Role of RNA directed DNA methylation in controlling genomic imprinting in arabidopsis thaliana

1


ROLE OF RNA-DIRECTED DNA METHYLATION IN
GENOMIC IMPRINTING IN ARABIDOPSIS THALIANA







VU MINH THIET
(B. Sc, Vietnam National University, Hanoi)









A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARMENT OF BIOLOGICAL SCIENCES
NATIONAL UNIVERSITY OF SINGAPORE
2010

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ACKNOWLEDGEMENT

I would like to take this opportunity to thank everyone involved who helped
me get to this place in my life and fulfill the long process of persuading the PhD
study.
I especially would like to express my deepest gratitude to my supervisor
Frederic Berger. I would never finished my study without his unending support for the
last few years. I believe that I have learnt a tremendous amount from him. I appreciate
him for believing in me along the way. I would also like to thank my supervisor Prof
Davis Ng for his help that allowed me to stay and work in TLL.
I have extremely lucky to work with hearty colleagues Pauline, Pei Qi, Jeanie,
Li Jing, Ramesh, Chen Zhong, Sarah, Heike, Nie Xin, Tomo, and also the former lab
members Mathieu, Tadashi, Jonathan, Arnold. I would like to thank my colleagues for
their sharing, accompanying me all the times during my study. A special thank I give
to Pauline for her excellent guidance when I started working in the lab, I have learnt a
lot from her discipline in doing experiment and her critical thinking in science.
I would like to thank my thesis advisory committee Dr Yu Hao, Dr Toshiro
Ito, and Dr Jose Dinneny for their support.
I would also like to thank DBS Graduate Office and Ms Reena, Ms Priscilla.
They are always there to answer my questions and help me to process my paper works
as fast as possible.
I also would like to thank Tam, Long, Hang, Hung, Ngoc, Trang for their
support and encouragement for last few years in Singapore.

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I would like to thank my parents for their support over the years and not

complaining me when I could not come home for several TETs. A special thank to my
brother for his presence in Singapore and his understanding of my hard time. Thanks
for your care of our parents.
Last but not least, I acknowledge NUS graduate scholarship and TLL for
financial support.
Thank you so much everyone.


September, 2010

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TABLE OF CONTENTS

SUMMARY vi

LIST OF TABLES x

LIST OF ABBREVIATIONS xi

CHAPTER 1: INTRODUCTION 1

Abstract 2

1. Introduction of imprinting 3

1.1.1 First reports of imprinting in plants 6

1.1.2 The impact of interploid crosses on imprinting discovery 6

1.2 Imprinted genes and their function 9

1.2.1 Arabidopsis imprinted genes 9

1.2.2 Conservation of Polycomb group imprinted genes in cereals 14

1.3 Conclusion 15

2. Molecular mechanisms controlling imprinting 15

2.1 Imprinting by DNA methylation 16

2.1.1 Maintenance of DNA methylation on the silent alleles 16

2.1.2 Two-step removal of DNA methylation in the central cell 18

2.2 Molecular controls of imprinting by Histone methylation 20

2.3. Cis-elements controlling imprinting 23

2.3.1 Cis-elements in the promoter 23

2.3.2. Evidence for imprinting regulation by long distance elements 25

2.4 Genomic imprinting and RNA-directed DNA methylation 27

2.5 Imprinting, a by-product of the global reprogramming? 30


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3. Biological significance and evolution of imprinting 31

3.1 Parental conflict 31

3.2 Maternal control 33

3.3 Imprinting, a factor of speciation 34

3.4 Transposons as the primer of imprinting evolution in a specific developmental
context 34

Aims of the research 36

CHAPTER 2: GENOMIC IMPRINTING AND RNA-DIRECTED DNA
METHYLATION IN ARABIDOPSIS 39

2.1 Introduction 40

2.2. Results 43

2.2.1 SDC is silenced in somatic tissues and expressed in seeds 43

2.2.2 SDC is a new imprinted gene in Arabidopsis 47

2.2.3 Silencing mechanism of SDC paternal allele 50

2.2.4 Mechanism of activation of SDC maternal allele 52

2.3 Discussion 59

2.4 Future work 65

Material and Methods 66

CHAPTER 3: Accession-dependent Imprinting of HAIKU2 is controlled by the
RdDM Pathway 73

3.1 Introduction 74

3.2 Results 78

3.2.1 IKU2 expression in gametes 78

3.2.2 Is IKU2 an imprinted gene? 80


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3.2.3 What mechanism controls silencing of the paternal IKU2 allele? 83

3.3 Discussion 86

3.4 Future work 90

Material and Methods 91

CHAPTER 4: GENERAL DISCUSSION 94

4.1 Main findings 95

4.2 Biological significance 96

4.2.1 Further expansion of the number of imprinted genes 96

4.2.3 Is imprinting the by-product of the asymmetrical activity of major controls
of DNA methylation? 98

4.3. Future perspective 100

References 103

Annex: Maternal effect of mutation in RdDM pathway on seed development 116




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SUMMARY

In flowering plants and placental mammals, a subset of genes are expressed
depending on their parent of origin and defined as imprinted genes. In Arabidopsis,
the non-expressed allele of imprinted genes is silenced by either DNA methylation or
Histone methylation by Polycomb repressive complex activity. The Arabidopsis
imprinted genes are expressed only in endosperm, which nurtures embryo
development. Imprinted expression is established in two steps involving the
maintenance of DNA methylation. The paternal silenced allele remains marked by
DNA methylation during spermatogenesis while gene silencing is released from the
maternal allele by a demethylation pathway active during female gametogenesis.

After fertilization, the expressed allele remains active and the inactive allele remains
silent by maintenance DNA methylation machinery.
DNA methylation can be deposited de novo through the RNA-directed DNA
methylation pathway (RdDM) or maintained by the methyltransferase MET1. The
maintenance methyltransferase MET1 plays a major role and is sufficient to establish
monoallelic expression of most imprinted genes identified so far. However, the
function of RdDM in genomic imprinting has remained largely unknown.
In contrast with MET1 activity, the RdDM pathway results in methylation of cytosine
residues in any context and in absence of a hemimethylated template. The RdDM
pathway comprises the de novo methyltransferase DRM2 and the RNA polymerases
POLIV and POLV. In the course of this thesis, we have identified a role of the RdDM
pathway in regulating genomic imprinting in Arabidopsis thaliana.

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First, we identified SDC (suppressor of drm1, drm2, and cmt3) as a maternally
imprinted gene. The SDC gene is primarily silenced by the RdDM pathway. We
showed that SDC is specifically expressed in endosperm from its maternal allele and
silencing of the paternal allele requires the RdDM pathway.
The absence of expression of key genes in the RdDM pathway during female
gametogenesis while it is maintained during spermatogenesis is sufficient to explain
the origin of the imprinted expression of SDC.
Second, our results showed that the RdDM pathway is also necessary for silencing the
paternal allele of HAIKU2, which is a maternally expressed imprinted gene in
endosperm. The imprinted expression of HAIKU2 is observed in a genetic context-
depending manner, relying on the accessions used in the reciprocal crosses. HAIKU2
controls endosperm growth.
In conclusion, we described two novel imprinted genes in Arabidopsis. More
importantly, we identified RdDM as a new silencing mechanism functioning in
imprinting establishment. The loss of RdDM activity during female gametogenesis is

predicted to cause a genome-wide demethylation. Our findings suggest that a new
class of RdDM-dependent imprinted genes remains to be characterized in plants.

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List of Figures

Figure
number
Figure title Page
1-1 Gametogenesis in flowering plants 4
1-2 Double fertilization in angiosperms 5
1-3 Analysis of MEDEA imprinting 6
1-4 Developmental feature of endosperm 13
1-5 Establishment of DNA methylation-dependent imprinted
genes throughout the plant life cycle
17
1-6 DNA methylation dependent mechanisms leading to
imprinting of maternally expressed genes in Arabidopsis
19
1-7 Polycomb Repressive Complex 2 (PRC2) dependent
mechanisms leading to imprinting of maternally expressed
genes in Arabidopsis
21
1-8 Long distance cis-elements of imprinted genes 26
1-9 Regulation of PHERES1 imprinting 26
1-10 RNA-directed DNA methylation pathway 29
2-1 De novo methylation controls SDC expression 45
2-2 Expression of SDC in gametophytes and seeds 46
2-3 Allele specific RT-PCR analysis of maternal SDC

expression
49
2-4 SDC imprint is controlled by RdDM pathway 51
2-5 Maternally expressed SDC is not controlled by DEMETER 54
2-6 Expression of NRPD2 gametophytes and developing seed 55
2-7 NRPD2a-RFP construct recues the loss of endogenous
nrpd2a function
56
2-8 Expression of NRPD1b-RFP (PolV) in gametophytes and
developing seed
57
2-9 A proposed model for mechanism activating SDC 58

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expression in central cell

2-10 A proposed model for RdDM function in controlling SDC
imprinting
63
3-1 Loss of IKU2 causes small seed size phenotype 77
3-2 HAIKU genetic pathway controls seed size 77
3-3 RT-PCR analysis of IKU2 expression 79
3-4 Allele-specific RT-PCR results show maternal expression
of IKU2
81
3-5 Origin parental expression of IKU2 in combination of
different Arabidopsis accessions
82
3-6 Expression of IKU2 in mutation of DNA methylation and

Polycomb group
85
3-7 Silencing of paternal IKU2 allele is controlled by a DNA-
dependent RNA polymerase IV
85



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LIST OF TABLES

Table
number
Title

page
1 List of plant imprinted genes and their function 12


xi

LIST OF ABBREVIATIONS


AtFH5 ARABIDOPSIS FORMIN HOMOLOGUE 5
CMT3 CHROMODOMAIN METHYLTRANSFERASE
DME DEMETER
MET1 DNA METHYLTRANSFERASE 1

POLIV DNA-DEPENDENT RNA POLYMERASE 4
POLV DNA-DEPENDENT RNA POLYMERASE 5
DRM2 DOMAIN REARRANGED METHYLTRANSFERASE
FIE FERTILIZATION INDEPENDENT ENDOSPERM
FIS FERTILIZATION INDEPENDENT SEED
FWA FLOWERING WAGENINGEN
GAPDH GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE
H3K27 Lysine 27 residue of HISTONE3
MPC MATERNALLY EXPRESSED PAB C-TERMINAL
m-RFP MONOMERIC RED FLUORESCENT PROTEIN
MSI1 MULTICOPY-SUPPRESSOR OF IRA1
NRPD1A NUCLEAR RNA POLYMERASE D1
NRPD2A NUCLEAR RNA POLYMERASE D2
NRPD1B NUCLEAR RNA POLYMERASE E
PRC2 Polycomb Repressive Complex 2
RdDM RNA-directed DNA methylation
SDC SUPPRESSOR OF drm1, drm1 and cmt3

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CHAPTER 1: INTRODUCTION






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Abstract

Although most genes are expressed equally from both parental alleles, imprinted
genes are differentially expressed depending on their parental origin. In flowering
plants, imprinting is regulated by DNA methylation and histone methylation. Most
imprinted genes are silenced by chromatin modifications during vegetative
development. During gametogenesis the male or the female allele is activated by
removal of chromatin modification and remains active after fertilization while the
other allele remains silenced, leading to imprinted gene expression. Imprinting
mechanisms are conserved across plant species and to a certain extent there is
evidence of convergent evolution of imprinting mechanisms between plants and
mammals. The physiological significance and evolutionary origin of imprinting are
still unclear but in plants, imprinting may be the consequence of global epigenetic
reprogramming during sexual reproduction.

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1. Introduction of imprinting
In plants male and female gametes are produced after meiosis following a series of
divisions of a haploid male or female spore (Figure 1-1). In flowering plants male
gametogenesis takes place in stamen to produce pollen. Meiosis produces four haploid
microspores. Each microspore experiences two mitotic divisions producing a pollen
grain comprising a large vegetative cell and two identical haploid sperm cells (Figure
1-1). Female gametogenesis takes place within the diploid tissues of the ovule
(Yadegari and Drews, 2004). Meiosis produces one surviving megaspore. The haploid
megaspore undergoes a series of three syncytial divisions, followed by cellularisation
producing the embryo sac. The embryo sac contains the haploid female gamete or egg
cell, and the central cell (Figure 1-1).
Plant reproduction is characterized by a double fertilization. Two sperms are delivered
by the pollen tube to the egg cell and the central cell. Fertilization of the egg cell leads
to embryogenesis (Figure 1-2). The second sperm cell activates division of the central
cell leading to production of the endosperm. The endosperm develops around the

embryo and allows transfer of maternal nutrients and physical protection to the
embryo (Figure 1-2). In most plant species, the central cell inherits two haploid nuclei
from the syncytial gametophyte. The endosperm genome thus contains two doses of
the maternal genome and one dose of the paternal genome. This specific parental
genomic dosage attracted interest in early studies of plant reproduction, which led to
the discovery of imprinting in plants. After an historical account, we will review in
this section the identity and function of imprinted genes in plants.


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Figure 1-1. Gametogenesis in flowering plants
In flowering plants, male gametogenesis takes place in the anther of the flower.
Diploid pollen mother cell undergoes meiosis to produce a tetrad structure carrying
four haploid nuclei. Each of them develops into a microspore. Each microspore
undergoes a mitotic division without cytokinesis to produce two haploid nuclei. The
two nuclei have different destiny, one become generative nucleus which later on
divide into two sperm cells; whereas the other haploid nucleus remains undivided and
become vegetative nucleus.
Female gametophytes are produced by meiosis division of megaspore mother cell in
the ovary of the flower. A single megaspore mother cell undergoes meiosis to produce
four megaspores which are linearly arranged. Out of these four megaspores, three
degenerate and the remaining undergoes three successive mitotic divisions without
cytokinesis to form a large cell with eight haploid nuclei. Three of them move to the
micropylar end to form two sygergid cells which degenerate soon. The remaining
develops into the egg cell. Another group of three nuclei migrate to the chalazal pole
and become antipodal cells. The remaining two nuclei in the centre of the embryo sac
unite to become central cell.

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Figure 1-2 Double fertilization in angiosperm.
Two sperm cells of the pollen grain are delivered into the embryo sac which contains
two female gametes, egg cell and central cell. One sperm cell fertilizes egg cell to
give rise embryo. The second sperm cell combine with the diploid central cell to
produce a triploid (3n) tissue called endosperm which develops surrounding the
embryo and supports embryogenesis. Two products of double fertilization develop
into the seed structure which is protected by maternally derived seed integument.

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1.1 Historical discovery of imprinting
1.1.1 First reports of imprinting in plants
The term imprinting was originally adopted to qualify the differential elimination of
paternal chromosomes in the mealybug Sciara (Crouse, 1960). The first example of
imprinted expression of a gene was identified in the study of pigmentation of the outer
layers of the endosperm in maize (Kermicle, 1970). Irregular anthocyanin
pigmentation was linked to certain alleles of the R gene and was conferred only when
the mutation was inherited from the mother. Kermicle proposed that expression of the
r allele depended on its parental origin, and he further showed that the effects
observed did not depend on gene dosage. Hence, imprinting was initially described in
plants several years before the concept was formulated in mice following the
discovery that certain chromosomal regions can lead to developmental abnormalities

when both copies are exclusively maternally or paternally derived (Cattanach and
Kirk, 1985). However parent of origin expression in maize was only observed for
certain r mutant alleles and did not reflect that in the wild type only the maternal R
allele is expressed. Today the mechanism causing allele-dependent imprinting is still
not understood.
1.1.2 The impact of interploid crosses on imprinting discovery
Evidence for parental genomic imprinting also came from the study of the seeds
developing from crosses between plants with different ploidy. As early as the middle
of the twentieth century crosses between tetraploid and diploid plants were shown to
result in seed abortion due to endosperm failure (Cooper, 1951; Randolph, 1935). It
was later shown in maize that a critical maternal : paternal genome dosage in the

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endosperm was required for seed survival (Lin, 1984). These experiments were
repeated in Arabidopsis using tetraploid and hexaploid plant lines (Scott et al., 1998).
Increased paternal contribution caused endosperm enlargement, whereas increased
maternal dosage had the opposite effect. These results could be explained by different
expression between paternal alleles and maternal alleles of certain genes important for
endosperm development. These experiments led to the model that two sets of
maternally expressed imprinted genes (MEG) and paternally expressed imprinted
genes (PEG) control endosperm development.
MEDEA (MEA), the first imprinted gene in Arabidopsis was identified more than a
decade ago (Kinoshita et al., 1999; Vielle-Calzada et al., 1999). It was shown clearly
that MEA was actively transcribed after fertilization (Baroux et al., 2006; Vielle-
Calzada et al., 1999). Maternal transcription of MEA was shown using a
polymorphism between two wild type strains of Arabidopsis (Figure 1-3). MEA
maternal expression was then confirmed using transcriptional reporters (Figure 1-3)
(Kinoshita et al., 1999; Luo et al., 2000; Wang et al., 2006).

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Figure 1-3: Analysis of MEDEA imprinting
A. Expression of MEA::GUS reporter construct in crosses, which involve the
transgenic line carrying the reporter construct as a mother or as a father.
MEA::GUS is expressed only when contributed maternally.
B. Schematic of the analysis of the imprinted expression of MEA endogenous
locus. A sequence polymorphism is used to distinguish MEA mRNA and
αVPE mRNA between the wild type strains Col and Rld. Seeds resulting from
crosses between the two parents express mRNAs from both parental alleles
and two bands are detected. In contrast MEA mRNA originates only from the
maternal allele and a single band is detected.



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1.2 Imprinted genes and their function
The endosperm is currently the only tissue where imprinted gene expression has been
identified in Arabidopsis (Berger and Chaudhury, 2009; Kinoshita et al., 2008) rice,
and maize with the exception of Maternally expressed in embryo 1 (Mee1) in maize
which is imprinted in the embryo as well as in the endosperm (Jahnke and Scholten,
2009) (Table 1).
1.2.1 Arabidopsis imprinted genes

In Arabidopsis, amongst the first maternally expressed imprinted genes identified
MEA (Kinoshita et al., 1999; Vielle-Calzada et al., 1999) and FERTILIZATION
INDEPENDENT SEED 2 (FIS2) (Jullien et al., 2006a; Luo et al., 2000; Luo et al.,
1999) are core members of the endosperm specific FERTILIZATION
INDEPENDENT SEED (FIS) Polycomb group Repressor Complex 2 (PRC2) also
including FERTILIZATION INDEPENDENT ENDOSPERM (FIE) (Ohad et al., 1999)
and MULTICOPY-SUPPRESSOR OF IRA1 (MSI1) (Guitton and Berger, 2005;
Guitton et al., 2004; Kohler et al., 2003b), which are not imprinted. PRC2 methylates
the lysine 27 residue of HISTONE3 (H3K27), and thereby represses transcription
(Hennig and Derkacheva, 2009; Schuettengruber et al., 2007).
The wild-type endosperm posterior pole (also defined as chalazal pole) is
distinguished from the peripheral and anterior (micropylar) domains of the endosperm
by a multinucleate structure identified as the cyst (Boisnard-Lorig et al., 2001; Brown
et al., 1999; Scott et al., 1998) (Figure 1-4). The endosperm of fis mutants is
characterized by multiple defects including enhanced proliferation, much enlarged

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posterior structures and absence of cellularization (Guitton et al., 2004; Kiyosue et al.,
1999; Kohler et al., 2003a; Luo et al., 1999). This pleiotropic phenotype might be the
consequence of the maintenance of a juvenile developmental program (Ingouff et al.,
2005a). Although some targets of the FIS PcG complex have been identified, the
pathways downstream of this transcriptional regulation are unknown and targets
whose functions explain the fis phenotype have not been fully understood. We will
detail below the function of two targets of the FIS PcG complex, which are
themselves imprinted, the ARABIDOPSIS FORMIN HOMOLOGUE 5 (AtFH5), and
PHERES1.
AtFH5 encodes an actin-nucleating agent (Ingouff et al., 2005b) and is maternally
expressed in the endosperm (Fitz Gerald et al., 2009). The posterior endosperm cyst
develops from the migration of nuclei from the peripheral endosperm (Guitton et al.,

2004) (Figure 1-4). The early endosperm syncytial development ends when
cellularization partitions the syncytium into mono-nucleate cells, but cellularization
does not occur in the posterior pole (Brown et al., 1999; Sorensen et al., 2002). AtFH5
expression is confined to the posterior pole and is required for nuclear migration to
this part of endosperm (Ingouff et al., 2005b). The restricted expression of AtFH5 in
the posterior endosperm depends on the FIS PRC2. In absence of FIS function AtFH5
is expressed ectopically, preventing proper development of the posterior pole (Fitz
Gerald et al., 2009).
PHERES1 (PHE1) is paternally expressed (Kohler et al., 2005; Makarevich et al.,
2006) in endosperm and encodes a type one MADS-box transcription factor of the
AGAMOUS-LIKE family (AGAMOUS-LIKE37). PHE1 antagonizes the role of FIS

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PRC2 on endosperm growth but its mechanism of action remains unclear (Kohler et
al., 2003a).
Two other imprinted genes identified in Arabidopsis have been studied in further
details, FLOWERING WAGENINGEN (FWA) and MATERNALLY EXPRESSED PAB
C-TERMINAL (MPC). FWA encodes an homeodomain leucine zipper (HD-ZIP)
protein (Soppe et al., 2000), is expressed maternally only in endosperm where its
function is not known (Kinoshita et al., 2004). When ectopically expressed in
vegetative tissues FWA binds and inhibits the function of FLOWERING LOCUS T
(FT), causing late flowering (Ikeda et al., 2007). Three other members of HD-ZIP
genes (HDG3, HDG8 and HDG9) also show imprinted expression in endosperm
(Gehring et al., 2009b). HDG8 and HDG9 are maternally expressed while HDG3 is
expressed predominantly from its paternal allele.
MPC encodes the C-terminal region of a poly(A) binding protein (PABP) (Tiwari et
al., 2008). MPC is able to bind CTC-interacting (CID) proteins but lacks the RNA
binding domain and its function is not known. MPC is also expressed but not
imprinted in vegetative tissues and in the embryo.

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Table 1. List of imprinted genes and their function in plants
Species Name Potential function Reference
Maize (Zea
mayz)




R (Certain alleles only) Transcription Factor (Kermicle, 1970)
Allele MO17 of the dzr1
locus
Reserve protein
(Chaudhuri and
Messing, 1994)
Fertilization independent
endosperm 1 (Fie1)
Pc-G chromatin
remodeling factor
(Danilevskaya et al.,
2003)
No apical meristem related
protein 1 (Nrp1)
Unknown
(Haun and Springer,
2008)
Maize Enhancer of Zeste1
(Mez1)

Pc-G chromatin
remodeling factor
(Haun et al., 2007)
Maternally expressed gene1
(Meg1)
Cys rich peptide
(Gutierrez-Marcos
et al., 2004)
Arabidopsis
thaliana




MEDEA (MEA)
Pc-G chromatin
remodeling factor
(Kinoshita et al.,
1999; Vielle-
Calzada et al., 1999)
FLOWERING
WAGENINGEN (FWA)
Homeobox
transcription factor
(Kinoshita et al.,
2004)
PHERES1 (PHE1)
Type1 MADS-box
transcription factor
(Kohler et al., 2005;

Makarevich et al.,
2008)
FERTILIZATION
INDEPENDENT SEED 2
(FIS2)
Pc-G chromatin
remodeling factor
(Jullien et al.,
2006b; Luo et al.,
2000; Luo et al.,
1999)
MATERNALLY
EXPRESSED PAB C-
TERMINAL (MPC) 43
C-terminal domain of
poly(A) binding
proteins (PABPs);
probably controls
mRNA stability and
translation
(Tiwari et al., 2008)
HD-ZIP GENE9 (HDG9) Transcription factor
(Gehring et al.,
2009b)
HD-ZIP GENE8 (HDG8) Transcription factor
(Gehring et al.,
2009b)
HD-ZIP GENE3 (HDG3) Transcription factor
(Gehring et al.,
2009b)

ATMYBR2 Transcription factor
(Gehring et al.,
2009b)
AT5G62110
Putative Transcription
factor
(Gehring et al.,
2009b)
MEE1


13





Figure 1-4 Developmental feature of endosperm.
Endosperm is a part of the developing seed and is thought to control the maternal
nutrient supply for the embryo development. (A) Endosperm development starts with
several rounds of synchronous syncytial divisions without cytokinesis to form three
endosperm domains defined as anterior micropylar, peripheral and posterior chalazal
domain. (B) At heart embryo stage, the endosperm cellularization take places at
anterior domain around the embryo, followed by the cellularization of peripheral
domain. In contrast, the posterior endosperm does not cellularise and consists of multi
nucleate masses of cytoplasm, defined as the cyst (cy) at the most posterior part, and
as nodules (no) when located at the anterior part of the cyst (Scott et al., 1998). (C)
AtFORMIN5 (AtFH5) encodes acting nucleating agent. Its expression is restricted to
the posterior pole during endosperm development. AtFH5 is required for completion
of cytokinesis (Ingouff et al., 2005b).