaf Senescence Database (Li et al., 2012) and selected genes with
promoters containing a consensus EIN3 family binding sequence A(C/T)G(A/T)A(C/T)CT
(Kosugi and Ohashi, 2000; Yamasaki et al., 2005). We selected 19 genes as candidates for
investigation (Table S1). Based on the e-FP website, three genes, SAG29 (SENESCENCE-
ASSOCIATED GENE 29)/SWEET15, ORE4 and SENESCENCE ASSOCIATED RECEPTOR
PROTEIN KINASE (SARK), showed high expression in ovules. Quantitative PCR analysis
was performed to examine their expression in the pistils of stages FG5-FG7 of wild-type and
ebf1-/-ebf2+/- plants (Figure 5a). The expression of SAG29 in ebf1-/-ebf2+/- ovules was about
four times higher than that in the wild type, whereas the expression of ORE4 was lower, and
that of SARK did not differ between the mutant and wild type (Figure 5b). SAG29 encodes a
member of the SWEET sucrose efflux transporter protein family. A previous investigation
showed that PSAG29::GUS was highly expressed in ovules (Seo et al., 2011). To test whether
EIN3 directly regulates SAG29, an electrophoretic mobility shift assay (EMSA) was

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 performed first. The EIN3 protein (amino acids 141 to 352) was able to bind to the labelled
probe, while an unlabelled competitor probe could dose-dependently abolish the binding
effect (Figure 5c), indicating that EIN3 directly bound the SAG29/SWEET15 promoter. To
Accepted Article
further examine whether EIN3 could directly activate the expression of SAG29/SWEET15 in
vivo, a transient dual luciferase assay in Arabidopsis protoplasts was also performed (Figure
5d). Overexpression of EIN3 in protoplasts significantly increased the activity of the
SAG29/SWEET15 promoter to approximately 4-fold that of the negative control (Figure 5d),
indicating that EIN3 directly activated the expression of SAG29/SWEET15. These results
suggest that SAG29 might be involved in pollen tube attraction and embryonic development.

Expression of SAG29 driven by the DD31 promoter disrupts pollen tube attraction
To further study whether SAG29/SWEET15 is involved in pollen tube attraction, we
overexpressed SAG29 in synergid cells by using the synergid-specific promoter proDD31.
We constructed PDD31::SAG29 and transformed it into wild-type plants (Figure 6a). Eight in
11 plants showed reduced seed set (Figure 6b). The siliques of these transgenic lines
contained a number of spot-like aborted ovules that failed to develop into seeds on the
placenta (Figure 6b). In transgenic line one, there were only 51.3% normal ovules per silique
(n=486, Figure 6c). Aniline blue staining showed that 46.29% of pollen tubes failed to enter
an ovule (Figure 6d and e). The ratio of abnormal ovules was consistent with that of aborted
seed number (Figure 6e). Therefore, overexpression of SAG29 in synergid cells affects pollen
tube attraction, suggesting that SAG29 regulated by the EIN3-dependent ethylene signaling
pathway plays a role in blocking pollen tube attraction.

To examine whether the disruption of SAG29 can rescue the defective pollen tube attraction
of ebf1 ebf2 ovules, we tried to generate an ebf1 ebf2 sag29 triple mutant. Unfortunately,
neither the heterozygous plants (ebf1-/-ebf2+/-sag29-/- and ebf1+/-ebf2-/-sag29-/-) nor the
homozygous plant (ebf1-/-ebf2-/-sag29-/-) was identified among the ebf1+/-ebf2+/-sag29+/-
progeny (n=385). One probable reason for this result might be the high linkage between the
gene loci of EBF2 and SAG29 (Figure S4). Additionally, SAG29 might require the expression
of other EIN3 target genes to commonly affect pollen tube attraction.

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 SAG29 expression is elevated after pollination at the micropylar end of the ovule

Previous studies showed that SAG29 transcripts are strongly expressed in ovules, and the
Accepted Article
SAG29 protein is expressed in seed coat cells (Seo et al., 2011; Chen et al., 2015). To
determine the detailed expression pattern of SAG29 in ovules, we made a construct of SAG29
fused with eGFP and driven by its native promoter, and transformed it into wild type. A weak
SAG29-eGFP signal could initially be detected at the cells surrounding the micropylar end in
un-pollinated ovules (Figure 7a). At 2 hours after pollination (HAP), the SAG29-eGFP signal
was similar to that of an un-pollinated ovule (Figure 7b). However, the signal was
significantly elevated at 4 HAP, and it continuously increased at the micropylar end to reach a
peak at 8 HAP (Figure 7c and d), suggesting that the SAG29 expression level may be
significantly activated by ethylene signaling at 8 HAP. Next, the signal started to decrease at
16 HAP (Figure 7e) and gradually disappeared at approximately 22 HAP (Figure 7f). The
expression dynamics of SAG29 in synergid cells are consistent with its function in blocking
pollen tube attraction.

DISCUSSION
EBF1 and EBF2 play redundant roles to ensure female gametophytic and embryonic
development
Ethylene is widely involved in plant senescence and stress responses. EBF1 and EBF2 have
been reported to control the degradation of EIN3, the key transcription factor of the ethylene
signaling pathway (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004).
Previous investigation indicates that ethylene signaling is also involved in plant reproduction,
as ebf1 ebf2 homozygous plants could not be identified (An et al., 2010). In this work, we
demonstrate that EBF1 and EBF2 affect female gametophytic and embryonic development,
while the ebf1 ebf2 male gametophyte is functionally normal.

The synergid cells of the female gametophyte play the role to attract the pollen tube. We
demonstrate that EIN3 over-accumulation in the synergid cells of ebf1 ebf2 blocks pollen
tube attraction (Figures 2d and f, 3a). However, genetic analysis reveals that about 33.3% of
ebf1 ebf2 female gametophytes (ovules) are still functional (Table 1). As ebf1 ebf2 pollen is
functional and ebf1-/-ebf2-/- plants could not be identified (Figure 1), this indicates that ebf1-/-
ebf2-/- is embryo lethal. Although we have no direct evidence of EIN3 accumulation in ebf1

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 ebf2 embryos, overexpression of the direct EIN3 target, SAG29, in synergid cells results in
reduced fertility, and the knockout of EIN3 in the triple mutant ebf1-/-ebf2-/-ein3-/- led to
restored fertility (Figure 1a and b). EIN3 might accumulate in the embryo and lead to
Accepted Article
developmental defects.

Pollen tube attraction by the female gametophyte and embryonic development are two
indispensable processes of plant reproduction. Ethylene plays an important role in integrating
environmental cues during plant stress responses and organ senescence (Abeles et al., 1992).
The repression of over-activated ethylene signaling can prevent abnormal senescence of the
synergid cells and, therefore, ensure appropriate pollen tube attraction and guarantee
successful sexual reproduction. ebf1 and ebf2 single mutant have no reproductive defects,
suggesting that either EBF1 or EBF2 is sufficient to block ethylene signaling in the synergid
cells and embryo. Therefore, possessing both EBF1 and EBF2 guarantees that synergid cells
will attract pollen tubes under different environmental conditions before fertilization.

SAG29 activated by EIN3 plays a role in synergid function for pollen tube attraction
SAG29/SWEET15 belongs to the SWEET family of sugar transporters (Chen et al., 2010;
Chen et al., 2012). Previous research has shown that the expression level of SAG29 is
significantly elevated during leaf senescence (Quirino et al., 1999; Seo et al., 2011). SAG29
might function with SWEET11 and SWEET12 in providing nutrition between the seed coat
and endosperm during embryonic development (Chen et al., 2015). In this work, the
expression of SAG29 was evidently increased in ebf1 ebf2, and EIN3 can directly activate the
expression of SAG29 (Figure 5). Transgenic lines expressing SAG29 in synergid cells showed
disrupted pollen tube attraction (Figure 6). Cytological observations showed that SAG29-
eGFP was expressed at the micropylar end of the ovule (Figure 7). Therefore, SAG29 plays a
role in ethylene signaling to block pollen tube attraction. As a sucrose transporter, SAG29
can transport polysaccharides (Chen et al., 2010). Sugar is the fundamental material for cell
development. Overexpression of SAG29 might lead to the excess inflow or outflow of sugars,
which disrupts the sugar metabolism and affects the osmotic pressure of the cell. In addition,
a recent study reported that the arabino-galactan polysaccharide material (AMOR) was
crucial for the acquisition of ovule attraction competence by the pollen tube (Mizukami et al.,
2016). The SAG29-eGFP location is similar to that of AMOR (Figure 7). SAG29 may be
associated with the secretion of AMOR sugar chains or other attractants. Over-accumulation
of SAG29 might interfere with the normal transportation of these attractants. However, the

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 statistical data of the EIN3 and SAG29 overexpression lines showed a lower efficiency of
blocking pollen tube attraction than that of the EIN3 overexpression lines (Figures 4f and 6e).
EIN3 may also activate the expression of other genes that participate in blocking pollen tube
Accepted Article
attraction.

Ethylene signaling blocks pollen tube attraction

Synergid cells play the major role in attracting a pollen tube to enter the embryo sac. After
the first pollen tube enters the embryo sac, the attraction of other pollen tubes is blocked, so
that only one pollen tube can be attracted into the ovule. Therefore, the plant has developed a
mechanism to repel the attraction of a second pollen tube. Previous investigations have
indicated that ethylene signaling is involved in the pollen tube block. In ein2, ein3, and ein3
eil1 mutants, multiple pollen tubes enter the embryo sac (Volz et al., 2013). In this work,
pollen tube attraction was found to be blocked in ebf1 ebf2 double mutants, and EIN3
accumulated at synergid cells, supporting the hypothesis that ethylene signaling is a key
factor for the establishment of the second pollen tube block system. A previous study
proposed that the second pollen tube block system occurs in a three-step process (Maruyama
et al., 2015). The first step is synergid-endosperm fusion, and the pollen tube attractants are
diluted after synergid-endosperm fusion. The second step is the activation of ethylene
signaling, and the third step is the disorganization of the synergid cell nucleus (Maruyama et
al., 2015). Cell fusion is a drastic process, which may trigger significant changes of materials
within cells. Ethylene signaling is quite sensitive to environmental change. EIN3 might
accumulate greatly in a short time due to this change and consequently activate the
expression of SAG29 (Figure 5). As a sugar transporter, the activated SAG29 might lead to
the loss of sugars in the synergid cells, which may be required for blocking the second pollen
tube.

EXPERIMENTAL PROCEDURES
Plant Material, Growth Conditions and Mutant Identification
The ebf1-1 and ebf2-1 single and double mutants, ein3-1 and ebf1 ebf2 ein3 triple mutants
and Arabidopsis ecotype Col-0 used in this report were previously described (Guo and Ecker,
2003; An et al., 2010). The sag29 mutant used in this study is a T-DNA tagged mutant
(Salk_116181) obtained from the Arabidopsis Biological Resource Center
(www.arabidopsis.org). Seeds were sown on vermiculite and allowed to vernalize for 3 days

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 in a 16 hours light/8 h dark photoperiod. All transgenic plants were screened on PNS or 1/2
MS medium with 50 mg/L hygromycin B. The T-DNA insertion sites in ebf1 and ebf2 were
identified by PCR (Guo and Ecker, 2003). The primers were summarized in Supplemental
Accepted Article
Table S2. The mutant ein3-1 is a point mutant identified by enzyme digestion after PCR.

Plasmid Construction and Transformation of Plants

The PDD31::GFP constructs were created by cloning the promoter following the method of a
previous study (Steffen et al., 2007). The PEBF1::EBF1-GFP and PEIN3::EIN3-GFP constructs
were created by cloning the EBF1 and EIN3 promoters and genomic sequences into the
KpnI/BamHI and EcoRI/NcoI sites of the binary vector pCAMBIA1300-GFP-NOS. The
primer sequences used for PCR are shown in Supplemental Table S2. All constructs were
verified by DNA sequencing. The constructs were introduced into Agrobacterium
tumefaciens strain GV3101 by exposure to liquid nitrogen for 1 min and a 37 °C water bath
for 5 min. Then, the strain was shaken at 28 °C for 3 hours and plated on solid LB medium.
The medium was then placed in a 28 °C incubator for 2 days. The strain containing the vector
was transformed into ebf1-/-ebf2+/- plants.

Confocal Analysis of Female Gametophyte Structure and the Expression Pattern of

Transgenic Plants
Confocal laser scanning microscopy was used to analyse ebf1-/-ebf2+/- and wild-type plants as
described (Christensen et al., 1998). For observing GFP expression in the female
gametophyte, we emasculated flowers at stage 12c (Smyth et al., 1990) and removed flowers
from plants. Then, the sepals, petals, and stamens were removed, and the carpel was dissected
from the walls by using a syringe needle. The ovules were mounted on a slide in 10 mM
phosphate buffer (pH 7.0). To accurately determine GFP expression in the female
gametophyte, we analysed T2 lines for each construct.

RNA Extraction from Ovules and qRT-PCR

Pistils were harvested from mutant and wild-type flowers at stages 12c and 13 (FG4-FG7) by
removing the surrounding sepals, petals and stamens under microscopy. Pistils were placed
without stigmas, styles or pedicels into 1.5-ml tubes (RNAse-free) and treated with liquid
nitrogen. Then, a steel ball treated overnight at 180 °C was placed into each tube, and 1 ml
TRIzol was added. The tubes were then placed in a tissue lyser and shaken for 1 min at 60
Hz. The first-strand cDNA was obtained by using the Toyobo First-Strand cDNA Synthesis

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 Kit. Quantitative PCR analysis was performed as described previously (Lou et al., 2014). The
β-tubulin gene was used as a positive control, and each sample was repeated with three
replicates. The relative expression levels were calculated according to cycle number. The
Accepted Article
related primer sequences are provided in Table S2.

Fluorescence Staining of Pollen Tubes

Wild-type, ebf1-/-ebf2+/-, and ebf1-/-ebf2-/-ein3-/- flowers were emasculated overnight and then
crossed with wild-type pollen grains for 16 h. Pistils were cleared in Carnoy fluid for 4 h and
then washed with phosphate buffered saline (PBS, pH 7.4) three times. The samples were
then softened with 7 M NaOH overnight and washed with PBS (pH 7.4). Finally, the pistils
were stained with a diluted aniline blue staining solution for ten minutes. An Olympus BX51
fluorescence microscope was used for observation.

Electrophoretic Mobility Shift Assay (EMSA)

Construction of a plasmid for the expression of recombinant EIN3 protein (amino acids 141
to 352) in the Rosetta E. coli strain and purification of the EIN3 protein were as described (Li
et al., 2013). EMSA involved use of a Light Shift Chemiluminescent EMSA kit (Thermo
Scientific). The DNA fragment containing the consensus sequence (ATGTAC) in the SAG29
regulatory region was generated by PCR amplification with specific primers (see supplement)
that were used to generate a biotin-labelled DNA fragment. The EMSA was performed with a
Light Shift Chemiluminescent EMSA Kit (Thermo Scientific,
http://www.thermoscientific.com). The binding reactions, which contained 10X binding
buffer, 0.05 mg mL-1 poly (dI-dC), EIN3-pMAL recombinant fusion protein and biotin-
labelled DNA, were performed at room temperature for 20 min. The subsequent processes
were performed according to the manufacturer’s instructions.

Dual transient expression assay in Arabidopsis protoplasts

To generate luciferase reporter constructs, the promoter of SAG29 (833 bp) was amplified
from Col-0 genomic DNA and cloned into pGreenII 0800-Luc, with the SAG29 promoter
driving a firefly luciferase (LUC) gene and a CaMV 35S promoter driving a Renilla
luciferase (REN) gene. The 35S:EIN3 effector construct was generated with the full-length
EIN3 coding sequence (CDS) and cloned into the vector pEarleyGate203 to obtain
pEarley203-EIN3 by recombination, as described in Qiu et al., 2015. The primers used for all

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 constructs are listed in Table S2. The protoplast isolation and transformation methods were
also described in Qiu et al., 2015.
Accepted Article
ACCESSION NUMBERS
Accession numbers for each of the gene referred to in this work are as follws: EBF1,
At2g25490; EBF2, At5g25350; EIN3, At3g20770; SAG29, At5g13170

ACKNOWLEDGEMENT

We thank Rita Groβ-Hardt for the EIN3-YFP plasmid. This work was supported by grants
from the Ministry of Science and Technology of China (2016YFD100902) and the National
Science Foundation of China (31300262).

The author declare no conflicts of interest.

SHORT LEGENDS FOR SUPPORTING INFORMATION

Figure S1. The fertility observation of ebf1 and ebf2 single mutants.

Figure S2. The characterization of ebf1 and ebf2 T-DNA insertion mutants. a. The T-DNA
position in ebf1, ebf2 mutants and the related identification primer positions. b. Construction
of PEBF2::EBF2-GFP and the phenotype of the related complementation lines in an ebf1-/-
ebf2-/-background.

Figure S3. Ovule development of the wild type and ebf1-/- ebf2+/- mutant.

Figure S4. Diagram of the gene location of EBF1, EBF2 and SAG29 on the Arabidopsis
chromosomes

Table S1. Putative EIN3 targets with high or specific expression in ovules.

Table S2. The primer sequences used in this study.

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 REFERENCES
Abeles FB, Morgan PW, Saltveit ME (1992). Ethylene in plant biology, Second edition (San
Accepted Article
Diego, California: Academic Press, Inc.).

Abeles FB, Dunn LJ, Morgens P, Callahan A, Dinterman RE, Schmidt J (1988) Induction of
33-kD and 60-kD peroxidases during ethylene-induced senescence of cucumber
cotyledons. Plant Physiol 87: 609-615

An F, Zhao Q, Ji Y, Li W, Jiang Z, Yu X, Zhang C, Han Y, He W, Liu Y, Zhang S, Ecker JR,

Guo H (2010) Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and
EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2
that requires EIN2 in Arabidopsis. Plant Cell 22: 2384-2401

Binder BM, Mortimore LA, Stepanova AN, Ecker JR, Bleecker AB (2004) Short-term
growth responses to ethylene in Arabidopsis seedlings are EIN3/EIL1 independent.
Plant Physiol 136: 2921-2927

Bleecker AB, Estelle MA, Somerville C, Kende H (1988) Insensitivity to ethylene conferred
by a dominant mutation in arabidopsis thaliana. Science 241: 1086-1089

Chang KN, Zhong S, Weirauch MT, Hon G, Pelizzola M, Li H, Huang SS, Schmitz RJ,
Urich MA, Kuo D, Nery JR, Qiao H, Yang A, Jamali A, Chen H, Ideker T, Ren B,
Bar-Joseph Z, Hughes, TR, Ecker JR (2013) Temporal transcriptional response to
ethylene gas drives growth hormone cross-regulation in Arabidopsis eLife 2: e00675

Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR (1997) Activation of the

ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-
INSENSITIVE3 and related proteins. Cell 89: 1133-1144

Chen LQ, Hou BH, Lalonde S, Takanaga H, Hartung ML, Qu XQ, Guo WJ, Kim JG,
Underwood W, Chaudhuri B, Chermak D, Antony G, White FF, Somerville SC,
Mudgett MB, Frommer WB (2010) Sugar transporters for intercellular exchange and
nutrition of pathogens. Nature 468: 527-532

Chen LQ, Lin IW, Qu XQ, Sosso D, McFarlane HE, Londono A, Samuels AL, Frommer WB

This article is protected by copyright. All rights reserved.

 (2015) A cascade of sequentially expressed sucrose transporters in the seed coat and
endosperm provides nutrition for the Arabidopsis embryo. Plant Cell 27: 607-619
Accepted Article
Chen LQ, Qu XQ, Hou BH, Sosso D, Osorio S, Fernie AR, Frommer WB (2012) Sucrose
efflux mediated by SWEET proteins as a key step for phloem transport. Science 335:
207-211

Christensen BB, Sternberg C, Andersen JB, Eberl L, Moller S, Givskov M, Molin S (1998)
Establishment of new genetic traits in a microbial biofilm community. Appl Environ
Microbiol 64: 2247-2255

Christensen CA, Subramanian S, Drews GN (1998) Identification of gametophytic mutations

affecting female gametophyte development in Arabidopsis. Dev Biol 202: 136-151

Clark KL, Larsen PB, Wang X, Chang C (1998) Association of the Arabidopsis CTR1 Raf-
like kinase with the ETR1 and ERS ethylene receptors. Proc Natl Acad Sci 95: 5401-
5406

Drews GN, Yadegari R (2002) Development and function of the angiosperm female
gametophyte. Annu Rev Genet 36: 99-124

Guo H, Ecker JR (2003) Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-
dependent proteolysis of EIN3 transcription factor. Cell 115: 667-677

Gagne JM, Smalle J, Gingerich DJ, Walker JM, Yoo SD, Yanagisawa S, Vierstra RD (2004)
Arabidopsis EIN3-binding F-box1 and 2 form ubiquitin-protein that repress ethylene
action and promote growth by directing EIN3 degradation. Proc Natl Acad Sci 101:
6803-6808

Higashiyama T (2002) The synergid cell: attractor and acceptor of the pollen tube for double
fertilization. J Plant Res 115: 149-160

Hua, J, Meyerowitz, EM (1998) Ethylene responses are negatively regulated by a receptor

gene family in Arabidopsis thaliana. Cell 94: 261-271

This article is protected by copyright. All rights reserved.

 Ju C, Yoon GM, Shemansky JM, Lin DY, Ying ZI, Chang J, Garrett WM, Kessenbrock M,
Groth G, Tucker ML, Cooper B, KieberJJ, Chang C (2012) CTR1 phosphorylates the
central regulator EIN2 to control ethylene hormone signaling from the ER membrane
Accepted Article
to the nucleus in Arabidopsis. Proc Natl Acad Sci 109: 19486-19491

Kim HJ, Hong SH, Kim YW, Lee IH, Jun JH, Phee BK, Rupak T, Jeong H, Lee Y, Hong BS,
Nam HG, Woo HR, Lim PO (2014) Gene regulatory cascade of senescence-associated
NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf
senescence signaling in Arabidopsis. J Exp Bot 65: 4023-4036

Kosugi S, Ohashi Y (2000) Cloning and DNA-binding properties of a tobacco Ethylene-

Insensitive3 (EIN3) homolog. Nucleic Acids Res 28: 960-967

Li Z, Peng J, Wen X, Guo H (2012) Gene network analysis and functional studies of
senescence-associated genes reveal novel regulators of Arabidopsis leaf senescence. J
Integr Plant Biol 54: 526-539

Li Z, Peng J, Wen X, Guo H (2013) Ethylene-insensitive3 is a senescence-associated gene

that accelerates age-dependent leaf senescence by directly repressing miR164
transcription in Arabidopsis. Plant Cell 25: 3311-3328

Li WY, Ma MD, Feng Y, Li HJ, Wang YC, Ma YT, Li MZ, An FY, Guo HW (2015) EIN2-
directed translational regulation of ethylene signaling in Arabidopsis. Cell 163: 670-
683

Lou Y, Xu XF, Zhu J, Gu JN, Blackmore S, Yang ZN (2014) The tapetal AHL family protein
TEK determines nexine formation in the pollen wall. Nat Commun 5: 3855

Maruyama D, Volz R, Takeuchi H, Mori T, Igawa T, Kurihara D, Kawashima T, Ueda M, Ito

M, Umeda M, Nishikawa S, Gross-Hardt R, Higashiyama T (2015) Rapid elimination
of the persistent synergid through a cell fusion mechanism. Cell 161: 907-918

Mizukami AG, Inatsugi R, Jiao J, Kotake T, Kuwata K, Ootani K, Okuda S,

This article is protected by copyright. All rights reserved.

 Sankaranarayanan S, Sato Y, Maruyama D, Iwai H, Garenaux E, Sato C, Kitajima K,
Tsumuraya Y, Mori H, Yamaguchi J, Itami K, Sasaki N, Higashiyama T (2016) The
AMOR arabinogalactan sugar chain induces pollen-tube competency to respond to
Accepted Article
ovular guidance. Curr Biol 26: 1091-1097

Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C, Genschik P (2003)

EIN3-dependent regulation of plant ethylene hormone signaling by two arabidopsis F-
box proteins: EBF1 and EBF2 Cell 115: 679-689

Qiao H, Shen Z, Huang SS, Schmitz RJ, Urich MA, Briggs SP, Ecker JR (2012) Processing
and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas
Science 338: 390-393

Qiu K, Li ZP, Yang Z, Chen JY, Wu SX, Zhu XY, Gao S, Gao J, Ren GD, Kuai BK, Zhou X
(2015) EIN3 and ORE1 accelerate degreening during ethylene-mediated leaf
Senescence by directly activating chlorophyll catabolic genes in Arabidopsis. PLoS
Genetics 11: e1005399

Quirino BF, Normanly J, Amasino RM (1999) Diverse range of gene activity during
Arabidopsis thaliana leaf senescence includes pathogen-independent induction of
defense-related genes. Plant Mol Biol 40: 267-278

Seo PJ, Park JM, Kang SK, Kim SG, Park CM (2011) An Arabidopsis senescence-associated
protein SAG29 regulates cell viability under high salinity. Planta 233: 189-200

Solano R, Stepanova A, Chao Q, Ecker JR (1998) Nuclear events in ethylene signaling: a

transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-
RESPONSE-FACTOR1 Gene & Dev 12: 3703-3714

Smyth DR, Bowman JL, Meyerowitz EM (1990) Early flower development in Arabidopsis.
Plant Cell 2: 755-767

Steffen JG, Kang IH, Macfarlane J, Drews GN (2007) Identification of genes expressed in the

This article is protected by copyright. All rights reserved.

 Arabidopsis female gametophyte. Plant J 51: 281-292

Volz R, Heydlauff J, Ripper D, von Lyncker L, Gross-Hardt R (2013) Ethylene signaling is

Accepted Article
required for synergid degeneration and the establishment of a pollen tube block. Dev
Cell 25: 310-316

Wen X, Zhang CL, Ji YS, Zhao Q, He WR, An FY, Jiang LW, Guo HW (2012) Activation of
ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl
terminus. Cell Research 22: 1613-1616

Yang WC, Shi DQ, Chen YH (2010) Female gametophyte development in flowering plants.
Annu Rev Plant Biol 61: 89-108

Yamasaki K, Kigawa T, Inoue M, Yamasaki T, Yabuki T, Aoki M, Seki E, Matsuda T, Tomo

Y, Terada T, Shirouzu M, Tanaka A, Seki M, Shinozaki K, Yokoyama S (2005)
Solution structure of the major DNA-binding domain of Arabidopsis thaliana
ethylene-insensitive3-like3. J Mol Biol 348: 253-264

Figures
Figure 1. Morphology and genetic analysis of the ebf1-/-ebf2+/- mutant
(a) A wild-type silique with full seed set. An ebf1-/-ebf2+/- silique with about half aborted
ovules and aborted seed. A PEBF1::EBF1-GFP complemented line (CM line) silique with
seeds. An ebf1-/-ebf2-/-ein3-/- silique with seeds. White Arrowheads, aborted ovules. Black
Arrowheads, aborted seed. Bar=2 mm.
(b) Quantification of mature and aborted seeds in wild type, ebf1-/-ebf2+/- mutant, CM line
and ebf1-/-ebf2-/-ein3-/- mutant. Data are means±SD percentage. Asterisks indicate significant
difference of the aborted ovules in ebf1-/-ebf2+/- compared with that of the wild type, the
PEBF1::EBF1-GFP-CM transgenic line, and the ebf1-/- ebf2-/- ein3-/- mutant (**: P<0.01,

Student’s t-test).

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 Figure 2. Pollen tube attraction was defective in ebf1-/-ebf2+/- ovules
(a) A pollen tube can be guided into a wild-type ovule. Arrowheads, pollen tubes entering
the micropylar ends of ovules.
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(b, c) Pollen tube attraction in ebf1-/-ebf2+/- ovules. (b) Normal pollen tube attraction into an
ovule (arrowhead). (c) Failure of pollen tube attraction into an ovule.
(d, e) Pollen tube attraction in ebf1-/-ebf2-/-ein3-/- ovules. (d) Normal pollen tube attraction
into an ovule (arrowhead). (e) Failure of pollen tube attraction into an ovule. All the pollen
tubes in (a-e) were visualized by aniline blue staining.
(f) Quantification of successful pollen tube attraction and no pollen tube attraction. Bars=50
μm. Data are means±SD percentage. Asterisks indicate significant difference of the pollen

tube entry in ebf1-/-ebf2+/- compared with that of the wild type (**: P<0.01, Student’s t-test).

Figure 3. Localization of native promoter driving EIN3-GFP and DD31 promoter

driving GFP in wild-type and ebf1-/-ebf2+/- ovules
(a) Construction of EIN3-GFP driven by its own promoter. EIN3 localization and
quantification in wild type and ebf1-/-ebf2+/-. Bar=50 μm. Data are means±SD percentage of
ovules. Asterisks indicate significant difference of the EIN3-GFP signal detection in ebf1-/-

ebf2+/- compared with that in the wild type ovule (**: P<0.01, Student’s t-test).

(b) Construction of GFP driven by the DD31 promoter. GFP signal and quantification in
ebf1-/- and ebf1-/-ebf2+/-. Bar=20 μm. Data are means±SD percentage of ovules. Asterisks
indicate significant difference of PDD31:GFP signal in the ebf1-/-ebf2+/- compared with that in

the ebf1-/- synergid cell (**: P<0.01, Student’s t-test).

Figure 4. Phenotype analysis of truncated EIN3 C-terminus transgenic lines

(a) Construction of EIN3 driven by the DD31 promoter. Graphic of the putative EIN3,
ΔEIN31~550 and ΔEIN31~450 transcripts.
(b) Pollen tube attraction analysis of ovules from PDD31:: ΔEIN31~450 transgenic lines. Left
image, ovules showing normal pollen tube attraction. Right image, ovules showing disrupted
pollen tube attraction. NPT, normally attracted pollen tube. Bars=50 μm.
(c) Quantification of mature seeds in developing siliques in three transgenic lines. Data are
means±SD percentage. Asterisks indicate significant difference of pollen tube entry in the

PDD31:: ΔEIN31~450 transgenic lines compared with that in the wild type (**: P<0.01, Student’s

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 t-test).
(d-f) Dissected siliques produced by EIN3, ΔEIN31~550 and ΔEIN31~450 transgenic plants.
EIN3 line siliques contained more aborted seeds than those of the other two lines. Bar=2mm .
Accepted Article
(g-i) Statistical analysis of pollen tube entry into the ovules of wild type and PDD31::
ΔEIN31~450 transgenic lines.

Figure 5. EIN3 can promote SAG29 expression by directly binding to its putative
binding site on a promoter
(a) Putative EIN3 binding sites on the SAG29, ORE4 and SARK promoters.
(b) Quantitative PCR analysis of SAG29, ORE4 and SARK expression in wild-type and ebf1-/-
ebf2+/- pistils at stages FG5-FG7. Three biological replicates were performed. Data are
means±SD percentage.
(c) EMSA of the direct binding of EIN3 (amino acids 141 to 352) to the SAG29 promoter bio-
labelled probe. Arrow, protein/DNA complex.
(d) Left: the schematic diagrams of effector and reporter constructs used in the transient dual
luciferase assays. The CaMV 35S promoter was used to drive the expression of EIN3; the
empty vector was used as a control. The reporter construct was composed of two parts: the
Renilla gene driven by the 35S promoter was used for internal normalization; the
SAG25/SWEET15 promoter was constructed to drive the expression of LUCIFERASE. Right:
the relative activity of LUC was significantly increased when EIN3 was overexpressed,
which indicates the activation of the SAG29/SWEET promoter by EIN3. Asterisks indicate
significant difference of the elevated LUC activity compared with that in the negative control

(**: P<0.01, Student’s t-test).

Figure 6. Phenotype analysis of PDD31::SAG29 transgenic lines in Arabidopsis

(a) Construction of SAG29-GFP driven by the DD31 promoter.
(b) Dissected siliques produced by three independent transgenic lines of PDD31::SAG29.
Bar=2 mm.
(c) Quantification of mature seeds produced in siliques from three transgenic plants. Data are
means±SD percentage. Asterisks indicate significant difference of normal seed production in

the PDD31:: SAG29 transgenic lines compared with that in the wild type (**: P<0.01, Student’s

t-test).
(d) Pollen tube attraction in transgenic plants. Pollen tube was normally attraced into an ovule

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 (left). No pollen tube guided into an abnormal ovule (right). NPT, normally attracted pollen
tube. Bar=50 μm.
(e) Quantification of successful pollen tube attraction and no pollen tube attraction. Data are
Accepted Article
means±SD percentage. Asterisks indicate significant difference of pollen tube entry in the

PDD31:: SAG29 transgenic lines compared with that in the wild type (**: P<0.01, Student’s t-

test).

Figure 7. Expression pattern of SAG29 protein in ovules after pollination

(A) Confocal image of SAG29-eGFP in un-pollinated mature ovule of the transgenic line
PSAG29::SAG29. me, micropylar end. Arrowhead, surrounding cells at the micropylar end.
(B) Confocal image of SAG29-eGFP in mature PSAG29::SAG29 ovule 2 hours after
pollination.
(C) Confocal image of SAG29-eGFP in mature PSAG29::SAG29 ovule 4 hours after
pollination.
(D) Confocal image of SAG29-eGFP in mature PSAG29::SAG29 ovule 8 hours after
pollination. Arrowhead, SAG29 expression is significantly activated in the surrounding
cells at the micropylar end.
(E) Confocal image of SAG29-eGFP in mature PSAG29::SAG29 ovule 16 hours after
pollination.
(F) Confocal image of SAG29-eGFP in mature PSAG29::SAG29 ovule 22 hours after
pollination. All the bars=20 μm.
Table 1. Genetic analysis of ebf1-/- ebf2+/- mutants
The number of different plant genotypes among selfed and reciprocally crossed progeny is
shown. The TEF and TEM represent the Transmission Efficiency of the Female and Male
gametophytes, respectively. The calculation of transmission efficiency was performed
according to the following formula: mutants/wild-type progeny x 100%. The chi-squared
calculation showed that the ratio of progeny (ebf1+/- ebf2+/+ and ebf1+/- ebf2+/-) using the wild
type as female parent and ebf1-/- ebf2+/- as female parent differed extremely significantly from
the expected ratio (1:1; double asterisks). P=4.26E-12<0.001. The ratio of progeny (ebf1+/-
ebf2+/+ and ebf1+/- ebf2+/-) using ebf1-/- ebf2+/- as female parent and the wild type as female
parent did not differ significantly from the expected ratio (1:1). P=0.835>0.05. The ratio of
selfed progeny (1:1.2) differs significantly from the expected ratio (1:1; asterisk).
P=0.015<0.05

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Accepted Article

Table 1. Genetic analysis of ebf1 -/- ebf2 +/- mutants

Parental Genotype Progeny
female x male +/- +/+ +/- +/- -/- +/+ -/- +/- total TEF TEM
ebf1 ebf2 ebf1 ebf2 ebf1 ebf2 ebf1 ebf2
-/- +/- -/- +/-
ebf1 ebf2 ebf1 ebf2 / / 205 257* 462 / /

-/- +/-
WT ebf1 ebf2 47 45 / / 92 / 95.7%

-/- +/- / /
ebf1 ebf2 WT 90 18 108 20%** /

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