Epigenetic Reprogramming in Plant Reproductive Lineages
1
School of Life Sciences, University of Warwick, Wellesbourne Campus, Coventry CV35 9EF, UK
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
*Corresponding author: E-mail, [email protected]; Fax, +44-247675000
(Received January 28, 2012; Accepted March 29, 2012)
2
Monoecious flowering plants produce both microgametophytes (pollen) and megagametophytes (embryo sacs) containing the male and female gametes, respectively, which
participate in double fertilization. Much is known about cellular and developmental processes giving rise to these reproductive structures and the formation of gametes. However,
little is known about the role played by changes in the
epigenome in dynamically shaping these defining events
during plant sexual reproduction. This has in part been hampered by the inaccessibility of these structures—especially
the female gametes, which are embedded within the female
reproductive tissues of the plant sporophyte. However, with
the recent development of new cellular isolation technologies that can be coupled to next-generation sequencing, a
new wave of epigenomic studies indicate that an intricate
epigenetic regulation takes place during the formation of
male and female reproductive lineages. In this mini review,
we assess the fast growing body of evidence for the epigenetic regulation of the developmental fate and function of
plant gametes. We describe how small interfereing RNAs and
DNA methylation machinery play a part in setting up unique
epigenetic landscapes in different gametes, which may be
responsible for their different fates and functions during
fertilization. Collectively these studies will shed light on
the dynamic epigenomic landscape of plant gametes or
‘epigametes’ and help to answer important unresolved questions on the sexual reproduction of flowering plants, especially those underpinning the formation of two products of
fertilization, the embryo and the endosperm.
of this cycle, most flowering plants establish their germline as
sporophytic development approaches maturity. The meiotic
products originate from separate male and female diploid
sporophytes but, rather than differentiating directly into gametes, as is the case in animals, they instead give rise to multicellular structures known as haploid gametophytes. This
developmental process is complex and its regulation is not
yet fully understood (Harrison et al. 2010). Nonetheless, the
final outcome is the formation of a male gametophyte or
pollen, consisting of one large vegetative cell and two gametic
sperm cells, and a megagametophyte or embryo sac with its
two gametes—the egg cell and the central cell—and five
accessory cells that provide support during double fertilization.
Although the cellular events that give rise to these male and
female gametophytes, respectively, are well understood, it is
becoming increasingly clear that the production of male and
female lineages is not only under genetic, but, more importantly, also under epigenetic control. Here we discuss recent
evidence supporting the view that dynamic changes in small
non-coding RNAs and DNA methylation are part of a complex
epigenetic reprogramming that takes place during the development of plant reproductive lineages that in Angiosperms
culminates with the formation of the two largely similar male
and two very different female gametes.
Keywords: Epigenetics Gametes Reproduction.
Sperm are formed within pollen grains, which are located in the
anthers of flowering plants. Meiosis in the young anther generates four haploid meiotic products (microspores), which first
undergo a cell cycle resulting in formation of bicellular pollen
grains containing a vegetative and a generative cell. In a second
cycle, the generative cell divides to form two haploid sperm
(Berger and Twell 2011). In some taxa, this second mitotic division can occur either in the anther (e.g. Arabidopsis) or during
pollen tube growth (e.g. Nicotiana). Thus the male germline
sensu stricto arises very late in higher plants, and the gametes
appear even later (Dickinson and Grant-Downton 2009).
However, this truncation of microgametophye development,
when compared with lower plants, indicates that epigenetic
Abbreviations: DDM1, DECREASE IN DNA METHYLATION
1; DME, DEMETER; DRM2, de novo DNA methyltransferase 2;
MET1, DNA methyltransferase 1; miRNA, microRNA; MMC,
megaspore mother cell; RdDM, RNA-dependent DNA methylation; siRNA, small interfering RNA; TE, transposable
element.
Introduction
The life cycle of flowering plants alternates between a diploid
sporophytic phase and a haploid gametophytic phase. As part
Special Focus Issue – Mini Review
Jose F. Gutierrez-Marcos1,* and Hugh G. Dickinson2
Epigenetic Reprogramming in the Male
Gametes
Plant Cell Physiol. 53(5): 817–823 (2012) doi:10.1093/pcp/pcs052, available online at www.pcp.oxfordjournals.org
! The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 53(5): 817–823 (2012) doi:10.1093/pcp/pcs052 ! The Author 2012.
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J. F. Gutierrez-Marcos and H. G. Dickinson
events taking place in the young gametophyte are likely to
impact on the epigenomic landscape of the sperm themselves.
To capture all the events that may contribute to epigenetic
reprogramming in male gametes, this section of the review
will discuss the epigenetic events taking place during microsporogenesis and microgametogenesis leading to the development of the tricellular pollen encapsulating two sperm cells.
A number of epigenetic pathway genes are required for male
gametophyte development in toto. In Arabidopsis, studies of
met1 mutants have shown that maintenance CG methylation is
required for the inheritance of epigenetic marks throughout gametophyte generation (Saze et al. 2003). Likewise, a
number of changes in chromatin structure are reported to be
required for successful development of the male gametophyte
(Berr et al. 2010).
The epigenetics of microsporogenesis
Little is known of the epigenetics of plant meiosis, but data
from other organisms suggest that recombination and synapsis
are guided by epigenetic marks (Borde et al. 2009, Takada et al.
2011). Furthermore, expression data from both rice and
Arabidopsis show that normally silenced transposable elements
(TEs) and neighboring sequences become transcribed during
meiotic prophase (Chen et al. 2010, Yang et al. 2011), indicating a partial release of epigenetic repression. In addition, recent
data indicate that DNA methylation and histone modifications
play a pivotal role in the regulation of meiosis (Perrella et al.
2010, Melamed-Bessudo and Levy 2012, Mirouze et al. 2012).
Further evidence for epigenetic reprogramming during meiosis
is provided by increased transcription of 55 genes of putative
mitochondrial origin inserted in a silenced centromeric
chromosomal region (Chen et al. 2010).
Interestingly, TE transcripts are not over-represented in the
microspore transcriptome (Honys and Twell 2004), indicating
that this phase of activation is temporary and that epigenetic
silencing pathways, presumably involving RNA-dependent
DNA methylation (RdDM), are restored post-meiosis. Transcriptional analyses do not often help to identify key epigenetic
regulators, instead mutant analyses have shown that in rice
MEIOSIS ARRESTED IN LEPTOTENE 1 and 2 (MEL1 and 2)
are required for meiosis (Nonomura et al. 2007, Nonomura
et al. 2011). MEL1 is the ortholog of Arabidopsis AGO5,
while MEL2 is an RNA recognition motif (RRF) protein, suggesting that interaction with intergenic and/or repeat-associated
small interfering RNAs (siRNAs) in the case of MEL1, and specific RNA processing in the case of MEL2, are required for meiotic progression.
There is indirect evidence that young microspores remain
developmentally labile, as both temperature and nutrient stress
(Dickinson 1994) can divert them to a sporophytic pathway
resulting in androgenic embryogenesis, a process greatly
exploited by industry (Dunwell 2010). Sequence data are now
available for rice microspores (Wei et al. 2011) and are showing
that these cells contain a number of novel microRNAs
818
(miRNAs) and stage-specific enrichment for certain
siRNAs—but surprisingly not those likely to be involved in TE
silencing. Likewise, small RNA sequence data are now available
for four stages of male gametophyte development in
Arabidopsis (Le Trionnaire et al. 2011), which has revealed
new putative miRNAs, most of them specific to the microspore
stage, suggesting that miRNA activity is essential for the establishment of male gametophytic ‘developmental commitment’.
The epigenetics of microgametogenesis
The first mitotic division of the pollen is unusual in that it is
asymmetric. Recent studies have identified a number of genes
including DUO1/3 (Brownfield et al. 2009), APC/C cell cycle
regulators (Zheng et al. 2011) and also an miRNA (Palatnik
et al. 2007) essential for this division. However, the processes
controlling the formation of the cytoskeleton that asymmetrically partitions the cytoplasm into two daughter cells (i.e. the
vegetative and generative cells), and why they pursue such different fates, remain unclear (Berger and Twell 2011). The
answer may lie with a changing epigenetic landscape accompanying these formative cell divisions. Certainly, unraveling epigenetic events during pollen development is complicated by
the fact that maturing male reproductive cell lines are invested
by numbers of different and very active tissues, and that following pollen mitosis I, pollen daughter cells follow radically
different developmental pathways. Immunohistochemical analysis of cytosine methylation has, however, shown differences
between the two nuclei of tobacco pollen (Oakeley et al. 1997),
but these chromatin differences are most probably established
after pollen mitosis I. More recently, fractionation strategies
have been developed to isolate both transcripts and small
RNA populations from pollen cells at key developmental
stages (Honys and Twell 2004, Le Trionnaire et al. 2011), as
well as sperm cells from mature pollen grains (Borges et al.
2008). Combining these technologies with large-scale sequencing has indicated that epigenetics may play a unique and
active role in plant male gametogenesis. From the earliest transcriptomic analyses it became clear that small RNA pathways
are very active early in pollen development, including those
mediating miRNA formation and action. The identification of
RDR6 and DCL3 at these early stages has also pointed to the
activity of siRNA processing pathways operating in pollen
(Grant-Downton et al. 2009a). Intriguingly, many key components of small RNA pathways persist through pollen development into the mature pollen grain and are particularly active in
the sperm (Borges et al. 2008, Borges et al. 2011), reinforcing
the view that cells of the male gametophyte are under strict
epigenetic control throughout development (summarized
in Fig. 1).
Expression data from both large-scale sequencing
(Grant-Downton et al. 2009b, Le Trionnaire et al. 2011) and
microarrays (Chambers and Shuai 2009) have confirmed the
presence of pollen non-coding small RNA populations and
that the majority of pollen miRNAs are also present in the
Plant Cell Physiol. 53(5): 817–823 (2012) doi:10.1093/pcp/pcs052 ! The Author 2012.
Epigenetic reprogramming in plant gametes
Sporogenesis
Meiosis
MALE
MEL1
MET1
DDM1
MEL1
MET1
DDM1
AGO9
FEMALE
Gametogenesis
DDM1
MET1
DME1
AGO5
SRRP1
Fig. 1 Overview of the epigenetic regulation taking place in male and female reproductive lineages of flowering plants. Top panel: during male
reproductive development, epigenetic signals from within the anther locule probably contribute to microspore specification. During male
meiosis, MEL1 epigenetically regulates meiotic progression of the meiocytes in rice, although it is not known whether other epigenetic signals also
regulate this process in other plants. In addition, MET1 and DDM1 regulate meiotic recombination. During pollen maturation, DDM1-dependent
small RNAs are derived in the vegetative nucleus and migrate to sperm cells to repress TE activity. However, it is not yet known if the sperm cells
play a role in regulating vegetative cell function. Bottom panel: during female reproductive development, a megaspore mother cell is specified in
the developing ovule by the independent action of AGO9 and AGO5 in surrounding sporophytic tissues to restrict megaspore initiation and
promote megasporogenesis, respectively. Meiotic progression is also regulated in rice female lineages by MEL1 and/or possibly by as yet unknown
factors from nucellar tissues. In addition, MET1 and DDM1 regulate meiotic recombination. Female gametes are formed within the embryo sac
and adopt distinct epigenetic features, such as specific DNA methylation patterns, that in Arabidopsis are mediated by the actions of the DNA
methyltransferase MET1, the DNA glycosylase DME1, and the histone chaperone SSRP1. It is possible that the sporophytic integuments may also
confer epigenetic control over female gametogenesis. Germline lineages are colored in orange, accessory surrounding cells are highlighted in
green, and other integument cells are colored in yellow. Solid arrows indicate known epigenetic factors and broken arrows indicate possible
epigenetic signals.
sporophyte. However, detailed expression analysis has revealed
that not all male gametophytic miRNAs are able to cleave their
target sequences, which suggests that they may act to regulate
gene expression by translational repression (Grant-Downton
et al. 2009b). This complex mode of regulation may be significant in pollen as it is maintained in a dormant state prior to
release from the anther. Further, sequence data from sperm
cells have also identified abundant small RNAs and, more importantly, miRNAs, specifically enriched in the sperm (Slotkin
et al. 2009, Borges et al. 2011). While molecular analyses have
shown the presence of both miRNAs and their ‘products’, evidence of their function is essentially circumstantial. However, it
has been shown recently that artificial miRNAs are able to
down-regulate endogenous target transcripts not only in
mature pollen (Coimbra et al. 2009) but also in the germline
(Slotkin et al. 2009). The presence of abundant trans-acting
siRNAs in developing pollen (Grant-Downton et al. 2009b, Le
Trionnaire et al. 2011) coupled with the impaired pollen tube
growth observed in mutants defective in the siRNA pathway
suggests that trans-acting siRNAs may also play an important
part in development pre- and post-germination. In addition,
natural antisense siRNAs (nat-siRNA) would also appear to be
important in male gametophyte as shown by the misregulation
of a nat-siRNA in Arabidopsis which results in impaired fertilization (Ron et al. 2010).
The very late formation of a germline, sensu stricto, in plants
suggests that the complex mechanisms evolved by animals to
suppress TE activity in the germline (Klattenhoff and Theurkauf
2008) may be absent in the male gametophyte. Analysis of
epigenetic pathways and small RNA populations in the sperm
and vegetative cells of Arabidopsis supports this hypothesis
(Slotkin et al. 2009). Late in pollen development the chromatin
remodeling factor DECREASE IN DNA METHYLATION 1 (DDM1)
becomes down-regulated in the vegetative nucleus with a concomitant increase in transcripts of retrotransposons. Strikingly,
analysis of sperm small RNAs revealed the presence of high
levels of TE-derived siRNAs, leading to the proposal that
siRNAs generated in the vegetative nucleus are translocated
to the sperm where they increase the silencing of TEs in the
gametic genome by reinforcing RdDM (Slotkin et al. 2009).
Plant Cell Physiol. 53(5): 817–823 (2012) doi:10.1093/pcp/pcs052 ! The Author 2012.
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J. F. Gutierrez-Marcos and H. G. Dickinson
In support of this intercellular movement of small RNAs, it has
been shown that expression of a transgenic sperm-specific
green fluorescent protein (GFP) was down-regulated by an artificial miRNA expressed in the vegetative cell under the LAT52
promoter (Slotkin et al. 2009). Importantly, the LAT52
promoter is expressed in both the microspore and binucelate
stages of pollen development (Eady et al. 1995), so it remains
possible that this silencing mechanism is established well before
pollen mitosis II, with vegetative cell siRNAs first silencing germline TEs in the generative cell. The possibility of siRNA translocation between the vegetative cell and the sperm is
supported by the evidence for sperm cell cytoplasmic projections that link the vegetative nucleus with the sperm within the
male germ unit (McCue et al. 2011).
Intriguingly, the DNA glycosylase DEMETER (DME) has
recently been found to be restricted to the pollen vegetative
cell (Schoft et al. 2011). In the female central cell, DME acts to
hypomethylate specific DNA sequences, and is responsible for
activating the maternal alleles of certain imprinted genes (Hsieh
et al. 2011). Transmission of the mutant dme allele through
pollen is reported to be reduced for some Arabidopsis ecotypes,
indicating that it is required for gametophyte and/or progametic development—presumably by establishing the DNA
methylation landscape of the vegetative cell. While DME is
expressed in the principal accessory cells of both male and
female germlines, its absence from the sperm indicates that it
has no influence on the methylome of male gametes, which
may play a key part in establishing the parent-of-origin methylation asymmetry observed at some imprinted loci in plant
gametes (Gutierrez-Marcos et al. 2006; see also Ikeda 2012).
It is possible that DME-induced hypomethylation of TEs in
the vegetative nucleus results in their transcriptional activation
and may contribute to an increase in TE-derived siRNAs.
Therefore, the abundant siRNAs reported in pollen vegetative
cells may be a product of both chromatin remodeling and
DNA hypomethylation. However, it is not yet clear whether
DME specifically targets TEs for demethylation in the
vegetative cell.
Collectively, these studies reveal that all components of the
epigenetic machinery play a pivotal role in the development
and reprogramming of the male germline.
Epigenetic Reprogramming in Female Gametes
The female gametes are formed within the female gametophyte, which is generated after a series of divisions from a
single surviving haploid megaspore cell (Berger and Twell
2011). The megaspore undergoes three syncytial divisions to
form an eight-nucleate embryo sac/female gametophyte that
then cellularizes into seven cells that differentiate into four
morphologically distinct cell types. Two of these adjacently
located cells differentiate into the haploid egg and the homodiploid central cell gametes. Although is not clearly understood
how these two genetically identical female gametes
820
differentiate during cellularization, it is possible to speculate
that this process probably involves epigenetic processes
initiated during the syncytial developmental phase of the
female gametophyte and that culminates in cellularization.
Although this hypothesis is not yet supported by experimental
data, the analysis of mutants which affect nuclear and cellular
proliferation in the embryo sac leading to the production of
extra gametes (Evans 2007, Purugganan and Fuller 2009)
suggests that the fate of female gametes is not only determined
by positional information but could also involve global epigenetic changes such as differences in chromatin structure
(Garcia-Aguilar et al. 2010, Pillot et al. 2010). This section of
the review will focus on the epigenetic events, mainly
non-coding RNAs and DNA methylation, that take place
during the formation of the female gametophyte and gametogenesis (summarized in Fig. 1).
The epigenetics of megagametogenesis
The megaspore mother cells (MMCs) are specified in a subepidermal position of the sporangia. Intriguingly, the identification
of an argonaute mutant in Arabidopsis that develops multiple
MMCs indicates that the fate of the female spore mother cell is
epigenetically regulated, most probaby by the action of small
non-coding RNAs (Olmedo-Monfil et al. 2010). However,
AGO9 is not expressed in the subepidermal layer where the
MMC is specified, but instead is present in the adjacent epidermal cell layer, which could indicate the non-cell-autonomous
action of AGO9. More recently, analysis of a mutant allele for
AGO5 has revealed that the small RNA pathway also acts in
somatic nucellar cells to promote megagametogenesis in megaspores (Tucker et al. 2012). This analysis also revealed that
AGO5 acts independently of AGO9, thus pointing to the complex role that small RNAs play in megagametophyte development (Fig. 1). These finding are perhaps not surprising as
non-coding small RNAs have recently been implicated in the
non-cell-autonomous regulation of gene expression in other
plant organs by the RdDM pathway (Dunoyer et al. 2010,
Molnar et al. 2010). The hypothesis that similar non-cellautonomous mechanisms may take place in the specification
of sporogenic cell fate is further supported by the recent analysis of maize transgenic lines with down-regulated homologs of
Arabidopsis de novo DNA methyltransferase 2 (DRM2), which
also exhibit ectopic embyo sac formation from supernumerary
MMCs (Garcia-Aguilar et al. 2010).
Intriguingly, AGO9 and de novo methyltransferases interact
with TE-derived siRNAs, whose activity is required to silence
TEs in the female gametophyte (Duran-Figueroa and
Vielle-Calzada 2010, Olmedo-Monfil et al. 2010, Singh et al.
2011). However, it remains to be shown if discrete proteincoding loci are affected by this epigenetic regulation and if
they directly participate in the development of the female
gametophyte.
Regulation of the transition from mitosis to meiosis in the
female gametophyte is also epigenetically regulated. This view is
Plant Cell Physiol. 53(5): 817–823 (2012) doi:10.1093/pcp/pcs052 ! The Author 2012.
Epigenetic reprogramming in plant gametes
supported by the discovery of a rice meiotic mutant, meiosis
arrested at leptotene 1 (mel-1), whose affected gene encodes a
sporogenous cell-specific Argonaute protein. However, the role
of non-coding RNAs in the mitotic to meiotic switch in other
species remains unclear. It is envisaged that further investigation to reveal the identity of MEL1 targets could lead to the
discovery of other key epigenetic factor(s) regulating the meiotic switch in plants.
In addition, several recent studies indicate that large-scale
epigenetic modifications take place during female gametophyte development. In particular, chromatin organization of
the egg and central cell gametes differs from that of accessory cells (Ingouff et al. 2010, Pillot et al. 2010), thus suggesting that distinct epigenetic features are established prior
to or during the cellularization of the female gametophyte.
Intriguingly, the epigenetic landscape of the two female gametes differs not just at the chromatin level (Garcia-Aguilar
et al. 2010, Ingouff et al. 2010, Pillot et al. 2010) but also in
terms of the DNA methylation patterns (Gutierrez-Marcos
et al. 2006, Jahnke and Scholten 2009). This asymmetry in
DNA methylation between female gametes is largely due to
the activity of DME in the central cell (Choi et al. 2002,
Gehring et al. 2006) and by the transcriptional repression
of DNA methyltransferases (namely MET1) by the retinoblastoma pathway during maturation of the female gametophyte (Jullien et al. 2006, Costa and Gutierrez-Marcos 2008).
This global epigenetic reprogramming taking place in central
cells may be associated with the high transcriptional activity
of TEs (Le et al. 2005, Yang et al. 2011), which probably may
contribute to an abundant siRNA population in the central
cell. It has been proposed that the global demethylation
found in the central cell may be directed by siRNAs, which
in turn could direct DME to discrete portions of the genome
(Bourc’his and Voinnet 2010). However, DME and DME-like
proteins appear preferentially to target gene-neighboring
chromosomal regions and not particularly TEs (Gehring
et al. 2009, Schoft et al. 2011). An alternative hypothesis is
that DME is directed to transcriptionally active regions of the
genome featuring a specific chromatin conformation. This
view is supported by the recent identification of an
Arabidopsis mutant (ssrp1), which codes for a chromatin
protein, FACT histone chaperone, that is required for DNA
demethylation in the central cell (Ikeda et al. 2011). Thus, an
attractive possibility raised by this study is that remodeling
of chromatin may precede genome-wide DNA demethylation
in the central cell.
Exactly why the central cell undergoes such a massive epigenetic reprogramming remains a mystery. One intriguing
explanation is that mobile small RNAs from the central cell
are able to act non-cell-autonomously to silence TEs in the
neighboring egg cell and later in the zygote (Mosher and
Melnyk 2010), but this theory awaits experimental confirmation. Epigenetic changes in the central cells could also be
pivotal for marking genes in the female central cell prior to
fertilization, which could later establish the asymmetric gene
expression observed at a significant number of imprinted gene
loci in plants (reviewed by Ikeda 2012). Molecular experimental
approaches such as a genome-wide epigenetic profiling of
female gametes could be easily performed to test this
hypothesis.
Future Challenges
While a combination of refined cell fractionation techniques
and large-scale sequencing has resulted in a clearer understanding of the epigenetic control of male and female gametophyte
development in higher plants, there are a number of areas
where lack of data is hampering significant advances.
For example, microspore-based systems for making double haploid plants would dramatically improve the rate of improvement of some crop species such as wheat, and a better
understanding of the role played by epigenetics in establishing
embryogenic developmental commitment would transform
our ability to achieve this goal. Also, the mechanism by which
the pollen exists in a transcriptionally dormant state in the
anther until it is activated on the stigma surface remains unknown. However, the observation that pollen tube growth is
compromised in dicer mutants indicates that the transcriptional activity of mature pollen may be regulated by
siRNAs. Further, the putative silencing of sperm TEs by
siRNAs generated in the vegetative cell seems to be reflected
by a similar gamete or accessory cell interaction in the female
gametophyte (Mosher and Melnyk 2010). It is important that
robust data are now obtained to ascertain the precise timing of
these events, and the process by which small RNAs regulate
gametogenesis. Another outstanding question is: do small
non-coding RNAs play a role in female gamete function? This
could be revealed by the systematic functional analysis of AGO
members that are specifically expressed in female gametes
(Wuest et al. 2010). Equally, mobile signals produced in the
female gametophytic accessory cells and/or surrounding maternal tissues may also influence gamete function and thus
warrant further investigation. Certainly, these are exciting
times for studying the dynamic nature of plant epigenomes
(see Kinoshita and Jacobsen 2012), and in particular the
part played by epigenetic factors in shaping the unique genomes of developing male and female reproductive lineages and
gametes.
Funding
This work was funded by the Royal Society; ESF/RTD
Framework COST action [FA0903]; the BBSRC [grants BB/
E008585/1 and BB/F008082/1] to J.G.-M.
Acknowledgements
We thank Liliana Costa for critical reading of the manuscript.
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